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.
Aspiring
to develop a positron emission tomography (PET) imaging
agent for the GluN2B subunits of the N-methyl-d-aspartate receptor (NMDAR), a key therapeutic target for drug
development toward several neurological disorders, we synthesized
a series of 2,3,4,5-tetrahydro-1H-3-benzazepine and
6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-amine analogues.
After in vitro testing via competition binding assay and autoradiography,
[18F]PF-NB1 emerged as the best performing tracer with
respect to specificity and selectivity over σ1 and σ2
receptors and was thus selected for further in vivo evaluation. Copper-mediated
radiofluorination was accomplished in good radiochemical yields and
high molar activities. Extensive in vivo characterization was performed
in Wistar rats comprising PET imaging, biodistribution, receptor occupancy,
and metabolites studies. [18F]PF-NB1 binding was selective
to GluN2B-rich forebrain regions and was specifically blocked by the
GluN2B antagonist, CP-101,606, in a dose-dependent manner with no
brain radiometabolites. [18F]PF-NB1 is a promising fluorine-18
PET tracer for imaging the GluN2B subunits of the NMDAR and has utility
for receptor occupancy studies.
This study addresses the question whether inductively coupled plasma mass spectrometry (ICP-MS) can be used as a method for the in vitro and in vivo characterization of non-radioactive metal conjugates to predict the properties of analogous radiopharmaceuticals. In a "proof-of-concept" study, the prostate-specific membrane antigen (PSMA)-targeting [ 175 Lu]Lu-PSMA-617 and [ 159 Tb]Tb-PSMA-617 were compared with their respective radiolabeled analogues, [ 177 Lu]Lu-PSMA-617 (PLU-VICTO, Novartis) and [ 161 Tb]Tb-PSMA-617. ICP-MS and conventional γ-counting of the cell samples revealed almost identical results (<6% absolute difference between the two technologies) for the in vitro uptake and internalization of the (radio)metal conjugates, irrespective of the employed methodology. In vivo, an equal uptake in PSMA-positive PC-3 PIP tumor xenografts was determined 1 h after the injection of [ 175 Lu]Lu-/[ 177 Lu]Lu-PSMA-617 (41 ± 6% ID/g and 44 ± 12% IA/g, respectively) and [ 159 Tb]Tb-/[ 161 Tb]Tb-PSMA-617 (44 ± 5% ID/g and 44 ± 5% IA/g, respectively). It was further revealed that it is crucial to use the same ratios of the (radio)metal-labeled and unlabeled ligands for both methodologies to obtain equal data in organs in which receptor saturation was reached such as the kidneys (12 ± 2% ID/g vs 10 ± 1% IA/g, 1 h after injection). The data of this study demonstrate that the use of high-sensitivity ICP-MS allows reliable and predictive quantification of compounds labeled with stable metal isotopes in cell and tissue samples obtained in preclinical studies. It can, hence, be employed as a valid alternative to the state-of-the-art γ-counting methodology to detect radioactive ligands.
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