Kidney on a Chip Model in conjunction with systems biology modelling to predict renal pharmacokinetic drug-drug interactions

肾脏芯片模型与系统生物学模型相结合，预测肾脏药代动力学药物相互作用

• 批准号: 2432019
• 金额: --
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2432019
• 关键词: Kidney; Chip; Model; conjunction; systems

A suite of hepatic metabolic in vitro tools, such as recombinant enzymes and hepatocytes, have been used in conjunction with systems biology modelling to predict drug pharmacokinetics (PK) in animals. This approach has become a stalwart in the discovery of new drugs over the last 5 years and has led to a reduction in the use of animals as well as giving a better understanding of the processes that drive the PK of a drug at the cellular level. However, limited research has been carried out on in vitro renal cellular tools in conjunction with systems biology modelling in order to predict the renal contribution to the PK of a drug. Currently, renal drug clearance is predicted in human by carrying out invasive PK studies in animals and investigating whether an empirical relationship exists between the different species. In many cases an empirical relationship is not established due to species differences in transporter activity and the prediction of the renal contribution to the PK of a drug for a target species becomes uncertain. In addition to the prediction of renal drug clearance, an established renal in vitro cellular set of tools in conjunction with systems biology modelling would allow the prediction of renal drug-drug interactions.Currently in the Paine lab, mixed primary proximal (PTC) and distal tubular cell (DTC) monolayers are established in a transwell format using established methods from rat and pig kidney tissues and the kinetics of drug movement in these transwell monolayer culture systems have been investigated. However, one of the major drawbacks of transwell systems is that vital 3D architecture is not present leading to "leaky" junctions and under expression of membrane transporters. Recently, The Wyss Institute at Harvard University have shown that including haemodynamic flow into an in vitro model leads to a more realistic representation of the blood brain barrier.Recent studies have highlighted the theoretical and experimental impact of haemodynamic flow on membrane recycling. Haemodynamic flow is therefore expected to drive the exocytosis of cytosolic membrane transporters that could, in turn, provide an explanation for the deficiency of transwell monolayer cultures and the potential morphological change of major membrane organelles. The objectives of the herein proposal are to develop mixed primary proximal cell (PTC) and distal tubular cell (DTC) cultures with an organ on a chip system using rat, pig and human (if available) tissue. Tight junction integrity will be assessed by TEER measurements and sodium fluorescein permeability and transporter (OATs,OCTs, MRPs, MDR1) protein levels assessed by mRNA, immunocytochemistry and western blotting (Years 1-2). The effect of haemodynamic flow will be assessed by varying the hydrostatic pressure load though the Kidney on a chip model via a micro pump. The kinetic activity of drugs known to undergo renal clearance will be measured using HPLC-MS/MS. Membrane transporter saturation parameters (Km; Vmax) will be determined for a range of pressures. Known drug transporter inhibitors will be investigated in the presence of the renally cleared drugs and IC50's determined (Years 2-3). Systems biology models using mathematical and kinetic modelling software such as Matlab, Berkeley Madonna and Phoenix, in conjunction with the organ on a chip kinetic data will be built to model and validate a) the effect of pressure change on transporter function b) predict existing in vivo renal clearance data and c) predict known in vivo renal drug-drug interactions for both marketed drugs and novel literature compounds (Years 3-4).

一套肝代谢体外工具，如重组酶和肝细胞，已与系统生物学建模结合使用，以预测动物中的药物药代动力学（PK）。在过去的5年中，这种方法已经成为新药发现的坚定支持者，并导致了动物使用的减少，以及更好地理解在细胞水平上驱动药物PK的过程。然而，为了预测肾脏对药物PK的贡献，对体外肾细胞工具结合系统生物学建模进行了有限的研究。目前，通过在动物中进行侵入性PK研究并研究不同种属之间是否存在经验关系来预测人体肾脏药物清除率。在许多情况下，由于转运蛋白活性的种属差异，无法建立经验关系，并且靶种属的肾脏对药物PK的贡献预测变得不确定。除了预测肾脏药物清除率外，一套已建立的肾脏体外细胞工具与系统生物学建模相结合，将允许预测肾脏药物-药物相互作用。混合原代近端和远端肾小管细胞使用已建立的方法从大鼠和猪肾组织建立transwell格式的单层，已经研究了transwell单层培养系统。然而，transwell系统的主要缺点之一是不存在重要的3D结构，导致“泄漏”连接和膜转运蛋白表达不足。最近，哈佛大学Wyndham研究所的研究表明，将血流动力学纳入体外模型可以更真实地再现血脑屏障，最近的研究强调了血流动力学对膜再循环的理论和实验影响。因此，血液动力学流动预计将驱动胞质膜转运蛋白的胞吐作用，这反过来可以为transwell单层培养的缺陷和主要膜细胞器的潜在形态变化提供解释。本文建议的目的是使用大鼠、猪和人（如果可用）组织开发具有器官芯片系统的混合原代近端细胞（PTC）和远端肾小管细胞（DTC）培养物。将通过TEER测量评估紧密连接完整性，并通过mRNA、免疫细胞化学和蛋白质印迹评估荧光素钠渗透性和转运蛋白（OAT、OCT、MRP、MDR 1）蛋白水平（第1-2年）。通过微泵改变肾芯片模型的流体静压负荷，评估血液动力学流量的影响。将使用HPLC-MS/MS测量已知经历肾清除的药物的动力学活性。将确定一系列压力下的膜转运蛋白饱和参数（Km; Vmax）。将在存在经肾脏清除的药物的情况下研究已知的药物转运蛋白抑制剂，并测定IC 50（第2-3年）。系统生物学模型使用数学和动力学建模软件，如Matlab，伯克利麦当娜和凤凰，结合芯片上器官，将建立动力学数据，以建模和验证a）压力变化对转运蛋白功能的影响B）预测现有的体内肾清除率数据和c）预测已知的上市药物和新文献化合物的体内肾药物相互作用（第3-4年）。