Morton A. Schwartz

Diazepam metabolites in the rat: Characterization by high-resolution mass spectrometry and nuclear magnetic resonance

This report illustrates the usefulness of combining thin-layer chromatography for metabolite separation and purification with high-resolution mass spectrometry and nuclear magnetic resonance for metabolite characterization. Four metabolites of diazepam (7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one) were identified in the intestinal tract of one rat that had been injected intraperitoneally with 25 mg of3H-diazepam. These are the glucuronide and/or sulfate conjugates of 3-hydroxy diazepam (metabolite A), and the conjugates of the 5-hydroxyphenyl analogues of diazepam, 3-hydroxy diazepam, and N-desmethyl diazepam (metabolites B, C, and D, respectively). In each of the phenolic metabolites (B, C, and D) the hydroxyl function is in the para position.

Blood levels and electroencephalographic effects of diazepam and bromazepam.

Blood levels and electroencephalographic (EEG) data were collected for 2 hr after single oral doses of bromazepam (9 mg), diazepam (10 mg), and placebo in 13 male adult volunteers. Both drugs caused an increase in beta activity (above 13 Hz) and a decrease in alpha activity (9 to 11 Hz) in the EEG. Blood levels of 100 nglml of diazepam or 50 ng Iml of bromazepam were associated with significant changes in EEG beta activity. Temporal changes in the EEG after administration of diazepam or bromazepam paralleled development of plasma levels of these drugs. Although a weakly significant correlation was found between measurable diazepam blood levels and amount of increased EEG beta activity, the relationship between measurable bromazepam blood levels and the degree of EEG changes was not significant. Quantitative EEG is a sensitive continuous response measure, useful in defining cerebral activity, response latency, and relative potency of psychoactive benzodiazepines.

Metabolism of14C-medazepam hydrochloride in dog, rat and man

Abstract Labeled medazepam (7-chloro-2,3-dihydro-1-methyl-5-phenyl-1H-1,4-benzo-diazepine-5-14C) and metabolites were identified and measured by two-dimensional thin-layer chromatography. In the dog, 2 mg/kg of medazepam-5-14C HCl given orally or i.v. did not yield measurable blood levels of intact drug. The close agreement of the amount of 14C excreted in the urine after oral and i.v. administration and of the rates of this excretion indicated that the labeled compound was well absorbed. Fractionation of the blood revealed that biotransformation of medazepam was a rapid process. After a higher dose, 20 mg/kg i.v., medazepam disappeared from the blood bi-exponentially with half-lives of 0.16 and 2.7 hr. Pathways of medazepam metabolism, which apparently did not include diazepam as an intermediate, were postulated on the basis of metabolites identified in experiments both in vivo and in vitro. In the rat, highest tissue concentrations of14C were seen 2–4 hr after an oral dose of 14C-medazepam HCl. It was clear from the metabolites identified that the formation of diazepam and phenolic diazepam metabolites represented an important pathway of medazepam metabolism in this species. The labeled compound was rapidly absorbed by two human subjects who received single 30-mg oral doses. The peak blood levels of medazepam (less than 0.2 μg/ml at 1–2 hr) declined rapidly with a half-life of 1–2 hr. A marked difference between the subjects in rates of elimination of metabolites from the blood and in excretion of urinary metabolites appeared related to body weight and the activity of drug-metabolizing enzymes. Preliminary evidence for diazepam formation in man was also obtained.

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).

Isocarboxazid Metabolism in Rat Liver Homogenate

Summary Isocarboxazid was extensively metabolized under aerobic or anaerobic conditions in an in vitro system containing rat liver homogenate as the enzyme source. Benzylhydrazine was a major product of this metabolism as determined by paper chromatography and reverse isotope dilution in studies with C14–isocarboxazid. The possible occurrence of this pathway in vivo was discussed.

Pharmacodynamic studies with (−)-3-phenoxy-N-methylmorphinan in rats

Analgesia and brain and plasma concentrations of (-)-3-phenoxy-N-methylmorphinan (PMM) and its metabolites were determined in rats administered 50 mg/kg of 3H-labeled PMM p.o., an approximate ED50. Unchanged PMM and two active metabolites, levorphanol and a different phenol, p-hydroxylated on the 3-phenoxy group (pOH-PMM), were present in brain at concentrations greater than in plasma. Analgesia was observed from 1 to 6 hr and was associated with brain concentrations of 400-1400 ng/g of PMM, 190-300 ng/g of pOH-PMM, and 16-27 ng/g of levorphanol. The presence of 58% of the administered dose as unchanged PMM in the gastrointestinal tract at 6 hr may reflect slow absorption and explain the persisting brain concentrations of PMM and its metabolites as well as the prolonged analgesia. Analgesia may have been due to the presence in brain of only PMM, pOH-PMM or levorphanol, or to the combined activity of two or three of these substances. Administration of the approximate ED50 of 3H-labeled levorphanol (0.1 mg/kg, s.c., or 6 mg/kg, p.o.) resulted in brain levorphanol concentrations (11-18 ng/g) close to those observed when PMM was administered p.o. at 50 mg/kg. After administration of an approximate subcutaneous ED50 of [3H]pOH-PMM of 24 mg/kg, the brains contained pOH-PMM (1500-4100 ng/g) and levorphanol (60-100 ng/g); these levorphanol concentrations were higher than those found after administration of the approximate ED50 of PMM or levorphanol. The findings indicate that brain levorphanol concentrations resulting from administration of PMM or pOH-PMM to rats may account for the analgesic activity observed, i.e. that PMM and pOH-PMM may act as prodrugs for levorphanol