School of Pharmacy and Chemistry, Liverpool John Moores University, Byrom Street, Liverpool, L33AF England.
Chem Res Toxicol. 2009 Mar 16;22(3):592-603. doi: 10.1021/tx800431x.
Research aimed at nonanimal approaches to provide the relevant information needed for the effective assessment of skin sensitization, for both hazard characterization and risk assessment purposes, is currently an area of high activity, stimulated by regulatory initiatives related to chemicals used in consumer products. The ability of a chemical to react covalently with protein or peptide nucleophiles in the skin is recognized as the key determinant in determining sensitization potency, and initiatives to develop peptide reactivity assays to replace animal testing have been undertaken recently. This paper describes a high throughput kinetic profiling (HTKP) approach, developed as an extension of a published standard assay, with the aim of providing a quantitatively robust end point in the form of a kinetic profile from which reactivity to a model peptide can be quantified in the form of second order rate constants. The approach allows solubility issues to be identified and overcome; these are frequently encountered, but can often go undetected, in aqueous reactivity assays with organic compounds of interest in the skin sensitization context. Using rate constants determined by the HTKP approach we have obtained a quantitative mechanistic model for the Michael acceptor reaction mechanistic domain, relating the sensitization potency in the murine local lymph node assay to the rate constant. The observation that the correlation is not improved by incorporation of a hydrophobicity term has implications regarding the nature and location of the skin nucleophile whose reaction leads to sensitization by Michael acceptor electrophiles.
目前，为了满足有效评估皮肤致敏的相关信息需求，针对非动物方法的研究非常活跃，这主要是受到与消费品中使用的化学物质相关的监管措施的推动。人们已经认识到，一种化学物质与皮肤中的蛋白质或肽亲核试剂发生共价反应的能力是决定致敏潜力的关键决定因素，最近已经采取了一些措施来开发肽反应性测定法以替代动物测试。本文描述了一种高通量动力学分析（HTKP）方法，它是在已发表的标准测定法的基础上开发的，旨在提供一个定量稳健的终点，即动力学图谱，从中可以定量确定模型肽的反应性，其形式为二级速率常数。该方法可以识别和解决溶解度问题；在皮肤致敏背景下，与有机化合物进行的水性反应性测定中，这些问题经常出现，但往往会被忽视。通过使用 HTKP 方法确定的速率常数，我们获得了一个定量的迈克尔受体反应机制域的机械模型，将小鼠局部淋巴结测定中的致敏能力与速率常数联系起来。观察到将疏水性项纳入其中并不能提高相关性，这表明对导致迈克尔受体亲电试剂致敏的皮肤亲核试剂的性质和位置有了新的认识。
