WO2020075860A1 - Pharmaceutical composition for treating chronic dermatitis - Google Patents

Pharmaceutical composition for treating chronic dermatitis Download PDF

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WO2020075860A1
WO2020075860A1 PCT/JP2019/040328 JP2019040328W WO2020075860A1 WO 2020075860 A1 WO2020075860 A1 WO 2020075860A1 JP 2019040328 W JP2019040328 W JP 2019040328W WO 2020075860 A1 WO2020075860 A1 WO 2020075860A1
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pharmaceutical composition
chronic dermatitis
dermatitis
inhibitor
cholesterol
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PCT/JP2019/040328
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French (fr)
Japanese (ja)
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亮治 久保
亜紀子 久保
知子 荒尾
雅行 天谷
誠 末松
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学校法人慶應義塾
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4174—Arylalkylimidazoles, e.g. oxymetazolin, naphazoline, miconazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a pharmaceutical composition for treating chronic dermatitis, a screening method for candidate compounds for treating chronic dermatitis, and the like.
  • Atopic dermatitis and psoriasis are known as chronic dermatitis.
  • Atopic dermatitis is a skin disease whose main lesion is eczema with itching that develops under the influence of various atopic factors against the background of atopic diathesis, and in many cases aggravation and remission are repeated.
  • Psoriasis is a skin disease mainly composed of silvery white scales and a clear erythema with infiltration and thickening, and most of them are intractable and chronic.
  • oral antihistamines are supplementarily used.
  • Patent Document 1 discloses the use of a lanosterol 14- ⁇ demethylase inhibitor as an angiogenesis inhibitor.
  • Non-Patent Document 1 discloses a study that an intermediate metabolite of sterol can be an endogenous ligand of ROR ⁇ .
  • Non-Patent Document 2 discloses a study on suppression of ear swelling in an IMQ-induced psoriasis model by ketoconazole.
  • Non-Patent Document 3 discloses an attempt to treat atopic dermatitis by oral administration of ketoconazole.
  • Non-Patent Document 4 discloses that intermediate products are accumulated in the cholesterol biosynthesis pathway. Further, Non-Patent Document 5 reports a cholesterol synthesis intermediate accumulated in a patient lacking a specific enzyme.
  • Patent Document 1 WO2008 / 124132
  • Non-Patent Document 1 Identification of Natural RORg Ligands that Regulate the Development of Lymphoid Cells (Santori et al., 2015, Cell Metabolism 21, 286-297).
  • Non-Patent Document 2 Sterol metabolism controls TH17 differentiation by generating endogenous RORg agonists (Nat Chem Biol. 2015 Feb; 11 (2): 141-7).
  • Non-Patent Document 3 Back O. et al., Arch Dermatol. Res (1995) 287: 448-451.
  • Non-Patent Document 4 Biochim Biophys Acta. 1991; 1086 (1): 115-124. Hashimoto F, Hayashi H. Identification of intermediates after inhibition of cholesterol synthesis by aminotriazole treatment in vivo.
  • Non-Patent Document 5 J Clin Invest. 2011 Mar; 121 (3): 976-84.
  • He M Kratz LE, Michel JJ, Vallejo AN, Ferris L, Kelley RI, Hoover JJ, Jukic D, Gibson KM, Wolfe LA, Ramachandran D, Zwick ME, Vockley J.
  • Mutations in the human SC4MOL gene encoding a methyl sterol oxidase cause psoriasiform dermatitis, microcephaly, and developmental delay.
  • the present invention relates to a pharmaceutical composition, a method for screening a candidate compound for treating chronic dermatitis, a method for diagnosing chronic dermatitis, etc. shown below.
  • Cholesterol biosynthetic precursor represented by the following formula (I) (hereinafter also referred to as C29A), cholesterol biosynthetic precursor represented by the following formula (II) (hereinafter also referred to as C29B), the following formula ( Production of a cholesterol biosynthetic precursor represented by III) (hereinafter also referred to as C28A) and a cholesterol biosynthetic precursor represented by formula (IV) (hereinafter also referred to as C28B)
  • a pharmaceutical composition for the treatment of chronic dermatitis which comprises an active ingredient that inhibits
  • the CYP51 inhibitor is at least selected from azalanstat, bifonazole, butoconazole, croconazole, econazole, metconazole, oxyconazole, sulconazole, tebuconazole, pharmaceutically acceptable salts thereof, and combinations thereof.
  • the HMGCR inhibitor is atorvastatin, lovastatin, simvastatin, a pharmaceutically acceptable salt thereof, or a combination thereof.
  • the pharmaceutical composition according to (1) above which is an external pharmaceutical composition.
  • the pharmaceutical composition according to (1) above, wherein the chronic dermatitis is atopic dermatitis or psoriasis.
  • the present invention it is possible to provide a new pharmaceutical composition which is excellent in efficacy, safety, convenience, economical efficiency and the like against chronic dermatitis such as atopic dermatitis and psoriasis.
  • the present invention also provides a method for screening candidate compounds for treating chronic dermatitis.
  • the invention further provides a method for the diagnosis of chronic dermatitis.
  • FIG. 3 is a diagram showing changes in the accumulated amount of a specific lipid metabolite before and after treatment with secukinumab (Cosentyx) on a plurality of lesions of a patient with psoriasis vulgaris. It is a figure which shows the accumulation suppression effect of a specific lipid metabolite in an atopic dermatitis patient by a cholesterol biosynthesis inhibitor. It is a figure which shows the accumulation suppression effect of the specific lipid metabolite in a psoriasis vulgaris patient by a cholesterol biosynthesis inhibitor.
  • the present invention will be described in detail below.
  • the pharmaceutical composition of the present invention is useful for treating chronic dermatitis and contains an active ingredient that inhibits the production of cholesterol biosynthetic precursors.
  • lipid metabolites represented by the following formulas (I) to (IV) (respectively, biosynthetic precursors of the formula (I) (compounds of the formula (I)) and formula (II) Also referred to as synthetic precursor (compound of formula (II)), biosynthetic precursor of formula (III) (compound of formula (III)) and biosynthetic precursor of formula (IV) (compound of formula (IV)). ) Is mentioned. It was newly confirmed that lipid metabolites represented by the formulas (I) to (IV) are accumulated in the lesions of chronic dermatitis.
  • the above-mentioned cholesterol precursor that is, the biosynthetic precursor (C29A) represented by the formula (I), the biosynthetic precursor (C29B) represented by the formula (II), the biosynthetic precursor represented by the formula (III)
  • the lipid metabolites of C28A) and the biosynthetic precursor (C28B) represented by the formula (IV) are also referred to as specific lipid metabolites.
  • the toxicity of these particular lipid metabolites associated with the development of dermatitis may be explained by the pathology of CHILD syndrome.
  • CHILD syndrome is a sex-linked dominant inherited disease that has a heterozygous mutation in the gene encoding NSDHL.
  • NSDHL is one of the enzymes responsible for the demethylation reaction at the 4-position in cholesterol biosynthesis. Since the NSDHL gene is present on the X chromosome and only one gene on two X chromosomes is activated in females, the cholesterol biosynthesis reaction occurs before NSDHL in cells in which the mutant NSDHL gene is activated. Stop. Since the specific lipid metabolite has a structure having one or two methyl groups at the 4-position, it is considered that the demethylation reaction failed due to the mutation of NSDHL and accumulated.
  • CHILD syndrome a psoriasis-like skin rash accompanied by erythema, keratinization, and desquamation on the left and right sides occurs, but the accumulation of specific lipid metabolites is found only at the site of the skin rash.
  • HMGCR inhibitors and CYP51 inhibitors suppress the production of specific lipid metabolites by blocking the cholesterol biosynthesis pathway upstream of NSDHL, but topical application of these inhibitors improves dermatitis.
  • cholesterol production is decreased by mutation of NSDHL gene, but skin rash is not improved by topical application of cholesterol. Based on these, it is strongly supported that a specific lipid metabolite is associated with dermatitis.
  • the molecular structures of the cholesterol biosynthetic precursors of formulas (I) to (IV), which are the specific lipid metabolites described above, were determined as follows. That is, for the compounds of formula (I) and formula (II), the results of GC / MS analysis of the synthesized 4,4-dimethylcholesta-8 (9) -en-3beta-ol and 14-Demethyllanosterol preparations were confirmed. The structure was determined from the agreement of time and confirming ion. On the other hand, regarding the compounds of formula (III) and formula (IV), the molecular structure was determined by collating the analysis results of GC / MS with the literature reports (Non-patent documents 4 and 5 described above). FIG.
  • FIG. 1 shows mass spectrum data of the compounds represented by the formulas (I) and (II).
  • FIG. 2 shows mass spectrum data of the compounds represented by the formulas (III) and (IV).
  • peaks appear at 1.21, 1.27, 1.12, and 1.17, respectively, as relative retention times when the retention time of cholesterol is 1 in the GC / MS analysis. It has been confirmed that these peaks appear specifically in chronic dermatitis (see Table 2 in Example 2 below).
  • the active ingredient (also referred to as cholesterol biosynthesis inhibitor) that inhibits the production of the cholesterol biosynthesis precursor is a CYP51 inhibitor, an HMGCR inhibitor, a bisphosphonate drug, amorolfine or a pharmaceutically acceptable salt thereof. Salts as well as fluazinam or a pharmaceutically acceptable salt thereof are mentioned.
  • the pharmaceutical composition of the present invention preferably contains, as an active ingredient, at least one of a CYP51 inhibitor and an HMGCR inhibitor.
  • CYP51 inhibitor examples include azalanstat, bifonazole, butoconazole, croconazole, everconazole, econazole, efinaconazole, fenticonazole, flutrimazole, phosfluconazole, isabconazole, metconazole, neticonazole, omoconazole, oxyconazole. , Sertaconazole, sulconazole, tebuconazole, terconazole and thioconazole or pharmaceutically acceptable salts thereof.
  • Preferred CYP51 inhibitors include azalanstat, bifonazole, butoconazole, croconazole, econazole, metconazole, oxyconazole, sulconazole and tebuconazole or pharmaceutically acceptable salts thereof.
  • a plurality of compounds selected from CYP51 inhibitor compounds and pharmaceutically acceptable salts thereof may be used in combination.
  • Examples of the pharmaceutically acceptable salt include alkali metals (eg, lithium, sodium, potassium etc.), alkaline earth metals (eg, magnesium, calcium, barium etc.), transition metals (eg, zinc, iron etc.). , Ammonia, organic bases (eg, trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, meglumine, ethylenediamine, pyridine, picoline, quinoline, etc.), and salts with amino acids, or inorganic acids (eg, hydrochloric acid, Sulfuric acid, nitric acid, carbonic acid, hydrobromic acid, phosphoric acid, hydroiodic acid, etc., and organic acids (for example, formic acid, acetic acid, propionic acid, trifluoroacetic acid, citric acid, lactic acid, tartaric acid, oxalic acid, maleic acid) , Fumaric acid, mandelic acid, glutaric acid,
  • HMGCR inhibitor Preferred specific examples of the HMGCR inhibitor include atorvastatin, lovastatin, simvastatin, pharmaceutically acceptable salts of these compounds, and the like.
  • a plurality of compounds selected from the compound which is an HMGCR inhibitor and pharmaceutically acceptable salts thereof may be used in combination.
  • bisphosphonate type drug examples include alendronate, risedronate, pharmaceutically acceptable salts of these compounds, and the like.
  • the pharmaceutical composition of the present invention comprises, as an active ingredient, amorolfine, alendronate, atorvastatin, azalanstat, bifonazole, butoconazole, croconazole, econazole, fluazinam, lovastatin, metconazole, oxyconazole, risedronate, simvastatin. , Sulconazole, tebuconazole, and / or terconazole or a pharmaceutically acceptable salt thereof is preferably contained.
  • a plurality of compounds selected from the above-mentioned compounds as active ingredients and pharmaceutically acceptable salts thereof may be used in combination.
  • the pharmaceutical composition of the present invention is useful as a therapeutic agent for chronic dermatitis.
  • Chronic dermatitis for which treatment with a pharmaceutical composition is effective is associated with atopic dermatitis, psoriasis, pustular psoriasis, palmoplantar pustulosis, ichthyosis group, chronic urticaria, xeroderma, hand eczema, CHILD syndrome Examples include chronic dermatitis and other skin diseases that exhibit chronic skin inflammation.
  • the pharmaceutical composition has an excellent therapeutic effect on atopic dermatitis and psoriasis.
  • the pharmaceutical composition of the present invention can be used as an external preparation intended for direct administration to a dermatitis site, and its dosage forms include, for example, ointments, creams, lotions, liniments, and poultices. , Plasters, patches, plasters, gels, liquids and the like.
  • additives and the like in pharmaceutical compositions in addition to the above compounds as active ingredients, absorption promoters, pH adjusters, preservatives, flavoring agents, dispersants, wetting agents, stabilizers, preservatives, suspending agents, surfactants, etc.
  • the additives for pharmaceutical preparations can be blended alone or in admixture of two or more.
  • the absorption promoter examples include monohydric alcohols having 20 or less carbon atoms (ethyl alcohol, isopropyl alcohol, stearyl alcohol, etc.), pyrrolidone derivatives (2-pyrrolidone, 1-methyl-2-pyrrolidone, etc.), ureas (urea, Thiourea), cyclodextrin ( ⁇ -cyclodextrin etc.), menthol, 1-dodecylazacycloheptan-2-one, calcium thioglycolate, limonene and the like.
  • monohydric alcohols having 20 or less carbon atoms ethyl alcohol, isopropyl alcohol, stearyl alcohol, etc.
  • pyrrolidone derivatives (2-pyrrolidone, 1-methyl-2-pyrrolidone, etc.
  • ureas urea, Thiourea
  • cyclodextrin ⁇ -cyclodextrin etc.
  • menthol 1-dodec
  • the content of the absorption enhancer varies depending on the dosage form, the base component, etc., but usually 0.1% by weight based on the total weight of the pharmaceutical composition from the viewpoint of effectively exhibiting the absorption enhancer action.
  • the above content is preferably 0.3% by weight or more, and from the viewpoint of suppressing the occurrence of side effects, it is 10% by weight or less, preferably 5% by weight or less.
  • the pH adjusting agent examples include inorganic acids such as hydrochloric acid, sulfuric acid or phosphoric acid, organic acids such as acetic acid, succinic acid, fumaric acid or malic acid, and metal salts of these acids.
  • the blending amount of the pH adjuster varies depending on the dosage form, base components and the like, but it is usually preferable to blend it in such a range that the pH of the preparation is 4 to 8.
  • preservatives or preservatives include paraoxybenzoic acid, methylparaben, chlorobutanol, benzyl alcohol, methyl paraoxybenzoate and the like.
  • flavoring agent examples include menthol, rose oil, eucalyptus oil, d-camphor and the like
  • specific examples of the dispersant include sodium metaphosphate, potassium polyphosphate, silicic acid anhydride and the like. Can be mentioned.
  • wetting agent examples include, for example, propylene glycol, glycerin, sorbitol, sodium lactate, sodium hyaluronate and the like
  • stabilizer examples include, for example, sodium hydrogen sulfite, tocopherol, ethylenediaminetetraacetic acid (EDTA). ), Citric acid and the like.
  • suspending agent examples include, for example, tragacanth powder, gum arabic powder, bentonite, sodium carboxymethyl cellulose and the like
  • surfactant examples include, for example, polyoxyethylene hydrogenated castor oil, sesquiolein. Examples thereof include sorbitan fatty acid esters such as acid sorbitan, and polyoxyl stearate.
  • an oily base or an emulsion base can be used as the base.
  • oily base examples include hydrocarbons (hydrocarbons having 12 to 32 carbon atoms, liquid paraffin, white petrolatum, squalene, squalane, plastibase, etc.), higher alcohols (lauryl alcohol, cetyl alcohol, stearyl alcohol or oleyl alcohol).
  • C12-C30 aliphatic monohydric alcohols, etc. higher fatty acids (C6-C32 saturated or unsaturated fatty acids such as palmitic acid or stearic acid), higher fatty acid esters (myristyl palmitate or stearyl stearate) Ester of fatty acid such as lanolin; Carnauba wax or the like ester of fatty acid having 10 to 32 carbon atoms and aliphatic monohydric alcohol having 14 to 32 carbon atoms; saturated or unsaturated fatty acid having 10 to 22 carbon atoms such as glyceryl monolaurate With glycerin Or their hydrogenated products, etc.), glycols (ethylene glycol, propylene glycol, polyethylene glycol, and the like), vegetable oils, animal oils, and the like.
  • C6-C32 saturated or unsaturated fatty acids such as palmitic acid or stearic acid
  • higher fatty acid esters myristyl palmitate or stearyl stearate
  • emulsion bases examples include oil-in-water bases, water-in-oil bases, and suspension bases.
  • oil-in-water base the components such as lanolin, propylene glycol, stearyl alcohol, petrolatum, silicone oil, liquid paraffin, glyceryl monostearate, and polyethylene glycol are added in the aqueous phase in the presence or absence of a surfactant.
  • examples include bases emulsified and dispersed therein.
  • examples of the water-in-oil type base include bases obtained by adding water to components such as petrolatum, higher aliphatic alcohols, liquid paraffin in the presence of a nonionic surfactant, and emulsifying and dispersing.
  • examples of the suspension-type base include an aqueous base that is made into a gel by adding a suspending agent such as starch, glycerin, high-viscosity carboxymethyl cellulose, and carboxyvinyl polymer to water.
  • a suspending agent such as starch, glycerin, high-viscosity carboxymethyl cellulose, and carboxyvinyl polymer
  • the pharmaceutical composition of the present invention can be produced by a conventional method for preparing an external preparation.
  • an ointment or cream is prepared by kneading, emulsifying or suspending the raw materials of the base according to each dosage form to prepare the base, then adding the active ingredient and various additives, and mixing with a screw mixer or the like. It can be produced by mixing in the machine.
  • the pharmaceutical composition of the present invention can be used in any form of suspension type, emulsion type or solution type lotion.
  • the base of the suspension lotion include gums such as gum arabic and tragacanth, celluloses such as methyl cellulose and hydroxyethyl cellulose, and clays such as bentonite, and a mixture of water and the like.
  • the emulsion type lotion base include a base obtained by emulsifying water and a fatty acid such as stearic acid or oleic acid, and an oily substance such as higher alcohols such as stearyl alcohol or cetyl alcohol.
  • the base of the solution type lotion include water, ethanol, glycerin, alcohols such as propylene glycol and the like.
  • the lotion can be produced, for example, by adding various base components to purified water, mixing and stirring, and then adding the active ingredients and additives, mixing, and filtering if desired. it can.
  • the base for the liniment include vegetable oils such as olive oil, alcohols such as ethanol or isopropanol, and mixtures thereof with water.
  • the liniment agent can be produced, for example, by dissolving the active ingredient in a base and, if desired, adding an additive for formulation thereto and mixing them.
  • the base for poultices includes, for example, water-soluble polymer compounds such as polyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, and the like.
  • the poultice can be produced, for example, by mixing an active ingredient, a base and a desired formulation additive, heating and cooling.
