4.2. Metabolic Stability of 25B-NBF in Human Hepatocytes

Pooled cryopreserved human hepatocytes were carefully thawed in thawing medium and resuspended in Krebs–Henseleit buffer to a final density of 1.0 × 10⁶ cells/mL. Then a 60 μL aliquot of this hepatocyte suspension and equal volume of 25B-NBF (4 μM) in Krebs–Henseleit buffer were mixed into 96-well plates and incubated in triplicate for 0, 5, 15, 30, 60, 120, or 180 min in a CO₂ incubator at 37 °C. Incubation was quenched by addition of 120 µL ice-cold acetonitrile to each well, and the samples were centrifuged at 15,000× g for 10 min at 4 °C after 5 min sonication. Then 100 µL of supernatants were vortex-mixed with equal volume of water and 2 μL aliquots were analyzed by an LC–MS/MS system. Honokiol (2 µM) was separately incubated for the positive control of this system. The peak area ratios of substances versus IS at each sampling point were used in subsequent calculations of parameters. The following equations were used in the calculation of elimination parameters, including t_1/2, Cl_int, Cl_hep, and hepatic extraction ratio of 25B-NBF.

Q_h refers to hepatic blood flow [29]. Comparing to the general classification of hepatic extraction ratio, the metabolic rate of 25B-NBF was evaluated [30].