Evaluation of cannabinoid type 2 receptor expression and pyridine-based radiotracers in brains from a mouse model of Alzheimer's disease.

Neuroinflammation plays an important role in the pathophysiology of Alzheimer's disease. The cannabinoid type 2 receptor (CB2R) is an emerging target for neuroinflammation and therapeutics of Alzheimer's disease. Here, we aim to assess the alterations in brain CB2R levels and evaluate novel CB2R imaging tracers in the arcAß mouse model of Alzheimer's disease amyloidosis. Immunohistochemical staining for amyloid-ß deposits (6E10), microgliosis (anti-Iba1 and anti-CD68 antibodies), astrocytes (GFAP) and the anti-CB2R antibody was performed on brain slices from 17-month-old arcAß mice. Autoradiography using the CB2R imaging probes [18F]RoSMA-18-d6, [11C]RSR-056, and [11C]RS-028 and mRNA analysis were performed in brain tissue from arcAß and non-transgenic littermate (NTL) mice at 6, 17, and 24 months of age. Specific increased CB2R immunofluorescence intensities on the increased number of GFAP-positive astrocytes and Iba1-positive microglia were detected in the hippocampus and cortex of 17-month-old arcAß mice compared to NTL mice. CB2R immunofluorescence was higher in glial cells inside 6E10-positive amyloid-ß deposits than peri-plaque glial cells, which showed low background immunofluorescence in the hippocampus and cortex of 17-month-old arcAß mice. Ex vivo autoradiography showed that the specific binding of [18F]RoSMA-18-d6 and [11C]RSR-056 was comparable in arcAß and NTL mice at 6, 17, and 24 months of age. The level of Cnr2 mRNA expression in the brain was not significantly different between arcAß and NTL mice at 6, 17, or 24 months of age. In conclusion, we demonstrated pronounced specific increases in microglial and astroglial CB2R expression levels in a mouse model of AD-related cerebral amyloidosis, emphasizing CB2R as a suitable target for imaging neuroinflammation.

Discovery, synthesis and evaluation of novel reversible monoacylglycerol lipase radioligands bearing a morpholine-3-one scaffold.

Monoacylglycerol lipase (MAGL) is a serine hydrolase that plays an important role in the endocannabinoid degradation in the brain. It has recently emerged as a promising therapeutic target in the treatment of neuroinflammatory and neurodegenerative diseases, such as multiple sclerosis, Alzheimer's disease and Parkinson's disease. Development of MAGL-specific radioligands for non-invasive imaging by positron-emission tomography (PET) would deepen our knowledge on the relevant pathological changes in diseased states and accelerate drug discovery. In this study, we report the selection and synthesis of two morpholine-3-one derivatives as potential reversible MAGL PET tracer candidates based on their multiparameter optimization scores. Both compounds ([11C]1, [11C]2) were radiolabeled by direct [11C]CO2 fixation and the in vitro autoradiographic studies demonstrated their specificity and selectivity towards MAGL. Dynamic PET imaging using MAGL knockout and wild-type mice confirmed the in vivo specificity of [11C]2. Our preliminary results indicate that morpholine-3-one derivative [11C]2 ([11C]RO7279991) binds to MAGL in vivo, and this molecular scaffold could serve as an alternative lead structure to image MAGL in the central nervous system.

In vivo Imaging of Cannabinoid Type 2 Receptors: Functional and Structural Alterations in Mouse Model of Cerebral Ischemia by PET and MRI.

