JP2008247898A - Agent for preventing or treating oxidative-stress-associated eye disease, containing triterpenoid as active ingredient - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new agent for preventing or treating oxidative-stress-associated eye diseases. <P>SOLUTION: A compound represented by formula (I) or a salt thereof can exhibit excellent capabilities of inducing a phase II xenobiotic metabolism enzyme in posterior ocular tissues such as a neuroretina, retinal pigment epithelium, and retina-choroid. Therefore, it is useful an active ingredient of an agent for preventing or treating oxidative-stress-associated eye diseases such as age-related macular degeneration, diabetic retinopathy, diabetic macular edema, and retinal pigment degeneration. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to age-related macular degeneration, diabetic retinopathy, diabetic macular edema, retinitis pigmentosa, proliferative vitreoretinopathy, retina, which contains a compound represented by the following general formula (I) or a salt thereof as an active ingredient Arterial occlusion, retinal vein occlusion, uveitis, labeling disease, retinopathy of prematurity, retinal detachment, retinal pigment epithelial detachment, optic neuropathy resulting from these diseases, optic neuropathy resulting from glaucoma, ischemic optic neuropathy, The present invention relates to a preventive or therapeutic agent for eye diseases associated with oxidative stress such as cataract.

[Where:
R _1, R _2, R _3, R _4, R _5, R ₆ and R ₇ are the same or different and each represents a hydrogen atom or an alkyl group;
X represents a cyano group or CO—Y;
Y is an aromatic selected from the group consisting of a hydrogen atom, a halogen atom, a hydroxyl group, an amino group, an alkyl group, an alkoxy group, an alkylamino group, a halogenoalkylamino group, or an imidazole ring, a pyrazole ring, a triazole ring, and a benzimidazole ring. A heterocyclic group;
The aromatic heterocyclic group defined above may be substituted with a halogen atom, an alkyl group, a phenyl group or a halogenophenyl group. ]

  The living body functions by regulating the balance of oxidation-antioxidation and maintaining a constant intracellular redox state. The state in which the balance between oxidation and antioxidant is inclined in the oxidation direction is called oxidative stress. Oxidative stress modifies and damages biopolymers such as lipids, DNA, and proteins, causing cell dysfunction and cell death, carcinogenesis, hypertension, arteriosclerosis, cranial neurodegenerative diseases, inflammation, asthma, skin diseases, aging It is known to be deeply involved in the onset and progression of chronic diseases associated with aging such as macular degeneration and cataracts. Therefore, a defense mechanism that maintains a constant oxidation-antioxidation balance is a vital defense system for the living body, and it is thought that enhancing the ability to defend against oxidative stress is useful for the prevention and treatment of these diseases. (Non-Patent Document 1).

In recent years, it has become clear that the Keap1-Nrf2 system plays a central role in mammalian oxidative stress defense mechanisms. Nrf2 (N F-E2 r elated f actor 2) is glutathione -S- transferase to detoxify electrophilic substances directly, the second phase enzyme and heme oxygenase xenobiotic metabolizing such quinone oxidoreductase, antioxidant enzymes such peroxiredoxin It is a transcription factor that induces and controls gene expression. Nrf2 is in steady state (unstimulated), but in the cytoplasm Keap1 (K elch-like E CH a ssociated p rotein 1) and forms a complex in an inactive state, the electrophilic agent and active oxygen When exposed to oxidative stress, such as species, the interaction with Keap1 is attenuated (dissociation is promoted) and transferred to the nucleus. Nrf2 which translocates to the nucleus is small Maf (M usculo a poneurotic F ibrosarcoma ) to form a group factors and heteroduplex mer, antioxidants response element which is a target sequence (ARE: a ntioxidant r esponsive e lement) the promoter sequence called To induce gene expression of the above-described enzymes. As described above, Nrf2 is known to uniformly control defense against oxidative stress in the living body and detoxification and excretion of xenobiotics (Non-patent Document 2).

  In addition, in mice in which the Keap1-Nrf2 system is broken (Nrf2 gene-deficient mice), the balance of intracellular oxidation-antioxidation is disrupted, and the cancer is easily developed (pronegastric cancer caused by benzpyrene, bladder cancer caused by nitrosamine, etc. ), Increased sensitivity to foreign substances / oxidative stress (liver damage due to acetaminophen, lung damage due to high oxygen exposure, etc.), inflammation / immune system abnormalities (glomerulonephritis, systemic autoimmune disease, tobacco-induced emphysema, etc.) It has been reported to show. Thus, it has been suggested that Nrf2 functional deficiency causes oxidative stress and induces diseases such as cancer, hypertension, cranial neurodegenerative diseases, and inflammation (Non-patent Document 3).

  From such knowledge, application to the prevention or improvement of diseases associated with oxidative stress using activation of Nrf2 has been attempted, and the usefulness of some Nrf2 activators has been reported. For example, it has been reported that oltipraz or sulforaphane known as substances that activate Nrf2 suppresses forepgastric carcinogenesis caused by benzpyrene, and that oltipraz, a substance that activates Nrf2, inhibits bladder carcinogenesis caused by nitrosamine. (Non-patent Document 3).

  Non-patent documents 4 to 5 and patent documents 1 to 3 are reports on the usefulness of Nrf2 activators for age-related macular degeneration and cataract, which are ophthalmic diseases related to oxidative stress. Age-related macular degeneration is a degeneration associated with age-related changes in the macular, and it is known that oxidative stress such as retinaldehyde (all-trans-retinaldehyde) accumulated by exposure to ultraviolet rays damages retinal pigment cells. Non-Patent Document 4 and Patent Document 1 report that sulforaphane and bis-2-hydroxybenzylideneacetone, which are Nrf2 activators, suppress this cell damage. Non-Patent Document 5 reports that curcumin, an Nrf2 activator, suppresses galactose-induced cataract. Patent Documents 2 and 3 describe treatment methods for age-related macular degeneration drusen formation, diabetic retinopathy, and retinopathy / optic neuropathy associated with glaucoma using quercetin, which is an Nrf2 activator.

