Drug Metabolism and Pharmacokinetics, Suven Life Sciences Ltd., Jeedimetla, Hyderabad, India (V.R.C.P., G.B., R.N.); Bio-analysis, Suven Life Sciences Ltd., Pashamylaram, Medak, India (P.C., D.R.A.); and In Vitro ADMET Laboratories Inc., Columbia, Maryland (A.P.L.).
Drug Metabolism and Pharmacokinetics, Suven Life Sciences Ltd., Jeedimetla, Hyderabad, India (V.R.C.P., G.B., R.N.); Bio-analysis, Suven Life Sciences Ltd., Pashamylaram, Medak, India (P.C., D.R.A.); and In Vitro ADMET Laboratories Inc., Columbia, Maryland (A.P.L.)
Drug Metab Dispos. 2019 Oct;47(10):1032-1039. doi: 10.1124/dmd.119.088237. Epub 2019 Aug 2.
Here, we report the application of a novel hepatocyte system, the cofactor-supplemented permeabilized cryopreserved human hepatocytes [MetMax human hepatocytes (MMHHs)] in a higher-throughput 384-well plate assay for the evaluation of cytochrome P450 (P450) inhibition. The assay was created to develop physiologically relevant P450 inhibition information, taking advantage of the complete organelle composition and their associated drug-metabolizing enzymes of the MMHH but with the ease of use of human liver microsomes, including storage at -80°C instead of in liquid nitrogen, and thaw and use without centrifugation and microscopic evaluation as required for intact hepatocytes. Nine key P450 isoforms for drug metabolism (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) were evaluated using multiple isoform-selective inhibitors. Results with MMHH were found to be comparable to those obtained with intact cryopreserved human hepatocytes (CHHs). Isoform-selective drug-metabolizing enzyme pathways evaluated were phenacetin -deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), bupropion hydroxylation (CYP2B6), amodiaquine -deethylation (CYP2C8), diclofenac 4-hydroxylation (CYP2C9), -mephenytoin 4'-hydroxylation (CYP2C19), dextromethorphan -demethylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), and midazolam 1'-hydroxylation and testosterone 6-hydroxylation (CYP3A4). The K values obtained with MMHHs were comparable with those reported in the literature for CHHs. Using substrate concentrations at or near K values, the IC values for the standard inhibitors against the P450 activities were found to be comparable between MMHHs and CHHs, with 73% and 84% of values falling within 2-fold and 3-fold, respectively, from the line of unity. The results indicate that MMHHs can be an efficient experimental system for the evaluation of P450 inhibition in hepatocytes. SIGNIFICANCE STATEMENT: MetMax human hepatocytes (MMHHs) are cofactor-supplemented cryopreserved human hepatocytes with the complete drug-metabolizing enzyme pathways of the conventional hepatocytes but with the convenience of human liver microsomes, including storage at -80°C instead of in liquid nitrogen, and direct thaw and use without a need for centrifugation and microscopic examination. Here, we report the application of MMHH in a high-throughput assay in a 384-well plate format for the evaluation of cytochrome P450 (P450) inhibition. Our results show that data obtained with MMHH are similar to those with conventional hepatocytes, suggesting that the MMHH 384-well P450 inhibition assay can be used routinely for the evaluation of drug-drug interaction potential of new chemical entities in drug development.
在这里，我们报告了一种新型肝细胞系统的应用，即补充辅因子的渗透冷冻保存人肝细胞[MetMax 人肝细胞（MMHHs）]在高通量 384 孔板测定中用于评估细胞色素 P450（P450）抑制。该测定方法旨在开发与生理相关的 P450 抑制信息，利用 MMHH 完整的细胞器组成及其相关的药物代谢酶，但使用人肝微粒体具有易用性，包括在-80°C 下储存，而不是在液氮中储存，以及解冻和使用，无需为完整的肝细胞进行离心和显微镜评估。使用多种同工酶选择性抑制剂评估了 9 种用于药物代谢的关键 P450 同工酶（CYP1A2、CYP2A6、CYP2B6、CYP2C8、CYP2C9、CYP2C19、CYP2D6、CYP2E1 和 CYP3A4）。结果发现，MMHH 的结果与使用冷冻保存的完整人肝细胞（CHHs）获得的结果相当。评估了同工酶选择性药物代谢酶途径，包括对乙酰氨基酚去乙基化（CYP1A2）、香豆素 7-羟化（CYP2A6）、丁丙诺啡羟化（CYP2B6）、阿莫地喹去乙基化（CYP2C8）、双氯芬酸 4-羟化（CYP2C9）、-美芬妥因 4'-羟化（CYP2C19）、右美沙芬去甲基化（CYP2D6）、氯唑沙宗 6-羟化（CYP2E1）以及咪达唑仑 1'-羟化和睾酮 6-羟化（CYP3A4）。与文献中报道的 CHHs 相比，使用 MMHHs 获得的 K 值是可比的。使用接近 K 值的底物浓度，标准抑制剂对 P450 活性的 IC 值在 MMHHs 和 CHHs 之间是可比的，分别有 73%和 84%的值在 2 倍和 3 倍以内，分别与单位线一致。结果表明，MMHHs 可以成为评估肝细胞 P450 抑制的有效实验系统。
MetMax 人肝细胞（MMHHs）是补充辅因子的冷冻保存人肝细胞，具有传统肝细胞的完整药物代谢酶途径，但具有人肝微粒体的便利性，包括在-80°C 下储存，而不是在液氮中储存，以及直接解冻和使用，无需离心和显微镜检查。在这里，我们报告了在高通量 384 孔板格式中使用 MMHH 进行细胞色素 P450（P450）抑制评估的应用。我们的结果表明，使用 MMHH 获得的数据与使用传统肝细胞获得的数据相似，这表明 MMHH 384 孔板 P450 抑制测定法可常规用于评估新药在药物开发中的药物相互作用潜力。
