Deconvoluting the in vitro to in vivo drug clearance gap: Questioning the predictive performance of traditional hepatic clearance models

In vitro to in vivo extrapolation (IVIVE) methods for hepatic clearance (CL H ) prediction often underpredict, partly due to reliance on mathematical liver disposition models such as the well-stirred model (WSM) or parallel tube model (PTM). The ex vivo isolated perfused rat liver (IPRL) model bridges in vitro and in vivo data, providing mechanistic insights into the predictive accuracy of IVIVE models. This study evaluates the IPRL model across a diverse selection of 16 compounds, and benchmarks results against in vitro and in vivo data to verify the predictive performance of the WSM and PTM. Results demonstrate that both the IPRL and in vivo clearance conflict with assumptions of the WSM (AAFE = 2.85) or PTM (AAFE = 1.74), which consider liver outlet concentration as a driver for hepatic elimination rate. However, except for terfenadine, IPRL clearance predictions were within two-fold (AAFE = 1.59) of in vivo clearance when the liver inlet concentration was utilized to calculate the CL H . When employing the WSM or PTM for in vitro to ex vivo extrapolation, underpredictions were observed for compounds with high plasma protein binding and subject to sinusoidal hepatic uptake, reflecting model oversimplification compared to in vivo dynamics. Our findings experimentally challenge the theoretical assumptions underlying the use of the WSM and PTM in IVIVE methods. Unique insights from the IPRL model point to the next steps needed to advance IVIVE: refining current liver disposition models through enhanced and next-generation in vitro assays, capturing dynamic in vivo disposition mechanisms, and exploring complementary models.