  • a support such as a non-woven fabric, an elastic body such as natural rubber or isoprene rubber, a filler such as zinc white and titanium oxide, an adhesive such as terpene resin
  • An imparting agent, a release agent such as vinyl acetate, a softening agent such as liquid paraffin, an antiaging agent such as dibutylhydroxytoluene (BHT), and the like can be used in appropriate combination.
  • the plaster, patch, plaster and the like can be produced by a conventional method such as a solution method or a hot pressing method.
  • the solvent for preparing the liquid agent examples include water, ethanol, isopropyl alcohol, benzyl alcohol, polyethylene glycol (PEG400 etc.), propylene glycol, propylene carbonate, or a mixture thereof. Further, the liquid agent can be used by impregnating it with gauze, a wound surface covering material and the like.
  • the amount of the active ingredient that is, the amount of the CYP51 inhibitor, HMGCR inhibitor, etc., added to the pharmaceutical composition (formulation) varies depending on the dosage form.
  • the pharmaceutical composition It can be 0.0025 to 5% by weight, more preferably 0.5 to 5% by weight, and particularly preferably 1 to 2% by weight, based on the total weight of
  • the total amount of the pharmaceutical composition is 0.1 to 200 mg / mL, more preferably 5 to 50 mg / mL, especially 10 to 20 mg / mL.
  • the pharmaceutical composition is 0.1 to 200 mg / mL, more preferably 5 to 50 mg / mL, especially 10 to 20 mg / mL.
  • the pharmaceutical composition is 0.1 to 200 mg / mL, more preferably 5 to 50 mg / mL, especially 10 to 20 mg / mL.
  • the pharmaceutical composition is 0.1 to 200 mg / mL, more preferably 5 to 50 mg / mL, especially 10 to 20 mg /
  • the dosage of the pharmaceutical composition of the present invention depends on the type of itch, the disease site, the degree of itch, etc., and an appropriate amount of the above formulation is administered once to several times a day by topical administration (by application to the affected area, etc.). Local administration).
  • the method for screening a candidate compound for treating chronic dermatitis in the present invention is a measurement for measuring an inhibitory activity of a target compound for inhibiting the production of at least one specific lipid metabolite (cholesterol biosynthetic precursor). And a determination step of determining whether or not the target compound is effective for treating chronic dermatitis based on the measured inhibitory activity value.
  • the inhibitory activity can be measured by the following method. For example, 1 ⁇ 10 5 (1 mL) of HaCaT cells suspended in DMEM (Dulbecco's modified Eagle) medium supplemented with cholesterol-depleted serum were seeded in a 12-well multiwell plate, and 17-hydroxyprogesterone at a final concentration of 30 ⁇ M was added thereto. Further, 1 mM of the target compound dissolved in dimethyl sulfoxide is added so as to have a final concentration of 1 ⁇ M, and the mixture is cultured for 48 hours.
  • DMEM Dynamic fetalated Eagle
  • the cells are washed twice with PBS (phosphate buffered saline), 200 ⁇ L of TrypLE Express is added, and the mixture is incubated at 37 ° C. for 13 minutes. After adding 0.5 mL of PBS and collecting the cells in a tube, centrifugation is performed at 4 ° C. and 500 g for 5 minutes to precipitate the cells, and after removing the supernatant, the cells are resuspended in 0.4 mL of PBS.
  • a diatomaceous earth column (ISOLUTE SLE + 400, Biotage) is set in a glass tube washed with 1 mL of hexane, and the whole amount of the cell suspension is added.
  • ⁇ GC / MS analysis conditions Capillary column: Rtx-5MS (length 30m, inner diameter 0.25mm, film thickness 0.25 ⁇ m) Oven temperature: 150 °C, hold 1 min -20 °C / min ⁇ 250 °C, 5 °C / min ⁇ 280 °C, hold 10 minutes -20 °C / min ⁇ 330 °C, hold 3 minutes.
  • the peak area of the specific lipid metabolite when the compound is not added is set to 0% inhibition.
  • the determination step for example, in order to formulate a compound having an activity of a predetermined threshold value or more, based on the value of the measured inhibitory activity, whether the compound is effective in treating chronic dermatitis by the following method. To determine. That is, when it is measured by the measuring method of the inhibitory activity in the above-mentioned measuring step, which is also used in Example 8 described later, at least one of the specific lipid metabolites is, for example, 50 at 10 ⁇ M.
  • a compound exhibiting an accumulation inhibitory activity of not less than%, more preferably a compound exhibiting an accumulation inhibitory activity of not less than 50% at 1 ⁇ M with respect to at least one of the specific lipid metabolites is determined to be effective for the treatment of chronic dermatitis.
  • the method of the present invention including the above measurement step and determination step is useful as a method for screening a candidate drug that is expected to be effective in treating chronic dermatitis.
  • the method for diagnosing chronic dermatitis in the present invention comprises a detection step of detecting at least one specific lipid metabolite from a skin sample, a detection amount of a cholesterol biosynthetic precursor detected from the sample, and a standard. And a comparison step of comparing the reference amount with
  • the specific lipid metabolite is detected by the following method. Approximately 1 mg of the keratin of the lesion is collected, and the lipid is extracted with 3 mL of hexane. To the extract, 10 ⁇ L of an ethanol solution containing 0.1 ⁇ g / ⁇ L of D7-cholesterol is added as an internal standard. The entire amount of this is loaded onto a silica gel column (Waters Sep-Pak Silica), and the fraction eluted with 1 mL of ethyl acetate is collected in a 2 mL glass vial. The contents are heated to 40 ° C. under a nitrogen stream to dryness, and then derivatized with trimethylsilyl.
  • silica gel column Waters Sep-Pak Silica
  • GC / MS analysis conditions are the same as the GC / MS analysis conditions in the above-described screening method for candidate compounds for treating chronic dermatitis.
  • quantification is possible by analyzing standard samples whose concentrations are known in advance under the same conditions, standardizing by the peak area ratio with the internal standard, and determining the area area per unit amount of each substance. .
  • the area of the peak of cholesterol and the peak of the substance to be analyzed are compared and evaluated as a relative amount. Specifically, the area of the peak of the substance to be analyzed with respect to the cholesterol peak 100000 is calculated, and the relative area obtained from the specimen obtained from the lesion and that obtained from the normally keratinized specimen are compared.
  • the actual measurement data obtained by the above-mentioned method are shown.
  • the following values are all measured by the above-mentioned method, and the peak area of each substance per 100000 peak area of cholesterol is expressed as an index.
  • the peak areas of the respective cholesterol biosynthesis precursors in the normal keratinized samples and the like were as follows. As shown below, in atopic dermatitis and psoriasis vulgaris, the accumulation of all specific lipid metabolites (cholesterol biosynthetic precursor) was 5 times or more that of normal keratinized keratin on average.
  • the amount of cholesterol biosynthesis precursor detected in the sample is a predetermined reference amount. More than that, the sample donor is likely to have chronic dermatitis.
  • the method of the present invention having the detection step and the comparison step is a method for diagnosing chronic dermatitis, or a method for assisting diagnosis of chronic dermatitis (evaluation of severity, determination of therapeutic effect). Etc.) is useful.
  • the reference amount is a value evaluated by the ratio of the peak area to cholesterol of a specific lipid metabolite detected when performing GC-MS analysis under predetermined analysis conditions, and keratinized normally.
  • C28A is 150 or less
  • C28B is 100 or less
  • C29A is 200 or less
  • C29B is 100 or less. Therefore, for the cholesterol peak area 100000, the peak area 150 can be the C28A reference amount, the peak area 100 can be the C28B reference amount, the peak area 200 can be the C29A reference amount, and the peak area 100 can be the C29B reference amount.
  • the method of the present invention including the detection step and the comparison step is useful as a method for diagnosing chronic dermatitis or a method for assisting diagnosis of chronic dermatitis.
  • Example 1 [Treatment of CHILD syndrome] To the lesion on the left side of a patient with chronic dermatitis associated with CHILD syndrome, 1% atorvastatin / 2% cholesterol aqueous solution was applied externally twice a day using atorvastatin as an HMGCR inhibitor. Improvement of the eruption was observed 1 month after the start of external application, and the eruption disappeared completely 7 months after the start of external application.
  • FIG. 3 (A1) shows the lesion area on the left side before the start of treatment with atorvastatin
  • FIG. 3 (A2) shows the left side area after the start of treatment.
  • 1% oxyconazole cream (trade name: quinazole cream) was used as a CYP51 inhibitor for topical lesions on the posterior side of the neck of patients with chronic dermatitis associated with CHILD syndrome, and external application was performed once a day. It was The skin rash disappeared completely 4 months after the start of external application.
  • FIG. 3 (B1) shows a lesion on the back side of the neck before the start of treatment with oxyconazole
  • FIG. 3 (B2) shows the back side of the neck after the start of treatment. From the above, it was confirmed that both the HMGCR inhibitor and the CYP51 inhibitor are effective against chronic dermatitis associated with CHILD syndrome.
  • Example 2 [Chronic dermatitis associated with CHILD syndrome]
  • CHILD syndrome chronic dermatitis associated with CHILD syndrome
  • FIG. 4 Specific lipid metabolites are accumulated only in the lesions.
  • 10 ⁇ L of an ethanol solution of 0.1 ⁇ g / ⁇ L of D7-cholesterol was added to the extract.
  • ⁇ GC / MS analysis conditions Capillary column: Rtx-5MS (length 30m, inner diameter 0.25mm, film thickness 0.25 ⁇ m) Oven temperature: 150 °C, hold 1 min -20 °C / min ⁇ 250 °C, 5 °C / min ⁇ 280 °C, hold 10 minutes -20 °C / min ⁇ 330 °C, hold 3 minutes.
  • Carrier gas He, Linear velocity 39.0 cm / sec Ion source temperature: 200 °C Interface temperature: 280 °C Vaporization chamber temperature: 250 ° C
  • the compounds of the above formulas (I) to (IV) all have a sterol skeleton and contain an OH group at the 3-position.
  • the compounds of formula (I) and formula (II) (having 29 carbon atoms) each have two methyl groups at the 4-position, and compounds of formula (III) and formula (IV) (having 28 carbon atoms) All have one methyl group at the 4-position.
  • the m / z values of the GC / MS spectrum data of the cholesterol biosynthetic precursors shown in Table 2 are theoretical values, and for example, the peak of 484.4 in the compound of formula (II) (C29B) is The hydroxyl group of the molecule of the cholesterol biosynthesis precursor (C29B) is a value derived from a —OSi (C 3 H 9 ) form produced by trimethylsilylation (TMS conversion), and 379.3 is a CH 4 value derived from the above TMS form. And OSi (C 3 H 9 ) are the values derived from the desorbed fragment, and 135.1 is the value derived from the C 10 H 15 fragment.
  • the theoretical m / z value of the above compound corresponds to the measured value shown in FIGS. 1 and 2, but a measurement error is also recognized.
  • a peak of 484.35 corresponding to the theoretical value of 444.4 for the compound (C29B) of the formula (II) and a theoretical value of A peak of 135.15 corresponding to a certain 135.1 is detected.
  • an error of up to about ⁇ 0.1 can be recognized.
  • a measurement error of up to about ⁇ 0.01 was observed.
  • the above-mentioned 1.21 was used in the compound (C29A) of the formula (I).
  • the measured value of 1 changes in the range of 1.20 to 1.22, and in the compound (C29B) of the formula (II), the measured value of 1.27 can change in the range of 1.26 to 1.28.
  • the measured value of 1.12 changes within the range of 1.11 to 1.13, and in the compound (C28B) of the formula (IV), the above 1. It is possible that the 17 measurements may vary from 1.16 to 1.18.
  • FIG. 4 is a diagram showing measured accumulation amounts of lipid metabolites in a lesion area of a chronic dermatitis patient associated with CHILD syndrome and a healthy area of the same patient.
  • specific lipid metabolites that were hardly detected in the normal part were detected in the keratin of the lesion part of the CHILD syndrome patient.
  • the molecular structure of the detected compound was confirmed to be represented by the formulas (I) to (IV) as described above.
  • Example 3 [Inhibition of lipid metabolite accumulation] A test system for artificially accumulating the lipid metabolite specified in Example 2 was constructed, and the production suppressing effect of various cholesterol biosynthesis inhibitors was examined. (experimental method) When the human keratinocyte cell line HaCaT is cultured in a medium containing 10% cholesterol-free serum, the HaCaT cells use the components in the medium to synthesize a large amount of cholesterol. At this time, by adding 17-hydroxyprogesterone to the medium at a concentration of 30 ⁇ M, the activity of the enzyme (NSDHL) that oxidatively demethylates the methyl group at the 4-position is inhibited, and as with the skin of CHILD syndrome, Lipid metabolites accumulate in cells.
  • NSDHL the enzyme
  • the m / z values of the substance-specific ions used for the evaluation of the relative amounts of specific lipids were C29A (compound of formula (I)): m / z 486.4, respectively.
  • the addition of each drug at the concentrations shown in the graph of FIG. 5 suppressed the accumulation of specific lipid metabolites.
  • an inhibitory effect was observed for all drugs in a dose-dependent manner.
  • Example 4 [Accumulation of lipid metabolites in chronic skin diseases] It was tested whether accumulation of lipid metabolites identified in Example 2 was confirmed in patients with chronic dermatitis. (experimental method) The method of implementation is the same as in Example 2 above. That is, keratin of healthy person, keratin of CHILD syndrome patient (lesion and healthy part), keratin of psoriasis vulgaris patient (lesion and healthy part), keratin of inherited keratosis patient (lesion), acute dermatitis Approximately 1 mg each of the keratin (lesion) of the patient, the keratin (lesion) of the patient with poisoning eruption, and the keratin (lesion) of the patient with atopic dermatitis were collected, and lipids were extracted in the same manner as in Example 2.
  • Example 4 In addition, in the lesions of patients with hereditary keratosis, contact dermatitis (acute dermatitis), toxic eruption patients, and callus patients, the accumulated amount of specific lipid metabolites may be the same as that of healthy individuals. all right.
  • the results of Example 4 are shown in FIGS. 6 (A) and 6 (B).
  • the peak area per unit weight of each lipid was calculated, and the peak area of each lipid per unit weight of a healthy person was set as a reference value of 1, and the accumulation amount of the specific lipid metabolite in the lesion site was calculated. The relative value indicating the multiple of the reference value was displayed.
  • Example 5 [Changes in lipid metabolites associated with treatment] Secukinumab (trade name: Cosentyx (human anti-human IL-17A monoclonal antibody preparation)) was subcutaneously injected once to the lesion site of a patient with psoriasis vulgaris (300 mg). Specifically, 300 mg was subcutaneously injected once at 0, 1, 2, 3, 4, and 5 weeks, and thereafter, 300 mg was subcutaneously injected once every 4 weeks.
  • Secukinumab trade name: Cosentyx (human anti-human IL-17A monoclonal antibody preparation)
  • keratin was collected from the same lesion site before and after administration of secukinumab for psoriasis vulgaris, and lipid extraction and analysis were performed in the same manner as in Example 2.
  • the skin rash with scales was relieved by the treatment, and when the skin rash was healed, the scales could not be collected. Comparing the two collected cases before and after treatment, the accumulation of specific lipid metabolites was reduced in both cases. The result is shown in FIG. 7.
  • the vertical axis of FIG. 7 represents the relative value of the accumulated amount of specific lipid metabolites, and more specifically, the peak area of each substance per 100000 peak areas when cholesterol was analyzed under the same conditions. There is.
  • Example 6 [Treatment of atopic dermatitis] Topical steroids for the lesions of patients with atopic dermatitis were discontinued, and atorvastatin (1 wt% atorvastatin solution prepared by dissolving atorvastatin bulk powder in water to a concentration of 1%) and quinazole (1% oxycostatin). Nazoru cream, brand name: quinazol cream) was applied twice a day at 350 mg (equivalent to 3.5 mg of bulk powder), healthy area, lesion area just before the start of treatment with atorvastatin and quinazole, lesion after 1 week from treatment.
  • atorvastatin 1 wt% atorvastatin solution prepared by dissolving atorvastatin bulk powder in water to a concentration of 1%)
  • quinazole 1% oxycostatin
  • the keratin was collected from the affected area and from the lesioned area after 3 weeks from the treatment, and the lipid was extracted and analyzed in the same manner as in Example 2.
  • the vertical axis represents the peak area of each substance per 100,000 cholesterol peak area.
  • Example 7 [Treatment of psoriasis vulgaris] Oquinazole (1% oxyconazole cream, trade name: quinazole cream) was applied to the lesion site of a patient with psoriasis vulgaris twice a day for 2 weeks. Then, keratin was collected from a healthy part, a lesion immediately before the start of treatment with quinazole, and a lesion 4 weeks after the treatment, and lipid extraction and analysis were carried out in the same manner as in Example 2. As a result, it was confirmed that, even in psoriasis vulgaris, the application of quinazole suppressed the accumulation of the lipid in the affected area (see FIG. 9). In FIG. 9, as in FIG. 8, the vertical axis represents the peak area of each substance per 100000 peak area of cholesterol. Then, after 4 weeks from the treatment, reduction of skin scales was observed, so it is considered that quinazole has an effect of suppressing skin symptoms.
  • Example 8 Evaluation of C29A accumulation inhibitory activity of compound
  • the C29A accumulation inhibitory activity of the evaluation compound was measured by the same method as described above, that is, the method adopted for the measuring step for measuring the inhibitory activity of inhibiting the production of a specific lipid metabolite.
  • alendronate and risedronate showed good C29A accumulation inhibitory activity of 50% or more at 10 ⁇ M.
  • amorolfine hydrochloride, atorvastatin, azalanstat, bifonazole, butoconazole nitrate, croconazole hydrochloride, econazole, fluazinam, lovastatin, metconazole, oxyconazole nitrate, simvastatin, sulconazole nitrate, tebuconazole and terconazole are 50% or more at 1 ⁇ M. It showed good C29A accumulation inhibitory activity.
  • Example 9 [Evaluation of human CYP51A1 inhibitory activity of compound] The inhibitory activity of the compound of the above formula (I) was tested as follows. (experimental method) The inhibitory activity of human CYP51A1 was measured by the following method. The evaluation compound was dissolved in DMSO so that the concentration was 2.5 mM. The dissolved evaluation compound was diluted with DMSO at a public ratio of 3 to prepare a dilution series. 1 ⁇ L of the evaluation compound diluted with DMSO was diluted with a mixed solution (12.5 ⁇ L) of 100 mM potassium phosphate, 3.3 mM magnesium chloride, and 0.38 mg / mL Triton X100.
  • the silkworm microsome fraction expressing the human CYP51A1 protein was obtained from Sysmex.
  • the enzymatic activity of CYP51A1 was calculated from the amount of NADPH used when metabolizing lanosterol as a substrate.
  • NADP / NADPH assay kit (ab65349) from Abcam was used for the measurement of NADPH. That is, the respective final concentrations were 100 mM potassium phosphate, 3.3 mM magnesium chloride, 0.38 mg / mL Triton X100, 0.564 U / mL NADPH-P450 reductase, 40 ⁇ M lanosterol, 260 ⁇ g / mL.
