Relative Activation of Human Pregnane X Receptor versus Constitutive Androstane Receptor Defines Distinct Classes of CYP2B6 and CYP3A4 Inducers

Abstract

Both the human pregnane X receptor (hPXR) and constitutive androstane receptor (hCAR) are capable of regulating CYP3A4 and CYP2B6 gene expression. However, the majority of currently identified CYP3A4 and CYP2B6 inducers are confirmed activators of hPXR but not hCAR. To compare these receptors with respect to their chemical selectivities, 16 drugs known to induce CYP3A4 and/or CYP2B expression were evaluated for relative activation of hPXR versus hCAR. Because of the high basal but low chemical-induced activation of hCAR in immortalized cells, alternative methods were used to evaluate hCAR activation potential. Thirteen of the 16 compounds were classified as moderate to strong hPXR activators. In contrast, carbamazepine (CMZ), efavirenz (EFV), and nevirapine (NVP) were classified as negligible or weak hPXR activators at concentrations associated with efficacious CYP2B6 reporter or endogenous gene induction in primary human hepatocytes, suggesting potential activation of hCAR. Subsequent experiments demonstrated that these three drugs efficiently induced nuclear accumulation of in vivo-transfected enhanced yellow fluorescent protein-hCAR and significantly increased expression of a CYP2B6 reporter gene when hCAR was expressed in CAR Ϫ/Ϫ mice. In addition, using a recently identified, chemically responsive splice variant of hCAR (hCAR3), the hCAR activation profiles of the 16 compounds were evaluated. By combining results from the hPXR-and hCAR3-based reporter gene assays, these inducers were classified as hPXR, hCAR, or hPXR/hCAR dual activators. Our results demonstrate that CMZ, EFV, and NVP induce CYP2B6 and CYP3A4 preferentially through hCAR and that hCAR3 represents a sensitive tool for in vitro prediction of chemical-mediated human CAR activation.CYP3A4 and CYP2B6 are induced at the mRNA, protein, and activity levels by the same compounds, including rifampin, phenobarbital, clotrimazole, cyclophosphamide, calcium channel antagonists, HMG-CoA reductase inhibitors, and thiazolidinediones (Drocourt et al., 2001;Kocarek et al., 2002;Lindley et al., 2002;Sahi et al., 2003;Faucette et al., 2004). Coinduction of these enzymes is mediated through transcriptional activation of the corresponding genes by the nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR), which are capable of binding to the same response elements in the promoter regions of the CYP3A4 and CYP2B6 genes (Goodwin et al

Comparison of Immortalized Fa2N-4 Cells and Human Hepatocytes as in Vitro Models for Cytochrome P450 Induction

“…Interestingly, the EC 50 (2.3 M) value generated in human hepatocytes was significantly lower than that observed in Fa2N-4 cells, whereas the E max value (12.9-fold) was significantly higher (Table 4). This finding further emphasizes the importance of CAR in the regulation of CYP2B6 (Faucette et al, 2006(Faucette et al, , 2007.…”

“…2 and 3). Among the compounds tested, rifampicin, bosentan, moricizine, and ritonavir are reported as selective PXR-activators (LeCluyse, 2001;Luo et al, 2002;van Giersbergen et al, 2002), phenobarbital, phenytoin, and efavirenz are PXR/CAR dual activators (Hariparsad et al, 2004;Wang et al, 2004;Trubetskoy et al, 2005;Bell and Michalopoulos, 2006;Faucette et al, 2007), and CITCO and artemisinin are selective CAR activators (Maglich et al, 2003;Simonsson et al, 2006). Rifampicin-mediated CYP3A4 induction was conducted in Fa2N-4 cells and four batches of human hepatocytes (DMQ, 527, 455, and LHO).…”

“…Therefore, the low inductive response of phenytoin in Fa2N-4 cells is probably due to the low CAR expression. Similarly, efavirenz a relatively potent CAR but weak PXR activator (Faucette et al, 2007) also showed low CYP3A4 induction in Fa2N-4 cells (Fig. 3H).…”

Quantitative Prediction of in Vivo Profiles of CYP3A4 Induction in Humans from in Vitro Results with a Reporter Gene Assay

“…For the induction of CYP3A4, in addition to PXR, CAR may be involved at least partly. The success of our prediction for rifampicin and omeprazole may be supported by the fact that these two drugs are selective PXR activators (Faucette et al, 2007), and, consequently, the induction effect was considered to be caused predominantly through the activation of PXR. In contrast, phenobarbital, phenytoin, and carbamazepine also activate CAR along with PXR (Wang et al, 2004).…”

“…To calculate the value of CC, the R predict (t) values were fitted to the reported data of R in vivo values due to rifampicin treatment (Table 2) using the SAAM II program (SAAM Institute, Seattle, WA). Rifampicin was used as a compound to determine CC values, because it is a strong PXR-selective inducer used as a positive control in numerous in vivo and in vitro studies (Lemaire et al, 2004;Faucette et al, 2007). In the analysis, we used some physiological parameters, the absolute values of which are listed in Table 6.…”