Evaluating idiosyncratic metabolism using a flow based tissue engineering and proteomics approach for drug induced toxicity

药物不良反应(ADR)是全球住院的一个主要原因。这些疾病可能是由多种药物引起的，包括常见的非处方药，如扑热息痛，非类固醇抗炎药物，如布洛芬，以及给患者开的治疗癌症、糖尿病和心律失常的药物。在我们老龄化的人口中，对慢性病的日益关注可能意味着长期接触某些药物，ADR可能会作为药物反应的直接原因或出于患者安全考虑而停药的需要而产生危及生命的后果。受ADR影响的两个主要器官是肝脏和心脏。药物代谢主要发生在肝脏，推测药物代谢酶的个体差异加上线粒体遗传可能在目前尚未确定的特殊药物所致肝损伤的机制中发挥重要作用。这两个因素继而影响药物的新陈代谢，然后药物可能或多或少容易转化为可能导致心脏毒性事件的代谢物。在这个项目中，我们的目标是在连续的介质流动下使用人类细胞培养系统来模拟血液流动所看到的纯粹的压力，以便直接开展翻译研究。为了整合线粒体变异，我们产生了带有传递线粒体杂交体的HepG2细胞，作为单倍组H、T、J、U个性化线粒体功能的体外模型，单倍组H、T、J、U代表了全球线粒体遗传的主要进化偏差，使我们能够描绘线粒体单倍组对任何药物特定的不良反应的影响。我们将进一步使用诱导多能干细胞来源(IPSC)肝细胞来监测代谢的个体间差异。在高潮中，我们的目标是阐明由线粒体遗传或代谢酶变异引起的DILI机制。该项目利用了Kirkstall的QV900 Flow系统，该系统允许器官特定模块的连接，允许我们通过Flow将肝脏模块直接连接到心脏模块，这进一步允许我们确定不同背景的肝脏代谢在心脏毒性事件进展中的影响。概述的研究将在利物浦大学MRC药物安全科学中心(CDSS)进行，该中心拥有最先进的设备、实验室和著名科学家，他们对药物不良反应的机制基础进行基础研究。这是一个多学科的项目，你将接受培训，培养大量的肝脏和心脏细胞，并在QV900系统中操作和剂量细胞。然后，我们将通过评估关键表型，如活性、形态、搏动频率和评估已知的细胞损伤生物标记物，来研究药物诱导的变化如何影响肝脏和心脏细胞的健康。我们将使用分子技术在基因(qPCR和微阵列)和蛋白质(蛋白质印迹)水平评估通路。免疫荧光将被用来确定形态变化，质谱学将被用来量化代谢物的水平以及蛋白质水平的全球蛋白质变化。这种新的模型设置将使我们能够站在个性化药物的前沿，并在多器官水平上直接评估患者特定的变化对治疗效果的影响，并在研究的初始阶段减少动物数量。这项研究将从机制上和翻译上深入了解特殊药物引起的器官损伤，以评估对肝脏和心血管病理生理学的影响，并确定这些机制在遗传变量患者中可能会发生什么变化，为更个性化地解决全球药物安全问题铺平道路。

Adverse drug reactions (ADRs) are a major cause of global hospitalisation. These can be caused by a wide range of drugs that include common over the counter medication such as paracetamol, non-steroidal anti-inflammatory such as ibuprofen as well as drugs given to patients for cancer, diabetes and cardiac arrhythmias. In our aging population where a growing concern of chronic illness can mean prolonged exposure to certain drugs, ADRs can have life threatening consequences either as a direct cause of drug reactions or as a need for drug withdrawal for patient safety concerns. The two major organs affected by ADRs are the liver and the heart. Drug metabolism occurs predominantly in the liver and it has been hypothesised that inter-individual variation in drug metabolising enzymes coupled with mitochondrial genetics may play a large role in the yet undetermined mechanisms involved in idiosyncratic drug induced liver injury. These two factors in turn then affect the metabolism of drugs that then maybe converted more or less readily into metabolites that can lead to cardiotoxic events. In this project we aim to use human cell culture systems under continuous flow of media to emulate the sheer stress seen by blood flow in order to develop directly translational research. To incorporate mitochondria variation we have generated HEPG2 cells with transmitochondrial cybrids as an in vitro model of personalised mitochondrial function for haplogroups H, T, J, U which represent the major evolutionary deviations in global mitochondria genetic allowing us to delineate the influence of mitochondrial haplogroup on any drug-specific adverse reaction. We further will use induced pluripotent stem cell derived (iPSC) hepatocytes to monitor inter-individual variation in metabolism. In culmination we aim to shed light on DILI mechanisms caused by mitochondrial genetic or variations in metabolising enzymes. This project utilises the QV900 flow system from Kirkstall, which allows the connection of organ specific modules allowing us to connect a liver module directly to a cardiac module by flow, which further allows us to determine the effects of varied backgrounds of liver metabolism in the progression of cardiotoxic events. The research outlined will be carried out at the MRC Centre for Drug Safety Science (CDSS) at the University of Liverpool, which houses state-of-the art equipment, laboratories and renowned scientists conducting fundamental research into the mechanistic basis of adverse drug reactions. This is a multi-disciplinary project in which you will be trained to culture a multitude of liver and cardiac cells and to operate and dose cells in the QV900 system. We will then examine how drug-induced changes affect both liver and cardiac cell health by assessing key phenotypes such as viability, morphology, beat rates and assessment of known biomarkers of cell damage. We will asses pathways at the gene (qPCR and microarray) and protein (Western blot) level using molecular techniques. Immunofluorescence will be performed to determine morphological changes and mass spectroscopy will be used to quantify the levels of metabolites as well as global protein changes in protein levels.This novel model set-up will allow us to be on the forefront of personalised medicine and to directly evaluate patient specific variation on the effect of treatments at a multi-organ level and reduce the number of animals in the initial phases of research. The research will gain mechanistic and translational insight into idiosyncratic drug induced organ injury in order to assess the effect on liver and cardiovascular pathophysiology and ascertain how these regimes may vary in genetically variable patients paving the way to a more personalised approach to global drug safety.