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Preclinical Evaluation of Benzazepine-Based PET Radioligands (R)- and (S)-¹¹C-Me-NB1 Reveals Distinct Enantiomeric Binding Patterns and a Tightrope Walk Between GluN2B- and σ₁-Receptor-Targeted PET Imaging.

ABSTRACT: The study aims to investigate the performance characteristics of the enantiomers of ¹¹C-Me-NB1, a recently reported PET imaging probe that targets the GluN2B subunit of N-methyl-d-aspartate (NMDA) receptors. Methods: Reference compound Me-NB1 (inhibition constant for hGluN1/GluN2B, 5.4 nM) and the phenolic precursor were prepared via multistep synthesis. Following chiral resolution by high-performance liquid chromatography, enantiopure precursor compounds, (R)-NB1 and (S)-NB1, were labeled with ¹¹C and validated in rodents using in vitro/ex vivo autoradiography, PET experiments, and dose-response studies. To illustrate the translational relevance, (R)- ¹¹C-Me-NB1 was validated in autoradiographic studies using postmortem human GluN2B-rich cortical and GluN2B-deficient cerebellar brain slices. To determine target engagement, receptor occupancy was assessed at different plasma concentrations of CP101,606, a GluN2B receptor antagonist. Results: The radiosynthesis of (R)- and (S)- ¹¹C-Me-NB1 was accomplished in 42% ± 9% (decay-corrected) radiochemical yields. Molar activity ranged from 40 to 336 GBq/μmol, and an excellent radiochemical purity of greater than 99% was achieved. Although (R)- ¹¹C-Me-NB1 displayed heterogeneous accumulation with high selectivity for the GluN2B-rich forebrain, (S)- ¹¹C-Me-NB1 revealed a homogeneous distribution across all brain regions in rodent brain autoradiograms and predominantly exhibited σ₁-receptor binding. Similar to rodent brain, (R)- ¹¹C-Me-NB1 showed in postmortem human brain tissues higher binding in the cortex than in the cerebellum. Coincubation of the GluN2B-antagonist CERC-301 (1 μM) reduced cortical but not cerebellar binding, demonstrating the specificity of (R)- ¹¹C-Me-NB1 binding to the human GluN2B-containing NMDA receptor. In vivo specificity of (R)- ¹¹C-Me-NB1 in the GluN2B-expressing cortex, striatum, thalamus, and hippocampus was demonstrated by PET imaging in rodents. Applying GluN2B-antagonist eliprodil, an evident dose-response behavior was observed with (R)- ¹¹C-Me-NB1 but not with (S)- ¹¹C-Me-NB1. Our findings further underline the tightrope walk between GluN2B- and σ₁-receptor-targeted imaging, illustrated by the entirely different receptor binding behavior of the 2 radioligand enantiomers. Conclusion: (R)- ¹¹C-Me-NB1 is a highly selective and specific PET radioligand for imaging the GluN2B subunit of the NMDA receptor. The entirely different receptor binding behavior of (R)- ¹¹C-Me-NB1 and (S)- ¹¹C-Me-NB1 raises awareness of a delicate balance that is underlying the selective targeting of either GluN2B-carrying NMDA or σ₁-receptors.

SUBMITTER: Haider A

PROVIDER: S-EPMC6681693 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Preclinical Evaluation of Benzazepine-Based PET Radioligands (R)- and (S)-11C-Me-NB1 Reveals Distinct Enantiomeric Binding Patterns and a Tightrope Walk Between GluN2B- and σ1-Receptor-Targeted PET Imaging.

Haider Achi A Herde Adrienne Müller AM Krämer Stefanie D SD Varisco Jasmine J Keller Claudia C Frauenknecht Katrin K Auberson Yves P YP Temme Louisa L Robaa Dina D Sippl Wolfgang W Schibli Roger R Wünsch Bernhard B Mu Linjing L Ametamey Simon M SM

Journal of nuclear medicine : official publication, Society of Nuclear Medicine 20190125 8

The study aims to investigate the performance characteristics of the enantiomers of 11C-Me-NB1, a recently reported PET imaging probe that targets the GluN2B subunit of N-methyl-d-aspartate (NMDA) receptors. Methods: Reference compound Me-NB1 (inhibition constant for hGluN1/GluN2B, 5.4 nM) and the phenolic precursor were prepared via multistep synthesis. Following chiral resolution by high-performance liquid chromatography, enantiopure precursor compounds, (R)- ...[more]

