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

Abstract

As part of our continuous efforts to develop a suitable 18 F-labeled PET radioligand with improved characteristics for imaging the N-methyl-D-aspartate receptors (NMDARs) subtype 2B (GluN1/2B), we investigated in the current work ortho-fluorinated (OF) and meta-fluorinated (MF) analogs of 18 F-para-fluorinated (PF)-NB1, a 3-benzazepine-based radiofluorinated probe. Methods: 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. 18 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, 18 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. Results: 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 18 F to afford 18 F-OF-NB1 in more than 95% radiochemical purity and molar activities of 192 ± 33 GBq/μmol. In autoradiography experiments, 18 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. Conclusion: 18 F-OF-NB1 is a highly promising PET probe for imaging the GluN2B subunits of the N-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.

Comparison of three novel radiotracers for GluN2B-containing NMDA receptors in non-human primates: (R)-[11C]NR2B-Me, (R)-[18F]of-Me-NB1, and (S)-[18F]of-NB1

“…As in previous studies performed in rodents using these and similar GluN2B-targeting radioligands, 13,16–20 displaceable binding was observed in the cerebellum. In situ hybridization studies performed show negligible expression of the mRNA encoding GluN2B subunits in this region during adulthood in rodents 41,42 and humans.…”

“…Radiosyntheses were performed as previously described for (R) -[ 11 C]NR2B-Me, 17,27 (R) -[ 18 F]OF-Me-NB1, 20,22 and (S) -[ 18 F]OF-NB1. 13,23 All PET scans were performed on the Focus 220 scanner (Siemens Medical Solutions, Knoxville, TN) after radiotracer injection. A transmission scan was collected before tracer injection for attenuation correction.…”

“…9 GluN2B-targeting allosteric modulators have been tested or proposed for the treatment of neurodegenerative disorders, neuropathic pain, schizophrenia, and depression. 5,[11][12][13] Despite extensive preclinical literature demonstrating the importance of GluN2B-containing NMDA receptors in fundamental and disease-related processes, there are few tools available to study these sites in humans. Quantification of GluN2B-containing NMDA receptors selectively in vivo using positron emission tomography (PET) can advance research into the specific role of this receptor subunit in neuropsychiatric disorders as well as the development of drugs targeting the GluN2B site.…”

“…15 Recently, our group and others have identified several promising candidates. 13,[16][17][18][19][20][21] The compounds (R)-[ 18 F] OF-Me-NB1 and [ 18 F]OF-NB1 were developed to target the GluN2B ifenprodil binding site and evaluated in vitro and in vivo in rodent models, showing good brain uptake, displacement by GluN2B-specific drugs, and no evidence of brain-penetrant radiometabolites. 13,20 Sensitivity to blocking by drugs targeting the structurally similar r receptor site was also evaluated.…”

Targeting Kca3.1 Channels in Cancer

“…Thus, PET tracers are available to image ligand-gated ion channels such as nicotinic acetylcholine receptor subtypes [74] and NMDA receptors. Addressing specifically the ifenprodil binding site allows the selective imaging of NMDA receptors containing the GluN2B subunit [75][76][77][78]. However, PET tracers for imaging of voltage-or Ca 2+ -activated ion channels such as the K ca 3.1 channel have not been developed so far.…”