  • aqueous solution (21 ⁇ L) containing human CYP51A1 microsomes was prepared, 2 ⁇ L of the diluted evaluation compound was added, and the mixture was allowed to stand for 5 minutes. 2 ⁇ L of 0.5 mM NADPH solution diluted with a mixed aqueous solution of 100 mM potassium phosphate, 3.3 mM magnesium chloride, and 0.38 mg / mL Triton X100 was added thereto, and the mixture was allowed to stand at room temperature for 20 minutes. The subsequent operation was performed according to the protocol of NADP / NADPH assay kit of Abcam.
  • the NADPH consumption when the above test was performed without adding the evaluation compound was defined as 100% activity, and the inhibitory activity (IC 50 ) of human CYP51A1 was calculated from the NADPH consumption in the presence of each concentration of the evaluation compound.
  • IC 50 inhibitory activity of human CYP51A1
  • azalanstat, bifonazole, butoconazole nitrate, croconazole hydrochloride, metconazole, oxyconazole nitrate, sulconazole nitrate and tebuconazole showed good IC 50 of 1 ⁇ M or less.
  • the production of a novel specific cholesterol biosynthetic precursor is inhibited, and a pharmaceutical composition having an excellent therapeutic effect on chronic dermatitis, and the detected amount of the specific cholesterol biosynthetic precursor are focused.
  • a method for screening a candidate drug for chronic dermatitis can be provided.

Abstract

The present invention addresses the problem of realizing a new pharmaceutical composition, etc., for chronic dermatitis having an adequate therapeutic effect. The above problem is solved by a pharmaceutical composition for treating chronic dermatitis, the pharmaceutical composition containing an active ingredient that inhibits the production of any cholesterol biosynthesis precursor represented by formulas (I)-(IV).

Description

慢性皮膚炎の治療のための医薬組成物Pharmaceutical composition for the treatment of chronic dermatitis
　本発明は、慢性皮膚炎の治療のための医薬組成物、慢性皮膚炎治療用の候補化合物のスクリーニング方法等に関する。 The present invention relates to a pharmaceutical composition for treating chronic dermatitis, a screening method for candidate compounds for treating chronic dermatitis, and the like.
　慢性皮膚炎として、アトピー性皮膚炎、乾癬等が知られている。アトピー性皮膚炎は、アトピー素因を背景に様々な環境因子による影響を受けて発症する、痒みを伴う湿疹を主病変とする皮膚疾患であり、多くは増悪と寛解を繰り返す。乾癬は、銀白色の鱗屑と浸潤・肥厚を伴う境界明瞭な紅斑を主病変とする皮膚疾患であり、多くは難治性であり慢性に推移する。アトピー性皮膚炎の治療には、ステロイド剤やカルシニューリン阻害剤などの外用に加え、補助的に経口抗ヒスタミン薬が用いられる。また、乾癬の治療は、ステロイド外用剤やビタミンD3外用剤及びそれらの合剤を含む局所療法と、シクロスポリン、レチノイド、メトトレキサートをはじめとする全身療法、UVAやUVBを用いた光線療法を組み合わせて行われる。最近では、TNF-αやIL-12/23、IL-17に対する生物学的製剤も用いられるようになった。 Atopic dermatitis and psoriasis are known as chronic dermatitis. Atopic dermatitis is a skin disease whose main lesion is eczema with itching that develops under the influence of various atopic factors against the background of atopic diathesis, and in many cases aggravation and remission are repeated. Psoriasis is a skin disease mainly composed of silvery white scales and a clear erythema with infiltration and thickening, and most of them are intractable and chronic. In the treatment of atopic dermatitis, in addition to external use such as steroids and calcineurin inhibitors, oral antihistamines are supplementarily used. For the treatment of psoriasis, local therapy including topical steroids, topical vitamin D3 and their combinations, systemic therapy including cyclosporine, retinoids and methotrexate, and phototherapy using UVA and UVB are combined. Be seen. Recently, biologics for TNF-α, IL-12 / 23, and IL-17 have also been used.
　しかしながら、これらはいずれも対症療法により症状を緩和することを目標としており、根治療法は存在しない。これらの疾患に対し、様々な対症療法が行われているが、全ての患者に同じ対症療法が有効とは限らない。そこで、今までの治療方法とは異なる、発症メカニズムに基づいた新たな対症療法が、治療の幅を広げるものとして期待される。
　また、これらの慢性炎症性皮膚疾患に対して用いられるステロイド外用剤は、長期使用では、皮膚萎縮、皮膚感染症の誘発などの副作用を生じ得る。アトピー性皮膚炎の治療におけるカルシニューリン阻害剤は、塗布部の刺激感が問題となっており、経口抗ヒスタミン薬によっても鎮静化されない痒みは患者のQOLを著しく低下させている。また、乾癬に対する局所療法は多くの場合、効果が緩徐であり、全身療法では副作用の発現、光線療法では頻回の受診による時間的な拘束が問題となっている。また、生物学的製剤では高額な医療費が問題となっている。 However, these all aim at alleviating the symptoms by symptomatic treatment, and there is no root treatment. Although various symptomatic treatments are used for these diseases, the same symptomatic treatment is not necessarily effective for all patients. Therefore, a new symptomatic treatment based on the onset mechanism, which is different from the conventional treatment methods, is expected to expand the range of treatment.
Moreover, the steroid external preparations used for these chronic inflammatory skin diseases may cause side effects such as skin atrophy and induction of skin infections when used for a long time. Calcineurin inhibitors in the treatment of atopic dermatitis have a problem of irritation at the application site, and itching that is not sedated by oral antihistamines significantly reduces the QOL of patients. Further, in many cases, local therapy for psoriasis has a slow effect, side effects occur in systemic therapy, and time restraint due to frequent visits in phototherapy poses a problem. In addition, high cost of medical care is a problem for biological products.
　なお、特許文献１には、ラノステロール１４－αデメチラーゼ阻害剤を血管新生の阻害剤として用いることが開示されている。非特許文献１には、ステロールの中間代謝物がＲＯＲγの内因性リガンドになり得るという研究について開示されている。非特許文献２には、ケトコナゾールによるＩＭＱ誘発乾癬モデルにおける耳介腫脹抑制の研究について開示されている。非特許文献３では、ケトコナゾールの経口投与によるアトピー性皮膚炎の治療の試みについて開示されている。非特許文献４では、コレステロール生合成経路にて中間産物が蓄積することが開示されている。また、非特許文献５では、特定の酵素を欠損した患者において蓄積したコレステロール合成中間体について報告されている。 Note that Patent Document 1 discloses the use of a lanosterol 14-α demethylase inhibitor as an angiogenesis inhibitor. Non-Patent Document 1 discloses a study that an intermediate metabolite of sterol can be an endogenous ligand of RORγ. Non-Patent Document 2 discloses a study on suppression of ear swelling in an IMQ-induced psoriasis model by ketoconazole. Non-Patent Document 3 discloses an attempt to treat atopic dermatitis by oral administration of ketoconazole. Non-Patent Document 4 discloses that intermediate products are accumulated in the cholesterol biosynthesis pathway. Further, Non-Patent Document 5 reports a cholesterol synthesis intermediate accumulated in a patient lacking a specific enzyme.
　特許文献１:ＷＯ２００８／１２４１３２ Patent Document 1: WO2008 / 124132
　非特許文献１: Identification of Natural RORg Ligands that Regulate the Development of Lymphoid Cells(Santori et al., 2015, Cell Metabolism 21, 286-297)
　非特許文献２: Sterol metabolism controls TH17 differentiation by generating endogenous RORg agonists（Nat Chem Biol. 2015 Feb;11(2):141-7）
　非特許文献３: Back O. et al., Arch Dermatol. Res (1995) 287: 448-451
　非特許文献４: Biochim Biophys Acta. 1991;1086(1):115-124.
Hashimoto F, Hayashi H. 
Identification of intermediates after inhibition of cholesterol synthesis by aminotriazole treatment in vivo. 
　非特許文献５: J Clin Invest. 2011 Mar;121(3):976-84. doi: 10.1172/JCI42650.
He M, Kratz LE, Michel JJ, Vallejo AN, Ferris L, Kelley RI, Hoover JJ, 
Jukic D, Gibson KM, Wolfe LA, Ramachandran D, Zwick ME, Vockley J.
Mutations in the human SC4MOL gene encoding a methyl sterol oxidase cause psoriasiform dermatitis, microcephaly, and developmental delay. Non-Patent Document 1: Identification of Natural RORg Ligands that Regulate the Development of Lymphoid Cells (Santori et al., 2015, Cell Metabolism 21, 286-297).
Non-Patent Document 2: Sterol metabolism controls TH17 differentiation by generating endogenous RORg agonists (Nat Chem Biol. 2015 Feb; 11 (2): 141-7).
Non-Patent Document 3: Back O. et al., Arch Dermatol. Res (1995) 287: 448-451.
Non-Patent Document 4: Biochim Biophys Acta. 1991; 1086 (1): 115-124.
Hashimoto F, Hayashi H.
Identification of intermediates after inhibition of cholesterol synthesis by aminotriazole treatment in vivo.
Non-Patent Document 5: J Clin Invest. 2011 Mar; 121 (3): 976-84. Doi: 10.1172 / JCI42650.
He M, Kratz LE, Michel JJ, Vallejo AN, Ferris L, Kelley RI, Hoover JJ,
Jukic D, Gibson KM, Wolfe LA, Ramachandran D, Zwick ME, Vockley J.
Mutations in the human SC4MOL gene encoding a methyl sterol oxidase cause psoriasiform dermatitis, microcephaly, and developmental delay.
　上記のような状況において、新たな慢性皮膚炎用の医薬組成物を提供することが望まれている。 In the above situation, it is desired to provide a new pharmaceutical composition for chronic dermatitis.
　本願発明は、以下に示す、医薬組成物、慢性皮膚炎治療用の候補化合物のスクリーニング方法、慢性皮膚炎の診断のための方法等に関する。 The present invention relates to a pharmaceutical composition, a method for screening a candidate compound for treating chronic dermatitis, a method for diagnosing chronic dermatitis, etc. shown below.
（１）下記式（Ｉ）で表されるコレステロール生合成前駆物質（以下、Ｃ２９Ａとも称する）、下記式（ＩＩ）で表されるコレステロール生合成前駆物質（以下、Ｃ２９Ｂとも称する）、下記式（ＩＩＩ）で表されるコレステロール生合成前駆物質（以下、Ｃ２８Ａとも称する）、及び、式（ＩＶ）で表されるコレステロール生合成前駆物質（以下、Ｃ２８Ｂとも称する）のいずれかのコレステロール前駆物質の産生を阻害する有効成分を含む、慢性皮膚炎の治療のための医薬組成物。
Figure JPOXMLDOC01-appb-C000004
 （２）前記有効成分が、ＣＹＰ５１阻害剤、又は、ＨＭＧＣＲ阻害剤である、上記（１）に記載の慢性皮膚炎の治療のための医薬組成物。
（３）前記有効成分が、前記ＣＹＰ５１阻害剤である、上記（２）に記載の医薬組成物。
（４）前記ＣＹＰ５１阻害剤が、アザランスタット、ビフォナゾール、ブトコナゾール、クロコナゾール、エコナゾール、メトコナゾール、オキシコナゾール、スルコナゾール、テブコナゾール、それらの薬学的に許容される塩、及び、それらの組み合わせから選ばれる少なくともいずれかを含む、上記（３）に記載の医薬組成物。
（５）前記有効成分が、前記ＨＭＧＣＲ阻害剤である、上記（２）に記載の医薬組成物。
（６）前記ＨＭＧＣＲ阻害剤が、アトルバスタチン、ロバスタチン、シンバスタチン、それらの薬学的に許容される塩、又はそれらの組み合わせである、上記（５）に記載の医薬組成物。
（７）外用医薬組成物である、上記（１）に記載の医薬組成物。
（８）前記慢性皮膚炎が、アトピー性皮膚炎、又は、乾癬である、上記（１）に記載の医薬組成物。
（９）アモロルフィン、アレンドロネート、アトルバスタチン、アザランスタット、ビフォナゾール、ブトコナゾール、クロコナゾール、エコナゾール、フルアジナム、ロバスタチン、メトコナゾール、オキシコナゾール、リセドロネート、シンバスタチン、スルコナゾール、テブコナゾール、テルコナゾール、それらの薬学的に許容される塩、及び、それらの組み合わせから選ばれる少なくともいずれかを含む、慢性皮膚炎の治療のための医薬組成物。（１０）（ａ）対象化合物について、下記式（Ｉ）～（ＩＶ）で表されるコレステロール生合成前駆物質のいずれかの産生を阻害する阻害活性を測定する測定工程と、
　（ｂ）測定された前記阻害活性の値に基づいて、前記対象化合物が慢性皮膚炎の治療に有効な候補化合物であるか否かを判定する判定工程とを含む、
　慢性皮膚炎治療用の候補化合物のスクリーニング方法。
Figure JPOXMLDOC01-appb-C000005
 （１１）（ｃ）下記式（Ｉ）～（ＩＶ）で表されるコレステロール生合成前駆物質のいずれかを皮膚のサンプルから検出する検出工程と、
　（ｄ）前記サンプルから検出された前記コレステロール生合成前駆物質の検出量と、基準となる基準量とを比較する比較工程とを有する、
　慢性皮膚炎の診断のための方法。
Figure JPOXMLDOC01-appb-C000006
 (1) Cholesterol biosynthetic precursor represented by the following formula (I) (hereinafter also referred to as C29A), cholesterol biosynthetic precursor represented by the following formula (II) (hereinafter also referred to as C29B), the following formula ( Production of a cholesterol biosynthetic precursor represented by III) (hereinafter also referred to as C28A) and a cholesterol biosynthetic precursor represented by formula (IV) (hereinafter also referred to as C28B) A pharmaceutical composition for the treatment of chronic dermatitis, which comprises an active ingredient that inhibits
Figure JPOXMLDOC01-appb-C000004
 (2) The pharmaceutical composition for treating chronic dermatitis according to (1) above, wherein the active ingredient is a CYP51 inhibitor or an HMGCR inhibitor.
(3) The pharmaceutical composition according to (2) above, wherein the active ingredient is the CYP51 inhibitor.
(4) The CYP51 inhibitor is at least selected from azalanstat, bifonazole, butoconazole, croconazole, econazole, metconazole, oxyconazole, sulconazole, tebuconazole, pharmaceutically acceptable salts thereof, and combinations thereof. The pharmaceutical composition according to (3) above, which comprises any of the above.
(5) The pharmaceutical composition according to (2) above, wherein the active ingredient is the HMGCR inhibitor.
(6) The pharmaceutical composition according to (5) above, wherein the HMGCR inhibitor is atorvastatin, lovastatin, simvastatin, a pharmaceutically acceptable salt thereof, or a combination thereof.
(7) The pharmaceutical composition according to (1) above, which is an external pharmaceutical composition.
(8) The pharmaceutical composition according to (1) above, wherein the chronic dermatitis is atopic dermatitis or psoriasis.
(9) Amorolfine, alendronate, atorvastatin, azalanstat, bifonazole, butoconazole, croconazole, econazole, fluazinam, lovastatin, metconazole, oxyconazole, risedronate, simvastatin, sulconazole, tebuconazole, terconazole, pharmaceutically acceptable thereof. A salt and / or a combination thereof, and a pharmaceutical composition for the treatment of chronic dermatitis. (10) A measurement step of measuring the inhibitory activity of the target compound (a) for inhibiting the production of any of the cholesterol biosynthetic precursors represented by the following formulas (I) to (IV),
(B) a determination step of determining whether or not the target compound is a candidate compound effective for the treatment of chronic dermatitis, based on the measured value of the inhibitory activity,
A method for screening a candidate compound for treating chronic dermatitis.
Figure JPOXMLDOC01-appb-C000005
 (11) (c) a detection step of detecting any of the cholesterol biosynthetic precursors represented by the following formulas (I) to (IV) from a skin sample,
(D) a comparison step of comparing a detected amount of the cholesterol biosynthetic precursor detected from the sample with a reference reference amount,
Methods for the diagnosis of chronic dermatitis.
Figure JPOXMLDOC01-appb-C000006
　上述の本発明によれば、アトピー性皮膚炎や乾癬などの慢性皮膚炎に対し、効果、安全性、利便性、及び経済性等において良好である、新たな医薬組成物を提供できる。また、本発明は、慢性皮膚炎治療用の候補化合物のスクリーニング方法を提供する。さらに本発明は、慢性皮膚炎の診断のための方法を提供する。 According to the present invention described above, it is possible to provide a new pharmaceutical composition which is excellent in efficacy, safety, convenience, economical efficiency and the like against chronic dermatitis such as atopic dermatitis and psoriasis. The present invention also provides a method for screening candidate compounds for treating chronic dermatitis. The invention further provides a method for the diagnosis of chronic dermatitis.
式（Ｉ）及び式（ＩＩ）で分子構造が表される脂質代謝物のマススペクトルデータを示す図である。It is a figure which shows the mass spectrum data of the lipid metabolite whose molecular structure is represented by Formula (I) and Formula (II). 式（ＩＩＩ）及び式（ＩＶ）で分子構造が表される脂質代謝物のマススペクトルデータを示す図である。It is a figure which shows the mass spectrum data of the lipid metabolite which molecular structure is represented by Formula (III) and Formula (IV). ＣＨＩＬＤ症候群に伴う慢性皮膚炎患者の病変部の治療前後の状態を示す図である。It is a figure which shows the state before and after the treatment of the lesion part of the chronic dermatitis patient with CHILD syndrome. ＣＨＩＬＤ症候群に伴う慢性皮膚炎患者の病変部と、同一患者の健常部とにおける脂質代謝物の蓄積量を示す図である。It is a figure which shows the accumulation amount of the lipid metabolite in the lesion part of the chronic dermatitis patient with CHILD syndrome, and the healthy part of the same patient. コレステロール生合成阻害剤による、特定の脂質代謝物の産生抑制効果を示す図である。It is a figure which shows the production suppression effect of a specific lipid metabolite by a cholesterol biosynthesis inhibitor. 慢性皮膚炎患者における特定の脂質代謝物の蓄積量の相対値を示す図である。It is a figure which shows the relative value of the accumulation amount of a specific lipid metabolite in a chronic dermatitis patient. 尋常性乾癬患者の複数の病変部に対する、セクキヌマブ（コセンティクス）による治療の前後における特定脂質代謝物の蓄積量の変化を示す図である。FIG. 3 is a diagram showing changes in the accumulated amount of a specific lipid metabolite before and after treatment with secukinumab (Cosentyx) on a plurality of lesions of a patient with psoriasis vulgaris. コレステロール生合成阻害剤による、アトピー性皮膚炎患者における特定の脂質代謝物の蓄積抑制効果を示す図である。It is a figure which shows the accumulation suppression effect of a specific lipid metabolite in an atopic dermatitis patient by a cholesterol biosynthesis inhibitor. コレステロール生合成阻害剤による、尋常性乾癬患者における特定の脂質代謝物の蓄積抑制効果を示す図である。It is a figure which shows the accumulation suppression effect of the specific lipid metabolite in a psoriasis vulgaris patient by a cholesterol biosynthesis inhibitor.
