Metabolism of halazepam by rat liver microsomes: Stereoselective formation and n‐dealkylation of 3‐hydroxyhalazepam

Abstract

Metabolism of halazepam [7-chloro-1,3-dihydro-5-phenyl-1-(2,2,2-trifluoroethyl)-2H-1,4-benzod iazepin- 2-one, HZ] was studied by incubation with liver microsomes prepared from untreated, phenobarbital (PB)-treated, and 3-methylcholanthrene (3MC)-treated male Sprague-Dawley rats. Metabolites of HZ were separated by normal-phase HPLC. Relative rates of HZ metabolism by liver microsomes prepared from untreated and treated rats were PB-treated much greater than untreated greater than 3MC-treated at low concentration of microsomal enzymes (0.25 mg protein per ml of incubation mixture) and PB-treated much greater than 3MC-treated approximately untreated at high concentration of microsomal enzymes (2 mg protein per ml of incubation mixture). The relative amounts of major metabolites were found to be 3-hydroxy-HZ (3-OH-HZ) greater than N-desalkylhalazepam (NDZ, also known as N-desmethyldiazepam and nordiazepam) much greater than oxazepam (OX) for all three rat liver microsomal preparations and the distribution of metabolites was independent of microsomal enzyme concentrations. Enantiomers of 3-OH-HZ were resolved by HPLC on a Chiralcel OC column (cellulose trisphenylcarbamate coated on silica gel, particle size 10 microns). 3-OH-HZ enantiomeres have racemization half-lives of approximately 150 min in pH 4, 7.5, and 10 aqueous solutions. 3-OH-HZ formed in the metabolism of HZ by liver microsomes prepared from untreated and treated rats were found to have 3R/3S enantiomer ratios of 37/63 (untreated), 55/45 (PB-treated), and 36/64 (3MC-treated), respectively. N-dealkylation of 3-OH-HZ by liver microsomes from PB-treated rats was substrate enantioselective; the 3R-enantiomer was N-dealkylated faster than 3S-enantiomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of diazepam and related benzodiazepines by human liver microsomes

“…However, not even this procedure enabled us to solubilize pinazepam, prazepam or balazepam in incubation media at concentrations bigh enough for enzyme kinetic studies. Recent reports with both prazepam (5) and halazepam (6) involved the use of methanolic solutions of substrates, which our results do not support for kinetic studies, though this may not have compromised the investigation of stereochemical factors for wbicb the cited studies were designed. The present method differed from that of Inaba et al (11) only in respect of the technique used for introduction of substrate, and in our use of an HPLC method for benzodiazepine analysis, as opposed to their GC method.…”

“…We bave demonstrated in the studies reported bere that diazepam, pinazepam, prazepam and halazepam do not bave sufficient solubility in microsomal incubates to render useful metabolic studies practicable with presently available product detection methodologies (Table III). Some workers (4)(5)(6) have utilized quantities of organic solvents to overcome this difficulty. We have shown, bowever, that a combination of two commonly used solvents, acetone and propylene glycol, substantially inhibits metabol-ism of diazepam by both the N-demethylation and the C3-bydroxylation pathways (Table IV).…”

“…However a review of the literature showed that many in vitro metabolism studies involved methodology which was open to criticism. Some examples are the use of organic solvents to solubilize benzodiazepines in the incubation medium (4)(5)(6), inappropriately long incubation times (e.g. 60 min (7», and addition of nicotinamide, a monooxygenase inhibitor, to the incubating medium (8,9).…”

Resolution and stability of oxazepam enantiomers

“…7,8 Following evaporation of D17P3, OX highly enriched in either (R)-or (S)-enantiomer was dissolved in either a pure solvent or a solvent mixture for the determination of racemization tlh. Racemization half-lives were determined using 23 organic solvents potentially useful in CSP-HPLC separation of OX enantiomers (Table 1).…”

“…Immediately afterward, changes of ellipticity (A@, in millidegrees) were recorded at a wavelength (255,310,320, or 330 nm) as a function of time at ambient temperature on a JASCO Model 500A spectropolarimeter uapan Scientific Co., Easton, MD). [7][8][9][10] The monitoring wavelength was chosen to avoid strong absorption by the solvent. The tlh was determined by plotting log (A@) versus time.…”