JP5547947B2 - Screening method for sensitizing substances - Google Patents
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Description
本発明は感作性物質のスクリーニング方法に関する。更に詳しくは本発明は、評価用のペプチドと被験物質との結合性を測定することにより当該被験物質の感作性を評価する感作性物質のスクリーニング方法に関する。 The present invention relates to a screening method for sensitizing substances. More specifically, the present invention relates to a method for screening a sensitizing substance, wherein the sensitizing property of the test substance is evaluated by measuring the binding property between the peptide for evaluation and the test substance.
医薬、農薬、化粧品等の商品の成分として、感作性物質、即ち生体にアレルギー反応等を誘発する化学物質が含有されないようにするため、それらの商品の成分について感作性の評価が求められる。 In order to prevent the inclusion of sensitizing substances, that is, chemical substances that induce allergic reactions in the living body, as components of products such as pharmaceuticals, agricultural chemicals and cosmetics, evaluation of sensitization is required for the components of those products. .
感作性を評価する有効な方法として、被験物質を実験動物に適用する in vivoの評価方法が考えられる。具体的に例示すれば、被験物質を実験動物の皮膚に適用し、適用部位の皮膚反応を観察するMaximization試験やBuehler試験、あるいは被験物質を実験動物の皮膚に適用してリンパ球の増殖を調べるLocal Lymph Node Assay等が挙げられる。しかし、これらの方法においては、実験動物の飼育・管理の手間を要する点に加え、実験動物への被験物質の適用作業が面倒であり、更に皮膚観察あるいは血液検査等の煩雑な作業も必要とし、多量の被験物質を準備する必要もある、等の種々の難点がある。 As an effective method for evaluating sensitization, an in vivo evaluation method in which a test substance is applied to an experimental animal can be considered. Specifically, the test substance is applied to the skin of a laboratory animal, and the Maximization test or Buehler test for observing the skin reaction at the application site, or the test substance is applied to the skin of a laboratory animal to examine lymphocyte proliferation. For example, Local Lymph Node Assay. However, in these methods, in addition to the time and labor required for breeding and managing laboratory animals, the work of applying the test substance to the laboratory animals is troublesome, and further complicated work such as skin observation or blood test is required. There are various difficulties such as the need to prepare a large amount of test substance.
このような点から、最終的に絞り込まれた被験物質については動物実験によるスクリーニングを行うとしても、商品開発の初期段階のように多数の被験物質を扱う場合には、少量の被験物質を用いて簡易・迅速に評価できるin vitroのスクリーニング方法が好ましい。もちろん、このような簡便法であっても、十分な信頼性が要求される。 From these points, even if screening of test substances that have been finally narrowed down is carried out by animal experiments, a small amount of test substances should be used when handling a large number of test substances as in the initial stage of product development. In vitro screening methods that allow simple and rapid evaluation are preferred. Of course, even such a simple method requires sufficient reliability.
代表的な in vitro スクリーニング方法として、感作性評価用のタンパク質又は低分子量ペプチドと被験物質との結合反応を測定することにより、被験物質の感作性をスクリーニングする方法が挙げられる。 As a typical in vitro screening method, there is a method of screening the sensitization property of a test substance by measuring a binding reaction between a test substance and a protein or low molecular weight peptide for sensitization evaluation.
ところで、特許文献１及び特許文献２に記載の方法では、いずれの実施例でも、評価用のタンパク質又は低分子量ペプチドを水又は水性緩衝液に溶解させ、一方では被験物質をメタノール、エタノール、アセトニトリル、アセトンといった有機溶剤に溶解させ、これらの溶解液を混和させた混和物中で両者を反応させている。そしてこれらの混和物における水又は水性緩衝液と有機溶剤との混和比率は、水又は水性緩衝液が過剰ないし大過剰である。 By the way, in the methods described in Patent Document 1 and Patent Document 2, in any of the Examples, the protein for evaluation or the low molecular weight peptide is dissolved in water or an aqueous buffer, while the test substance is methanol, ethanol, acetonitrile, Both are made to react in the mixture which melt | dissolved in organic solvents, such as acetone, and mixed these solution. The mixing ratio of water or aqueous buffer solution to organic solvent in these admixtures is excessive or large excess of water or aqueous buffer solution.
これに対して、例えば化粧品原料においては、親水性の物質から親油性の物質まで数多くの種類の成分が配合される。感作性物質としては、親水性及び親油性のものが知られているので、当然ながら親油性の成分も被験物質となり得る。 On the other hand, for example, in cosmetic raw materials, many types of ingredients are blended from hydrophilic substances to lipophilic substances. As sensitizing substances, hydrophilic and lipophilic substances are known, and naturally, lipophilic components can also serve as test substances.
しかし、特許文献１及び特許文献２に記載の方法のように、水又は水性緩衝液が過剰ないし大過剰である混和液中では、親油性の被験物質の感作性を正しく評価できない恐れがある。本願発明者が実験的に確認したところ、後述の実施例欄における比較例として示すように、感作性が陽性であることが既知の親油性被験物質について、水又は水性緩衝液が過剰ないし大過剰である混和液中で反応させたところ、既知の情報とは一致しない評価結果が得られている。 However, as in the methods described in Patent Document 1 and Patent Document 2, there is a possibility that the sensitization property of a lipophilic test substance cannot be correctly evaluated in a mixed solution in which water or an aqueous buffer is excessive or large excess. . As a result of experimental confirmation by the inventors of the present application, as shown as a comparative example in the Examples section described below, an excess or large amount of water or aqueous buffer solution is used for a lipophilic test substance known to be positive for sensitization. When the reaction is carried out in an excessive mixture, evaluation results that do not match the known information are obtained.
