Preclinical Development of F-OF-NB1 for Imaging GluN2B-Containing -Methyl-d-Aspartate Receptors and Its Utility as a Biomarker for Amyotrophic Lateral Sclerosis.

As part of our continuous efforts to develop a suitable F-labeled PET radioligand with improved characteristics for imaging the -methyl-d-aspartate receptors (NMDARs) subtype 2B (GluN1/2B), we investigated in the current work -fluorinated (OF) and -fluorinated (MF) analogs of F--fluorinated (PF)-NB1, a 3-benzazepine-based radiofluorinated probe. OF-NB1 and MF-NB1 were prepared using a multistep synthesis, and their binding affinities toward GluN2B subunits and selectivity over σ1 receptors (σ1Rs) were determined via competitive binding assays. F-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, F-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. The binding affinity values for OF-NB1 and MF-NB1 toward the GluN2B subunits were 10.4 ± 4.7 and 590 ± 36 nM, respectively. For σ1R binding, OF-NB1 and MF-NB1 exhibited inhibition constants of 410 and 2,700 nM, respectively. OF-NB1, which outperformed MF-NB1, was radiolabeled with F to afford F-OF-NB1 in more than 95% radiochemical purity and molar activities of 192 ± 33 GBq/μmol. In autoradiography experiments, F-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 50% receptor occupancy of 8.1 μmol/kg. Postmortem autoradiography results revealed lower expression of the GluN2B subunits in ALS brain tissue sections than in healthy controls, in line with immunohistochemistry results. F-OF-NB1 is a highly promising PET probe for imaging the GluN2B subunits of the methyl-d-aspartate receptor. It possesses utility for receptor occupancy studies and has potential for PET imaging studies in ALS patients and possibly other brain disorders.