Incubation was conducted in 96-well cell culture plate format. The plates were labeled as T0, T15, T30, T60, T90, T0-MC, T90-MC and Blank, respectively. All plates were pre-warmed for 10 to 20 mins at 37±1°C in an incubator of 5% CO2 and saturated humidity. The vials of cryopreserved SD rat, beagle dog, cynomolgus monkey and human hepatocyte were removed from the liquid nitrogen container and immediately immersed in a water bath (the setting temperature was 37°C) for approximately 90 secs to allow the ice pellets melted. The melted ice pellets were transferred into pre-warmed 40 mL of Thawing Medium tubes mixed well by gently inverting the tubes then centrifuged at 100 ×g for 5 mins at room temperature. The supernatants were discarded and cell pellets were re-suspended by adding appropriate volumes of pre-warmed WEM. The cells viability of each species was determined using Trypan Blue exclusion. The viabilities of rat, dog, cynomolgus and human hepatocytes were 89.0%, 94.3%, 88.0% and 91.8%, respectively. Cells were finally diluted to 0.625×106 cells/mL with pre-warmed WEM. At each corresponding time point, the sample reactions were stopped by adding 150 μL of acetonitrile containing 200 ng/mL tolbutamide and 200 ng/mL labetalol as internal standards. All sample plates were thoroughly mixed and centrifuged at 3220 ×g for 20 mins. The supernatants were diluted at ratio of 1:3 with ultra-pure water for STP0404 and control samples then submitted for LC/MS/MS analysis (Wuxi AppTec, China).

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