CYP3A4 activity in four different animal species liver microsomes using 7-benzyloxyquinoline and HPLC/spectrofluorometric determination.

Some microplate-based direct assays with different fluorometric substrates have been developed, among which 7-benzyloxyquinoline (BOQ) has demonstrated the highest degree of selectivity for CYP3A subfamily. In our study, we firstly developed and validated an efficient, fast and cheap HPLC/spectrofluorometric analytical method to quantify 7-hydroxyquinoline (BOQ metabolite). Secondly, BOQ oxidation rate (1.95±0.24μM/mg protein/min) was compared to that of midazolam (MDZ) (1.4±0.21μM/mg protein/min), an other specific CYP3A probe. However, the difference did not reach statistically significance (test of Sign; p=0.125, two tailed). Thirdly, the potential use of BOQ in other species than the rat (mouse, dog and monkey) was studied. The highest BOQ activity was observed in rat microsomes (3.75μmol/mg protein/min) with lower P450 content (0.3nmol/mg protein) compared to other species. Finally, the effect of CYP3A enzymes-selective inhibitor ketoconazole on the dealkylation of BOQ in control and dexamethasone (DM)-treated rat microsomes was studied. Ketoconazole inhibition potency was greater in control (IC50≈21.6μM) compared to DM induced (IC50≈32.3μM) microsomes. At concentrations greater than that considered to be enzyme-selective (e.g., 10–30μM), ketoconazole inhibitory activity did not rise significantly, and at the maximal concentration tested (1000μM) a nearly similar inhibition (76%) was observed than that at 50μM concentration (68.2%).