  On the other hand, triterpenoid is a general term for terpenes consisting of 30 carbons and compounds derived therefrom. The compound represented by the general formula (I) of the present invention is a kind of triterpenoid and is a derivative of oleanane having a pentacyclic structure. Patent Documents 4 and 5 include 2-cyano-3,12-dioxooleana-1,9 (11) -diene-28-oic acid, 1- [2-cyano-3,12-dioxooleana-1,9 (11 ) -Diene-28-oil] nitrile, 1- [2-cyano-3,12-dioxooleana-1,9 (11) -diene-28-oil] imidazole, and the like, which are interferons in mouse macrophages. It shows inhibitory activity against the production of nitric oxide induced by γ and is useful for the treatment of diseases such as cancer, Alzheimer's disease and Parkinson's disease. Non-patent documents 6 and 7 suggest that 1- [2-cyano-3,12-dioxooleana-1,9 (11) -diene-28-oil] imidazole has Nrf2 activation ability. It has been reported that expression of heme oxygenase 1, which is an antioxidant enzyme, was induced in organs (stomach, lung, etc.) of mice administered with the compound.

However, as a report on the compound represented by the general formula (I), a report examining the activation ability of Nrf2 in the ocular tissue and the expression of the foreign phase metabolism enzyme and antioxidant enzyme which are induced and controlled by Nrf2. There is no report examining the induction ability. In addition, there are no reports on the pharmacological action of the compound on eye diseases, and in particular, aging-related macular degeneration, diabetic retinopathy, diabetic macular edema, optic neuropathy caused by glaucoma, ischemic There are no reports on the prevention or improvement of optic nerve disorders.
International Publication No. 03/51313 Pamphlet International Publication No. 2005/063249 Pamphlet International Publication No. 2005/063295 Pamphlet WO99 / 65478 pamphlet International Publication No. 2004/064723 Pamphlet Biomed. Pharmacother. 57, 251-60 (2003) J. Biol. Chem., 278, 14, 12029-38 (2003) Separate book "Ayumi of Medicine" Redox-Medicine of Stress Protection, 46-9 (2005) Proc. Natl. Acad. Sci., 101, 10446-51 (2004) Mol. Vis., 9, 223-30 (2003) Cancer Res., 65 (11), 4789-98 (2005) Mol. Cancer Ther., 6 (1), 154-62 (2007)

  Therefore, it is an interesting problem to search for new pharmaceutical uses for the compound represented by the general formula (I).

  The inventors of the present invention conducted intensive studies to find a new pharmaceutical use of the compound represented by the general formula (I). As a result, the compound was found in the posterior ocular tissues such as the neural retina, retinal pigment epithelium, and retina choroid. The present inventors have found that it has an excellent ability to induce a foreign substance metabolic system phase II enzyme, and has reached the present invention. The same test was performed for a known compound having the same action as the compound, but the compound showed a much higher induction ability than the known compound, and the excellent usefulness of the compound was confirmed. there were.

That is, the present invention comprises a compound represented by the general formula (I) or a salt thereof (hereinafter collectively referred to as “the present compound”) as an active ingredient, age-related macular degeneration, diabetic retinopathy, Diabetic macular edema, retinitis pigmentosa, proliferative vitreoretinopathy, retinal artery occlusion, retinal vein occlusion, uveitis, label disease, retinopathy of prematurity, retinal detachment, retinal pigment epithelial detachment, for these diseases It is a prophylactic or therapeutic agent for ocular diseases associated with oxidative stress such as optic neuropathy caused by, optic neuropathy caused by glaucoma, ischemic optic neuropathy, and cataract.

[Where:
R _1, R _2, R _3, R _4, R _5, R ₆ and R ₇ are the same or different and each represents a hydrogen atom or an alkyl group;
X represents a cyano group or CO—Y;
Y is an aromatic complex selected from the group consisting of a hydrogen atom, a halogen atom, a hydroxyl group, an amino group, an alkyl group, an alkoxy group, an alkylamino group, a halogenoalkylamino group, or an imidazole ring, a pyrazole ring, a triazole ring, and a benzimidazole ring. A ring group;
The aromatic heterocyclic group defined above may be substituted with a halogen atom, an alkyl group, a phenyl group or a halogenophenyl group. ]
The present invention also includes administering to a patient a pharmacologically effective amount of the present compound as an active ingredient, age-related macular degeneration, diabetic retinopathy, diabetic macular edema, retinitis pigmentosa, proliferative vitreoretinopathy, Retinal artery occlusion, retinal vein occlusion, uveitis, Label disease, retinopathy of prematurity, retinal detachment, retinal pigment epithelial detachment, optic neuropathy caused by these diseases, optic neuropathy caused by glaucoma, ischemic optic neuropathy This is a method for preventing or treating eye diseases associated with oxidative stress such as cataract.

  The present invention also relates to age-related macular degeneration, diabetic retinopathy, diabetic macular edema, retinitis pigmentosa, proliferative vitreoretinopathy, retinal artery occlusion, retinal vein occlusion, uveitis, label disease, premature infant Book for preventing or treating eye diseases associated with oxidative stress such as retinopathy, retinal detachment, retinal pigment epithelial detachment, optic neuropathy caused by these diseases, optic neuropathy caused by glaucoma, ischemic optic neuropathy, cataract A compound.

