Flunitrazepam oxidative metabolism in human liver microsomes: involvement of CYP2C19 and CYP3A4

Abstract

1. The aims were to examine the kinetics of the oxidative metabolism of flunitrazepam in vitro when flunitrazepam was dissolved in dimethylformamide and acetonitrile, and to determine which cytochrome P450 isoform(s) are involved. 2. The kinetics of the formations of 3'-hydroxyflunitrazepam and desmethylflunitrazepam were non-linear and best estimated using the Hill equation. Inhibition of their formation was studied using specific chemical inhibitors, expressed enzyme systems and specific antibodies. 3. K_s, V_max, Cl_max and n (slope factor) for the formation of 3'-hydroxyflunitrazepam and desmethylflunitrazepam had ranges of 165-338 and 179-391 μM, 22-81 and 3-10 nmol.mg protein⁻¹.h⁻¹, 6-17 and 0.9-1.9 μl.mg protein⁻¹.h⁻¹, and 2.3-3.6 and 1.6-2.6 respectively when dimethylformamide was the organic solvent. 4. When acetonitrile was the solvent, K_s, V_max, Cl_max and n (slope factor) for the formation of 3'-hydroxyflunitrazepam and desmethylflunitrazepam had ranges of 173-231 and 74-597 μM, 35-198 and 2.7-48 nmol.mg protein⁻¹.h⁻¹, 13-47 and 0.7-6.3 μl.mg protein⁻¹.h⁻¹, and 1.5-3.6 and 1.1-2.7 respectively. 5. CYP2C19, CYP3A4 and CYP1A2 mediated the formation of both 3'-hydroxyflunitrazepam and desmethylflunitrazepam. 6. Investigators need carefully to consider the choice of organic solvent to avoid false CYP identification.