Identification of human cytochrome P450 enzymes involved in the formation of 4-hydroxyestazolam from estazolam

Abstract

To predict drug interactions with estazolam, the biotransformation of estazolam to its major hydoxylated metabolite, 4-hydroxyestazolam was studied in vitro using pooled human liver microsomes and individual expressed human cytochrome P450 (CYP) enzymes. Estazolam was metabolized to 4-hydroxyestazolam according to the Hill kinetic model in pooled human liver microsomes. The K_m value for the 4-hydroxylation of estazolam was 24.1 µM, and the V_max value was 52.6 pmol min⁻¹ mg⁻¹ protein. The formation of 4-hydroxyestazolam from estazolam in pooled human liver microsomes was significantly inhibited by itraconazole and erythromycin, specific CYP3A4 inhibitors, in a dose-dependent manner, with IC₅₀ values of 1.1 and 12.8 µM, respectively. When estazolam was incubated with expressed human CYP enzymes (CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4), it was metabolized only by CYP3A4. In conclusion, the biotransformation of estazolam to 4-hydroxyestazolam was catalyzed by CYP3A4.

Key words::

• Estazolam
• 4-Hydroxyestazolam
• CYP3A4