Zhang J.G.,Corning Gentest Contract Research Services |
Ho T.,Corning Gentest Contract Research Services |
Callendrello A.L.,Corning Gentest Contract Research Services |
Clark R.J.,Corning Gentest Contract Research Services |
And 6 more authors.
Drug Metabolism and Disposition | Year: 2014
Cytochrome P450 (P450) induction is often considered a liability in drug development. Using calibration curve-based approaches, we assessed the induction parameters R3 (a term indicating the amount of P450 induction in the liver, expressed as a ratio between 0 and 1), relative induction score, C max/EC50, and area under the curve (AUC)/F2 (the concentration causing 2-fold increase from baseline of the dose-response curve), derived from concentration-response curves of CYP3A4 mRNA and enzyme activity data in vitro, as predictors of CYP3A4 induction potential in vivo. Plated cryopreserved human hepatocytes from three donors were treated with 20 test compounds, including several clinical inducers and noninducers of CYP3A4. After the 2-day treatment, CYP3A4 mRNA levels and testosterone 6β-hydroxylase activity were determined by real-time reverse transcription polymerase chain reaction and liquid chromatography-tandem mass spectrometry analysis, respectively. Our results demonstrated a strong and predictive relationship between the extent of midazolam AUC change in humans and the various parameters calculated from both CYP3A4 mRNA and enzyme activity. The relationships exhibited with non-midazolam in vivo probes, in aggregate, were unsatisfactory. In general, the models yielded better fits when unbound rather than total plasma Cmax was used to calculate the induction parameters, as evidenced by higher R2 and lower root mean square error (RMSE) and geometric mean fold error. With midazolam, the R3 cut-off value of 0.9, as suggested by US Food and Drug Administration guidance, effectively categorized strong inducers but was less effective in classifying midrange or weak inducers. This study supports the use of calibration curves generated from in vitro mRNA induction response curves to predict CYP3A4 induction potential in human. With the caveat that most compounds evaluated here were not strong inhibitors of enzyme activity, testosterone 6β-hydroxylase activity was also demonstrated to be a strong predictor of CYP3A4 induction potential in this assay model. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.
PubMed | Corning Gentest Contract Research Services
Type: Journal Article | Journal: Expert opinion on drug metabolism & toxicology | Year: 2014
Evaluation of time-dependent inhibition (TDI) properties in drug candidates is generally required for any compound entering development. Methods to evaluate TDI, particularly in abbreviated formats, differ widely among laboratories and there appears to be lack of consensus how to address certain assay shortcomings.As a first objective of this work, we provide commentary on experimental and theoretical considerations in the conduct of abbreviated TDI testing. Methods considered are the single K(obs), the progress curve, the 2 + 2 method, the measurement of partition ratios and the IC shift assay. The merits of multiple experimental variations in the IC shift assay, including in depth discussion on the use of a dilution step are explored. Growing evidence suggests that the use of hepatocytes provides certain advantages over liver microsomes. Therefore, a second major objective of this work is to consider merits of the use of hepatocytes in TDI testing.An in-depth technical understanding of methods to evaluate TDI is critical to enable a selection of an assay aimed at efficiency while minimizing erroneous classification of TDI properties.