JP2022036341A - Pharmaceutical composition for treating chronic dermatitis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To realize a new pharmaceutical composition for chronic dermatitis having a sufficient therapeutic effect. The above-mentioned problem is a pharmaceutical composition for the treatment of chronic dermatitis, which comprises an active ingredient that inhibits the production of any of the cholesterol biosynthesis precursors represented by the following formulas (I) to (IV). It was solved by the thing. [Chemical formula 1] JPEG2022036341000011.jpg100146 [Selection diagram] Fig. 4

Description

The present invention relates to a pharmaceutical composition for the treatment of chronic dermatitis, a method for screening a candidate compound for the treatment of chronic dermatitis, and the like.

Atopic dermatitis, psoriasis and the like are known as chronic dermatitis. Atopic dermatitis is a skin disease whose main lesion is itchy eczema, which develops under the influence of various environmental factors against the background of atopic predisposition, and often exacerbates and relieves repeatedly. Psoriasis is a skin disease whose main lesion is silvery white scales and well-defined erythema with infiltration and thickening, and most of them are intractable and chronic. For the treatment of atopic dermatitis, in addition to external use such as steroids and calcineurin inhibitors, supplementary oral antihistamines are used. In addition, psoriasis is treated by combining topical therapy including topical steroids, topical vitamin D3, and their combinations with systemic therapy such as cyclosporin, retinoid, and methotrexate, and phototherapy using UVA and UVB. .. Recently, biopharmacy against TNF-α, IL-12 / 23, and IL-17 has also been used.

However, all of these are aimed at alleviating the symptoms by symptomatic treatment, and there is no curative treatment. Although various symptomatic treatments are used for these diseases, the same symptomatic treatment is not 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 broaden the range of treatment.
In addition, topical steroids used for these chronic inflammatory skin diseases cause side effects such as skin atrophy and induction of skin infections when used for a long period of time. For the treatment of atopic dermatitis, calcineurin inhibitors have a problem of irritation at the application site, and itching that is not relieved by oral antihistamines significantly lowers the QOL of patients. In addition, topical therapy for psoriasis is often slow in effect, systemic therapy has side effects, and phototherapy has problems of time constraints due to frequent visits. In addition, high medical costs are a problem for biopharmacy.

In addition, Patent Document 1 discloses that a lanosterol 14-α demethylase inhibitor is used as an inhibitor of angiogenesis. Non-Patent Document 1 discloses a study in which an intermediate metabolite of sterol can be an endogenous ligand for RORγ. Non-Patent Document 2 discloses a study on suppression of auricular 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 accumulate in the cholesterol biosynthesis pathway. In addition, Non-Patent Document 5 reports on cholesterol synthesis intermediates accumulated in patients 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. 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.

Under the above circumstances, it is desired to provide a new pharmaceutical composition for chronic dermatitis.

The present invention relates to the following pharmaceutical compositions, a method for screening candidate compounds for the treatment of chronic dermatitis, a method for diagnosing chronic dermatitis, and the like.

（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の少なくともいずれかの化合物を含む、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、又はそれらの薬学的に許容される塩である、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、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の化合物を含む、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。

（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。

(1) Cholesterol biosynthesis precursor represented by the following formula (I) (hereinafter, also referred to as C29A), cholesterol biosynthesis precursor represented by the following formula (II) (hereinafter, also referred to as C29B), the following formula (hereinafter, also referred to as C29B). Production of either cholesterol biosynthesis precursor represented by III) (hereinafter, also referred to as C28A) or cholesterol biosynthesis 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 cholesterol.

(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 comprises at least one compound of azalanstat, biphonazole, butoconazole, croconazole, econazole, metconazole, oxyconazole, sulconazole and tebuconazole or pharmaceutically acceptable salts thereof. The pharmaceutical composition according to (3).
(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, or a pharmaceutically acceptable salt thereof.
(7) The pharmaceutical composition according to (1) above, which is a pharmaceutical composition for external use.
(8) The pharmaceutical composition according to (1) above, wherein the chronic dermatitis is atopic dermatitis or psoriasis.
(9) Amorolfine, alendronate, atorvastatin, azalanstat, biphonazole, butoconazole, croconazole, econazole, fluazinum, lovastatin, methconazole, oxyconazole, lysedronate, simvastatin, sulconazole, tebuconazole, and telconazole or their pharmaceuticals. A pharmaceutical composition for the treatment of chronic dermatitis, comprising at least one compound of an acceptable salt.
(10) (a) A measurement step for measuring the inhibitory activity of the target compound to inhibit the production of any of the cholesterol biosynthesis precursors represented by the following formulas (I) to (IV).
(B) A determination step of determining whether or not the target compound is an effective candidate compound for the treatment of chronic dermatitis based on the measured value of the inhibitory activity.
A method for screening candidate compounds for the treatment of chronic dermatitis.

(11) (c) A detection step of detecting any of the cholesterol biosynthesis precursors represented by the following formulas (I) to (IV) from the skin sample, and
(D) The present invention comprises a comparative step of comparing the detected amount of the cholesterol biosynthesis precursor detected from the sample with the reference amount as a reference.
A method for diagnosing chronic dermatitis.

According to the above-mentioned invention, it is possible to provide a new pharmaceutical composition which is good in efficacy, safety, convenience, economy and the like for chronic dermatitis such as atopic dermatitis and psoriasis. The present invention also provides a method for screening candidate compounds for the treatment of chronic dermatitis. Furthermore, the present invention provides a method for diagnosing chronic dermatitis.

