Hepatocyte Uptake and Loss Assay (HUpLA): Proof-of-Concept of an All-in-One System for Measuring Hepatic Influx, Egress, and Metabolic Clearance Based on the Extended Clearance Concept.

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 ADME assays, resulting in non-valid 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 (ECM). In this study, we demonstrate proof-of-concept of a novel two-step assay enabling measurement of multiple kinetic parameters from a single experiment in plated human primary hepatocytes with and without transporter and CYP inhibitors - the Hepatocyte Uptake and Loss Assay (HUpLA). HUpLA accurately predicted the CLH of 8 of the 9 drugs (within 2-fold of the observed CLH). Distribution clearances were within 3-fold of observed literature values in standard uptake and efflux assays. In comparison, the conventional suspension hepatocyte stability assay poorly predicted the CLH CLH of only 2 drugs were predicted within 2-fold 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 HUpLA 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 advance is the concept of removing drug-containing media following intracellular drug loading, enabling measurement of drug reappearance rate in media, as well as measurement of loss of total drug in the test system unencumbered by background quantities of drug in media otherwise present in a conventional assay.