Modulation of major human liver microsomal cytochromes p450 by component alkaloids of goldenseal: Time-dependent inhibition and allosteric effects

Abstract

Botanical and other natural products (NPs) are often coconsumed with prescription medications, presenting a risk for cytochrome P450 (P450)-mediated NP-drug interactions. The NP goldenseal (Hydrastis canadensis) has exhibited antimicrobial activities in vitro attributed to isoquinoline alkaloids contained in the plant, primarily berberine, (2)-β-hydrastine, and to a lesser extent, hydrastinine. These alkaloids contain methylenedioxyphenyl rings, structural alertswith potential to inactivate P450s through formation of metabolic intermediate complexes. Time-dependent inhibition experiments were conducted to evaluate their ability to inhibit major P450 activities in human liver microsomes by using a cocktail of isozyme-specific substrate probes. Berberine inhibited CYP2D6 (dextromethorphan O-demethylation; KI= 2.7 μM, kinact= 0.065 minute-1) and CYP3A4/5 (midazolam 19-hydroxylation; KI= 14.8 μM, kinact= 0.019 minute-1); (2)-β-hydrastine inhibited CYP2C9 (diclofenac 49-hydroxylation; KI= 49 μM, kinact= 0.036 minute-1), CYP2D6 (KI> 250 μM, kinact> 0.06 minute-1), and CYP3A4/5 (KI=28μM, kinact= 0.056minute-1); and hydrastinine inhibited CYP2D6 (KI= 37 μM, kinact= 0.049 minute-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 19-hydroxylation by CYP3A5, lowering Km(app), but showed mixed inhibition and negative cooperativity toward this reaction when catalyzed by CYP3A4. Berberine inactivated CYP3A4 at a much faster rate than CYP3A5 and was a noncompetitive 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 NPdrug interactions involving goldenseal.