It is a figure which shows the mass spectrum data of the lipid metabolite whose molecular structure is represented by the formula (I) and the formula (II). It is a figure which shows the mass spectrum data of the lipid metabolite whose molecular structure is expressed by the formula (III) and the formula (IV). 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. 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. It is a figure which shows the effect of suppressing the production of a specific lipid metabolite by a cholesterol biosynthesis inhibitor. It is a figure which shows the relative value of the accumulation amount of a specific lipid metabolite in a patient with chronic dermatitis. It is a figure which shows the change of the accumulation amount of the specific lipid metabolite before and after the treatment with secukinumab (cosentyx) for a plurality of lesions of a psoriasis vulgaris patient. It is a figure which shows the effect of suppressing the accumulation of a specific lipid metabolite in atopic dermatitis patient by a cholesterol biosynthesis inhibitor. It is a figure which shows the effect of suppressing the accumulation of a 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 the treatment of chronic dermatitis and contains an active ingredient that inhibits the production of cholesterol biosynthesis precursors.

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 biosynthesis precursor]
As the cholesterol biosynthesis precursor, the lipid metabolites represented by the following formulas (I) to (IV) (the biosynthesis precursor of the formula (I) (the compound of the formula (I)) and the raw of the formula (II), respectively). Also referred to as a synthetic precursor (compound of formula (II)), a biosynthetic precursor of formula (III) (compound of formula (III)), and a biosynthetic precursor of formula (IV) (compound of formula (IV)). ).

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 precursors, that is, the biosynthetic precursor (C29A) represented by the formula (I), the biosynthetic precursor (C29B) represented by the formula (II), and 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 hereinafter referred to as specific lipid metabolites.
The toxicity associated with the development of dermatitis by these specific lipid metabolites can be explained by the pathophysiology of CHILD syndrome. CHILD syndrome is a concomitant dominant inherited disorder with a heterozygous functional deletion mutation in the gene encoding NSDHL. NSDHL is one of the enzymes responsible for the demethylation reaction at position 4 in cholesterol biosynthesis. Since the NSDHL gene is located on the X chromosome and only one gene on the two X chromosomes is activated in women, the cholesterol biosynthesis reaction is stopped before NSDHL in cells in which the mutant NSDHL gene is activated. do. Since the specific lipid metabolite shows a structure having one or two methyl groups at the 4-position, it is considered that the demethylation reaction was inadequate and accumulated due to the mutation of NSDHL. CHILD syndrome causes psoriasis-like eruptions with erythema, keratinization, and desquamation on both sides, but accumulation of specific lipid metabolites is found only in the area of the eruption. HMGCR inhibitors and CYP51 inhibitors suppress the production of specific lipid metabolites by blocking the cholesterol biosynthesis pathway upstream of NSDHL, but when these inhibitors are applied externally to the rash area, dermatitis is improved.
On the other hand, it has been reported that the mutation of the NSDHL gene reduces cholesterol production, but the external application of cholesterol does not improve the eruption.
Based on these, it is strongly supported that specific lipid metabolites are associated with dermatitis. Specific lipid metabolites with dermatitis-related toxicity accumulate in lesions of chronic inflammatory skin diseases such as atopic dermatitis and psoriasis, whereas such accumulation occurs in acute dermatitis and toxic eruptions. I can't. Therefore, specific lipid metabolites are considered to contribute to the chronicity of dermatitis. It has also been confirmed that in the treatment of psoriasis, the accumulation of specific lipid metabolites decreases as the eruption improves.
Thus, it can be said that the accumulation of specific lipid metabolites is related to the therapeutic effect of chronic dermatitis. Therefore, by suppressing the accumulation of specific lipid metabolites, the symptoms of chronic dermatitis can be alleviated, and as described in detail below, specific enzyme inhibitors are effective in treating chronic dermatitis. It was also confirmed that.

The molecular structure of the cholesterol biosynthesis precursors of the formulas (I) to (IV), which are the above-mentioned specific lipid metabolites, was determined as follows.
That is, for the compounds of formula (I) and formula (II), the synthesized 4,4-dimethylcholesta-8 (9) -en-3beta-ol and 14-Demethyllanosterol preparations were retained as a result of GC / MS analysis. The structure was determined from the coincidence of time and confirmed ions.
On the other hand, for 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 compounds whose molecular structures are represented by the formulas (I) and (II). Further, FIG. 2 shows mass spectrum data of compounds whose molecular structures are represented by the formulas (III) and (IV).
For the compounds of formulas (I) to (IV), peaks appeared at 1.21, 1.27, 1.12, and 1.17, respectively, as the relative retention time when the retention time of cholesterol was 1 in GC / MS analysis. These peaks have been confirmed to appear specifically in chronic dermatitis (see Table 2 of Example 2 below).

Further, as shown in Table 2 of Example 2, in the spectral data of each compound showing the response amount to the m / z value detected in the relative retention time described above, the m / z value is 50 or more. It was confirmed that at least the following peaks with m / z values were included in the 15 peaks selected in descending order of response amount.
Compound (C29A) of formula (I): 486.4, 381.3, 135.1
Compound (C29B) of formula (II): 484.4, 379.3, 135.1
Compound (C28A) of formula (III): 472.4, 367.3, 227.2
Compound (C28B) of formula (IV): 472.4, 269.2, 147.1

In the present invention, the active ingredient (also referred to as a cholesterol biosynthesis inhibitor) that inhibits the production of the cholesterol biosynthesis precursor is a CYP51 inhibitor, an HMGCR inhibitor, a bisphosphonate drug, amorolfine or pharmaceutically acceptable thereof. Examples include salts and fluazinum or pharmaceutically acceptable salts thereof.

The pharmaceutical composition of the present invention preferably contains at least one of a CYP51 inhibitor and an HMGCR inhibitor as an active ingredient.

