Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences , University of Toyama , Sugitani , 2630 Toyama , Japan.
Sanders-Brown Center on Aging , University of Kentucky , Lexington , KY 40536 , United States.
Mol Pharm. 2018 Jun 4;15(6):2327-2337. doi: 10.1021/acs.molpharmaceut.8b00179. Epub 2018 May 2.
Gabapentin is an antiseizure drug that is known to also have beneficial effects on the retinal cells. To use gabapentin in retinal pharmacotherapy, it is critical to understand gabapentin distribution in the retina. The purpose of this study was to clarify the kinetics of gabapentin influx transport across the inner and outer blood-retinal barrier (BRB), which regulates the exchange of compounds/drugs between the circulating blood and the retina. In vivo blood-to-retina gabapentin transfer was evaluated by the rat carotid artery injection technique. In addition, gabapentin transport was examined using in vitro models of the inner (TR-iBRB2 cells) and outer BRB (RPE-J cells). The in vivo [H]gabapentin transfer to the rat retina across the BRB was significantly reduced in the presence of unlabeled gabapentin, suggesting transporter-mediated blood-to-retina distribution of gabapentin. Substrates of the Na-independent l-type amino acid transporter 1 (LAT1), such as 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), also significantly inhibited the in vivo [H]gabapentin transfer. [H]Gabapentin uptake in TR-iBRB2 and RPE-J cells exhibited Na-independent and saturable kinetics with a K of 735 and 507 μM, respectively. Regarding the effect of various transporter substrates/inhibitors on gabapentin transport in these cells, LAT1 substrates significantly inhibited [H]gabapentin uptake in TR-iBRB2 and RPE-J cells. In addition, preloaded [H]gabapentin release from TR-iBRB2 and RPE-J cells was trans-stimulated by LAT1 substrates through the obligatory exchange mechanism as LAT1. Immunoblot analysis indicates the protein expression of LAT1 in TR-iBRB2 and RPE-J cells. These results imply that LAT1 at the inner and outer BRB takes part in gabapentin transport between the circulating blood and retina. Moreover, treatment of LAT1-targeted small interfering RNA to TR-iBRB2 cells significantly reduced both the level of LAT1 protein expression and [H]gabapentin uptake activities in TR-iBRB2 cells. In conclusion, data from the present study indicate that LAT1 at the inner BRB is involved in retinal gabapentin transfer, and also suggest that LAT1 mediates gabapentin transport in the RPE cells.
加巴喷丁是一种抗癫痫药物，已知对视网膜细胞也有有益的作用。为了在视网膜药物治疗中使用加巴喷丁，了解加巴喷丁在视网膜中的分布至关重要。本研究的目的是阐明内、外血视网膜屏障（BRB）中加巴喷丁流入转运的动力学，该转运调节了循环血液和视网膜之间的化合物/药物的交换。通过大鼠颈动脉注射技术评估体内血液至视网膜加巴喷丁的转移。此外，使用内（TR-iBRB2 细胞）和外 BRB（RPE-J 细胞）的体外模型检查了加巴喷丁的转运。在存在未标记的加巴喷丁的情况下，体内[H]加巴喷丁向大鼠视网膜的转移穿过 BRB 明显减少，表明加巴喷丁通过转运蛋白介导的血液至视网膜分布。L 型氨基酸转运蛋白 1（LAT1）的底物，如 2-氨基双环[2.2.1]庚烷-2-羧酸（BCH），也显著抑制体内[H]加巴喷丁的转移。TR-iBRB2 和 RPE-J 细胞中[H]加巴喷丁的摄取表现出与 Na 无关且可饱和的动力学，K 值分别为 735 和 507 μM。关于各种转运体底物/抑制剂对这些细胞中加巴喷丁转运的影响，LAT1 底物显著抑制了 TR-iBRB2 和 RPE-J 细胞中[H]加巴喷丁的摄取。此外，通过 obligatory exchange mechanism as LAT1，LAT1 底物可从 TR-iBRB2 和 RPE-J 细胞中预加载的[H]加巴喷丁释放中反式刺激。免疫印迹分析表明，LAT1 在 TR-iBRB2 和 RPE-J 细胞中表达蛋白。这些结果表明，内、外 BRB 处的 LAT1 参与了循环血液和视网膜之间的加巴喷丁转运。此外，用 LAT1 靶向的小干扰 RNA 处理 TR-iBRB2 细胞可显著降低 TR-iBRB2 细胞中 LAT1 蛋白表达水平和[H]加巴喷丁摄取活性。总之，本研究的数据表明，内 BRB 处的 LAT1 参与了视网膜加巴喷丁的转移，并提示 LAT1 介导了 RPE 细胞中的加巴喷丁转运。
