Microsomal Protein Concentration Modifies the Apparent Inhibitory Potency of CYP3A Inhibitors

Abstract

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 ...

Quantitative Prediction of the in Vivo Inhibition of Diazepam Metabolism by Omeprazole Using Rat Liver Microsomes and Hepatocytes

“…The 4-fold increase in IC 50 with addition of 4% HSA indicates that a decrease in the unbound concentration of OMP due to albumin binding results in a reduction in the extent of inhibition. An analogous effect was reported by Tran et al (2002) in microsomes; the inhibitory capacity of ketoconazole and OH-itraconazole on 3-HDZ formation from DZ was reduced using heat-inactivated protein.…”

“…To quantitatively predict inhibition of metabolism in vivo, the concentration of inhibitor in the in vitro incubation should relate to the concentration of inhibitor in the plasma. Differences may result from the extensive binding of the inhibitor to microsomes, as demonstrated by Tran et al (2002) and Margolis and Obach (2003). In addition, differences may occur as a consequence of accumulation of the inhibitor in the hepatocyte as a result of intracellular binding or active cell uptake.…”

“…The predicted degree of interaction caused by a reversibly acting inhibitor can then be determined based on the ratio of the inhibitor concentration at the active site in vivo to the K i determined in vitro (Ito et al, 1998a,b). The majority of inhibition predictions in the literature have been performed using microsomal data (Yamano et al, 1999a(Yamano et al, ,b, 2000(Yamano et al, , 2001Komatsu et al, 2000;Ishigam et al, 2001;Tran et al, 2002;Yao et al, 2003 vivo clearance compared with microsomes (Houston, 1994), there is, at present, a lack of evidence to demonstrate that hepatocytes are better predictors of inhibition potential. There has been much controversy in the literature as to whether the total plasma, unbound plasma, or liver inhibitor concentration should be used for in vitro-in vivo extrapolations of inhibition (Bertz and Granneman, 1997;Ito et al, 1998a,b;Lin, 2000;Weaver, 2001).…”

Validated Assays for Human Cytochrome P450 Activities

“…Also, the high selectivity of tandem mass spectrometry reduces the potential for the compound being tested as an inhibitor to interfere in the analysis. Previous work has shown that inhibition constants can be altered by increasing microsomal protein concentration, due to nonspecific binding of the inhibitor to the (Margolis and Obach, 2003) and specific binding to the enzyme depleting the inhibitor (Gibbs et al, 1999;Tran et al, 2002). The use of very low microsomal protein concentrations should obviate the need to measure the free fraction of inhibitor in the in vitro matrices used.…”

In Silico Modeling of Nonspecific Binding to Human Liver Microsomes

“…It has now been well established in the scientific literature that nonspecific binding of compounds into microsomal phospholipids will confound the experimental estimation of CL int and IC 50 by decreasing the fraction of unbound drug (fu mic ) to metabolizing enzymes within the incubation (Kalvass et al, 2001;Margolis and Obach, 2003;Obach, 1997Obach, , 1999Tran et al, 2002). In such cases, estimates of CL int and IC 50 derived from microsomes are best deemed "apparent" (i.e., CL int, app and IC 50, app ) because they represent a mixture of kinetic determinants with both relevant (CL int and IC 50 ) and irrelevant (fu mic ) implications for drug design by eqs.…”