[18F]Fluoroethylharmol: Improved Radiosynthesis and Evaluation of Monoamine Oxidase-A inAlzheimer’s and Parkinson’s Disease

1628 Objectives: Monoamine oxidases (MAO) play a significant role in the pathogenesis of neurodegeneration. The role of the brain isozyme, MAO-A, in neurodegeneration is less known. Here we report improved radiosynthesis and evaluation of [18F]fluoroethylharmol ([18F]FEH) as an imaging agent for MAO-A in postmortem Alzheimer’s disease (AD) and Parkinson’s disease (PD) brains. Methods: N-BOC-harmol-O-ethyl tosylate precursor (prepared in-house) was radiolabeled using approximately 10-20 mCi of [18F]fluoride in acetonitrile containing Kryptofix/K2CO3. The N-BOC-[18F]FEH was purified by HPLC (60%CH3CN:40% 0.1%aqueous Et3N; 2.5 mL/min, retention time 18 mins) and purity was confirmed by radioTLC. Deprotection of N-BOC-[18F]FEH was carried out using trifluoroacetic acid in CH2Cl2 to provide [18F]FEH in modest radiochemical yields. Human post-mortem brain tissues (10 μm thick) consisting of anterior cingulate (AC) and corpus callosum (CC) (controls (CN), AD and PD) were obtained on a Leica 1850 cryotome. Controls (CN), n=6; age 81-90; PD, n=6, age 77-89; AD, n=6, age 77-89. Brain slices were incubated [18F]FEH (0.1 μCi/cc) in Tris (pH 7.4) buffer at 25oC for 60 min. Nonspecific binding was measured in the presence of 100 μM clorgyline. Using the Optiquant program (Packard Instruments Co), regions of interest were drawn and digital light units/ mm2 (DLU/mm2) were used to quantify the percentage binding of [18F]FEH. Results: Two-step radiosynthesis using the N-BOC protected harmol precursor was found more suitable for production of [18F]FEH, compared to the unprotected precursor. [18F]FEH was found to be stable and was used for in vitro studies. The [18F]FEH labeled gray matter (AC) and white matter (CC) were clearly delineated in the autoradiographic images. All CN subjects exhibited lower [18F]FEH binding in the AC compared to the AD and PD subjects. Clorgyline displaced binding of [18F]FEH from the gray matter, consistent with specific binding of [18F]FEH to MAO-A. In the AD subjects, [18F]FEH ratio of binding, AC/CC = 2.34. In the case of the PD subjects, [18F]FEH binding ratio AC/CC = 1.60. The CN subjects showed a [18F]FEH binding ratio, AC/CC = 1.40. Conclusion: Increased [18F]FEH binding in PD and AD brains compared to CN brain samples suggests a significant upregulation of MAO-A in neurodegeneration. In vivo studies of [18F]FEH in 5XFAD mice for AD expressing Aβplaques and Hualpha-Syn(A53T) PD mouse model of α-synucleinopathy are currently underway. Figure 1 Legend: New synthesis of [18F]FEH is depicted. Harmol was reacted with ethyl ditosylate followed by di-tert-butyl dicarbonate to provide the N-BOC-harmol-O-ethyl tosylate precursor. N-BOC was incorporated so that the pyrrole nitrogen does not interfere with the fluorination process. In step A, harmol ethyltosylate is fluorinated, with the N-BOC protecting the pyrrole nitrogen. After the fluorination is complete, the N-BOC is removed in step B. Figure 2 Legend: Images A-C are autoradiographic images in which the total binding of [18F]FEH was quantified by the Optiquant program. Images D-F are the respective scanned images of the brain slices, in which the gray matter (GM) and white matter (WM) regions can be visualized more easily. AD subject (labeled A and D) displayed an AC/CC [18F]FEH binding ratio of 1.50, PD subject (labeled B and E) showed a binding ratio of 2.41, and CN subject (labeled C and F) had a binding ratio of 1.03. These values are consistent with AD and PD brain slices having more binding of [18F]FEH in the GM relative to the WM; whereas, the CN slice showed lower amounts of binding in the GM and WM regions.