Cytochrome P4502B6 and 2C9 do not metabolize midazolam: kinetic analysis and inhibition study with monoclonal antibodies

We determined the contribution of cytochrome P450 (CYP) isoformsto the metabolism of midazolam by kinetic analysis of humanliver microsomes and CYP isoforms and by examining the effectof chemical inhibitors and monoclonal antibodies against CYPisoforms in vitro. Midazolam was metabolized to 1'-hydroxymidazolam(1'-OH MDZ) by human liver microsomes with a Michaelis–Mentenconstant (K_m) of 4.1 (1.0) (mean (SD)) µmol litre^–1and a maximum rate of metabolism (V_max) of 5.5 (1.1) nmol min^–1mg protein^–1 (n=6). Of the nine representative human liverCYP isoforms, CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and3A5, three (CYP2B6, 3A4 and 3A5) showed midazolam 1'-hydroxylationactivity, with K_ms of 40.7, 1.7 and 3.0 µmol litre^–1,respectively, and V_max values of 12.0, 3.3 and 13.2 nmol min^–1nmol P450^–1, respectively (n=4). Midazolam 1'-hydroxylationactivity of human liver microsomes correlated significantlywith testosterone 6ß-hydroxylation activity, a markerof CYP3A activity (r²=0.77, P=0.0001), but not with S-mephenytoinN-demethylation activity, a marker of CYP2B6 activity (r²<0.01,P=0.84) (n=11). Troleandomycin and orphenadrine, chemical inhibitorsof CYP isoforms, inhibited the formation of 1'-OH MDZ by humanliver microsomes. Monoclonal antibody against CYP3A4 inhibitedthe formation of 1'-OH MDZ by 79%, whereas monoclonal antibodyagainst CYP2B6 had no effect on midazolam 1'-hydroxylation byhuman liver microsomes (n=5). These results indicate that onlyCYP3A4, but not CYP2B6 or CYP2C, is involved in the metabolismof midazolam in vitro. Br J Anaesth 2001; 86: 540–4