Cryopreserved human hepatocytes: characterization of drug-metabolizing enzyme activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability, and drug-drug interaction potential

Cryopreserved human hepatocytes were extensively characterized in our labor atory. The post-thaw viability, measured via dye exclusion, ranged from 55 to 83% for hepatocytes cryopreserved from 17 donors. Post-thaw viability an d yield (viable cells per vial) were found to be stable up to the longest s torage duration evaluated of 120 days. Drug-metabolizing enzyme activities of the cryopreserved hepatocytes (mean of ten donors) as percentages of the freshly isolated cells were: 97% for cytochrome P450 isoform (CYP) 1A2, 78 % for CYP2A6, 96% for CYP2C9, 86% for CYP2C19, 90% for CYP2D6, 164% for CYP 3A4, 76% for UDP-glucuronidase, and 88% for umbelliferone sulfotransferase. Known species-differences in 7-ethoxycoumarin (7-EC) metabolism were repro duced by cryopreserved hepatocytes from human, rat, rabbit, dog, and monkey , illustrating the utility of cryopreserved hepatocytes from multiple anima l species in the evaluation of species-differences in drug metabolism. High er throughput screening (HTS) assays were developed using cryopreserved hum an hepatocytes for hepatotoxicity, metabolic stability, and inhibitory drug -drug interactions. Dose-dependent cytotoxicity, measured using MTT metabol ism as an endpoint, was observed for the known hepatotoxic chemicals tamoxi fen, clozapine. cadmium chloride! diclofenac, amiodarone, tranylcypromine, precocene II, but not for 2-thiouracil. Cell density- and time-dependent me tabolism of 7-EC and dextromethorphan were observed in the FITS assay for m etabolic stability. Known CYP isoform-specific inhibitors were evaluated in the HTS assay for inhibitory drug-drug interactions. Furafylline, sulfaphe nazole. quinidine. and ketoconazole were found to be specific inhibitors of CYP1A2, CYP1C9, CYP2D6, and CYP3A4, respectively. Tranylcypromine and diet hyldithiocarbamate were found to be less specific, with inhibitory effects towards several CYP isoforms, including CYP2A6, CYP2C9, CYP2C19. and CYP2E1 . These results suggest that cryopreserved human hepatocytes represent a us eful experimental tool for the evaluation of drug metabolism, toxicity, and inhibitory drug-drug interaction potential. (C) 1999 Elsevier Science Irel and Ltd. All rights reserved.