[CYP51 inhibitor]
Examples of the CYP51 inhibitor include azalanstat, biphonazole, butconazole, croconazole, everconazole, econazole, efinaconazole, fenticonazole, flutrimazole, fosfluconazole, isabconazole, metconazole, neticonazole, omoconazole, and oxyconazole. , Sertaconazole, sulconazole, tebuconazole, terconazole and tioconazole or pharmaceutically acceptable salts thereof.
Preferred CYP51 inhibitors include azalanstat, biphonazole, butoconazole, croconazole, econazole, metconazole, oxyconazole, sulconazole and tebuconazole or pharmaceutically acceptable salts thereof.

Pharmaceutically acceptable salts include, for example, alkali metals (eg, lithium, sodium, potassium, etc.), alkaline earth metals (eg, calcium, barium, etc.), magnesium, transition metals (eg, zinc, iron, etc.). , Salts with ammonia, organic bases (eg, trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, meglumin, ethylenediamine, pyridine, picolin, quinoline, etc.) and amino acids, or inorganic acids (eg, hydrochloric acid, sulfuric acid). , Nitrate, carbonic acid, hydrobromic acid, phosphoric acid, hydroiodic acid, etc.), and organic acids (eg, formic acid, acetic acid, propionic acid, trifluoroacetic acid, citric acid, lactic acid, tartrate acid, oxalic acid, maleic acid, Examples thereof include salts with fumaric acid, mandelic acid, glutaric acid, malic acid, benzoic acid, phthalic acid, ascorbic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, etc.). In particular, salts with hydrochloric acid, sulfuric acid, phosphoric acid, tartaric acid, methanesulfonic acid and the like can be mentioned.

[HMGCR inhibitor]
Preferred specific examples of the HMGCR inhibitor include atorvastatin, lovastatin, simvastatin, pharmaceutically acceptable salts of these compounds and the like.

Examples of the bisphosphonate-based drug include alendronate, lysedronate, and pharmaceutically acceptable salts of these compounds.

In another embodiment, the pharmaceutical composition of the present invention comprises, as active ingredients, amorolfine, alendronate, atorvastatin, azalanstat, biphonazole, butoconazole, croconazole, econazole, fluazinum, lovastatin, metconazole, oxyconazole, lysedronate, simvastatin. , Sulconazole, tebuconazole, and at least one of telconazole or a pharmaceutically acceptable salt thereof.

[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 psoriasis, fish scales, chronic urticaria, psoriasis, hand eczema, and CHILD syndrome. Chronic dermatitis and other skin disorders presenting with chronic skin inflammation symptoms can be mentioned. The pharmaceutical composition is particularly excellent in the therapeutic effect on atopic dermatitis and psoriasis among the above-mentioned dermatitis.

[Dosage form of pharmaceutical composition]
The pharmaceutical composition of the present invention can be used as an external preparation for direct administration to a dermatitis site, and the dosage form thereof includes, for example, ointments, creams, lotions, liniments, and poultices. , Plasters, patches, plasters, gels, liquids and the like.

[Additives in pharmaceutical compositions, etc.]
In pharmaceutical compositions, in addition to the above compounds as active ingredients, absorption promoters, pH regulators, preservatives, fragrances, dispersants, wetting agents, stabilizers, preservatives, suspending agents, surfactants, etc. Additives for pharmaceutical preparations can be blended alone or in admixture of two or more.

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, etc.). Thiourea etc.), cyclodextrin (α-cyclodextrin etc.), menthol, 1-dodecylazacycloheptan-2-one, calcium thioglycolate, limonene and the like can be mentioned. The content of the absorption-promoting agent varies depending on the dosage form, the base component and the like, but usually, from the viewpoint of effectively exhibiting the absorption-promoting action, it is 0.1% by weight or more, preferably 0.3% by weight or more. From the viewpoint of suppressing the occurrence of side effects, it is preferably 10% by weight or less, preferably 5% by weight or less.

Specific examples of the pH adjuster include inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid, organic acids such as acetic acid, succinic acid, fumaric acid and malic acid, and metal salts of these acids. The blending amount of the pH adjuster varies depending on the dosage form, the base component and the like, but it is usually preferable to blend the pH adjuster in a range of 4 to 8 in the pH of the preparation.

Specific examples of the preservative or preservative include paraoxybenzoic acid, methylparaben, chlorobutanol, benzyl alcohol, methyl paraoxybenzoate and the like.

Specific examples of the flavoring agent include, for example, menthol, rose oil, eucalyptus oil, d-camfur, etc., and specific examples of the dispersant include, for example, sodium metaphosphate, potassium polyphosphate, silicic acid anhydride and the like. Can be mentioned.

Specific examples of the wetting agent include propylene glycol, glycerin, sorbitol, sodium lactate, sodium hyaluronate and the like, and specific examples of the stabilizer include sodium hydrogen sulfite, tocopherol and ethylenediaminetetraacetic acid (EDTA). ), Citric acid and the like.

Specific examples of the suspending agent include, for example, tragant powder, gum arabic powder, bentonite, sodium carboxymethyl cellulose and the like, and specific examples of the surfactant include, for example, polyoxyethylene hydrogenated castor oil and sesquiolein. Examples thereof include sorbitan fatty acid esters such as sorbitan acid and polyoxyl stearate.

When the pharmaceutical composition is an ointment or a cream, an oily base or an emulsion base can be used as the base.

