Journal
BIOPHARMACEUTICS & DRUG DISPOSITION
Volume 30, Issue 1, Pages 9-20Publisher
WILEY
DOI: 10.1002/bdd.641
Keywords
occupancy; benzodiazepines; saturation binding; LC/MS/MS; GABA-A
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Typically, the dose-occupancy curves for GABA-A receptor ligands are determined using in vivo binding of [H-3]flumazenil. This study describes in vivo binding experiments without the use of tracer ligands. Bound and free fractions were measured directly using a highly sensitive LC/MS/MS detection method after in vivo administration of the GABA-A ligands zolpidem, (RS)-zopidone, L-838417 and flumazenil, to demonstrate affinity and saturation of the filter-retained, membrane-bound fraction. The in vivo binding of flumazenil and L-838417 both saturated around 200 nm, at a similar level to the specific binding of (S)-zopiclone after doses of the racemic zopiclone, using (R)-zopiclone to estimate non-specific binding. This saturable component represented an estimate of benzodiazepine binding sites available on GABA-A receptors in vivo (200 nM). Dose-occupancy curves were constructed to estimate the dose required to achieve 50% occupancy and matched estimates obtained with tracer methods. In contrast to tracer methods, this method is uniquely suitable to the demonstration of stereoselective binding of the (S)-isomer in vivo after doses of racemic zopiclone. These results demonstrate that the LC/MS/MS measurements of total drug concentrations typically used in early drug development can be adapted to provide information about receptor occupancy in vivo. Copyright (C) 2009 John Wiley & Sons, Ltd.
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