Imaging of α7 nicotinic acetylcholine receptors (α7nAChRs): A challenge for PET radiotracer development.

Objectives: Pre- and postsynaptic modulation of different neurotransmitter systems and further non-neuronal mediators connect α7nAChRs to brain development, learning and memory, sleep, pain perception, reward, drug dependency and various neuropsychiatric diseases. Recent approaches to use α7 nAChR as target for neuroimaging interfere with insufficiencies of the available radiotracers connected to a rather low receptor density in brain¹. From a series of highly affine and α7nAChR-specific oxadiazolyl-diazabicyclononanes we have chosen fluoro-phenyl- and N-fluoroethyl-indole-substituted ligands for radiolabelling and comparative preclinical investigation of the respective radiotracers [¹⁸F]NS107432 and [¹⁸F]NS14490.

Methods: [¹⁸F]NS14490 was synthesized by one-step nucleophilic substitution of an unlabelled tosylate precursor (Fig.). Stability against defluorination and partition coefficient (shake-flask method, pH 7.2) and in vitro affinity towards α7nAChR and other nAChR were investigated. [¹⁸F]NS14490 receptor autoradiography of mice (Fig.) and pig brain was compared with [¹²⁵I]bungarotoxin as reference. In female CD-1 mice biodistribution (5´, 15´, 30´, 60´ p.i.) was investigated, and [¹⁸F]NS14490 metabolism was studied by radio-HPLC (plasma, brain, urine; 30´, 60´ p.i.). Target specific binding of [¹⁸F]NS14490 was demonstrated by pre-administration (10´ before radioligand) of highly specific α7nAChR ligands (SSR180711, NS6740; 10 mg/kg). A preliminary dynamic PET study was performed in female pig (Fig.).

Results: [¹⁸F]NS14490 was obtained within 2-2.5h with RCY of 36%±3% (n=13), RCP >98%, and A_s ~150 GBq/µmol. It was stable for >2h in TRIS-buffer and PBS. LogD_octanol/PBS of 1.11±0.02 (n=12) and logD_{cyclohexane/PBS} of -2.05±0.03 (n=3) were determined. The α7 binding affinity of NS14490 (2.5 nM) is considerably higher than assessed for NS10743 (11.6 nM²). [¹⁸F]NS14490 binding in mice and pig brain matches the expression of α7nAChR in these species. No receptor-free reference region was identified. Almost similar striatum-to-cerebellum ratios (~1.3) were obtained in pig autoradiography and dynamic PET studies with [¹⁸F]NS14490 and [¹⁸F]NS10743². However, compared to [¹⁸F]NS10743, a much lower fraction of [¹⁸F]NS14490 passed the blood-brain barrier (BBB) in mice (SUV 1.22 vs. 0.05 at 5´ p.i.). Notably, pre-administration of NS6740 significantly reduced the brain uptake of [¹⁸F]NS14490 in mice (30% at 60´ p.i.; p< 0.005) and the maximum SUV of [¹⁸F]NS14490 in pig brain (~0.6) was an order of magnitude higher than in mice. [¹⁸F]NS14490 is metabolically more stable than [¹⁸F]NS10743² with 55% vs. 40% of total activity in plasma accounting for parent compound at 60´ p.i. Chromatographic analysis of brain samples revealed that no radiometabolites of [¹⁸F]NS14490 crossed the BBB.

Conclusion: Radiofluorination of the novel oxadiazolyl-diazabicyclononane analogue NS14490 was achieved and optimized. [¹⁸F]NS14490 was obtained with high RCY, RCP and A_s. Although biodistribution data indicate lower brain uptake of [¹⁸F]NS14490 in comparison to [¹⁸F]NS10743 in mice, the more than ten-fold higher SUV in pig and the target specific binding shown by blocking studies in mice prompt us to continue with the preclinical evaluation of [¹⁸F]NS14490.
Acknowledgements: Supported by DFG (DE1165/2-1).

References: ¹Brust P, Peters D, Deuther-Conrad W (2012) Curr Drug Targets 13, 594-601.
²Deuther-Conrad W et al. (2009) Eur J Nucl Med Molec Imaging, 36, 791-800.