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

使用基于流的组织工程和蛋白质组学方法评估药物引起的毒性的特殊代谢

• 批准号: 2108500
• 金额: --
• 依托单位: University of Liverpool
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2108500
• 关键词: Evaluating; idiosyncratic; metabolism; using; flow

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.

药物不良反应（ADR）是全球住院的主要原因。这些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）、非甾体类抗炎药（如布洛芬）以及用于治疗癌症、糖尿病和心律失常的药物。在我们的老龄化人口中，对慢性疾病的日益关注可能意味着长期接触某些药物，ADR 可能会产生危及生命的后果，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，并且假设药物代谢酶的个体间变异与线粒体遗传学相结合可能在涉及特殊药物引起的肝损伤的尚未确定的机制中发挥重要作用。这两个因素反过来影响药物的代谢，然后这些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培养基流下使用人类细胞培养系统来模拟血流所看到的纯粹应力，以便开展直接转化研究。为了纳入线粒体变异，我们生成了具有传输软骨细胞杂种的 HEPG2 细胞，作为单倍群 H、T、J、U 的个性化线粒体功能的体外模型，这些单倍群代表了全球线粒体遗传中的主要进化偏差，使我们能够描述线粒体单倍群对任何药物特异性不良反应。我们进一步将使用诱导多能干细胞衍生（iPSC）肝细胞来监测个体间代谢变化。最终，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 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在进展中的影响心脏毒性事件。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，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，您将接受培训以培养大量肝细胞和心肌细胞，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（例如活力、形态、搏动率）和评估已知的细胞损伤生物标志物，研究药物引起的变化如何影响肝脏和心肌细胞的健康。我们将使用分子技术评估基因（qPCR 和微阵列）和蛋白质（蛋白质印迹）水平的途径。将进行免疫荧光来确定形态变化，并使用质谱来量化代谢物的水平以及蛋白质水平的整体蛋白质变化。这种新颖的模型设置将使我们能够站在个性化医疗的最前沿，并直接在多器官水平上评估患者特定的治疗效果差异，并减少研究初始阶段的动物数量。该研究将获得对特殊药物引起的器官损伤的机制和转化洞察，以评估其对肝脏和心血管病理生理学的影响，并确定这些机制在基因变异患者中可能如何变化，从而为全球药物安全的更个性化方法铺平道路。