Development and validation of an integrated tiered in vitro screening approach for predicting drug induced liver injury (DILI)

肝损伤是临床前毒性导致药物磨损的主要原因，通常发生在临床试验的后期。目前，肝损伤主要是通过动物(啮齿动物)模型来评估的。然而，迫切需要开发替代的体外和硅胶模型，一旦得到验证，将具有许多重要的潜在优势。由于已知多种行动模式很重要，因此需要一种基于机械性理解的综合分级筛选方法。该项目旨在利用肝细胞系(如HepG2)和原代肝细胞建立和评估包括胆盐输出泵和多药耐药蛋白2抑制试验、UDP-葡萄糖醛酸基转移酶1A1和1A3酶抑制、细胞毒性(四甲基偶氮唑蓝)、细胞毒性(四甲基偶氮唑蓝)、线粒体膜电位损伤(TMRE)、线粒体(有丝分裂)和细胞ROS(DCF-DA)的体外检测方法，并结合先进的技术包括Hi Content Image Express、Incucyte Cell Imager用于培养细胞的实时分析和成像，以及LC-MS-TOF用于反应代谢物的评估。此外，我们将探索将共聚焦成像与标记报告蛋白相结合来开发通过特定转录激活途径(例如，Nrf2核转位作为“抗氧化反应”的标志)介导的应激反应的分析方法。这些检测将使用已知有毒(如四氯化碳)和无毒化合物的“训练集”进行验证，以建立一个对已知肝毒物质的反应数据库，这些毒物通过不同的机制发挥作用。一旦得到验证，将使用一组化学物质(以及代谢物，例如酰基、N-，O-葡萄糖醛酸苷)来测试开发的分析的预测能力，这些化学物质几乎没有关于它们导致肝脏损伤的能力的信息，或者目前正在进行体内评估。还将评估与潜在全身药物水平的联系，

Liver injury is a major cause of drug attrition as a result of preclinical toxicity and usually occurs late on in clinical trials. Currently, liver injury is largely assessed using animal (rodent) models. However, there is an urgent need to develop alternative in vitro and in silico models that once validated would have a number of important potential advantages. Because multiple modes of action are known to be important an integrated tiered screening approach based on a mechanistic understanding is required. The aim of the project is o develop and assess in vitro assays including bile salt export pump and multidrug resistance protein 2 inhibition assay, UDP-glucuronosyl transferase 1A1 and 1A3 enzyme inhibition, cell toxicity (MTT, adenylate kinase), impairment of mitochondrial membrane potential (TMRE), mitochondrial (mitosox) and cellular ROS (DCF-DA) using liver cell lines (e.g. HepG2) and primary hepatocytes in combination with state-of-the-art technologies including the Hi Content Imaging Express, Incucyte Cell Imager for real time analysis and imaging of cells in culture and LC-MS-TOF for reactive metabolite assessment. In addition we will explore use of confocal-imaging in combination with tagged-reporter proteins to develop assays for stress-response mediated by specific transcriptional activation pathways (e.g. Nrf2 nuclear translocation as a marker of the "anti-oxidant response"). These assays will be validated using a "training set" of known toxic (e.g. carbon tetrachloride) and non-toxic compounds to build a database of responses to known hepatoxicants that exert their effects by different mechanisms. Once validated the predictive power of developed assays will be tested using a panel of chemicals (and metabolites, acyl,N-,O-glucuronides for example) where little information exists on their ability to cause liver injury or which are currently under in vivo assessment. A link to potential systemic drug levels will also be assessed,