In Vitro Method for Gut Absorption and Cytotoxicity

说明(申请人提供)：评估急性全身毒性的体外方法国际研讨会的报告得出结论认为，没有一种可用于评估急性全身毒性的体外方法被充分评估以取代使用动物。此外，该文件建议开发一种简单的肠道吸收预测系统，这将优化体外测试预测体内LD50值的能力。因此，这项建议概述了一系列研究，其目的是开发一种细胞培养技术，有可能筛选出具有代表性的化学物质在急性细胞毒性的同时对胃肠道吸收(GIA)的影响。使用细胞旁通透性的标记物，包括荧光黄、FITC-葡聚糖、[~3H]-甘露醇通透性和跨皮细胞电阻(TEER)测量，在体外测定化学物质对GIA的影响。急性细胞毒性通过四甲基偶氮唑蓝和NRU分析进行监测，这两种方法是细胞活力的推荐细胞毒性指标。将对ICCVAM指导文件建议的细胞毒性登记处中的20种化学制剂进行评估。急性3小时和24小时暴露是用连续的Caco-2单层进行的。生成剂量-反应曲线；外推GIA的50%有效浓度(EC50s)和急性毒性的50%抑制浓度(IC50s)。根据指导文件计算两组数据的回归曲线。然后将EC50和IC50彼此比较，并与动物LD50、人类毒性和致死浓度以及这些化学品的人体口服生物利用度数据进行比较。可以预期，一个可以同时系统地筛选急性毒性并与GIA的影响相区分的细胞培养模型，可以用于预测体内致死性研究的起始剂量，并提高体外细胞毒性数据预测体内LD50值的能力。

DESCRIPTION (provided by applicant): The Report of the International Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity concludes that none of the available in vitro methods for assessing acute systemic toxicity have been evaluated adequately to replace the use of animals. In addition, the document recommends the development of a simple predictive system for gut absorption, which would optimize the ability of in vitro assays to predict in vivo LD50 values. Consequently, this proposal outlines a series of studies whose aim is to develop a cell culture technique with the potential to screen representative chemicals for their effect on gastrointestinal absorption (GIA) concomitantly with acute cytotoxicity. Effect of chemicals on GIA in vitro is determined using markers for paracellular permeability, including Lucifer yellow, FITC-dextran, [3H]-mannitol permeability, and transepithelial electrical resistance (TEER) measurements. Acute cytotoxicity is monitored with the MTT and NRU assays, recommended cytotoxic indicators for cell viability. Twenty chemical agents in the Registry of Cytotoxicity, suggested by the ICCVAM Guidance Document will be evaluated. Acute 3-hour and 24-hour exposures are performed with continuous Caco-2 monolayers. Dose-response curves are generated; 50% effective concentrations (EC50s) for GIA and 50% inhibitory concentrations (IC50s) for acute toxicity are extrapolated. Regression curves for both sets of data are calculated according to the Guidance Document. EC50s and IC50s are then compared to each other and to animal LD50s, human toxic and lethal concentrations, and human oral bioavailability data available for these chemicals. It is anticipated that a cell culture model that can simultaneously and systematically screen for acute toxicity and distinguish from the effect on GIA, may be used to predict starting doses for in vivo lethality studies and to improve the ability of in vitro cytotoxicity data to predict in vivo LD50 values.