PMID: 30683765

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Project description:BackgroundThe NMDA receptor (NMDAR) plays a key role in the central nervous system, e.g., for synaptic transmission. While synaptic NMDARs are thought to have protective characteristics, activation of extrasynaptic NMDARs might trigger excitotoxic processes linked to neuropsychiatric disorders. Since extrasynaptic NMDARs are typically GluN2B-enriched, the subunit is an interesting target for drug development and treatment monitoring. Recently, the novel GluN2B-specific PET radioligand (R)-[11C]Me-NB1 was investigated in rodents and for the first time successfully translated to humans. To assess whether (R)-[11C]Me-NB1 is a valuable radioligand for (repeated) clinical applications, we evaluated its safety, biodistribution and dosimetry.MethodsFour healthy subjects (two females, two males) underwent one whole-body PET/MR measurement lasting for more than 120 min. The GluN2B-specific radioligand (R)-[11C]Me-NB1 was administered simultaneously with the PET start. Subjects were measured in nine passes and six bed positions from head to mid-thigh. Regions of interest was anatomically defined for the brain, thyroid, lungs, heart wall, spleen, stomach contents, pancreas, liver, kidneys, bone marrow and urinary bladder contents, using both PET and MR images. Time-integrated activity coefficients were estimated to calculate organ equivalent dose coefficients and the effective dose coefficient. Additionally, standardized uptake values (SUV) were computed to visualize the biodistribution.ResultsAdministration of the radioligand was safe without adverse events. The organs with the highest uptake were the urinary bladder, spleen and pancreas. Organ equivalent dose coefficients were higher in female in almost all organs, except for the urinary bladder of male. The effective dose coefficient was 6.0 µSv/MBq.ConclusionThe GluN2B-specific radioligand (R)-[11C]Me-NB1 was well-tolerated without reported side effects. Effective dose was estimated to 1.8 mSv when using 300 MBq of presented radioligand. The critical organ was the urinary bladder. Due to the low effective dose coefficient of this radioligand, longitudinal studies for drug development and treatment monitoring of neuropsychiatric disorders including neurodegenerative diseases are possible. Trial registration Registered on 11th of June 2019 at https://www.basg.gv.at (EudraCT: 2018-002933-39).

Project description:IntroductionWe recently reported 11C-NR2B-SMe ([S-methyl-11C](R,S)-7-thiomethoxy-3-(4-(4-methyl-phenyl)butyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepin-1-ol) and its enantiomers as candidate radioligands for imaging the GluN2B subunit within rat N-methyl-D-aspartate receptors. However, these radioligands gave unexpectedly high and displaceable binding in rat cerebellum, possibly due to cross-reactivity with sigma-1 (σ1) receptors. This study investigated 11C-labeled enantiomers of a close analogue (7-methoxy-3-(4-(p-tolyl)butyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepin-1-ol; NR2B-Me) of 11C-NR2B-SMe as new candidate GluN2B radioligands. PET was used to evaluate these radioligands in rats and to assess potential cross-reactivity to σ1 receptors.MethodsNR2B-Me was assayed for binding affinity and selectivity to GluN2B in vitro. 11C-NR2B-Me and its enantiomers were prepared by Pd-mediated treatment of boronic ester precursors with 11C-iodomethane. Brain PET scans were conducted after radioligand intravenous injection into rats. Various ligands for GluN2B receptors or σ1 receptors were administered at set doses in pre-blocking or displacement experiments to assess their impact on imaging data. 18F-FTC146 and enantiomers of 11C-NR2B-SMe were used for comparison. Radiometabolites from brain and plasma were measured ex vivo and in vitro.ResultsNR2B-Me enantiomers showed high GluN2B affinity and selectivity in vitro. 11C-NR2B-Me enantiomers gave high early whole rat brain uptake of radioactivity, including high uptake in cerebellum, followed by slower decline. Radioactivity in brain at 30 min ex vivo was virtually all unchanged radioligand. Only less lipophilic radiometabolites appeared in plasma. When 11C-(R)-NR2B-Me was used, three high-affinity GluN2B ligands-NR2B-SMe, Ro25-6981, and CO101,244-showed increasing pre-block of whole brain radioactivity retention with increasing dose. Two σ1 receptor antagonists, FTC146 and BD1407, were ineffective pre-blocking agents. Together, these results strongly resemble those obtained with 11C-NR2B-SMe enantiomers, except that 11C-NR2B-Me enantiomers showed faster reversibility of binding. When 18F-FTC146 was used as a radioligand, FTC146 and BD1407 showed strong pre-blocking effects whereas GluN2B ligands showed only weak blocking effects.Conclusion11C-NR2B-Me enantiomers showed specific binding to GluN2B receptors in rat brain in vivo. High unexpected specific binding in cerebellum was not due to σ1 receptors. Additional investigation is needed to identify the source of the high specific binding.

