In vivo saturation binding of GABA‐A receptor ligands to estimate receptor occupancy using liquid chromatography/tandem mass spectrometry

Abstract

Typically, the dose-occupancy curves for GABA-A receptor ligands are determined using in vivo binding of [3H]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 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.

Pharmacokinetic evaluation of eszopiclone: clinical and therapeutic implications

“…Eszopiclone has higher receptor affinity at the GABAbenzodiazepine (GABA-BZ) receptor complex than either R, S-zopiclone or R-zopiclone [13,14,[16][17][18]. This likely accounts for the conclusion that eszopiclone explains most of the clinical hypnotic activity of R,S-zopiclone.…”

Antagonism of the Ethanol-Like Discriminative Stimulus Effects of Ethanol, Pentobarbital, and Midazolam in Cynomolgus Monkeys Reveals Involvement of Specific GABAAReceptor Subtypes

“…In mouse forebrain, saturating concentrations of flumazenil (ϳ240 nM) are reached 10 min after 2.0 mg/kg i.p. flumazenil (Hopkins et al, 2009). …”

Development and Validation of a Reversed‐Phase HPLC Method for the Estimation of Zolpidem in Bulk Drug and Tablets

“…It has been measured in various biological specimens using a variety of different analytical techniques. Several analytical methods have been reported for quanti�cation of zolpidem in human body �uids and organ samples including capillary electrophoresis (CE) [7], GC [8][9][10], HPLC [1,3,[11][12][13][14][15][16][17], GC/MS [9,18,19], LC/MS [20], LC/MS/MS [21][22][23], and radioimmunoassay [24]. In the HPLC methods, the sensitivity of zolpidem is higher with �uorimetric detection than with UV detection [25].…”