そこで本発明は、評価用ペプチドと被験物質との結合性を測定して被験物質の感作性を評価する感作性物質のスクリーニング方法において、被験物質の親水性／親油性の別に関わらず正確な評価が得られるようにすることを、解決すべき技術的課題とする。 Therefore, the present invention provides a screening method for a sensitizing substance that evaluates the sensitization property of a test substance by measuring the binding property between the evaluation peptide and the test substance, regardless of whether the test substance is hydrophilic or lipophilic. The technical problem to be solved is to ensure that a good evaluation is obtained.
本願発明者は、上記課題の解決手段を追求する過程で、次の第１〜第２の点に着眼するに至り、これらの着眼点に基づく試行錯誤の結果、本発明を完成した。 The inventor of the present application came to focus on the following first and second points in the process of pursuing the means for solving the above problems, and completed the present invention as a result of trial and error based on these points of focus.
第１の着眼点：従来のin vitroスクリーニング法では、有機溶剤は単に被験物質を予備的に溶解させる目的で用いているに過ぎず、評価用のタンパク質又はペプチドと被験物質との反応は実質的に水性溶媒中で行っている。 First point: In the conventional in vitro screening method, the organic solvent is merely used for the purpose of preliminarily dissolving the test substance, and the reaction between the test protein or peptide and the test substance is substantial. In an aqueous solvent.
第２の着眼点：評価用ペプチドと被験物質との反応を従来のように実質的な水性溶媒中で行う場合と比較して、実質的な有機溶媒中で行う場合、ペプチドと被験物質の両成分の反応挙動が変わる可能性がある。又、そのことが親水性の被験物質及び親油性の被験物質の感作性の評価に意外な好影響をもたらすかも知れない。 Second point of focus: When the reaction between the peptide for evaluation and the test substance is carried out in a substantial organic solvent as compared with the conventional case, both the peptide and the test substance are reacted. The reaction behavior of the components may change. It may also have a surprisingly positive effect on the assessment of sensitization of hydrophilic and lipophilic test substances.
（第１発明）
上記課題を解決するための本願第１発明の構成は、評価用ペプチドと被験物質とを反応させる反応ステップと、前記反応ステップにおける評価用ペプチドと被験物質との結合性を測定して被験物質の感作性を評価する評価ステップとを含む感作性物質のスクリーニング方法であって、前記反応ステップを有機溶剤を５０質量％以上含有する反応溶媒中で行う、感作性物質のスクリーニング方法である。
(First invention)
The configuration of the first invention of the present application for solving the above-described problem is a reaction step of reacting an evaluation peptide with a test substance, and measuring the binding between the evaluation peptide and the test substance in the reaction step, A screening method for a sensitizing substance comprising an evaluation step for evaluating sensitization, wherein the reaction step is performed in a reaction solvent containing 50% by mass or more of an organic solvent. .
上記の第１発明において「評価用ペプチド」とは、感作性物質と特異的に結合する性質を持つため、被験物質が感作性物質であるか否かを判定する指標として利用できるペプチドをいう。従って、評価用ペプチドと結合し易い被験物質ほど、感作性が高いと判定される。又、「評価用ペプチド」というときの「ペプチド」とは、アミノ酸の重合度が３〜１０のオリゴペプチドをいう。 In the first invention, the “evaluation peptide” is a peptide that can be used as an index for determining whether or not a test substance is a sensitizer because it has a property of specifically binding to a sensitizer. Say. Therefore, it is determined that the test substance that easily binds to the evaluation peptide has higher sensitization. In addition, “peptide” when referred to as “evaluation peptide” refers to an oligopeptide having an amino acid polymerization degree of 3 to 10.
（第２発明）
上記課題を解決するための本願第２発明の構成は、前記第１発明に係る反応ステップで用いる有機溶剤がジメチルスルホキシド、ジメチルホルムアミド及び炭素数１〜４の一価アルコールから選ばれる１種以上である、感作性物質のスクリーニング方法である。
(Second invention)
The structure of the second invention of the present application for solving the above problem is that the organic solvent used in the reaction step according to the first invention is one or more selected from dimethyl sulfoxide, dimethylformamide and a monohydric alcohol having 1 to 4 carbon atoms. This is a screening method for sensitizing substances.
（第３発明）
上記課題を解決するための本願第３発明の構成は、前記第１発明又は第２発明に係る反応ステップの反応溶媒が有機溶剤を６０質量％以上含有するものである、感作性物質のスクリーニング方法である。
(Third invention)
The configuration of the third invention of the present application for solving the above-described problem is that the reaction solvent of the reaction step according to the first invention or the second invention contains an organic solvent in an amount of 60% by mass or more. Is the method.
（第４発明）
上記課題を解決するための本願第４発明の構成は、前記第１発明〜第３発明のいずれかに係る評価用ペプチドが下記の（１）〜（３）のいずれかに示すものである、感作性物質のスクリーニング方法である。
(Fourth invention)
The structure of the fourth invention of the present application for solving the above-mentioned problem is that the peptide for evaluation according to any one of the first invention to the third invention is one of the following (1) to (3): This is a screening method for sensitizing substances.
