In Vitro Inhibition of Tofacitinib Metabolism by Baohuoside I

Based on the evidence from in vivo experiments demonstrating that baohuoside I inhibits tofacitinib metabolism, we aimed to investigate the inhibitory effects of baohuoside I in vitro. RLMs were prepared and the inhibitory effect of baohuoside I on tofacitinib metabolism was assessed using methods similar to those previously described.15 (link) The volume of incubation system was 200 µL, containing 100 mM pH 7.4 potassium phosphate buffer, 0.5 mg/mL RLMs, and the varying concentration of tofacitinib (1, 2.5, 5, 10, 25, 50, 100, 200 and 400 μM). The system was preincubated at 37 °C for 5 min. The reaction was then initiated by the addition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) at a final concentration of 1 mM, and the incubation was continued for 30 min. To determine the half-maximal inhibitory concentration (IC₅₀) of baohuoside I, various concentrations of baohuoside I (100, 50, 10, 5, 1, 0.1, 0.01, and 0 μM) were selected along with 40 µM of tofacitinib, which is close to its K_m value. In order to investigate the inhibitory mechanism of baohuoside I on tofacitinib, the two drugs were subjected to incubation at various concentration gradients, including 1/4 K_m, 1/2 K_m, K_m, and 2K_m of tofacitinib, as well as 0, 1/4 IC₅₀, 1/2 IC₅₀, IC₅₀, and 2IC₅₀ of baohuoside I. The incubation procedure remained consistent with the afore mentioned method.