Evaluation of Normalization Methods To Predict CYP3A4 Induction in Six Fully Characterized Cryopreserved Human Hepatocyte Preparations and HepaRG Cells.

Prediction of drug-drug interactions due to cytochrome P450 isoform 3A4 (CYP3A4) overexpression is important because this CYP isoform is involved in the metabolism of about 30% of clinically used drugs from almost all therapeutic categories. Therefore, it is mandatory to attempt to predict the potential of a new compound to induce CYP3A4. Among several in vitro-in vivo extrapolation methods recently proposed in the literature, an approach using a scaling factor, called a d factor, for a given hepatocyte batch to provide extrapolation between in vitro induction data and clinical outcome has been adopted by leading health authorities. We challenged the relevance of the calibration factor determined using a set of 15 well-known clinical CYP3A4 inducers or the potent CYP3A4 inducer rifampicin only. These investigations were conducted using six batches of human hepatocytes and an established HepaRG cell line. Our findings show that use of a calibration factor is preferable for clinical predictions, as shown previously by other investigators. Moreover, the present results also suggest that the accuracy of prediction through calculation of this factor is sufficient when rifampicin is considered alone, and the use of a larger set of fully characterized CYP3A4 clinical inducers is not required. For the established HepaRG cell line, the findings obtained in three experiments using a single batch of cells show a good prediction accuracy with or without the d factor. Additional investigations with different batches of HepaRG cell lines are needed to confirm these results.

Evaluation of human hepatocytes under prolonged culture in a novel medium for the maintenance of hepatic differentiation: results with the model pro-inflammatory cytokine interleukin 6.

A major challenge for the evaluation of cytokine-induced down regulation of CYP gene expression in primary cultured hepatocytes is the spontaneous decrease in expression of the genes with culture duration. Based on our recent discovery that hepatocytes cultured for 7 days in a novel medium, Li's Differentiation Maintenance Medium (LDMM), would retain gene expression for markers of differentiation and most CYP isoforms at levels similar to those of the first day of culture, we examined the effects of the prototypical pro-inflammatory cytokine IL-6 in the "LDMM-stabilized (LS)" human hepatocyte model. The LS-human hepatocyte cultures were found to be responsive to IL-6 induction of the inflammatory gene marker, C-reactive protein (CRP), suggesting the expression of IL-6 receptors and the subsequent signaling pathways. Results from two independent laboratories with human hepatocytes from three donors demonstrated dose-dependent down regulation of the gene expression of several CYPs, i.e. 1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4. The results suggest that the LS-human hepatocytes may represent a physiologically relevant experimental model for mechanistic investigation of the down-regulatory effects of inflammatory cytokines.