JP2011059102A - Skin sensitization measuring reagent - Google Patents
Skin sensitization measuring reagent Download PDFInfo
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- JP2011059102A JP2011059102A JP2010168054A JP2010168054A JP2011059102A JP 2011059102 A JP2011059102 A JP 2011059102A JP 2010168054 A JP2010168054 A JP 2010168054A JP 2010168054 A JP2010168054 A JP 2010168054A JP 2011059102 A JP2011059102 A JP 2011059102A
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- 206010070835 Skin sensitisation Diseases 0.000 title claims abstract description 48
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
Description
本発明は、皮膚感作性測定試薬に関する。 The present invention relates to a reagent for measuring skin sensitization.
医薬、農薬、及び化粧品等の製品に含まれる化学物質はアレルギー反応を引き起こさない物質であることが肝要であり、製品開発に当っては、使用する化学物質の皮膚感作性を確認する必要がある。皮膚感作性の測定として最終的には、Maximization試験、Buehler試験、Local Lymph Node Assay等の動物実験による試験結果が必要となるが、動物実験は煩雑な作業や、多大の時間を必要とする。そのため、開発初期段階等において簡便で迅速な方法で化学物質の皮膚感作性を測定できる手段が求められていた。
煩雑な動物実験を行うことなく、被験物質の皮膚感作性を簡便かつ迅速に検定する方法として、グルタチオンを用いた皮膚感作試験が提案されている(特許文献1,2)。しかし、当該試験で用いることができる検出方法が質量分析に限られるという問題があった。
It is important that chemical substances contained in products such as pharmaceuticals, agricultural chemicals, and cosmetics are substances that do not cause allergic reactions, and it is necessary to confirm the skin sensitization of the chemical substances used in product development. is there. Eventually, the results of animal experiments such as the Maximization test, Buehler test, and Local Lymph Node Assay are required for measuring skin sensitization, but animal experiments require complicated work and a lot of time. . Therefore, there has been a demand for means capable of measuring the skin sensitization of a chemical substance by a simple and rapid method at an early stage of development.
A skin sensitization test using glutathione has been proposed as a method for easily and rapidly assaying the skin sensitization of a test substance without conducting complicated animal experiments (
本発明の目的は、化学物質の皮膚感作性を簡便かつ迅速に測定するための手段を提供することである。また、本発明の目的は、簡易な分析装置により測定が可能な皮膚感作性測定試薬を提供することである。 An object of the present invention is to provide a means for easily and rapidly measuring the skin sensitization of a chemical substance. Moreover, the objective of this invention is providing the skin sensitization measuring reagent which can be measured with a simple analyzer.
本発明者らは、簡易な分析装置の検出に用いられている検出器により検出可能な化合物を皮膚感作性測定試薬として用いることが上記課題の解決手段になりうると考え、鋭意研究の結果、本発明の完成に至った。
すなわち、本発明は以下[1]〜[11]を提供するものである。
The present inventors believe that using a compound that can be detected by a detector used for detection of a simple analyzer as a skin sensitization measuring reagent can be a solution to the above problem, and as a result of earnest research The present invention has been completed.
That is, the present invention provides the following [1] to [11].
[1]チオール基を1個以上有する構造を有する有機化合物であって紫外、可視光又は近赤外域に吸収を有する有機化合物を測定主薬として含む皮膚感作性測定試薬。
[2]前記有機化合物が芳香族基を有するシステインの誘導体である[1]に記載の試薬。
[3]前記有機化合物がN−(アリールアルキルカルボニル)システインである[1]に記載の試薬。
[4]前記有機化合物がN−(2−フェニルアセチル)システイン又はN−[2−(ナフタレン−1−イル)アセチル]システインである[1]に記載の試薬。
[1] A reagent for measuring skin sensitization comprising an organic compound having a structure having at least one thiol group and having absorption in the ultraviolet, visible light, or near infrared region as a measuring agent.
[2] The reagent according to [1], wherein the organic compound is a derivative of cysteine having an aromatic group.
[3] The reagent according to [1], wherein the organic compound is N- (arylalkylcarbonyl) cysteine.
[4] The reagent according to [1], wherein the organic compound is N- (2-phenylacetyl) cysteine or N- [2- (naphthalen-1-yl) acetyl] cysteine.
[5]粉末形態で提供される[1]〜[4]のいずれか一項に記載の試薬。
[6]前記有機化合物が、有機酸塩類を含む水性緩衝液もしくは水又はこれらと有機溶媒との混合溶媒に溶解した形態で提供される[1]〜[4]のいずれか一項に記載の試薬。
[7]前記有機化合物の濃度が1mM〜500mMである[6]に記載の試薬。
[5] The reagent according to any one of [1] to [4] provided in a powder form.
