Abstract

We used human liver microsomes (HLMs) and recombinant cytochromes P450 (P450s) to identify the routes of efavirenz metabolism and the P450s involved. In HLMs, efavirenz undergoes primary oxidative hydroxylation to 8-hydroxyefavirenz (major) and 7-hydroxyefavirenz (minor) and secondary metabolism to 8,14-dihydroxyefavirenz. The formation of 8-hydroxyefavirenz in two HLMs showed sigmoidal kinetics (average apparent K_m, 20.2 μM; V_max, 140 pmol/min/mg protein; and Hill coefficient, 1.5), whereas that of 7-hydroxyefavirenz formation was characterized by hyperbolic kinetics (K_m, 40.1 μM and V_max, 20.5 pmol/min/mg protein). In a panel of 10 P450s, CYP2B6 formed 8-hydroxyefavirenz and 8,14-dihydroxyefavirenz from efavirenz (10 μM) at the highest rate. The K_m value for the formation of 8-hydroxyefavirenz in CYP2B6 derived from hyperbolic Eq. 12.4 μM) was close to that obtained in HLMs (K_m, 20.2 μM). None of the P450s tested showed activity toward 7-hydroxylation of efavirenz. When 8-hydroxyefavirenz (2.5 μM) was used as a substrate, 8,14-dihydroxyefavirenz was formed by CYP2B6 at the highest rate, and its kinetics showed substrate inhibition (K_si, ∼94 μM in HLMs and ∼234 μM in CYP2B6). In a panel of 11 HLMs, 8-hydroxyefavirenz and 8,14-dihydroxyefavirenz formation rates from efavirenz (10 μM) correlated significantly with the activity of CYP2B6 and CYP3A. N,N',N”-Triethylenethiophosphoramide (thioTEPA; 50 μM) inhibited the formation rates of 8-hydroxyefavirenz and 8,14-dihydroxyefavirenz from efavirenz (10 μM) by ≥60% in HLMs) and CYP2B6, with K_i values < 4 μM. In conclusion, CYP2B6 is the principal catalyst of efavirenz sequential hydroxylation. Efavirenz systemic exposure is likely to be subject to interindividual variability in CYP2B6 activity and to drug interactions involving this isoform. Efavirenz may be a valuable phenotyping tool to study the role of CYP2B6 in human drug metabolism.