Comparison of Lorazepam [7-Chloro-5-(2-chlorophenyl)-1,3- dihydro-3-hydroxy-2H-1,4-benzodiazepin-2-one] Occupancy of Rat Brain -Aminobutyric AcidA Receptors Measured Using in Vivo [H]Flumazenil (8-Fluoro 5,6-dihydro-5-methyl-6-oxo-4H- imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic Acid Ethyl Ester) Binding and [C]Flumazenil Micro-Positron Emission Tomography

The occupancy by lorazepam of the benzodiazepine binding site of rat brain GABAA receptors was compared when measured using either in vivo binding of [H]flumazenil (8-fluoro 5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester) in terminal studies or [C]flumazenil binding in anesthetized animals assessed using a small animal positron emission tomography (PET) scanner (micro-PET). In addition, as a bridging study, lorazepam occupancy was measured using [H]flumazenil in vivo binding in rats anesthetized and dosed under micro-PET conditions. Plasma lorazepam concentrations were also determined, and for each occupancy method, the concentration required to produce 50% occupancy (EC50) was calculated because this parameter is independent of the route of lorazepam administration. For the in vivo binding assay, lorazepam was dosed orally (0.1–10 mg/kg), whereas for the micro-PET study, lorazepam was given via the i.v. route as a low dose (0.75 mg/kg bolus) and then a high dose (0.5 mg/kg bolus then 0.2 mg/ml infusion). The lorazepam plasma EC50 in the [ C]flumazenil micro-PET study was 96 ng/ml [95% confidence intervals (CIs) 74–124 ng/ml], which was very similar to the [H]flumazenil micro-PET simulation study (94 ng/ml; 95% CI 63–139 ng/ml), which in turn was comparable with the [H]flumazenil in vivo binding study (134 ng/ml; 95% CI 119–151 ng/ml). These data clearly show that despite the differences in dosing (i.v. in anesthetized versus orally in conscious rats) and detection (in vivo dynamic PET images versus ex vivo measurements in filtered and washed brain homogenates), [C]flumazenil micro-PET produces results similar to [H]flumazenil in vivo binding. The pharmacological effect of any drug is a function of its intrinsic efficacy and the extent to which it occupies the target, whether that is an enzyme, transporter, or receptor. The extent to which a drug occupies its target is related to its affinity as well as its concentration. In peripheral tissues for which there is a negligible plasma-tissue barrier, drug concentrations in the plasma can be used as a surrogate of those in the target tissue. However, this is not the case for the CNS because the blood-brain barrier restricts penetration of drug into the CNS. Consequently, plasma drug concentrations may be misleading when interpreting the pharmacological effects of CNS drugs; therefore, it is necessary to measure the Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.114884. ABBREVIATIONS: CNS, central nervous system; PET, positron emission tomography; lorazepam, 7-chloro-5-(2-chlorophenyl)-1,3-dihydro-3hydroxy-2H-1,4-benzodiazepin-2-one; flumazenil, 8-fluoro 5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester; DMSO, dimethyl sulfoxide; 3D, three-dimensional; ROI, region of interest; TAC, time-activity curve. 0022-3565/07/3203-1030–1037$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 320, No. 3 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 114884/3181338 JPET 320:1030–1037, 2007 Printed in U.S.A.