Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa, 920-1192, Japan.
Faculty of Pharma-Sciences, Teikyo University, 2-11-1 Kaga, Itabashi, Tokyo, 173-8605, Japan.
Pharm Res. 2022 Jul;39(7):1549-1559. doi: 10.1007/s11095-022-03223-z. Epub 2022 Mar 21.
Identification of blood-brain barrier (BBB) uptake transporters is a major challenge in the research and development of central nervous system (CNS) drugs. However, conventional methods that consider known drug uptake characteristics have failed at identifying the responsible transporter molecule. The present study aimed at identifying aripiprazole uptake transporters in BBB model hCMEC/D3 cells using a knockdown screening study targeting various transporters, including uncharacterized ones.
We evaluated the effect of 214 types of siRNA targeting transporters on the uptake of aripiprazole, an atypical antipsychotic drug, in hCMEC/D3 cells. Aripiprazole uptake was determined using Xenopus oocytes expressing the candidate genes extracted from the siRNA screening assay.
The estimated unbound brain to plasma concentration ratio (K) of aripiprazole was estimated as 0.67 in wild-type mice and 1.94 in abcb1a/1b/abcg2 knockout mice, suggesting the involvement of both uptake and efflux transporters in BBB permeation. According to siRNA knockdown screening studies, organic cation/carnitine transporter 2 (OCTN2) and long-chain fatty acid transporter 1 (FATP1) were identified as candidate genes. The uptake of aripiprazole by hCMEC/D3 cells was decreased by OCTN2 inhibitors, but not by FATP1 inhibitors. A partially increased uptake of aripiprazole was observed in OCTN2-expressing Xenopus oocytes. Finally, to evaluate transporter-mediated BBB permeation of drugs, the reported and estimated K values were summarized.
A knockdown screening study in combination with K values showed that aripiprazole was a potential substrate of OCTN2. The technique described in this study can be applied to identifying novel BBB transporters for CNS drugs.
鉴定血脑屏障（BBB）摄取转运体是中枢神经系统（CNS）药物研发的主要挑战。然而，考虑已知药物摄取特征的传统方法未能鉴定出负责的转运体分子。本研究旨在使用针对各种转运体（包括未鉴定的转运体）的敲低筛选研究，鉴定 BBB 模型 hCMEC/D3 细胞中的阿立哌唑摄取转运体。
我们评估了针对 214 种转运体的 siRNA 对 hCMEC/D3 细胞中阿立哌唑摄取的影响，阿立哌唑是一种非典型抗精神病药物。使用从 siRNA 筛选试验中提取的候选基因表达的 Xenopus oocytes 来测定阿立哌唑的摄取。
在野生型小鼠中，阿立哌唑的未结合脑/血浆浓度比（K）估计值为 0.67，在 abcb1a/1b/abcg2 敲除小鼠中为 1.94，表明摄取和外排转运体均参与 BBB 渗透。根据 siRNA 敲低筛选研究，鉴定出有机阳离子/肉碱转运体 2（OCTN2）和长链脂肪酸转运体 1（FATP1）为候选基因。hCMEC/D3 细胞中阿立哌唑的摄取被 OCTN2 抑制剂降低，但不受 FATP1 抑制剂的影响。在表达 OCTN2 的 Xenopus oocytes 中观察到阿立哌唑摄取的部分增加。最后，为了评估药物经转运体介导的 BBB 渗透，总结了报告和估计的 K 值。
结合 K 值的敲低筛选研究表明，阿立哌唑可能是 OCTN2 的底物。本研究中描述的技术可用于鉴定 CNS 药物的新型 BBB 转运体。
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