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

一套肝脏体外代谢工具，如重组酶和肝细胞，已被用于预测药物在动物体内的药代动力学(PK)。在过去的5年里，这种方法已经成为发现新药的坚定力量，并导致了动物使用的减少，以及在细胞水平上更好地理解了驱动药物PK的过程。然而，为了预测肾脏对药物PK的贡献，将体外肾细胞工具与系统生物学模型相结合进行的研究有限。目前，通过在动物身上进行侵入性PK研究并调查不同物种之间是否存在经验关系，可以预测肾脏药物在人体内的清除量。在许多情况下，由于转运蛋白活性的物种差异，没有建立经验关系，并且对于目标物种肾脏对药物PK的贡献的预测变得不确定。除了肾脏药物清除的预测外，建立的肾脏体外细胞集结合系统生物学模型将使预测肾脏药物-药物相互作用成为可能。目前在Paine实验室，使用已建立的方法从大鼠和猪肾组织中建立了初级近端(PTC)和远端肾小管细胞(DTC)混合单层，并研究了这些跨孔单层培养系统中药物运动的动力学。然而，Transwell系统的一个主要缺点是没有重要的3D结构，导致膜转运蛋白的“泄漏”连接和表达不足。最近，哈佛大学的Wyss研究所发现，在体外模型中加入血液动力学流动可以更真实地表示血脑屏障。最近的研究强调了血液动力学流动对膜循环的理论和实验影响。因此，血流动力学有望驱动胞膜转运体的胞吐作用，这反过来又可以解释Transwell单层培养的缺陷和主要膜细胞器潜在的形态变化。本文建议的目标是利用大鼠、猪和人(如果可用)组织开发具有芯片系统上的器官的原代近端细胞(PTC)和远端小管细胞(DTC)的混合培养。紧密连接的完整性将通过TEER测量和钠荧光素通透性和转运蛋白(燕麦、OCTs、MRPs、mdr1)蛋白水平的mRNA、免疫细胞化学和Western blotting来评估(1-2岁)。通过微泵在芯片模型上改变肾脏的静水压力负荷来评估血流的影响。已知可进行肾清除的药物的动力学活性将使用高效液相-质谱仪/质谱仪进行测量，膜转运体饱和参数(Km；Vmax)将在一定压力范围内确定。已知的药物转运蛋白抑制剂将在肾清除药物存在的情况下进行调查，并确定S的IC50(第2-3年)。系统生物学模型将使用数学和动力学建模软件，如Matlab、Berkeley Madonna和Phoenix，结合芯片上器官的动力学数据来建立，以建模和验证a)压力变化对转运体功能的影响b)预测现有的体内肾脏清除数据，以及c)预测上市药物和新文献化合物的已知的体内肾脏药物相互作用(第3-4年)。

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).