Preclinical Development of 18F-OF-NB1 for Imaging GluN2B-Containing N-Methyl-D-Aspartate Receptors and its Utility as a Biomarker for Amyotrophic Lateral Sclerosis

As part of our continuous efforts to develop a suitable fluorine-18 labeled positron emission tomography (PET) radioligand with improved imaging characteristics for imaging the GluN2B-bearing N-Methyl-D-aspartate receptors (NMDARs), we investigated in the current work ortho- and meta-fluorinated analogues of 18F-PF-NB1, a 3-benzazepine-based radiofluorinated probe. Methods: OF-NB1 and MF-NB1 were prepared using a multi-step synthesis and their binding affinities towards GluN2B subunits and selectivity over sigma-1 receptors (σ1Rs) were determined via competitive binding assays. 18F-OF-NB1 was synthesized via copper-mediated radiofluorination, and was evaluated in Wistar rats by in vitro autoradiography, PET imaging, ex vivo biodistribution, metabolite experiments and receptor occupancy studies using CP-101,606, an established GluN2B antagonist. To determine in vivo selectivity, 18F-OF-NB1 was validated in wild-type and σ1R knock-out mice. Translational relevance was assessed in autoradiographic studies using postmortem human brain tissues from healthy individuals and ALS patients, the results of which were corroborated by immunohistochemistry. Results: The binding affinity values for OF-NB1 and MF-NB1 towards the GluN2B subunits were 10.4 ± 4.7 nM and 590 ± 36 nM, respectively. For σ1R binding, OF-NB1 and MF-NB1 exhibited Ki values of 410 nM and 2700 nM, respectively. OF-NB1, which outperformed MF-NB1, was radiolabeled with 18F to afford 18F-OF-NB1 in > 95% radiochemical purity and molar activities of 192±33 GBq/μmol. In autoradiography experiments, 18F-OF-NB1 displayed a heterogeneous and specific binding in GluN2B subunit-rich brain regions such as the cortex, striatum, hypothalamus and hippocampus. PET imaging studies in Wistar rats showed a similar heterogeneous uptake, and no brain radiometabolites were detected. A dose-dependent blocking effect was observed with CP-101,606 (0.5-15 mg/kg) and resulted in a D50 of 8.1 µmol/kg. Postmortem autoradiography results revealed a lower expression level of the GluN2B subunits in ALS brain tissue sections compared to healthy controls, in line with immunohistochemistry results. Conclusion: 18F-OF-NB1 is a highly promising PET imaging probe for imaging the GluN2B subunits of the NMDAR. It possesses utility for receptor occupancy studies and has potential for PET imaging studies in ALS patients and possibly other brain disorders.ISSN:0097-9058ISSN:0022-3123ISSN:0161-5505ISSN:2159-662XISSN:1535-566