  The present invention also relates to age-related macular degeneration, diabetic retinopathy, diabetic macular edema, retinitis pigmentosa, proliferative vitreoretinopathy, retinal artery occlusion, retinal vein occlusion, uveitis, label disease, premature infant Manufactures prophylactic or therapeutic agents for retinopathy, retinal detachment, retinal pigment epithelial detachment, optic neuropathy caused by these diseases, optic neuropathy caused by glaucoma, ischemic optic neuropathy, cataract and other eye diseases associated with oxidative stress For the use of this compound.

  Each group used in the claims and specification shall have the following meaning throughout the claims and specification.

  “Halogen atom” means fluorine, chlorine, bromine or iodine.

  “Alkyl” refers to linear or branched alkyl having 1 to 6 carbon atoms. Specific examples include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl and the like.

  “Alkoxy” represents straight-chain or branched alkoxy having 1 to 6 carbon atoms. Specific examples include methoxy, ethoxy, n-propoxy, n-butoxy, n-pentoxy, n-hexyloxy, iopropoxy, isobutoxy, sec-butoxy, tert-butoxy, isopentyloxy and the like.

  “Alkylamino” refers to monoalkylamino having 1 to 6 carbon atoms or dialkylamino having 2 to 12 carbon atoms. Specific examples of monoalkylamino include methylamino, ethylamino, propylamino, butylamino, hexylamino and the like, and specific examples of dialkylamino include dimethylamino, diethylamino, dipropylamino, dibutylamino, dihexylamino and the like.

  “Halogenoalkyl” refers to an alkyl having one or a plurality of the same or different halogen atoms as substituents.

  “Halogenophenyl” refers to phenyl having one or more of the same or different halogen atoms as substituents.

  “Halogenoalkylamino” refers to amino having one or a plurality of the same or different halogenoalkyl as a substituent.

  When this compound has a free amino group, a free alkylamino group or a free halogenoalkylamino group as a substituent, these substituents may be protected with a protecting group. Also, when the aromatic heterocyclic ring has a free nitrogen atom, the nitrogen atom may be protected with a protecting group.

  “A protective group for a free amino group, a free alkylamino group, a free halogenoalkylamino group or an aromatic heterocyclic group having a free nitrogen atom” refers to an unsubstituted alkenyl group such as an allyl group; a formyl group, etc. Hydrocarbonyl group; substituted or unsubstituted alkylcarbonyl group such as acetyl group, trichloroacetyl group, trifluoroacetyl group, benzoyl group, 4-chlorobenzoyl group, picolinoyl group, substituted or unsubstituted arylcarbonyl group, or unsubstituted aromatic Heterocyclic carbonyl group; methoxycarbonyl group, isobutoxycarbonyl group, tert-butoxycarbonyl group, 2,2,2-trichloroethoxycarbonyl group, benzyloxycarbonyl group, diphenylmethoxycarbonyl group, phenoxycarbonyl group, m-nitrophenoxycarbonyl Basics Substituted or unsubstituted alkyloxycarbonyl, or substituted or unsubstituted aryloxycarbonyl group; methylsulfonyl group, benzylsulfonyl group, phenylsulfonyl group, 4-chlorophenylsulfonyl group, tolylsulfonyl group, 2,4,6-trimethylphenylsulfonyl group A substituted or unsubstituted alkylsulfonyl group, or a substituted or unsubstituted arylsulfonyl group; a free amino group, a free alkylamino group, a free halogenoalkylamino group, or an aromatic heterocyclic group having a free nitrogen atom Those commonly used as protecting groups for.

  The substituted alkyl group, substituted alkylcarbonyl group, substituted arylcarbonyl group, substituted alkyloxycarbonyl group, substituted aryloxycarbonyl group, substituted alkylsulfonyl group or substituted arylsulfonyl group are each a halogen atom, an alkoxy group, an alkyl group, An alkyl group, an alkylcarbonyl group, an arylcarbonyl group, an alkyloxycarbonyl group, an aryloxycarbonyl group, an alkylsulfonyl substituted with one or more groups selected from an aryl group, a halogenoaryl group, an alkoxyaryl group, and a nitro group Represents a group or an arylsulfonyl group.

  In the “multiple groups” in the present invention, each group may be the same or different, and preferably represents 2 or 3 groups, more preferably 2 groups.

  Further, the “group” in the present invention includes a hydrogen atom, a halogen atom and an oxo ligand.

  The “salt” in the present compound is not particularly limited as long as it is a pharmaceutically acceptable salt. Salts with inorganic acids 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, naphthalenesulfonic acid, sulfosalicylic acid Salts with acids, quaternary ammonium salts such as methyl bromide and methyl iodide, bromide ions, chloride ions, iodine ions Salts with halogen ions such as lithium, salts with alkali metals such as lithium, sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, metal salts with iron and zinc, salts with ammonia, Ethylenediamine, 2-aminoethanol, 2,2-iminobis (ethanol), 1-deoxy-1- (methylamino) -2-D-sorbitol, 2-amino-2- (hydroxymethyl) -1,3-propanediol And salts with organic amines such as procaine and N, N-bis (phenylmethyl) -1,2-ethanediamine.

  When geometric isomers or optical isomers are present in the present compound, these isomers are also included in the scope of the present invention.

  Moreover, this compound may take the form of the hydrate or the solvate.

  Furthermore, when proton tautomerism exists in this compound, those tautomers are also included in the scope of the present invention.

