Mapping the Expression and Biodistribution of Class-IIa Histone Deacetylases (HDACs) in the Central Nervous System with Positron Emission Tomography.

1595 Introduction: The Human histone deacetylases (HDACs) form a large family of 18 members classified into four classes (I to IV). The class-IIa HDACs is a sub family comprised of four enzymes: HDAC4, HDAC5, HDAC7 and HDAC9. There is a mounting evidence on a key role for the class-IIa HDACs in the disorders of the central nervous system (CNS). Specifically, the class-IIa HDAC4 and 5 are expressed throughout the brain and their silencing or truncation led to defect in spatial learning and memory, cognitive impairment and dementia. Furthermore, postmortem studies established a link between expression of class-IIa HDAC and neurodegenerative diseases such as Alzheimer’s and Huntington’s diseases, among others. However, despite intense studies, relatively little is known regarding the expression level and distribution of class-IIa HDACs in the human brain in its entirety. Therefore, we developed positron emission tomography (PET) tracers that target the class-IIa HDACs for in vivo imaging of the CNS to support clinical translation of class-IIa HDAC disease-modifying therapeutics and monitor response to therapy. Methods: Small molecules containing the 5-trifluoromethyl-1,2,4-oxadiazole (TFMO) moiety were reported to exhibit potent and specific class-IIa HDACs inhibition owing to the TFMO moiety interaction with the zinc ion at the bottom of the catalytic pocket of the class-IIa HDAC enzymes. We performed a focused structure activity relationship (SAR) study and identified NT160 among other compounds that exhibited potent class-IIa HDAC inhibition (IC50 = low to sub nM) and high selectivity over other HDAC classes and isoforms (IC50 >600 nM). We then incorporated the Fluorine-18 (18F-) in the exact location as it appears on the TFMO moiety of class-IIa HDAC inhibitors, thus enabling the radiosynthesis of an identical radioactive homolog maintaining the exact affinity and specificity to class-IIa HDACs. Moreover, this universal labeling site allowed for in vivo SAR studies to delineate and optimize the in vivo characteristics of our lead PET tracer (i.e metabolic and pharmacokinetic profile) without changing the radiolabeling method or radiolabeling site. We performed radiochemistry initially by treating the bromodifluoromethyl-precursor with potassium 18F-fluoride (K18F) to produce the 18F-NT160 albeit the radiochemical yield was very low ( Results: We validated the utility of our late-stage radiochemical synthesis and radiolabeled a variety of class-IIa HDACs inhibitors, thus producing identical radiolabeled homologs in a single radiosynthetic step. The radiosynthesis, HPLC purification and formulation of the final product was accomplished in less than 90 min. Moreover, in vivo dynamic PET imaging studies in healthy Sprague Dawley rats with the lead tracer 18F-NT160 demonstrated a high uptake in the brain indicating a successful CNS penetration and excellent pharmacokinetic profile. uPET imaging with 18F-NT160 provided a high-quality images and clear contrast between the various regions of the brain with high brain uptake in gray matter regions. Conclusion: We demonstrated a successful radiolabeling and subsequent in vivo imaging with our new TFMO containing class-IIa HDAC targeting PET tracer (18F-NT160). Our data indicate a successful and high-quality CNS PET imaging and heterogenic accumulation of radioactivity in the brain. Acknowledgements: Support for this work includes NIH grant R01AG067417 from the National Institute on Aging (Awarded to N.T.). The authors acknowledge the start-up support to N.T from the Stony Brook Cancer Center. This work was also supported in part by Stony Brook Renaissance School of Medicine and the Office of the Vice President for Research (Targeted Research Opportunity Program-FUSION Awarded to N.T).