Automated Screening With Confirmation of Mechanism-Based Inactivation of Cyp3a4, Cyp2c9, Cyp2c19, Cyp2d6, and Cyp1a2 in Pooled Human Liver Microsomes

Abstract

ABSTRACT:A strategy is proposed to profile compounds for mechanism-based inactivation of CYP3A4, CYP2C19, CYP2C9, CYP2D6, and CYP1A2 based on an apparent partition ratio screen. Potent positives from the screen are confirmed by time-and concentration-dependent inactivation assays. Quasi-irreversible inhibitions are then differentiated from irreversible inactivations by oxidation with potassium ferricyanide and/or dialysis. The three-step screening procedure has been validated with acceptable accuracy and precision for detection and confirmation of mechanism-based inactivators in drug discovery. We report here the apparent partition ratios for 19 mechanism-based inactivators and four quasi-irreversible inhibitors obtained under the same experimental conditions. The apparent partition ratio screen was automated to provide throughput for determining structure-mechanism-based inactivation relationships. Information about reversibility can be used to assess potential toxicity mediated by covalent adducts, as well as the potential for pharmacokinetic drug-drug interactions. Direct comparison of known mechanism-based inactivators and quasi-irreversible inhibitors, based on our screening of apparent partition ratios, has identified ritonavir, mibefradil, and azamulin as highly effective mechanism-based inactivators; e.g., 1 mol of CYP3A4 was inactivated on turnover of about 2 mol of compound. Other mechanismbased inactivators we identified include bergamottin (CYP1A2 besides previously reported CYP3A4), troglitazone (CYP3A4), rosiglitazone (CYP3A4), and pioglitazone (CYP3A4). Comparison of the apparent partition ratios and inactivation clearance data for the three glitazones suggests that the chromane moiety on troglitazone contributes to its greater potency for mechanism-based inactivation.

In Vitro and In Vivo Drug-Drug Interaction Studies to Assess the Effect of Abiraterone Acetate, Abiraterone, and Metabolites of Abiraterone on CYP2C8 Activity

“…Gemfibrozil 1-Ob-glucuronide produced substantial inhibition of CYP2C8 activity in the presence of NADPH but minimal enzyme activity loss in the absence of NADPH; the apparent partition ratio (APR), which represents the ratio of substrate turnover per molecule of enzyme being inactivated, was 78, indicating strong and rapid time-dependent inhibition of CYP2C8. In cases of time-dependent inhibition, we defined APR values less than 100 as strong inhibition and values between 100 and 500 as moderate inhibition (Lim et al, 2005). Conversely, there was no NADPHdependent loss of CYP2C8 activity with quercetin, abiraterone, abiraterone sulfate, or abiraterone sulfate N-oxide, indicating no timedependent inhibition of CYP2C8 (Fig.…”

CYP3A Time-Dependent Inhibition Risk Assessment Validated with 400 Reference Drugs

“…Discrimination between TDI-positive drugs forming a metabolic intermediate complex versus those being authentic mechanism-based inactivators would be of great interest because the clinical risk is higher for the later (Lim et al, 2005;Kalgutkar et al, 2007). Combining the high-throughput screen described here with recently validated methods to distinguish a metabolic intermediate from mechanism-based inactivators seems to be possible and is currently under investigation.…”

“…The characteristic parameters for TDI, k inact , the maximal inactivation rate, and K I , the concentration at half k inact , and their ratio k inact /K I are typically used for risk evaluation. Results of attempts to use excerpts of these experiments to screen a large number of compounds have been published (Atkinson et al, 2005;Lim et al, 2005;Obach et al, 2007;Perloff et al, 2009). The IC 50 shift method, which is essentially an extension of the classic highthroughput testing for reversible inhibition, is the most popular because of its easy setup.…”

“…However, Obach et al, 2007 have recently proposed a risk ranking model based on the shifted IC 50 . As an alternative, single concentration, single time point assays were reported for which percentage activity remaining or apparent partition ratio was used for risk evaluation (Lim et al, 2005;Watanabe et al, 2007).…”

Risk Assessment for Drug-Drug Interaction Caused by Metabolism-Based Inhibition of CYP3A Using Automated in Vitro Assay Systems and Its Application in the Early Drug Discovery Process

“…Many pharmaceutical companies have established MBI assay systems to detect either enzymatic activity loss at a single concentration or IC 50 shift caused by preincubation. Other assays are designed to obtain MBI-related kinetic parameters Naritomi et al, 2004;Yamamoto et al, 2004;Atkinson et al, 2005;Lim et al, 2005;Zhao et al, 2005). Such systems use pooled human liver microsomes (HLMs), recombinant P450 isozymes, and human hepatocytes with P450 probe substrates and fluorescent substrates.…”