PURPOSE: Stroke is one of the most prevalent vascular diseases. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and follow alterations of receptor expression and metabolism in ischemic stroke. The aim of this study was to assess the cannabinoid type 2 receptor (CB2R) levels in transient middle cerebral artery occlusion (tMCAO) mouse models at subacute stage using positron emission tomography (PET) with our novel tracer [18F]RoSMA-18-d6 and structural imaging by magnetic resonance imaging (MRI). PROCEDURES: Our recently developed CB2R PET tracer [18F]RoSMA-18-d6 was used for imaging neuroinflammation at 24 h after reperfusion in tMCAO mice. The RNA expression levels of CB2R and other inflammatory markers were analyzed by quantitative real-time polymerase chain reaction using brain tissues from tMCAO (1 h occlusion) and sham-operated mice. [18F]fluorodeoxyglucose (FDG) was included for evaluation of the cerebral metabolic rate of glucose (CMRglc). In addition, diffusion-weighted imaging and T2-weighted imaging were performed for anatomical reference and delineating the lesion in tMCAO mice. RESULTS: mRNA expressions of inflammatory markers TNF-α, Iba1, MMP9 and GFAP, CNR2 were increased to 1.3-2.5 fold at 24 h after reperfusion in the ipsilateral compared to contralateral hemisphere of tMCAO mice, while mRNA expression of the neuronal marker MAP-2 was markedly reduced to ca. 50 %. Reduced [18F]FDG uptake was observed in the ischemic striatum of tMCAO mouse brain at 24 h after reperfusion. Although higher activity of [18F]RoSMA-18-d6 in ex vivo biodistribution studies and higher standard uptake value ratio (SUVR) were detected in the ischemic ipsilateral compared to contralateral striatum in tMCAO mice, the in vivo specificity of [18F]RoSMA-18-d6 was confirmed only in the CB2R-rich spleen. CONCLUSIONS: This study revealed an increased [18F]RoSMA-18-d6 measure of CB2R and a reduced [18F]FDG measure of CMRglc in the ischemic striatum of tMCAO mice at subacute stage. [18F]RoSMA-18-d6 might be a promising PET tracer for detecting CB2R alterations in animal models of neuroinflammation without neuronal loss.

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.

As part of our continuous efforts to develop a suitable 18F-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 18F-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. 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. 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 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. Conclusion:18F-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.

Identification and Preclinical Development of a 2,5,6-Trisubstituted Fluorinated Pyridine Derivative as a Radioligand for the Positron Emission Tomography Imaging of Cannabinoid Type 2 Receptors.

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 (Ki for CB2) of 0.8 nM and a remarkable selectivity factor of >12,000 over CB1, RoSMA-18-d6 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-d6 showed exceptional CB2 attributes as demonstrated by in vitro autoradiography, ex vivo biodistribution, and positron emission tomography (PET). Further, [18F]RoSMA-18-d6 was used to detect CB2 upregulation on postmortem human ALS spinal cord tissues. Overall, these results suggest that [18F]RoSMA-18-d6 is a promising CB2 PET radioligand for clinical translation.

Chronic social stress in mice alters energy status including higher glucose need but lower brain utilization.

Chronic stress leads to changes in energy status and is a major risk factor for depression, with common symptoms of reductions in body weight and effortful motivation for reward. Indeed, stress-induced disturbed energy status could be a major aetio-pathogenic factor for depression. Improved understanding of these putative inter-relationships requires animal model studies of effects of stress on both peripheral and central energy-status measures and determinants. Here we conducted a study in mice fed on a standard low-fat diet and exposed to either 15-day chronic social stress (CSS) or control handling (CON). Relative to CON mice, CSS mice had attenuated body weight maintenance/gain despite consuming the same amount of food and expending the same amount of energy at any given body weight. The low weight of CSS mice was associated with less white and brown adipose tissues, and with a high respiratory exchange ratio consistent with increased dependence on glucose as energy substrate. Basal plasma insulin was low in CSS mice and exogenous glucose challenge resulted in a relatively prolonged elevation of blood glucose. With regard to hunger and satiety hormones, respectively, CSS mice had higher levels of acylated ghrelin in plasma and of ghrelin receptor gene expression in ventromedial hypothalamus and lower levels of plasma leptin, relative to CON mice. However, whilst CSS mice displayed this constellation of peripheral changes consistent with increases in energy need and glucose utilization relative to CON mice, they also displayed attenuated uptake of [18F]FDG in brain tissue specifically. Reduced brain glucose utilization in CSS mice could contribute to the reduced effortful motivation for reward in the form of sweet-tasting food that we have reported previously for CSS mice. It will now be important to utilize this model to further understanding of the mechanisms via which chronic stress can increase energy need but decrease brain glucose utilization and how this relates to regional and cellular changes in neural circuits for reward processing relevant to depression.

Structure-Activity Relationship Studies of Pyridine-Based Ligands and Identification of a Fluorinated Derivative for Positron Emission Tomography Imaging of Cannabinoid Type 2 Receptors.

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 Ki (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.

