In vitro simulation of clinical pharmacokinetics uncovers crucial hurdles of in vivo HDAC treatment. a reduction of class I HDAC activity after in vitro treatment of MED8A cells with vorinostat or MS-275 measured at times indicated after washout of HDACi as determined by HDAC activity assay. HDAC activity is strongly reduced immediately upon vorinostat treatment, however immediately returns to normal after a 90 min incubation period followed by washout. HDAC activity is reduced in a time-dependent manner upon treatment with MS275, and returns to normal approx. 24 h after a 90 min incubation period followed by washout. b Incubation with vorinostat induces immediate and strong acetylation of histone 4 (acH4), which is quickly reduced after a 90 min incubation period followed by washout. Incubation with MS275 induces acetylation of histone 4 (H4) in a time dependent manner, which is stable even after a 90 min incubation period followed by washout. Actin served as loading control. c Incubation for 72 h with vorinostat or MS-275 induces strong reduction of metabolic activity, but this effect is nullified if metabolic activity is measured after 72 h with an incubation period of 90 min followed by washout once every 24 h. Bars depict mean and standard error of three independent experiments, significant differences are indicated: * = p < 0.05 (unpaired t-test).