　以下に本発明について詳細に説明する。
　本発明の医薬組成物は、慢性皮膚炎の治療に有用であり、コレステロール生合成前駆物質の産生を阻害する有効成分を含む。 The present invention will be described in detail below.
The pharmaceutical composition of the present invention is useful for treating chronic dermatitis and contains an active ingredient that inhibits the production of cholesterol biosynthetic precursors.
［コレステロール生合成前駆物質］
　上記コレステロール生合成前駆物質として、下記式（Ｉ）～（ＩＶ）で示される脂質代謝物（それぞれ、式（Ｉ）の生合成前駆物質（式（Ｉ）の化合物）、式（ＩＩ）の生合成前駆物質（式（ＩＩ）の化合物）、式（ＩＩＩ）の生合成前駆物質（式（ＩＩＩ）の化合物）及び、式（ＩＶ）の生合成前駆物質（式（ＩＶ）の化合物）ともいう）が挙げられる。
Figure JPOXMLDOC01-appb-C000007
 　慢性皮膚炎の病変部において、式（Ｉ）～（ＩＶ）で示される脂質代謝物が蓄積されることが、新たに確認された。
　上述のコレステロール前駆体、すなわち、式（Ｉ）で示される生合成前駆物質（Ｃ２９Ａ）、式（ＩＩ）で示される生合成前駆物質（Ｃ２９Ｂ）、式（ＩＩＩ）で示される生合成前駆物質（Ｃ２８Ａ）、及び、式（ＩＶ）で示される生合成前駆物質（Ｃ２８Ｂ）の脂質代謝物を、以下、特定脂質代謝物ともいう。
　これらの特定脂質代謝物が皮膚炎発症に関連した毒性を有することは、ＣＨＩＬＤ症候群の病態から説明し得る。ＣＨＩＬＤ症候群は、ＮＳＤＨＬをコードする遺伝子の機能欠失性変異をヘテロに持つ伴性優性遺伝性疾患である。ＮＳＤＨＬはコレステロールの生合成において４位の脱メチル化反応を担う酵素の一つである。ＮＳＤＨＬ遺伝子はＸ染色体上に存在し、女性では二つあるＸ染色体上の一つの遺伝子のみが活性化するため、変異型ＮＳＤＨＬ遺伝子が活性化した細胞では、コレステロールの生合成反応がＮＳＤＨＬの手前で停止する。特定脂質代謝物は、４位に１つあるいは２つのメチル基を有す構造を示すため、ＮＳＤＨＬの変異によって脱メチル化反応が不全となって蓄積したものと考えられる。ＣＨＩＬＤ症候群では、左右片側性に紅斑、角化、落屑を伴う乾癬様の皮疹を生じるが、特定脂質代謝物の蓄積は、皮疹のある部位においてのみにみられる。ＨＭＧＣＲ阻害剤やＣＹＰ５１阻害剤はＮＳＤＨＬよりも上流でコレステロール生合成経路を遮断することで特定脂質代謝物の産生を抑制するが、これらの阻害剤を皮疹部に外用塗布すると皮膚炎が改善する。
　一方で、ＮＳＤＨＬ遺伝子の変異によってコレステロール産生が低下するが、コレステロールを外用塗布しても皮疹が改善しないことが報告されている。
　これらを踏まえると、特定脂質代謝物が皮膚炎に関連していることが強く支持される。アトピー性皮膚炎や乾癬といった慢性炎症性皮膚疾患の病変部では、皮膚炎に関連した毒性を有する特定脂質代謝物が蓄積している一方で、急性皮膚炎や中毒疹ではそのような蓄積はみられない。そのため、特定脂質代謝物は皮膚炎の慢性化に寄与すると考えられる。また、乾癬治療において、皮疹の改善に伴い特定脂質代謝物の蓄積が低下することも確認されている。
　このように、特定脂質代謝物の蓄積と、慢性皮膚炎の治療効果とが互いに関連しているといえる。このため、特定脂質代謝物の蓄積を抑制することにより、慢性皮膚炎の症状を軽減できるのであり、以下に詳述するように、特定の酵素阻害剤が、慢性皮膚炎の治療に有効であることも確認された。 [Cholesterol biosynthetic precursor]
As the cholesterol biosynthetic precursors, lipid metabolites represented by the following formulas (I) to (IV) (respectively, biosynthetic precursors of the formula (I) (compounds of the formula (I)) and formula (II) Also referred to as synthetic precursor (compound of formula (II)), biosynthetic precursor of formula (III) (compound of formula (III)) and biosynthetic precursor of formula (IV) (compound of formula (IV)). ) Is mentioned.
Figure JPOXMLDOC01-appb-C000007
 It was newly confirmed that lipid metabolites represented by the formulas (I) to (IV) are accumulated in the lesions of chronic dermatitis.
The above-mentioned cholesterol precursor, that is, the biosynthetic precursor (C29A) represented by the formula (I), the biosynthetic precursor (C29B) represented by the formula (II), the biosynthetic precursor represented by the formula (III) ( Hereinafter, the lipid metabolites of C28A) and the biosynthetic precursor (C28B) represented by the formula (IV) are also referred to as specific lipid metabolites.
The toxicity of these particular lipid metabolites associated with the development of dermatitis may be explained by the pathology of CHILD syndrome. CHILD syndrome is a sex-linked dominant inherited disease that has a heterozygous mutation in the gene encoding NSDHL. NSDHL is one of the enzymes responsible for the demethylation reaction at the 4-position in cholesterol biosynthesis. Since the NSDHL gene is present on the X chromosome and only one gene on two X chromosomes is activated in females, the cholesterol biosynthesis reaction occurs before NSDHL in cells in which the mutant NSDHL gene is activated. Stop. Since the specific lipid metabolite has a structure having one or two methyl groups at the 4-position, it is considered that the demethylation reaction failed due to the mutation of NSDHL and accumulated. In CHILD syndrome, a psoriasis-like skin rash accompanied by erythema, keratinization, and desquamation on the left and right sides occurs, but the accumulation of specific lipid metabolites is found only at the site of the skin rash. HMGCR inhibitors and CYP51 inhibitors suppress the production of specific lipid metabolites by blocking the cholesterol biosynthesis pathway upstream of NSDHL, but topical application of these inhibitors improves dermatitis.
On the other hand, it has been reported that cholesterol production is decreased by mutation of NSDHL gene, but skin rash is not improved by topical application of cholesterol.
Based on these, it is strongly supported that a specific lipid metabolite is associated with dermatitis. In the lesions of chronic inflammatory skin diseases such as atopic dermatitis and psoriasis, specific lipid metabolites with toxic properties related to dermatitis are accumulated, while in acute dermatitis and toxic eruption, such accumulation is not observed. I can't. Therefore, it is considered that specific lipid metabolites contribute to chronic dermatitis. In addition, it has been confirmed that in the treatment of psoriasis, the accumulation of specific lipid metabolites decreases as the skin rash improves.
Thus, it can be said that the accumulation of specific lipid metabolites and the therapeutic effect on chronic dermatitis are related to each other. Therefore, by suppressing the accumulation of specific lipid metabolites, it is possible to reduce the symptoms of chronic dermatitis, and as described in detail below, specific enzyme inhibitors are effective in treating chronic dermatitis. It was also confirmed.
　上述の特定脂質代謝物である、式（Ｉ）～（ＩＶ）のコレステロール生合成前駆物質の分子構造は、以下のように決定した。
　すなわち、式（Ｉ）及び式（ＩＩ）の化合物については、合成した4,4-dimethylcholesta-8(9)-en-3beta-ol 、及び14-Demethyllanosterol標品をＧＣ／ＭＳ解析した結果、保持時間と確認イオンの一致から構造を決定した。
　一方、式（ＩＩＩ）及び式（ＩＶ）の化合物については、ＧＣ／ＭＳの解析結果と文献報告（上述の非特許文献４及び５）を照合することで分子構造を決定した。図１に、式（Ｉ）及び式（ＩＩ）で分子構造が表される化合物のマススペクトルデータを示す。また、図２に、式（ＩＩＩ）及び式（ＩＶ）で分子構造が表される化合物のマススペクトルデータを示す。
　なお、式（Ｉ）～（ＩＶ）の化合物については、ＧＣ／ＭＳ解析にてコレステロールの保持時間を1としたときの相対的な保持時間としてそれぞれ1.21，1.27，1.12，及び1.17にピークが出現することが確認されており、これらのピークは、慢性皮膚炎に特異的に出現するものとして確認されている（下記実施例２の表２参照）。 The molecular structures of the cholesterol biosynthetic precursors of formulas (I) to (IV), which are the specific lipid metabolites described above, were determined as follows.
That is, for the compounds of formula (I) and formula (II), the results of GC / MS analysis of the synthesized 4,4-dimethylcholesta-8 (9) -en-3beta-ol and 14-Demethyllanosterol preparations were confirmed. The structure was determined from the agreement of time and confirming ion.
On the other hand, regarding the compounds of formula (III) and formula (IV), the molecular structure was determined by collating the analysis results of GC / MS with the literature reports (Non-patent documents 4 and 5 described above). FIG. 1 shows mass spectrum data of the compounds represented by the formulas (I) and (II). Further, FIG. 2 shows mass spectrum data of the compounds represented by the formulas (III) and (IV).
For the compounds of formulas (I) to (IV), peaks appear at 1.21, 1.27, 1.12, and 1.17, respectively, as relative retention times when the retention time of cholesterol is 1 in the GC / MS analysis. It has been confirmed that these peaks appear specifically in chronic dermatitis (see Table 2 in Example 2 below).
　さらに、実施例２の表２にて示されるように、上述の相対保持時間で検出されるｍ／ｚ値に対するレスポンス量を示す各化合物のスペクトルデータにおいて、ｍ／ｚ値が５０以上であってレスポンス量が多い順から選択した１５のピーク中に、少なくとも下記のｍ／ｚ値のピークが含まれることが確認された。
　式（Ｉ）の化合物（Ｃ２９Ａ）　　：４８６．４；３８１．３；１３５．１
　式（ＩＩ）の化合物（Ｃ２９Ｂ）　：４８４．４；３７９．３；１３５．１
　式（ＩＩＩ）の化合物（Ｃ２８Ａ）：４７２．４；３６７．３；２２７．２
　式（ＩＶ）の化合物（Ｃ２８Ｂ）  ：４７２．４；２６９．２；１４７．１ Furthermore, as shown in Table 2 of Example 2, in the spectrum data of each compound showing the response amount with respect to the m / z value detected at the above-mentioned relative retention time, the m / z value was 50 or more. It was confirmed that at least the following peaks of m / z values were included in the 15 peaks selected from the order of large response amount.
Compound of formula (I) (C29A): 486.4; 381.3; 135.1
Compound of formula (II) (C29B): 484.4; 379.3; 135.1
Compound of formula (III) (C28A): 472.4; 367.3; 227.2
Compound of formula (IV) (C28B): 472.4; 269.2; 147.1.
　本発明における、前記コレステロール生合成前駆物質の産生を阻害する有効成分（コレステロール生合成阻害剤ともいう）としては、ＣＹＰ５１阻害剤、ＨＭＧＣＲ阻害剤、ビスホスホネート系薬剤、アモロルフィン又はその薬学的に許容される塩、並びにフルアジナム又はその薬学的に許容される塩が挙げられる。 In the present invention, the active ingredient (also referred to as cholesterol biosynthesis inhibitor) that inhibits the production of the cholesterol biosynthesis precursor is a CYP51 inhibitor, an HMGCR inhibitor, a bisphosphonate drug, amorolfine or a pharmaceutically acceptable salt thereof. Salts as well as fluazinam or a pharmaceutically acceptable salt thereof are mentioned.
　本発明の医薬組成物は、有効成分として、ＣＹＰ５１阻害剤、及び、ＨＭＧＣＲ阻害剤の少なくともいずれかを含有することが好ましい。 The pharmaceutical composition of the present invention preferably contains, as an active ingredient, at least one of a CYP51 inhibitor and an HMGCR inhibitor.
［ＣＹＰ５１阻害剤］
　ＣＹＰ５１阻害剤としては、例えば、アザランスタット、ビフォナゾール、ブトコナゾール、クロコナゾール、エベルコナゾール、エコナゾール、エフィナコナゾール、フェンチコナゾール、フルトリマゾール、ホスフルコナゾール、イサブコナゾール、メトコナゾール、ネチコナゾール、オモコナゾール、オキシコナゾール、セルタコナゾール、スルコナゾール、テブコナゾール、テルコナゾール及びチオコナゾール又はそれらの薬学的に許容される塩が挙げられる。
　好ましいＣＹＰ５１阻害剤としては、アザランスタット、ビフォナゾール、ブトコナゾール、クロコナゾール、エコナゾール、メトコナゾール、オキシコナゾール、スルコナゾール及びテブコナゾール又はそれらの薬学的に許容される塩が挙げられる。
　医薬組成物においては、ＣＹＰ５１阻害剤である化合物と薬学的に許容されるそれらの塩から選択される、いずれかを複数、組み合わせて用いてもよい。 [CYP51 inhibitor]
Examples of CYP51 inhibitors include azalanstat, bifonazole, butoconazole, croconazole, everconazole, econazole, efinaconazole, fenticonazole, flutrimazole, phosfluconazole, isabconazole, metconazole, neticonazole, omoconazole, oxyconazole. , Sertaconazole, sulconazole, tebuconazole, terconazole and thioconazole or pharmaceutically acceptable salts thereof.
Preferred CYP51 inhibitors include azalanstat, bifonazole, butoconazole, croconazole, econazole, metconazole, oxyconazole, sulconazole and tebuconazole or pharmaceutically acceptable salts thereof.
In the pharmaceutical composition, a plurality of compounds selected from CYP51 inhibitor compounds and pharmaceutically acceptable salts thereof may be used in combination.
　薬学的に許容される塩としては、例えば、アルカリ金属（例えば、リチウム、ナトリウム、カリウム等）、アルカリ土類金属（例えば、マグネシウム、カルシウム、バリウム等）、遷移金属（例えば、亜鉛、鉄等）、アンモニア、有機塩基（例えば、トリメチルアミン、トリエチルアミン、ジシクロヘキシルアミン、エタノールアミン、ジエタノールアミン、トリエタノールアミン、メグルミン、エチレンジアミン、ピリジン、ピコリン、キノリン等）、およびアミノ酸との塩、または無機酸（例えば、塩酸、硫酸、硝酸、炭酸、臭化水素酸、リン酸、ヨウ化水素酸等）、および有機酸（例えば、ギ酸、酢酸、プロピオン酸、トリフルオロ酢酸、クエン酸、乳酸、酒石酸、シュウ酸、マレイン酸、フマル酸、マンデル酸、グルタル酸、リンゴ酸、安息香酸、フタル酸、アスコルビン酸、ベンゼンスルホン酸、ｐ－トルエンスルホン酸、メタンスルホン酸、エタンスルホン酸等）との塩が挙げられる。特に塩酸、硫酸、リン酸、酒石酸、メタンスルホン酸との塩等が挙げられる。 Examples of the pharmaceutically acceptable salt include alkali metals (eg, lithium, sodium, potassium etc.), alkaline earth metals (eg, magnesium, calcium, barium etc.), transition metals (eg, zinc, iron etc.). , Ammonia, organic bases (eg, trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, meglumine, ethylenediamine, pyridine, picoline, quinoline, etc.), and salts with amino acids, or inorganic acids (eg, hydrochloric acid, Sulfuric acid, nitric acid, carbonic acid, hydrobromic acid, phosphoric acid, hydroiodic acid, etc., and organic acids (for example, formic acid, acetic acid, propionic acid, trifluoroacetic acid, citric acid, lactic acid, tartaric acid, oxalic acid, maleic acid) , Fumaric acid, mandelic acid, glutaric acid, phosphorus Acid, benzoic acid, phthalic acid, ascorbic acid, benzenesulfonic acid, p- toluenesulfonic acid, methanesulfonic acid, and salts with ethanesulfonic acid, etc.). Particularly, salts with hydrochloric acid, sulfuric acid, phosphoric acid, tartaric acid, methanesulfonic acid and the like can be mentioned.
［ＨＭＧＣＲ阻害剤］
　ＨＭＧＣＲ阻害剤の好ましい具体例としては、アトルバスタチン、ロバスタチン、シンバスタチン、これらの化合物の薬学的に許容される塩等が挙げられる。
　医薬組成物においては、ＨＭＧＣＲ阻害剤である化合物と薬学的に許容されるそれらの塩から選択される、いずれかを複数、組み合わせて用いてもよい。 [HMGCR inhibitor]
Preferred specific examples of the HMGCR inhibitor include atorvastatin, lovastatin, simvastatin, pharmaceutically acceptable salts of these compounds, and the like.
In the pharmaceutical composition, a plurality of compounds selected from the compound which is an HMGCR inhibitor and pharmaceutically acceptable salts thereof may be used in combination.
　ビスホスホネート系薬剤としては、例えば、アレンドロネート、リセドロネート、これらの化合物の薬学的に許容される塩等が挙げられる。 Examples of the bisphosphonate type drug include alendronate, risedronate, pharmaceutically acceptable salts of these compounds, and the like.
　別の態様として、本発明の医薬組成物は、有効成分として、アモロルフィン、アレンドロネート、アトルバスタチン、アザランスタット、ビフォナゾール、ブトコナゾール、クロコナゾール、エコナゾール、フルアジナム、ロバスタチン、メトコナゾール、オキシコナゾール、リセドロネート、シンバスタチン、スルコナゾール、テブコナゾール、及び、テルコナゾール、又はそれらの薬学的に許容される塩の少なくともいずれかを含有することが好ましい。また、医薬組成物においては、上述の有効成分としての化合物とそれらの薬学的に許容される塩から選択される、いずれかを複数、組み合わせて用いてもよい。 In another aspect, the pharmaceutical composition of the present invention comprises, as an active ingredient, amorolfine, alendronate, atorvastatin, azalanstat, bifonazole, butoconazole, croconazole, econazole, fluazinam, lovastatin, metconazole, oxyconazole, risedronate, simvastatin. , Sulconazole, tebuconazole, and / or terconazole or a pharmaceutically acceptable salt thereof is preferably contained. In the pharmaceutical composition, a plurality of compounds selected from the above-mentioned compounds as active ingredients and pharmaceutically acceptable salts thereof may be used in combination.
［慢性皮膚炎］
　本発明の医薬組成物は、慢性皮膚炎の治療剤として有用である。医薬組成物による治療が有効である慢性皮膚炎としては、アトピー性皮膚炎、乾癬、膿疱性乾癬、掌蹠膿疱症、魚鱗癬群、慢性蕁麻疹、乾皮症、手湿疹、ＣＨＩＬＤ症候群に伴う慢性皮膚炎、その他、慢性的な皮膚炎症状を呈する皮膚疾患が挙げられる。医薬組成物は、上記皮膚炎の中でも特に、アトピー性皮膚炎、及び、乾癬に対する治療効果に優れている。 [Chronic dermatitis]
The pharmaceutical composition of the present invention is useful as a therapeutic agent for chronic dermatitis. Chronic dermatitis for which treatment with a pharmaceutical composition is effective is associated with atopic dermatitis, psoriasis, pustular psoriasis, palmoplantar pustulosis, ichthyosis group, chronic urticaria, xeroderma, hand eczema, CHILD syndrome Examples include chronic dermatitis and other skin diseases that exhibit chronic skin inflammation. Among the above dermatitis, the pharmaceutical composition has an excellent therapeutic effect on atopic dermatitis and psoriasis.
