The Combination of Chemical and Antibody Inhibitors for Superior P450 3A Inhibition in Reaction Phenotyping Studies

Abstract

ABSTRACT:Cytochrome P450 (P450) reaction phenotyping is a key process toward accurately determining the contribution of different P450s to the metabolism of new chemical entities. The significance of P450s to drug disposition has led to the identification of selective chemical and antibody inhibitors for individual P450 enzymes. Despite these advances, the maximal inhibition attainable is limited by the use of inhibitor concentrations that maintain selectivity for the individual P450s. Thus, most commercially available inhibitors produce a maximal inhibition of ϳ80%. Herein, the combination of chemical plus antibody inhibitors is explored to find whether P450 3A could be selectively and completely (>99%) inhibited by using both inhibitors simultaneously.The majority of marketed drugs are metabolized by members of the cytochrome P450 (P450) superfamily of enzymes (Wienkers and Heath, 2005). Given the limited number of P450 enzymes that contribute to the clearance of commercial therapeutics, P450s represent the primary source of clinically observed drug interactions in patients. In vitro techniques have evolved to enable the early estimation of drug interaction potential with preclinical drug candidates either as perpetrator or victim prior to clinical evaluation (Lu et al., 2003).The improved extrapolation of in vitro data to humans over the last decade is partially attributed to the inclusion of additional parameters that impact the scaling of in vitro data. For example, considering unbound fraction of drug during in vitro microsomal incubations and attempts to approximate the most appropriate inhibitor concentration in vivo can improve in vitro extrapolations (Margolis and Obach, 2003;Obach et al., 2005Obach et al., , 2006. Furthermore, a retrospective analysis of disparate in vitro estimates of drug interactions compared with clinically observed interactions illustrates that predictions can be improved when the fraction of drug metabolized through individual P450 pathways is accurately determined (Fig. 1) (Ito et al., 2005). P450 reaction phenotyping is used to determine fraction of drug metabolized by P450s and is possible in part because of the accumulation of compounds identified as selective inhibitors of individual P450 enzymes (Cai et al., 2004;Stresser et al., 2004;Walsky et al., 2005b). Additionally, the introduction of selective antibody inhibitors has expanded the diversity of inhibitors available to determine the involvement of different P450 pathways to a drug's clearance. However, commercial antibodies and chemical inhibitors typically do not yield complete inhibition of the P450 enzymes, especially at concentrations that maintain selectivity for the individual P450 enzymes. In the balance to achieve maximal yet selective inhibition of individual P450 enzymes the effective inhibitor concentrations typically result in inhibition up to 80%. The remaining 20% activity reduces the predictive nature of in vitro extrapolation techniques and increases the uncertainty regarding the potential activity from...

Evaluation of CYP2C8 Inhibition In Vitro: Utility of Montelukast as a Selective CYP2C8 Probe Substrate

“…4, A-C). The formation of M2 (sulfoxidation) and M5 (21-hydroxylation) was inhibited with the CYP3A inhibitor ketoconazole (500 nM) plus a CYP3A-selective antibody, a combination known to produce a more complete and selective inhibition than either alone (Rock et al, 2008) (Fig. 4, E and F).…”

“…The incubation was then diluted to 0.2 mg/ml microsomal protein in 100 mM potassium phosphate buffer (pH 7.4). Ketoconazole (500 nM) was added to the CYP3A4 incubation (Rock et al, 2008). Montelukast was added to the incubation mixture at a final concentration of 0.1 M. Control P450 substrate marker reactions, as reported previously (Walsky and Obach, 2004), were run to verify the effectiveness of the P450 antibodies.…”

Reaction Phenotyping

“…Furthermore, remaining activity can bring into question the P450 activity from less abundant hepatic P450s such as 2B6 and 2C8, which are showing increased metabolic activity toward numerous commercial drugs (Walsky et al, 2005 ). An approach using the combination of commercial antibody and chemical inhibitor against P450 3A has been reported (Rock et al, 2008 ), and showed a superior inhibition profi le of CYP3A4 compared with the use of antibody and chemical inhibitors individually.…”