[6] The organic compound according to any one of [1] to [4], wherein the organic compound is provided in a form dissolved in an aqueous buffer solution or water containing an organic acid salt or a mixed solvent of these and an organic solvent. reagent.
[7] The reagent according to [6], wherein the concentration of the organic compound is 1 mM to 500 mM.
[8](1)チオール基を1個以上有する構造を有する有機化合物であって紫外、可視光又は近赤外域に吸収を有する有機化合物と被験物質とを反応させること、及び
(2)前記反応による前記有機化合物の量の低下を光学的測定により検出することを含む皮膚感作性の測定方法。
[9](1)チオール基を1個以上有する構造を有する有機化合物であって紫外、可視光又は近赤外域に吸収を有する有機化合物と被験物質とを反応させること、及び、
(12)前記反応により生じた化合物を光学的測定により検出することを含む
皮膚感作性の測定方法。
[8] (1) reacting an organic compound having a structure having one or more thiol groups and having absorption in the ultraviolet, visible light, or near infrared region with a test substance, and (2) the reaction A method for measuring skin sensitization, which comprises detecting a decrease in the amount of the organic compound by optical measurement by optical measurement.
[9] (1) reacting an organic compound having a structure having one or more thiol groups and having absorption in the ultraviolet, visible light, or near infrared region with a test substance; and
(12) A method for measuring skin sensitization comprising detecting a compound produced by the reaction by optical measurement.
[10]前記有機化合物がN−(2−フェニルアセチル)システイン又はN−[2−(ナフタレン−1−イル)アセチル]システインである[8]又は[9]に記載の方法。
[11]前記工程(1)で得られる反応物をクロマトグラフィー処理することを含む[8]〜[10]のいずれか一項に記載の方法。
[10] The method according to [8] or [9], wherein the organic compound is N- (2-phenylacetyl) cysteine or N- [2- (naphthalen-1-yl) acetyl] cysteine.
[11] The method according to any one of [8] to [10], wherein the reaction product obtained in the step (1) is chromatographed.
本発明により、汎用の簡易な分析装置により測定が可能な皮膚感作性測定試薬が提供される。本発明の試薬により化学物質の皮膚感作性を簡便かつ迅速に測定することができる。 According to the present invention, a reagent for measuring skin sensitization that can be measured by a general-purpose simple analyzer is provided. With the reagent of the present invention, the skin sensitization of a chemical substance can be measured easily and rapidly.
本明細書において、「〜」とはその前後に記載される数値を下限値及び上限値として含む意味で使用される。
本明細書において皮膚感作性の測定とは皮膚感作性の検定を含む意味であり、また、一定の基準の皮膚感作性の有無の判断、及び皮膚感作性の定量的測定を含む意味である。
In the present specification, “to” is used to mean that the numerical values described before and after it are included as a lower limit value and an upper limit value.
In the present specification, the measurement of skin sensitization is meant to include an assay of skin sensitization, and includes determination of the presence or absence of a certain standard of skin sensitization and quantitative measurement of skin sensitization. Meaning.
本発明の皮膚感作性測定試薬は、チオール基を1個以上有する構造を有する有機化合物であって紫外、可視光又は近赤外域に吸収を有する有機化合物を含む。本発明の皮膚感作性測定試薬に含まれる有機化合物はそのままの状態、又は溶液状態で、好ましくは、波長190〜2500nmの領域で吸収を示す化合物であり、さらに好ましくは、波長200〜700nmの領域に吸収を示す化合物である。さらに、該波長領域に吸収極大を有する化合物が好ましい。また、本発明の皮膚感作性測定試薬に含まれる有機化合物は、好ましくは、その吸収極大のモル吸光係数(L/mol・cm)が10以上の吸収を有する化合物であり、さらに好ましくは、モル吸光係数が100以上の吸収を有する化合物である。特に好ましくは、波長200〜700nmの領域に吸収極大を有し、そのモル吸光係数が100以上の吸収を有する化合物である。 The skin sensitization measuring reagent of the present invention is an organic compound having a structure having at least one thiol group, and includes an organic compound having absorption in the ultraviolet, visible light, or near infrared region. The organic compound contained in the skin sensitization measuring reagent of the present invention is in a state as it is or in a solution state, preferably a compound exhibiting absorption in a wavelength range of 190 to 2500 nm, more preferably a wavelength of 200 to 700 nm. It is a compound that absorbs in the region. Furthermore, a compound having an absorption maximum in the wavelength region is preferable. The organic compound contained in the skin sensitization measuring reagent of the present invention is preferably a compound having an absorption maximum molar absorption coefficient (L / mol · cm) of 10 or more, more preferably A compound having an absorption with a molar extinction coefficient of 100 or more. Particularly preferred is a compound having an absorption maximum in a wavelength range of 200 to 700 nm and an absorption having a molar extinction coefficient of 100 or more.
