Abstract

In vitro - in vivo extrapolation (IVIVE) to predict human hepatic clearance including metabolism and transport requires extensive experimental resources. In addition, there may be technical challenges to measure low clearance values. Therefore, prospective identification of rate-determining step(s) in hepatic clearance through application of the Extended Clearance Classification System (ECCS) could be beneficial for optimal compound characterization. IVIVE for hepatic intrinsic clearance (CLint,h) prediction is conducted for a set of 36 marketed drugs with low-to-high in vivo clearance, which are substrates of metabolic enzymes and active uptake transporters in the liver. The compounds were assigned to the ECCS classes and CLint,h, estimated with HepatoPac® (a micro-patterned hepatocyte co-culture system), was compared to values calculated based on suspended hepatocyte incubates. A P_app threshold (apical to basal) of 50 nm/s in LLC-PK1 cells proved optimal for ECCS classification. A reasonable performance of the IVIVE for compounds across multiple classes using HepatoPac® was achieved (with 2- to 3-fold error), except for substrates of uptake transporters (class 3b), where scaling of uptake clearance using plated hepatocytes is more appropriate. Irrespective of the ECCS assignment, metabolic clearance can be estimated well using HepatoPac®. The validation and approach elaborated in the present study can result in proposed decision trees for the selection of the optimal in vitro assays guided by ECCS class assignment, to support compound optimization and candidate selection.

Significance Statement Characterization of the rate-determining step(s) in hepatic elimination could be on the critical path of compound optimization during drug discovery. This study demonstrated that HepatoPac® and plated hepatocytes are suitable tools for the estimation of metabolic and active uptake clearance, respectively, for a larger set of marketed drugs, supporting a comprehensive strategy to select optimal in vitro tools and to achieve ECCS-dependent IVIVE for human clearance prediction.