培高利特经 L 型氨基酸转运蛋白 1（SLC7A5）在人脑微血管内皮细胞系中的转运。
Transport of Pregabalin Via L-Type Amino Acid Transporter 1 (SLC7A5) in Human Brain Capillary Endothelial Cell Line.
机构信息
Division of Pharmaceutics, Faculty of Pharmacy, Keio University, 1-5-30, Shibakoen, Minato-ku, Tokyo, 108-8512, Japan.
Laboratory of Drug Disposition & Pharmacokinetics, Faculty of Pharma-Sciences, Teikyo University, 2-11-1, Kaga Itabashi-ku, Tokyo, 173-8605, Japan.
出版信息
Pharm Res. 2018 Oct 29;35(12):246. doi: 10.1007/s11095-018-2532-0.
PURPOSE
The anti-epileptic drug pregabalin crosses the blood-brain barrier (BBB) in spite of its low lipophilicity. This study was performed to determine whether L-type amino acid transporters (LAT1/SLC7A5 and LAT2/SLC7A8) contribute to the uptake of pregabalin.
METHODS
Pregabalin uptake by LATs-transfected HEK293 cells or hCMEC/D3 cells, an in vitro human BBB model, was measured by LC-MS/MS analysis. Expression of LAT1 mRNA in hCMEC/D3 cells was determined by quantitative RT-PCR analysis.
RESULTS
Overexpression of LAT1, but not LAT2, in HEK293 cells significantly increased the cellular uptake of pregabalin, and the LAT1-mediated uptake was saturable with a K of 0.288 mM. LAT1-mediated amino acid uptake was inhibited specifically and almost completely in the presence of 1 mM pregabalin. The uptake of pregabalin by hCMEC/D3 cells was sodium-independent, saturable (K = 0.854 mM), and strongly inhibited by large amino acids at 1 mM, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, a specific system L inhibitor, at 1 mM and by JPH203, a LAT1-selective inhibitor, at 10 μM. Pregabalin uptake in hCMEC/D3 cells was also decreased by 75% by the silencing of LAT1 gene using LAT1 siRNA.
CONCLUSIONS
Our results indicate that LAT1, but not LAT2, recognizes pregabalin as a substrate. It is suggested that LAT1 mediates pregabalin transport at the BBB.
目的
尽管抗癫痫药物普瑞巴林的脂溶性较低，但它仍能穿过血脑屏障（BBB）。本研究旨在确定 L 型氨基酸转运体（LAT1/SLC7A5 和 LAT2/SLC7A8）是否有助于普瑞巴林的摄取。
方法
通过 LC-MS/MS 分析测定 LATs 转染的 HEK293 细胞或体外 BBB 模型 hCMEC/D3 细胞对普瑞巴林的摄取情况。通过定量 RT-PCR 分析测定 hCMEC/D3 细胞中 LAT1 mRNA 的表达。
结果
在 HEK293 细胞中过表达 LAT1，但不是 LAT2，可显著增加普瑞巴林的细胞摄取，LAT1 介导的摄取具有 0.288 mM 的 K 值。在存在 1 mM 普瑞巴林的情况下，LAT1 介导的氨基酸摄取被特异性且几乎完全抑制。hCMEC/D3 细胞对普瑞巴林的摄取是钠离子非依赖性的、饱和的（K=0.854 mM），并且在 1 mM 时被大氨基酸强烈抑制，在 1 mM 时被系统 L 的特异性抑制剂 2-氨基双环[2.2.1]庚烷-2-羧酸强烈抑制，在 10 μM 时被 LAT1 选择性抑制剂 JPH203 强烈抑制。用 LAT1 siRNA 沉默 LAT1 基因也使 hCMEC/D3 细胞中普瑞巴林的摄取减少了 75%。
结论
我们的结果表明，LAT1 而不是 LAT2 将普瑞巴林识别为底物。提示 LAT1 介导普瑞巴林在 BBB 的转运。
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