上記の有機化合物としては例えば、システインの誘導体であって、芳香族基を有するものが挙げられる。芳香族基は、フェニル基、ナフチル基などの炭化水素系芳香族基のほか、ピリジル基、フリル基、チオフェニル基などのヘテロ元素を有するものであってもよい。上記の有機化合物として、さらに具体的には、システインのアミノ基又はカルボキシル基にアミド結合などによりベンゼン環やナフタレン環などのアリール基を結合させた化合物が挙げられる。このうち、N−(アリールアルキルカルボニル)システイン等が特に好ましい。N−(アリールアルキルカルボニル)システインにおいて、アリール基は炭素数6〜16程度であればよい。またアルキルカルボニル基における炭素数は2〜11程度であればよい。カルボニル基に結合するアルキル基は直鎖状、分岐鎖状、環状のいずれでもよく、例えば、メチル基、エチル基、プロピル基、イソプロピル基、ブチル基、イソブチル基、sec−ブチル基、tert−ブチル基、ペンチル基、イソペンチル基、ネオペンチル基、tert−ペンチル基、ヘキシル基、へプチル基、オクチル基、ノニル基、デシル基、2−エチルヘキシル基、シクロプロピル基などを挙げることができる。上記の有機化合物としての具体例としては、N−(2−フェニルアセチル)システイン、及びN−[2−(ナフタレン−1−イル)アセチル]システインが挙げられる。 Examples of the organic compound include cysteine derivatives having an aromatic group. The aromatic group may have a hetero element such as a pyridyl group, a furyl group, or a thiophenyl group in addition to a hydrocarbon aromatic group such as a phenyl group or a naphthyl group. More specifically, examples of the organic compound include compounds in which an aryl group such as a benzene ring or a naphthalene ring is bonded to an amino group or a carboxyl group of cysteine through an amide bond or the like. Of these, N- (arylalkylcarbonyl) cysteine and the like are particularly preferable. In N- (arylalkylcarbonyl) cysteine, the aryl group may have about 6 to 16 carbon atoms. Moreover, the carbon number in an alkylcarbonyl group should just be about 2-11. The alkyl group bonded to the carbonyl group may be linear, branched or cyclic, for example, methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl. Group, pentyl group, isopentyl group, neopentyl group, tert-pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, 2-ethylhexyl group, cyclopropyl group and the like. Specific examples of the organic compound include N- (2-phenylacetyl) cysteine and N- [2- (naphthalen-1-yl) acetyl] cysteine.
上記有機化合物は公知の方法により製造することが可能であり、例えば、N−(2−フェニルアセチル)システインは以下のように合成することができる。 The organic compound can be produced by a known method. For example, N- (2-phenylacetyl) cysteine can be synthesized as follows.
水酸化ナトリウム5.1g、水126gの水溶液にL−シスチン7.3gを添加し、溶解する。溶液を氷水浴にて冷却し、ここへ、フェニルアセチルクロリド10.2gを滴下する。反応混合物を氷水浴下で1時間撹拌し、さらに、室温下1時間撹拌したのち、濃塩酸6mLを添加する。析出した結晶を濾取し、水で洗浄、乾燥し、N,N’−ビス(2−フェニルアセチル)シスチンを得る(9.8g)。
N,N’−ビス(2−フェニルアセチル)シスチン4.8g、亜鉛粉末2.0g、メタノール20mL、水5mLの混合物を30℃に加温し、濃硫酸3.0gを2時間かけて滴下する。反応混合物を30℃で1時間撹拌したのち、濾過により不溶物を濾別し、水60mL、塩化ナトリウム1gを加える。溶液を酢酸エチル80mLで抽出し、有機層を飽和食塩水30mLで洗浄し、硫酸マグネシウムで乾燥後、濾過、濃縮する。得られた残渣を酢酸エチル/ヘキサン=1/1から再結晶し、乾燥後しN−(2−フェニルアセチル)システインを得る(3.1g)。
7.3 g of L-cystine is added to an aqueous solution of 5.1 g of sodium hydroxide and 126 g of water and dissolved. The solution is cooled in an ice-water bath, and 10.2 g of phenylacetyl chloride is added dropwise thereto. The reaction mixture is stirred for 1 hour in an ice-water bath, and further stirred for 1 hour at room temperature, and then 6 mL of concentrated hydrochloric acid is added. The precipitated crystals are collected by filtration, washed with water and dried to obtain N, N′-bis (2-phenylacetyl) cystine (9.8 g).
