Abstract Histone deacetylase (HDAC) enzymes exert control over gene transcription and cell cycle progression and their inhibition has recently emerged as an efficacious strategy to treat cancer. However, current HDAC inhibitors have been linked to a shared undesirable toxicological profile that prompts the development of new entities. The present study evaluates the in vitro and in vivo PK/PD of a newly developed HDAC inhibitor, HDAC-C1A. HDAC-C1A inhibited class I, II and sirtuins, with highest affinity for HDAC6 (IC50 = 63 ng/mL), an HDAC subtype thought to be associated with low toxicity; HDAC6 knockout does not lead to embryonic lethality. The drug irreversibly inhibited HDAC from HeLa cell extract; in HCT116 cells inhibition of enzyme activity as assessed by levels of acetyl-histone H3, H4 and acetyl-tubulin was maintained after washout demonstrating an irreversible mechanism. HDAC-C1A treatment was associated with a dose and time dependent increase of histone and non-histone targets that was maintained at 4 hours after washout; acetylation was lost by 4 h with clinically licensed HDAC inhibitor SAHA. HDAC-C1A inhibited the growth of a panel of 7 cancer cell lines with a mean GI50 of 1.6 ± 0.6 µg/mL. The drug was relatively stable after parenteral administration for 4 h. The Cmax and AUC0-4h following i.p. injection (160 mg/kg) were 20.4 µg/mL and 50 µg/mL*h, respectively. Regarding efficacy, HDAC-C1A treatment was associated with a Tumor Growth Delay (TGD2x) of 5.7 ± 1.4 days and a Tumor Growth Inhibition (TGI) of 78% compared with vehicle when given i.p. at 20 mg/kg b.i.d. in a HCT116 human colon cancer xenograft model. This dose was non-toxic (no reduction in body weight). We also assessed the potential of [18F]fluorothymidine positron emission tomography ([18F]FLT-PET) to measure early response to HDAC-C1A treatment in HCT116 xenograft bearing mice. There was a 2-fold decrease in tumor [18F]FLT uptake compared to vehicle treated animals at 48 h post-treatment; the area under the normalized [18F]FLT time versus activity curve was 117 ± 6.9 at before treatment and decreased to 106 ± 5.5 (P = 0.02) at 24 hours and 54 ± 5 (P = 0.0001) at 48 h after initiating treatment. In summary, HDAC-C1A combines irreversible HDAC inhibition with a favourable pharmacokinetic profile leading to significant anti-tumor activity in the HCT116 tumor model. Early response to the drug was detectable by [18F]FLT-PET. These preclinical data support further development of HDAC-C1A. This work was supported in part by Cancer Research U.K.-Engineering and Physical Sciences Research Council Grant C2536/A10337. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2608. doi:10.1158/1538-7445.AM2011-2608