Quantitative, Translational, and ADME Sciences, Abbvie Inc., North Chicago, Illinois.
Quantitative, Translational, and ADME Sciences, Abbvie Inc., North Chicago, Illinois
Drug Metab Dispos. 2024 Sep 16;52(10):1048-1059. doi: 10.1124/dmd.124.001768.
Hepatic clearance ( ) prediction is a critical parameter to estimate human dose. However, underpredictions are common, especially for slowly metabolized drugs, and may be attributable to drug properties that pose challenges for conventional in vitro absorption, distribution, metabolism, and elimination (ADME) assays, resulting in nonvalid data, which prevents in vitro to in vivo extrapolation and predictions. Other processes, including hepatocyte and biliary distribution via transporters, can also play significant roles in Recent advances in understanding the interplay of metabolism and drug transport for clearance processes have aided in developing the extended clearance model. In this study, we demonstrate proof of concept of a novel two-step assay enabling the measurement of multiple kinetic parameters from a single experiment in plated human primary hepatocytes with and without transporter and cytochrome P450 inhibitors-the hepatocyte uptake and loss assay (HUpLA). HUpLA accurately predicted the of eight of the nine drugs (within twofold of the observed ). Distribution clearances were within threefold of observed literature values in standard uptake and efflux assays. In comparison, the conventional suspension hepatocyte stability assay poorly predicted the The of only two drugs was predicted within twofold of the observed Therefore, HUpLA is advantageous by enabling the measurement of enzymatic and transport processes concurrently within the same system, alleviating the need for applying scaling factors independently. The use of primary human hepatocytes enables physiologically relevant exploration of transporter-enzyme interplay. Most importantly, HUpLA shows promise as a sensitive measure for low-turnover drugs. Further evaluation across different drug characteristics is needed to demonstrate method robustness. SIGNIFICANCE STATEMENT: The hepatocyte uptake and loss assay involves measuring four commonly derived in vitro hepatic clearance endpoints. Since endpoints are generated within a single test system, it blunts experimental error originating from assays otherwise conducted independently. A key advantage is the concept of removing drug-containing media following intracellular drug loading, enabling the measurement of drug reappearance rate in media as well as the measurement of loss of total drug in the test system unencumbered by background quantities of drug in media otherwise present in a conventional assay.
肝清除率（）预测是估计人体剂量的关键参数。然而， 预测往往不足，尤其是对于代谢缓慢的药物，这可能归因于药物性质对传统的体外吸收、分布、代谢和消除（ADME）测定构成挑战，导致数据无效，从而无法进行体外到体内的外推和预测。其他过程，包括通过转运体的肝细胞和胆汁分布，也可以在 中发挥重要作用。最近在理解代谢和药物转运对清除过程的相互作用方面的进展，有助于开发扩展的清除模型。在这项研究中，我们证明了一种新的两步测定法的概念验证，该方法能够在单层培养的人原代肝细胞中从单次实验中测量多个动力学参数，同时使用和不使用转运体和细胞色素 P450 抑制剂——肝细胞摄取和损失测定法（HUpLA）。HUpLA 准确预测了九种药物中的八种药物的（与观察到的 相差两倍以内）。在标准摄取和流出测定中，分布清除率与文献值相差三倍以内。相比之下，传统的悬浮肝细胞稳定性测定法对 的预测较差。只有两种药物的 预测值与观察到的 相差两倍以内。因此，HUpLA 的优势在于能够在同一系统中同时测量酶和转运体过程，无需独立应用缩放因子。使用人原代肝细胞能够在生理相关的范围内探索转运体-酶相互作用。最重要的是，HUpLA 有望成为低周转率药物的敏感测量方法。需要进一步评估不同的药物特性，以证明方法的稳健性。 意义：肝细胞摄取和损失测定法涉及测量四个通常衍生的体外肝清除率终点。由于终点在单个测试系统中生成，因此它削弱了源自否则独立进行的测定的实验误差。一个关键优势是在细胞内药物加载后去除含药物的培养基的概念，这使得能够测量药物在培养基中的再出现率以及在测试系统中未受药物的总损失的测量，而无需考虑常规测定中否则存在的培养基中药物的背景量。
