Development of [¹⁸F]FAZIN3 for PET imaging of neurofibrillary tangles in Alzheimer’s Disease

Objectives: Neurofibrillary tangles (NFT) are aggregates of hyperphosphorylated tau proteins and the density of NFT has a significant correlation to the severity of Alzheimer’s disease. New second generation PET radiotracers such as [¹⁸F]MK-6240 offer greater selectivity for NFT and are currently being evaluated in human studies. In an effort to further enhance selectivity for 3R Tau and 4R Tau, reduce nonspecific binding, and simplify radiosynthesis to a single step, we have designed novel fluoroalkylated azaindoles (FAZIN) as potential PET imaging agents. Here we report radiosynthesis, rodent PET studies and human postmortem brain autoradiographic studies of [¹⁸F]FAZIN3.

Methods: For radiolabeling, 2.5 mg of AZIN3-Tosyl was dissolved in acetonitrile. To a reaction vial, 10-20 mCi of [¹⁸F]fluoride containing Kryptofix (8 mg) and K₂CO₃ (2.5 mg) was added and heated for 10 min at 100^oC to dryness. Acetonitrile (1 mL) was added and heating continued for another 10 min to ensure anhydrous conditions. AZIN3-Tosyl precursor was added and the reaction mixture was heated for 20 minutes at 100^oC. After unreacted [¹⁸F]fluoride removal using neutral Al₂O₃ seppak, RadioTLC confirmed product and reaction mixture was purified on C₁₈ reverse-phase HPLC using 60% CH₃CN-40% 0.1% aqueous Et₃N, flow rate of 2.5 mL/min. Pure [¹⁸F]FAZIN3 was collected at a retention time of 10 mins decay-corr radiochemical yield of 20%. Male C57BL/6 mice were used for in vivo Inveon PET/CT studies and analyzed using Inveon Research Workplace (IRW) software. Human 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) consisting of both anterior cingulate and corpus callosum (Figure 1A and 1B) were obtained on a Leica 1850 cryotome.

Results: Radiosynthesis of [¹⁸F]FAZIN3 proceeded smoothly, with no other major side-products. Purified radiochemical yield was 20% and purity was confirmed by HPLC. [¹⁸F]FAZIN3 was found to be stable for in vitro and in vivo studies. After intraperitoneal administration of [¹⁸F]FAZIN3 (approx. 50 μCi), a 30 minute dynamic scan showed uptake and clearance from the brain with little retention in any brain region. Uptake within the kidneys, liver, and eyes were observed. No radioactivity was observed in the bones suggested that [¹⁸F]FAZIN3 was resistant to defluorination. AC binding of [¹⁸F]FAZIN3 was clearly evident in the AD brains (Figure 1A) and was found to be significantly greater than the control subjects. Ratio of AC/CC= 2.27 in AD subjects.

Conclusions: [¹⁸F]FAZIN3 is a novel PET radiotracer suitable for imaging NFT. Radiosynthesis is a single step, it is stable in vivo, does not show significant retention in the normal mouse brain and binds to NFT in the AD brain slices containing AC. Further studies are underway to establish its selectivity for 3R Tau and 4R Tau. RESEARCH SUPPORT: NIH/NIA RF1 AG029479 (JM)

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