Integrated assessment of rifampicin‐mediated induction of transporters and drug‐metabolizing enzymes in a long‐term human liver tissue chip system

Rifampicin is a prototypical clinical inducer used in drug–drug interaction (DDI) studies. However, the clinical relevance and predictability of rifampicin‐mediated hepatic transporter induction remain poorly defined. Conventional hepatocyte culture models fail to capture clinically relevant regulation of transporters and drug‐metabolizing enzymes (DMEs), particularly at the protein level. In this study, we characterized a liver tissue chip (LTC) as a new approach methodology (NAM) for long‐term transporter‐ and DME‐mediated induction risk assessment. Transporter‐certified® cryopreserved primary human hepatocytes from three donors were cultured in the LTC under recirculating flow with or without 10 μM rifampicin for 7 days. The LTC facilitated simultaneous mRNA‐protein quantification for transporters and mRNA‐protein‐activity profiling for cytochrome P450s (CYPs) within individual chips across treatment and control conditions. In addition, extracellular bile acid profiling and intracellular rifampicin concentrations were measured by LC–MS/MS. Rifampicin induced multiple transporters and enzymes at the protein level, including organic anion transporting polypeptide (OATP)2B1, P‐glycoprotein, CYP3A4, CYP2C19, and uridine 5’‐diphospho‐glucuronosyltransferase (UGT)1A4. Induction patterns differed between transporter mRNA and protein in many cases, suggesting regulatory mechanisms beyond PXR‐mediated activation. Matched mRNA‐protein‐activity measurements of five CYP enzymes also revealed discrepancies in rifampicin‐mediated induction magnitude. Rifampicin altered bile acid composition, increasing taurine‐conjugated bile acids and inducing bile acid‐related genes. Measured intracellular rifampicin concentrations differed substantially from experimentally employed media concentrations. Overall, these findings demonstrate that the LTC enables integrated assessment of mRNA, protein, and activity measurements in addition to bile acid profiles and intracellular drug exposure, which may improve mechanistic understanding of induction processes.