Evaluation of [18F]Flotaza, a newβ-amyloid PET imaging agent in post-mortem human Alzheimer’s disease brain and 5XFAD transgenic mice

1627 Objectives: PET studies of amyloid β (Aβ ) accumulation in Alzheimer’s disease (AD) have shown clinical utility. Several fluorine-18 labeled PET radiotracers are now being clinically used. The aim of this study was to develop and evaluate the effectiveness of a new fluorine-18 analog of [11C]TAZA, [18F]Flotaza (2-{2-[2-[18F]fluoroethoxy]ethoxy}ethoxy)-4’-N,N-dimethylaminoazobenzene, Fig-1), for Aβ plaque imaging in postmortem AD brain slices. Preliminary PET/CT evaluation of [18F]Flotaza for in vivo stability was also assessed in transgenic 5XFAD mice, expressing Aβ plaques. Methods: Precursor, (2-{2-[2-tosylethoxy]ethoxy}ethoxy)-4’-N,N-dimethylaminoazobenzene) was synthesized using commercially available starting materials. Binding affinity of flotaza was measured in AD brain slices using [3H]PIB for Aβ plaques and [125I]IPPI for Tau. Fluorine-18 in H218O from PETNET was rendered anhydrous by drying of the [18F]fluoride, Kryptofix, and K2CO3 mixture at 120 oC for 10 mins. The precursor, tosylate (1-2 mg in 0.5 mL of anhydrous acetonitrile) was added and the reaction went for 30 min at 96 oC. Human AD (n=6) post-mortem brain tissues consisting of anterior cingulate (AC) and corpus callosum CC (AD and controls (CN)) were obtained from Banner Health, Sun City, Arizona. Brain slices (10 μm thick) were treated with [18F]Flotaza in 40% ethanol PBS buffer pH 7.4 (60 mL; 2 μCi/mL) were incubated at 25 oC for 1.25 hr. Nonspecific binding was measured in the presence of 10 μM PIB. The slices were then washed with cold PBS buffer, 50% ethanolic PBS buffer twice, PBS buffer and cold water. The brain sections were air dried, exposed overnight on a phosphor film, and then placed on the Phosphor Autoradiographic Imaging System/ Cyclone Storage Phosphor System and extent of binding of [18F]Flotaza was measured in DLU/mm2. Mice, C57BL/6 and 5XFAD mice were injected ip (25 μCi) for in vivo Inveon PET/CT studies. Results: The single step radiosynthesis of [18F]Flotaza was very efficient. Product was purified in a reverse-phase HPLC C18 Econosil column 250x10 mm with 60% acetonitrile: 40% water containing 0.1% triethylamine with a flow rate of 2.5 mL/min. The retention time of [18F]Flotaza was found to be 18 minutes.and was produced in high radiochemical yields (>25%) with specific activities >74 GBq/μmol. This radiosynthesis is currently being optimized using the GE Tracerlab FX N. For Aβ plaques, slices from all subjects were positively immunostained with anti-Aβ. Using [3H]PIB, flotaza exhibited nanomolar affinity for Aβ whereas no displacement of [125I]IPPI bound to Tau was observed, thus confirming selectivity for Aβ plaques. Very high ratios (>100) of [18F]Flotaza in anterior cingulate to corpus callosum was observed in all the 6 subjects. Very little white matter binding was seen. [18F]Flotaza binding in the anterior cingulate strongly correlated with anti-Aβ immunostains. [18F]Flotaza was found to be stable in vivo and no bone uptake confirmed lack of defluorination. Conclusion: [18F]Flotaza is a new β-amyloid PET imaging agent which can be made in one-step radiosynthesis followed by HPLC purification. It exhibited high binding to Aβ plaques in postmortem human AD brains. Detailed quantitative brain PET/CT studies in transgenic 5XFAD mice models are underway to evaluate the utility of [18F]Flotaza in mice imaging and their potential use in AD drug development.