title: Midazolam and triazolam biotransformation in mouse and human liver microsomes: relative contribution of CYP3A and CYP2C isoforms.
abstract: Midazolam (MDZ) and triazolam (TRZ) hydroxylation, reactions considered to be cytochrome P-4503A (CYP3A)-mediated in humans, were examined in mouse and human liver microsomes. In both species, alpha- and 4-hydroxy metabolites were the principal products. Western blotting with anti-CYP3A1 antibody detected a single band of immunoreactive protein in both human and mouse samples: 0.45 +/- 0. 12 and 2.02 +/- 0.24 pmol/mg protein (mean +/- S.E., n = 3), respectively. Ketoconazole potently inhibited MDZ and TRZ metabolite formation in human liver microsomes (IC(50) range, 0.038-0.049 microM). Ketoconazole also inhibited the formation of both TRZ metabolites and of 4-OH-MDZ formation in mouse liver microsomes (IC(50) range, 0.0076-0.025 microM). However, ketoconazole (10 microM) did not produce 50% inhibition of alpha-OH-MDZ formation in mouse liver microsomes. Anti-CYP3A1 antibodies produced concentration-dependent inhibition of MDZ and TRZ metabolite formation in human liver microsomes and of TRZ metabolite and 4-OH-MDZ formation in mouse liver microsomes to less than 20% of control values but reduced alpha-OH-MDZ formation to only 66% of control values in mouse liver microsomes. Anti-CYP2C11 antibodies inhibited alpha-OH-MDZ metabolite formation in a concentration-dependent manner to 58% of control values in mouse liver microsomes but did not inhibit 4-OH-MDZ formation. Thus, TRZ hydroxylation appears to be CYP3A specific in mice and humans. alpha-Hydroxylation of MDZ has a major CYP2C component in addition to CYP3A in mice, demonstrating that metabolic profiles of drugs in animals cannot be assumed to reflect human metabolic patterns, even with closely related substrates.
authors.affiliation: Department of Pharmacology, Tufts University School of Medicine, Boston, Massachusetts, USA.
authors.forename: M D
authors.initials: MD
authors.lastname: Perloff
authors.forename: L L
authors.initials: LL
authors.lastname: von Moltke
authors.forename: M H
authors.initials: MH
authors.lastname: Court
authors.forename: T
authors.initials: T
authors.lastname: Kotegawa
authors.forename: R I
authors.initials: RI
authors.lastname: Shader
authors.forename: D J
authors.initials: DJ
authors.lastname: Greenblatt
mesh_terms: D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases; D001711:Biotransformation; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007120:Immunochemistry; D066298:In Vitro Techniques; D020128:Inhibitory Concentration 50; D007654:Ketoconazole / Q000494:pharmacology; D051379:Mice; D008862:Microsomes, Liver / Q000378:metabolism*; D008874:Midazolam / Q000493:pharmacokinetics*; D006899:Mixed Function Oxygenases / Q000276:immunology; D010089:Oxidoreductases, N-Demethylating / Q000502:physiology*; D020033:Protein Isoforms / Q000502:physiology; D013045:Species Specificity; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000276:immunology; D014229:Triazolam / Q000493:pharmacokinetics*
chemical_list: D000906:Antibodies; D020033:Protein Isoforms; C400322:cytochrome P-450 CYP2C subfamily; D014229:Triazolam; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; D039181:Steroid 16-alpha-Hydroxylase; D010089:Oxidoreductases, N-Demethylating; D008874:Midazolam; D007654:Ketoconazole