title: 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.
authors.affiliation: Sepracor Inc., Marlborough, MA 01752, USA. seth.hopkins@sepracor.com
authors.forename: Seth C
authors.initials: SC
authors.lastname: Hopkins
authors.forename: J
authors.initials: J
authors.lastname: Brian Nofsinger
authors.forename: Michael S
authors.initials: MS
authors.lastname: Allen
authors.forename: Patrick
authors.initials: P
authors.lastname: Koch
authors.forename: Mark A
authors.initials: MA
authors.lastname: Varney
mesh_terms: D000465:Algorithms; D000818:Animals; D053961:Azabicyclo Compounds / Q000493:pharmacokinetics; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D005442:Flumazenil / Q000493:pharmacokinetics; D005464:Fluorobenzenes / Q000493:pharmacokinetics; D018755:GABA Agonists / Q000493:pharmacokinetics; D018757:GABA Modulators / Q000493:pharmacokinetics; D006993:Hypnotics and Sedatives / Q000493:pharmacokinetics; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D008024:Ligands; D008297:Male; D051379:Mice; D015233:Models, Statistical; D010879:Piperazines / Q000493:pharmacokinetics; D011725:Pyridines / Q000493:pharmacokinetics; D011963:Receptors, GABA-A / Q000378:metabolism*; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry; D014230:Triazoles / Q000493:pharmacokinetics; D000077334:Zolpidem
chemical_list: D053961:Azabicyclo Compounds; D005464:Fluorobenzenes; D018755:GABA Agonists; D018757:GABA Modulators; D006993:Hypnotics and Sedatives; D007202:Indicators and Reagents; D008024:Ligands; D010879:Piperazines; D011725:Pyridines; D011963:Receptors, GABA-A; D014230:Triazoles; C515050:zopiclone; D005442:Flumazenil; D000077334:Zolpidem; C409903:L 838,417