ABSTRACT.Early prediction of human clearance is often challenging, in particular for the growing number of low-clearance compounds. Long-term in vitro models have been developed which enable sophisticated hepatic drug disposition studies and improved clearance predictions. Here, the cell line HepG2, iPSC-derived hepatocytes (iCell®), the hepatic stem cell line HepaRG™, and human hepatocyte co-cultures (HμREL™ and HepatoPac®) were compared to primary hepatocyte suspension cultures with respect to their key metabolic activities. Similar metabolic activities were found for the long-term models HepaRG™, HμREL™, and HepatoPac® and the short-term suspension cultures when averaged across all 11 enzyme markers, although differences were seen in the activities of CYP2D6 and non-CYP enzymes. For iCell® and HepG2, the metabolic activity was more than tenfold lower. The micropatterned HepatoPac® model was further evaluated with respect to clearance prediction. To assess the in vitro parameters, pharmacokinetic modeling was applied. The determination of intrinsic clearance by nonlinear mixed-effects modeling in a long-term model significantly increased the confidence in the parameter estimation and extended the sensitive range towards 3% of liver blood flow, i.e., >10-fold lower as compared to suspension cultures. For in vitro to in vivo extrapolation, the well-stirred model was used. The micropatterned model gave rise to clearance prediction in man within a twofold error for the majority of low-clearance compounds. Further research is needed to understand whether transporter activity and drug metabolism by non-CYP enzymes, such as UGTs, SULTs, AO, and FMO, is comparable to the in vivo situation in these long-term culture models.KEY WORDS: in vitro clearance; in vitro liver models; IVIVE; nonlinear mixed-effects modeling.

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. Significant 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 This article has not been copyedited and formatted. The final version may differ from this version.

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