Nuclear Receptor Activation

• The primary mechanism of cytochrome P450 induction is via increased gene transcription which typically occurs through nuclear receptor activation.
• The most common nuclear receptors involved in the induction of drug metabolizing enzymes include the pregnane X receptor (PXR), the aryl hydrocarbon receptor (AhR), and the constitutive androstane receptor (CAR) which are known to regulate CYP3A4, CYP1A2 and CYP2B6, respectively.
• An industry survey of current practices and recommendations (¹Chu et al., (2009) Drug Metab Dispos 37; 1339) indicates 64% of survey respondents routinely use nuclear receptor transactivation assays to assess the potential of test compounds to cause enzyme induction.
• Cyprotex can evaluate PXR, AhR and CAR3 using INDIGO Bioscience PXR, AhR and CAR3 reporter cells (human cells engineered to express human PXR, AhR and CAR3) with luciferase activity measured via luminescence and cell viability assessed through fluorescence.

Known activator of PXR was selected and screened in the PXR nuclear receptor activation assay. Data generated were compared to those published in the literature.

Data analysis of the nuclear receptor activation assay:

Transcriptional activation is monitored by luminescence. Data are expressed as fold activation relative to the vehicle control, following normalization of luciferase activity against cell viability. The use of 5 or more doses of test article and positive control allows for the derivation of EC₅₀ and E_max values from nonlinear regression analysis of the log dose‑response curves. These outputs may not be determined with test articles exhibiting atypical dose‑response curves or with poorly defined parameters. The average observed fold activation is calculated as a percentage of the positive control to allow for inter‑day comparison and ranking of test articles, using the equation below:

_{1) Chu V et al., (2009) Drug Metab Dispos 37; 1339-1354
2) Yueh M-F et al., (2005) Drug Metab Dispos 33; 38-48}