Defining the use of midazolam as a probe of CYP3A4 activity: sensitive quantitation of the metabolites and characterization of mechanism-based inactivation

Abstract

A highly sensitive GC NCI/MS assay was developed for midazolam metabolites. The limit of quantitation was 0.3 ng/mL. This assay allowed the determination of kinetic parameters for metabolite formation from in vitro incubations that contained only 250 fmol/mL CYP3A4 and included midazolam concentrations as low as 200 nM (similar to the in vivo concentrations of this drug). Time-dependent metabolite formation was found to be limited to only 4 minutes for the calculation of initial rates of product formation, and the reasons for this were investigated.During these investigations, evidence that midazolam is a selective mechanism-based inactivator of CYP3A4 was obtained and can be summarized as follows: (1) Active site titration experiments gave linear results in both human liver microsomes and a cDNA-expressed CYP3A4 preparation, and the partition ratio was approximately 200 in both systems. (2) Pseudo-first order, saturable inactivation kinetics were observed, and a $\rm K\sb{I}=15.0\pm3.4\ \mu$M and a k$\rm\sb{inact}=0.338\pm0.135\ min\sp{-1}$ were determined. (3) The requirement of catalytic turnover for inactivation was inferred from the observation that inactivation required NADPH. (4) Inactivation was not attenuated in the presence of superoxide dismutase, catalase, glutathione, or N-acetyl cysteine. (5) Inactivation was attenuated in the presence of a competing substrate, alfentanil. (6) A 1 to 1 correspondence between the level of NADPH-dependent $\sp3$H-midazolam irreversible binding to microsomal protein and the level of inactivation of CYP3A4 was observed. Therefore, all of the established criteria for mechanism-based inactivation were addressed.Despite mechanism-based inactivation, midazolam can be used as an in vitro probe of CYP3A4 activity. When the midazolam concentrations used to determine K$\rm\sb{m}$ and V$\rm\sb{max}$ are $\leq$4 $\mu$M, the ratio, K$\rm\sb{m}/V\sb{max},$ for 1$\sp\prime$-hydroxylation was determined by the content of CYP3A4 in hepatic microsomes from a population of organ donors. Under these conditions inactivation did not significantly alter the kinetics of metabolite formation. However, when the concentrations of midazolam were $\geq$10 $\mu$M, inactivation significantly effected the relative rates observed for 1$\sp\prime$- and 4-hydroxylation. The gross kinetic characteristics of 1$\sp\prime$- and 4-hydroxylation at high midazolam concentrations were predicted by calculating the time-averaged levels of CYP3A4 remaining during the incubation.