（１）H-Phe-Thr-Leu-Cys-Phe-Arg-NH2
（２）H-Phe-Thr-Leu-His-Phe-Arg-NH2
（３）H-Phe-Thr-Leu-Lys-Phe-Arg-NH2
（第５発明）
上記課題を解決するための本願第５発明の構成は、前記第１発明〜第４発明のいずれかに係る被験物質が親油性物質である、感作性物質のスクリーニング方法である。
(1) H-Phe-Thr-Leu-Cys-Phe-Arg-NH 2
(2) H-Phe-Thr-Leu-His-Phe-Arg-NH 2
(3) H-Phe-Thr-Leu-Lys-Phe-Arg-NH 2
(Fifth invention)
The configuration of the fifth invention of the present application for solving the above problem is a method for screening a sensitizing substance, wherein the test substance according to any one of the first to fourth inventions is a lipophilic substance.
上記の第５発明において「親油性物質」とは、水に溶けにくい物質を指し、詳しくはオクタノール／水分配係数（ｌｏｇＫＯ／Ｗ）が１以上、好ましくは２以上の物質をいう。 In the above fifth invention, the “lipophilic substance” refers to a substance that is hardly soluble in water, and specifically refers to a substance having an octanol / water partition coefficient (log K 2 O / W 2 ) of 1 or more, preferably 2 or more.
本願発明者は、感作性物質と特異的に結合する性質が知られており、あるいはそのような性質を自ら見出した低分子量ペプチドを評価用に用いるという前提のもとに、評価用ペプチドと被験物質の反応挙動が反応溶媒（結合反応の環境）によって影響される可能性を考慮しつつ、反応溶媒を種々に変更して検討した結果、第１発明を完成するに至った。 The inventor of the present application is known to have a property of specifically binding to a sensitizing substance, or on the premise that a low molecular weight peptide that has found such a property is used for evaluation, Considering the possibility that the reaction behavior of the test substance is affected by the reaction solvent (binding reaction environment), the first invention was completed as a result of various changes in the reaction solvent.
第１発明のように、有機溶剤を５０質量％以上含有する反応溶媒中で評価用ペプチドと被験物質を反応させると、被験物質の親水性／親油性の別あるいはそれらの程度に関わらず、感作性の高い被験物質ほど評価用ペプチドと結合し易いことが分かった。従って、被験物質が親水性の物質であっても、親油性の物質であっても、その感作性の評価を通じて、感作性物質を十分な信頼性のもとにスクリーニングすることができる。しかもこの方法は、少量の被験物質を用いて簡易・迅速にスクリーニングすることができるin vitro スクリーニング方法である。 As in the first invention, when the evaluation peptide and the test substance are reacted in a reaction solvent containing 50% by mass or more of an organic solvent, the sensitivity is increased regardless of the hydrophilicity / lipophilicity of the test substance or their degree. It was found that the test substance with higher productivity was more likely to bind to the evaluation peptide. Therefore, regardless of whether the test substance is a hydrophilic substance or a lipophilic substance, the sensitizing substance can be screened with sufficient reliability through evaluation of the sensitization. Moreover, this method is an in vitro screening method that allows simple and rapid screening using a small amount of test substance.
反応溶媒が水や緩衝液（水溶液）である場合、あるいは反応溶媒が有機溶剤を含有してもその含有量が５０質量％未満である場合は、被験物質、特に親油性の被験物質の感作性を正しく評価できない場合がある。 When the reaction solvent is water or a buffer solution (aqueous solution), or when the reaction solvent contains an organic solvent and its content is less than 50% by mass, sensitization of the test substance, particularly a lipophilic test substance Sexuality may not be evaluated correctly.
第２発明によれば、本発明の反応ステップで用いるに特に適した有機溶剤が提供される。又、反応ステップにおける評価用ペプチドと被験物質の反応溶媒としては、第３発明に規定するように、有機溶剤を６０質量％以上含有するものが特に好ましい。 According to the second invention, an organic solvent particularly suitable for use in the reaction step of the present invention is provided. Further, as the reaction solvent for the peptide for evaluation and the test substance in the reaction step, those containing 60% by mass or more of an organic solvent are particularly preferable as defined in the third invention.
評価用ペプチドは、感作性物質と特異的に結合する性質を持ち、重合度が３〜１０の範囲内の低分子量ペプチドである限りにおいて限定されないが、第４発明の（１）〜（３）に規定するペプチドが特に好ましい。これらのペプチドの内、（１）、（２）に規定するものは評価用ペプチドとして公知であるが、（３）に規定するものは、評価用ペプチドとして利用できることを本願発明者が新たに見出したペプチドである。 The evaluation peptide is not limited as long as it is a low molecular weight peptide having a property of specifically binding to a sensitizing substance and having a polymerization degree in the range of 3 to 10, but the (1) to (3) of the fourth invention. The peptide defined in) is particularly preferred. Among these peptides, those defined in (1) and (2) are known as evaluation peptides, but the present inventor newly found that those defined in (3) can be used as evaluation peptides. Peptide.
本発明に係る感作性物質のスクリーニング方法は、被験物質が親水性物質であっても有効に適用できるが、第５発明に規定するように、被験物質が親油性物質である場合に特に好ましく適用される。 The screening method for a sensitizing substance according to the present invention can be effectively applied even if the test substance is a hydrophilic substance, but is particularly preferable when the test substance is a lipophilic substance as defined in the fifth invention. Applied.
次に、本発明を実施するための形態を、その最良の形態を含めて説明する。 Next, modes for carrying out the present invention will be described including the best mode.
