Noncompetitive inhibition of human CYP2C9 in vitro by a commercial Rhodiola rosea product

Abstract

A commercial Rhodiola rosea (R. rosea) product has previously demonstrated CYP2C9 inhibition in humans. The purpose of this study was to provide in vitro inhibitory data for this particular interaction and to classify the mechanism of the interaction. Another aim was to examine the in vitro influence of ethanol on the CYP2C9 activity. Human CYP2C9 (wild type) isolated from a baculovirus-infected cell system was incubated with 0.8 μmol/L losartan for 20 min. Sulfaphenazole was used as a positive control. The commercial R. rosea product “Arctic Root” was used as test inhibitor. Formation of the CYP2C9-produced losartan metabolite EXP-3174 was determined by validated LC-MS/MS methodology. Possible mechanism-based (irreversible) inhibition was evaluated using time- and NADPH-dependent inhibition assays. Kinetic constants (Km, Vmax, and Ki) were calculated from a Lineweaver-Burk plot. Mode of inhibition was determined. CYP2C9 was inhibited by “Arctic Root” with an IC50 (extract concentration yielding 50% reduction in enzyme activity) of 19.2 ± 2.7 μg/mL. Inhibitor concentrations of 20 μg/mL and 40 μg/mL yielded Ki values of 16.37 μg/mL and 5.59 μg/mL, respectively. The Lineweaver-Burk plot showed noncompetitive inhibition mode. No time- or NADPH-dependent inhibition was observed. The presence of ethanol inhibited CYP2C9 activity in a concentration-dependent manner. In conclusion, the commercial R. rosea product “Arctic Root” demonstrated noncompetitive inhibition of CYP2C9 in vitro. Further work identifying the constituents responsible for this inhibition is needed.