［医薬組成物の剤形］
　本発明の医薬組成物は、皮膚炎部位への直接投与を目的とした外用剤として使用することができ、その剤形としては、例えば、軟膏剤、クリーム剤、ローション剤、リニメント剤、パップ剤、プラスター剤、パッチ剤、硬膏剤、ゲル剤、液剤等が挙げられる。 [Dosage Form of Pharmaceutical Composition]
The pharmaceutical composition of the present invention can be used as an external preparation intended for direct administration to a dermatitis site, and its dosage forms include, for example, ointments, creams, lotions, liniments, and poultices. , Plasters, patches, plasters, gels, liquids and the like.
［医薬組成物における添加剤など］
　医薬組成物においては、有効成分としての上記化合物以外に、吸収促進剤、ｐＨ調整剤、保存剤、着香料、分散剤、湿潤剤、安定剤、防腐剤、懸濁剤、界面活性剤等の医薬製剤用添加剤を単独もしくは２種以上を混合して配合することができる。 [Additives and the like in pharmaceutical compositions]
In the pharmaceutical composition, in addition to the above compounds as active ingredients, absorption promoters, pH adjusters, preservatives, flavoring agents, dispersants, wetting agents, stabilizers, preservatives, suspending agents, surfactants, etc. The additives for pharmaceutical preparations can be blended alone or in admixture of two or more.
　吸収促進剤としては、例えば、炭素数２０以下の１価アルコール（エチルアルコール、イソプロピルアルコール、ステアリルアルコール等）、ピロリドン誘導体（２－ピロリドン、１－メチル－２－ピロリドン等）、尿素類（尿素、チオ尿素等）、シクロデキストリン（α－シクロデキストリン等）、メントール、１－ドデシルアザシクロヘプタン－２－オン、チオグリコール酸カルシウム、リモネン等が挙げられる。該吸収促進剤の含有量は、剤形や基剤成分等によって異なるが、通常、吸収促進作用を効果的に発現させる観点からは、医薬組成物の全体重量を基準として、０．１重量％以上、好ましくは０．３重量％以上とし、副作用発現抑制の観点からは、１０重量％以下、好ましくは５重量％以下とする。 Examples of the absorption promoter include monohydric alcohols having 20 or less carbon atoms (ethyl alcohol, isopropyl alcohol, stearyl alcohol, etc.), pyrrolidone derivatives (2-pyrrolidone, 1-methyl-2-pyrrolidone, etc.), ureas (urea, Thiourea), cyclodextrin (α-cyclodextrin etc.), menthol, 1-dodecylazacycloheptan-2-one, calcium thioglycolate, limonene and the like. The content of the absorption enhancer varies depending on the dosage form, the base component, etc., but usually 0.1% by weight based on the total weight of the pharmaceutical composition from the viewpoint of effectively exhibiting the absorption enhancer action. The above content is preferably 0.3% by weight or more, and from the viewpoint of suppressing the occurrence of side effects, it is 10% by weight or less, preferably 5% by weight or less.
　ｐＨ調整剤の具体例としては、例えば、塩酸、硫酸又はリン酸等の無機酸、酢酸、コハク酸、フマル酸又はリンゴ酸等の有機酸、或いはこれら酸の金属塩等が挙げられる。該ｐＨ調整剤の配合量は、剤形や基剤成分等により異なるが、通常、製剤のｐＨが４～８となるような範囲で配合することが好ましい。 Specific examples of the pH adjusting agent include inorganic acids such as hydrochloric acid, sulfuric acid or phosphoric acid, organic acids such as acetic acid, succinic acid, fumaric acid or malic acid, and metal salts of these acids. The blending amount of the pH adjuster varies depending on the dosage form, base components and the like, but it is usually preferable to blend it in such a range that the pH of the preparation is 4 to 8.
　保存剤又は防腐剤の具体例としては、例えば、パラオキシ安息香酸、メチルパラベン、クロロブタノール、ベンジルアルコール、パラオキシ安息香酸メチル等が挙げられる。 Specific examples of preservatives or preservatives include paraoxybenzoic acid, methylparaben, chlorobutanol, benzyl alcohol, methyl paraoxybenzoate and the like.
　着香料の具体例としては、例えば、メントール、ローズ油、ユーカリ油、ｄ－カンフル等が挙げられ、また分散剤の具体例としては、例えば、メタリン酸ナトリウム、ポリリン酸カリウム、無水ケイ酸等が挙げられる。 Specific examples of the flavoring agent include menthol, rose oil, eucalyptus oil, d-camphor and the like, and specific examples of the dispersant include sodium metaphosphate, potassium polyphosphate, silicic acid anhydride and the like. Can be mentioned.
　湿潤剤の具体例としては、例えば、プロピレングリコール、グリセリン、ソルビトール、乳酸ナトリウム、ヒアルロン酸ナトリウム等が挙げられ、また安定剤の具体例としては、例えば、亜硫酸水素ナトリウム、トコフェロール、エチレンジアミン四酢酸（ＥＤＴＡ）、クエン酸等が挙げられる。 Specific examples of the wetting agent include, for example, propylene glycol, glycerin, sorbitol, sodium lactate, sodium hyaluronate and the like, and specific examples of the stabilizer include, for example, sodium hydrogen sulfite, tocopherol, ethylenediaminetetraacetic acid (EDTA). ), Citric acid and the like.
　懸濁剤の具体例としては、例えば、トラガント末、アラビアゴム末、ベントナイト、カルボキシメチルセルロースナトリウム等が挙げられ、また、界面活性剤の具体例としては、例えば、ポリオキシエチレン硬化ヒマシ油、セスキオレイン酸ソルビタン等のソルビタン脂肪酸エステル、ステアリン酸ポリオキシル等が挙げられる。 Specific examples of the suspending agent include, for example, tragacanth powder, gum arabic powder, bentonite, sodium carboxymethyl cellulose and the like, and specific examples of the surfactant include, for example, polyoxyethylene hydrogenated castor oil, sesquiolein. Examples thereof include sorbitan fatty acid esters such as acid sorbitan, and polyoxyl stearate.
　医薬組成物が、軟膏剤又はクリーム剤である場合には、基剤として、油脂性基剤又は乳剤性基剤を用いることができる。 When the pharmaceutical composition is an ointment or cream, an oily base or an emulsion base can be used as the base.
　油脂性基剤としては、例えば、炭化水素類（炭素数１２～３２の炭化水素、流動パラフィン、白色ワセリン、スクワレン、スクワラン又はプラスチベース等）、高級アルコール（ラウリルアルコール、セチルアルコール、ステアリルアルコール又はオレイルアルコール等の炭素数１２～３０の脂肪族１価アルコール等）、高級脂肪酸（パルミチン酸又はステアリン酸等の炭素数６～３２の飽和又は不飽和脂肪酸）、高級脂肪酸エステル（パルミチン酸ミリスチル又はステアリン酸ステアリル等の脂肪酸エステル；ラノリン又はカルナウバロウ等の炭素数１０～３２の脂肪酸と炭素数１４～３２の脂肪族１価アルコールとのエステル；グリセリルモノラウリレート等の炭素数１０～２２の飽和もしくは不飽和脂肪酸とグリセリンとのエステル、又はそれらの水素添加物等）、グリコール類（エチレングリコール、プロピレングリコール、ポリエチレングリコール等）、植物油、動物油等が挙げられる。 Examples of the oily base include hydrocarbons (hydrocarbons having 12 to 32 carbon atoms, liquid paraffin, white petrolatum, squalene, squalane, plastibase, etc.), higher alcohols (lauryl alcohol, cetyl alcohol, stearyl alcohol or oleyl alcohol). C12-C30 aliphatic monohydric alcohols, etc.), higher fatty acids (C6-C32 saturated or unsaturated fatty acids such as palmitic acid or stearic acid), higher fatty acid esters (myristyl palmitate or stearyl stearate) Ester of fatty acid such as lanolin; Carnauba wax or the like ester of fatty acid having 10 to 32 carbon atoms and aliphatic monohydric alcohol having 14 to 32 carbon atoms; saturated or unsaturated fatty acid having 10 to 22 carbon atoms such as glyceryl monolaurate With glycerin Or their hydrogenated products, etc.), glycols (ethylene glycol, propylene glycol, polyethylene glycol, and the like), vegetable oils, animal oils, and the like.
　乳剤性基剤としては、例えば、水中油型基剤、油中水型基剤、懸濁型基剤等が挙げられる。水中油型基剤としては、界面活性剤の存在下又は非存在下で、前記ラノリン、プロピレングリコール、ステアリルアルコール、ワセリン、シリコン油、流動パラフィン、グリセリルモノステアレート、ポリエチレングリコール等の成分を水相中に乳化、分散せしめた基剤等が挙げられる。油中水型基剤としては、ワセリン、高級脂肪族アルコール、流動パラフィン等の成分に、非イオン性界面活性剤の存在下で、水を加え、乳化、分散せしめた基剤等が挙げられる。また、懸濁型基剤としては、水に、デンプン、グリセリン、高粘度カルボキシメチルセルロース、カルボキシビニルポリマーなどの懸濁化剤を加えてゲル状にした水性基剤等が挙げられる。 Examples of emulsion bases include oil-in-water bases, water-in-oil bases, and suspension bases. As the oil-in-water base, the components such as lanolin, propylene glycol, stearyl alcohol, petrolatum, silicone oil, liquid paraffin, glyceryl monostearate, and polyethylene glycol are added in the aqueous phase in the presence or absence of a surfactant. Examples include bases emulsified and dispersed therein. Examples of the water-in-oil type base include bases obtained by adding water to components such as petrolatum, higher aliphatic alcohols, liquid paraffin in the presence of a nonionic surfactant, and emulsifying and dispersing. In addition, examples of the suspension-type base include an aqueous base that is made into a gel by adding a suspending agent such as starch, glycerin, high-viscosity carboxymethyl cellulose, and carboxyvinyl polymer to water.
［医薬組成物の製造方法］
　本発明の医薬組成物は、慣用の外用剤調製方法によって製造することができる。例えば、軟膏剤又はクリーム剤は、それぞれの剤形に応じて基剤の原料を混練、乳化又は懸濁せしめて基剤を調製した後、有効成分及び各種添加剤を加え、スクリューミキサー等の混合機中で混合することにより製造することができる。 [Method for producing pharmaceutical composition]
The pharmaceutical composition of the present invention can be produced by a conventional method for preparing an external preparation. For example, an ointment or cream is prepared by kneading, emulsifying or suspending the raw materials of the base according to each dosage form to prepare the base, then adding the active ingredient and various additives, and mixing with a screw mixer or the like. It can be produced by mixing in the machine.
　本発明の医薬組成物は、懸濁型、乳剤型もしくは溶液型ローション剤のいずれの剤形でも使用することができる。懸濁型ローションの基剤としては、アラビアゴム、トラガントゴム等のゴム類、メチルセルロース、ヒドロキシエチルセルロース等のセルロース類、ベントナイト等の粘土類の懸濁剤と水の混合物等が挙げられる。乳剤型ローションの基剤としては、水とステアリン酸又はオレイン酸等の脂肪酸、ステアリルアルコール又はセチルアルコール等の高級アルコール等の油性物質を乳化させた基剤等が挙げられる。溶液型ローションの基剤としては、水、エタノール、グリセリン、プロピレングリコール等のアルコール等が挙げられる。該ローション剤は、例えば、精製水に種々の基剤成分を添加して混合、攪拌した後、有効成分及び添加剤を加えて混合し、所望に応じて濾過を行なうことにより、製造することができる。
　リニメント剤のための基剤としては、例えば、オリーブ油等の植物油類、エタノールもしくはイソプロパノール等のアルコール類、或いはそれらと水との混合物等が挙げられる。該リニメント剤は、例えば、基剤に有効成分を溶解し、所望により、これに製剤用添加物を加えて混合することにより、製造することができる。 The pharmaceutical composition of the present invention can be used in any form of suspension type, emulsion type or solution type lotion. Examples of the base of the suspension lotion include gums such as gum arabic and tragacanth, celluloses such as methyl cellulose and hydroxyethyl cellulose, and clays such as bentonite, and a mixture of water and the like. Examples of the emulsion type lotion base include a base obtained by emulsifying water and a fatty acid such as stearic acid or oleic acid, and an oily substance such as higher alcohols such as stearyl alcohol or cetyl alcohol. Examples of the base of the solution type lotion include water, ethanol, glycerin, alcohols such as propylene glycol and the like. The lotion can be produced, for example, by adding various base components to purified water, mixing and stirring, and then adding the active ingredients and additives, mixing, and filtering if desired. it can.
Examples of the base for the liniment include vegetable oils such as olive oil, alcohols such as ethanol or isopropanol, and mixtures thereof with water. The liniment agent can be produced, for example, by dissolving the active ingredient in a base and, if desired, adding an additive for formulation thereto and mixing them.
　パップ剤のための基剤としては、例えば、ポリアクリル酸、ポリビニルアルコールもしくはポリビニルピロリドン等の水溶性高分子化合物等が挙げられる。該パップ剤は、例えば、有効成分、基剤及び所望の製剤用添加物を混合し、加熱後冷却することにより、製造することができる。 The base for poultices includes, for example, water-soluble polymer compounds such as polyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, and the like. The poultice can be produced, for example, by mixing an active ingredient, a base and a desired formulation additive, heating and cooling.
　プラスター剤、パッチ剤、又は硬膏剤のための基剤としては、例えば、不織布等の支持体、天然ゴム又はイソプレンゴム等の弾性体、亜鉛華、酸化チタン等の充填剤、テルペン樹脂等の粘着付与剤、酢酸ビニル等の剥離処理剤、流動パラフィン等の軟化剤、ジブチルヒドロキシトルエン（ＢＨＴ）等の老化防止剤等を適宜、組合せて使用することができる。該プラスター剤、パッチ剤、硬膏剤等は、溶液法や熱圧法などの常法により製造することができる。 As a base for plasters, patches, or plasters, for example, a support such as a non-woven fabric, an elastic body such as natural rubber or isoprene rubber, a filler such as zinc white and titanium oxide, an adhesive such as terpene resin An imparting agent, a release agent such as vinyl acetate, a softening agent such as liquid paraffin, an antiaging agent such as dibutylhydroxytoluene (BHT), and the like can be used in appropriate combination. The plaster, patch, plaster and the like can be produced by a conventional method such as a solution method or a hot pressing method.
　液剤調製のための溶媒としては、例えば、水、エタノール、イソプロピルアルコール、ベンジルアルコール、ポリエチレングリコール（ＰＥＧ４００等）、プロピレングリコール、プロピレンカーボネート、又はこれらの混合物等があげられる。また、該液剤は、ガーゼ、創面被覆材等に含浸させて使用することもできる。 Examples of the solvent for preparing the liquid agent include water, ethanol, isopropyl alcohol, benzyl alcohol, polyethylene glycol (PEG400 etc.), propylene glycol, propylene carbonate, or a mixture thereof. Further, the liquid agent can be used by impregnating it with gauze, a wound surface covering material and the like.
　上記医薬組成物（製剤）中への有効成分、すなわち、ＣＹＰ５１阻害剤、ＨＭＧＣＲ阻害剤等の配合量は、剤形によっても異なるが、例えば、軟膏剤もしくはクリーム剤の場合には、医薬組成物の全体重量を基準として、０．００２５～５重量％、より好ましくは０．５～５重量％、とりわけ好ましくは１～２重量％とすることができる。また、医薬組成物を含む医薬が液剤である場合には、医薬組成物の全体量を基準として、０.１～２００mg/mL、より好ましくは５～５０mg/mL、とりわけ１０～２０mg/mLとすることが好ましい。 The amount of the active ingredient, that is, the amount of the CYP51 inhibitor, HMGCR inhibitor, etc., added to the pharmaceutical composition (formulation) varies depending on the dosage form. For example, in the case of an ointment or cream, the pharmaceutical composition It can be 0.0025 to 5% by weight, more preferably 0.5 to 5% by weight, and particularly preferably 1 to 2% by weight, based on the total weight of When the drug containing the pharmaceutical composition is a liquid, the total amount of the pharmaceutical composition is 0.1 to 200 mg / mL, more preferably 5 to 50 mg / mL, especially 10 to 20 mg / mL. Preferably.
［医薬組成物の適用］
　なお、本発明の医薬組成物の投与量は、痒みの種類、疾患部位、痒みの程度等に応じて、上記製剤の適量を１日当り１回～数回、局所投与（患部への塗布等による局所投与）すればよい。 [Application of pharmaceutical composition]
The dosage of the pharmaceutical composition of the present invention depends on the type of itch, the disease site, the degree of itch, etc., and an appropriate amount of the above formulation is administered once to several times a day by topical administration (by application to the affected area, etc.). Local administration).
［慢性皮膚炎治療用の候補化合物のスクリーニング方法］
　本発明における、慢性皮膚炎治療用の候補化合物のスクリーニング方法は、対象となる化合物が有する、特定脂質代謝物（コレステロール生合成前駆物質）の少なくとも１種の産生を阻害する阻害活性を測定する測定工程と、測定された阻害活性の値に基づいて、対象化合物が慢性皮膚炎の治療に有効か否かを判定する判定工程とを含む。 [Screening Method of Candidate Compounds for Treatment of Chronic Dermatitis]
The method for screening a candidate compound for treating chronic dermatitis in the present invention is a measurement for measuring an inhibitory activity of a target compound for inhibiting the production of at least one specific lipid metabolite (cholesterol biosynthetic precursor). And a determination step of determining whether or not the target compound is effective for treating chronic dermatitis based on the measured inhibitory activity value.