A mixture of 4.8 g of N, N′-bis (2-phenylacetyl) cystine, 2.0 g of zinc powder, 20 mL of methanol and 5 mL of water is heated to 30 ° C., and 3.0 g of concentrated sulfuric acid is added dropwise over 2 hours. After stirring the reaction mixture at 30 ° C. for 1 hour, insoluble materials are filtered off by filtration, and 60 mL of water and 1 g of sodium chloride are added. The solution is extracted with 80 mL of ethyl acetate, and the organic layer is washed with 30 mL of saturated brine, dried over magnesium sulfate, filtered and concentrated. The obtained residue is recrystallized from ethyl acetate / hexane = 1/1 and dried to obtain N- (2-phenylacetyl) cysteine (3.1 g).
本発明の皮膚感作性測定試薬は、上記の有機化合物のみからなるものであってもよく、測定主薬である上記の有機化合物のほかに1又は2以上の添加剤を含んでいるものであってもよい。添加剤の例としては、pH調整剤、安定化剤、等が挙げられる。また、本発明の皮膚感作性測定試薬は、上記の測定主薬及び必要に応じて上記の添加剤を、水、水性緩衝液、有機溶媒、又はこれらいずれかの混合溶媒等に溶解させたものであってもよい。
本発明の皮膚感作性測定試薬は、溶液、液体状、固体状(粉末、顆粒、凍結乾燥物、錠剤等)のいずれの形態で提供されてもよい。
The skin sensitization measuring reagent of the present invention may be composed only of the above-mentioned organic compound, and contains one or more additives in addition to the above-mentioned organic compound which is a measuring agent. May be. Examples of the additive include a pH adjuster and a stabilizer. The skin sensitization measuring reagent of the present invention is obtained by dissolving the above-mentioned measuring agent and, if necessary, the above-mentioned additives in water, an aqueous buffer solution, an organic solvent, or any mixed solvent thereof. It may be.
The reagent for measuring skin sensitization of the present invention may be provided in any form of a solution, liquid, or solid (powder, granule, lyophilized product, tablet, etc.).
本発明の皮膚感作性測定試薬は、例えば、酢酸アンモニウムなどの有機酸塩類を含む水性緩衝液もしくは水又はこれらと有機溶媒との混合溶媒に溶解した形態で、例えば約0.01μM〜約1M程度、通常約1mM〜約500mM程度の上記有機化合物の濃度として使用すればよい。被験物質は、例えば、メタノール、エタノール、アセトニトリル、アセトンなどの有機溶媒又はこれらの混合溶媒に、例えば約0.01μM〜約1M程度の濃度、通常約1mM〜約500mM程度の濃度となるように溶解すればよい。次いで、本発明の皮膚感作性測定試薬の測定主薬である上記有機化合物と被験物質溶液とを、上記有機化合物と被験物質のモル濃度比が例えば1:100〜20:1となるように混合し反応させればよい。反応は、上記有機化合物と被験物質とを含む溶液を、例えば約4℃〜約60℃程度の温度範囲にて保温しながら、通常約1分〜約2日間程度攪拌又は静置することによって行うことができる。 The skin sensitization measuring reagent of the present invention is, for example, in an aqueous buffer solution containing an organic acid salt such as ammonium acetate or water or a form dissolved in a mixed solvent of these with an organic solvent, for example, about 0.01 μM to about 1 M. The concentration of the organic compound is usually about 1 mM to about 500 mM. The test substance is dissolved in, for example, an organic solvent such as methanol, ethanol, acetonitrile, acetone, or a mixed solvent thereof so as to have a concentration of about 0.01 μM to about 1 M, usually about 1 mM to about 500 mM. That's fine. Next, the organic compound and the test substance solution, which are the measurement agents of the skin sensitization measuring reagent of the present invention, are mixed so that the molar concentration ratio of the organic compound and the test substance is, for example, 1: 100 to 20: 1. And react. The reaction is carried out by stirring or leaving the solution containing the organic compound and the test substance in a temperature range of about 4 ° C. to about 60 ° C. for about 1 minute to about 2 days. be able to.
