Ex vivo assessment of binding site occupancy of monoamine reuptake inhibitors: methodology and biological significance.
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.
Neuroscience Biology Department, Bristol-Myers Squibb Research and Development Institute, 5 Research Parkway, Wallingford, CT 06492-7660, USA.
Kelly
K
Lengyel
Rick
R
Pieschl
Todd
T
Strong
Thaddeus
T
Molski
Gail
G
Mattson
Nicholas J
NJ
Lodge
Yu-Wen
YW
Li
D000818:Animals; D001345:Autoradiography / Q000379:methods; D001665:Binding Sites / Q000187:drug effects; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D001931:Brain Mapping; D004305:Dose-Response Relationship, Drug; D008297:Male; D026901:Membrane Transport Proteins / Q000378:metabolism*; D014179:Neurotransmitter Uptake Inhibitors / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D011868:Radioisotopes / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D014018:Tissue Distribution / Q000187:drug effects
D026901:Membrane Transport Proteins; D014179:Neurotransmitter Uptake Inhibitors; D011868:Radioisotopes