Structure-Affinity Relationships of 2,3,4,5-Tetrahydro-1H-3-benzazepine and 6,7,8,9-Tetrahydro-5H-benzo[7]annulen-7-amine Analogues and the Discovery of a Radiofluorinated 2,3,4,5-Tetrahydro-1H-3-benzazepine Congener for Imaging GluN2B Subunit-Containing N-Methyl-d-aspartate Receptors.

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.

Chronic Nicotine Exposure Alters Metabotropic Glutamate Receptor 5: Longitudinal PET Study and Behavioural Assessment in Rats.

Using positron emission tomography (PET), a profound alteration of the metabotropic glutamate receptor 5 (mGluR5) was found in human smoking addiction and abstinence. As human PET data either reflect the impact of chronic nicotine exposure or a pre-existing vulnerability to nicotine addiction, we designed a preclinical, longitudinal study to investigate the effect of chronic nicotine exposure on mGluR5 with the novel radiotracer [18F]PSS232 using PET. Twelve male dark Agouti rats at the age of 6 weeks were assigned randomly to three groups. From day 0 to day 250 the groups received 0 mg/L, 4 mg/L, or 8 mg/L nicotine solution in the drinking water. From day 250 to 320 all groups received nicotine-free drinking water. PET scans with [18F]PSS232 were performed in all animals on days 0, 250, and 320. To assess locomotion, seven tests in square open field arenas were carried out 72 days after the last PET scan. During the first four tests, rats received 0 mg/L nicotine and for the last three tests 4 mg/L nicotine in the drinking water. After 250 days of nicotine consumption [18F]PSS232 binding was reduced in the striatum, hippocampus, thalamus, and midbrain. At day 320, after nicotine withdrawal, [18F]PSS232 binding increased. These effects were more pronounced in the 4 mg/L nicotine group. Chronic administration of nicotine through the drinking water reduced exploratory behaviour. This preliminary longitudinal PET study demonstrates that chronic nicotine administration alters behaviour and mGluR5 availability. Chronic nicotine administration leads to decreased [18F]PSS232 binding which normalizes after prolonged nicotine withdrawal.

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.

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.

Metabotropic glutamate receptor subtype 5 is altered in LPS-induced murine neuroinflammation model and in the brains of AD and ALS patients.

PURPOSE: The aim of the present study was to determine the expression levels of mGluR5 in different mouse strains after induction of neuroinflammation by lipopolysaccharide (LPS) challenge and in the brains of patients with Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS) post mortem to investigate mGluR5 expression in human neurodegenerative diseases. METHODS: C57BL/6 and CD1 mice were injected intraperitoneally with either 10 mg/kg LPS or saline. mGluR5 and TSPO mRNA levels were measured after 1 and 5 days by qPCR, and mGluR5 protein levels were determined by PET imaging with the mGluR5-specific radiotracer [18F]PSS232. mGluR5 expression was evaluated in the post-mortem brain slices from AD and ALS patients using in vitro autoradiography. RESULTS: mGluR5 and TSPO mRNA levels were increased in brains of C57BL/6 and CD1 mice 1 day after LPS treatment and remained significantly increased after 5 days in C57BL/6 mice but not in CD1 mice. Brain PET imaging with [18F]PSS232 confirmed increased mGluR5 levels in the brains of both mouse strains 1 day after LPS treatment. After 5 days, mGluR5 levels in CD1 mice declined to the levels in vehicle-treated mice but remained high in C57BL/6 mice. Autoradiograms revealed a severalfold higher binding of [18F]PSS232 in post-mortem brain slices from AD and ALS patients compared with the binding in control brains. CONCLUSION: LPS-induced neuroinflammation increased mGluR5 levels in mouse brain and is dependent on the mouse strain and time after LPS treatment. mGluR5 levels were also increased in human AD and ALS brains in vitro. PET imaging of mGluR5 levels could potentially be used to diagnose and monitor therapy outcomes in patients with AD and ALS.

Ketamine and Ceftriaxone-Induced Alterations in Glutamate Levels Do Not Impact the Specific Binding of Metabotropic Glutamate Receptor Subtype 5 Radioligand [18F]PSS232 in the Rat Brain.

