Preclinical Development of ¹⁸F-OF-NB1 for Imaging GluN2B-Containing <i>N</i>-Methyl-d-Aspartate Receptors and Its Utility as a Biomarker for Amyotrophic Lateral Sclerosis.

Abstract

As part of our continuous efforts to develop a suitable ¹⁸F-labeled PET radioligand with improved characteristics for imaging the <i>N</i>-methyl-d-aspartate receptors (NMDARs) subtype 2B (GluN1/2B), we investigated in the current work <i>ortho</i>-fluorinated (OF) and <i>meta</i>-fluorinated (MF) analogs of ¹⁸F-<i>para</i>-fluorinated (PF)-NB1, a 3-benzazepine-based radiofluorinated probe. <b>Methods:</b> 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. 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. <b>Results:</b> 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 ¹⁸F to afford ¹⁸F-OF-NB1 in more than 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 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. <b>Conclusion:</b>¹⁸F-OF-NB1 is a highly promising PET probe for imaging the GluN2B subunits of the <i>N-</i>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.