Investigation of the binding kinetics of nicotinic acetylcholine receptors by PET/CT using fluorine-18 labeled Nifene and 2-Fluoro-3-(2(S)-azetidinylmethoxy)pyridine

1310 Objectives: The α4β2 subtype of nicotinic acetylcholine receptors (nAChRs) is upregulated in response to nicotine exposure. Varenicline, an FDA approved medication for nicotine addiction, is a high affinity-weak base α4β2R ligand. However, the mechanisms of its action are yet to be fully investigated. One of the proposed mechanisms suggests that varenicline is trapped within intracellular acidic vesicles and slowly released, which is thought to be the neurobiological basis for its smoking cessation properties. Currently available PET tracers for α4β2Rs, all of which are weak bases, can be classified according to their differential kinetics based on their ligand pKas and receptor affinity. These differences in kinetics and displaceable binding should provide insight into the cellular mechanism of nicotinic receptor upregulation and how varenicline alters this process. The purpose of this study is to compare the differential kinetics and selective binding in two [18F]labeled α4β2R PET probes, i.e., 2-[18F]A85380 and [18F]Nifene, in mouse models. Methods: 2-[18F]A85380 (2-[18F]Fluoro-3-(2(S)-azetidinylmethoxy)pyridine), a radiotracer known with slow kinetics, was synthesized from the commercially available precursor, 2-TMA-A85380. [18F]Nifene, a radiotracer with relatively fast kinetics, was synthesized from the precursor N-Boc-nitronifene. An IBA Synthera V2 automatic synthesis module equipped with Synthera preparative HPLC was used for the radiolabeling inside a Comecer Hotcell. To establish ligand binding kinetics, wild type C57BL/J6 mice were used for both PET probes, while their littermates of β2-subunit knockout mice were used to confirm the specificity of α4β2Rs. [18F]labeled ligands were injected into anesthetized mice via tail vein (~250 μCi/100 μl), immediately followed by PET/CT imaging using the Molecubes preclinical imaging system. Dynamic PET acquisition was done in a 7 hour continuous scanning for 2-[18F]A85380 and in 2 hour for [18F]Nifene. High resolution CT imaging was performed after the PET scan for the purpose of anatomic co-registration. The binding kinetics of different brain regions were analyzed using Invicro Vivoquant software on CT-fused PET images co-registered with the available 3D brain atlas. Results: Synthesis of both radioligands was carried out at the cyclotron facility of the University of Chicago. Typical yields for 2-[18F]A85380 were 34% (decay corrected) with a specific activity of 8,300 mCi/μmole and higher than 99% radiochemical purity, while representative radiochemical yield was 6.3% (decay corrected) with higher than 99% purity for [18F]Nifene. The dynamic analyses demonstrated a fast binding kinetics of [18F]Nifene, peaking at ~10 minutes after tracer injection and a slow binding kinetics of 2-[18F]A85380, peaking at ~2 hours post injection. In wild type mice, both 2-[18F]A85380 and [18F]Nifene showed the highest binding as measured by %ID/g tissue in the thalamus (2.5% and 11.5%, respectively) and lowest binding in the cerebellum (1.1% & 5.9%). This regional difference of binding was eliminated in β2-subunit knockout mice, further confirming the specificity of the two ligands. Conclusions: Both 2-[18F]A85380 and [18F]Nifene were successfully employed in PET/CT mouse brain imaging for the kinetic study of α4β2Rs nicotine receptor, making this study the first to use 2-[18F]A85380 in mouse models. The selectivity of the synthesized probes is consistent with other publications showing the distribution of the α4β2 nicotine receptor in the brain of human and mice. With an understanding of the differences in probe kinetics, we will be able to better study the precise mechanism of nicotine receptor upregulation and the displaceable binding of nicotine and varenicline, which has implications for not only smoking cessation but other neurological conditions such as dementia, anxiety, and attention deficits.