title: Metabolism of a 14C/3H-labeled GABAA receptor partial agonist in rat, dog and human liver microsomes: evaluation of a dual-radiolabel strategy.
abstract: 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 GABA(A) receptor partial agonist, was investigated in rat, dog and human liver microsomes. Due to its significant metabolic cleavage at C(8) observed in preliminary biotransformation studies with non-radiolabeled 1, both [(14)C]1 and [(3)H]1 were synthesized with respective radioisotopes placed on either side of C(8) to determine if all microsomal metabolites formed after C(8)N-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 (14)C-only metabolites equaled that of (3)H-only metabolites in all incubations. In-line radiometric analysis worked extremely well (and was very reproducible) for quantifying either (14)C- or (3)H-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 (14)C- from (3)H-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 (14)C- and (3)H-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.
authors.affiliation: Department of Pharmacokinetics, Pharmacodynamics and Metabolism, Pfizer Global Research and Development, Groton/New London Laboratories, Pfizer Inc., Groton, CT 06340, USA. Christopher.L.Shaffer@pfizer.com
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Shaffer
authors.forename: Connie S
authors.initials: CS
authors.lastname: Langer
mesh_terms: D000818:Animals; D001711:Biotransformation; D002250:Carbon Radioisotopes; D002853:Chromatography, Liquid; D004285:Dogs; D005069:Evaluation Studies as Topic; D018755:GABA Agonists / Q000737:chemistry / Q000378:metabolism*; D058785:GABA-A Receptor Agonists; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000378:metabolism*; D015394:Molecular Structure; D051381:Rats; D017207:Rats, Sprague-Dawley; D011963:Receptors, GABA-A / Q000235:genetics*; D012588:Scintillation Counting; D013997:Time Factors; D014316:Tritium / Q000378:metabolism*
chemical_list: D002250:Carbon Radioisotopes; D018755:GABA Agonists; D058785:GABA-A Receptor Agonists; D011963:Receptors, GABA-A; D014316:Tritium