Current In Vitro Methods to Determine Hepatic Kp_uu: A Comparison of Their Usefulness and Limitations

Unbound intrahepatic drug concentrations determine the interaction potential with intracellular targets related to toxicity, pharmacokinetics, or pharmacodynamics. Recently, the unbound liver-to-blood partition coefficient (Kp_uu) based on the Extended Clearance Model (ECM) has been developed providing indirect estimates of unbound intrahepatic drug concentrations. This study aimed to determine Kp_uu for 18 diverse drug compounds by 3 alternative in vitro methods and to compare the outcome with the ECM approach. Kp_uu was calculated from independent measurements of hepatocellular drug accumulation (Kp) and unbound fraction in hepatocytes (fu_hep) either assessed from steady-state accumulation at 4°C (temperature method), using equilibrium dialysis (homogenization method), or empirically from logD_7.4 (logD_7.4 method). Deviations to ECM-based Kp_uu data were closely linked to the absence of intrinsic clearance processes (metabolism, biliary secretion) in the investigated methods. Differences in fu_hep additionally contributed to deviations in Kp_uu. The homogenization method generally provided lowest fu_hep values, especially for compounds with high molecular weight or low logD_7.4. Kp_uu values of compounds with low intrinsic clearance correlated well between the ECM and temperature methods independent of physicochemical properties. Therefore, only the ECM provides an integrated quantitative determination of hepatic Kp_uu. Temperature and homogenization methods, however, represent useful alternatives if compound properties are appropriately considered.