Examples of the oily base include hydrocarbons (hydrocarbons having 12 to 32 carbon atoms, liquid paraffin, white vaseline, squalane, squalane or plastibase, etc.) and higher alcohols (lauryl alcohol, cetyl alcohol, stearyl alcohol or oleyl alcohol). Fatty acid monohydric alcohols having 12 to 30 carbon atoms such as), higher fatty acids (saturated or unsaturated fatty acids having 6 to 32 carbon atoms such as palmitic acid or stearic acid), higher fatty acid esters (myristyl palmitate or stearyl stearate). Fatty acid ester such as; ester of fatty acid having 10 to 32 carbon atoms such as lanolin or carnauba wax and an aliphatic monohydric alcohol having 14 to 32 carbon atoms; saturated or unsaturated fatty acid having 10 to 22 carbon atoms such as glyceryl monolaurylate. Esters of and glycerin or their hydrocarbons, etc.), glycols (ethylene glycol, propylene glycol, polyethylene glycol, etc.), vegetable oils, animal oils, etc. may be mentioned.

Examples of the emulsion base include an oil-in-water base, a water-in-oil base, and a suspension base. As the oil-in-water base, the components such as lanolin, propylene glycol, stearyl alcohol, petrolatum, silicon oil, liquid paraffin, glyceryl monostearate, and polyethylene glycol are added to the aqueous phase in the presence or absence of a surfactant. Examples thereof include a base that has been emulsified and dispersed. Examples of the water-in-oil base include a base obtained by adding water to components such as petrolatum, higher fatty alcohol, and liquid paraffin in the presence of a nonionic surfactant, and emulsifying and dispersing the base. Examples of the suspension type base include an aqueous base obtained by adding a suspending agent such as starch, glycerin, high-viscosity carboxymethyl cellulose, and carboxyvinyl polymer to water to form a gel.

[Manufacturing method of pharmaceutical composition]
The pharmaceutical composition of the present invention can be produced by a conventional method for preparing an external preparation. For example, for ointments or creams, the raw materials for the base are kneaded, emulsified or suspended according to the respective dosage forms to prepare the base, then the active ingredient and various additives are added, and the mixture is mixed with a screw mixer or the like. It can be manufactured by mixing in the machine.

The pharmaceutical composition of the present invention can be used in any dosage form of suspension type, emulsion type or solution type lotion. Examples of the base of the suspension type lotion include rubbers such as Arabica rubber and tragant rubber, celluloses such as methyl cellulose and hydroxyethyl cellulose, and a mixture of a suspension agent for clays such as bentonite and water. Examples of the base of the emulsion type lotion include water and a base obtained by emulsifying an oily substance such as a fatty acid such as stearic acid or oleic acid, and a higher alcohol 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, then adding the active ingredient and additives, mixing, and filtering if desired. can.
Examples of the base for the liniment agent 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 a pharmaceutical additive to the base and mixing them.

Examples of the base for the poultice include water-soluble polymer compounds such as polyacrylic acid, polyvinyl alcohol and polyvinylpyrrolidone. The poultice can be produced, for example, by mixing an active ingredient, a base and a desired pharmaceutical additive, heating and then cooling.

Examples of the base for plasters, patches or plasters include supports such as non-woven fabrics, elastic materials such as natural rubber or isoprene rubber, fillers such as zinc flower and titanium oxide, and adhesion of terpene resin and the like. An agent, a stripping agent such as vinyl acetate, a softening agent such as liquid paraffin, an antiaging agent such as dibutylhydroxytoluene (BHT), and the like can be appropriately combined and used. The plaster agent, patch agent, plaster agent and the like can be produced by a conventional method such as a solution method or a thermal pressure method.

Examples of the solvent for preparing the liquid preparation include water, ethanol, isopropyl alcohol, benzyl alcohol, polyethylene glycol (PEG400 and the like), propylene glycol, propylene carbonate or a mixture thereof. Further, the liquid agent can also be used by impregnating it with gauze, a wound surface covering material or the like.

The amount of the active ingredient, that is, the CYP51 inhibitor, the HMGCR inhibitor, etc., in the pharmaceutical composition (formulation) varies depending on the dosage form, but in the case of an ointment or a cream, for example, the pharmaceutical composition. Based on the total weight of the above, 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. When the drug containing the pharmaceutical composition is a liquid preparation, the total amount of the pharmaceutical composition is 0.1 to 200 mg / mL, more preferably 5 to 50 mg / mL, and particularly 10 to 20 mg / mL. It is preferable to do so.

[Application of pharmaceutical composition]
The dose of the pharmaceutical composition of the present invention depends on the type of itch, the site of the disease, the degree of itch, etc., and the appropriate amount of the above-mentioned preparation is locally administered once to several times a day (by application to the affected area, etc.). Local administration) may be performed.

[Screening method for candidate compounds for the treatment of chronic dermatitis]
The method for screening a candidate compound for the treatment of chronic dermatitis in the present invention measures the inhibitory activity of the target compound to inhibit the production of at least one specific lipid metabolite (cholesterol biosynthesis precursor). It comprises a step and a determination step of determining whether the target compound is effective in treating chronic dermatitis based on the measured inhibitory activity value.

