TY - JOUR T1 - Modulation of Major Human Liver Microsomal Cytochromes P450 by Component Alkaloids of Goldenseal: Time-Dependent Inhibition and Allosteric Effects JF - Drug Metabolism and Disposition JO - Drug Metab Dispos DO - 10.1124/dmd.120.091041 SP - dmd.120.091041 AU - Matthew G. McDonald AU - Dan-Dan Tian AU - Kenneth E. Thummel AU - Mary F Paine AU - Allan E. Rettie Y1 - 2020/01/01 UR - http://dmd.aspetjournals.org/content/early/2020/06/26/dmd.120.091041.abstract N2 - Botanical and other natural products (NPs) are often co-consumed with prescription medications, presenting a risk for cytochrome P450 (CYP)-mediated NP-drug interactions. The NP goldenseal (Hydrastis canadensis) has exhibited anti-microbial activities in vitro attributed to isoquinoline alkaloids contained in the plant, primarily berberine, (-)-β-hydrastine and to a lesser extent, hydrastinine. These alkaloids contain methylenedioxyphenyl rings, structural alerts with potential to inactivate CYPs through formation of metabolic intermediate complexes. Time-dependent inhibition experiments were conducted to evaluate their ability to inhibit major CYP activities in human liver microsomes using a cocktail of isozyme-specific substrate probes. Berberine inhibited CYP2D6 (dextromethorphan O-demethylation; KI = 2.7 μM, kinact = 0.065 min-1) and CYP3A4/5 (midazolam 1'-hydroxylation; KI = 14.8 μM, kinact = 0.019 min-1); (-)-β-hydrastine inhibited CYP2C9 (diclofenac 4'-hydroxylation; KI = 49 μM, kinact = 0.036 min-1), CYP2D6 (KI > 250 μM, kinact > 0.06 min-1) and CYP3A4/5 (KI = 28 μM, kinact = 0.056 min-1); and hydrastinine inhibited CYP2D6 (KI = 37 μM, kinact = 0.049 min-1) activity. Berberine additionally exhibited allosteric effects on midazolam hydroxylation, showing both positive and negative heterotropic cooperativity. Experiments with recombinant isozymes showed that berberine activated midazolam 1'-hydroxylation by CYP3A5, lowering Km(app), but showed mixed inhibition and negative cooperativity towards this reaction when catalyzed by CYP3A4. Berberine inactivated CYP3A4 at a much faster rate than CYP3A5 and was a non-competitive inhibitor of midazolam 4-hydroxylation by CYP3A4 but a strong mixed inhibitor of the CYP3A5 catalyzed reaction. These complex kinetics should be considered when extrapolating the risk for NP-drug interactions involving goldenseal.SIGNIFICANCE STATEMENT Robust kinetic parameters were determined for the reversible and time-dependent inhibition of CYP2C9, CYP2D6 and CYP3A4/5 activities in HLMs by major component isoquinoline alkaloids contained in the botanical natural product (NP) goldenseal. The alkaloid berberine also exhibited opposing, isozyme-specific, allosteric effects on midazolam hydroxylation mediated by recombinant CYP3A4 (inhibition) and CYP3A5 (activation). These data will inform the development of a physiologically-based pharmacokinetic model that can be used to predict potential clinically relevant goldenseal-drug interactions. ER -