TABLE 3

Kinetic parameters of midazolam 1′- and 4-hydroxylation by recombinant P450 3A enzymes and tissue microsomes from marmosets, cynomolgus monkeys, and humans

Kinetic parameters were determined by nonlinear regression analysis (mean ± standard error, n = 17 points of substrate concentrations of 0.5–625 μM) using the equation v = Vmax × [S]/(Km + [S]) for Michaelis-Menten or v = Vmax × [S]/(Km + [S] + [S]2/Ks) for substrate inhibition (Shimizu et al., 2007; Okada et al., 2009). Units of enzyme activities for tissue microsomes and recombinant P450 proteins are nmol/min/mg protein and nmol/min/nmol of P450, respectively. Units of Vmax/Km for tissue microsomes and recombinant P450 proteins are ml/min/nmol and ml/min/mg protein, respectively. The units of Km and Ks values are μM.

Enzyme SourceMidazolam 1′-HydroxylationMidazolam 4- hydroxylation
KmVmaxVmax/KmKSKmVmaxVmax/KmKS
Microsomes
 Marmoset liver8.1 ± 0.56.0 ± 0.10.74640 ± 5037 ± 21.5 ± 0.10.041
 Cynomolgus monkey liver15 ± 15.0 ± 0.10.332200 ± 6067 ± 44.2 ± 0.10.063
 Human liver6.6 ± 0.42.5 ± 0.10.381300 ± 13064 ± 31.1 ± 0.10.017
 Marmoset small intestine6.6 ± 0.51.7 ± 0.10.263200 ± 82033 ± 60.35 ± 0.020.011
 Cynomolgus monkey small intestine7.0 ± 0.22.1 ± 0.10.3039 ± 11.4 ± 0.10.036
 Human small intestine1.7 ± 0.10.88 ± 0.010.52930 ± 6827 ± 20.34 ± 0.010.013
Recombinant P450
 Marmoset 3A4 ortholog2.5 ± 0.435 ± 114710 ± 15021 ± 225 ± 11.2
 Marmoset 3A5 ortholog170 ± 443.7 ± 0.40.020190 ± 214.7 ± 0.20.025
 Marmoset 3A9034 ± 116 ± 12000 ± 100310 ± 213.4 ± 0.10.011
 Cynomolgus monkey 3A42.5 ± 0.221 ± 18.4600 ± 7916 ± 140 ± 12.51200 ± 100
 Cynomolgus monkey 3A53.2 ± 0.528 ± 18.81900 ± 6808.2 ± 0.124 ± 12.9
 Human 3A41.7 ± 0.212 ± 17.1660 ± 10025 ± 38.6 ± 0.50.322600 ± 1100
 Human 3A53.8 ± 0.547 ± 2121900 ± 56030 ± 25.9 ± 0.10.20