Cytochrome P450 Induction Assays

The cytochrome P450 (CYP450) induction assay provides a means to assess whether a test compound increases the production of specific CYP450 enzymes. We provide CYP450 induction assays for all small molecule formulations such as pharmaceuticals, industrial chemicals, and consumer products.

Our CYP450 induction assays use genetically modified versions of the immortalized human liver cell line HepaRG™, created with our proprietary CompoZr® zinc finger nuclease (ZFN) technology. Apart from fresh human hepatocytes, HepaRG cells are the most metabolically active liver cell line described to date and have the potential for use as a viable surrogate in many functional liver assays, including cytochrome P450 induction, with none of the drawbacks of limited availability and donor-to-donor variation. The modified lines include clone 5F, which has enhanced functional properties, including response to CYP inducers, and knockout lines of individual nuclear receptors (PXR, CAR and AhR), which can be used with control cells to identify nuclear receptor interactions.

Cytochrome P450 (CYP) enzymes can be induced by many xenobiotics and drugs through the activation of nuclear receptors such as constitutive androstane receptor (CAR), pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR). Understanding the potential for CYP induction by a new chemical entity (NCE) is important to minimize adverse drug-drug interactions. CYP induction may be determined by measuring changes in either enzyme activity or mRNA levels. Because enzyme inhibition and cytotoxicity may affect results from activity assays, while effects on mRNA stabilization may be missed if just mRNA levels are assessed, using both endpoints provides the most comprehensive assessment of CYP induction potential.

Our CYP450 induction assays are available in two formats.

Standard Assessment
Here the ability of a test compound to induce the three main metabolically relevant cytochrome P450 enzymes (CYP1A2, CYP2B6 and CYP3A4) is assessed in the HepaRG liver cell line, clone 5F, alongside control compounds.

Nuclear Receptor Interaction Assessment
Follow-up options for “positives” from the standard assay include confirmation of interaction with a specific nuclear receptor (PXR, CAR or AhR) by using individual HepaRG liver knockout cell lines (PXR KO, CAR KO and AhR KO) and comparing induction results with control cells.
 

 Cytochrome P450 Induction Assay Protocol

 
Test System HepaRG liver cell line, clone 5F (human)
Test Compound Concentration 0.1, 1 and 10 µM (or custom)
CYP Isoforms Assessed CYP1A2, CYP2B6 and CYP3A4
Number of Replicates 3
Negative Control Vehicle (0.1% DMSO)
Positive Controls Omeprazole (CYP1A2)
CITCO (CYP2B6)
Rifampin (CYP3A4)
Compound Requirements 30 µL of 10 mM solution
Exposure Period 72 hours (media changed every 24 hours)
Probe Substrates Phenacetin (CYP1A2)
Bupropion (CYP2B6)
Testosterone (CYP3A4)
Analysis Method LC-MS/MS quantification of probe substrate metabolites: acetaminophen (CYP1A2), hydroxybupropion (CYP2B6) and
6β-hydroxytestosterone (CYP3A4)
RT-PCR of mRNA expression (CYP1A2, CYP2B6 and CYP3A4)
Data Delivery Assay methods
Basal and induced activity (pmol/min/mg protein)
mRNA levels
Fold induction above vehicle control
% of positive control induction

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