該反応により、上記有機化合物と被験物質との反応性を調べることによって、被験物質の皮膚感作性を測定することができる。上記の反応性を調べるためには、皮膚感作性測定試薬溶液と被験物質溶液との混合液中における上記有機化合物の残存量および/又は上記有機化合物と被験物質との反応生成物の生成量を分析すればよい。この分析を経時的に行うことにより、上記有機化合物と被験物質との反応速度定数を求め、この値から皮膚感作性を評価することができる。 By examining the reactivity between the organic compound and the test substance, the skin sensitization property of the test substance can be measured. In order to examine the reactivity, the remaining amount of the organic compound in the mixed solution of the skin sensitization measuring reagent solution and the test substance solution and / or the amount of reaction product produced between the organic compound and the test substance Should be analyzed. By performing this analysis over time, a reaction rate constant between the organic compound and the test substance can be obtained, and skin sensitization can be evaluated from this value.
なお、残存量を分析する場合、皮膚感作性測定試薬はチオール基を有しているため、容易に酸化されて上記化合物のジスルフィド体(2量体)になることがある。そのため、上記有機化合物の残存量を定量する際には、必要に応じてジスルフィド体も含めて分析を行い、これらの総計により上記有機化合物の残存量としてもよい。 When analyzing the residual amount, since the skin sensitization measuring reagent has a thiol group, it may be easily oxidized to a disulfide form (dimer) of the above compound. Therefore, when the remaining amount of the organic compound is quantified, an analysis including the disulfide is performed as necessary, and the total amount of these may be used as the remaining amount of the organic compound.
化合物および上記反応により生成した化合物の分析方法としては、特に限定されないが、例えば、高速液体クロマトグラフィー(HPLC)、ガスクロマトグラフィー(GC)、薄層クロマトグラフィー(TLC)などにより、上記反応により生成した化合物、上記有機化合物及び被験物質を分離して分析する方法が挙げられる。上記HPLC、GC又はTLCに用いることのできるクロマトグラフ手法としては、逆相、順相、イオン交換などを挙げることができる。このようなクロマトグラフ手法に使用可能な市販のカラムやTLCとしては、例えば、LCカラムとしてはCAPCELL-PAK(資生堂製)、L-column ODS(化学品評価研究機構製)、Shodex Asahipak (昭和電工製)などを挙げることができ、TLCプレートではシリカゲル60F254(メルク社製)、Silica Gel Plate (ナカライテスク社製)などを挙げることができる。 The method for analyzing the compound and the compound produced by the above reaction is not particularly limited, but it is produced by the above reaction by, for example, high performance liquid chromatography (HPLC), gas chromatography (GC), thin layer chromatography (TLC), etc. And a method of separating and analyzing the compound, the organic compound and the test substance. Examples of chromatographic techniques that can be used for the HPLC, GC, or TLC include reverse phase, normal phase, and ion exchange. Commercially available columns and TLC that can be used for such chromatographic methods include, for example, LC columns such as CAPCELL-PAK (manufactured by Shiseido), L-column ODS (manufactured by Chemicals Evaluation and Research Organization), Shodex Asahipak (Showa Denko) Examples of TLC plates include silica gel 60F254 (Merck) and Silica Gel Plate (Nacalai Tesque).
上記反応により生成した化合物又は残存する上記有機化合物の検出方法は、特に限定されないが、例えば上記HPLC分析で利用することのできる検出器としては、紫外可視検出器、近赤外検出器、蛍光検出器、示差屈折率検出器、電気伝導度検出器、蒸発光散乱検出器などが挙げられる。紫外可視検出器としては、例えば、単波長紫外可視検出器、二波長紫外可視検出器、フォトダイオードアレイ検出器などが挙げることができる。また、このような検出法に使用可能な市販の検出器としては、紫外可視検出器、示差屈折率検出器、電気伝導度検出器の場合、島津製作所製、日立製作所製、ウォーターズ製、資生堂製などの検出器、蒸発光散乱検出器としては島津製作所製などが挙げられる。 The detection method of the compound produced by the reaction or the remaining organic compound is not particularly limited. For example, a detector that can be used in the HPLC analysis includes an ultraviolet-visible detector, a near-infrared detector, and fluorescence detection. , Differential refractive index detector, electrical conductivity detector, evaporative light scattering detector, and the like. Examples of the ultraviolet visible detector include a single wavelength ultraviolet visible detector, a dual wavelength ultraviolet visible detector, and a photodiode array detector. In addition, as a commercially available detector that can be used for such a detection method, in the case of an ultraviolet-visible detector, a differential refractive index detector, and an electrical conductivity detector, Shimadzu, Hitachi, Waters, Shiseido Examples of such detectors and evaporative light scattering detectors include those manufactured by Shimadzu Corporation.
本発明の皮膚感作性測定試薬を用いた測定方法における検出は、上記に限定されず、例えば、特開2003-14761号公報又は特開2008-139275号公報に記載の方法を参照して、分子量等に基づく、特定の質量のイオン検出により行ってもよい。 Detection in the measurement method using the skin sensitization measurement reagent of the present invention is not limited to the above, for example, refer to the method described in JP2003-14761A or JP2008-139275A, You may carry out by ion detection of the specific mass based on molecular weight etc.
