title: Current In Vitro Methods to Determine Hepatic Kpuu: A Comparison of Their Usefulness and Limitations.
abstract: 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 Kpuufor 18 diverse drug compounds by 3 alternative in vitro methods and to compare the outcome with the ECM approach. Kpuuwas 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(logD7.4method). Deviations to ECM-based Kpuudata were closely linked to the absence of intrinsic clearance processes (metabolism, biliary secretion) in the investigated methods. Differences in fuhepadditionally contributed to deviations in Kpuu. The homogenization method generally provided lowest fuhepvalues, especially for compounds with high molecular weight or low logD7.4. Kpuuvalues 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.
authors.affiliation: Department of PK Sciences, ADME Section, Novartis Institutes for BioMedical Research, Basel, Switzerland; Department of Pharmaceutical Sciences, Division of Pharmaceutical Technology, University of Basel, Basel, Switzerland.
authors.forename: Julia
authors.initials: J
authors.lastname: Riede
authors.affiliation: Department of PK Sciences, ADME Section, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Gian
authors.initials: G
authors.lastname: Camenisch
authors.affiliation: Department of Pharmaceutical Sciences, Division of Pharmaceutical Technology, University of Basel, Basel, Switzerland.
authors.forename: Jörg
authors.initials: J
authors.lastname: Huwyler
authors.affiliation: Department of PK Sciences, ADME Section, Novartis Institutes for BioMedical Research, Basel, Switzerland. Electronic address: birk.poller@novartis.com.
authors.forename: Birk
authors.initials: B
authors.lastname: Poller
mesh_terms: D002478:Cells, Cultured; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008657:Metabolic Clearance Rate; D008954:Models, Biological; D004364:Pharmaceutical Preparations / Q000097:blood* / Q000378:metabolism*; D013696:Temperature
chemical_list: D004364:Pharmaceutical Preparations
keywords: active transport; biliary excretion; disposition; distribution; hepatic metabolism; hepatocytes; human liver microsomes; passive diffusion/transport; protein binding; tissue partition