Premium
In vivo saturation binding of GABA‐A receptor ligands to estimate receptor occupancy using liquid chromatography/tandem mass spectrometry
Author(s) -
Hopkins Seth C.,
Brian Nofsinger J.,
Allen Michael S.,
Koch Patrick,
Varney Mark A.
Publication year - 2009
Publication title -
biopharmaceutics and drug disposition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.419
H-Index - 58
eISSN - 1099-081X
pISSN - 0142-2782
DOI - 10.1002/bdd.641
Subject(s) - in vivo , flumazenil , chemistry , zopiclone , receptor , chromatography , pharmacology , gabaa receptor , biochemistry , hypnotic , biology , microbiology and biotechnology
Abstract Typically, the dose‐occupancy curves for GABA‐A receptor ligands are determined using in vivo binding of [ 3 H]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)‐zopiclone, 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 n m , 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 n m ). 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 © 2009 John Wiley & Sons, Ltd.