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

J Pharm Pharmacol. 1998 Mar;50(3):265-74. doi: 10.1111/j.2042-7158.1998.tb06859.x.


The kinetics of the N-demethylation of adinazolam to N-desmethyladinazolam (NDMAD), and of NDMAD to didesmethyladinazolam (DDMAD), were studied with human liver microsomes using substrate concentrations in the range 10-1000 microM. The specific cytochrome P450 (CYP) isoforms mediating the biotransformations were identified using microsomes containing specific recombinant CYP isozymes expressed in human lymphoblastoid cells, and by the use of CYP isoform-selective chemical inhibitors. Adinazolam was demethylated by human liver microsomes to NDMAD, and NDMAD was demethylated to DDMAD; the substrate concentrations, Km, at which the reaction velocities were 50% of the maximum were 92 and 259 microM, respectively. Another metabolite of yet undetermined identity (U) was also formed from NDMAD (Km 498 microM). Adinazolam was demethylated by cDNA-expressed CYP 2C19 (Km 39 microM) and CYP 3A4 (Km 83 microM); no detectable activity was observed for CYPs 1A2, 2C9, 2D6 and 2E1. Ketoconazole, a relatively specific CYP 3A4 inhibitor, inhibited the reaction; the concentration resulting in 50% of maximum inhibition, IC50, was 0.15 microM and the inhibition constant, Ki, was < 0.04 microM in five of six livers tested. Troleandomycin, a specific inhibitor of CYP 3A4, inhibited adinazolam N-demethylation with an IC50 of 1.96 microM. The CYP 2C19-inhibitor omeprazole resulted in only partial inhibition (IC50 21 microM) and sulphaphenazole, alpha-naphthoflavone, quinidine and diethyldithiocarbamate did not inhibit the reaction. NDMAD was demethylated by cDNA-expressed CYP 3A4 (Km 220 microM, Hill number A 1.21), CYP 2C19 (Km 187 microM, Hill number A 1.29) and CYP 2C9 (Km 1068 microM). Formation of U was catalysed by CYP 3A4 alone. Ketoconazole strongly inhibited NDMAD demethylation (IC50 0.14 microM) and formation of U (IC50 < 0.1 microM) whereas omeprazole and sulphaphenazole had no effect on reaction rates. These results show that CYP 3A4 is the primary hepatic CYP isoform mediating the N-demethylation of adinazolam and NDMAD. Co-administration of adinazolam with CYP 3A4 inhibitors such as ketoconazole or erythromycin might lead to reduced efficacy, since adinazolam by itself has relatively weak benzodiazepine agonist activity, with much of the pharmacological activity of adinazolam being attributable to its active metabolite NDMAD.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Anti-Anxiety Agents / metabolism*
  • Aryl Hydrocarbon Hydroxylases*
  • Benzodiazepines / metabolism*
  • Biotransformation
  • Cytochrome P-450 CYP1A2 / metabolism
  • Cytochrome P-450 CYP2C19
  • Cytochrome P-450 CYP2D6 / metabolism
  • Cytochrome P-450 CYP2E1 / metabolism
  • Cytochrome P-450 CYP3A
  • Cytochrome P-450 Enzyme Inhibitors
  • Cytochrome P-450 Enzyme System / metabolism*
  • Enzyme Inhibitors / pharmacology
  • Humans
  • In Vitro Techniques
  • Ketoconazole / pharmacology
  • Kinetics
  • Macrolides
  • Microsomes, Liver / enzymology*
  • Mixed Function Oxygenases / antagonists & inhibitors
  • Mixed Function Oxygenases / metabolism*
  • Omeprazole / pharmacology
  • Steroid 16-alpha-Hydroxylase*
  • Steroid Hydroxylases / metabolism


  • Anti-Anxiety Agents
  • Cytochrome P-450 Enzyme Inhibitors
  • Enzyme Inhibitors
  • Macrolides
  • didesmethyladinazolam
  • Benzodiazepines
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • Steroid Hydroxylases
  • Cytochrome P-450 CYP2E1
  • Aryl Hydrocarbon Hydroxylases
  • CYP1A2 protein, human
  • CYP2C19 protein, human
  • CYP3A protein, human
  • Cytochrome P-450 CYP1A2
  • Cytochrome P-450 CYP2C19
  • Cytochrome P-450 CYP2D6
  • Cytochrome P-450 CYP3A
  • Steroid 16-alpha-Hydroxylase
  • Omeprazole
  • adinazolam
  • mono-N-demethyladinazolam
  • Ketoconazole