〔感作性物質のスクリーニング方法〕
本発明に係る感作性物質のスクリーニング方法は、（ａ）評価用ペプチドと被験物質とを反応させる反応ステップと、（ｂ）前記反応ステップにおける評価用ペプチドと被験物質との結合性を測定して被験物質の感作性を評価する評価ステップとを含む、一種のin vitroスクリーニング方法である。
[Screening method for sensitizing substances]
The screening method for a sensitizing substance according to the present invention comprises (a) a reaction step in which an evaluation peptide and a test substance are reacted, and (b) a binding property between the evaluation peptide and the test substance in the reaction step. And an evaluation step for evaluating the sensitization of the test substance.
〔反応ステップ〕
本発明の最大の特徴は、評価用ペプチドと被験物質との反応を、有機溶剤を５０質量％以上、より好ましくは６０質量％以上含有する反応溶媒中で行う点にある。有機溶剤が８０質量％以上ないし１００質量％を占める反応溶媒も特に好ましく用いられる。
[Reaction step]
The greatest feature of the present invention is that the reaction between the evaluation peptide and the test substance is carried out in a reaction solvent containing an organic solvent in an amount of 50% by mass or more, more preferably 60% by mass or more. A reaction solvent in which the organic solvent occupies 80% by mass to 100% by mass is particularly preferably used.
反応ステップにおいて反応溶媒に含有させる有機溶剤の種類は限定されないが、特に好ましくはジメチルスルホキシド（ＤＭＳＯ）、ジメチルホルムアミド（ＤＭＦ）及び炭素数１〜４の一価アルコールが例示され、炭素数１〜４の一価アルコールとしては、メタノール、エタノール、n-プロパノール、イソプロパノール、n-ブタノール、イソブタノール、sec-ブタノール、tert-ブタノールが例示される。これらの有機溶剤の内の２種以上の併用、あるいはこれらの有機溶剤と他種の有機溶剤との２種以上の併用も好ましく例示される。 The kind of the organic solvent to be contained in the reaction solvent in the reaction step is not limited, but particularly preferable examples include dimethyl sulfoxide (DMSO), dimethylformamide (DMF) and monohydric alcohol having 1 to 4 carbon atoms, and 1 to 4 carbon atoms. Examples of monohydric alcohols include methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, and tert-butanol. A combination of two or more of these organic solvents or a combination of two or more of these organic solvents and another organic solvent is also preferred.
反応溶媒は５０質量％未満、より好ましくは４０質量％未満の水系溶媒を含有することができる。水系溶媒としては水、水性緩衝液が挙げられる。水性緩衝液としてリン酸ナトリウム、リン酸カリウム等のリン酸アルカリ金属塩等の無機酸塩や、酢酸ナトリウム、酢酸カリウム、酢酸アンモニウム等の酢酸塩等の有機酸塩等を含む水性緩衝溶液が例示される。反応溶媒はその他にも例えば少量の酢酸等を含有することができる。 The reaction solvent can contain less than 50% by weight, more preferably less than 40% by weight aqueous solvent. Examples of the aqueous solvent include water and an aqueous buffer solution. Examples of aqueous buffer solutions include aqueous acid buffer solutions containing inorganic acid salts such as alkali metal phosphates such as sodium phosphate and potassium phosphate, and organic acid salts such as sodium acetate, potassium acetate and ammonium acetate. Is done. In addition, the reaction solvent can contain a small amount of acetic acid, for example.
前記した特許文献１、２に記載の方法では、評価用のタンパク質又は低分子量ペプチドを水性溶媒に溶解させる一方で被験物質を有機溶剤に溶解させ、これらの溶解液を混和させて反応ステップを行っているが、本発明においては、このような手順に従っても良いし、従わなくても良い。 In the methods described in Patent Documents 1 and 2 described above, the test protein or low molecular weight peptide is dissolved in an aqueous solvent while the test substance is dissolved in an organic solvent, and these dissolved solutions are mixed to perform the reaction step. However, in the present invention, such a procedure may be followed or may not be followed.