(A) As a preferable example in this compound, in the compound represented by the general formula (I), a compound in which each group is a group shown below or a salt thereof can be mentioned.

_{(A1) R 1, R 2} , R 3, R 4, R 5, R 6 and _{R 7} represents an alkyl group; and / or (a2) X represents a cyano group or a CO-Y; and / Or (a3) Y represents a hydroxyl group, an amino group, an alkoxy group, an alkylamino group, a halogenoalkylamino group or an aromatic heterocyclic group selected from the group consisting of an imidazole ring and a pyrazole ring; and / or (a4) The aromatic heterocycle defined above may be substituted with an alkyl group, a phenyl group or a halogenophenyl group.

  That is, in the compound represented by the general formula (I), a compound or a salt thereof comprising one or more combinations selected from the above (a1), (a2), (a3) and (a4).

(B) As a more preferable example in this compound, the compound or its salt in which each group is a group shown below in the compound shown by general formula (I).

_{(B1) R 1, R 2} , R 3, R 4, R 5, R 6 and _{R 7} represents an alkyl group; and / or (b2) X represents a cyano group or a CO-Y; and / Or (b3) Y represents a hydroxyl group, an amino group, an alkoxy group, an alkylamino group, a halogenoalkylamino group or an aromatic heterocyclic group selected from the group consisting of an imidazole ring and a pyrazole ring; and / or (b4) When Y is an imidazole ring, the imidazole ring may be substituted with an alkyl group, a phenyl group, or a halogenophenyl group.

  That is, in the compound represented by the general formula (I), a compound or a salt thereof comprising one or more combinations selected from the above (b1), (b2), (b3) and (b4).

(C) As a more preferable example in the present compound, a compound represented by the general formula (I) defined as described above, or a salt thereof, each of which is a group shown below can be mentioned.

_{(C1) R 1, R 2} , R 3, R 4, R 5, R 6 and _{R 7} represents a methyl group; and / or (c2) X is a cyano group, a carboxyl group, a methyl oxycarbonyl group, ethyl Oxycarbonyl group, butyloxycarbonyl group, aminocarbonyl group, methylaminocarbonyl group, ethylaminocarbonyl group, dimethylaminocarbonyl group, 2,2,2-trifluoroethylaminocarbonyl group, imidazol-1-ylcarbonyl group, 2 -A methylimidazol-1-ylcarbonyl group, a 2-phenylimidazol-1-ylcarbonyl group, a 2- (4-fluorophenyl) imidazol-1-ylcarbonyl group or a pyrazol-1-yl group.

  That is, in the compound represented by the general formula (I) defined as described above, a compound or a salt thereof comprising one or more combinations selected from the above (c1) and (c2).

The most preferred examples of the compound include 2-cyano-3,12-dioxooleana-1,9 (11) -diene-28-oic acid represented by the following formula (II) and 2 represented by the following formula (III). -Cyano-3,12-dioxooleana-1,9 (11) -diene-28-oic acid methyl ester, or 1- [2-cyano-3,12-dioxooleana-1, represented by the following formula (IV) 9 (11) -Diene-28-oil] imidazole.

  This compound can be manufactured according to the normal method in the field | area of synthetic organic chemistry, and can also be manufactured based on the method described in WO99 / 65478 or WO2004 / 064723.

  In the present invention, oxidative stress refers to stress experienced by individuals or cells due to reactive oxygen species, ultraviolet rays, radiation, metals, chemical substances, or stimuli that cause production of reactive oxygen species in cells. In addition, an oxidative stress-related eye disease refers to an eye disease caused by such oxidative stress, particularly a disease caused by oxidative stress in the vitreous body, retina, choroid, sclera or optic nerve, for example, , Age-related macular degeneration (drusen formation in early age-related macular degeneration, atrophic age-related macular degeneration, wet age-related macular degeneration), diabetic retinopathy, diabetic macular edema, retinitis pigmentosa, proliferative vitreoretinopathy, Retinal artery occlusion, retinal vein occlusion, uveitis, Label disease, retinopathy of prematurity, retinal detachment, retinal pigment epithelial detachment, optic neuropathy caused by these diseases, optic neuropathy caused by glaucoma, ischemic optic neuropathy And eye diseases such as cataracts.

  The compound can be formulated as necessary by adding a pharmaceutically acceptable additive and using a technique widely used as a single preparation or a combined preparation.

  When this compound is used for the prevention or treatment of the aforementioned eye diseases, it can be administered to patients orally or parenterally. As the dosage form, oral administration, topical administration to the eye (instillation administration) , Intravitreal administration, subconjunctival administration, subtenon administration, etc.), intravenous administration, transdermal administration, etc., and dosage forms suitable for administration with pharmaceutically acceptable additives as necessary To be formulated. Examples of dosage forms suitable for oral administration include tablets, capsules, granules, powders, etc. Examples of dosage forms suitable for parenteral administration include injections, eye drops, eye ointments, gels, An insertion agent etc. are mentioned. These can be prepared using conventional techniques widely used in the field. In addition to these preparations, this compound can also be made into preparations for DDS (drug delivery system) such as preparations for intraocular implants and microspheres.

  For example, the tablet is an excipient such as lactose, glucose, D-mannitol, anhydrous calcium hydrogen phosphate, starch, sucrose; carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, crospovidone, starch, partially pregelatinized starch , Disintegrating agents such as low-substituted hydroxypropylcellulose; binders such as hydroxypropylcellulose, ethylcellulose, gum arabic, starch, partially pregelatinized starch, polyvinylpyrrolidone, polyvinyl alcohol; magnesium stearate, calcium stearate, talc, hydrous silicon dioxide , Lubricants such as hydrogenated oils; refined sucrose, hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, ethylcellulose, polyvinyl chloride Coating agents pyrrolidone; citric acid, aspartame, using ascorbic acid, appropriately selected and the like flavoring agent such as menthol, can be prepared.

