Kinetic parameters for efavirenz 8-oxidation
Efavirenz (0.5, 2, 5, 20, and 50 μM) was incubated with monkey liver microsomes (0.1 mg/ml) or recombinant monkey CYP2C or CYP2B6 enzymes (10 pmol/ml) at 37°C for 10 minutes in the presence of an NADPH-generating system. 8-Hydroxyefavirenz formation was quantified by LC-MS/MS using mefenamic acid as internal standard and showed linearity between 0.1–0.5 mg protein/ml in monkey liver microsomes, 10–50 pmol of P450 in monkey CYP2C9, and reaction time range of 10–20 minutes. Kinetic analysis was done using nonlinear regression analysis employing the Michaelis-Menten equation or the equation for substrate inhibition: v = Vmax • [S]/(Km + [S]), or v = Vmax • [S]/(Km + [S] + [S]2/Ks).
| Enzyme | Efavirenz 8-Oxidation | |||
|---|---|---|---|---|
| Km | Vmax | Vmax/Km | Ks | |
| Monkey liver microsomes | 2.5 ± 0.3 μM | 0.23 ± 0.01a | 0.09 | — |
| Monkey CYP2C9 | 9.9 ± 2.2 μM | 5.4 ± 1.0a | 0.54 | 3.3 ± 0.7 μM |
| Monkey CYP2C19 | — | <0.05a | — | — |
| Monkey CYP2C76 | — | <0.05a | — | — |
| Monkey CYP2B6 | — | <0.05a | — | — |
↵a nmol/min per nanomole of P450.