Several studies showed that [11C]ABP688 binding is altered following drug-induced perturbation of glutamate levels in brains of humans, non-human primates and rats. We evaluated whether the fluorinated derivative [18F]PSS232 can be used to assess metabotropic glutamate receptor 5 (mGluR5) availability in rats after pharmacological challenge with ketamine, known to increase glutamate, or ceftriaxone, known to decrease glutamate. In vitro autoradiography was performed on rat brain slices with [18F]PSS232 to prove direct competition of the drugs for mGluR5. One group of rats were challenged with a bolus injection of either vehicle, racemic ketamine, S-ketamine or ceftriaxone followed by positron emission tomography PET imaging with [18F]PSS232. The other group received an infusion of the drugs during the PET scan. Distribution volume ratios (DVRs) were calculated using a reference tissue model. In vitro autoradiography showed no direct competition of the drugs with [18F]PSS232 for the allosteric binding site of mGluR5. DVRs of [18F]PSS232 binding in vivo did not change in any brain region neither after bolus injection nor after infusion. We conclude that [18F]PSS232 has utility for measuring mGluR5 density or occupancy of the allosteric site in vivo, but it cannot be used to measure in vivo fluctuations of glutamate levels in the rat brain.

Evaluation of 4-oxo-quinoline-based CB2 PET radioligands in R6/2 chorea huntington mouse model and human ALS spinal cord tissue.

The cannabinoid receptor 2 (CB2) has been implicated in a series of neurodegenerative disorders and has emerged as an interesting biological target for therapeutic as well as diagnostic purposes. In the present work, we describe an improved radiosynthetic approach to obtain the previously reported CB2-specific PET radioligand [18F]RS-126 in higher radiochemical yields and molar activities. Additionally, the study revealed that prolongation of the [18F]RS-126 fluoroalkyl side chain ultimately leads to an improved stability towards mouse liver enzymes but is accompanied by a reduction in selectivity over the cannabinoid receptor 1 (CB1). Huntington-related phenotypic changes as well as striatal D2R downregulation were confirmed for the transgenic R6/2 mouse model. CB2 upregulation in R6/2 Chorea Huntington mice was observed in hippocampus, cortex, striatum and cerebellum by qPCR, however, these results could not be confirmed at the protein level by PET imaging. Furthermore, we evaluated the utility of the newly developed [11C]RS-028, a potent [18F]RS-126 derivative with increased polarity and high selectivity over CB1 in post-mortem human ALS spinal cord and control tissue. Applying in vitro autoradiography, the translational relevance of CB2 imaging was demonstrated by the specific binding of [11C]RS-028 to post-mortem human ALS spinal cord tissue.

Evaluation of 11C-Me-NB1 as a Potential PET Radioligand for Measuring GluN2B-Containing NMDA Receptors, Drug Occupancy, and Receptor Cross Talk.

Clinical and preclinical research with modulators at the N-methyl-d-aspartate (NMDA) receptor GluN2B N-terminal domain (NTD) aims for the treatment of various neurologic diseases. The interpretation of the results is hampered by the lack of a suitable NMDA PET tracer for assessing the receptor occupancy of potential drugs. We have developed 11C-Me-NB1 as a PET tracer for imaging GluN1/GluN2B-containing NMDA receptors and used it to investigate in rats the dose-dependent receptor occupancy of eliprodil, a GluN2B NTD modulator. Methods:11C-Me-NB1 was synthesized and characterized by in vitro displacement binding experiments with rat brain membranes, in vitro autoradiography, and blocking and displacement experiments by PET and PET kinetic modeling. Receptor occupancy by eliprodil was studied by PET with 11C-Me-NB1. Results:11C-Me-NB1 was synthesized at 290 ± 90 GBq/µmol molar activity, 7.4 ± 1.9 GBq total activity at the end of synthesis (n = 17), and more than 99% radiochemical purity. 11C-Me-NB1 binding in rat brain was blocked in vitro and in vivo by the NTD modulators Ro-25-6981 and eliprodil. Half-maximal receptor occupancy by eliprodil occurred at 1.5 µg/kg. At 1 mg/kg of eliprodil, a dose with reported neuroprotective effects, more than 99.5% of binding sites were occupied. In vitro, 11C-Me-NB1 binding was independent of the σ-1 receptor (Sigma1R), and the Sigma1R agonist (+)-pentazocine did not compete for high-affinity binding. In vivo, a 2.5 mg/kg dose of (+)-pentazocine abolished 11C-Me-NB1-specific binding, indicating an indirect effect of Sigma1R on 11C-Me-NB1 binding. Conclusion:11C-Me-NB1 is suitable for the in vivo imaging of NMDA GluN1/GluN2B receptors and the assessment of receptor occupancy by NTD modulators. GluN1/GluN2B NMDA receptors are fully occupied at neuroprotective doses of eliprodil. Furthermore, 11C-Me-NB1 enables imaging of GluN1/GluN2B NMDA receptor cross talk.