また、本発明の皮膚感作性測定試薬を用いた方法においては、好ましくは光学的検出方法を用いることができる。好ましくは、上述の紫外可視検出器、近赤外検出器を用いるとよい。 In the method using the skin sensitization measuring reagent of the present invention, an optical detection method can be preferably used. Preferably, the above-described ultraviolet-visible detector and near-infrared detector are used.
以下に実施例により本発明を詳細に説明するが、本発明はこれらに限定されるものではない。 EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.
[例1]
被検物質の溶液(32.5mMのアセトン溶液)1400μlと、以下に記載のN−(2−フェニルアセチル)システイン(以下、化合物A)溶液を700μl混合し、25℃で攪拌した。この混合液を1時間後、2時間後、4時間後、24時間後にそれぞれ100μlずつサンプリングして、溶離液Aを900μl添加して混合した後に以下に記載の条件にてHPLCに供した。また、化合物Aの標準溶液を調製後に以下に記載の条件にてHPLCに供した。得られたクロマトグラムから化合物Aを定量して、混合液中における化合物Aの残存量を求めた。
[Example 1]
1400 μl of the test substance solution (32.5 mM acetone solution) and 700 μl of the following N- (2-phenylacetyl) cysteine (hereinafter, compound A) solution were mixed and stirred at 25 ° C. This mixed solution was sampled 100 μl after 1 hour, 2 hours, 4 hours, and 24 hours, 900 μl of eluent A was added and mixed, and then subjected to HPLC under the conditions described below. Further, a standard solution of Compound A was prepared and subjected to HPLC under the conditions described below. Compound A was quantified from the obtained chromatogram, and the remaining amount of Compound A in the mixed solution was determined.
[化合物A溶液調製]
化合物A(分子量239.06)を77.7 mg秤量して、アセトン5 mlを加えて溶解し、次に窒素ガス置換した0.2M酢酸アンモニウム水溶液を加えて50 mlに定容した(6.5 mM溶液)。
[Compound A solution preparation]
77.7 mg of compound A (molecular weight 239.06) was weighed and dissolved by adding 5 ml of acetone, and then a volume of 0.2M ammonium acetate aqueous solution substituted with nitrogen gas was added to make up to 50 ml (6.5 mM solution).
[HPLC測定条件]
装置:LC-20AD(島津製作所製)
カラム:CAPCELL-PAK ODS(3.0×150 mm)
カラム温度:40℃
流速:0.4 ml/min.
検出波長:210 nm
溶離液A:蒸留水/アセトニトリル=98/2(0.1%トリフルオロ酢酸)
溶離液B:アセトニトリル/蒸留水=90/10(0.1%トリフルオロ酢酸)
溶出条件:A/B=70/30
注入量:10μl
分析時間:5分間
[HPLC measurement conditions]
Equipment: LC-20AD (manufactured by Shimadzu Corporation)
Column: CAPCELL-PAK ODS (3.0 x 150 mm)
Column temperature: 40 ° C
Flow rate: 0.4 ml / min.
Detection wavelength: 210 nm
Eluent A: distilled water / acetonitrile = 98/2 (0.1% trifluoroacetic acid)
Eluent B: acetonitrile / distilled water = 90/10 (0.1% trifluoroacetic acid)
Elution condition: A / B = 70/30
Injection volume: 10μl
Analysis time: 5 minutes
[比較例1]
化合物Aの代わりにグルタチオン(GSH)を用いて、例1と同様にGSHを定量して、混合液中におけるGSHの残存量を求めた。ただし、GSH 溶液の調製は以下のように行い、測定においてはLC/MS/MSを用いた。測定条件は、以下に示す。
LC条件
装置:LC-20AD(島津製作所製)
カラム:CAPCELL-PAK ODS(3.0×50 mm)
カラム温度:40℃
流速:0.4 ml/min.
溶離液A:蒸留水/アセトニトリル=98/2(0.1%トリフルオロ酢酸)
溶離液B:アセトニトリル/蒸留水=90/10(0.1%トリフルオロ酢酸)
溶出条件:A/B=70/30
[Comparative Example 1]
Using glutathione (GSH) instead of compound A, GSH was quantified in the same manner as in Example 1 to determine the residual amount of GSH in the mixed solution. However, the GSH solution was prepared as follows, and LC / MS / MS was used for the measurement. The measurement conditions are shown below.
LC conditions Equipment: LC-20AD (manufactured by Shimadzu Corporation)
Column: CAPCELL-PAK ODS (3.0 x 50 mm)
Column temperature: 40 ° C
Flow rate: 0.4 ml / min.