Project description:The N-methyl-d-aspartate receptor (NMDAR) plays a crucial role in neurodegenerative diseases such as Alzheimer disease and in the treatment of major depression by fast-acting antidepressants such as ketamine. Given their broad implications, GluN2B-containing NMDARs have been of interest as diagnostic and therapeutic targets. Recently, (R)-11C-Me-NB1 was investigated preclinically and shown to be a promising radioligand for imaging GluN2B subunits. Here, we report on the performance characteristics of this radioligand in a first-in-humans PET study. Methods: Six healthy male subjects were scanned twice on a fully integrated PET/MR scanner with (R)-11C-Me-NB1 for 120 min. Brain uptake and tracer distribution over time were investigated by SUVs. Test-retest reliability was assessed with the absolute percentage difference and the coefficient of variation. Exploratory total volumes of distribution (VT) were computed using an arterial input function and the Logan plot as well as a constrained 2-tissue-compartment model with the ratio of rate constants between plasma and tissue compartments (K1/k2) coupled (2TCM). SUV was correlated with VT to investigate its potential as a surrogate marker of GluN2B expression. Results: High and heterogeneous radioligand uptake was observed across the entire gray matter with reversible kinetics within the scan time. SUV absolute percentage difference ranged from 6.9% to 8.5% and coefficient of variation from 4.9% to 6.0%, indicating a high test-retest reliability. A moderate correlation was found between SUV averaged from 70 to 90 min and VT using Logan plot (Spearman ρ = 0.44). Correlation between VT Logan and 2TCM was r = 0.76. Conclusion: The radioligand (R)-11C-Me-NB1 was highly effective in mapping GluN2B-enriched NMDARs in the human brain. With a heterogeneous uptake and a high test-retest reliability, this radioligand offers promise to deepen our understanding of the GluN2B-containing NMDAR in the pathophysiology and treatment of neuropsychiatric disease such as Alzheimer disease and major depression. Additionally, it could help in the selection of appropriate doses of GluN2B-targeting drugs.

Project description:GluN2B subunit-containing N-methyl-d-aspartate (NMDA) receptors have been implicated in various neurological disorders. Nonetheless, a validated fluorine-18 labeled positron emission tomography (PET) ligand for GluN2B imaging in the living human brain is currently lacking. As part of our PET ligand development program, we have recently reported on the preclinical evaluation of [18F]OF-NB1 - a GluN2B PET ligand with promising attributes for potential clinical translation. However, the further development of [18F]OF-NB1 is currently precluded by major limitations in the radiolabeling procedure. These limitations include the use of highly corrosive reactants and racemization during the radiosynthesis. As such, the aim of this study was to develop a synthetic approach that allows an enantiomerically pure radiosynthesis of (R)-[18F]OF-NB1 and (S)-[18F]OF-NB1, as well as to assess their in vitro and in vivo performance characteristics for imaging the GluN2B subunit-containing NMDA receptor in rodents. A two-step radiosynthesis involving radiofluorination of the boronic acid pinacol ester, followed by coupling to the 3-benzazepine core structure via reductive amination was employed. The new synthetic approach yielded enantiomerically pure (R)-[18F]OF-NB1 and (S)-[18F]OF-NB1, while concurrently circumventing the use of corrosive reactants. In vitro autoradiograms with mouse and rat brain sections revealed a higher selectivity of (R)-[18F]OF-NB1 over (S)-[18F]OFNB1 for GluN2B-rich brain regions. In concert with these observations, blockade studies with commercially available GluN2B antagonist, CP101606, showed a significant signal reduction, which was more pronounced for (R)-[18F]OF-NB1 than for (S)-[18F]OF-NB1. Conversely, blockade experiments with sigma2 ligand, FA10, did not result in a significant reduction of tracer binding for both enantiomers. PET imaging experiments with CD1 mice revealed a higher brain uptake and retention for (R)-[18F]OF-NB1, as assessed by visual inspection and volumes of distribution from Logan graphical analyses. In vivo blocking experiments with sigma2 ligand, FA10, did not result in a significant reduction of the brain signal for both enantiomers, thus corroborating the selectivity over sigma2 receptors. In conclusion, we have developed a novel synthetic approach that is suitable for upscale to human use and allows the enantiomerically pure radiosynthesis of (R)-[18F]OF-NB1 and (S)-[18F]OF-NB1. While both enantiomers were selective over sigma2 receptors in vitro and in vivo, (R)-[18F]OF-NB1 showed superior GluN2B subunit specificity by in vitro autoradiography and higher volumes of distribution in small animal PET studies.

Dataset Information

Preclinical Evaluation of Benzazepine-Based PET Radioligands (R)- and (S)-¹¹C-Me-NB1 Reveals Distinct Enantiomeric Binding Patterns and a Tightrope Walk Between GluN2B- and σ₁-Receptor-Targeted PET Imaging.

Publications

Preclinical Evaluation of Benzazepine-Based PET Radioligands (<i>R</i>)- and (<i>S</i>)-<sup>11</sup>C-Me-NB1 Reveals Distinct Enantiomeric Binding Patterns and a Tightrope Walk Between GluN2B- and σ<sub>1</sub>-Receptor-Targeted PET Imaging.

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