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In Vitro Method for Gut Absorption and Cytotoxicity

肠道吸收和细胞毒性的体外方法

基本信息

批准号：
6595805
负责人：
FRANK A BARILE
金额：
$ 16.19万
依托单位：
ST. JOHN'S UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-06-01 至 2006-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6595805
关键词：
cell line cytotoxicity fluorescent dye /probe gastrointestinal toxin absorption membrane permeability method development pharmacokinetics statistics /biometry tissue /cell culture toxicant screening

项目摘要

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.

描述（由申请人提供）：评估急性全身毒性的体外方法国际研讨会报告得出结论，评估急性全身毒性的现有体外方法均未经过充分评估以取代动物试验。此外，该文件建议开发一种简单的肠道吸收预测系统，这将优化体外检测预测体内LD50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，以筛选具有代表性的化学物质对胃肠道吸收（GIA）伴随急性细胞毒性的影响。化学物质对体外GIA的影响是通过细胞旁渗透性标记物来确定的，包括路西法黄、fitc -葡聚糖、[3H]-甘露醇渗透性和经上皮电阻（TEER）测量。急性细胞毒性监测与MTT和NRU测定，推荐的细胞毒性指标的细胞活力。将对ICCVAM指导文件建议的细胞毒性登记中的20种化学制剂进行评估。急性3小时和24小时暴露于连续Caco-2单层。生成剂量-反应曲线；GIA的50%有效浓度（ec50）和急性毒性的50%抑制浓度（ic50）是外推的。根据指导文件计算两组数据的回归曲线。然后相互比较ec50和ic50，并与动物ld50、人类毒性和致死浓度以及这些化学品的人类口服生物利用度数据进行比较。可以预期，一个细胞培养模型可以同时和系统地筛选急性毒性和区分对GIA的影响，可用于预测体内致死研究的起始剂量，并提高体外细胞毒性数据预测体内LD50值的能力。