Eluent A: distilled water / acetonitrile = 98/2 (0.1% trifluoroacetic acid)
Eluent B: acetonitrile / distilled water = 90/10 (0.1% trifluoroacetic acid)
Elution condition: A / B = 70/30
MS/MS条件
装置:ABI 3200QTRAP(アプライドバイオシステムズ社製)
Ionization mode:negative(-)
Source/Gas
CUR(curtain gas):20(psi)
CAD(collision gas):5
IS(ionspray voltage):-4000(volts)
GAS1(ion source gas1):70
GAS2(ion source gas2):80
Compound
DP((declustering potential):-40(volts)
EP(entrance potential):-4.5(volts)
CE(collision energy):-26(volts)
CXP(collision cell exit potential):0(volts)
検出:m/z 306.1 → m/z 143.1 (transition)
MS / MS conditions Equipment: ABI 3200QTRAP (Applied Biosystems)
Ionization mode: negative (-)
Source / Gas
CUR (curtain gas): 20 (psi)
CAD (collision gas): 5
IS (ionspray voltage): -4000 (volts)
GAS1 (ion source gas1): 70
GAS2 (ion source gas2): 80
Compound
DP ((declustering potential):-40 (volts)
EP (entrance potential):-4.5 (volts)
CE (collision energy): -26 (volts)
CXP (collision cell exit potential): 0 (volts)
Detection: m / z 306.1 → m / z 143.1 (transition)
[GSH溶液調製]
グルタチオン(GSH 分子量307.32)を100 mg秤量して、窒素ガス置換した0.2M酢酸アンモニウム水溶液を加えて50 mlに定容した(6.5 mM溶液)。
[GSH solution preparation]
100 mg of glutathione (GSH molecular weight 307.32) was weighed, and 0.2 M ammonium acetate aqueous solution substituted with nitrogen gas was added to adjust the volume to 50 ml (6.5 mM solution).
[測定結果]
被験化合物として以下の6化合物を使用して、上記例1および比較例1に従った皮膚感作性の測定を行った。その結果を表1及び図1に示す。
(被験化合物)
BQ:p−ベンゾキノン(皮膚感作性:極めて強度)(分子量108.10)
DNCB:1−クロロ−2,4−ジニトロベンゼン(皮膚感作性:極めて強度)(分子量202.55)
CA: 桂皮アルデヒド(皮膚感作性:中程度)(分子量132.16)
CTR: シトラール(皮膚感作性:中程度)(分子量:152.26)
EDM: エチレングリコールジメタクリレート(皮膚感作性:弱)(分子量198.24)
HPM: 2−ヒドロキシプロピルメタクリレート(皮膚感作性:なし)(分子量144.19)
[Measurement result]
Using the following 6 compounds as test compounds, skin sensitization was measured according to Example 1 and Comparative Example 1. The results are shown in Table 1 and FIG.
(Test compound)
BQ: p-benzoquinone (skin sensitization: extremely strong) (molecular weight 108.10)
DNCB: 1-chloro-2,4-dinitrobenzene (skin sensitization: extremely strong) (molecular weight 202.55)
CA: cinnamaldehyde (skin sensitization: moderate) (molecular weight 132.16)
CTR: Citral (Skin sensitization: Moderate) (Molecular weight: 152.26)
EDM: Ethylene glycol dimethacrylate (skin sensitization: weak) (molecular weight 198.24)
HPM: 2-hydroxypropyl methacrylate (skin sensitization: none) (molecular weight 144.19)
上記結果において、皮膚感作性は各時間におけるGSH又は化合物Aの残存率(%)が低いほど高いと判断される。
上記の結果、GSHを用いた例と化合物Aを用いた例で得られる結果は、類似していることがわかる。
In the above results, the skin sensitization is judged to be higher as the residual rate (%) of GSH or Compound A at each time is lower.
From the above results, it can be seen that the results obtained in the example using GSH and the example using Compound A are similar.
[例2]
上記と同様に調製した被検物質の溶液と、化合物Aの溶液を用い、例1と同様に化合物Aを定量して、混合液中における化合物Aの残存量を求めた(「被検物質/化合物A」=10)。ただし、測定方法は例1と同様とした。
反応した化合物Aの自然対数値を縦軸、反応時間を横軸にしてグラフ作成し、作成したグラフの傾きから反応速度定数を算出した。
さらに、被検物質の溶液の濃度を65mM、130mMに変更した例(それぞれ「被検物質/化合物A」が20および40)で同様に、反応速度定数を算出した。さらに、反応速度定数を縦軸、化合物Aと感作性物質との比率を横軸にしてグラフを作成した。結果を図2に示す。
[Example 2]
Using a solution of the test substance prepared in the same manner as above and a solution of compound A, compound A was quantified in the same manner as in Example 1 to determine the remaining amount of compound A in the mixture (“test substance / Compound A "= 10). However, the measurement method was the same as in Example 1.
