Hepatic zonation in a microfluidic liver model: application to drug metabolism

描述(申请人提供)：新药的肝毒性在药物发现的临床前阶段往往是不可预测的，导致50%进入I期临床试验的药物失败，导致每种药物造成~2-6年的时间损失和~1500万美元的资源损失。目前的体外药物筛选平台通常不考虑肝细胞代谢的区带，这对于准确模拟体内功能至关重要，因为不同区带发生不同程度的生物转化，影响肝脏毒性。虽然已经研究了许多代谢区带诱导剂，尤其是O2，但我们仍然缺乏对什么浓度和浓度梯度的化学诱导剂导致生理区带的系统了解。我们的长期目标是创建一种生理上相关的、高通量的系统，用于体外预测药物筛选。我们的目标是系统地了解化学制剂中的浓度梯度是如何引起肝脏代谢分区的，并将这种理解整合到一个高通量微流控筛选平台中，用于快速药物毒性筛选。中心假设是，可以使用区带诱导剂(O2、激素、胆汁酸和乙醇)中的浓度梯度来产生肝细胞代谢的动态区带，以指导微流控肝细胞培养中形态和代谢上不同区带的发展，以阐明异种肝毒性。这一建议的基本原理是，目前的微制造技术允许使用微流体来创建更多包含肝细胞异质性的生理性肝脏模型。此外，这种微流控平台可以被多路复用以进行高通量分析，从而实现精确控制和高效的药物筛选。目的1：设计和制作一种用于肝细胞培养的复合微流控平台，以系统地测定带状诱导剂对细胞代谢的浓度效应。目的2：建立微流控平台，评价单一和多个区带诱导剂浓度梯度对肝细胞动态代谢的影响。目的3：通过细胞消耗，模拟肝脏生理，沿流向建立肝细胞代谢区带，长期培养检测各种药物的肝毒性。这些AIMS的成功结果将提供能够复制带状肝细胞形态和代谢的新型体外微流控培养系统。我们期望阐明沿着腺泡的带状诱导物的梯度与肝细胞动态代谢反应之间的联系，并将代谢反应与药物代谢和毒性联系起来。这些研究的更广泛影响将是开发一个预测性的、高通量的体外药物筛选平台，以取代预测能力较差的动物模型。

DESCRIPTION (provided by applicant): Hepatotoxicity of new drugs, which is often unpredictable in the preclinical phase of drug discovery, causes failure of 50% of pharmaceuticals that make it to Phase I clinical trials, resulting in ~2-6yr loss in time and ~$15 million in resources per drug. Current in vitro drug screening platforms typically do not consider zonation in hepatocyte metabolism, which is critical for accurate modeling of in vivo function as biotransformation occurs to different extents in different zones, affecting hepatotoxicity. Although many metabolic zonal inducers have been studied, most notably O2, we still lack a systematic understanding of what concentration and concentration gradients of the chemical inducers lead to physiological zonation. Our long-term goal is to create a physiologically relevant, high-throughput system for predictive in vitro drug-screening. Our objective is to develop a systematic understanding of how concentration gradients in chemical agents induce hepatic metabolic zonation and incorporate this understanding into a high-throughput microfluidic screening platform for rapid drug toxicity screening. The central hypothesis is that dynamic zonation of hepatocyte metabolism can be generated using concentration gradients in zonation induction agents (O2, hormones, bile acids, and ethanol) to direct the development of morphologically and metabolically distinct zones within a microfluidic hepatocyte cell culture to elucidate xenobiotic hepatotoxicity. The rationale for this proposal is that current microfabrication technology allows the use of microfluidics to create more physiological liver models that incorporate hepatocyte heterogeneity. Furthermore, such microfluidic platforms can be multiplexed for high- throughput analysis, allowing precisely controlled and efficient drug screening. Aim 1: Design and fabricate a multiplexed microfluidic platform for hepatocyte culture to systematically determine the concentration effects of zonation inducers on cell metabolism. Aim 2: Create a microfluidic platform to assess the effects of concentration gradients of single and multiple zonation induction agents on hepatocyte dynamic metabolism. Aim 3: Create hepatocyte metabolic zonation in the flow-direction via cellular consumption, mimicking liver physiology, and test the hepatotoxicity of various drugs in long-term culture. The successful outcome of these aims will provide novel in vitro microfluidic culture systems capable of reproducing zonal hepatocyte morphology and metabolism. We expect to elucidate the connection between gradients of zonal inducers along the acinus and hepatocyte dynamic metabolic responses, and to correlate metabolic response to drug metabolism and toxicity. The broader impact of the studies will be the development of a predictive, high- throughput in vitro drug-screening platform to replace poorly predictive animal models.