TABLE 2

Kinetic analyses for midazolam hydroxylation by recombinant CYP3A enzymes

Midazolam (1.4–100 μM) was incubated with recombinant CYP3A enzymes (40 pmol equivalent/mL) at 37°C for 15 min. Kinetic parameters were calculated from curves fitted by nonlinear regression (mean ± standard error, n = 10 substrate concentrations, in duplicate) using the substrate inhibition equation: v = Vmax × [S]/(Km + [S] + [S]2/Ks). Dog CYP3A98 and CYP3A12 catalyzed midazolam 1′-hydroxylation with statistically similar Km and Vmax values among four dog CYP3A enzymes tested.

P450Midazolam hydroxylationKm,μMKs,μMVmax, nmol/min/nmol P450Vmax/Km, ml/min/nmol
3A981-hydroxylation5.3 ± 3.3425 ± 3047 ± 158.9
4-hydroxylation27 ± 6N.A.18 ± 20.67
3A991-hydroxylation130 ± 120N.A.8.6 ± 5.10.05
4-hydroxylation160 ± 130N.A.8.3 ± 4.60.05
3A121-hydroxylation3.3 ± 1.472 ± 3463 ± 1119
4-hydroxylation35 ± 8N.A.20 ± 20.57
3A261-hydroxylation27 ± 6N.A.2.1 ± 0.20.08
4-hydroxylation25 ± 4N.A.4.1 ± 0.20.16
Human 3A41-hydroxylation4.0 ± 0.9185 ± 6760 ± 515
4-hydroxylation52 ± 9N.A.41 ± 40.79
  • N.A., not available.