即ち、第１の手順例として、被験物質の有機溶媒溶液（第１溶液）と評価用ペプチドの水性溶媒溶液（第２溶液）を別途に準備し、両者の溶液を、有機溶媒が全溶媒（有機溶媒及び水性溶媒）中の５０質量％以上を占めることとなる比率で混和させて、被験物質と評価用ペプチドを反応させることができる。第２の手順例として、上記の第１の手順例において第１溶液と第２溶液の溶媒を逆にし、被験物質の水性溶媒溶液と評価用ペプチドの有機溶媒溶液を別途に準備してから、両者の溶液を混和させることができる。第３の手順例として、被験物質と評価用ペプチドをそれぞれ同一組成の溶解液に溶解させた上で、これらの溶解液を混合して被験物質と評価用ペプチドを反応させることができる。第４の手順例として、被験物質と評価用ペプチドとの共通の溶解液を予め準備し、この溶解液に被験物質と評価用ペプチドを順不同で溶解させて反応させることができる。上記した「同一組成の溶解液」あるいは「共通の溶解液」は、有機溶剤が５０質量％以上を占める限りにおいて、例えば１種又は２種以上の有機溶剤のみからなる溶解液でも良いし、１種又は２種以上の有機溶剤と、水又は水性緩衝液との混和液であっても良い。本発明の反応ステップでいう「反応溶媒」とは、上記の第１、第２の手順例においては第１溶液と第２溶液とを混和した後の溶媒を言い、第３の手順例においては上記した混合後の「同一組成の溶解液」を言い、第４の手順例においては「共通の溶解液」を言う。 That is, as a first procedure example, an organic solvent solution (first solution) of a test substance and an aqueous solvent solution (second solution) of a peptide for evaluation are separately prepared. The test substance and the peptide for evaluation can be reacted by mixing at a ratio of 50% by mass or more in the organic solvent and the aqueous solvent. As a second procedure example, in the above first procedure example, the solvents of the first solution and the second solution are reversed, and an aqueous solvent solution of the test substance and an organic solvent solution of the peptide for evaluation are separately prepared, Both solutions can be mixed. As a third procedure example, the test substance and the evaluation peptide can be dissolved in a solution having the same composition, and then the test substance and the evaluation peptide can be reacted by mixing these solutions. As a fourth procedure example, a common solution of the test substance and the evaluation peptide can be prepared in advance, and the test substance and the evaluation peptide can be dissolved in this solution in any order and reacted. The above-mentioned “solution having the same composition” or “common solution” may be, for example, a solution consisting of only one or two or more organic solvents as long as the organic solvent occupies 50% by mass or more. A mixed solution of seeds or two or more organic solvents and water or an aqueous buffer may be used. The “reaction solvent” as used in the reaction step of the present invention refers to the solvent after the first solution and the second solution are mixed in the first and second procedure examples, and in the third procedure example. The above-mentioned “solution having the same composition” after mixing is referred to as “common solution” in the fourth procedure example.
反応ステップにおける評価用ペプチドと被験物質との反応条件は限定されないが、例えば、４℃から６０℃程度の温度範囲、好ましくは２５〜４５℃程度に保温しながら、１０分間〜約２日間程度、好ましくは２時間〜２日程度、より好ましくは２４時間程度かけて行う。 The reaction conditions of the peptide for evaluation and the test substance in the reaction step are not limited. For example, while maintaining a temperature range of about 4 ° C. to 60 ° C., preferably about 25 to 45 ° C., about 10 minutes to about 2 days, Preferably it is performed for about 2 hours to 2 days, more preferably over about 24 hours.
〔評価用ペプチドと被験物質〕
本発明で用いる評価用ペプチドは、感作性物質と特異的に結合する性質を持つため、被験物質が感作性物質であるか否かを判定する指標として利用できるペプチドである。このような評価用ペプチドとして、具体的にはアミノ酸の重合度が３〜１０のオリゴペプチドが挙げられ、更に好ましくは、限定はされないが、下記の（１）〜（３）に示すものが挙げられる。
[Evaluation peptide and test substance]
The peptide for evaluation used in the present invention is a peptide that can be used as an index for determining whether or not a test substance is a sensitizing substance because it has a property of specifically binding to a sensitizing substance. Specific examples of such peptides for evaluation include oligopeptides having a degree of polymerization of amino acids of 3 to 10, and more preferably, the peptides shown in the following (1) to (3) are not limited. It is done.
（１）H-Phe-Thr-Leu-Cys-Phe-Arg-NH2（以下「ペプチドＣ」と称する）
（２）H-Phe-Thr-Leu-His-Phe-Arg-NH2（以下「ペプチドＨ」と称する）
（３）H-Phe-Thr-Leu-Lys-Phe-Arg-NH2（以下「ペプチドＫ」と称する）
本発明においては、被験物質として、親水性の物質も親油性の物質もスクリーニングの対象とすることができる。とりわけ親油性の物質がスクリーニングに適する。反応ステップにおける反応溶媒中の評価用ペプチド、被験物質の濃度は限定されないが、評価用ペプチドについては、例えば０．０１μＭ〜１Ｍ程度、通常は１０μＭ〜１００ｍＭ程度の濃度となるように溶解していることが好ましい。被験物質については、例えば０．０１μＭ〜１Ｍ程度、通常は１ｍＭ〜５００ｍＭ程度の濃度となるよう溶解していることが好ましい。
(1) H-Phe-Thr-Leu-Cys-Phe-Arg-NH 2 (hereinafter referred to as “Peptide C”)
(2) H-Phe-Thr-Leu-His-Phe-Arg-NH 2 (hereinafter referred to as “Peptide H”)
(3) H-Phe-Thr-Leu-Lys-Phe-Arg-NH 2 (hereinafter referred to as “Peptide K”)
In the present invention, both a hydrophilic substance and a lipophilic substance can be screened as test substances. In particular, lipophilic substances are suitable for screening. The concentration of the peptide for evaluation and the test substance in the reaction solvent in the reaction step is not limited, but the peptide for evaluation is dissolved to have a concentration of, for example, about 0.01 μM to 1 M, usually about 10 μM to 100 mM. It is preferable. About a test substance, it is preferable to melt | dissolve, for example so that it may become a density | concentration of about 0.01 micromol-1M, normally about 1 mM-500 mM.
反応溶媒中での被験物質と評価用ペプチドの混合比は限定されないが、反応を確実に進行させる必要があり、被験物質：評価用ペプチドのモル比は１：１〜１０００：１程度、特に１０：１〜２００：１程度とすることが好ましい。 The mixing ratio of the test substance and the evaluation peptide in the reaction solvent is not limited, but it is necessary to allow the reaction to proceed reliably. The molar ratio of the test substance to the evaluation peptide is about 1: 1 to 1000: 1, particularly 10 : About 1 to 200: 1 is preferable.