GABAA receptor subtypes in the mouse brain: Regional mapping and diazepam receptor occupancy by in vivo [18F]flumazenil PET.

Classical benzodiazepines, which are widely used as sedatives, anxiolytics and anticonvulsants, exert their therapeutic effects through interactions with heteropentameric GABAA receptors composed of two α, two ß and one γ2 subunit. Their high affinity binding site is located at the interface between the γ2 and the adjacent α subunit. The α-subunit gene family consists of six members and receptors can be homomeric or mixed with respect to the α-subunits. Previous work has suggested that benzodiazepine binding site ligands with selectivity for individual GABAA receptor subtypes, as defined by the benzodiazepine-binding α subunit, may have fewer side effects and may even be effective in diseases, such as schizophrenia, autism or chronic pain, that do not respond well to classical benzodiazepines. The distributions of the individual α subunits across the CNS have been extensively characterized. However, as GABAA receptors may contain two different α subunits, the distribution of the subunits does not necessarily reflect the distribution of receptor subtypes with respect to benzodiazepine pharmacology. In the present study, we have used in vivo [18F]flumazenil PET and in vitro [3H]flumazenil autoradiography in combination with GABAA receptor point-mutated mice to characterize the distribution of the two most prevalent GABAA receptor subtypes (α1 and α2) throughout the mouse brain. The results were in agreement with published in vitro data. High levels of α2-containing receptors were found in brain regions of the neuronal network of anxiety. The α1/α2 subunit combinations were predictable from the individual subunit levels. In additional experiments, we explored in vivo [18F]flumazenil PET to determine the degree of receptor occupancy at GABAA receptor subtypes following oral administration of diazepam. The dose to occupy 50% of sensitive receptors, independent of the receptor subtype(s), was 1-2mg/kg, in agreement with published data from ex vivo studies with wild type mice. In conclusion, we have resolved the quantitative distribution of α1- and α2-containing homomeric and mixed GABAA receptors in vivo at the millimeter scale and demonstrate that the regional drug receptor occupancy in vivo at these GABAA receptor subtypes can be determined by [18F]flumazenil PET. Such information should be valuable for drug development programs aiming for subtype-selective benzodiazepine site ligands for new therapeutic indications.

Cannabinoid receptor type 2 (CB2) as one of the candidate genes in human carotid plaque imaging: Evaluation of the novel radiotracer [11C]RS-016 targeting CB2 in atherosclerosis.

INTRODUCTION: Endarterectomized human atherosclerotic plaques are a valuable basis for gene expression studies to disclose novel imaging biomarkers and therapeutic targets, such as the cannabinoid receptor type 2 (CB2). In this work, CB2 is expressed on activated immune cells, which are abundant in inflamed plaques. We evaluated the CB2-specific radiotracer [11C]RS-016 for imaging vascular inflammation in human and mouse atherosclerotic lesions. METHODS: The differential gene expression of microscopically classified human carotid plaques was evaluated using quantitative polymerase chain reaction. In addition, CB2 expression levels in human plaques were investigated by in vitro autoradiography. As an appropriate animal model we used apolipoprotein E knockout mice (ApoE KO) with shear stress-induced atherosclerosis to evaluate CB2 levels in vivo. Positron emission tomography (PET) was performed with both the CB2 radioligand [11C]RS-016 and the metabolic radiotracer [18F]fluorodeoxyglucose ([18F]FDG) at various time points. Retrospectively, carotids were dissected for histopathology and gene expression analysis. RESULTS: We identified 28 human genes differentially expressed in atherosclerotic plaques compared to normal arteries of which 12 were upregulated preferentially in vulnerable plaques. The latter group included members of matrix metalloproteinase family and the T-lymphocyte activation antigens CD80 and CD86. CB2 was upregulated by 2-fold in human atherosclerotic plaques correlating with CD68 expression levels. Specific in vitro binding of [11C]RS-016 was predominantly observed to plaques. In vivo PET imaging of ApoE KO mice revealed accumulation of [11C]RS-016 and [18F]FDG in atherosclerotic plaques. Development of advanced plaques with elevated CB2 and CD68 levels were found in vitro in ApoE KO mice resembling human vulnerable plaques. CONCLUSION: We identified human genes associated with plaque vulnerability, which potentially could serve as novel imaging or therapeutic targets. The CB2-specific radiotracer [11C]RS-016 detected human plaques by in vitro autoradiography and accumulated in vivo in plaques of ApoE KO mice, however not exclusively in vulnerable plaques.

