A unified methodology enabling simultaneous measurements of CYP3A4 mRNA, protein, and activity induction responses in...

Pregnane X receptor (PXR) is a key transcriptional regulator of drug-metabolizing enzymes and transporters, notably CYP3A4, which metabolizes a significant proportion of clinically used drugs. PXR activation can induce CYP3A4 expression, potentially leading to drug-drug interactions (DDIs) by altering the pharmacokinetics of CYP3A4 substrates, particularly for narrow therapeutic index drugs. Conventional induction assays rely on measuring CYP3A4 mRNA and enzyme activity, but mRNA levels often do not correlate with enzyme activity, which can lead to mispredictions of DDIs. To address this gap, we incorporated our newly established Fast and Surfactant-Treated proteomic workflow into the current in vitro induction assay to enable simultaneous quantification of CYP3A4 mRNA, protein, and enzyme activity induction from a single experiment. Using rifampicin as a PXR agonist, we demonstrated that the unified All-in-One assay provided consistent induction parameters with discrete assays, offering a robust method for assessing CYP3A4 induction. We also applied this approach to the tyrosine kinase inhibitors pazopanib and crizotinib, revealing nonuniformities in their induction profiles across mRNA, protein, and enzyme activity endpoints. Specifically, although both tyrosine kinase inhibitors induced CYP3A4 mRNA expression in a dose-dependent manner, they do not lead to protein induction, suggesting that the in vitro induction observed at the mRNA level may not translate to clinical induction. Collectively, these preliminary findings suggest that protein measurements may provide a more holistic representation of CYP3A4 induction and can potentially improve the predictability of clinical DDIs in drug development. SIGNIFICANCE STATEMENT: We described and validated a unified assay that can simultaneously measure CYP3A4 mRNA, protein, and enzyme activity induction from a single human hepatocyte experiment. This unified All-in-One approach has the potential to improve in vitro-in vivo correlation and translation of CYP3A4-mediated induction drug-drug interactions for new chemical entities. However, further work, including the integration of static or dynamic physiologically based pharmacokinetic modeling with protein induction data, will be required to fully confirm these insights.