Kinetic characterization and identification of the enzymes responsible forthe hepatic biotransformation of adinazolam and N-desmethyladinazolam in man

The kinetics of the N-demethylation of adinazolam to N-desmethyladinazolam (NDMAD), and of NDMAD to didesmethyladinazolam (DDMAD), were studied with h uman liver microsomes using substrate concentrations in the range 10-1000 m u M. The specific cytochrome P450 (CYP) isoforms mediating the biotransform ations were identified using microsomes containing specific recombinant CYP isozymes expressed in human lymphoblastoid cells, and by the use of CYP is oform-selective chemical inhibitors. Adinazolam was demethylated by human liver microsomes to NDMAD, and NDMAD w as demethylated to DDMAD; the substrate concentrations, K-m, at which the r eaction velocities were 50% of the maximum were 92 and 259 mu M, respective ly. Another metabolite of yet undetermined identity (U) was also formed fro m NDMAD (K-m 498 mu M). Adinazolam was demethylated by cDNA-expressed CYP 2 C19 (K-m 39 mu M) and CYP 3A4 (K-m 83 mu M); no detectable activity was obs erved for CYPs 1A2, 2C9, 2D6 and 2E1. Ketoconazole, a relatively specific C YP 3A4 inhibitor, inhibited the reaction; the concentration resulting in 50 % of maximum inhibition, IC50, was 0.15 mu M and the inhibition constant, K -i, was < 0.04 mu M in five of six livers tested. Troleandomycin, a specifi c inhibitor of CYP 3A4, inhibited adinazolam N-demethylation with an IC50 o f 1.96 mu M. The CYP 2C19-inhibitor omeprazole resulted in only partial inh ibition (IC50 21 mu M) and sulphaphenazole, alpha-naphthoflavone, quinidine and diethyldithiocarbamate did not inhibit the reaction. NDMAD was demethy lated by cDNA-expressed CYP 3A4 (K-m 220 mu M, Hill number A 1.21), CYP 2C1 9 (K-m 187 mu M, Hill number A 1.29) and CYP 2C9 (K-m 1068 mu M). Formation of U was catalysed by CYP 3A4 alone. Ketoconazole strongly inhibited NDMAD demethylation (IC50 0.14 mu M) and formation of U (IC50 < 0.1 mu M) wherea s omeprazole and sulphaphenazole had no effect on reaction rates. These results show that CYP 3A4 is the primary hepatic CYP isoform mediatin g the N-demethylation of adinazolam and NDMAD. Go-administration of adinazo lam with CYP 3A4 inhibitors such as ketoconazole or erythromycin might lead to reduced efficacy, since adinazolam by itself has relatively weak benzod iazepine agonist activity, with much of the pharmacological activity of adi nazolam being attributable to its active metabolite NDMAD.