Current In Vitro Methods to Determine Hepatic Kpuu: 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 (Kpuu) based on the Extended Clearance Model (ECM) has been developed providing indirect estimates of unbound intrahepatic drug concentrations. This study aimed to determine Kpuu for 18 diverse drug compounds by 3 alternative in vitro methods and to compare the outcome with the ECM approach. Kpuu was calculated from independent measurements of hepatocellular drug accumulation (Kp) and unbound fraction in hepatocytes (fuhep) either assessed from steady-state accumulation at 4°C (temperature method), using equilibrium dialysis (homogenization method), or empirically from logD7.4 (logD7.4 method). Deviations to ECM-based Kpuu data were closely linked to the absence of intrinsic clearance processes (metabolism, biliary secretion) in the investigated methods. Differences in fuhep additionally contributed to deviations in Kpuu. The homogenization method generally provided lowest fuhep values, especially for compounds with high molecular weight or low logD7.4. Kpuu 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 Kpuu. Temperature and homogenization methods, however, represent useful alternatives if compound properties are appropriately considered.