Induction of human cytochrome P450 enzymes : predictive in vitro models and rifampicin induction in vivo

Abstract: The cytochrome P450 (P450) enzymes comprise the most important enzyme system with regard to phase I metabolism of drugs. Induction of P450s can result in decreased plasma concentrations of the drug itself or a coadminstered drug, followed by lack of effect. In the present study different in vitro models have been investigated for their ability to predict P450 induction in humans. It was found that human liver slices respond to prototypical inducers, although the model is not applicable to screening of large sets of compounds. High throughput screening can however be performed in a reporter gene assay. The study showed that results from a PXR reporter gene assay could be used to classify compounds as CYP3A in vivo inducers or non-inducers when relating in vivo AUC to PXR EC50 values. Subsequently it was shown that instead of EC50 values, the concentration giving a 2-fold increase of baseline levels (F2 values) could be used for the classification of compounds. A new cell line, HepaRG cells, was also investigated for prediction of P450 induction. Results from experiments in HepaRG cells did not only classify compounds as inducers or non-inducers, but gave a strong correlation (R2=0.863) to in vivo CYP3A induction, and could hence be used to quantitatively predict the extent of CYP3A induction in vivo. In addition, the drug metabolising properties of the HepaRG cells were evaluated. Stable mRNA expression of drug metabolising enzymes, transporters, and liver specific factors in HepaRG cells were shown for up to six weeks in culture. Although the mRNA expression of drug metabolising P450s were lower in HepaRG cells as compared to human hepatocytes, the relative levels of the P450s were similar. The HepaRG cells could thus be used not only for induction studies but also for investigation of metabolic pattern of drugs and new chemical entities. Furthermore, the in vivo induction of P450s by three different daily doses (20, 100, and 500 mg) of rifampicin was investigated. Rifampicin is perhaps the most well documented CYP3A inducer in vivo, and is used as a positive control in induction studies in vitro. Rifampicin is also an inducer of CYP1A, CYP2B6, and CYP2C enzymes. By the use of the Karolinska cocktail, the response of four P450s could be investigated at one time point in the same subject. CYP1A2 and CYP2C9 were induced after 500 mg rifampicin daily, and CYP2C19 after 100 mg rifampicin daily. A strong 4-fold induction of CYP3A4 was seen at 500 mg rifampicin daily for both quinine/3 -hydroxyquinine and 4ß-hydroxycholesterol measurements (p<0.001). CYP3A4 was also induced at the two lower doses of rifampicin measured by either of these two markers (p<0.01). A strong correlation (Spearman rank rs=0.71; 95% C.I.=0.52-0.90; p<0.001; n=22) of the two CYP3A4 markers indicates that the cholesterol metabolite 4β-hydroxycholesterol could be used as an endogenous marker for CYP3A4 induction. By the use of 4β-hydroxycholesterol, CYP3A4 induction can be investigated concurrently with the pharmacokinetics of the drug candidate in vivo, and no separate CYP3A induction study is needed.