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 +/- 0.24 microM/mg protein/min) was compared to that of midazolam (MDZ) (1.4 +/- 0.21 microM/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 (3.75 micromol/mg protein/min) with lower P450 content (0.3 nmol/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 (IC(50) approximately 21.6 microM) compared to DM induced (IC(50) approximately 32.3 microM) microsomes. At concentrations greater than that considered to be enzyme-selective (e.g., 10-30 microM), ketoconazole inhibitory activity did not rise significantly, and at the maximal concentration tested (1,000 microM) a nearly similar inhibition (76%) was observed than that at 50 microM concentration (68.2%).
Baririan
Narine
N
Clinical Pharmacology Unit, Hôpital Universitaire St. Luc, Université Catholique de Louvain, UCL, Avenue Hippocrate, 10 MD/GAEN, 1200 Brussels, Belgium.
Desager
Jean-Pierre
JP
Petit
Martine
M
Horsmans
Yves
Y
D000818:Animals; D002138:Calibration; D002623:Chemistry Techniques, Analytical / Q000379:methods; D002626:Chemistry, Pharmaceutical / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000378:metabolism*; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D000882:Haplorhini; D006912:Hydroxyquinolines / Q000032:analysis; D020128:Inhibitory Concentration 50; D007654:Ketoconazole / Q000032:analysis; D007700:Kinetics; D008297:Male; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology*; D008874:Midazolam / Q000032:analysis; D011804:Quinolines / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D013045:Species Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013250:Steroid Hydroxylases / Q000737:chemistry; D013379:Substrate Specificity
D006912:Hydroxyquinolines; D011804:Quinolines; C065956:7-benzyloxyquinoline; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D008874:Midazolam; D007654:Ketoconazole