  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.

  Eye drops include isotonic agents such as sodium chloride and concentrated glycerin; buffering agents such as sodium phosphate and sodium acetate; surface activity such as polyoxyethylene sorbitan monooleate, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil Agents; Stabilizers such as sodium citrate and sodium edetate; can be selected and used as needed from preservatives such as benzalkonium chloride and paraben, and pH is acceptable for ophthalmic preparations Although it may be in the range, it is usually preferably in the range of 4-8. The eye ointment can be prepared using a commonly used base such as white petrolatum or liquid paraffin.

  The preparation for intraocular implant 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 present invention also relates to a method for preventing or treating an eye disease associated with oxidative stress comprising administering a pharmacologically effective amount of the present compound as an active ingredient to a patient (human).

  The dose of this compound can be appropriately changed depending on the dosage form, the severity of the patient's symptoms, age, weight, doctor's judgment, etc. 0.01-5000 mg per day, preferably 0.1-2500 mg, more preferably 1-1000 mg can be administered in one or several divided doses. 0.01 to 200 mg per dose can be administered in one or several divided doses. In the case of eye drops, 0.000001 to 10% (W / V), preferably 0.00001 to 1% (W / V), more preferably 0.0001-0.1% (W / V) active ingredient concentration can be instilled once or several times a day. Furthermore, in the case of an intraocular implant preparation, an intraocular implant preparation containing 0.01 to 2000 mg for an adult can be implanted in the eye.

  When a test described below was performed, in Test 1, the compound was converted to a foreign substance metabolism phase 2 enzyme whose expression is controlled by Nrf2, NAD (P) H quinone oxidoreductase (NAD (P) H quinone oxidoreductase 1 : Hereinafter also referred to as “NQO1”) was shown to be induced in nervous system cells. In Test 2, the present compound was shown to induce NQO1 in the neural retina and retinal pigment epithelium. That is, this compound induces and controls the gene expression of foreign phase 2 metabolizing phase enzymes in the posterior ocular tissues such as the neural retina, retinal pigment epithelium, and retina choroid, and enhances the ability to protect against oxidative stress. Prevent or reduce Furthermore, in Test 3, the compound was shown to improve photodamage in a rat photodamage model. In Test 4, it was shown that this compound has an excellent choroidal neovascularization inhibitory effect in a laser-induced rat choroidal neovascularization model. Based on the above, this compound is age-related macular degeneration (drusen formation in early age-related macular degeneration, atrophic age-related macular degeneration, wet age-related macular degeneration), diabetic retinopathy, diabetic macular edema, retinitis pigmentosa, proliferation Vitreoretinopathy, retinal artery occlusion, retinal vein occlusion, uveitis, label disease, retinopathy of prematurity, retinal detachment, retinal pigment epithelial detachment, optic neuropathy resulting from these diseases, optic nerve resulting from glaucoma It is useful as a prophylactic or therapeutic agent for ophthalmic diseases associated with oxidative stress such as injury, ischemic optic neuropathy and cataract.

  Although the result of Tests 1-4 and a formulation example is shown below, these examples are for understanding this invention better, and do not limit the scope of the present invention.

Test 1. NQO1 Inducibility Evaluation Test and Cell Viability Evaluation Test Using Nervous System Cells NQO1 induction ability of the present compounds and compounds with reported Nrf2 activation ability was evaluated using neural cells. In addition, the cell viability of these compounds was evaluated.

(Experiment overview)
Using PC12 (rat adrenal brown cell type), which is widely used as a nervous system cell, this compound, 2-cyano-3,12-dioxooleana-1,9 (11) -diene-28-oiic acid (hereinafter, "CDDO"), 2-cyano-3,12-dioxooleana-1,9 (11) -diene-28-oic acid methyl ester (hereinafter also referred to as "CDDO-Me") and 1- [2-cyano −3,12-dioxooleana-1,9 (11) -diene-28-oil] imidazole (hereinafter also referred to as “CDDO-Im”), and Nrf2 of quercetin, which is a compound that has been reported to activate Nrf2. Activation ability was evaluated. In addition, the cell viability of these compounds was also evaluated. As an index of Nrf2 activation, the enzyme activity of NQO1 (foreign substance metabolic phase 2 enzyme), which is known to increase in expression due to Nrf2 activation, was used.

(NQO1 activation ability evaluation test method)
PC12 was seeded in a 96-well plate at 3 × 10 ⁴ cells / well and cultured under conditions of 37 ° C., 5% CO ₂ /95% air for 24 hours. As a culture solution of PC12, RPMI1640 containing 5% fetal bovine serum (FBS) and 10% horse serum (HS) was used. Next, the culture solution was removed and replaced with a culture solution of different concentration containing CDDO, CDDO-Me, CDDO-Im or quercetin (the preparation method will be described later). The NQO1 activity of each cell was measured after culturing for 24 hours under conditions of 37 ° C. and 5% CO ₂ /95% air. NQO1 activity was assayed with reference to the method of Prochaska et al. (Anal Biochem. 1988 Mar; 169 (2): 328-36). Specifically, a reaction mixture containing menadione and MTT (methyl thiazole tetrazoleum) was added to a cell extract obtained by lysing cells with a surfactant, reacted for 5 minutes, and then the absorbance at 550 nm was measured. NQO1 activity was calculated.

