In vivo receptor occupation by benzodiazepines and correlation with the pharmacological effect

The existence of specific receptor sites for benzodiazepines has been well documented by in vitro binding studies. In this study, using a highly radiolabelled [3H]-flunitrazepam, we investigated the binding of benzodiazepines to their receptor sites under in vivo conditions. Tracer doses of [3H]flunitrazepam (0.001 mg/kg) were injected i.v. into mice and the concentration of the drug in the brain was monitored. The accumulation of [3H]flunitrazepam 20 min after injection was found to be highest in the hippocampus, cortex, hypothalamus; to be intermediate in the striatum, medulla oblongata/pons and midbrain and to be lowest in the cerebellum. This corresponds well with the different densities of benzodiazepine receptors which we found in in vitro studies, with the exception of medulla oblongata/pons and cerebellum. When increasing doses (0.01--10 mg/kg) of non-labelled benzodiazepine derivatives (flunitrazepam, clonazepam, the 3S and 3R enantiomers of 5-(o-fluorophenyl)-1,3-dihyrdo-1,3-dimethyl-7-nitro-2H-1,4-benzodiazepine-2-one, and chlordiazepoxide) were injected simultaneously with [3H]flunitrazepam, a dose-dependant, saturable and and stereo-specific decrease of [3H]flunitrazepam concentration in the mouse hippocampus was observed. The dose range in which the unlabelled benzodiazepines decreases the levels of [3H]flunitrazepam in the hippocampus corresponds closely to that which inhibited pentylenetetrazol- or picrotoxin-induced seizures, indicating that this in vivo method determines the occupation of pharmacologically relevant receptors.