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.