CONCENTRATION-DEPENDENT METABOLISM OF DIAZEPAM IN MOUSE-LIVER

Previous mouse liver studies with diazepam (DZ), N-desmethyldiazepam ( NZ), and temazepam (TZ) confirmed that under first-order conditions, D Z formed NZ and TZ in parallel. Oxazepam (OZ) was generated via NZ and not TZ despite that performed NZ and TZ were both capable of forming OZ. In the present studies, the concentration-dependent sequential met abolism of DZ was studied in perfused mouse livers and microsomes, wit h the aim of distinguishing the relative importance of NZ and TZ as pr ecursors of OZ. In microsomal studies, the K(m)s and V(max)s, correcte d for binding to microsomal proteins, were 34 mu M and 3.6 nmole/min p er mg and 239 mu M and 18 nmole/min per mg, respectively, for N-demeth ylation and C-3-hydroxylation of DZ. The K(m)s and V(max)s for N-demet hylation and C-3-hydroxylation of TZ and NZ, respectively, to form OZ, were 58 mu M and 2.5 nmole/min per mg and 311 mu M and 2 nmole/min pe r mg, respectively. The constants suggest that at low DZ concentration s, NZ formation predominates and is a major source of OZ, whereas at h igher, DZ concentrations, TZ is the important source of OZ. In livers perfused will DZ at input concentrations of 13 to 35 mu M, the extract ion 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 disproportionately with increasing DZ concentration, wher eas that for OZ decreased and paralleled the behavior of NZ. Computer simulations based on a tubular flow model and the in vitro enzymatic p arameters provided a poor in vitro-organ correlation. The E{DZ}, appea rance rates of the metabolites, and tire extraction ratio of formed NZ (E{NZ, DZ}) were poorly predicted; TZ was incorrectly identified as t he major precursor of OZ. Simulations with optimized parameters improv ed the correlations and identified NZ as the major contributor of OZ. Saturation of DZ N-demethylation at higher DZ concentrations increased the role of TZ in the formation of OZ. The poor aqueous solubility (l imiting the concentration range of substrates used in vitro), avid tis sue binding mid the coupling of enzymatic reactions in liver favoring sequential metabolism, are possible explanations for the poor in vitro -organ correlation. This work emphasizes tire complexity of tire hepat ic intracellular milieu for drug metabolism and the need for additiona l modeling efforts to adequately describe metabolite kinetics.