In the measurement step, for example, the inhibitory activity can be measured by the following method.
For example, 1x10 ⁵ (1 mL) HaCaT cells suspended in DMEM (Dalvecco's Modified Eagle) medium supplemented with cholesterol-free serum were seeded in a 12-well multi-well plate, wherein the final concentration of 17-hydroxyprogesterone was 30 μM. Further, 1 mM of the target compound dissolved in dimethylsulfoxide is added to a final concentration of 1 μM, and the mixture is cultured for 48 hours.
After washing the above cells twice with PBS, 200 μL of TrypLE Express is added and the cells are incubated at 37 ° C. for 13 minutes. After adding 0.5 mL of PBS and collecting the cells in a tube, centrifuge at 4 ° C. at 500 g for 5 minutes to precipitate the cells, remove the supernatant, and then resuspend 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 cell suspension is added. After filling the column with the cell suspension using a syringe, 750 μL dichloromethane is added to the top of the column and the effluent is collected. Repeat the same operation 3 times to obtain a sample solution.
Add 10 μL of 0.1 μg / μL of D7-cholesterol ethanol solution to the recovered sample solution, mix well, and then nitrogen-substitute at 40 ° C. for 30 minutes to dry. To the dried sample, add 50 μL BSTFA + TMCS, 99: 1 (Supelco) and 10 μL pyridine, immediately cap the glass vial and allow to stand at 40 ° C. 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 ° C, holding 1 minute-20 ° C / min → 250 ° C, 5 ° C / min → 280 ° C, holding 10 minutes-20 ° C / min → 330 ° C, holding 3 minutes. That is, hold at 150 ° C for 1 minute, raise the temperature to 250 ° C at a heating rate of 20 ° C / min, raise the temperature to 280 ° C at a heating rate of 5 ° C / min, and hold at 280 ° C for 10 minutes. , Raise to 330 ° C at a heating rate of 20 ° C / min and hold for 3 minutes.
Carrier gas: He, linear velocity 39.0 cm / sec Ion source temperature: 200 ° C
Interface temperature: 280 ℃
Vaporization chamber temperature: 250 ℃
Then, at least one peak area of the specific lipid metabolite (cholesterol biosynthesis precursor) is measured, and the inhibitory activity of the target compound is calculated. At that time, the peak area of the specific lipid metabolite when no compound is added is set to 0% inhibition.
Further, in the determination step, for example, in order to formulate a compound having an activity equal to or higher than a predetermined threshold value, whether or not the compound is effective for the treatment of chronic dermatitis by the following method based on the measured inhibitory activity value. Is determined. That is, when measured by the measuring method adopted in Example 8 described later, which is the method for measuring the inhibitory activity in the above-mentioned measuring step, at least one of the specific lipid metabolites is, for example, 50 at 10 μM. A compound exhibiting an accumulation inhibitory activity of% or more, more preferably a compound exhibiting an accumulation inhibitory activity of 50% or more at 1 μM for at least one specific lipid metabolite is determined to be effective for the treatment of chronic dermatitis.
As described above, the method of the present invention having the above-mentioned measurement step and determination step is useful as a screening method for a candidate drug expected to be effective for the treatment of chronic dermatitis.

[Method for diagnosing chronic dermatitis]
The method for diagnosing chronic dermatitis in the present invention includes a detection step of detecting at least one specific lipid metabolite from a skin sample, a detection amount of a cholesterol biosynthesis precursor detected from the sample, and a reference. It has a comparison step of comparing with a reference amount to be.

In the detection step, for example, a specific lipid metabolite is detected by the following method.
Approximately 1 mg of keratin at the lesion is collected and lipids are extracted with 3 mL of hexane. To the extract, add 10 μL of 0.1 μg / μL of D7-cholesterol ethanol solution as an internal standard. The whole amount is loaded on 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 dry, and then trimethylsilyl derivatization is performed. Specifically, 50 μL of BSTFA-TMCS (99: 1) solution and 10 μL of pyridine are added to the vial after drying, mixed, tightly closed, and kept warm at 40 ° C. for 40 minutes. The GC / MS analysis conditions here are the same as the GC / MS analysis conditions in the above-mentioned screening method for candidate compounds for the treatment of chronic dermatitis.

Furthermore, it is possible to quantify by analyzing a standard sample whose concentration is clear in advance under the same conditions, standardizing it by the peak area ratio with the internal standard, and obtaining the area area per unit amount of each substance. ..
For peaks for which standard samples are not available, compare the area with the cholesterol peak and evaluate as a relative amount. Specifically, the peak area of the substance to be analyzed is calculated for the cholesterol peak 100000, and the relative area obtained from the sample obtained from the lesion and the normally keratinized sample is compared.

The actual measurement data obtained by the above method is shown below. The following values are all measured by the above-mentioned method, and the peak area of each substance per 100,000 peak area of cholesterol is expressed as an index. The peak areas of each cholesterol biosynthesis precursor in normal keratinized samples were as follows. As shown below, in atopic dermatitis and psoriasis vulgaris, all specific lipid metabolites (precursors of cholesterol biosynthesis) accumulated more than 5 times as much as normal keratinized keratin on average.

As described above, since it was newly confirmed that specific lipid metabolites are accumulated in the lesion area of chronic dermatitis, the detected amount of cholesterol biosynthesis precursor detected from the sample is a predetermined reference amount. If more, the donor of the sample is likely to have chronic dermatitis.
As described above, the method of the present invention having the above-mentioned detection step and comparison step is a method for diagnosing chronic dermatitis or a method for assisting the diagnosis of chronic dermatitis (evaluation of severity, determination of therapeutic effect). ), Which is useful.
Here, the reference amount is a value evaluated by the ratio of the peak area of the specific lipid metabolite detected when performing GC-MS analysis under predetermined analysis conditions to cholesterol, and is normally keratinized keratin. Then, for the peak area of cholesterol of 100,000, C28A is 150 or less, C28B is 100 or less, C29A is 200 or less, and C29B is 100 or less. Therefore, for a peak area of cholesterol of 100,000, a peak area of 150 can be a reference amount of C28A, a peak area of 100 can be a reference amount of C28B, a peak area of 200 can be a reference amount of C29A, and a peak area of 100 can be a reference amount of C29B.

As described above, since it was newly confirmed that specific lipid metabolites are accumulated in the lesion area of chronic dermatitis, the detected amount of cholesterol biosynthesis precursor detected from the sample is a predetermined reference amount. If more, the donor of the sample is likely to have chronic dermatitis.
As described above, the method of the present invention having the above-mentioned detection step and comparison step is useful as a method for diagnosing chronic dermatitis or a method for assisting the diagnosis of chronic dermatitis.

