In Vitro Method for Gut Absorption and Cytotoxicity

描述（由申请方提供）：急性全身毒性体外评估方法国际研讨会的报告得出结论，评估急性全身毒性的现有体外方法均未得到充分评价，无法取代动物。此外，该文件建议开发一种简单的肠道吸收预测系统，这将优化体外试验预测体内LD 50值的能力。因此，本提案概述了一系列研究，其目的是开发一种细胞培养技术，该技术有可能筛选代表性化学品对胃肠道吸收（GIA）的影响，同时伴有急性细胞毒性。使用细胞旁渗透性标记物（包括荧光黄、FITC-葡聚糖、[3 H]-甘露醇渗透性和跨上皮电阻（TEER）测量）测定化学品对体外GIA的影响。用MTT和NRU试验（推荐的细胞活力细胞毒性指标）监测急性细胞毒性。将评价ICCVAM指南文件建议的细胞毒性登记研究中的20种化学试剂。使用连续Caco-2单层进行急性3小时和24小时暴露。 生成剂量-反应曲线;外推GIA的50%有效浓度（EC 50）和急性毒性的50%抑制浓度（IC 50）。根据指南文件计算两组数据的回归曲线。然后将EC 50和IC 50相互比较，并与这些化学品的动物LD 50、人类毒性和致死浓度以及人类口服生物利用度数据进行比较。预计可同时系统筛选急性毒性并区分对GIA影响的细胞培养模型可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LD 50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.