〔評価ステップ〕
評価ステップでは、前記反応ステップにおける評価用ペプチドと被験物質との結合性を測定して、被験物質の感作性を評価する。この目的を十分な信頼性のもとに達成できる限りにおいて評価ステップの具体的手法は限定されないが、以下の代表的手法を例示できる。
[Evaluation step]
In the evaluation step, the binding property between the evaluation peptide and the test substance in the reaction step is measured to evaluate the sensitization property of the test substance. The specific method of the evaluation step is not limited as long as this object can be achieved with sufficient reliability, but the following representative methods can be exemplified.
第１の手法は、特許文献１、２に記載の方法に準ずるもので、評価用ペプチドと被験物質を混合・反応させた反応液をＨＰＬＣ（高速液体クロマトグラフィー）等で分析し、混合前の評価用ペプチド溶液及び被験物質溶液からは検出されず反応液からのみ検出される成分（評価用ペプチドと被験物質の結合物）のピークを調べて両者の結合性を調べる方法であって、特定のピークの有無により判定をするものである。 The first method is based on the method described in Patent Documents 1 and 2, and the reaction solution obtained by mixing and reacting the evaluation peptide and the test substance is analyzed by HPLC (high performance liquid chromatography) or the like, and before mixing. A method of investigating the peak of a component (a conjugate of an evaluation peptide and a test substance) that is not detected from an evaluation peptide solution and a test substance solution but only detected from a reaction solution, Judgment is based on the presence or absence of a peak.
第２の、特に好ましい手法は、反応ステップにおいて評価用ペプチドに対して過剰量の被験物質を反応させることを前提として、評価ステップにおいてはＨＰＬＣ等を利用して評価用ペプチドの残存量を測定し、減少したペプチドは被験物質との結合により消費されたものと見做して、評価用ペプチドと被験物質との結合率を求める、という方法であって、例えばG. Frank Gerberick et al., Toxicological Sciences 81,
332-343 (2004)に記載された方法が例示される。
A second and particularly preferable method is based on the premise that an excessive amount of a test substance is reacted with the evaluation peptide in the reaction step, and the residual amount of the evaluation peptide is measured using HPLC or the like in the evaluation step. In this method, it is assumed that the reduced peptide is consumed by the binding to the test substance, and the binding rate between the evaluation peptide and the test substance is obtained, for example, G. Frank Gerberick et al., Toxicological Sciences 81,
The method described in 332-343 (2004) is illustrated.
以上の第１、第２の手法において、評価用ペプチドと被験物質を反応させた反応液の分析方法としては、上記ＨＰＬＣの他に、ガスクロマトグラフィー（ＧＣ）、薄層クロマトグラフィー（ＴＬＣ）、質量分析（ＭＳ）等をあげることができ、ＨＰＬＣ、ＧＣ、又はＴＬＣのいずれかとＭＳとを組み合わせた分析方法（ＬＣ−ＭＳ，ＧＣ−ＭＳ，ＴＬＣ−ＭＳ）も用いることもできる。この方法によれば、試料に評価用ペプチドと被験物質との複数種類の結合物が含まれていても、それらを個々に分離し、それぞれについて分析することができる。 In the first and second methods described above, as a method for analyzing the reaction solution obtained by reacting the evaluation peptide and the test substance, in addition to the HPLC, gas chromatography (GC), thin layer chromatography (TLC), Mass spectrometry (MS) etc. can be mentioned, The analysis method (LC-MS, GC-MS, TLC-MS) which combined MS with either HPLC, GC, or TLC can also be used. According to this method, even if the sample contains a plurality of types of conjugates of the evaluation peptide and the test substance, they can be individually separated and analyzed.
上記のＨＰＬＣ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。質量分析で利用できるイオン化法として、例えば、マトリクス支援レーザーイオン化（ＭＡＬＤＩ）法、エレクトロスプレーイオン化（ＥＳＩ）法、大気圧化学イオン化（ＡＰＣＩ）法、電子衝撃イオン化（ＥＩ）法、高速原子衝撃イオン化（ＦＡＢ）法等が挙げられる。 Examples of chromatographic techniques that can be used for the above HPLC include reverse phase, normal phase, and ion exchange. Examples of ionization methods that can be used in mass spectrometry include matrix-assisted laser ionization (MALDI), electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), electron impact ionization (EI), and fast atom bombardment ionization ( FAB) method and the like.
以下に本発明の実施例を説明する。本発明の技術的範囲は、以下の実施例によって限定されない。 Examples of the present invention will be described below. The technical scope of the present invention is not limited by the following examples.
〔被験物質〕
本実施例に係る感作性物質の評価試験においては、感作性の有無が既知である５種類の被験物質を試験に供した。これらの被験物質を末尾の表１に列挙した。表１には、それらの被験物質のオクタノール／水分配係数（ｌｏｇＫＯ／Ｗ）と油溶性／親水性の区別、及び感作性の有無（「＋」が感作性有り、「−」が感作性無し）を併せて示した。
[Test substance]
In the evaluation test for sensitizing substances according to this example, five types of test substances whose presence or absence of sensitization were known were used for the test. These test substances are listed in Table 1 at the end. Table 1 shows the distinction between octanol / water partition coefficient (log K 2 O / W 2 ) and oil solubility / hydrophilicity of these test substances, and the presence or absence of sensitization (“+” indicates sensitization, “−” indicates The sensitization property was also shown.