Example 1
[Treatment for CHILD syndrome]
Atorvastatin was used as an HMGCR inhibitor for the lesion on the left side of a patient with chronic dermatitis associated with CHILD syndrome, and external application of 1% atorvastatin and 2% cholesterol aqueous solution was performed twice a day. Improvement of the rash was observed 1 month after the start of external use, and the rash completely disappeared 7 months after the start of external use. The lesion on the left armpit before the start of treatment with atorvastatin is shown in FIG. 3 (A1), and the lesion on the left armpit after the start of treatment is shown in FIG. 3 (A2).
In addition, 1% oxyconazole cream (trade name: Okinawal cream) was used as a CYP51 inhibitor for the lesion on the posterior side of the neck of a patient with chronic dermatitis associated with CHILD syndrome, and external application was performed once a day. rice field. The rash disappeared completely 4 months after the start of external use. The lesion on the posterior side of the neck before the start of treatment with oxiconazole is shown in FIG. 3 (B1), and the lesion on the posterior side of the neck after the start of treatment is shown in FIG. 3 (B2).
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]
Comparing the keratin of the lesioned part of a patient with chronic dermatitis associated with CHILD syndrome and the keratinized skin of a healthy part of the same patient, as shown in FIG. 4, specific lipid metabolites are accumulated only in the lesioned part. Was confirmed.
(experimental method)
Approximately 1 mg of keratin at the lesion was collected, and lipids were extracted with 3 mL of hexane. To the extract, 10 μL of 0.1 μg / μL of D7-cholesterol ethanol solution was added as an internal standard substance. 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 dry, and then trimethylsilyl derivatization was performed. Specifically, 50 μL of BSTFA-TMCS (99: 1) solution and 10 μL of pyridine were added to the vial after drying, mixed, sealed, and kept warm 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 ° C, holding 1 minute-20 ° C / min → 250 ° C, 5 ° C / min → 280 ° C, holding 10 minutes-20 ° C / min → 330 ° C, holding 3 minutes.
Carrier gas: He, linear velocity 39.0 cm / sec Ion source temperature: 200 ° C
Interface temperature: 280 ℃
Vaporization chamber temperature: 250 ℃

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、コレステロール生合成前駆物質Ａ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（Ｃ_３Ｈ_９）体に由来する値であり、３７９．３は、上記ＴＭＳ体からＣＨ_４とＯＳｉ（Ｃ_３Ｈ_９）が脱離した断片に由来する値であり、１３５．１は、Ｃ_１０Ｈ_１５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（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。

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 the formulas (I) and (II) (the number of carbon atoms are 29) both have two methyl groups at the 4-position, and the compounds of the formulas (III) and the formula (IV) (the number of carbon atoms is 28). All have one methyl group at the 4-position.
The m / z values of the GC / MS spectral data of the cholesterol biosynthesis precursor shown in Table 2 are all theoretical values. For example, the peak of 484.4 in the compound (C29B) of the formula (II) is set. It is a value derived from the -OSi (C ₃ H ₉ ) compound produced by trimethylsilylation (TMS) of the hydroxyl group of the molecule of the cholesterol biosynthesis precursor A, and 379.3 is a value derived from the above TMS compound to CH ₄ and OSi ( C ₃ H ₉ ) is a value derived from the desorbed fragment, and 135.1 is a value derived from the fragment of C ₁₀ H ₁₅ .
The theoretical value of m / z 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 measurement data of C29B in FIG. 1, due to a slight measurement error, the peak of 484.35 corresponding to the theoretical value of 484.4 for the compound (C29B) of the formula (II) and the theoretical value are used. A peak of 135.15 corresponding to a certain 135.1 has been detected respectively. As described above, in the m / z value of the GC / MS spectral data, an error of up to about ± 0.1 can be observed.
Further, regarding the value of the relative retention time with respect to cholesterol, which is an internal standard substance, a measurement error of about ± 0.01 is observed. For example, in the compound (C29B) of the formula (II), the above 1.27. The measured value of is also likely to vary in the range of 1.26 to 1.28.

FIG. 4 is a diagram showing the measured accumulation amount of lipid metabolites in a lesion portion of a patient with chronic dermatitis associated with CHILD syndrome and a healthy portion of the same patient. Thus, in the keratin of the lesion of CHILD syndrome patients, specific lipid metabolites that were hardly detected in the normal part were detected.
Then, it was confirmed that the molecular structure of the detected compound was represented by the formulas (I) to (IV) as described above.

Example 3
[Effect of suppressing accumulation of lipid metabolites]
A test system for artificially accumulating the lipid metabolites identified in Example 2 was constructed, and the production inhibitory effect of various cholesterol biosynthesis inhibitors was investigated.
(experimental method)
When the human keratinocyte cultured cell line HaCaT is cultured in a medium containing 10% cholesterol-free serum, HaCaT cells will synthesize a large amount of cholesterol using the components in the medium. 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 was inhibited, and it was identified as in the case of CHILD syndrome skin. Lipid metabolites accumulate in cells.
A test was conducted to investigate whether the accumulation of specific lipid metabolites could be suppressed by adding various cholesterol biosynthesis inhibitors to the culture system of HaCaT cells simulating CHILD syndrome.
To evaluate whether the added drug suppresses specific lipid accumulation, after culturing, lipids are extracted from each cell, and the relative 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 (-) to examine the effect of the administered drug.
The m / z values of substance-specific ions used to evaluate the relative amount of a particular lipid are, respectively.

