Cytochrome P4502B6 and 2C9 do not metabolize midazolam: kinetic analysis and inhibition study with monoclonal antibodies.
We determined the contribution of cytochrome P450 (CYP) isoforms to the metabolism of midazolam by kinetic analysis of human liver microsomes and CYP isoforms and by examining the effect of chemical inhibitors and monoclonal antibodies against CYP isoforms in vitro. Midazolam was metabolized to 1'-hydroxymidazolam (1'-OH MDZ) by human liver microsomes with a Michaelis-Menten constant (Km) of 4.1 (1.0) (mean (SD)) micromol litre(-1) and a maximum rate of metabolism (Vmax) of 5.5 (1.1) nmol min(-1) mg protein(-1) (n = 6). Of the nine representative human liver CYP isoforms, CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5, three (CYP2B6, 3A4 and 3A5) showed midazolam 1'-hydroxylation activity, with Kms of 40.7, 1.7 and 3.0 micromol litre(-1), respectively, and Vmax values of 12.0, 3.3 and 13.2 nmol min(-1) nmol P450(-1), respectively (n = 4). Midazolam 1'-hydroxylation activity of human liver microsomes correlated significantly with testosterone 6beta-hydroxylation activity, a marker of CYP3A activity (r2 = 0.77, P = 0.0001), but not with S-mephenytoin N-demethylation activity, a marker of CYP2B6 activity (r2 < 0.01, P = 0.84) (n = 11). Troleandomycin and orphenadrine, chemical inhibitors of CYP isoforms, inhibited the formation of 1'-OH MDZ by human liver microsomes. Monoclonal antibody against CYP3A4 inhibited the formation of 1'-OH MDZ by 79%, whereas monoclonal antibody against CYP2B6 had no effect on midazolam 1'-hydroxylation by human liver microsomes (n = 5). These results indicate that only CYP3A4, but not CYP2B6 or CYP2C, is involved in the metabolism of midazolam in vitro.
Hamaoka
N
N
Department of Anesthesiology and Intensive Care Medicine, Osaka City University Medical School, Osaka, Japan.
Oda
Y
Y
Hase
I
I
Asada
A
A
D018686:Anesthetics, Intravenous / Q000378:metabolism*; D014151:Anti-Anxiety Agents / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000276:immunology; D001189:Aryl Hydrocarbon Hydroxylases; D019388:Cytochrome P-450 CYP1A2; D065702:Cytochrome P-450 CYP2B6; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006900:Hydroxylation; D008862:Microsomes, Liver / Q000378:metabolism; D008874:Midazolam / Q000378:metabolism*; D009966:Orphenadrine / Q000494:pharmacology; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D014217:Troleandomycin / Q000494:pharmacology
D018686:Anesthetics, Intravenous; D014151:Anti-Anxiety Agents; D000911:Antibodies, Monoclonal; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System; D009966:Orphenadrine; D014217:Troleandomycin; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C504573:CYP1A2 protein, human; C585599:CYP2B6 protein, human; D019388:Cytochrome P-450 CYP1A2; D065702:Cytochrome P-450 CYP2B6; D039181:Steroid 16-alpha-Hydroxylase; C022657:steroid hormone 6-beta-hydroxylase; D010089:Oxidoreductases, N-Demethylating; D008874:Midazolam