Development of new PET radioligands for α7 nicotinic acetylcholine receptors: A highly challenging task

Objectives: The particular properties of intracellular signalling triggered by activation of neuronal and non-neuronal α7 nicotinic acetylcholine receptors (nAChR) establishes the involvement α7 nAChR in a host of pathological conditions including brain and cancer diseases. Recent approaches to clinically use the potential of α7 nAChR as diagnostic target by quantitative imaging by PET interfere with insufficient characteristics of the available radiotracers. From a series of highly affine and α7 nAChR-specific oxadiazolyl-diazabicyclononanes we have chosen fluoro-phenyl- and N-fluoroethyl-indole-substituted ligands for radio-labelling and comparative preclinical investigation of the respective radiotracers [¹⁸F]NS10743 [1] and [¹⁸F]NS14490.

Methods: [¹⁸F]NS14490 was synthesized by both a two-step labelling process starting from the indole NS14540 via [¹⁸F]fluoroethyltosylate 1 as well as a one-step nucleophilic substitution of a tosylate precursor 2 using K[¹⁸F]F-Kryptofix 222-carbonate complex (Fig.1). Stability and partition coefficient (shake-flask method) at pH 7.2 as well as in vitro affinity towards α7 nAChR were analyzed. The specific in vitro binding of [¹⁸F]NS14490 assessed by autoradiography of mice and pig brain was compared with [¹²⁵I]α-bungarotoxin as reference. Biodistribution of [¹⁸F]NS14490 was investigated in female CD-1 mice at 5, 15, 30, and 60 min p.i. Radiotracer metabolism in vivo was studied by radio HPLC analysis of mice plasma, brain, and urine samples (30 and 60 min p.i.). Target specific binding of [¹⁸F]NS14490 in brain was shown by pre-administration of specific α7 nAChR ligands SSR180711 and NS6740 (10 mg/kg i.p., 10 min before [¹⁸F]NS14490). A preliminary dynamic PET study was performed in one female piglet.

Results: [¹⁸F]NS14490 was obtained by two-step and one-step radiosyntheses with labelling yields of ~7% and ~70%, respectively. The product of the further used one-step procedure was obtained with 36 ± 3% RCY at A_s ~150 GBq/µmol and a radiochemical purity of >98% within ~2.5 hours (n=13). [¹⁸F]NS14490 was stable for at least 120 min in both TRIS buffer and PBS. LogD_octanol/PBS of 1.11 ± 0.02 (n=12) and logD_{cyclohexane/PBS} = -2.05 ± 0.03 (n=3) were determined. The α7 nAChR binding affinity of NS14490 (K_i=2.5 nM) is considerably higher than assessed for NS10743 (K_i=11.6 nM [1]). The distribution of [¹⁸F]NS14490 binding in mice and pig brain matches the expression of α7 nAChR in these species. Compared to [¹⁸F]NS10743, a much lower fraction of [¹⁸F]NS14490 passed the blood-brain barrier (BBB) in mice (SUV 1.76 [2] vs. 0.05 at 5 min p.i.). Notably, pre-administration of NS6740 significantly reduced brain uptake of [¹⁸F]NS14490 in mice (-30% at 60 min p.i.; p-value <0.005), and the maximum SUV of [¹⁸F]NS14490 in pig brain (~0.6) was about tenfold higher than in mice. [¹⁸F]NS14490 is metabolically more stable than [¹⁸F]NS10743 [2] with 55% vs. 40% of total activity in plasma accounting for the respective parent radiotracer at 60 min p.i. , and the chromatographic analysis of brain samples revealed that none of the radiometabolites of [¹⁸F]NS14490 detected in plasma crossed the BBB.

Conclusion: Radiofluorination of the novel oxadiazolyl-diazabicyclononane analogue NS14490 was achieved and optimized. [¹⁸F]NS14490 was obtained with high RCY and A_s. Although biodistribution data indicate lower brain uptake of [¹⁸F]NS14490 in comparison to [¹⁸F]NS10743 in mice, the tenfold higher SUV in pig as well as the target specific binding shown by blocking studies in mice prompt us to continue with the preclinical evaluation of [¹⁸F]NS14490.

Acknowledgements: The work was financially supported by DFG (DE 1165/2-1). We would like to thank Jonni Heberg, Tove Thomsen, and Gitte Friberg for technical assistance.
References: [1] Deuther-Conrad, W, et al (2009) Eur J Nucl Med Mol Imaging, 36, 791-800.