　測定工程においては、例えば、以下の方法で阻害活性を測定可能である。
　例えば、コレステロール除去血清を添加したＤＭＥＭ（ダルベッコ改変イーグル）培地に懸濁した１ｘ１０５個（１ｍＬ）のＨａＣａＴ細胞を１２ウェルのマルチウェルプレートに播種し、そこに最終濃度３０μＭの１７－ヒドロキシプロゲステロン、さらにジメチルスルホキシドに溶解した１ｍＭの対象化合物を最終濃度１μＭになるように添加し、４８時間培養する。
　上記細胞をＰＢＳ（リン酸緩衝生理食塩水）で２回洗浄したのち、２００μＬのＴｒｙｐＬＥ　Ｅｘｐｒｅｓｓを添加し３７℃で１３分間インキュベートする。０．５ｍＬのＰＢＳを添加して細胞をチューブに回収した後、４℃、５００ｇで５分間、遠心し、細胞を沈殿させ、上清を除去した後に０．４ｍＬのＰＢＳで再懸濁する。１ ｍＬのヘキサンで洗浄したガラスチューブにケイソウ土カラム（ＩＳＯＬＵＴＥ　ＳＬＥ＋４００、Ｂｉｏｔａｇｅ）をセットし、細胞懸濁液を全量添加する。シリンジを用いて細胞懸濁液をカラムに充填させたのち、７５０μＬのジクロロメタンをカラム上部に添加し、流出液を回収する。同じ作業を３回繰り返し、サンプル溶液を得る。
　回収したサンプル溶液に０．１μｇ／μＬのＤ７－コレステロールのエタノール溶液を１０μＬ添加し、よく混合した後に４０℃で３０分間、窒素置換を行い乾燥させる。乾燥したサンプルに、５０μＬのＢＳＴＦＡ＋ＴＭＣＳ（Ｎ，Ｏ－ビス（トリメチルシリル）トリフルオロアセトアミド＋トリメチルクロロシラン），９９：１（Ｓｕｐｅｌｃｏ社）および１０μＬのピリジンを加え、ガラスバイアルにすぐにキャップをして４０℃で３０分間静置する。２μＬのサンプルをガスクロマトグラフィー／マススペクトロメトリー（ＧＣ／ＭＳ）で分析する。ＧＣ／ＭＳ分析条件は、以下に示す通りである。
＜ＧＣ／ＭＳ分析条件＞
　キャピラリーカラム：Rtx-5MS (長さ30m, 内径0.25mm, 膜厚0.25μm)
　オーブン温度：150 ℃, 保持1分-20℃／分→250℃, 5℃／分→280℃, 保持10分- 20℃／分→330℃, 保持3分。すなわち、150 ℃で1分間保持し、20℃／分の昇温速度で250℃まで昇温し、 5℃／分の昇温速度で280℃まで昇温し、280℃で10分間、保持し、20℃／分の昇温速度で330℃まで昇温し、3分間、保持する。
　キャリアガス：He, 線速度39.0 cm/秒
　イオン源温度：　200℃
　インターフェース温度：280℃
　気化室温度：　250℃
　そして特定脂質代謝物（コレステロール生合成前駆物質）の少なくとも１種のピークエリアを測定し、対象化合物の阻害活性を算出する。その際、化合物を添加しない場合の特定脂質代謝物のピークエリアを０％阻害とする。
　また、判定工程においては、例えば、所定の閾値以上の活性を有する化合物を製剤化すべく、測定された阻害活性の値に基づいて、以下の方法で当該化合物が慢性皮膚炎の治療に有効か否かを判定する。すなわち、上述の測定工程における阻害活性の測定方法であって、後述の実施例８においても採用されている測定方法で測定したときに、少なくともいずれかの特定脂質代謝物について、例えば、１０μＭで50%以上の蓄積阻害活性を示す化合物、より好ましくは、少なくともいずれかの特定脂質代謝物について、１μＭで50%以上の蓄積阻害活性を示す化合物を、慢性皮膚炎の治療に有効と判定する。
　このように、上記測定工程と判定工程とを有する本願発明の方法は、慢性皮膚炎の治療に有効と予想される候補医薬のスクリーニング方法として有用である。 In the measurement step, for example, the inhibitory activity can be measured by the following method.
For example, 1 × 10 5 (1 mL) of HaCaT cells suspended in DMEM (Dulbecco's modified Eagle) medium supplemented with cholesterol-depleted serum were seeded in a 12-well multiwell plate, and 17-hydroxyprogesterone at a final concentration of 30 μM was added thereto. Further, 1 mM of the target compound dissolved in dimethyl sulfoxide is added so as to have a final concentration of 1 μM, and the mixture is cultured for 48 hours.
The cells are washed twice with PBS (phosphate buffered saline), 200 μL of TrypLE Express is added, and the mixture is incubated at 37 ° C. for 13 minutes. After adding 0.5 mL of PBS and collecting the cells in a tube, centrifugation is performed at 4 ° C. and 500 g for 5 minutes to precipitate the cells, and after removing the supernatant, the cells are resuspended in 0.4 mL of PBS. A diatomaceous earth column (ISOLUTE SLE + 400, Biotage) is set in a glass tube washed with 1 mL of hexane, and the whole amount of the cell suspension is added. After filling the column with the cell suspension using a syringe, 750 μL of dichloromethane is added to the upper part of the column to collect the effluent. The same operation is repeated 3 times to obtain a sample solution.
To the recovered sample solution, 10 μL of 0.1 μg / μL D7-cholesterol ethanol solution was added, mixed well, and replaced with nitrogen at 40 ° C. for 30 minutes to dry. To the dried sample, 50 μL BSTFA + TMCS (N, O-bis (trimethylsilyl) trifluoroacetamide + trimethylchlorosilane), 99: 1 (Supelco) and 10 μL pyridine were added and the glass vial was immediately capped at 40 ° C. Let stand for 30 minutes. 2 μL of sample is analyzed by gas chromatography / mass spectrometry (GC / MS). The GC / MS analysis conditions are as shown below.
<GC / MS analysis conditions>
Capillary column: Rtx-5MS (length 30m, inner diameter 0.25mm, film thickness 0.25μm)
Oven temperature: 150 ℃, hold 1 min -20 ℃ / min → 250 ℃, 5 ℃ / min → 280 ℃, hold 10 minutes -20 ℃ / min → 330 ℃, hold 3 minutes. That is, hold at 150 ° C for 1 minute, raise to 250 ° C at a heating rate of 20 ° C / min, raise to 280 ° C at a heating rate of 5 ° C / min, and hold at 280 ° C for 10 minutes. The temperature is raised to 330 ° C. at a heating rate of 20 ° C./min and held for 3 minutes.
Carrier gas: He, Linear velocity 39.0 cm / sec Ion source temperature: 200 ℃
Interface temperature: 280 ℃
Vaporization chamber temperature: 250 ° C
Then, at least one peak area of the specific lipid metabolite (precursor of cholesterol biosynthesis) is measured, and the inhibitory activity of the target compound is calculated. At that time, the peak area of the specific lipid metabolite when the compound is not added is set to 0% inhibition.
Further, in the determination step, for example, in order to formulate a compound having an activity of a predetermined threshold value or more, based on the value of the measured inhibitory activity, whether the compound is effective in treating chronic dermatitis by the following method. To determine. That is, when it is measured by the measuring method of the inhibitory activity in the above-mentioned measuring step, which is also used in Example 8 described later, at least one of the specific lipid metabolites is, for example, 50 at 10 μM. A compound exhibiting an accumulation inhibitory activity of not less than%, more preferably a compound exhibiting an accumulation inhibitory activity of not less than 50% at 1 μM with respect to at least one of the specific lipid metabolites is determined to be effective for the treatment of chronic dermatitis.
As described above, the method of the present invention including the above measurement step and determination step is useful as a method for screening a candidate drug that is expected to be effective in treating chronic dermatitis.
［慢性皮膚炎の診断のための方法］
　本発明における、慢性皮膚炎の診断のための方法は、特定脂質代謝物の少なくとも１種を皮膚のサンプルから検出する検出工程と、サンプルから検出されたコレステロール生合成前駆物質の検出量と、基準となる基準量とを比較する比較工程とを有する。 [Methods for diagnosis of chronic dermatitis]
The method for diagnosing chronic dermatitis in the present invention comprises a detection step of detecting at least one specific lipid metabolite from a skin sample, a detection amount of a cholesterol biosynthetic precursor detected from the sample, and a standard. And a comparison step of comparing the reference amount with
　検出工程においては、例えば、以下の方法で特定脂質代謝物を検出する。
　病変部の角質を約１ｍｇ採取し、３ｍＬのヘキサンで脂質を抽出する。抽出物に対し、内標準物質（Ｉｎｔｅｒｎａｌ　ｓｔａｎｄａｒｄ）として、０．１μｇ／μＬのＤ７－コレステロールのエタノール溶液を１０μＬ添加する。これをシリカゲルカラム（Ｗａｔｅｒｓ　Ｓｅｐ－Ｐａｋ　Ｓｉｌｉｃａ）に全量ロードし、１ｍＬの酢酸エチルで溶出する画分を２ｍＬのガラスバイアルに回収する。内容物を窒素気流下４０℃に加温して乾固させた後、トリメチルシリル誘導体化を行う。具体的には、乾固後のバイアルに５０μＬのＢＳＴＦＡ－ＴＭＣＳ（９９：１）液と１０μＬのｐｙｒｉｄｉｎｅを加え、混和したのち密栓して４０℃で４０分間保温する。ここでのＧＣ／ＭＳ分析条件は、上述の慢性皮膚炎治療用の候補化合物のスクリーニング方法におけるＧＣ／ＭＳ分析条件と同一である。 In the detection step, for example, the specific lipid metabolite is detected by the following method.
Approximately 1 mg of the keratin of the lesion is collected, and the lipid is extracted with 3 mL of hexane. To the extract, 10 μL of an ethanol solution containing 0.1 μg / μL of D7-cholesterol is added as an internal standard. The entire amount of this is loaded onto a silica gel column (Waters Sep-Pak Silica), and the fraction eluted with 1 mL of ethyl acetate is collected in a 2 mL glass vial. The contents are heated to 40 ° C. under a nitrogen stream to dryness, and then derivatized with trimethylsilyl. Specifically, 50 μL of BSTFA-TMCS (99: 1) solution and 10 μL of pyridine are added to the dried vial, and after mixing, the container is sealed and kept at 40 ° C. for 40 minutes. The GC / MS analysis conditions here are the same as the GC / MS analysis conditions in the above-described screening method for candidate compounds for treating chronic dermatitis.
　さらに、あらかじめ濃度が明らかである標準試料を同じ条件で分析しておき、内部標準とのピーク面積比により標準化し、各物質の単位量あたりのエリア面積を求めておくことによる定量も可能である。
　標準試料が入手できない場合のピークに関しては、コレステロールのピークと分析対象物質のピークとの面積の比較を行って相対量として評価する。具体的にはコレステロールのピーク100000に対する、分析対象物質のピークの面積を算出し、病変部から得られた検体と、正常に角化した検体から得られた相対面積の比較を行う。 Furthermore, quantification is possible by analyzing standard samples whose concentrations are known in advance under the same conditions, standardizing by the peak area ratio with the internal standard, and determining the area area per unit amount of each substance. .
Regarding the peak when the standard sample is not available, the area of the peak of cholesterol and the peak of the substance to be analyzed are compared and evaluated as a relative amount. Specifically, the area of the peak of the substance to be analyzed with respect to the cholesterol peak 100000 is calculated, and the relative area obtained from the specimen obtained from the lesion and that obtained from the normally keratinized specimen are compared.
　以下に、上述の方法により得られた実際の測定データを示す。下記値は、すべて上述の方法により測定をおこない、コレステロールのピーク面積100000当たりの各物質のピーク面積を指数として表したものである。正常角化検体等におけるそれぞれのコレステロール生合成前駆物質のピーク面積は以下の通りであった。以下に示すように、アトピー性皮膚炎や尋常性乾癬ではすべての特定脂質代謝物（コレステロール生合成前駆物質）で、平均して正常角化角質の５倍以上の蓄積を認めた。
Figure JPOXMLDOC01-appb-T000008
 Below, the actual measurement data obtained by the above-mentioned method are shown. The following values are all measured by the above-mentioned method, and the peak area of each substance per 100000 peak area of cholesterol is expressed as an index. The peak areas of the respective cholesterol biosynthesis precursors in the normal keratinized samples and the like were as follows. As shown below, in atopic dermatitis and psoriasis vulgaris, the accumulation of all specific lipid metabolites (cholesterol biosynthetic precursor) was 5 times or more that of normal keratinized keratin on average.
Figure JPOXMLDOC01-appb-T000008
　上述のように、慢性皮膚炎の病変部においては、特定脂質代謝物が蓄積されることが、新たに確認されたため、サンプルから検出されたコレステロール生合成前駆物質の検出量が、所定の基準量よりも多い場合、当該サンプルの提供者は、慢性皮膚炎に罹患している可能性が高いと考えられる。
　このように、上記検出工程と比較工程とを有する本願発明の方法は、慢性皮膚炎の診断のための方法、あるいは、慢性皮膚炎の診断を補助する方法（重症度の評価、治療効果の判定等の方法）として、有用である。
　ここで基準量とは、所定の分析条件でGC-MS分析を行った際に検出される特定脂質代謝物の、コレステロールに対するピーク面積の比率で評価される値であり、正常に角化した角質ではコレステロールのピーク面積100000に対し、C28Aが150以下、C28Bが100以下、C29Aが200以下、C29Bが100以下である。よってコレステロールのピーク面積100000に対して、ピーク面積150がC28Aの基準量、ピーク面積100がC28Bの基準量、ピーク面積200がC29Aの基準量、ピーク面積100がC29Bの基準量となり得る。 As described above, it is newly confirmed that specific lipid metabolites are accumulated in the lesions of chronic dermatitis, and therefore the amount of cholesterol biosynthesis precursor detected in the sample is a predetermined reference amount. More than that, the sample donor is likely to have chronic dermatitis.
As described above, the method of the present invention having the detection step and the comparison step is a method for diagnosing chronic dermatitis, or a method for assisting diagnosis of chronic dermatitis (evaluation of severity, determination of therapeutic effect). Etc.) is useful.
Here, the reference amount is a value evaluated by the ratio of the peak area to cholesterol of a specific lipid metabolite detected when performing GC-MS analysis under predetermined analysis conditions, and keratinized normally. For Cholesterol peak area 100000, C28A is 150 or less, C28B is 100 or less, C29A is 200 or less, and C29B is 100 or less. Therefore, for the cholesterol peak area 100000, the peak area 150 can be the C28A reference amount, the peak area 100 can be the C28B reference amount, the peak area 200 can be the C29A reference amount, and the peak area 100 can be the C29B reference amount.
　上述のように、慢性皮膚炎の病変部においては、特定脂質代謝物が蓄積されることが、新たに確認されたため、サンプルから検出されたコレステロール生合成前駆物質の検出量が、所定の基準量よりも多い場合、当該サンプルの提供者は、慢性皮膚炎に罹患している可能性が高いと考えられる。
　このように、上記検出工程と比較工程とを有する本願発明の方法は、慢性皮膚炎の診断のための方法、あるいは、慢性皮膚炎の診断を補助する方法として、有用である。 As described above, it is newly confirmed that specific lipid metabolites are accumulated in the lesions of chronic dermatitis, and therefore the amount of cholesterol biosynthesis precursor detected in the sample is a predetermined reference amount. More than that, the sample donor is likely to have chronic dermatitis.
As described above, the method of the present invention including the detection step and the comparison step is useful as a method for diagnosing chronic dermatitis or a method for assisting diagnosis of chronic dermatitis.
　実施例１
［ＣＨＩＬＤ症候群の治療］
　ＣＨＩＬＤ症候群に伴う慢性皮膚炎の患者の左脇の病変部に対して、ＨＭＧＣＲ阻害剤としてアトルバスタチンを用い、１％アトルバスタチン・２％コレステロール水溶液の外用を１日２回行った。外用開始後１ヶ月で皮疹の改善が認められ、外用開始後７ヶ月で完全に皮疹は消失した。アトルバスタチンによる治療開始前の左脇の病変部を図３（Ａ１）に、治療開始後の左脇部位を図３（Ａ２）に示す。
　また、ＣＨＩＬＤ症候群に伴う慢性皮膚炎の患者の首の後側の病変部に対して、ＣＹＰ５１阻害剤として１％オキシコナゾールクリーム（販売名：オキナゾールクリーム）を用い、外用を１日１回行った。外用開始後４ヶ月で完全に皮疹は消滅した。オキシコナゾールによる治療開始前の首の後側の病変部を図３（Ｂ１）に、治療開始後の首の後側部位を図３（Ｂ２）に示す。
　以上より、ＣＨＩＬＤ症候群に伴う慢性皮膚炎に対して、ＨＭＧＣＲ阻害剤、及び、ＣＹＰ５１阻害剤が共に有効であることが確認された。 Example 1
[Treatment of CHILD syndrome]
To the lesion on the left side of a patient with chronic dermatitis associated with CHILD syndrome, 1% atorvastatin / 2% cholesterol aqueous solution was applied externally twice a day using atorvastatin as an HMGCR inhibitor. Improvement of the eruption was observed 1 month after the start of external application, and the eruption disappeared completely 7 months after the start of external application. FIG. 3 (A1) shows the lesion area on the left side before the start of treatment with atorvastatin, and FIG. 3 (A2) shows the left side area after the start of treatment.
In addition, 1% oxyconazole cream (trade name: quinazole cream) was used as a CYP51 inhibitor for topical lesions on the posterior side of the neck of patients with chronic dermatitis associated with CHILD syndrome, and external application was performed once a day. It was The skin rash disappeared completely 4 months after the start of external application. FIG. 3 (B1) shows a lesion on the back side of the neck before the start of treatment with oxyconazole, and FIG. 3 (B2) shows the back side of the neck after the start of treatment.
From the above, it was confirmed that both the HMGCR inhibitor and the CYP51 inhibitor are effective against chronic dermatitis associated with CHILD syndrome.
　実施例２
［ＣＨＩＬＤ症候群に伴う慢性皮膚炎］
　ＣＨＩＬＤ症候群に伴う慢性皮膚炎患者の病変部角質と、同一患者の健常部角質皮膚とを比較したところ、図４に示されるように、病変部においてのみ特定の脂質代謝物が蓄積していることが確認された。
（実験方法）
　病変部の角質を約1mg採取し、3mLのヘキサンで脂質を抽出した。抽出物に対し、内標準物質として、０．１μｇ／μＬのＤ７－コレステロールのエタノール溶液１０μＬを添加した。シリカゲルカラム(Waters Sep-Pak Silica)に全量ロードし、1mLの酢酸エチルで溶出する画分を２mLのガラスバイアルに回収した。内容物を窒素気流下40℃に加温して乾固させたあと、トリメチルシリル誘導体化を行った。具体的には、乾固後のバイアルに50μLのBSTFA-TMCS （99:1）液と10μLのpyridineを加え、混和したのち密栓して40℃で40分間保温した。ＧＣ／ＭＳ分析条件は、以下に示す通りであり、ＧＣ／ＭＳ分析の結果は表２に示す通りである。
＜ＧＣ／ＭＳ分析条件＞
　キャピラリーカラム：Rtx-5MS (長さ30m, 内径0.25mm, 膜厚0.25μm)
　オーブン温度：150 ℃, 保持1分-20℃／分→250℃, 5℃／分→280℃, 保持10分- 20℃／分→330℃, 保持3分。
　キャリアガス：He, 線速度39.0 cm/秒
　イオン源温度：　200℃
　インターフェース温度：280℃
　気化室温度：　250℃ Example 2
[Chronic dermatitis associated with CHILD syndrome]
When the keratinous lesions of a patient with chronic dermatitis associated with CHILD syndrome are compared with the keratinous skin of a healthy subject of the same patient, as shown in FIG. 4, specific lipid metabolites are accumulated only in the lesions. Was confirmed.
