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

药物不良反应（ADR）是全球住院治疗的主要原因。这些可能是由广泛的药物引起的，包括常见的非处方药，如扑热息痛，非甾体抗炎药，如布洛芬，以及给癌症，糖尿病和心律失常患者的药物。在我们的老龄化人群中，对慢性疾病的日益关注可能意味着长期暴露于某些药物，ADR可能具有危及生命的后果，无论是作为药物反应的直接原因，还是出于患者安全考虑而需要停药。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中，并且已经假设药物代谢酶的个体间变异与线粒体遗传学结合可能在特异质药物诱导的肝损伤中涉及的尚未确定的机制中发挥重要作用。这两个因素反过来又影响药物的代谢，然后药物可能或多或少容易转化为代谢产物，导致心脏毒性事件。在这个项目中，我们的目标是使用人类细胞培养系统在连续流动的媒体，以模拟纯粹的压力，看到血流，以发展直接的转化研究。为了整合线粒体变异，我们产生了具有线粒体胞质杂交体的HEPG2细胞，作为单倍型群H、T、J、U的个性化线粒体功能的体外模型，这些单倍型群代表了全球线粒体遗传的主要进化偏差，使我们能够描述线粒体单倍型群对任何药物特异性不良反应的影响。我们还将使用诱导多能干细胞衍生（iPSC）肝细胞来监测代谢的个体间差异。最后，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。该项目利用来自Kirkstall的QV900流量系统，该系统允许连接器官特定模块，使我们能够通过流量将肝脏模块直接连接到心脏模块，这进一步使我们能够确定肝脏代谢的不同背景对心脏毒性事件进展的影响。概述的研究将在利物浦大学的MRC药物安全科学中心（CDSS）进行，该中心拥有最先进的设备，实验室和着名的科学家，他们对药物不良反应的机制基础进行基础研究。这是一个多学科项目，您将接受培训，培养大量肝脏和心脏细胞，并在QV900系统中操作和计量细胞。然后，我们将研究药物诱导的变化如何通过评估关键表型（如活力、形态学、心率）和评估已知的细胞损伤生物标志物来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（Western印迹）水平上评估途径。免疫荧光将用于确定形态学变化，质谱将用于量化代谢物水平以及蛋白质水平的整体蛋白质变化。这种新的模型设置将使我们能够走在个性化医疗的最前沿，并在多器官水平上直接评估治疗效果的患者特异性差异，并减少研究初始阶段的动物数量。该研究将获得对特异质药物诱导的器官损伤的机制和转化见解，以评估对肝脏和心血管病理生理学的影响，并确定这些方案如何在遗传变异患者中变化，为全球药物安全性的更个性化方法铺平道路。

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.