〔評価用ペプチド溶液の調製〕
（ペプチドＫ溶液）
末尾の表２に示す実施例１〜１５及び比較例１〜３の内、「ペプチド」の欄に「Ｋ」と表記した実施例及び比較例については、前記のペプチドＫ（ＳＩＧＭＡ製、分子量８１０．９８）１ｍｇに対して、当該実施例又は比較例における「溶媒」の欄にそれぞれ示す溶媒１ｍＬを加え、必要があれば超音波分散して、ペプチドＫを溶解した。
[Preparation of peptide solution for evaluation]
(Peptide K solution)
Of Examples 1 to 15 and Comparative Examples 1 to 3 shown in Table 2 at the end, for Examples and Comparative Examples in which “K” is written in the “Peptide” column, the peptide K (manufactured by SIGMA, molecular weight 810) is used. .98) To 1 mg, 1 mL of the solvent shown in the “Solvent” column in the Examples or Comparative Examples was added, and if necessary, ultrasonically dispersed to dissolve peptide K.
（ペプチドＣ溶液）
末尾の表２において「ペプチド」の欄に「Ｃ」と表記した実施例については、前記のペプチドＣ（ＳＩＧＭＡ製、分子量７８５．９５）１ｍｇに対して、当該実施例の「溶媒」の欄にそれぞれ示す溶媒１ｍＬを加え、必要があれば超音波分散して、ペプチドＫを溶解した。
(Peptide C solution)
In the example of “C” in the column of “peptide” in Table 2 at the end, 1 mg of the peptide C (manufactured by SIGMA, molecular weight of 785.95) is 1 mg in the “solvent” column of the example. Peptide K was dissolved by adding 1 mL of the indicated solvent and ultrasonically dispersing if necessary.
（ペプチドＨ溶液）
末尾の表２において「ペプチド」の欄に「Ｈ」と表記した実施例については、前記のペプチドＨ(ＳＩＧＭＡ製、分子量８１９．９５)１ｍｇに対して、当該実施例の「溶媒」の欄に示す溶媒１ｍＬを加え、必要があれば超音波分散して、ペプチドＨを溶解した。
(Peptide H solution)
For the examples where “H” is written in the “peptide” column in the last Table 2, with respect to 1 mg of the peptide H (manufactured by SIGMA, molecular weight 819.95), the “solvent” column of the example is used. Peptide H was dissolved by adding 1 mL of the indicated solvent and ultrasonically dispersing if necessary.
（使用した溶媒）
なお、表２において溶媒が混合溶媒である場合にはその混合比率を質量比で表記している。例えば実施例７において溶媒を「ＤＭＳＯ：水＝８０：２０」と表記しているのは、８０質量部のＤＭＳＯと２０質量部の水との混合溶媒であることを示す。又、比較例１、２においては溶媒としてエタノール１６v/v％を使用しているが、これをエタノールの質量％に換算し、「エタノール:水＝１３：８７」と表記している。末尾の表３には、各実施例及び比較例で利用した溶媒のメーカー、グレード及び純度の一覧表を示している。
(Solvent used)
In Table 2, when the solvent is a mixed solvent, the mixing ratio is expressed as a mass ratio. For example, the expression “DMSO: water = 80: 20” in Example 7 indicates that the solvent is a mixed solvent of 80 parts by mass of DMSO and 20 parts by mass of water. In Comparative Examples 1 and 2, ethanol 16 v / v% is used as a solvent, and this is converted to ethanol mass% and expressed as “ethanol: water = 13: 87”. Table 3 at the end shows a list of manufacturers, grades and purities of the solvents used in the examples and comparative examples.
〔被験物質溶液の調製〕
表２の実施例１〜１５及び比較例１〜３について、それぞれの「被験物質」の欄に示す各被験物質を５０ｍｇ程度精秤し、後述の「ペプチドと被験物質との反応」の項で述べるペプチド溶液との混合後の濃度が結果的に表２の「被験物質濃度」の欄に示す濃度となるような濃度で、溶媒によく溶解させ被験物質溶液とした。なお、その際に用いた溶媒は当該実施例又は比較例における「溶媒」の欄にそれぞれ示す溶媒である。従って、各実施例及び比較例において、その評価用ペプチド溶液と被験物質溶液とは同一組成の溶媒を用いている。
[Preparation of test substance solution]
About Examples 1 to 15 and Comparative Examples 1 to 3 in Table 2, about 50 mg of each test substance shown in the column of “test substance” is precisely weighed, and in the “reaction between peptide and test substance” described later. The concentration after mixing with the peptide solution to be described is a concentration that results in the concentration shown in the column “Test substance concentration” in Table 2. In addition, the solvent used in that case is a solvent respectively shown in the column of the "solvent" in the said Example or a comparative example. Therefore, in each of the examples and comparative examples, the evaluation peptide solution and the test substance solution use a solvent having the same composition.
〔ペプチドと被験物質との反応〕
５ｍＬエッペンチューブに、マイクロピペッター等を用い２００μＬの上記ペプチド溶液を添加した。次に３００μＬの上記被験物質溶液を添加して、チューブに蓋をし、４０℃、遮光下で２４時間放置したものを試験溶液とした。なお、上記被験物質溶液の代わりに各例に係る溶媒を用い、これと各例に係る評価用ペプチド溶液とを用いて上記と同様の操作を行い、各実施例及び各比較例についてのコントロールの試験溶液を調製した。試験は同じ被験物質について３回行った。
[Reaction between peptide and test substance]
200 μL of the above peptide solution was added to a 5 mL Eppendorf tube using a micropipette or the like. Next, 300 μL of the above test substance solution was added, the tube was capped, and the test solution was allowed to stand for 24 hours at 40 ° C. under light shielding. In addition, using the solvent according to each example in place of the test substance solution, using this and the peptide solution for evaluation according to each example, the same operation as described above was performed, and control for each example and each comparative example was performed. A test solution was prepared. The test was performed three times on the same test substance.
