Short Communication A 96-Well Plate Assay for CYP4503A Induction Using Cryopreserved Human Hepatocytes
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چکیده
A reliable and practical CYP3A 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 carbamazepine. Thin-layer (TL) Matrigel was found to yield the highest basal and induced levels of CYP3A activity as determined by testosterone 6 -hydroxylation. Concentration-dependent CYP3A induction of rifampicin was reproducible with the EC50 values of 0.36 0.28 M from four batches of human hepatocytes using the 96-well plate with TL Matrigel. The rank order of induction potency for nine inducers or noninducers at a concentration of 10 M were well comparable among the multiple donors, by expressing the results as percentage of change compared with the positive control, 10 M rifampicin. Cotreatment of avasimibe or efavirenz with 10 M rifampicin was found to reduce CYP3A activities induced by rifampicin at a lower rate than treatment with rifampicin alone, whereas treatment with 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 cotreatment 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 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.
منابع مشابه
A 96-well plate assay for CYP4503A induction using cryopreserved human hepatocytes.
A reliable and practical CYP3A 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 carbamazepine. Thin-layer (TL) Matrigel was found to yield the highest basal and induced levels of CYP3A activity as determined by testosterone 6β-hydr...
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Due to the importance of in vitro cytochrome P450 (P450) induction assay to assess the possible drug-drug interaction events, the recent US Food and Drug Administration draft guidance and European Medicines Agency guideline recommend to assess P450 induction using fresh or cryopreserved hepatocytes at mRNA level and/or enzyme activity level. Although cryopreserved hepatocytes are commercially a...
متن کاملDmd056804 867..871
Due to the importance of in vitro cytochrome P450 (P450) induction assay to assess the possible drug-drug interaction events, the recent US Food and Drug Administration draft guidance and European Medicines Agency guideline recommend to assess P450 induction using fresh or cryopreserved hepatocytes at mRNA level and/or enzyme activity level. Although cryopreserved hepatocytes are commercially a...
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تاریخ انتشار 2010