Biliary and Intestinal Cell Models of Drug Toxicity

描述（由申请人提供）： 最近，候选药物的前所未有的扩展已经产生了对通过快速、灵敏、可再现和成本有效的体外测定进行早期毒性测试的迫切需求。特别需要监测对胆管细胞、胆管上皮细胞（BDC）或肠细胞、小肠上皮细胞（IEC）的损伤的细胞毒性测定。虽然这些部位是广泛使用的抗癌药物的已知靶点，但由于缺乏适当的细胞系统，尚未建立体外试验。拟议研究的目标是使用新获得的细胞系统和药物治疗动物的胆汁开发细胞毒性试验，作为将细胞暴露于反应性药物/代谢物的生理相关方式，这是最合理的暴露途径。选择用于验证这些体外细胞毒性模型的抗癌药物[碘莫司汀（CCNU）、甲氨蝶呤（MTX）、多西他赛（DCT）]在体内对BDC或IEC具有明确的毒性，代表了通过不同机制发挥作用的药物类别，并广泛经胆汁排泄。 我们的方法是基于初步的研究表明，从大鼠胆汁与胆管破坏剂4，4 '-二氨基二苯基甲烷（DAPM）是细胞毒性的原代大鼠BDC在体外。体内DAPM诱导的胆管损伤的功能和形态学特征与体外产生的BDC损伤几乎相同。据报道，用NSAID吲哚美辛处理的大鼠胆汁在体外损伤大鼠肠细胞系。提出了形成绒毛样肠上皮细胞（Cdx-IEC-6细胞）的更分化的细胞系作为本申请中的模型IEC系统。 我们的具体目标是（1）通过细胞损伤的活力测定和功能指数，在体外验证胆汁药物/代谢产物对BDC与IEC的靶器官特异性，（2）表征线粒体损伤、细胞旁通透性、ATP含量和膜渗漏敏感测定的特异性、重现性和高通量可能性，作为胆汁药物/代谢产物诱导功能障碍的潜在生物标志物，和（3）使用激光捕获显微切割和基因芯片分析，确定胆汁药物/代谢物是否在体内与体外对BDC和IEC中的基因表达诱导可比较的早期效应。本研究中开发的基本技术将导致筛选潜在药物损伤靶位点的新方法，这些靶位点在体内不易研究。

DESCRIPTION (provided by applicant): Recent, unprecedented expansion of candidate drugs has generated a critical need for early phase toxicity testing by in vitro assays that are rapid, sensitive, reproducible and cost effective There is a particular need for cytotoxicity assays that monitor injury to cholangiocytes, epithelial cells of bile ducts (BDC), or to enterocytes, epithelial cells of the small intestine (IEC). Although these sites are known targets of widely used anticancer drugs, in vitro assays have not been established due to the lack of appropriate cell systems The goal of the proposed research is to develop cytotoxicity assays using newly available cell systems and bile from drug-treated animals as a physiologically relevant way to expose the cells to reactive drug/metabolites, which is the most plausible route of exposure. The anticancer drugs [Iomustine (CCNU), methotrexate (MTX), docetaxel (DCT)] chosen to validate these in vitro cytotoxicity models have well-established toxicities to BDC or IEC in vivo, represent classes of drugs that work by different mechanisms, and are extensively excreted in bile. Our approach is based on preliminary studies indicating that bile from rats treated with the cholangiodestructive agent 4,4'-diaminodiphenylmethane (DAPM) is cytotoxic to primary rat BDC in vitro. Functional and morphological characteristics of DAPM-induced injury to bile ducts in vivo are virtually identical to the BDC injury produced in vitro. Bile from rats treated with the NSAID indomethacin has been reported to injure a rat intestinal cell line in vitro. A more differentiated cell line that forms villus-like enterocytes (Cdx-IEC-6 cells) is proposed as the model IEC system in this application. Our specific aims are (1) validate the target organ specificity of biliary drug/ metabolites to BDC versus IEC in vitro by viability assays and functional indices of cell injury, (2) characterize the specificity, reproducibility and high-throughput possibilities of sensitive assays for mitochondrial injury, paracellular permeability, ATP content, and membrane leakage as potential biomarkers of biliary drug/ metabolite-induced dysfunction, and (3) determine if biliary drug/metabolites induce comparable early effects on gene expression in BDC and IEC in vivo versus in vitro using laser capture microdissection and gene chip analyses. The basic techniques developed in this research will lead to new methods for screening potential drug injury to target sites that are not easily investigated in vivo.