C29A (compound of formula (I)): m / z 486.4
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.
Accumulation of specific lipid metabolites was suppressed by adding each drug at the concentration shown in the graph. In addition, in the results of the test in which the concentration was changed by 10 times, a dose-dependent inhibitory effect was observed for all the drugs.
As a result, it was found that, in particular, oxiconazole suppressed the accumulation of specific lipid metabolites (see [Fig. 5]). Therefore, it was suggested that an inhibitor of the accumulation of specific lipid metabolites could be a therapeutic agent.

Example 4
[Accumulation of lipid metabolites in chronic skin diseases]
It was tested whether the accumulation of lipid metabolites identified in Example 1 was also confirmed in patients with chronic dermatitis.
(experimental method)
The method of implementation is the same as that of the second embodiment.
That is, keratin of healthy people, keratin of CHILD syndrome patients (lesion and healthy parts), keratin of psoriasis vulgaris patients (lesions and healthy parts), keratin of hereditary keratosis patients (lesions), acute dermatitis. Approximately 1 mg of each of the patient's keratin (lesion), the keratin of the toxic eczema patient (lesion), and the keratin of the atopic dermatitis patient (lesion) were collected, and the lipid was extracted by the same method as in Example 2. ,Analysis was carried out. The peak area of each specific lipid metabolite per tissue weight was evaluated in comparison with that of the keratin of healthy subjects. In the lesions of CHILD syndrome patients, the rash part of psoriasis vulgaris patients, and the lesion part of atopic dermatitis patients, accumulation of specific lipid metabolites 100 times or more per tissue weight was observed as compared with the keratin of healthy subjects. On the other hand, the keratin of the healthy part of CHILD syndrome patients and the non-rash part of psoriasis vulgaris patients was not different from that of healthy subjects. In addition, in the lesions of hereditary keratoderma patients, contact dermatitis (acute dermatitis) patients, toxic eruptions patients, and callus patients, the accumulated amount of specific lipid metabolites may be the same as that of healthy human keratin. all right. The results of Example 4 are shown in FIG. In FIG. 6, the peak area of each lipid per unit weight is calculated, and the peak area of each lipid per unit weight of a healthy person is set as a reference value of 1, and the amount of accumulated specific lipid metabolite in the lesion. Displayed a relative value indicating how many times the reference value was.

Example 5
[Changes in lipid metabolites associated with treatment]
Secukinumab (trade name: Cosentyx (human anti-human IL-17A monoclonal antibody preparation) was subcutaneously injected at a dose of 300 mg to the lesion of a patient with psoriasis vulgaris.
Specifically, 300 mg was subcutaneously injected once every 0, 1, 2, 3, 4, and 5 weeks, and thereafter, 300 mg was subcutaneously injected once every 4 weeks.

For the above patients, keratin was collected from the same site of the lesion before and after administration of secukinumab vulgaris, and lipid extraction and analysis were performed by the same method as in Example 1. The eruption with scales was relieved by the treatment, and when the eruption healed, the scales could not be collected. Comparing before and after treatment in the two cases that could be collected, the accumulation of specific lipid metabolites decreased in both cases. The result is shown in FIG. The vertical axis of FIG. 7 represents the relative value of the accumulated amount of the specific lipid metabolite, and more specifically, represents the peak area of each substance per 100,000 peak area when cholesterol is analyzed under the same conditions. There is.
From the above, as a result of measuring the specific lipid metabolites in the keratin 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. 7 (A) and 7 (B)).
Thus, in skin lesions with chronic symptoms such as psoriasis and atopic dermatitis, which will be described in detail later, specific lipid metabolites having toxicity associated with dermatitis are accumulated (FIGS. 7A and 7B). ) On the other hand, no such accumulation was observed in skin lesions without chronic inflammation (such as acute dermatitis and psoriasis). Therefore, specific lipid metabolites are considered to contribute to the chronicity of dermatitis.
As described above, it has been confirmed that the accumulation of specific lipid metabolites decreases as the skin symptoms are improved by the treatment of psoriasis. Therefore, the accumulation of specific lipid metabolites is related to the therapeutic effect of chronic dermatitis. It can be said that it is doing. Therefore, it was suggested that an inhibitor of the accumulation of specific lipid metabolites could be a therapeutic agent for these chronic skin diseases.

Example 6
[Treatment for atopic dermatitis]
The external application of steroids to the lesions of patients with atopic dermatitis was discontinued, and atorvastatin (1 wt% atorvastatin solution in which atorvastatin powder was dissolved in water to a concentration of 1%) and oxiconazole (1% oxiconazole) were discontinued. Nazole cream (brand name: oxiconazole cream) is applied twice a day at 350 mg (equivalent to 3.5 mg of raw powder) to a healthy part, a lesion immediately before the start of treatment with atorvastatin and oxiconazole, and a lesion one week after the treatment. The horny substance was collected from the part and the lesion part 3 weeks after the treatment, and the lipid was extracted and analyzed by the same method as in Example 2. As a result, it was confirmed that in atopic dermatitis, application of atorvastatin and oxiconazole suppresses 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 peak area of cholesterol.
When steroid application is stopped for atopic dermatitis, the dermatitis usually tends to worsen. On the other hand, when atorvastatin and oxiconazole were applied after discontinuation of steroid application as in this example, the affected area, which usually deteriorates, was maintained in almost the same state as when the steroid application was discontinued. rice field.

Example 7
[Treatment for psoriasis vulgaris]
Oxiconazole (1% oxiconazole cream, trade name: oxiconazole cream) was applied to the lesions of patients with psoriasis vulgaris twice a day for 2 weeks. Then, keratin was collected from a healthy part, a lesion part immediately before the start of treatment with oxiconazole, and a lesion part 4 weeks after the treatment, and lipid extraction and analysis were performed by the same method as in Example 2. As a result, it was confirmed that even in psoriasis vulgaris, application of oxiconazole suppresses 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 100,000 peak area of cholesterol.
Since a decrease in skin scales was observed 4 weeks after the treatment, it is recognized that oxiconazole has an effect of suppressing skin symptoms.

