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

J Pharm Biomed Anal. 2007 Mar 12;43(4):1195-205. doi: 10.1016/j.jpba.2006.11.022. Epub 2006 Dec 5.


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.

Publication types

  • Comparative Study
  • Evaluation Study

MeSH terms

  • Animals
  • Biotransformation
  • Carbon Radioisotopes
  • Chromatography, Liquid
  • Dogs
  • Evaluation Studies as Topic
  • GABA Agonists / chemistry
  • GABA Agonists / metabolism*
  • GABA-A Receptor Agonists*
  • Humans
  • In Vitro Techniques
  • Kinetics
  • Male
  • Mass Spectrometry
  • Microsomes, Liver / metabolism*
  • Molecular Structure
  • Rats
  • Rats, Sprague-Dawley
  • Receptors, GABA-A / genetics*
  • Scintillation Counting
  • Time Factors
  • Tritium / metabolism*


  • Carbon Radioisotopes
  • GABA Agonists
  • GABA-A Receptor Agonists
  • Receptors, GABA-A
  • Tritium