A 96-well Plate Assay for CYP3A Induction Using Cryopreserved Human Hepatocytes

Abstract A reliable and practical cytochrome P450 (CYP) 3A induction assay with cryopreserved human hepatocytes in a 96-well format was developed. Various 96-well plates with different basement membrane were evaluated using prototypical inducers, rifampicin, phenytoin, and carbamazepin. Thin-layer (TL) matrigel was found to yield the highest basal and induced levels of CYP3A activity as determined by testosterone 6β-hydroxylation. Concentration-dependent CYP3A induction of μ M rifampicin. Co-treatment of avasimibe or efavirenz with 10 μ M rifampicin was found to reduce CYP3A activities induced by rifampicin lower than that treated rifampicin alone, whereas the phenobarbital and carbamazepine had no effect. From a comparison of induced CYP3A activities and gene expression levels, there were compounds that would cause induction of CYP3A4 mRNA but not activity, presumably due to their inhibitory effect on CYP3A activity. The co-treatment assay of test compound with rifampicin allows us to exclude the false negative results caused by the cytotoxicity and/or the mechanism-based inactivation, when the drug candidate's ability for CYP3A induction is evaluating by the enzyme activity. This 96-well plate assay, which is robust, reproducible and convenient, has demonstrated the paramount applicability to the early drug discovery stage