Drug Tox Testing
Testing for CYP450 induction is part of drug-drug interaction (DDI) studies, and cell viability assays serve as a control in such studies. Cytochrome P450s are a class of enzymes that catalyze metabolism of drugs, but only a small number of these enzymes catalyze the majority of drug metabolism. Some CYP450 genes are induced by drug compounds. For example, CYP3A4 and -1A2 are important to the FDA because they metabolize a substantial number of drugs and represent two distinct induction pathways.
CYP450 gene inductions are important because they can cause adverse drug-drug interactions. In other words, if two drugs are co-administered and one drug causes the accelerated metabolism of the second one by way of P450 induction, reduced efficacy of the second drug is the result.
Recently, Promega, BioTek Instruments, and Celsis In Vitro Technologies collaborated to produce a multifunctional assay for the measurement of CYP450 induction. “Our assay design lends itself to high-throughput screening,” said Timothy Moeller, scientific advisor at Celsis, who added that an important novelty of the collaborative assay design is that these assays have not previously been performed in 384-well plates.
“As DDI studies are moved into a 384-well format, and a greater number of compounds are evaluated, it is essential to incorporate automation into the assay process,” said Brad Larson, principal scientist at BioTek. Two instruments from BioTek were used in the new design to handle all liquid handling functions.
“Each liquid handler has a small footprint enabling it to be placed into existing laminar flow hoods. Due to the fact that induction assays are typically performed over multiple days, sample sterility is extremely important. Automation such as that used here allows the researcher to greatly reduce or eliminate the risk of contamination without having to exhaust the monetary resources of the research lab,” added Larson.
Celsis supplied the human hepatocytes that are the third component of this collaborative assay design effort. Hepatocytes give all the individual variation of real liver cells including variable induction of cytochrome P450s, making them the gold standard for this cell-based tox testing. “Toxicity in cell culture is well established as a predictor of toxicity in the whole animal or human being,” said James Cali, Ph.D., R&D director, assay design at Promega.
The cell viability and CYP3A4 assay technologies are Promega’s CellTiterGlo and P450-Glo, respectively. “Our CellTiterGlo product correlates ATP level to cell viability.” The CYP3A4/1A2 multiplex relies on the combined strength of a luminescent substrate for CYP3A4 and a fluorescent one for CYP1A2. The addition of the CellTiterGlo viability assay adds a parameter for normalization to cell number and simultaneous toxicity testing.
“Our assay configuration was designed for discovery-phase ADME testing. This method allows the drug company to characterize multiple compounds for DDI and toxicity liabilities so as to fail problematic compounds early and cheap.” Dr. Cali added that by increasing throughput, adding automation, and reducing well volume, drug companies are able to apply what has traditionally been late-stage analysis to an early secondary profiling stage.