Transactivation Assays to Assess Canine and Rodent Pregnane X Receptor (PXR) and Constitutive Androstane Receptor (CAR) Activation.

The pregnane X receptor (PXR/SXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3) are nuclear receptors (NRs) involved in the regulation of many genes including cytochrome P450 enzymes (CYPs) and transporters important in metabolism and uptake of both endogenous substrates and xenobiotics. Activation of these receptors can lead to adverse drug effects as well as drug-drug interactions. Depending on which nuclear receptor is activated will determine which adverse effect could occur, making identification important. Screening for NR activation by New Molecular Entities (NMEs) using cell-based transactivation assays is the singular high throughput method currently available for identifying the activation of a particular NR. Moreover, screening for species-specific NR activation can minimize the use of animals in drug development and toxicology studies. With this in mind, we have developed in vitro transactivation assays to identify compounds that activate canine and rat PXR and CAR3. We found differences in specificity for canine and rat PXR, with the best activator for canine PXR being 10 µM SR12813 (60.1 ± 3.1-fold) and for rat PXR, 10 µM dexamethasone (60.9 ± 8.4 fold). Of the 19 test agents examined, 10 and 9 significantly activated rat and canine PXR at varying degrees, respectively. In contrast, 5 compounds exhibited statistically significant activation of rat CAR3 and 4 activated the canine receptor. For canine CAR3, 50 µM artemisinin proved to be the best activator (7.3 ± 1.8 and 10.5 ± 2.2 fold) while clotrimazole (10 µM) was the primary activator of the rat variant (13.7 ± 0.8 and 26.9 ± 1.3 fold). Results from these studies demonstrated that cell-based transactivation assays can detect species-specific activators and revealed that PXR was activated by at least twice as many compounds as was CAR3, suggesting that there are many more agonists for PXR than CAR.

Current in vitro high throughput screening approaches to assess nuclear receptor activation.

The screening of new drug candidates for nuclear receptor activation can identify agents with the potential to produce drug-drug interactions or elicit adverse drug effects. The nuclear receptors of interest are those that control the expression of drug metabolizing enzymes and drug transporters, and include the constitutive androstane receptor (CAR, NR1I3), the pregnane X receptor (PXR, NR1I2) and the aryl hydrocarbon receptor (AhR). This review will focus on the methods currently used to assess activation of these receptors. Assessment of nuclear receptor activation can be accomplished using direct or indirect approaches. Indirect methods quantify specific gene products that result from nuclear receptor activation while direct approaches measure either the binding of ligands to the receptors or the transcriptional events produced by ligand binding. Assays that directly quantify nuclear receptor activation are growing in popularity and, importantly, are amenable to high throughput screening (HTS). Several ligand binding assays are currently being utilized, including radioligand competition binding, where compounds compete with radiolabelled ligand for binding to PXR or CAR, such as the scintillation proximity binding assay that measures the reaction of ligands with receptor-coated beads. A fluorescence resonance energy transfer assay has also been developed, where the fluorescent signal is generated via the ligand-dependent interaction between the fluorescently-labeled ligand binding domain of a nuclear receptor and co-activator proteins. Other in vitro activation assays include transient- and stably-transfected cell lines incorporating an expression vector for PXR, CAR or AhR plus a reporter gene vector containing response elements. The methods focused on in this review will be limited to the more direct in vitro approaches that are amenable to high throughput screening.

Regulation of CYP3A4 expression in human hepatocytes by pharmaceuticals and natural products.

Human CYP3A4 metabolizes a majority of clinically important substrates at variable rates. Accounting for these unpredictable rates is the wide variation noted in expression of this enzyme that is due, in part, to xenobiotic exposure. We used primary cultures of human hepatocytes from 17 individuals to assess the inducibility of CYP3A4 mRNA by prototypical inducers, dietary flavonoids, and botanicals. Those agents producing the greatest mRNA accumulation were 10 microM RIF (699 +/- 307% of control levels) 100 microM phenytoin (707 +/- 188% of control), 1 mM phenobarbital (536 +/- 207% of control), and 100 microM omeprazole (404 +/- 8% of control). Various concentrations of RIF were found to exhibit a typical dose-response curve for CYP3A4 mRNA content. A reporter gene assay using the human pregnane X receptor (hPXR) and promoter regions of CYP3A4 transiently transfected into HepG2 cells, exhibited inductive properties by the aforementioned therapeutics that were similar to those observed in hepatocytes. Several flavonoids including quercetin, resveratrol, and curcumin were also examined for their ability to induce CYP3A4 in human hepatocytes. Only quercetin produced accumulation of CYP3A4 mRNA (230 +/- 73% of control). When examined in a reporter gene assay, this flavonoid exhibited negligible increases in luciferase activity suggesting that quercetin induced CYP3A4 by mechanisms that may not involve PXR. We also examined the effects of herbals on CYP3A4 expression in human hepatocytes. Grapeseed extract, ginseng, silymarin, and kava-kava produced 270 +/- 73, 155 +/- 83, 100 +/- 10, and 386 +/- 185% of control CYP3A4 mRNA, respectively. Of these botanicals only kava-kava produced enhanced luciferase activity (11.6 +/- 2.1 fold above DMSO treated cells). Such results indicate that kava-kava required PXR to mediate CYP3A4 induction. Collectively, results demonstrated that several botancials induce CYP3A4, suggesting the potential for drug-herbal interactions.