title: Verapamil hepatic clearance in four preclinical rat models: towards activity-based scaling.
abstract: The current study was designed to cross-validate rat liver microsomes (RLM), suspended rat hepatocytes (SRH) and the isolated perfused rat liver (IPRL) model against in vivo pharmacokinetic data, using verapamil as a model drug. Michaelis-Menten constants (Km), for the metabolic disappearance kinetics of verapamil in RLM and SRH (freshly isolated and cryopreserved), were determined and corrected for non-specific binding. The 'unbound' Km determined with RLM (2.8 µM) was divided by the 'unbound' Km determined with fresh and cryopreserved SRH (3.9 µM and 2.1 µM, respectively) to calculate the ratio of intracellular to extracellular unbound concentration (Kpu,u). Kpu,u was significantly different between freshly isolated (0.71) and cryopreserved (1.31) SRH, but intracellular capacity for verapamil metabolism was maintained after cryopreservation (200 vs. 191 µl/min/million cells). Direct comparison of intrinsic clearance values (Clint) in RLM versus SRH, yielded an activity-based scaling factor (SF) of 0.28-0.30 mg microsomal protein/million cells (MPPMC). Merging the IPRL-derived Clint with the MPPMC and SRH data, resulted in scaling factors for MPPGL (80 and 43 mg microsomal protein/g liver) and HPGL (269 and 153 million cells/g liver), respectively. Likewise, the hepatic blood flow (61 ml/min/kg b.wt) was calculated using IPRL Clint and the in vivo Cl. The scaling factors determined here are consistent with previously reported CYP450-content based scaling factors. Overall, the results show that integrated interpretation of data obtained with multiple preclinical tools (i.e. RLM, SRH, IPRL) can contribute to more reliable estimates for scaling factors and ultimately to improved in vivo clearance predictions based on in vitro experimentation.
authors.affiliation: Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, O&N2, Leuven, Belgium.
authors.forename: J
authors.initials: J
authors.lastname: Nicolaï
authors.affiliation: Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, O&N2, Leuven, Belgium.
authors.forename: T
authors.initials: T
authors.lastname: De Bruyn
authors.affiliation: Laboratory of Lipid Biochemistry and Protein Interactions, KU Leuven Department of Cellular and Molecular Medicine, O&N1, Leuven, Belgium.
authors.forename: P P
authors.initials: PP
authors.lastname: Van Veldhoven
authors.affiliation: Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, O&N2, Leuven, Belgium.
authors.forename: J
authors.initials: J
authors.lastname: Keemink
authors.affiliation: Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, O&N2, Leuven, Belgium.
authors.forename: P
authors.initials: P
authors.lastname: Augustijns
authors.affiliation: Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, O&N2, Leuven, Belgium.
authors.forename: P
authors.initials: P
authors.lastname: Annaert
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000379:methods; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008657:Metabolic Clearance Rate / Q000187:drug effects / Q000502:physiology; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D023421:Models, Animal; D051381:Rats; D014700:Verapamil / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D014700:Verapamil
keywords: Kpu,u; hepatic clearance; isolated perfused liver; liver microsomes; scaling factors; suspended hepatocytes; verapamil