(Cell viability evaluation test method)
PC12 is cultured in the same procedure as in the (NQO1 activation ability evaluation test method), and the culture solution is replaced with a culture solution of different concentrations containing CDDO, CDDO-Me, CDDO-Im or quercetin at 37 ° C., 5 Cell viability was measured after 24 hours of culturing under conditions of% CO ₂ /95% air. For the measurement, Cell Counting Kit-8 (Dojindo Laboratories) was used.

(Data analysis)
The NQO1 activity (%) of CDDO, CDDO-Me, CDDO-Im and quercetin was expressed with the NQO1 activity of the cells treated with the base medium described later as 100%. The maximum value of the NQO1 induction rate was Emax, and the 50% induction concentration (EC ₅₀ ) of NQO1 activity was calculated with EXSAS (Ver. 7.1.6; Arm Co., Ltd.). Moreover, the survival rate (%) of CDDO, CDDO-Me, CDDO-Im, and quercetin was represented by setting the survival rate of the cells treated with the base culture solution as 100%. The 50% cytotoxic concentration (LC ₅₀ ) was calculated by EXSAS.

(Preparation of culture solution containing test compound)
Each compound was dissolved in DMSO, and the concentration of each compound was adjusted as follows by diluting these solutions 1000 times with the culture solution of each cell. In addition, a culture solution containing only DMSO was used as a base culture solution.

CDDO: 0.3, 1, 3, 10, 30, 100, 300, 1000, 3000, 10000 and 30000 (nM)
CDDO-Me: 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000, 3000, 10000 and 30000 (nM)
CDDO-Im: 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000, 3000, 10000 and 30000 (nM)
Quercetin: 1.56, 3.13, 6.25, 12.5, 25, 50, 100 and 200 (μM)
(result)
Table 1 shows the EC ₅₀ , Emax, LC ₅₀ and LC ₅₀ / EC ₅₀ of each compound in PC12.

(Discussion)
As is clear from Table 1, it was shown that CDDO, CDDO-Me, CDDO-Im and quercetin induce NQO1 in PC12. Here, although quercetin was found to induce NQO1 in PC12, its inducibility are _{The EC 50} values = 6600nM. On the other hand, CDDO, CDDO-Me and CDDO-Im have excellent NQO1 inducing ability in PC12, and it is particularly surprising that CDDO-Im was an extremely excellent EC ₅₀ value = 11.4 nM. Should be the result.

  From the above results, the present compounds represented by CDDO, CDDO-Me and CDDO-Im have an excellent ability to induce Nrf2 in nervous system cells, and are significantly prevented or improved for ocular diseases involving oxidative stress. It was shown to have an effect.

Test 2. NQO1 induction ability evaluation test using rats Rats were used to evaluate the ability of CDDO-Me to activate NQO1 in the retina choroid including the neural retina and retinal pigment epithelium by oral administration.

(experimental method)
CDDO-Me solution suspended in 1% (W / V) methylcellulose aqueous solution (prepared by dissolving methylcellulose in ultrapure water) as a base for 6-week-old Wistar male rats (Nippon Charles River) Was administered orally once daily for 5 days at doses of 3, 10 and 30 mg / kg. In addition, the number of examples in each administration group is 4 eyes (2 animals). One, three and five days after administration, a choroid fraction containing the rat neural retina and retinal pigment epithelium was removed and homogenized to obtain a tissue extract. NQO1 activity in the extract was measured in the same manner as in Test 1. Further, the total protein concentration in the extract was measured, and NQO1 activity per 1 mg of total protein was calculated.

(result)
The time course of NQO1 activity in each administration group is shown in FIG. 1 (neural retina) and FIG. 2 (retina choroid including retinal pigment epithelium).

(Discussion)
As is clear from FIGS. 1 and 2, NQO1 activity was induced in any of the choroidal fractions including the neural retina and retinal pigment epithelium depending on the dose of CDDO-Me. From this result, it was shown that CDDO-Me reaches the nerve retina, retinal pigment epithelium and reticulochoroid by oral administration, and induces NQO1 in each tissue, that is, activates Nrf2. Here, the retinal tissue has an inner blood-retinal barrier (inner BRB) composed of retinal vascular endothelial cells and an outer blood retinal barrier (outer BRB) composed of retinal pigment epithelial cells. Thus, it is known that drug transport to the posterior eye region such as membrane tissue is strictly controlled. That is, even if the triterpenoid compound, which is the present compound, is administered systemically, it is not clear whether it actually reaches the neural retina, retinal pigment epithelium or reticulochoroid. However, the results of Test 2 showed that CDDO-Me, which is the present compound, reached the neural retina, retinal pigment epithelium and retina choroid, and induced NQO1 in each tissue.

  From the above results, this compound represented by CDDO-Me has an excellent Nrf2 inducing ability in the neural retina, retinal pigment epithelium and retina choroid, and has an effect of preventing or improving eye diseases involving oxidative stress. It was shown to have.

Test 3. Pharmacological test using rat photo-damage model The pharmacological effect of CDDO-Me was evaluated using a rat photo-damage model. The rat light damage model is a model animal in which oxidative stress is induced mainly in the photoreceptor cells and the retinal pigment epithelial cell layer by light exposure, and mainly as a model animal of retinal degeneration (for example, age-related macular degeneration). It is widely used.

