Concentration-dependent metabolism of diazepam in mouse liver

  • Marie V. St-Pierre
  • K. Sandy Pang


Previous mouse liver studies with diazepam (DZ),N-desmethyldiazepam (NZ), and temazepam (TZ) confirmed that under first-order conditions, DZ formed NZ and TZ in parallel. Oxazepam (OZ) was generatedvia NZ and not TZ despite that preformed NZ and TZ were both capable of forming OZ. In the present studies, the concentration-dependent sequential metabolism of DZ was studied in perfused mouse livers and microsomes, with the aim of distinguishing the relative importance of NZ and TZ as precusors of OZ. In microsomal studies, theKms andVmaxs, corrected for binding to microsomal proteins, were 34 μM and 3.6 nmole/min per mg and 239 μM and 18 nmole/min per mg, respectively, forN-demthylation andC3-hydroxylation of DZ. TheKms andVmaxs forN-demethylation andC3-hydroxylation of TZ and NZ, respectively, to form OZ, were 58 μM and 2.5 nmole/min per mg and 311 μM and 2 nmole/min per mg, respectively. The constants suggest that at low DZ concentrations, NZ formation predominates and is a major source of OZ, whereas at higher DZ concentrations, TZ is the important source of OZ. In livers perfused with DZ at input concentrations of 13 to 35 μM, the extraction ratio of DZ (E{DZ}) decreased from 0.83 to 0.60. NZ was the major metabolite formed although its appearance was less than proportionate with increasing DZ input concentration. By contrast, the formation of TZ increased disporportionately with increasing DZ concentration, whereas that for OZ decreased and paralleled the behavior of NZ. Computer simulations based on a tubular flow model and thein vitro enzymatic parameters provided a poorin vitro-organ correlation. TheE{DZ}, appearance rates of the metabolites, and the extraction ratio of formed NZ (E{NZ, DZ}) were poorly predicted; TZ was incorrectly identified as the major precursor of OZ. Simulations with optimized parameters imporved the correlations and identified NZ as the major contributor of OZ. Saturation of DZN-demethylation at higher DZ concentrations increased the role of TZ in the formation of OZ. The poor aqueous solubility (limiting the concentration range of substrates usedin vitro), avid tissue binding and the coupling of enzymatic reactions in liver, favoring sequential metabolism, are possible explanations for the poorin vitro-organ correlation. This work emphasizes the complexity of the hepatic intracellular milieu for drug metabolism and the need for additional modeling efforts to adequately describe metabolite kinetics.

Key Words

benzodiazpines diazepam C3-hydroxylation andN-demethylation N-desmethyldiazepam nordiazepam temazepam oxazepam tubular flow-model Km andVmax metabolite kinetics perfused mouse liver mouse liver microsomes in vitro-organ correlation 


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Copyright information

© Plenum Publishing Corporation 1995

Authors and Affiliations

  • Marie V. St-Pierre
    • 1
  • K. Sandy Pang
    • 1
    • 2
  1. 1.Faculty of PharmacyUniversity of TorontoTorontoCanada
  2. 2.Department of Pharmacology, Faculty of MedicineUniversity of TorontoTorontoCanada

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