Lengyel Kelly, Pieschl Rick, Strong Todd, Molski Thaddeus, Mattson Gail, Lodge Nicholas J, Li Yu-Wen
Neuroscience Biology Department, Bristol-Myers Squibb Research and Development Institute, 5 Research Parkway, Wallingford, CT 06492-7660, USA.
Neuropharmacology. 2008 Jul;55(1):63-70. doi: 10.1016/j.neuropharm.2008.04.014. Epub 2008 Apr 25.
The goal of this study was to develop and validate ex vivo binding assays for serotonin (SERT), norepinephrine (NET) and dopamine (DAT) transporters, and to use these assays to evaluate the binding site occupancy of triple and double monoamine reuptake inhibitors in rat brains. This study demonstrated that while autoradiographic methods provided anatomic precision and regional resolution, the homogenate binding method for site occupancy assessment yielded comparable sensitivity with markedly improved throughput. For ex vivo binding assays, the reduction of temperature and time during the in vitro process (primarily incubation with a radioligand) markedly decreased the dissociation of test agents from binding sites in brain tissues. This reduction, in turn, minimized the potential for underestimation of site occupancy in vivo especially for test compounds with affinity >10nM. The ratios of measured occupancy ED(50) values (doses at which 50% occupancy occurs) among SERT, NET and DAT sites for duloxetine, venlafaxine, nomifensine, indatraline, DOV 21,947 and DOV 216,303 were consistent with the ratios of the in vitro affinities between these target binding sites. The biological relevance of the monoamine transporter occupancy for these compounds is discussed.
本研究的目的是开发并验证用于5-羟色胺（SERT）、去甲肾上腺素（NET）和多巴胺（DAT）转运体的体外结合测定法，并使用这些测定法评估大鼠脑中三重和双重单胺再摄取抑制剂的结合位点占有率。本研究表明，虽然放射自显影方法提供了解剖学精度和区域分辨率，但用于位点占有率评估的匀浆结合方法具有相当的灵敏度，且通量显著提高。对于体外结合测定，体外过程中温度和时间的降低（主要是与放射性配体孵育）显著减少了测试剂从脑组织结合位点的解离。这种减少反过来又最大限度地降低了体内位点占有率被低估的可能性，尤其是对于亲和力>10nM的测试化合物。度洛西汀、文拉法辛、诺米芬辛、茚达曲林、DOV 21,947和DOV 216,303在SERT、NET和DAT位点的测量占有率ED(50)值（发生50%占有率的剂量）之比与这些靶结合位点之间的体外亲和力之比一致。讨论了这些化合物单胺转运体占有率的生物学相关性。
