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, (−)-β-hydrastine, and to a lesser extent, hydrastinine. These alkaloids contain methylenedioxyphenyl rings, structural alerts with 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; K_I = 2.7 μM, k_inact = 0.065 minute⁻¹) and CYP3A4/5 (midazolam 1′-hydroxylation; K_I = 14.8 μM, k_inact = 0.019 minute⁻¹); (−)-β-hydrastine inhibited CYP2C9 (diclofenac 4′-hydroxylation; K_I = 49 μM, k_inact = 0.036 minute⁻¹), CYP2D6 (K_I > 250 μM, k_inact > 0.06 minute⁻¹), and CYP3A4/5 (K_I = 28 μM, k_inact = 0.056 minute⁻¹); and hydrastinine inhibited CYP2D6 (K_I = 37 μM, k_inact = 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 1′-hydroxylation by CYP3A5, lowering K_m(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 NP-drug interactions involving goldenseal.