Metabolism of a 14C/3H-labeled GABAA receptor partial agonist in rat, dog and human liver microsomes: Evaluation of a dual-radiolabel strategy

The metabolism of 2-{[2-(3-fluoropyrid-2-yl)-1H-imidazol-1-yl]methyl}-1-propyl-5-cyano-1H-benzimidazole (1), a potent subtype-selective GABAA receptor partial agonist, was investigated in rat, dog and human liver microsomes. Due to its significant metabolic cleavage at C8 observed in preliminary biotransformation studies with non-radiolabeled 1, both [14C]1 and [3H]1 were synthesized with respective radioisotopes placed on either side of C8 to determine if all microsomal metabolites formed after C8N-dealkylation of 1 (or its core-intact metabolites) could be detected and quantified adequately. Both radiolabeled forms of 1, used separately in mono-radiolabel studies in cross-species microsomes and concomitantly in dual-radiolabel studies in rat microsomes, permitted the detection and quantification of all metabolites of 1, and a combination of radioactive and mass spectral data allowed structural elucidation of its Phase I metabolites. As expected, the sum of 14C-only metabolites equaled that of 3H-only metabolites in all incubations. In-line radiometric analysis worked extremely well (and was very reproducible) for quantifying either 14C- or 3H-compounds within separate incubations when using mono-radiolabeled 1. However, although the in-line radiodetector provided a comprehensive qualitative metabolic profile using dual-radiolabled 1, its inability to exclude completely 14C- from 3H-generated counts caused a degree of ambiguity pertaining to metabolite quantification. Thus, off-line liquid scintillation counting of collected dual-radiolabeled incubation LC-fractions was employed to quantify both 14C- and 3H-metabolites simultaneously, while in-line radiodetection was only used for qualitative analyses accompanying MS and MS/MS experiments. These studies demonstrated the analytical feasibility of using a dual-radiolabel approach for subsequent in vivo ADME studies with 1.