Received for publication March 21, 2007.
Revised July 16, 2007.
Accepted for publication July 19, 2007.

In vitro Prediction and in vivo Verification of Enantioselective Human Tofisopam Metabolite Profiles

Abstract

In vitro studies were conducted to elucidate the metabolic profiles of and the responsible enzymes for the metabolism of (R)- and (S)-tofisopam (1-(3,4-dimethoxyphenyl)-5-ethyl-7,8-dimethoxy-4-methyl-5H-2,3-benzodiazepine). Large differences were observed between the two enantiomers. The major metabolite in incubations of 500 ng/ml (1.3 µM) (R)-tofisopam in human liver microsomes corresponded to demethylation of the methoxy group at the 4 position of the phenyl ring (M3). Incubating (R)-tofisopam with recombinant P450 or with human liver microsomes and isoform selective P450 chemical inhibitors indicated that M3 was primarily catalyzed by CYP2C9. Similar incubations with S-tofisopam indicated that the primary metabolite was due to demethylation of the methoxy group at the 7 position of the benzodiazepine ring (M1), and this reaction was catalyzed primarily by CYP3A4. The primary metabolites of both enantiomers were further demethylated to form a common di-demethylated metabolite (M5), where the methoxy groups at positions 4 and 7 are demethylated. Analysis of plasma and urine samples from human clinical trials confirmed the in vitro observations. Subjects orally treated with 200 mg BID (R)-tofisopam had a 2 hr M1:M3 plasma ratio of 1:29 and a ratio of 1:123 in urine; whereas, patients orally administered (S)-tofisopam at 150 mg TID mg/kg had opposite M1 to M3 ratios of 8:1 in plasma and 6:1 in urine.

Key words: cytochrome P450 catalyzed oxidations, drug disposition, in vitro-in vivo prediction, metabolite kinetics