(experimental method)
Approximately 1 mg of the keratin of the lesion was collected and lipid was extracted with 3 mL of hexane. As an internal standard substance, 10 μL of an ethanol solution of 0.1 μg / μL of D7-cholesterol was added to the extract. The entire amount was loaded on a silica gel column (Waters Sep-Pak Silica), and the fraction eluted with 1 mL of ethyl acetate was collected in a 2 mL glass vial. The contents were heated to 40 ° C. under a nitrogen stream to dryness, and then derivatized with trimethylsilyl. Specifically, 50 μL of BSTFA-TMCS (99: 1) solution and 10 μL of pyridine were added to a vial after being dried and mixed, and the mixture was sealed and kept at 40 ° C. for 40 minutes. The GC / MS analysis conditions are as shown below, and the results of the GC / MS analysis are as shown in Table 2.
<GC / MS analysis conditions>
Capillary column: Rtx-5MS (length 30m, inner diameter 0.25mm, film thickness 0.25μm)
Oven temperature: 150 ℃, hold 1 min -20 ℃ / min → 250 ℃, 5 ℃ / min → 280 ℃, hold 10 minutes -20 ℃ / min → 330 ℃, hold 3 minutes.
Carrier gas: He, Linear velocity 39.0 cm / sec Ion source temperature: 200 ℃
Interface temperature: 280 ℃
Vaporization chamber temperature: 250 ° C
Figure JPOXMLDOC01-appb-T000009
 　上記式（Ｉ）～（ＩＶ）の化合物は、いずれもステロール骨格を有し、3位にOH基を含むものである。なお、式（Ｉ）及び式（ＩＩ）の化合物（炭素数は29）はいずれも4位に2つのメチル基を有し、式（ＩＩＩ）及び式（ＩＶ）の化合物（炭素数は28）はいずれも4位に1つのメチル基を有する。
　なお、表２に示すコレステロール生合成前駆物質のＧＣ／ＭＳのスペクトルデータのｍ／ｚ値はいずれも理論値であり、例えば、式（ＩＩ）の化合物（Ｃ２９Ｂ）における４８４．４のピークは、当該コレステロール生合成前駆物質（Ｃ２９Ｂ）の分子の水酸基がトリメチルシリル化（ＴＭＳ化）されて生じる－ＯＳｉ（Ｃ３Ｈ９）体に由来する値であり、３７９．３は、上記ＴＭＳ体からＣＨ４とＯＳｉ（Ｃ３Ｈ９）が脱離した断片に由来する値であり、１３５．１は、Ｃ１０Ｈ１５の断片に由来する値である。
　そして上記化合物のｍ／ｚの理論値は、図１、及び、図２において示される実測値と対応しているものの測定誤差も認められる。例えば、図１のＣ２９Ｂの測定データにおいては、若干の測定誤差により、式（ＩＩ）の化合物（Ｃ２９Ｂ）についての理論値である４８４．４に対応する４８４．３５のピーク、及び、理論値である１３５．１に対応する１３５．１５のピークが、それぞれ検出されている。このように、ＧＣ／ＭＳのスペクトルデータのｍ／ｚ値においては、概ね、±０．１程度までの誤差が認められ得る。
　また、内部標準物質であるコレステロールに対する上記相対保持時間の値についても、概ね±０．０１程度までの測定誤差が認められ、例えば、式（Ｉ）の化合物（Ｃ２９Ａ）においては、上記１．２１の測定値が１．２０～１．２２の範囲で変化し、式（ＩＩ）の化合物（Ｃ２９Ｂ）においては、上記１．２７の測定値が１．２６～１．２８の範囲で変化する可能性もある。また、式（ＩＩＩ）の化合物（Ｃ２８Ａ）においては、上記１．１２の測定値が１．１１～１．１３の範囲で変化し、式（ＩＶ）の化合物（Ｃ２８Ｂ）においては、上記１．１７の測定値が１．１６～１．１８の範囲で変化する可能性もある。
Figure JPOXMLDOC01-appb-T000009
 The compounds of the above formulas (I) to (IV) all have a sterol skeleton and contain an OH group at the 3-position. The compounds of formula (I) and formula (II) (having 29 carbon atoms) each have two methyl groups at the 4-position, and compounds of formula (III) and formula (IV) (having 28 carbon atoms) All have one methyl group at the 4-position.
The m / z values of the GC / MS spectrum data of the cholesterol biosynthetic precursors shown in Table 2 are theoretical values, and for example, the peak of 484.4 in the compound of formula (II) (C29B) is The hydroxyl group of the molecule of the cholesterol biosynthesis precursor (C29B) is a value derived from a —OSi (C 3 H 9 ) form produced by trimethylsilylation (TMS conversion), and 379.3 is a CH 4 value derived from the above TMS form. And OSi (C 3 H 9 ) are the values derived from the desorbed fragment, and 135.1 is the value derived from the C 10 H 15 fragment.
The theoretical m / z value of the above compound corresponds to the measured value shown in FIGS. 1 and 2, but a measurement error is also recognized. For example, in the measured data of C29B in FIG. 1, due to some measurement error, a peak of 484.35 corresponding to the theoretical value of 444.4 for the compound (C29B) of the formula (II) and a theoretical value of A peak of 135.15 corresponding to a certain 135.1 is detected. As described above, in the m / z value of the GC / MS spectrum data, an error of up to about ± 0.1 can be recognized.
Also, with respect to the value of the relative retention time with respect to cholesterol as an internal standard substance, a measurement error of up to about ± 0.01 was observed. For example, in the compound (C29A) of the formula (I), the above-mentioned 1.21 was used. The measured value of 1 changes in the range of 1.20 to 1.22, and in the compound (C29B) of the formula (II), the measured value of 1.27 can change in the range of 1.26 to 1.28. There is also a nature. In addition, in the compound (C28A) of the formula (III), the measured value of 1.12 changes within the range of 1.11 to 1.13, and in the compound (C28B) of the formula (IV), the above 1. It is possible that the 17 measurements may vary from 1.16 to 1.18.
　図４は、ＣＨＩＬＤ症候群に伴う慢性皮膚炎患者の病変部と、同一患者の健常部とにおける脂質代謝物の測定された蓄積量を示す図である。このように、ＣＨＩＬＤ症候群患者の病変部の角質では、正常部分ではほとんど検出されない特定脂質代謝物が検出された。
　そして検出された化合物の分子構造は、上述のように、式（Ｉ）～（ＩＶ）で表されることが確認された。 FIG. 4 is a diagram showing measured accumulation amounts of lipid metabolites in a lesion area of a chronic dermatitis patient associated with CHILD syndrome and a healthy area of the same patient. As described above, specific lipid metabolites that were hardly detected in the normal part were detected in the keratin of the lesion part of the CHILD syndrome patient.
The molecular structure of the detected compound was confirmed to be represented by the formulas (I) to (IV) as described above.
　実施例３
［脂質代謝物の蓄積抑制効果］
　実施例２で特定された脂質代謝物を人為的に蓄積させる試験系を構築し、各種のコレステロール生合成阻害剤による、産生抑制効果を調べた。
（実験方法）
　ヒトケラチノサイト培養細胞株ＨａＣａＴをコレステロールフリーの血清１０％を含む培地で培養すると、ＨａＣａＴ細胞は培地中の成分を用いて、多量のコレステロールを合成するようになる。この時、培地に１７－ヒドロキシプロゲステロンを３０μＭの濃度で加えることで、４位のメチル基を酸化的に脱メチル化する酵素（ＮＳＤＨＬ）の活性が阻害され、ＣＨＩＬＤ症候群の皮膚と同様に、特定脂質代謝物が細胞に蓄積する。
　このＣＨＩＬＤ症候群を模したＨａＣａＴ細胞の培養系に、様々なコレステロール生合成阻害剤を加えることで、特定の脂質代謝物の蓄積が抑えられるかどうかを検討するための試験を行った。
　添加した薬剤が特定の脂質蓄積を抑制するかどうかの評価は、培養後、それぞれの細胞から脂質を抽出し、コレステロールのピーク（ｍ／ｚ ４５８．４）に対する特定の脂質の相対的なピーク面積を計測し、１７－ヒドロキシプロゲステロンのみ加えた（－）場合の相対ピーク面積と比較することで行い、こうして、投与した薬剤の効果を検討した。
　特定の脂質の相対量の評価に用いた、物質に特異的なイオンのｍ／ｚ値はそれぞれ、　Ｃ２９Ａ（式（Ｉ）の化合物）　　：　ｍ／ｚ　４８６．４，
　Ｃ２９Ｂ（式（ＩＩ）の化合物）　：　ｍ／ｚ　４８４．４
　Ｃ２８Ａ（式（ＩＩＩ）の化合物）：　ｍ／ｚ　２２７．２，及び、　
　Ｃ２８Ｂ（式（ＩＶ）の化合物）　：　ｍ／ｚ　４７２．４であった（上記表２参照）。
　図５のグラフに示した濃度の各薬物を添加することで特定の脂質代謝物の蓄積は抑制された。また、濃度を１０倍ずつ変化させた試験の結果においても、すべての薬物に関して、用量依存的に阻害効果が認められた。
　その結果、特に、オキシコナゾールによって特定の脂質代謝物の蓄積量が抑制されることが見出された（［図５］参照）。従って、特定の脂質代謝物の蓄積阻害剤が治療薬になり得ることが示唆された。 Example 3
[Inhibition of lipid metabolite accumulation]
A test system for artificially accumulating the lipid metabolite specified in Example 2 was constructed, and the production suppressing effect of various cholesterol biosynthesis inhibitors was examined.
(experimental method)
When the human keratinocyte cell line HaCaT is cultured in a medium containing 10% cholesterol-free serum, the HaCaT cells use the components in the medium to synthesize a large amount of cholesterol. At this time, by adding 17-hydroxyprogesterone to the medium at a concentration of 30 μM, the activity of the enzyme (NSDHL) that oxidatively demethylates the methyl group at the 4-position is inhibited, and as with the skin of CHILD syndrome, Lipid metabolites accumulate in cells.
A test was conducted to examine whether or not the accumulation of specific lipid metabolites can be suppressed by adding various cholesterol biosynthesis inhibitors to the HaCaT cell culture system simulating the CHILD syndrome.
Whether or not the added drug inhibits specific lipid accumulation was evaluated by extracting lipids from each cell after culturing and comparing the peak area of the specific lipid with respect to the cholesterol peak (m / z 458.4). Was measured and compared with the relative peak area when only 17-hydroxyprogesterone was added (−), and thus the effect of the administered drug was examined.
The m / z values of the substance-specific ions used for the evaluation of the relative amounts of specific lipids were C29A (compound of formula (I)): m / z 486.4, respectively.
C29B (compound of formula (II)): m / z 484.4
C28A (compound of formula (III)): m / z 227.2, and
C28B (compound of formula (IV)): m / z 472.4 (see Table 2 above).
The addition of each drug at the concentrations shown in the graph of FIG. 5 suppressed the accumulation of specific lipid metabolites. In addition, in the results of the test in which the concentration was changed by 10 times, an inhibitory effect was observed for all drugs in a dose-dependent manner.
As a result, in particular, it was found that oxyconazole suppressed the accumulated amount of specific lipid metabolites (see [FIG. 5]). Therefore, it was suggested that a specific lipid metabolite accumulation inhibitor could be a therapeutic agent.
　実施例４
［慢性皮膚疾患における脂質代謝物の蓄積］
　実施例２で特定された脂質代謝物の蓄積が、慢性皮膚炎の患者においても確認されるか実験した。
（実験方法）
　実施方法は、上記実施例２と同じである。
　すなわち、健常人の角質、ＣＨＩＬＤ症候群患者の角質（病変部及び健常部）、尋常性乾癬患者の角質（病変部及び健常部）、遺伝性角化症患者の角質（病変部）、急性皮膚炎患者の角質（病変部）、中毒疹患者の角質（病変部）、及び、アトピー性皮膚炎患者の角質（病変部）をそれぞれ約１ｍｇ採取し、実施例２と同様の方法で脂質を抽出し、分析を行った。組織重量あたりの各特定脂質代謝物のピーク面積を、健常人の角質のそれと比較して評価した。ＣＨＩＬＤ症候群患者の病変部、尋常性乾癬患者の皮疹部、アトピー性皮膚炎患者の病変部では、健常人の角質に比べて組織重量あたり１００倍以上の特定脂質代謝物の蓄積が認められた。一方、ＣＨＩＬＤ症候群患者の健常部、尋常性乾癬患者の非皮疹部の角質では、健常人の角質と差がなかった。また、遺伝性角化症患者、接触性皮膚炎（急性皮膚炎）患者、中毒疹患者、及び、胼胝患者の病変部では、特定脂質代謝物の蓄積量は健常人の角質と変わらないことがわかった。実施例４の結果を図６（Ａ）と（Ｂ）に示す。なお、図６では、それぞれの脂質の単位重量あたりのピーク面積を算出し、健常人の単位重量あたりの各脂質のピーク面積を基準値である１として、病変部における特定脂質代謝物の蓄積量が基準値の何倍になったかを示す相対値を表示した。 Example 4
[Accumulation of lipid metabolites in chronic skin diseases]
It was tested whether accumulation of lipid metabolites identified in Example 2 was confirmed in patients with chronic dermatitis.
(experimental method)
The method of implementation is the same as in Example 2 above.
That is, keratin of healthy person, keratin of CHILD syndrome patient (lesion and healthy part), keratin of psoriasis vulgaris patient (lesion and healthy part), keratin of inherited keratosis patient (lesion), acute dermatitis Approximately 1 mg each of the keratin (lesion) of the patient, the keratin (lesion) of the patient with poisoning eruption, and the keratin (lesion) of the patient with atopic dermatitis were collected, and lipids were extracted in the same manner as in Example 2. ,Analysis was carried out. The peak area of each specific lipid metabolite per tissue weight was evaluated by comparison with that of the keratin of a healthy person. In the lesions of patients with CHILD syndrome, the skin lesions of patients with psoriasis vulgaris, and the lesions of patients with atopic dermatitis, accumulation of 100 or more specific lipid metabolites per tissue weight was observed as compared to the keratin of healthy individuals. On the other hand, in the keratin of the healthy part of the CHILD syndrome patient and the non-rash site of the psoriasis vulgaris patient, there was no difference from that of the healthy person. In addition, in the lesions of patients with hereditary keratosis, contact dermatitis (acute dermatitis), toxic eruption patients, and callus patients, the accumulated amount of specific lipid metabolites may be the same as that of healthy individuals. all right. The results of Example 4 are shown in FIGS. 6 (A) and 6 (B). In addition, in FIG. 6, the peak area per unit weight of each lipid was calculated, and the peak area of each lipid per unit weight of a healthy person was set as a reference value of 1, and the accumulation amount of the specific lipid metabolite in the lesion site was calculated. The relative value indicating the multiple of the reference value was displayed.
　実施例５
［治療に伴う脂質代謝物の変化］
　尋常性乾癬の患者の病変部に対して、セクキヌマブ（販売名：コセンティクス（ヒト型抗ヒトIL-17Aモノクローナル抗体製剤）を1回300mg、皮下注射した。
　具体的には、０，１，２，３，４，５週目に1回300mg、皮下注射し、以後は４週間ごとに1回300mg、皮下注射を行った。 Example 5
[Changes in lipid metabolites associated with treatment]
Secukinumab (trade name: Cosentyx (human anti-human IL-17A monoclonal antibody preparation)) was subcutaneously injected once to the lesion site of a patient with psoriasis vulgaris (300 mg).
Specifically, 300 mg was subcutaneously injected once at 0, 1, 2, 3, 4, and 5 weeks, and thereafter, 300 mg was subcutaneously injected once every 4 weeks.
　上記の患者に対し、尋常性乾癬のセクキヌマブ投与前、及び、投与後に病変部の同じ箇所から角質を採取し、実施例２と同じ方法で脂質の抽出と分析を行った。鱗屑を伴う皮疹は治療によって軽快し、皮疹が治癒した場合は鱗屑の採取ができなくなった。採取ができた２例で治療前後を比較すると、２例とも特定脂質代謝物の蓄積は低減した。この結果を図７に示す。図７の縦軸は、特定脂質代謝物の蓄積量の相対値を表していて、より具体的には、同一条件でコレステロールを分析したときのピーク面積１０００００あたりの各物質のピーク面積を表している。
　以上より、乾癬の治療前後で病変部の角質中の特定脂質代謝物を測定した結果、治療前に蓄積していた特定脂質代謝物が、いずれもセクキヌマブによる治療で低減したことが確認された（図７（Ａ）と（Ｂ）参照）。 
　このように、乾癬、及び詳細を後述するアトピー性皮膚炎といった慢性症状を伴う皮膚病変では、皮膚炎に関連した毒性を有する特定脂質代謝物が蓄積している（図７（Ａ）と（Ｂ）のＢｅｆｏｒｅ欄参照）一方で、慢性炎症を伴わない皮膚病変（急性皮膚炎や中毒疹など）ではそのような蓄積はみられなかった。このため、特定脂質代謝物は皮膚炎の慢性化に寄与すると考えられる。
　そして上述のように、乾癬治療による皮膚症状の改善に伴い、特定脂質代謝物の蓄積が低下することも確認されているため、特定脂質代謝物の蓄積と、慢性皮膚炎の治療効果とは互いに関連しているといえる。従って、特定の脂質代謝物の蓄積阻害剤が、これらの慢性皮膚疾患の治療薬になり得ることが示唆された。 For the above patients, keratin was collected from the same lesion site before and after administration of secukinumab for psoriasis vulgaris, and lipid extraction and analysis were performed in the same manner as in Example 2. The skin rash with scales was relieved by the treatment, and when the skin rash was healed, the scales could not be collected. Comparing the two collected cases before and after treatment, the accumulation of specific lipid metabolites was reduced in both cases. The result is shown in FIG. 7. The vertical axis of FIG. 7 represents the relative value of the accumulated amount of specific lipid metabolites, and more specifically, the peak area of each substance per 100000 peak areas when cholesterol was analyzed under the same conditions. There is.
From the above, as a result of measuring the specific lipid metabolites in the corneum of the lesion before and after the treatment of psoriasis, it was confirmed that the specific lipid metabolites accumulated before the treatment were all reduced by the treatment with Secukinumab ( See FIGS. 7A and 7B).
Thus, in skin lesions with chronic symptoms such as psoriasis and atopic dermatitis described in detail later, specific lipid metabolites having toxicity associated with dermatitis are accumulated (FIGS. 7 (A) and (B)). On the other hand, such accumulation was not observed in skin lesions (acute dermatitis, toxic eruption, etc.) without chronic inflammation. Therefore, specific lipid metabolites are considered to contribute to chronic dermatitis.
And as described above, it has been confirmed that the accumulation of specific lipid metabolites decreases with the improvement of skin symptoms due to the treatment of psoriasis. Therefore, the accumulation of specific lipid metabolites and the therapeutic effect on chronic dermatitis are mutually related. It can be said that they are related. Therefore, it was suggested that a specific lipid metabolite accumulation inhibitor could be a therapeutic agent for these chronic skin diseases.
