Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki FI-00014, Finland.
Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki FI-00014, Finland.
Eur J Pharm Sci. 2024 Sep 1;200:106852. doi: 10.1016/j.ejps.2024.106852. Epub 2024 Jul 15.
Preincubation with inhibitor in organic anion transporting polypeptide (OATP) in vitro assays may increase the inhibition potency of inhibitors compared to conventional inhibition assays with only short inhibitor coincubation with substrate. The decrease in IC may affect prediction of drug-drug interactions (DDI) involving these transporters and inhibitors. Only few drugs, however, have been assessed for the preincubation-dependent inhibition of the OATP2B1 transporter. Therefore, we studied the effect of preincubation on OATP2B1 inhibition with five known OATP2B1 inhibitors (atorvastatin, erlotinib, ezetimibe, ticagrelor and simeprevir) in HEK293 cells transiently overexpressing OATP2B1. IC values were determined with and without inhibitor preincubation for 20 min with three different OATP2B1 substrates (dibromofluorescein, DBF; 5-carboxyfluorescein, 5-CF; estrone sulfate). Atorvastatin, ezetimibe, and simeprevir displayed more than 2-fold lower IC values after preincubation with at least one of the tested substrates. Altogether, 4 out of 15 inhibitor/substrate combinations exhibited more than 2-fold potentiation of IC after inhibitor preincubation. In addition, preincubation by itself, without inhibitor present with the substrate, resulted in more than 50% inhibition of OATP2B1-mediated uptake of DBF and/or 5-CF by atorvastatin, ticagrelor and simeprevir. Thus, erlotinib was the only inhibitor with no indication of potentiation of inhibition by preincubation with any of the tested substrates. In conclusion, preincubation resulted in inhibitor- and substrate-dependent inhibition of OATP2B1. These results support the conclusion that to reduce the risk of false negative DDI prediction, preincubation should be considered also in OATP2B1 inhibition assays.
在体外有机阴离子转运多肽（OATP）试验中，与仅用短时间抑制剂与底物共孵育的传统抑制试验相比，抑制剂的预孵育可能会增加抑制剂的抑制效力。IC 值的降低可能会影响涉及这些转运体和抑制剂的药物相互作用（DDI）的预测。然而，只有少数药物已被评估用于 OATP2B1 转运体的预孵育依赖性抑制。因此，我们在瞬时过表达 OATP2B1 的 HEK293 细胞中研究了五种已知的 OATP2B1 抑制剂（阿托伐他汀、厄洛替尼、依折麦布、替格瑞洛和simeprevir）的预孵育对 OATP2B1 抑制的影响。使用三种不同的 OATP2B1 底物（二溴荧光素、DBF；5-羧基荧光素、5-CF；雌酮硫酸酯），在有无抑制剂预孵育 20 分钟的情况下，测定 IC 值。阿托伐他汀、依折麦布和simeprevir 在与至少一种测试底物预孵育后，IC 值降低了 2 倍以上。总共，在抑制剂预孵育后，有 15 个抑制剂/底物组合中的 4 个表现出 IC 值的增强超过 2 倍。此外，即使没有抑制剂与底物一起存在，预孵育本身也会导致阿托伐他汀、替格瑞洛和simeprevir 对 OATP2B1 介导的 DBF 和/或 5-CF 摄取的抑制率超过 50%。因此，厄洛替尼是唯一一种没有迹象表明与任何测试底物预孵育后抑制作用增强的抑制剂。总之，预孵育导致 OATP2B1 的抑制剂和底物依赖性抑制。这些结果支持这样的结论，即为了降低假阴性 DDI 预测的风险，也应考虑在 OATP2B1 抑制试验中进行预孵育。
