Julia A Schulz Pauly, Elizabeth Sande, Mei Feng, Yue-Ting Wang, David M Stresser, John Cory Kalvass
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引用次数: 0
Abstract
Hepatic clearance (CLH ) prediction is a critical parameter to estimate human dose. However, CLH 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 CLH predictions. Other processes, including hepatocyte and biliary distribution via transporters, can also play significant roles in CLH 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 CLH of eight of the nine drugs (within twofold of the observed CLH ). 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 CLH The CLH of only two drugs was predicted within twofold of the observed CLH 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.
期刊介绍:
An important reference for all pharmacology and toxicology departments, DMD is also a valuable resource for medicinal chemists involved in drug design and biochemists with an interest in drug metabolism, expression of drug metabolizing enzymes, and regulation of drug metabolizing enzyme gene expression. Articles provide experimental results from in vitro and in vivo systems that bring you significant and original information on metabolism and disposition of endogenous and exogenous compounds, including pharmacologic agents and environmental chemicals.