Drug Safety Research and Evaluation, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
Drug Safety Research and Evaluation, Takeda Pharmaceutical Company Limited, 35 Landsdowne Street, Cambridge, MA 02139, USA.
Toxicol In Vitro. 2021 Aug;74:105159. doi: 10.1016/j.tiv.2021.105159. Epub 2021 Apr 3.
Here, we established a high-throughput in vitro assay system to predict reactive metabolite (RM) formation. First, we performed the glutathione (GSH) consumption assay to monitor GSH levels as an index of RM formation potential using HepaRG cells pretreated with 500 μM D,L-buthionine-(S,R)-sulfoximine (BSO) and then treated with ticlopidine and diclofenac. Both drugs, under GSH-reduced conditions, significantly decreased relative cellular GSH content by 70% and 34%, respectively, compared with that in cells not pretreated with BSO. Next, we examined the correlation between GSH consumption and covalent binding assays; the results showed good correlation (correlation coefficient = 0.818). We then optimized the test compound concentration for evaluating RM formation potential using 76 validation compound sets, and the highest sensitivity (53%) was observed at 100 μM. Finally, using HepG2 cells, PXB-cells, and human primary hepatocytes, we examined the cell types suitable for evaluating RM formation potential. The expression of CYP3A4 was highest in HepaRG cells, suggesting the highest sensitivity (56.4%) of the GSH consumption assay. Moreover, a co-culture model of PXB-cells and HepaRG cells showed high sensitivity (72.7%) with sufficient specificity (85.7%). Thus, the GSH consumption assay can be used to effectively evaluate RM formation potential in the early stages of drug discovery.
在这里，我们建立了一种高通量的体外测定系统来预测反应性代谢物（RM）的形成。首先，我们进行了谷胱甘肽（GSH）消耗测定，以监测 GSH 水平作为 RM 形成潜力的指标，使用经 500 μM D,L-丁硫氨酸-(S,R)-亚砜（BSO）预处理的 HepaRG 细胞，然后用噻氯匹定和双氯芬酸处理。在 GSH 还原条件下，这两种药物分别使相对细胞 GSH 含量显著降低 70%和 34%，与未用 BSO 预处理的细胞相比。接下来，我们检查了 GSH 消耗与共价结合测定之间的相关性；结果表明相关性良好（相关系数=0.818）。然后，我们使用 76 个验证化合物集优化了用于评估 RM 形成潜力的测试化合物浓度，在 100 μM 时观察到最高的灵敏度（53%）。最后，使用 HepG2 细胞、PXB 细胞和人原代肝细胞，我们检查了适合评估 RM 形成潜力的细胞类型。HepaRG 细胞中 CYP3A4 的表达最高，表明 GSH 消耗测定的灵敏度最高（56.4%）。此外，PXB 细胞和 HepaRG 细胞的共培养模型显示出高灵敏度（72.7%）和足够的特异性（85.7%）。因此，GSH 消耗测定可用于有效评估药物发现早期的 RM 形成潜力。
