Edward Postma

Metabolism of diazepam in vitro

Abstract 3 H-diazepam (7-chloro-1,3-dihydro-1-metbyl-5-phenyl-2H-1,4-benzodiazepin-2-one), labeled randomly in the 5-phenyl ring, was incubated for 1 hr at 37° with fortified 9000 g supernatants prepared from liver of control and phenobarbital-treated dogs and rats. Liver supernatants from 5 control dogs metabolized diazepam to roughly equal amounts of 7-chloro-1,3-dihydro-3-hydroxy-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one (I) and 7-chloro-1,3-dihydro-5-phenyl-2H-1,4-benzodia2epin-2-one (II) and only minimal amounts of 7-chloro-1, 3-dihydro-3-hydroxy-5-phenyl-2H-1 ,4-benzodiazepin-2-one (oxazepam). Pretreatment of 5 dogs with phenobarbital resulted in an increased conversion of diazepam to II and oxazepam. 3 H-labeled I and II produced in vitro were isolated and used as substrates in a 2-hr incubation with liver supernatant from a phenobarbital-treated dog; each was converted about equally well to oxazepam. No metabolism of diazepam was detected on incubation with dog brain homogenate or 9000 g supernatant. The metabolism of diazepam by a pooled 9000 g liver supernatant from 4 control rats was very similar to that seen with control dog liver supernatants. The pooled liver supernatants from 4 phenobarbital-treated rats yielded a much greater metabolism of diazepam with not only a marked increase in oxazepam, but also the production of polar unidentified metabolites.

N-desmethyldiazepam: a new metabolite of chlordiazepoxide in man.

Following administration of chlordiazepoxide HCI to man, N‐desmethyldiazepam, a known metabolite of diazepam (Valium), was identified in plasma. The metabolite was identified on the basis of its thin‐layer chromatographic (TLC) mobility, electron‐capture gas‐chromatographic (EC‐GC) retention time, and mass spectrum relative to authentic N‐desmethyldiazepam. Plasma levels of N‐desmethyldiazepam in subjects receiving both single and chronic doses of chlordiazepoxide were determined by an EC‐GC method with a limit of sensitivity of 10 ng/ml using 2‐ml samples and by a radioimmunoassay procedure which had a limit of sensitivity of 20 ng/ml using a 0.1‐ml sample. Both assay methods gave good agreement for the levels of N‐desmethyldiazepam. In subjects receiving a single 30‐mg oral or intravenous dose of chlordiazepoxide, measurable levels of N‐desmethyldiazepam in plasma (10 to 60 ng/ml) were obtained 24 to 72 hr after administration. In 5 subjects receiving 10 mg of chlordiazepoxide three times a day, steady‐state levels of N‐desmethyldiazepam in plasma were reached after about 1 wk of administration. The mean maximum and minimum steady‐state levels of N‐desmethyldiazepam were 260 and 220 ng/ml of plasma, respectively. Similar steady‐state levels were observed on treatment with 30 mg of chlordiazepoxide over 24 hr.

Chlordiazepoxide metabolites in the rat. Characterization by high resolution mass spectrometry

Abstract Four rat urinary metabolites of chlordiazepoxide (7-chloro-2-methylamino-5-phenyl-3H-1,4-benzodiazepine 4-oxide) labeled at C-2 with 14 C were separated by solvent extraction and thin-layer chromatography (TLC) and characterized by high resolution mass spectrometry. Each metabolite was found to have a hydroxyl function in the C-5 phenyl ring, and two-dimensional TLC of metabolites with reference compounds indicated that the most probable location of the phenolic function was para to the diazepine ring. The four metabolites were: 7-chloro-1,3-dihydro-5-(4-hydroxyphenyl)-2H-1,4-benzodiazepin-2-one 4-oxide (Metabolite 1); 2-amino-7-chloro-5-(4-hydroxyphenyl)-3H-1,4-benzodiazepine 4-oxide (Metabolite 2); 7-chloro-5-(4-hydroxyphenyl)-2-mwthylamino-3H-1,4-benzodiazepine 4-oxide (Metabolite 3); and 7-chloro-1,3-dihydro5-(4-hydroxyphenyl)-2H-1,4-benzodiazepin-2-one (Metabolite 4), which differs from the others in that it no longer retains the N-oxide function).