A Fluorescent Receptor Assay for Benzodiazepines Using Coumarin-Labeled Desethylflumazenil as Ligand

Abstract

This article describes a novel nonisotopic receptor assay for benzodiazepines with fluorescence detection. As labeled ligand (coumarin-labeled desethylflumazenil, CLDEF), a metabolite of the benzodiazepine antagonist flumazenil (desetheylflumazenil, Ro15-3890) has been coupled to a coumarin fluorophore, via a spacer. CLDEF had a Ki of 6.5 nM. To avoid the interference of the background fluorescence of the receptors in the measurement step, the bound CLDEF was dissociated from the receptors after the filtration step. This dissociation was achieved by incubating the CLDEF-bound to the receptors on the filters-with a weakly acetate buffer. The second filtrates then contained the previously bound CLDEF, which was then quantitated with a RP-HPLC system with a fluorescence detector. The results with a fluorescent receptor assay were very similar to those with a radioreceptor assay, in that the IC50 values of lorazepam were 7.2 +/- 0.5 and 6.6 +/- 0.7 nM, respectively.