〔分析〕
反応後の溶液（上記した２４時間放置後の試験溶液及びコントロール）をＨＰＬＣ移動相溶液Ａ：Ｂ＝９５：５にて１０倍希釈し、これを測定用サンプル液としてＨＰＬＣ分析に供した。使用した測定機器及び分析条件は、以下の通りである。
〔analysis〕
The solution after the reaction (the test solution and the control after being left for 24 hours as described above) was diluted 10-fold with HPLC mobile phase solution A: B = 95: 5, and this was subjected to HPLC analysis as a sample solution for measurement. The measuring instruments and analysis conditions used are as follows.
（測定機器）
ＨＰＬＣ：Ｗａｔｅｒｓ ａｌｌｉａｎｃｅ ２６９５
（分析条件）
カラム：Capcell pak C18 ACR １.０ｍｍΦ×３５ｍｍ, 粒径３μｍ（（株）資生堂）
カラム温度：４０℃
流量：２００μＬ／分
移動相：Ａ＝０.１％ギ酸／水 Ｂ＝０.０９５％ギ酸／アセトニトリル
グラジエント：５％（１分）−４０％（３０分）
検出器：Waters 2996 Photodiode Array Detector
検出波長：２２０ｎｍ
〔分析結果の判定〕
各実施例及び各比較例について、コントロール中の評価用ペプチドと試験溶液中の評価用ペプチドを比較して被験物質と結合していない評価用ペプチドの減少率を求めたとき、３回の試験結果の標準偏差が１０未満であれば、それら３回の結果の平均値を減少率として採用した。この減少率が１０％以上であれば感作性が陽性、１０％未満であれば陰性と判定した。
(measuring equipment)
HPLC: Waters alliance 2695
(Analysis conditions)
Column: Capcell pak C18 ACR 1.0 mmΦ × 35 mm, particle size 3 μm (Shiseido Co., Ltd.)
Column temperature: 40 ° C
Flow rate: 200 μL / min Mobile phase: A = 0.1% formic acid / water B = 0.095% formic acid / acetonitrile Gradient: 5% (1 minute) -40% (30 minutes)
Detector: Waters 2996 Photodiode Array Detector
Detection wavelength: 220 nm
[Judgment of analysis results]
For each Example and each Comparative Example, when the rate of decrease in the evaluation peptide not bound to the test substance was determined by comparing the evaluation peptide in the control and the evaluation peptide in the test solution, the results of three tests If the standard deviation was less than 10, the average value of these three results was adopted as the reduction rate. If this reduction rate was 10% or more, the sensitization was positive, and if it was less than 10%, it was judged negative.
こうして得られた各実施例及び各比較例における評価用ペプチドの減少率と陽性／陰性の判定結果を表２の「ペプチド減少率／判定結果」の欄に示す。更に、各被験物質について、in vivo試験の結果として既に知られている感作性と比較し、一致するかどうかを確認した。その結果を表２の「vivo結果との対比」の欄に示す。 The reduction rate of the peptide for evaluation and the positive / negative determination result in each Example and each Comparative Example thus obtained are shown in the column of “peptide reduction rate / determination result” in Table 2. Furthermore, each test substance was compared with the sensitization already known as a result of the in vivo test, and it was confirmed whether or not they matched. The results are shown in the column “Comparison with vivo results” in Table 2.
本発明によって、評価用ペプチドと被験物質との結合性を測定して被験物質の感作性を評価する感作性物質のスクリーニング方法において、被験物質の親水性／親油性の区別に関わらず正確な評価が得られる。 According to the present invention, in the method for screening a sensitizing substance, in which the binding property between the evaluation peptide and the test substance is measured to evaluate the sensitization of the test substance, the test substance is accurate regardless of whether the test substance is hydrophilic or lipophilic. Can be evaluated.
Claims (5)
前記反応ステップを有機溶剤を５０質量％以上含有する反応溶媒中で行うことを特徴とする感作性物質のスクリーニング方法。 A sensitizing substance comprising: a reaction step for reacting an evaluation peptide with a test substance; and an evaluation step for measuring the sensitization property of the test substance by measuring the binding property between the evaluation peptide and the test substance in the reaction step. Screening method,
A screening method for a sensitizing substance, wherein the reaction step is performed in a reaction solvent containing 50% by mass or more of an organic solvent.
（１）H-Phe-Thr-Leu-Cys-Phe-Arg-NH2
（２）H-Phe-Thr-Leu-His-Phe-Arg-NH2
（３）H-Phe-Thr-Leu-Lys-Phe-Arg-NH2 The method for screening a sensitizing substance according to any one of claims 1 to 3, wherein the peptide for evaluation is one of the following (1) to (3).
(1) H-Phe-Thr-Leu-Cys-Phe-Arg-NH 2
(2) H-Phe-Thr-Leu-His-Phe-Arg-NH 2
(3) H-Phe-Thr-Leu-Lys-Phe-Arg-NH 2
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