One can’t have a discussion on drug-drug interactions without addressing cytochrome P450 (CYP). The metabolism and clearance of most drugs are primarily mediated by CYP. Adverse drug-drug interactions frequently occur because of altered P450 activities, explains Jim Cali, Ph.D., senior scientist at Promega (www.promega.com).
“For example, nuclear receptor-mediated induction of a P450 gene by a component of St. John’s Wort reduces efficacy of oral contraceptives by increasing their rate of metabolism.”
One of Promega’s areas of strength, he states, is in bioluminescent luciferase reporter gene assays, which are ideal for studying gene regulation by nuclear receptors. According to Dr. Cali, Promega has also developed luminogenic P450 enzyme assays that use luciferin derivatives as P450 substrates. By combining reporter gene assays with enzyme assays, one can predict P450 induction at both the transcriptional level and at the level of enzyme activity during the earliest stages of drug discovery.
“We have developed three assays for the CYP3A4 enzyme, which accounts for about 30% of the P450 in human liver and oxidizes about half of all drugs that are eliminated after prior metabolism,” explains Dr. Cali. “Luciferin-PFBE is ideal for cell-based measurement of CYP3A gene inductions by drugs. The cell-based assay is rapid and simple and can be performed in a nonlytic mode that leaves cells intact for additional analysis.”
In addition to its utility for measuring induction in cell-based assays each substrate can be used as a probe for in vitro biochemical assays of recombinant CYP3A4 to detect P450 inhibition by test compounds. Dr. Cali points out that running a reporter assay is only doing part of the job. “We get a more complete picture by pairing reporter assays with enzyme assays.”
The value of this approach is illustrated by considering the effects of bergamottin, a component of grapefruit juice, on CYP3A4 expression. Although bergamottin activates CYP3A4 gene transcription, its net effect is to reduce activity, because it is also an inhibitor of the CYP3A4 enzyme. This explains how grapefruit juice consumption slows clearance of CYP3A4-metabolized drugs such as the cholesterol-lowering statins.
“Reporter gene technology paired with P450 enzyme assays is a good way to get a full picture of how certain compounds impact drug disposition through nuclear receptor interactions,” notes Dr. Cali.