To put these results in the context of the previous study, the subcutaneous administration is easier to interpret, since both studies used 4b in soluble form for acute administration, which largely sets aside any salt versus free-base stability disparities. Indeed, a comparative stability analysis of 4b and 4bNTFA prepared at 1 mg/ml in DMSO showed both are soluble, but the TFA salt again showed signs of degradation. According to Thomas et al, verification of an acute pharmacodynamic response was CMPD101 achieved by the repeated once daily injection for three consecutive days. Assuming linear kinetics and based on our values when 4b was dosed at 4.22 mg/kg, the brain Cmax achieved in that study would be in the region, sufficient to potentially fully inhibit Class I HDAC cellular activity, based on our cellular in vitro potency values, and consistent with the positive results obtained. However, given the fold lower exposures shown here from the oral administration versus the scroute via equivalent oral bolus administration of 150 mg/kg. This estimated Cmax is approximately lower than the most potent cellular HDAC in vitro IC50 value we measured. With the slower continuous administration of 4b via a drinking water study, we would expect the Cmax achieved to be significantly lower. The lack of any pharmacodynamic response predictive of central Class I HDAC CDP 840 hydrochloride inhibition when 4b was dosed orally at 150 mg/kg twice daily for 5 days confirmed this prediction. In conclusion, our findings demonstrate that the physicochemical properties, metabolic and P-glycoprotein substrate liabilities of 4b render it unsuitable as a molecular tool to investigate central Class I HDAC inhibition in vivo in mouse by oral administration. In the pivotal proof of concept trial, 4b was given to R6/2 mice in drinking water, leading to improved behavioral phenotypes. We conclude that this is highly unlikely to be due to HDAC inhibition in the CNS or related to the finding of the reversal of transcriptional dysregulation detected in the acute pharmacodynamic trial, where brain concentration of 4b was most likely,65 fold higher. Our results cast serious doubts on the validation of CNS HDAC3 as a target for the treatment of HD. Our findings are consistent with Moumne�� et al, who demonstrated that a genetic cross of Hdac heterozygotes with R6/2 mice effectively reduced nuclear HDAC3 levels, but did not ameliorate physiological or behavioural phenotypes and had no effect on molecular changes including dysregulated transcripts. We cannot rule out that a metabolite of 4b or C1 was responsible for the therapeutic benefit seen in R6/2 mice per oral dosing of 4b in the previous study. To our knowledge, no data has been published on the ADME properties of 4b or related compounds used in the FRDA mouse models. Our results underscore the absolute necessity for appropriate ADME evaluation of compounds prior to in vivo target validation.