Determining the Fraction Unbound of Herbal Product Constituents in Human Liver Microsomes to Improve Herb‐Drug Interaction Predictions

Adverse interactions between herbal products and conventional medications are a growing public health concern. Inhibition of drug metabolizing enzyme activity by herbal constituents is a common mechanism underlying these interactions. Inhibition constants (K i s) for isolated constituents of the top‐selling herbal product, milk thistle, towards multiple drug metabolizing enzymes have been assessed. The fraction unbound (f u ) for each constituent was determined in the current work to calculate ‘true' K i to improve interaction prediction accuracy. Incubation mixtures consisting of HLM (0.4 mg/mL), milk thistle constituent or binding control (1 µM), and potassium phosphate (KP i ) buffer (0.1 M, pH 7.4) were prepared. Aliquots (100 µL) were added to the donor side of 12‐14 kDa semipermeable membranes of a 96‐well equilibrium dialysis device; KP i (100 μL) was added to the receiver side. After 6 h at 37°C, aliquots (50 µL) were collected from each side, and internal standard (naringin) was added; contents were analyzed by LC‐MS/MS. f u was calculated by dividing the peak area ratio (analyte/naringin) of the receiver to that of the donor side. Constituent f u ranged from 0.77 ± 0.03 to 1.02 ± 0.03 (n = 3). High, medium, and low binding control f u was 0.29 ± 0.06, 0.63 ± 0.05, and 0.78 ± 0.05 (n = 9), respectively. The high milk thistle constituent f u will not substantially impact true K i . The equilibrium dialysis technique, routinely used to determine the f u of conventional drug molecules, was applied successfully to milk thistle constituents and could be applied to other herbal product constituents. Supported by the NIH (R01 GM077482) . DA was a SURF recipient.