(experimental method)
CDDO-Me solution suspended in 1% (W / V) methylcellulose aqueous solution (prepared by dissolving methylcellulose in ultrapure water) as a base for 6-week-old Wistar male rats (Nippon Charles River) Was orally administered once a day at doses of 1, 3 and 10 mg / kg for a total of 6 times from 3 days before light irradiation to immediately after light irradiation. In addition, radical scavenger phenyl-N-tert-butylnitrone (PBN) was administered once a day at a dose of 100 mg / kg, and repeated intraperitoneally three times in total from immediately before light irradiation to immediately after light irradiation, and used as a positive control. The number of cases in each administration group is 8 to 10 eyes (4 to 5 animals). Light damage was induced by irradiating with 2000 lux for 48 hours. After completion of light irradiation, dark adaptation for 4 hours was performed in a dark room, and electroretinogram (ERG) was measured. The amplitudes of the a wave and the b wave were calculated from the obtained waveform.

(result)
FIG. 3 (a wave) and FIG. 4 (b wave) show the amplitudes of the ERG a wave and b wave in the base administration group, respectively, with 100%.

(Discussion)
As is apparent from FIGS. 3 and 4, CDDO-Me has an improvement effect on attenuation of ERG a-wave and b-wave as compared to the base administration group, and a very small dose of 3 mg / kg. However, it was shown to have the same therapeutic effect as 100 mg / kg PBN. From the above result, it was shown that this compound represented by CDDO-Me has the prevention or improvement effect with respect to the eye disease in which oxidative stress is concerned. In particular, it has been shown to have a significant preventive or ameliorating effect on age-related macular degeneration.

Test 4. Pharmacological test using rat laser-induced choroidal neovascularization model The usefulness of CDDO-Me was evaluated using a laser-induced rat choroidal neovascularization model.

(Production method of krypton laser-induced rat choroidal neovascularization model)
Eight-week-old male BN rats (Nippon Charles River) were administered intramuscularly with 1 ml / kg of a mixed solution (7: 1) of 5% (W / V) ketamine hydrochloride injection and 2% xylazine hydrochloride injection. After anesthesia, 0.5% (W / V) tropicamide-0.5% phenylephrine hydrochloride ophthalmic solution was instilled to make mydriasis, and photocoagulation was performed using a krypton laser photocoagulator. Photocoagulation was performed in the posterior region of the fundus, avoiding thick retinal blood vessels, and focusing on the deep retina at 8 spots per eye (coagulation conditions: spot size 100 μm, output 100 mW, coagulation time 0.1 seconds). . After photocoagulation, fundus photography was performed to confirm the laser irradiation site.

(Drug administration method)
CDDO-Me was suspended in 1% (W / V) methylcellulose solution (prepared by dissolving methylcellulose in purified water) to 2 mg / ml or 6 mg / ml, and 10 mg / kg or 30 mg / ml each time. Oral administration was performed once a day for 10 days including the operation day from 10 days before the photocoagulation operation day (10 mg / kg or 30 mg / kg per day) at a dose of kg.

(Evaluation methods)
On day 7 after photocoagulation, the rats were given general anesthesia by intramuscular administration of 1 ml / kg of a mixed solution (7: 1) of 5% (W / V) ketamine hydrochloride injection and 2% xylazine hydrochloride injection. After 5% (W / V) tropicamide-0.5% phenylephrine hydrochloride ophthalmic solution was instilled to make mydriatic, 0.1 ml of 10% fluorescein solution was injected from the tail vein, and fluorescence fundus imaging was performed. In fluorescence fundus angiography, a spot where no fluorescent exposure was observed was judged negative (no angiogenesis), and a spot where fluorescent exposure was seen was judged positive. In addition, a photocoagulation site where slight fluorescence exposure was observed was determined to be positive (with angiogenesis) when two sites were present. Thereafter, the rate of choroidal neovascularization (%) was calculated from the number of positive spots with respect to 8 spots irradiated with laser according to Formula 1, and the rate of inhibition (%) of the evaluation drug was calculated according to Formula 2. The evaluation results of CDDO-Me are shown in Table 2. The number of cases in each administration group is 8.

[Formula 1]
Incidence rate of choroidal neovascularization (%) = (number of positive spots / total number of photocoagulation sites) × 100
[Formula 2]
Inhibition rate (%) = (A0−AX) / A0 × 100
A0: incidence of choroidal neovascularization in the base administration group AX: incidence of choroidal neovascularization in the drug administration group

(Discussion)
As is apparent from Table 2, CDDO-Me was shown to inhibit choroidal neovascularization in a laser-induced rat choroidal neovascularization model. That is, it was shown that this compound represented by CDDO-Me has an excellent angiogenesis inhibitory action in the choroid and has a remarkable preventive or ameliorating effect on age-related macular degeneration.

[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.

Formulation Example 1 Eyedrops 100ml CDDO 10mg
Sodium chloride 900mg
Polysorbate 80 Appropriate amount Disodium hydrogen phosphate Appropriate amount Sodium dihydrogen phosphate Appropriate amount Sterile purified water Appropriate amount Add CDDO and the above-mentioned components to sterilized purified water and mix them well to prepare an ophthalmic solution. Concentration of 0.05% (W / V), 0.1% (W / V), 0.5% (W / V), 1% (W / V) by changing the amount of CDDO added Agent can be prepared.

Formulation Example 2 Eye Ointment 100g CDDO-Me 0.3g
Liquid paraffin 10.0g
Appropriate amount of white petrolatum An eye ointment is prepared by adding CDDO-Me to uniformly melted white petrolatum and liquid paraffin, thoroughly mixing them, and then gradually cooling them. By changing the amount of CDDO-Me added, the concentration is 0.05% (W / V), 0.1% (W / V), 0.5% (W / V), 1% (W / W) Eye ointments can be prepared.

