Quantitative biodistribution of class-IIa histone deacetylases in rat brain with novel PET tracers

Introduction: The class-IIa histone deacetylases (HDACs) is a sub-family of four HDAC members (HDACs: 4, 5, 7 and 9) of the larger HDAC family of 18 enzymes. Class-IIa HDAC dysregulation in the brain is relevant across diverse human disorders of the central nervous system (CNS) such as stroke, Huntington’s and Alzheimer’s diseases which underscore their immense potential for molecular imaging and targeted therapy. Currently, non-invasive imaging biomarkers for quantifying the class-IIa HDAC expression in tumors and in the CNS are lacking. Therefore, our current work is aimed at developing class-IIa HDAC-specific positron emission tomography (PET) radiotracer for non-invasive quantification and mapping the biodistribution of class-IIa HDAC protein expression in cancer and brain diseases. Also, with the push for personalized medicine, our probe may identify a subset of patient population who may benefit the most from anti-class-IIa therapy and monitor their response to treatment.

Methods: The radiosyntheses of [¹⁸F]-NT160 and [¹⁸F]-NT2160 were performed by treating the bromodifluoromethyl-precursors with cesium [¹⁸F]fluoride (Cs[¹⁸F]). Radiochemical yield was 2-5%, radiochemical purity was >98% and molar activity was ranged from 0.33-0.49 GBq/umol. In vivo microPET/CT imaging studies were performed with healthy Sprague Dawley rats (200-250 g, N=6) using Inveon uPET (Siemens, Knoxville, TN). Dynamic PET images were obtained over 60 minutes followed by CT imaging (15 min scan). PET image analysis was performed using the AMIDE software by using manual segmentation of regions of interest (ROI), including whole brain, cortex, hippocampus, thalamus, cerebellum, and brain stem. Rat Brain Atlas was used for alignment and identification of specific anatomical markers of the brain. We performed self-blocking experiment to confirm the specificity of [¹⁸F]-2NT160 to class-IIa HDAC in the brain. Extent of tracer’s metabolism in the rat brain were determined ex-vivo from brain homogenates at 15 and 30 min after injection.

Results: PET imaging with [¹⁸F]-NT160 and [¹⁸F]-NT2160 produced high-quality PET images with heterogenous uptake in the brain and excellent imaging characteristics: brain uptake is high in the grey matter regions; tissue kinetics are appropriate for an [¹⁸F]-tracer and specific binding for class-IIa HDAC was demonstrated by self-blockade. Both tracers exhibited fast blood clearance and low level of uptake in the muscle (background tissue). Dynamic PET data over 60 minutes revealed higher uptake in the cortex, hippocampus, and thalamus compared to the cerebellum and the rest of the brain (Figure 1). Notably, [¹⁸F]-NT2160 showed an improved metabolic stability in rat brain homogenates over [¹⁸F]-NT160. Our PET imaging studies provide quantitative and accurate data on class-IIa HDAC expression in brain regions which underscore the significance of our approach to non-invasively provide quantitative information on the level of class-IIa HDAC in vivo in real time.

Conclusions: This is the first accurate and quantitative information on the biodistribution of class-IIa HDACs expression in the brain. Our new lead radiotracers displayed excellent pharmacokinetic and imaging characteristics with high tracer uptake in grey matter regions, leading to high-quality PET images. Studies are ongoing in our lab to overcome the low specific activity associated with [¹⁸F]-trifluoromethyl moiety. These studies are ongoing and will be reported in due course.

Support: This work is funded by the National Institutes of Health/National Institute on Aging (Grant#: R01AG067417) and by the Stony Brook Cancer Center.

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