The previously reported carbon-11 labeled GluN2B PET radioligand C-Me-NB1 served as a starting point for derivatization and led to the successful development of a radiofluorinated analogue designated (R)-F-OF-Me-NB1. Given the short physical half-life of 20.3 min for carbon-11, (R)-F-OF-Me-NB1 with a physical half-life of 109.8 min would allow satellite distribution to nuclear medicine facilities without an on-site cyclotron. Two fluorinated Me-NB1 derivatives, OF-Me-NB1 and PF-Me-NB1, were synthesized. Upon chiral resolution, the respective enantiomers were radiolabeled with carbon-11 and assessed in a proof-of-concept study by applying in vitro autoradiography on rodent brain sections. Based on the autoradiograms, (R)-OF-Me-NB1 was selected for radiofluorination and preclinical evaluation by ex vivo autoradiography, PET imaging, biodistribution and metabolite studies in Wistar rats. To rule out off-target binding to the σ1 receptor, the brain uptake of (R)-F-OF-Me-NB1 in wild-type mice was compared with σ1 receptor knock-out mice. Autoradiographic assessment revealed that both enantiomers ofC-PF-Me-NB1 distributed homogenously across all brain regions on rodent brain sections. In contrast, the two enantiomers of C-OF-Me-NB1 exhibited an entirely different behaviour. While (S)-C-OF-Me-NB1 bound virtually to all brain regions with considerable σ1 receptor binding, (R)-C-OF-Me-NB1 exhibited high selectivity and specificity for the GluN2B-rich rat forebrain. These findings were confirmed for the radiofluorinated analogue (R)-C-OF-Me-NB1, which was obtained via copper-mediated radiofluorination in radiochemical yields of 13-25% and molar activities ranging from 61-168 GBq/µmol. PET imaging and biodistribution studies in Wistar rats indicated appropriate pharmacokinetic profile and high in vivo specific binding of (R)-F-OF-Me-NB1 as revealed by blocking studies with GluN2B-antagonist CP101,606. Off-target binding to the σ1 receptor was excluded by PET imaging with σ1 receptor knock-out mice. Receptor occupancy experiments with CP101,606 revealed a D50-value of 8.3 µmol/kg (intravenous). (R)-F-OF-Me-NB1 is a promising radiofluorinated probe that exhibits specificity and selectivity for the GluN2B-containing N-methyl-D-aspartate (NMDA) complex and enables in vivo target occupancy studies in rodents.

Despite the broad implications of the cannabinoid type 2 receptor (CB2) in neuroinflammatory processes, a suitable CB2-targeted probe is currently lacking in clinical routine. In this work, we synthesized 15 fluorinated pyridine derivatives and tested their binding affinities toward CB2 and CB1. With a sub-nanomolar affinity (K i for CB2) of 0.8 nM and a remarkable selectivity factor of >12,000 over CB1, RoSMA-18-d 6 exhibited outstanding in vitro performance characteristics and was radiofluorinated with an average radiochemical yield of 10.6 ± 3.8% (n = 16) and molar activities ranging from 52 to 65 GBq/μmol (radiochemical purity > 99%). [18F]­RoSMA-18-d 6 showed exceptional CB2 attributes as demonstrated by in vitro autoradiography, ex vivo biodistribution, and positron emission tomography (PET). Further, [18F]­RoSMA-18-d 6 was used to detect CB2 upregulation on postmortem human ALS spinal cord tissues. Overall, these results suggest that [18F]­RoSMA-18-d 6 is a promising CB2 PET radioligand for clinical translation.

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.

The cannabinoid type 2 (CB2) receptor has emerged as a valuable target for therapy and imaging of immune-mediated pathologies. With the aim to find a suitable radiofluorinated analogue of the previously reported CB2 positron emission tomography (PET) radioligand [11C]­RSR-056, 38 fluorinated derivatives were synthesized and tested by in vitro binding assays. With a K i (hCB2) of 6 nM and a selectivity factor of nearly 700 over cannabinoid type 1 receptors, target compound 3 exhibited optimal in vitro properties and was selected for evaluation as a PET radioligand. [18F]3 was obtained in an average radiochemical yield of 11 ± 4% and molar activities between 33 and 114 GBq/μmol. Specific binding of [18F]3 to CB2 was demonstrated by in vitro autoradiography and in vivo PET experiments using the CB2 ligand GW-405 833. Metabolite analysis revealed only intact [18F]3 in the rat brain. [18F]3 detected CB2 upregulation in human amyotrophic lateral sclerosis spinal cord tissue and may thus become a candidate for diagnostic use in humans.

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