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ADME PK

PXR and AhR 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 and AhR nuclear receptor activation utilizing stably-transfected human hepatoma cell lines (DPX2™ for PXR and 1A2-DRE™ for AhR) and a luciferase reporter gene assay.
Cyprotex also offers cytochrome P450 induction using human hepatocytes to meet with regulatory recommendations.

 

Because reporter assays are relatively high throughput and cost effective, they can be a valuable tool in drug discovery.

1Chu V, Einolf HJ, Evers R, Kumar G, Moore D, Ripp S, Silva J, Sinha V, Sinz M, and Skerjanec A (2009) Drug Metab Dispos 37; 1339-1354.

Protocol

PXR and AhR nuclear receptor activation assay protocol

Method Luciferase reporter gene assay
Cell Lines DPX2™ for PXR and 1A2-DRE™ cells for AhR (supplied by Puracyp)
Test Article Concentrations 0.1, 0.4, 1, 4, 10, 40, 100 µM (different concentrations available)
Quality Controls Vehicle control

Positive control (rifampicin for PXR and omeprazole for AhR)
Number of Replicates 2 replicates per concentration
Test Article Requirements 50 µL of a 100 mM DMSO solution
Data Delivery Fold activation relative to vehicle control

EC50 and Emax (if appropriate)

% Cell loss at each concentration and EC50 (if appropriate)

Data

Data from Cyprotex's PXR and AhR nuclear receptor activation assay

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

 
Figure 1
PXR nuclear receptor activation by rifampicin.

Data show the mean fold activation relative to the vehicle control. Error bars represent the standard deviation of 3 replicate incubations.
CompoundCyprotex EC50 (µM)Cyprotex Emax
(fold activation)
Literature Emax
(fold activation)
Rifampicin 1.1 25.7 at 10 µM 30.6 at 10 µM
Dexamethasone 24.2 9.9 at 100 µM 7.1 at 250 µM
Troglitazone 3.7 9.4 at 20 µM 8.3 at 50 µM
Omeprazole 22.4 12.5 at 100 µM 25.9 at 100 µM
Phenobarbital 323 19.3 at 1000 µM 13.5 at 1000 µM
Mifepristone 0.8 17.5 at 10 µM 9.3 at 10 µM
Table 1
PXR activation data for a set of 6 compounds and comparison against literature data2. The results show that data generated at Cyprotex compare well with data generated in the literature. No cytotoxicity was observed for any of the compounds at the concentration range tested with the exception of troglitazone for which cytotoxicity was observed at the highest concentration of 50 µM. This data point was excluded in this instance and not used for calculating the Emax or EC50.

References

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

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