Received for publication March 8, 2005.
Revised July 26, 2005.
Accepted for publication August 3, 2005.

Biotransformation of a GABA_A Partial Agonist In Sprague-Dawley Rats and Cynomolgus Monkeys: Identification of Two Unique N-Carbamoyl Metabolites

Abstract

The absorption, metabolism and excretion of 1, a GABA_A receptor partial agonist potentially useful in treating Generalized Anxiety Disorder, have been evaluated in both Sprague-Dawley rats and Cynomolgus monkeys using [¹⁴C]1. In both species, mass balance was achieved within 48 h post-dose with the majority of drug-related material excreted within the feces; the clearance of 1 in each species had both metabolic and renal components. In addition to the metabolites produced by aliphatic hydroxylation and/or N-dealkylation of 1, two unique metabolites were detected: a putative carbamic acid (M7) in rat plasma and monkey bile, and an N-carbamoyl glucuronide (M8) in both rat and monkey bile. Metabolite M8 was structurally deciphered by LC-MS/MS and NMR, and was readily generated in vitro upon incubation of [¹⁴C]1 with rat liver microsomes fortified with UDPGA and alamethicin under a CO₂ atmosphere. Treatment of M8 with -glucuronidase afforded 1 directly. The presence of M8 in bile and its notable absence from other matrices suggests the enterohepatic cycling of 1 via M8. Although the structure of M7 was not elucidated unequivocally due to its inability to be formed in vitro and its minimal absolute quantities in limited biological matrices, data herein clearly support its structural rationalization. Furthermore, since M7 is the precursor of M8, detection of M8 is indirect evidence of its existence. It is proposed that M7 arises from an equilibrium between 1 and dissolved CO₂-equivalents both in vivo and in vitro, similar to carbamino bonds observed in hemoglobin and certain amino acids, respectively.

Key words: analytical chemistry, biliary excretion, enterohepatic circulation, glucuronidation, liver microsomes, mass spectrometry, metabolite identification, phase II drug metabolism, structure elucidation

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