A graph was created with the natural logarithm of the reacted compound A as the vertical axis and the reaction time as the horizontal axis, and the reaction rate constant was calculated from the slope of the created graph.
Furthermore, reaction rate constants were calculated in the same manner in examples in which the concentration of the test substance solution was changed to 65 mM and 130 mM (“test substance / compound A” was 20 and 40, respectively). Further, a graph was prepared with the reaction rate constant on the vertical axis and the ratio of compound A and sensitizer on the horizontal axis. The results are shown in FIG.
[例3]
化合物Aの代わりにN−[2−(ナフタレン−1−イル)アセチル]システイン(以下、化合物B)を用い、例2と同様の手順で測定および計算を行いグラフを作成した。なお、化合物Bの測定方法は、化合物Aの測定条件に準じたが、測定波長は281nmとした。結果を図3に示す。
[Example 3]
Using N- [2- (naphthalen-1-yl) acetyl] cysteine (hereinafter referred to as Compound B) instead of Compound A, measurement and calculation were performed in the same procedure as in Example 2 to prepare a graph. The measurement method for Compound B was in accordance with the measurement conditions for Compound A, but the measurement wavelength was 281 nm. The results are shown in FIG.
[比較例2]
化合物Aの代わりにGSHを用い、例2と同様の手順で測定および計算を行いグラフを作成した。ただし、測定においては波長210 nmでの検出の代わりにLC/MS/MSを用いた。LC/MS/MSの条件は比較例1と同様である。結果を図4に示す。
[Comparative Example 2]
Using GSH instead of Compound A, measurement and calculation were performed in the same procedure as in Example 2 to prepare a graph. However, in the measurement, LC / MS / MS was used instead of detection at a wavelength of 210 nm. LC / MS / MS conditions are the same as in Comparative Example 1. The results are shown in FIG.
Claims (11)
(2)前記反応による前記有機化合物の量の低下を光学的測定により検出することを含む
皮膚感作性の測定方法。 (1) reacting an organic compound having a structure having at least one thiol group and having absorption in the ultraviolet, visible light, or near infrared region with a test substance; and (2) the organic by the reaction. A method for measuring skin sensitization comprising detecting a decrease in the amount of a compound by optical measurement.
(12)前記反応により生じた化合物を光学的測定により検出することを含む
皮膚感作性の測定方法。 (1) reacting a test substance with an organic compound having a structure having one or more thiol groups and having absorption in the ultraviolet, visible light, or near infrared region; and
(12) A method for measuring skin sensitization comprising detecting a compound produced by the reaction by optical measurement.
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JP2014037995A (en) * | 2012-08-13 | 2014-02-27 | Fujifilm Corp | Skin sensitization measuring reagent |
WO2020045621A1 (en) | 2018-08-31 | 2020-03-05 | 富士フイルム株式会社 | Skin sensitization measuring method and skin sensitization measuring reagent |
WO2020129509A1 (en) | 2018-12-21 | 2020-06-25 | 富士フイルム株式会社 | Phototoxicity or photoallergy measurement method and reagent for use in said measurement method |
JPWO2021060477A1 (en) * | 2019-09-26 | 2021-04-01 | ||
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JP2003014761A (en) * | 2001-06-29 | 2003-01-15 | Sumitomo Chem Co Ltd | Skin sensitivity test method |
JP2007183208A (en) * | 2006-01-10 | 2007-07-19 | Shiseido Co Ltd | In vitro evaluation method of sensitive material |
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JP2007183208A (en) * | 2006-01-10 | 2007-07-19 | Shiseido Co Ltd | In vitro evaluation method of sensitive material |
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JP2014037995A (en) * | 2012-08-13 | 2014-02-27 | Fujifilm Corp | Skin sensitization measuring reagent |
WO2020045621A1 (en) | 2018-08-31 | 2020-03-05 | 富士フイルム株式会社 | Skin sensitization measuring method and skin sensitization measuring reagent |
JPWO2020045621A1 (en) * | 2018-08-31 | 2021-09-16 | 富士フイルム株式会社 | Skin sensitization measurement method and skin sensitization measurement reagent |
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JP7273173B2 (en) | 2019-09-26 | 2023-05-12 | 富士フイルム株式会社 | Reagent for measuring skin sensitization, method for measuring skin sensitization, and compound |
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