ABSTRACT:The effect of microsomal protein concentration on the inhibitory potency of a series of CYP3A inhibitors was assessed in vitro using Microsomal binding of drug substrates and/or metabolic inhibitors is increasingly recognized as a potential source of artifact arising in the course of in vitro studies of drug metabolism. Nonspecific binding of substrate to microsomal protein can influence availability of the substrate to the metabolizing enzyme or enzymes in vitro, and thereby yield biased estimates of enzyme kinetic parameters. These, in turn, may produce inaccurate predictions when in vitro data are used to estimate in vivo pharmacokinetics (Obach, 1996(Obach, , 1997Obach et al., 1997;McLure et al., 2000;Venkatakrishnan et al., 2000c Venkatakrishnan et al., , 2001Kalvass et al., 2001). Inhibitor binding to microsomal systems may likewise influence estimation of potency of metabolic inhibitors (Gibbs et al., 1999a). The influence of binding has been termed "inhibitor depletion" when the inhibitor interacts with the active site (Gibbs et al., 1999a). However, inhibitor depletion could also refer to nonspecific inhibitor binding to microsomal preparations, as well as to actual microsomal consumption of the inhibitor itself through biotransformation.The present study evaluated the influence of microsomal protein concentration on the inhibitory capacity of known CYP3A inhibitors. In vitro 3-hydroxylation of diazepam to temazepam was used as an index reaction to profile CYP3A activity. Two selective serotonin reuptake inhibitors (fluvoxamine and norfluoxetine), three antifungal azole agents (itraconazole, ketoconazole, and fluconazole), a metabolite of itraconazole (OH-itraconazole), and the human immunodeficiency virus protease inhibitor ritonavir were used as representative CYP3A inhibitors. These agents have different intrinsic inhibitory capacities and different binding affinities. Materials and MethodsIncubation Procedures and Index Reaction Characteristics. Liver samples from individual human donors with no known liver disease were provided by the International Institute for the Advancement of Medicine (Exton, PA), the Liver Tissue Procurement and Distribution System (University of Minnesota, Minneapolis, MN), or the National Disease Research Interchange (Philadelphia, PA). All samples were of the CYP2D6 and CYP2C19 normal metabolizer phenotype based on prior in vitro phenotyping studies. Microsomes were prepared by ultracentrifugation; microsomal pellets were suspended in 0.1 M potassium phosphate buffer containing 20% glycerol and stored at Ϫ80°C until use. Chemical reagents and drug entities were purchased from commercial sources or kindly provided by their pharmaceutical manufacturers (von Moltke et al., 1993(von Moltke et al., , 1994a(von Moltke et al., ,b, 1996aVenkatakrishnan et al., 2000a Venkatakrishnan et al., ,b,c, 2001a.Diazepam 3-hydroxylation to form temazepam was used as the index reaction for CYP3A activity (Andersson et al., 1994;Ono et al., 1996;Jung et al., 1997; Venkatakrishnan ...
A healthy 40-year-old Caucasian male volunteer displayed unusually low clearance and long elimination half-life of alprazolam and trazodone, two CYP3A substrate drugs, following single-dose oral administration in clinical pharmacokinetic studies. Genomic DNA isolated from the individual's peripheral blood was subjected to polymerase chain reaction amplification and subsequent sequence analysis of a 592 base-pair segment upstream from the CYP3A coding region. The analysis revealed no variation from wild-type in the nucleotide present at position -290, previously suggested to influence expression and/or activity of CYP3A. The functional significance of this promoter region mutation is unclear and requires further evaluation.
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