Cytochrome P450 (CYP) induction plays an important role in the pharmacokinetics of a drug and can have consequences for drug efficacy through the reduction of plasma half-life, or drug toxicity if elevated levels of toxic metabolites are formed. These effects are commonly observed when one drug has an effect on a co-administered medication – a term known as drug-drug interactions or DDI.
Transcriptional gene activation, mediated by nuclear receptors such as the aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR), is the most common mechanism of CYP induction. These receptors correlate directly to the expression of CYP1A2 (AhR), CYP2B6 (CAR), and CYP3A4 plus the CYP2C enzymes (PXR). Therefore, receptor activation can be used as an early indicator of potential changes in CYP enzyme expression.
A less common mechanism for CYP induction is through mRNA or enzyme stabilisation. In this case, certain drugs don’t necessarily stimulate CYP enzyme expression, but rather, slow down CYP protein degradation.
In drug discovery, cell-based transactivation assays can be used for identifying CYP induction potential. In this assay, stably or transiently transfected cell lines containing the nuclear receptor to be evaluated and reporter gene vectors are used. Activation of the response elements following receptor heterodimerisation serves as a suitable proxy for CYP induction. Results are typically reported as Emax and EC50, or a concentration-dependent fold activation relative to vehicle control.
For more advanced drug development, such as IND-enabling studies and NDA submission, CYP induction is typically evaluated as part of a more extensive DDI package. At this stage, cryopreserved human hepatocytes are the preferred model with at least three donors assessed to account for inter-individual variability in response. The hepatocytes are typically incubated with the test compound over 48 to 72 hours, and CYP enzyme induction is evaluated by measuring mRNA levels and/or measuring the catalytic activity of an isoform-specific probe substrate. Because mRNA detection isn’t subjected to the masking effects of time dependent inhibition, regulatory authorities such as the US FDA and EMA recommend this method. However, if protein stabilisation is expected, catalytic activity analysis should also be conducted. Once again, results are typically reported as an Emax and EC50, or concentration-dependent fold increase in response relative to the control. In addition, measurement of test compound over several time points on the last day of incubation is encouraged.