　実施例６
［アトピー性皮膚炎の治療］
　アトピー性皮膚炎の患者の病変部に対するステロイドの外用を中止し、アトルバスタチン（アトルバスタチン原末を１％濃度になるように水に溶解させた１重量％アトルバスタチン液）、及び、オキナゾール（１％オキシコナゾールクリーム、販売名：オキナゾールクリーム）を１日２回、３５０ｍｇ（原末３．５ｍｇ相当）ずつ塗布し、健常部、アトルバスタチンとオキナゾールによる治療開始直前の病変部、治療から１週間経過後の病変部、及び、治療から３週間経過後の病変部から角質を採取し、実施例２と同じ方法で脂質の抽出と分析を行った。この結果、アトピー性皮膚炎では、アトルバスタチン、及び、オキナゾールの塗布により、患部の当該脂質の蓄積が抑制されることが確認された（図８参照）。図８においては、縦軸が、コレステロールのピーク面積１０００００あたりの各物質のピーク面積を表している。
　なお、アトピー性皮膚炎に対してステロイド塗布を中止すると、通常、皮膚炎は悪化する傾向にある。これに対し、本実施例のように、ステロイド塗布の中止後にアトルバスタチン、及び、オキナゾールを塗布したところ、通常は悪化することの多い患部が、ステロイドの塗付の中止時とほぼ同じ状態に保持された。 Example 6
[Treatment of atopic dermatitis]
Topical steroids for the lesions of patients with atopic dermatitis were discontinued, and atorvastatin (1 wt% atorvastatin solution prepared by dissolving atorvastatin bulk powder in water to a concentration of 1%) and quinazole (1% oxycostatin). Nazoru cream, brand name: quinazol cream) was applied twice a day at 350 mg (equivalent to 3.5 mg of bulk powder), healthy area, lesion area just before the start of treatment with atorvastatin and quinazole, lesion after 1 week from treatment. The keratin was collected from the affected area and from the lesioned area after 3 weeks from the treatment, and the lipid was extracted and analyzed in the same manner as in Example 2. As a result, in atopic dermatitis, it was confirmed that the application of atorvastatin and quinazole suppressed the accumulation of the lipid in the affected area (see FIG. 8). In FIG. 8, the vertical axis represents the peak area of each substance per 100,000 cholesterol peak area.
When steroid application is stopped for atopic dermatitis, dermatitis usually tends to be exacerbated. On the other hand, as in the present example, when atorvastatin and quinazole were applied after the steroid application was stopped, the affected area, which is often aggravated, was maintained in almost the same state as when the steroid application was stopped. It was
　実施例７
［尋常性乾癬の治療］
　尋常性乾癬の患者の病変部に対してオキナゾール（１％オキシコナゾールクリーム、販売名：オキナゾールクリーム）を１日２回、２週間に渡り塗布した。そして、健常部、オキナゾールによる治療開始直前の病変部、及び、治療から４週間経過後の病変部から角質を採取し、実施例２と同じ方法で脂質の抽出と分析を行った。この結果、尋常性乾癬においても、オキナゾールの塗布により、患部の当該脂質の蓄積が抑制されることが確認された（図９参照）。図９においても図８と同様に、縦軸が、コレステロールのピーク面積１０００００あたりの各物質のピーク面積を表している。
　そして、治療から４週間経過後には皮膚鱗屑の減少が認められたため、オキナゾールには、皮膚症状の抑制効果があると認められる。 Example 7
[Treatment of psoriasis vulgaris]
Oquinazole (1% oxyconazole cream, trade name: quinazole cream) was applied to the lesion site of a patient with psoriasis vulgaris twice a day for 2 weeks. Then, keratin was collected from a healthy part, a lesion immediately before the start of treatment with quinazole, and a lesion 4 weeks after the treatment, and lipid extraction and analysis were carried out in the same manner as in Example 2. As a result, it was confirmed that, even in psoriasis vulgaris, the application of quinazole suppressed the accumulation of the lipid in the affected area (see FIG. 9). In FIG. 9, as in FIG. 8, the vertical axis represents the peak area of each substance per 100000 peak area of cholesterol.
Then, after 4 weeks from the treatment, reduction of skin scales was observed, so it is considered that quinazole has an effect of suppressing skin symptoms.
　以上より、ＣＨＩＬＤ症候群に伴う慢性皮膚炎、尋常性乾癬、及び、アトピー性皮膚炎の病変部においては特定脂質代謝物が蓄積されること（実施例２及び４）、少なくともＣＨＩＬＤ症候群においては特定脂質代謝物の産生を阻害する阻害剤により、特定脂質代謝物の蓄積量を減少させられること（実施例３）、及び、乾癬において、治療による皮疹の改善に伴い特定脂質代謝物の蓄積量が減少すること（実施例５）が確認された。
　さらに、少なくとも、ＣＨＩＬＤ症候群に伴う慢性皮膚炎、尋常性乾癬、及び、アトピー性皮膚炎については、特定脂質代謝物の産生を阻害する阻害剤による、皮膚症状の改善、又は、特定脂質代謝物の蓄積の防止が確認された（実施例１、６、及び、７）。
　以上のことから、上記特定脂質代謝物は皮膚炎の慢性化に寄与すると考えられるのであり、特定脂質代謝物の蓄積が確認された慢性皮膚炎が（［図６］参照）、上記特定の脂質代謝物の産生を阻害する化合物による治療ターゲットとなり得ることが見出された。従って、特定の脂質代謝物の蓄積阻害剤が、これらの慢性皮膚疾患の治療薬になり得ることが示唆された。 From the above, specific lipid metabolites are accumulated in the lesions of chronic dermatitis, psoriasis vulgaris, and atopic dermatitis associated with CHILD syndrome (Examples 2 and 4). Inhibitors that inhibit the production of metabolites can reduce the amount of specific lipid metabolites accumulated (Example 3), and in psoriasis, the amount of specific lipid metabolites accumulated decreases as treatment improves the eruption. It was confirmed that this was done (Example 5).
Furthermore, at least for chronic dermatitis, psoriasis vulgaris, and atopic dermatitis associated with CHILD syndrome, improvement of skin symptoms or inhibition of specific lipid metabolites by an inhibitor that inhibits production of specific lipid metabolites. The prevention of accumulation was confirmed (Examples 1, 6, and 7).
From the above, it is considered that the specific lipid metabolite contributes to chronicity of dermatitis, and chronic dermatitis in which accumulation of the specific lipid metabolite was confirmed (see [Fig. 6]) It has been discovered that compounds that inhibit the production of metabolites can be therapeutic targets. Therefore, it was suggested that a specific lipid metabolite accumulation inhibitor could be a therapeutic agent for these chronic skin diseases.
　実施例８
［化合物のＣ２９Ａ蓄積阻害活性の評価］
　評価化合物のC29A蓄積阻害活性については、上述の方法、すなわち、特定脂質代謝物の産生を阻害する阻害活性を測定する測定工程について採用した方法と同じ方法で測定した。その結果、アレンドロネート及びリセドロネートは、１０μＭで50%以上の良好なC29A蓄積阻害活性を示した。また、アモロルフィン塩酸塩、アトルバスタチン、アザランスタット、ビフォナゾール、ブトコナゾール硝酸塩、クロコナゾール塩酸塩、エコナゾール、フルアジナム、ロバスタチン、メトコナゾール、オキシコナゾール硝酸塩、シンバスタチン、スルコナゾール硝酸塩、テブコナゾールおよびテルコナゾールは、１μＭで50%以上の良好なC29A蓄積阻害活性を示した。 Example 8
[Evaluation of C29A accumulation inhibitory activity of compound]
The C29A accumulation inhibitory activity of the evaluation compound was measured by the same method as described above, that is, the method adopted for the measuring step for measuring the inhibitory activity of inhibiting the production of a specific lipid metabolite. As a result, alendronate and risedronate showed good C29A accumulation inhibitory activity of 50% or more at 10 μM. Also, amorolfine hydrochloride, atorvastatin, azalanstat, bifonazole, butoconazole nitrate, croconazole hydrochloride, econazole, fluazinam, lovastatin, metconazole, oxyconazole nitrate, simvastatin, sulconazole nitrate, tebuconazole and terconazole are 50% or more at 1 μM. It showed good C29A accumulation inhibitory activity.
　実施例９
［化合物のヒトＣＹＰ５１Ａ１阻害活性の評価］
　上記式（Ｉ）の化合物の阻害活性について、以下のように実験した。
（実験方法）
　ヒトＣＹＰ５１Ａ１の阻害活性について、以下の方法で測定した。評価化合物は2.5ｍＭとなるようにＤＭＳＯで溶解した。溶解した評価化合物は公比３でＤＭＳＯに希釈し、希釈系列を用意した。ＤＭＳＯで希釈した評価化合物1μＬを１００ｍＭのリン酸カリウム、３．３ｍＭの塩化マグネシウム、０．３８ｍｇ／ｍＬのトリトンＸ１００の混合溶液（１２．５μＬ）で希釈した。
　ヒトＣＹＰ５１Ａ１タンパクを発現させたカイコのミクロソーム分画をシスメックス社から入手した。ＣＹＰ５１Ａ１の酵素活性を、基質となるラノステロールを代謝した際に使用するＮＡＤＰＨ量から算出した。ＮＡＤＰＨの測定にはＡｂｃａｍ社のＮＡＤＰ／ＮＡＤＰＨ　ａｓｓａｙ　ｋｉｔ（ａｂ６５３４９）を用いた。すなわち、それぞれ最終濃度が、１００ｍＭのリン酸カリウム、３．３　ｍＭの塩化マグネシウム、０．３８ｍｇ／ｍＬのトリトンＸ１００、０．５６４ Ｕ／ｍＬのＮＡＤＰＨ－Ｐ４５０レダクターゼ、４０μＭのラノステロール、２６０μｇ／ｍＬのヒトＣＹＰ５１Ａ１ミクロソームを含む水溶液（２１μＬ）を用意し、希釈した評価化合物２μＬを添加し、５分間静置した。そこに、１００ｍＭのリン酸カリウム、３．３　ｍＭの塩化マグネシウム、０．３８ｍｇ／ｍＬのトリトンＸ１００の混合水溶液で希釈した０．５ｍＭ　ＮＡＤＰＨ溶液を２μＬ添加し、室温で２０分間静置した。以降の操作はＡｂｃａｍ社のＮＡＤＰ／ＮＡＤＰＨ　ａｓｓａｙ　ｋｉｔのプロトコールに準じて行った。
　評価化合物を添加せずに上記試験を行った際のＮＡＤＰＨ消費量を１００％活性とし、各濃度の評価化合物存在下でのＮＡＤＰＨ消費量からヒトＣＹＰ５１Ａ１の阻害活性（ＩＣ５０）を算出した。
　その結果、アザランスタット、ビフォナゾール、ブトコナゾール硝酸塩、クロコナゾール塩酸塩、メトコナゾール、オキシコナゾール硝酸塩、スルコナゾール硝酸塩及びテブコナゾールは、1μＭ以下の良好なＩＣ５０を示した。 Example 9
[Evaluation of human CYP51A1 inhibitory activity of compound]
The inhibitory activity of the compound of the above formula (I) was tested as follows.
(experimental method)
The inhibitory activity of human CYP51A1 was measured by the following method. The evaluation compound was dissolved in DMSO so that the concentration was 2.5 mM. The dissolved evaluation compound was diluted with DMSO at a public ratio of 3 to prepare a dilution series. 1 μL of the evaluation compound diluted with DMSO was diluted with a mixed solution (12.5 μL) of 100 mM potassium phosphate, 3.3 mM magnesium chloride, and 0.38 mg / mL Triton X100.
The silkworm microsome fraction expressing the human CYP51A1 protein was obtained from Sysmex. The enzymatic activity of CYP51A1 was calculated from the amount of NADPH used when metabolizing lanosterol as a substrate. NADP / NADPH assay kit (ab65349) from Abcam was used for the measurement of NADPH. That is, the respective final concentrations were 100 mM potassium phosphate, 3.3 mM magnesium chloride, 0.38 mg / mL Triton X100, 0.564 U / mL NADPH-P450 reductase, 40 μM lanosterol, 260 μg / mL. An aqueous solution (21 μL) containing human CYP51A1 microsomes was prepared, 2 μL of the diluted evaluation compound was added, and the mixture was allowed to stand for 5 minutes. 2 μL of 0.5 mM NADPH solution diluted with a mixed aqueous solution of 100 mM potassium phosphate, 3.3 mM magnesium chloride, and 0.38 mg / mL Triton X100 was added thereto, and the mixture was allowed to stand at room temperature for 20 minutes. The subsequent operation was performed according to the protocol of NADP / NADPH assay kit of Abcam.
The NADPH consumption when the above test was performed without adding the evaluation compound was defined as 100% activity, and the inhibitory activity (IC 50 ) of human CYP51A1 was calculated from the NADPH consumption in the presence of each concentration of the evaluation compound.
As a result, azalanstat, bifonazole, butoconazole nitrate, croconazole hydrochloride, metconazole, oxyconazole nitrate, sulconazole nitrate and tebuconazole showed good IC 50 of 1 μM or less.
　このように、本発明の医薬組成物における有効成分である化合物のうち少なくともＣＹＰ５１阻害剤、及び、ＨＭＧＣＲ阻害剤が、慢性皮膚疾患の抑制に効果を奏することが示された。 Thus, it was shown that at least the CYP51 inhibitor and the HMGCR inhibitor among the compounds which are the active ingredients in the pharmaceutical composition of the present invention are effective in suppressing chronic skin diseases.
　本発明によれば、新規な特定のコレステロール生合成前駆物質の産生を阻害するとともに、慢性皮膚炎に対する治療効果の優れた医薬組成物、及び、特定のコレステロール生合成前駆物質の検出量に着目した、慢性皮膚炎の候補医薬のスクリーニング方法等を提供することができる。
  According to the present invention, the production of a novel specific cholesterol biosynthetic precursor is inhibited, and a pharmaceutical composition having an excellent therapeutic effect on chronic dermatitis, and the detected amount of the specific cholesterol biosynthetic precursor are focused. , A method for screening a candidate drug for chronic dermatitis can be provided.

Claims (11)

  1. 　下記式（Ｉ）～（ＩＶ）で表されるコレステロール生合成前駆物質のいずれかの産生を阻害する有効成分を含む、慢性皮膚炎の治療のための医薬組成物。
    Figure JPOXMLDOC01-appb-C000001
         A pharmaceutical composition for treating chronic dermatitis, which comprises an active ingredient that inhibits the production of any of the cholesterol biosynthetic precursors represented by the following formulas (I) to (IV).
    Figure JPOXMLDOC01-appb-C000001
  2. 　前記有効成分が、ＣＹＰ５１阻害剤、又は、ＨＭＧＣＲ阻害剤である、請求項１に記載の慢性皮膚炎の治療のための医薬組成物。 The pharmaceutical composition for treating chronic dermatitis according to claim 1, wherein the active ingredient is a CYP51 inhibitor or an HMGCR inhibitor.
  3. 　前記有効成分が、前記ＣＹＰ５１阻害剤である、請求項２に記載の医薬組成物。 The pharmaceutical composition according to claim 2, wherein the active ingredient is the CYP51 inhibitor.
  4. 　前記ＣＹＰ５１阻害剤が、アザランスタット、ビフォナゾール、ブトコナゾール、クロコナゾール、エコナゾール、メトコナゾール、オキシコナゾール、スルコナゾール、テブコナゾール、それらの薬学的に許容される塩、及び、それらの組み合わせから選ばれる少なくともいずれかを含む、請求項３に記載の医薬組成物。 The CYP51 inhibitor is at least any one selected from azalanstat, bifonazole, butoconazole, croconazole, econazole, metconazole, oxyconazole, sulconazole, tebuconazole, pharmaceutically acceptable salts thereof, and combinations thereof. The pharmaceutical composition according to claim 3, comprising:
  5. 　前記有効成分が、前記ＨＭＧＣＲ阻害剤である、請求項２に記載の医薬組成物。 The pharmaceutical composition according to claim 2, wherein the active ingredient is the HMGCR inhibitor.
  6. 　前記ＨＭＧＣＲ阻害剤が、アトルバスタチン、ロバスタチン、シンバスタチン、それらの薬学的に許容される塩、又は、それらの組み合わせである、請求項５に記載の医薬組成物。 The pharmaceutical composition according to claim 5, wherein the HMGCR inhibitor is atorvastatin, lovastatin, simvastatin, a pharmaceutically acceptable salt thereof, or a combination thereof.
  7. 　外用医薬組成物である、請求項１に記載の医薬組成物。 The pharmaceutical composition according to claim 1, which is an external pharmaceutical composition.
  8. 　前記慢性皮膚炎が、アトピー性皮膚炎、又は、乾癬である、請求項１に記載の医薬組成物。 The pharmaceutical composition according to claim 1, wherein the chronic dermatitis is atopic dermatitis or psoriasis.
  9. 　アモロルフィン、アレンドロネート、アトルバスタチン、アザランスタット、ビフォナゾール、ブトコナゾール、クロコナゾール、エコナゾール、フルアジナム、ロバスタチン、メトコナゾール、オキシコナゾール、リセドロネート、シンバスタチン、スルコナゾール、テブコナゾール、テルコナゾール、それらの薬学的に許容される塩、及び、それらの組み合わせから選ばれる少なくともいずれかを含む、慢性皮膚炎の治療のための医薬組成物。 Amorolfine, alendronate, atorvastatin, azalanstat, bifonazole, butoconazole, croconazole, econazole, fluazinam, lovastatin, metconazole, oxyconazole, risedronate, simvastatin, sulconazole, tebuconazole, terconazole, pharmaceutically acceptable salts thereof, And a pharmaceutical composition for the treatment of chronic dermatitis, which comprises at least one selected from the combination thereof.
  10. 　（ａ）対象化合物について、下記式（Ｉ）～（ＩＶ）で表されるコレステロール生合成前駆物質のいずれかの産生を阻害する阻害活性を測定する測定工程と、
    　（ｂ）測定された前記阻害活性の値に基づいて、前記対象化合物が慢性皮膚炎の治療に有効な候補化合物であるか否かを判定する判定工程とを含む、
    　慢性皮膚炎治療用の候補化合物のスクリーニング方法。
    Figure JPOXMLDOC01-appb-C000002
         (A) a measuring step of measuring the inhibitory activity of the target compound for inhibiting the production of any of the cholesterol biosynthetic precursors represented by the following formulas (I) to (IV),
    (B) a determination step of determining whether or not the target compound is a candidate compound effective for the treatment of chronic dermatitis, based on the measured value of the inhibitory activity,
    A method for screening a candidate compound for treating chronic dermatitis.
    Figure JPOXMLDOC01-appb-C000002
  11. 　（ｃ）下記式（Ｉ）～（ＩＶ）で表されるコレステロール生合成前駆物質のいずれかを皮膚のサンプルから検出する検出工程と、
    　（ｄ）前記サンプルから検出された前記コレステロール生合成前駆物質の検出量と、基準となる基準量とを比較する比較工程とを有する、
    　慢性皮膚炎の診断のための方法。
    Figure JPOXMLDOC01-appb-C000003
           (C) a detection step of detecting any of the cholesterol biosynthetic precursors represented by the following formulas (I) to (IV) from a skin sample,
    (D) a comparison step of comparing a detected amount of the cholesterol biosynthetic precursor detected from the sample with a reference reference amount,
    Methods for the diagnosis of chronic dermatitis.
    Figure JPOXMLDOC01-appb-C000003
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