Based on 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), and at least in CHILD syndrome, specific lipids are accumulated. An inhibitor that inhibits the production of metabolites can reduce the accumulation amount of a specific lipid metabolite (Example 3), and in psoriasis, the accumulation amount of a specific lipid metabolite decreases as the eruption is improved by treatment. What to do (Example 5) was confirmed.
Furthermore, at least for chronic dermatitis, psoriasis vulgaris, and atopic dermatitis associated with CHILD syndrome, improvement of skin symptoms or specific lipid metabolites by an inhibitor that inhibits the production of specific lipid metabolites Prevention of accumulation was confirmed (Examples 1, 6, and 7).
From the above, it is considered that the specific lipid metabolite contributes to the chronicity of dermatitis, and the chronic dermatitis in which the accumulation of the specific lipid metabolite is confirmed (see [FIG. 6]) is the specific lipid. It has been found that it can be a therapeutic target with compounds that inhibit the production of metabolites. Therefore, it was suggested that an inhibitor of the accumulation of specific lipid metabolites could be a therapeutic agent for these chronic skin diseases.

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 lysedronate showed good C29A accumulation inhibitory activity of 50% or more at 10 μM. In addition, amorolfine hydrochloride, atorvastatin, azaranstat, biphonazole, butconazole nitrate, croconazole hydrochloride, econazole, fluazinum, lovastatin, methconazole, oxyconazole nitrate, simvastatin, sulconazole nitrate, tebuconazole and terconazole are 50% or more at 1 μM. 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 to 2.5 mM. The dissolved evaluation compound was diluted to DMSO at a 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.
A microsome fraction of silk moth expressing the human CYP51A1 protein was obtained from Sysmex Corporation. The enzyme activity of CYP51A1 was calculated from the amount of NADPH used when metabolizing lanosterol as a substrate. A NADP / NADPH assay kit (ab65349) manufactured by Abcam was used for the measurement of NADPH. That is, the final concentrations are 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, respectively. 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. To this, 2 μL of a 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, and the mixture was allowed to stand at room temperature for 20 minutes. Subsequent operations were performed according to the protocol of NADP / NADPH assay kit manufactured by Abcam.
The inhibitory activity (IC ₅₀ ) of human CYP51A1 was calculated from the NADPH consumption in the presence of the evaluation compound at each concentration, assuming that the NADPH consumption when the above test was performed without adding the evaluation compound was 100% activity.
As a result, azalanstat, biphonazole, butoconazole nitrate, croconazole hydrochloride, metconazole, oxyconazole nitrate, sulconazole nitrate and tebuconazole showed good _IC50 of 1 μM or less.

Thus, it has been shown that the compound which is the active ingredient of the present invention, that is, at least a CYP51 inhibitor and an HMGCR inhibitor, is effective in suppressing chronic skin diseases.

According to the present invention, attention has been paid to a pharmaceutical composition which inhibits the production of a novel specific cholesterol biosynthesis precursor and has an excellent therapeutic effect on chronic dermatitis, and the amount of a specific cholesterol biosynthesis precursor detected. , A method for screening a candidate drug for chronic dermatitis and the like can be provided.

Claims (11)

A pharmaceutical composition for the treatment of chronic dermatitis, which comprises an active ingredient that inhibits the production of any of the cholesterol biosynthesis precursors represented by the following formulas (I) to (IV).

The pharmaceutical composition for the treatment of chronic dermatitis according to claim 1, wherein the active ingredient is a CYP51 inhibitor or an HMGCR inhibitor. The pharmaceutical composition according to claim 2, wherein the active ingredient is the CYP51 inhibitor. 3. The third aspect of the present invention, wherein the CYP51 inhibitor comprises any one selected from azalanstat, biphonazole, butoconazole, croconazole, econazole, metconazole, oxyconazole, sulconazole and tebuconazole or pharmaceutically acceptable salts thereof. Pharmaceutical composition. The pharmaceutical composition according to claim 2, wherein the active ingredient is the HMGCR inhibitor. The pharmaceutical composition according to claim 5, wherein the HMGCR inhibitor is atorvastatin, lovastatin, simvastatin, or a pharmaceutically acceptable salt thereof. The pharmaceutical composition according to claim 1, which is an external pharmaceutical composition. The pharmaceutical composition according to claim 1, wherein the chronic dermatitis is atopic dermatitis or psoriasis. Amorolfine, alendronate, atorvastatin, azaranstat, biphonazole, butoconazole, croconazole, econazole, fluazinum, lovastatin, methconazole, oxyconazole, lysedronate, simvastatin, sulconazole, tebuconazole, and telconazole or their pharmaceutically acceptable A pharmaceutical composition for the treatment of chronic dermatitis, comprising at least one compound of salt. (A) A measurement step for measuring the inhibitory activity of the target compound to inhibit the production of any of the cholesterol biosynthesis precursors represented by the following formulas (I) to (IV).
(B) A determination step of determining whether or not the target compound is an effective candidate compound for the treatment of chronic dermatitis based on the measured value of the inhibitory activity.
A method for screening candidate compounds for the treatment of chronic dermatitis.

(C) A detection step for detecting any of the cholesterol biosynthesis precursors represented by the following formulas (I) to (IV) from the skin sample, and
(D) The present invention comprises a comparative step of comparing the detected amount of the cholesterol biosynthesis precursor detected from the sample with the reference amount as a reference.
A method for diagnosing chronic dermatitis.

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