Thus, an in vivo proof of concept study linking functional central inhibition of Class I HDACs to efficacy in a disease state should ideally provide confirmation of at least some correlation between significant target engagement and phenotypic outcome, to support pursuit of this approach in a clinically afflicted population. We further characterized the in vitro ADME profile of 4b to explore any metabolic liabilities that would inform subsequent in vivo efficacy testing and dosing schedule. Our findings indicate that 4b is very unstable in plasma and in liver microsomes resulting in a very high predicted in vivo plasma clearance of 2.6 L/h/kg. The main hydrolysis product in plasma is M1, which accounts for 78% of the metabolism of parent, followed by M2, which accounts for 10% of the entire metabolism. Our biochemical and cellular profiling of these two metabolites confirmed that neither inhibits HDACs in a cellular setting, confirming that these metabolic products of 4b would not inhibit HDACs in vivo. In summary, based on our in vitro ADME data, we predict that 4b will be rapidly metabolised by plasma and hepatic amidases and other hepatic enzymes in vivo, significantly limiting systemic and CNS availability. Additionally, we show that 4b is a substrate for Pgp in MDCK cells overexpressing MDR1 of 4.9), in agreement with the efflux determined in Caco2 monolayers. This in vitro data predicts a limited oral bioavailability and very minimal CNS exposure for compound 4b. Despite an unfavourable predicted in vivo pharmacokinetic profile, we pursued further in vivo studies in light of the published positive efficacy study. Surprisingly, 4b did not dissolve in the previously described formulation. Unlike SAHA, which dissolves in cyclodextrins upon heating and is stable for seven days at room temperature, BAY-X 1005 investigation of both free-base and salt forms of 4b showed that neither was capable of full dissolution to 1 mg/mL as previously described. However, the physicochemical instability of the acidic salts of 4b, combined with the efficient and CID 5951923 irreversible conversion of the parent compound to the inactive benzimidazole C1, might explain the disparity between the published studies and our data. The benzimidazole C1 is readily soluble in aqueous media and the conversion process is exacerbated under acidic conditions and at elevated temperature. Hence, it is likely that there was very significant contamination of C1 in the chronic drinking water preparations previously used. In agreement with our in vitro ADME predictions, 4b reaching the systemic circulation was rapidly cleared, which resulted in a plasma half-life of approximately 40 min after subcutaneous dosing. Subcutaneous dosing resulted in much higher relative concentrations in plasma and brain tissue as the impact of first pass metabolism and efflux by MDR1 was reduced via this route.