Formulation Example 3 CDDO-Im 1 mg in 100 mg tablet
Lactose 66.4mg
Corn starch 20mg
Carboxymethylcellulose calcium 6mg
Hydroxypropylcellulose 6mg
Magnesium stearate 0.6mg
CDDO-Im and lactose are mixed in a blender, carboxymethylcellulose calcium and hydroxypropylcellulose are added to the mixture and granulated. The resulting granules are dried and sized, and magnesium stearate is added to the sized granules. In addition, mix and tablet with a tableting machine. Moreover, the tablet whose content in 100 mg is 0.1 mg, 10 mg, and 50 mg can be prepared by changing the addition amount of CDDO-Im.

Formulation Example 4 CDDO-Im 10 mg in 10 ml of injection
Sodium chloride 90mg
Polysorbate 80 appropriate amount sterile purified water appropriate amount CDDO-Im and sodium chloride are dissolved in sterile purified water to prepare an injection. By changing the amount of CDDO-Im added, tablets with a content of 0.1 mg, 10 mg, and 50 mg in 10 ml can be prepared.

It is a graph which shows a time-dependent change of NQO1 activity in the nerve retina of each administration group. It is a graph which shows a time-dependent change of NQO1 activity in the retina choroid including the retinal pigment epithelium of each administration group. It is a graph which shows the amplitude of each administration group by making the amplitude of the a-wave of ERG in a base administration group into 100%, respectively. It is a graph which shows the amplitude of each administration group by making the amplitude of the b wave of ERG in a base administration group into 100%, respectively.

Claims (10)

A prophylactic or therapeutic agent for eye diseases associated with oxidative stress, comprising a compound represented by the following general formula (I) or a salt thereof as an active ingredient.

[Where:
R _1, R _2, R _3, R _4, R _5, R ₆ and R ₇ are the same or different and each represents a hydrogen atom or an alkyl group;
X represents a cyano group or CO—Y;
Y is an aromatic selected from the group consisting of a hydrogen atom, a halogen atom, a hydroxyl group, an amino group, an alkyl group, an alkoxy group, an alkylamino group, a halogenoalkylamino group, or an imidazole ring, a pyrazole ring, a triazole ring, and a benzimidazole ring. A heterocyclic group;
The aromatic heterocyclic group defined above may be substituted with a halogen atom, an alkyl group, a phenyl group or a halogenophenyl group. ] In general formula (I):
_{R 1, R 2, R 3} , R 4, R 5, R 6 and _{R 7} represents an alkyl group;
X represents a cyano group or CO—Y;
Y represents a hydroxyl group, an amino group, an alkoxy group, an alkylamino group, a halogenoalkylamino group, or an aromatic heterocyclic group selected from the group consisting of an imidazole ring and a pyrazole ring;
The preventive or therapeutic agent according to claim 1, wherein the aromatic heterocyclic ring defined above may be substituted with an alkyl group, a phenyl group or a halogenophenyl group. In general formula (I):
_{R 1, R 2, R 3} , R 4, R 5, R 6 and _{R 7} represents an alkyl group;
X represents a cyano group or CO—Y;
Y represents a hydroxyl group, an amino group, an alkoxy group, an alkylamino group, a halogenoalkylamino group, or an aromatic heterocyclic group selected from the group consisting of an imidazole ring and a pyrazole ring;
The prophylactic or therapeutic agent according to claim 1, wherein when Y is an imidazole ring, the imidazole ring may be substituted with an alkyl group, a phenyl group or a halogenophenyl group. In general formula (I):
R _1, R _2, R _3, R _4, R _5, R ₆ and R ₇ represent a methyl group;
X is cyano group, carboxyl group, methyloxycarbonyl group, ethyloxycarbonyl group, butyloxycarbonyl group, aminocarbonyl group, methylaminocarbonyl group, ethylaminocarbonyl group, dimethylaminocarbonyl group, 2,2,2-trifluoro Ethylaminocarbonyl group, imidazol-1-ylcarbonyl group, 2-methylimidazol-1-ylcarbonyl group, 2-phenylimidazol-1-ylcarbonyl group, 2- (4-fluorophenyl) imidazol-1-ylcarbonyl group Or the preventive or therapeutic agent of Claim 1 which shows a pyrazol-1-yl group. The compound represented by the general formula [I] is 2-cyano-3,12-dioxooleana-1,9 (11) -diene-28-oic acid, 2-cyano-3,12-dioxooleana-1,9 (11 The preventive or therapeutic agent according to claim 1, which is methyl))-diene-28-oic acid methyl ester or 1- [2-cyano-3,12-dioxooleana-1,9 (11) -diene-28-oil] imidazole. The preventive or therapeutic agent according to any one of claims 1 to 5, wherein the eye disease associated with oxidative stress is a disease in the vitreous body, retina, choroid, sclera or optic nerve. Ocular diseases related to oxidative stress are age-related macular degeneration, diabetic retinopathy, diabetic macular edema, retinitis pigmentosa, proliferative vitreoretinopathy, retinal artery occlusion, retinal vein occlusion, uveitis, label disease Prevention of retinopathy of prematurity, retinal detachment, retinal pigment epithelial detachment, optic neuropathy resulting from these diseases, optic neuropathy resulting from glaucoma, ischemic optic neuropathy or cataract Or a therapeutic agent. The prophylactic or therapeutic agent according to any one of claims 1 to 5, wherein the ocular disease associated with oxidative stress is age-related macular degeneration, diabetic retinopathy or glaucoma. The prophylactic or therapeutic agent according to claim 7, wherein the administration form is ophthalmic administration, intravitreal administration, subconjunctival administration, subtenon administration or oral administration. The prophylactic or therapeutic agent according to claim 7, wherein the dosage form is eye drops, eye ointments, injections, tablets, fine granules or capsules.

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