A Multi-Endpoint Evaluation of Cytochrome P450 1A2, 2B6 and 3A4 Induction Response in Human Hepatocyte Cultures After Treatment with β - Naphthoflavone, Phenobarbital and Rifampicin

J.      G. Zhang; Thuy      Ho; Alanna      L. Callendrello; Charles      L. Crespi; David      M. Stresser

doi:10.2174/187231210792928224

Abstract

US FDA and EMEA guidance recommend that the preferred in vitro model for cytochrome P450 induction testing is human hepatocytes coupled with acceptable inducers as controls. However, there are surprisingly few published studies characterizing this model system for dose and time-dependence response to model inducing compounds. The concentration- dependent response and time-course for the induction of CYP1A2, CYP2B6 and CYP3A4 by inducing agents β-naphthoflavone, phenobarbital and rifampicin, respectively were examined in two or more donors using multiple endpoints (mRNA, enzyme activity and Western blot analysis). Depending on the endpoint, exposure time for maximal response of CYP induction potential for the three enzymes ranged from 24 to 72 hours. Of the concentrations of BNF, PB and RIF tested, those which gave the maximal response were found to be 33 μM, > 2 mM and 10 μM, respectively.

Keywords: Cytochrome P450, induction, human hepatocytes, concentration and time-dependence, Naphthoflavone, Phenobarbital, Rifampicin, hepatocytes, time-dependence response, CYP1A2, CYP2B6, CYP3A4, multiple end-points, concentration, time-dependence, drug-metabolizing enzymes, enzyme activity, Hepatocyte Culture, Hepatocytes plated, Induction Response, CYP Activity, Prototypical Inducers, Reverse Tran-scriptase Polymerase Chain Reaction (RT-PCR), Western Blot Analysis, Cell lysate, lysate protein, immunoblot response, chemiluminescence, fold-induction response, Time-Dependent Induction, Inhibitory Effect, carcinogenic polycyclic aromatic hydro-carbon, Aryl hydrocarbon receptor, Constitutive androstane receptor, Pregnane X receptor