Development of pharmacokinetic assays utilizing an organ-on-chip model of the human kidney proximal tubule

项目摘要 所提出的应用集中于开发即插即用的活体人类肾脏芯片， 测定药物候选物通过肾近端小管的分泌。一旦商业化，该芯片将使 药物开发人员更好地筛选临床前候选药物的肾毒性副作用。这意味着 药物在临床试验中的高损耗率，考虑到约20%因肾毒性而失败， 使药物开发更快、更经济、更安全。传统的临床前模型使用有限 用于评估肾小管分泌。在动物模型中，肾脏上皮细胞中的转运蛋白质 2D体外模型，即使使用人细胞，也不能提供适当的 培养条件，使适当的细胞功能。拟议中的肾脏芯片将利用商业 现有的Nortis器官芯片平台目前用于许多器官应用-包括 第一代肾脏模型，包含人肾近端小管，由原代细胞组织工程化 细胞为了使肾芯片在肾分泌的临床前评估中广泛使用， 有必要作出重大改进。可销售的产品需要稳定的细胞来源， 性能，将化合物精确递送到芯片中近端小管的基底外侧，以及 从芯片上采集精确的液体样本此外，预先装载有活的近端小管的芯片将代表 为客户带来显著的价值提升。该项目的第一阶段将旨在证明， 转运蛋白OAT 1/3和OCT 2对肾脏分泌至关重要，功能正常（I期/AIM 1）。 为了证明这一点，将芯片中的近端小管暴露于特定底物（阿德福韦酯用于OAT 1/3，西咪替丁 对于OCT 2），与这些转运蛋白的特异性抑制剂（阿德福韦酯的对氨基马尿酸盐和 二甲双胍用于西咪替丁）。用于生长小管的细胞将来自永生化的人类 肾近端小管细胞系，已显示在正确的细胞中表达OAT 1/3和OCT 2 当在Nortis芯片中培养时，第一阶段/AIM 2将实现芯片功能， 对小管上游和下游的微升规模的流体体积进行采样，这是 评估称为渗透性-表面积乘积（Pa）的分泌通量参数。在第二阶段， 芯片将配备有用于严格控制化合物递送到近端基底外侧的特征， 小管（II期/AIM 1）。随后，以下目标（II期/AIM 2）将侧重于肾近端的合格试验 OAT 1/3和OCT 2的小管分泌清除率具有足够的统计把握度，以确定耐用性， 再现性第二阶段/AIM 3旨在确定活体肾脏芯片可以运送给客户， 不受影响的检测性能，将由独立实验室进行验证。一旦发育完全 并且商业化的所提出的肾芯片可能适用于药物以外的应用。 例如环境健康与安全、精准医疗和再生医学。

Project Summary The proposed application focuses on the development of a plug-and-play living human kidney-on-chip for assaying secretion of drug candidates by the kidney proximal tubule. Once commercialized, the chip will enable drug developers to better screen pre-clinical drug candidates for nephrotoxic side effects. This stands to reduce the high attrition rate of drugs in clinical trials, given that about 20% fail because of nephrotoxicity, and would make drug development faster, more cost efficient, and safer. Conventional preclinical models are of limited use for assessing tubular secretion. In animal models, transporter proteins in the kidney epithelium differ significantly from those present in human kidney; 2D in-vitro models, even when using human cells, fail to provide appropriate culture conditions that enable appropriate cell function. The proposed kidney chip will leverage the commercially available Nortis organ-on-chip platform that is currently in use for a number of organ applications--including a first-generation kidney model that contains a human kidney proximal tubule, tissue-engineered from primary cells. In order to make the kidney chip useful for wide-spread use in pre-clinical assessment of renal secretion, substantial improvements are necessary. A marketable product requires a stable source of cells with sufficient performance, precision delivery of compounds to the basolateral side of the proximal tubule in the chip, and precise fluid sampling from the chip. Further, chips arriving pre-loaded with living proximal tubules will represent a significant value increase to the customer. Phase I of the project will aim to demonstrate that specific transporter proteins OAT1/3 and OCT2, which are crucial to renal secretion, function correctly (Phase I/AIM1). To prove this, proximal tubules in the chips will be exposed to specific substrates (adefovir for OAT1/3, cimetidine for OCT2), in conjunction with specific inhibitors of these transporters (para-aminohippurate for adefovir and metformin for cimetidine). The cells used for growing the tubules will be derived from an immortalized human kidney proximal tubule cell line that has been shown to express OAT1/3 and OCT2 in the correct cell compartments when cultured in the Nortis chip. Phase I/AIM2 will be to implement chip features for precise sampling of microliter-scale fluid volumes upstream and downstream of the tubule, which is required for assessing the secretory flux parameter termed the permeability-surface area product (Pa). During Phase II the chips will be equipped with features for tight control of compound delivery to the basolateral side of the proximal tubule (Phase II/AIM1). The following aim (Phase II/AIM2) will then focus on qualifying assays of kidney proximal tubule secretory clearance for OAT1/3 and OCT2 with sufficient statistical power to establish robustness and reproducibility. Phase II/AIM3 is designed to establish that living kidney chips can be shipped to customers with uncompromised assay performance, which will be validated by an independent laboratory. Once fully developed and commercialized the proposed kidney chip might be suitable for applications beyond the pharmaceutical sector, such as environmental health & safety, precision medicine, and regenerative medicine.