Pharmaceutical Research Division, Discovery Research Center, Takeda Pharmaceutical Company Limited, Yodogawa-ku, Osaka 532-8686, Japan.
Drug Metab Dispos. 2010 Nov;38(11):1912-6. doi: 10.1124/dmd.109.028613. Epub 2010 Aug 10.
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-hydroxylation. Concentration-dependent CYP3A induction of rifampicin was reproducible with the EC(50) values of 0.36 ± 0.28 μM 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 μM were well comparable among the multiple donors, by expressing the results as percentage of change compared with the positive control, 10 μM rifampicin. Cotreatment of avasimibe or efavirenz with 10 μM 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.
建立了一种可靠且实用的适用于 96 孔板的冷冻人原代肝细胞 CYP3A 诱导测定法。使用典型诱导剂利福平、苯妥英和卡马西平对具有不同基底膜的各种 96 孔板进行了评估。研究发现，薄层（TL）Matrigel 在睾酮 6β-羟化酶测定中产生的 CYP3A 活性的基础和诱导水平最高。使用 TL Matrigel 的 96 孔板，从 4 个人肝细胞批次中，利福平的 EC50 值为 0.36±0.28μM，可重复检测到利福平的浓度依赖性 CYP3A 诱导。在 10μM 浓度下，用 9 种诱导剂或非诱导剂处理时，以与阳性对照（10μM 利福平）相比的百分比变化表示结果，多个供体之间的诱导效力排序非常相似。与单独用利福平处理相比，阿伐麦布或依法韦仑与 10μM 利福平共同处理时，降低利福平诱导的 CYP3A 活性的速度较慢，而用苯巴比妥和卡马西平处理则没有影响。从诱导的 CYP3A 活性和基因表达水平的比较来看，有些化合物会导致 CYP3A4mRNA 的诱导，但不会导致活性的诱导，可能是因为它们对 CYP3A 活性具有抑制作用。在评估候选药物对 CYP3A 诱导的能力时，用利福平对测试化合物进行联合处理可以排除因细胞毒性和/或基于机制的失活而导致的假阴性结果。该 96 孔板测定法具有稳健、可重复和方便的特点，已证明在早期药物发现阶段具有重要的适用性。
