Kubo Yoshiyuki, Tsuchiyama Ai, Shimizu Yoshimi, Akanuma Shin-Ichi, Hosoya Ken-Ichi
Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama , Toyama, Japan.
Mol Pharm. 2014 Oct 6;11(10):3747-53. doi: 10.1021/mp500516j. Epub 2014 Sep 26.
In the present study, the blood-to-retina transport across the inner BRB was investigated for clonidine, a compound which is expected to exhibit a neuroprotective effect for the treatment of severe retinal diseases. In the in vivo study, the integration plot analysis for [(3)H]clonidine exhibited an apparent influx permeability clearance of 457 μL/(min·g retina) in the retina. The in vivo inhibition study suggests that the blood-to-retina transport of clonidine at the BRB is organic cation-sensitive since clonidine, pyrilamine, and propranolol, at a concentration of 40 mM, significantly reduced the retinal uptake index (RUI) of [(3)H]clonidine, and an inhibitory effect on the RUI was also exhibited by verapamil, at a concentration of 3 mM. The in vitro study with TR-iBRB2 cells, an in vitro model cell line of the inner BRB, suggests that carrier-mediated transport is involved in the blood-to-retina transport of clonidine at the inner BRB since the results obtained demonstrated time-, temperature-, pH-, and concentration-dependent [(3)H]clonidine uptake, with a Km of 286 μM. In the in vitro inhibition study, the [(3)H]clonidine uptake was significantly reduced by several organic cations, such as clonidine, verapamil, pyrilamine, and propranolol, and was competitively inhibited by 200 μM verapamil, in spite of slight or no significant alteration being produced with organic anions. Furthermore, the typical substrates and inhibitors of well-known organic cation transporters had no significant effect on the uptake of [(3)H]clonidine to suggest the involvement of novel transporter molecules in the transport of clonidine across the inner BRB. These results suggest that the blood-to-retina transport of clonidine across the inner BRB involves a carrier-mediated transport manner, suggesting the contribution of a novel organic cation transporter expressed by the retinal capillary endothelial cells.
在本研究中，对可乐定进行了跨内血视网膜屏障的血视网膜转运研究，可乐定是一种有望对严重视网膜疾病治疗发挥神经保护作用的化合物。在体内研究中，[³H]可乐定的积分图分析显示，视网膜中其表观流入通透性清除率为457 μL/(min·g视网膜)。体内抑制研究表明，可乐定在血视网膜屏障处的血视网膜转运对有机阳离子敏感，因为浓度为40 mM的可乐定、吡苄明和普萘洛尔显著降低了[³H]可乐定的视网膜摄取指数（RUI），浓度为3 mM的维拉帕米也对RUI有抑制作用。利用TR-iBRB2细胞（内血视网膜屏障的体外模型细胞系）进行的体外研究表明，载体介导的转运参与了可乐定在内血视网膜屏障处的血视网膜转运，因为所获得的结果显示[³H]可乐定摄取具有时间、温度、pH和浓度依赖性，米氏常数为286 μM。在体外抑制研究中，几种有机阳离子，如可乐定、维拉帕米、吡苄明和普萘洛尔，显著降低了[³H]可乐定的摄取，200 μM维拉帕米对其有竞争性抑制作用，而有机阴离子对其摄取的影响轻微或无显著改变。此外，已知有机阳离子转运体的典型底物和抑制剂对[³H]可乐定的摄取无显著影响表明，新型转运体分子参与了可乐定跨内血视网膜屏障的转运。这些结果表明，可乐定跨内血视网膜屏障的血视网膜转运涉及载体介导的转运方式，提示视网膜毛细血管内皮细胞表达的新型有机阳离子转运体发挥了作用。