CD80 Is Upregulated in a Mouse Model with Shear Stress-Induced Atherosclerosis and Allows for Evaluating CD80-Targeting PET Tracers.

PURPOSE: A shear stress-induced atherosclerosis mouse model was characterized for its expression of inflammation markers with focus on CD80. With this model, we evaluated two positron emission tomography (PET) radiotracers targeting CD80 as well as 2-deoxy-2-[18F]fluoro-D-mannose ([18F]FDM) in comparison with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG). PROCEDURE: A flow constrictive cuff implanted around the common carotid artery in apolipoprotein E knockout mice resulted in plaque formation. CD80 expression levels and plaque histopathology were evaluated. Serial PET/X-ray computed tomography scans were performed to follow inflammation. RESULTS: Plaque formation with increased levels of CD80 was observed. Histologically, plaques presented macrophage-rich and large necrotic areas covered by a thin fibrous cap. Of the CD80-specific tracers, one displayed an increased uptake in plaques by PET. Both [18F]FDG and [18F]FDM accumulated in atherosclerotic plaques. CONCLUSION: This mouse model presented, similar to humans, an increased expression of CD80 which renders it suitable for non-invasively targeting CD80-positive immune cells and evaluating CD80-specific radiotracers.

Synthesis and Biological Evaluation of Thiophene-Based Cannabinoid Receptor Type 2 Radiotracers for PET Imaging.

Over the past two decades, our understanding of the endocannabinoid system has greatly improved due to the wealth of results obtained from exploratory studies. Currently, two cannabinoid receptor subtypes have been well-characterized. The cannabinoid receptor type 1 (CB1) is widely expressed in the central nervous system, while the levels of the cannabinoid receptor type 2 (CB2) in the brain and spinal cord of healthy individuals are relatively low. However, recent studies demonstrated a CB2 upregulation on activated microglia upon neuroinflammation, an indicator of neurodegeneration. Our research group aims to develop a suitable positron emission tomography (PET) tracer to visualize the CB2 receptor in patients suffering from neurodegenerative diseases. Herein we report two novel thiophene-based (11)C-labeled PET ligands designated [(11)C]AAT-015 and [(11)C]AAT-778. The reference compounds were synthesized using Gewald reaction conditions to obtain the aminothiophene intermediates, followed by amide formation. Saponification of the esters provided their corresponding precursors. Binding affinity studies revealed Ki-values of 3.3 ± 0.5 nM (CB2) and 1.0 ± 0.2 µM (CB1) for AAT-015. AAT-778 showed similar Ki-values of 4.3 ± 0.7 nM (CB2) and 1.1 ± 0.1 µM (CB1). Radiosynthesis was carried out under basic conditions using [(11)C]iodomethane as methylating agent. After semi-preparative HPLC purification both radiolabeled compounds were obtained in 99% radiochemical purity and the radiochemical yields ranged from 12 to 37%. Specific activity was between 96 and 449 GBq/µmol for both tracers. In order to demonstrate CB2 specificity of [(11)C]AAT-015 and [(11)C]AAT-778, we carried out autoradiography studies using CB2-positive mouse/rat spleen tissues. The obtained results revealed unspecific binding in spleen tissue that was not blocked by an excess of CB2-specific ligand GW402833. For in vivo analysis, [(11)C]AAT-015 was administered to healthy rats via tail-vein injection. Evaluation of the CB2-positive spleen, however, showed no accumulation of the radiotracer. Despite the promising in vitro binding affinities, specific binding of [(11)C]AAT-015, and [(11)C]AAT-778 could not be demonstrated.

Synthesis and Pharmacological Evaluation of [11C]Granisetron and [18F]Fluoropalonosetron as PET Probes for 5-HT3 Receptor Imaging.

Serotonin-gated ionotropic 5-HT3 receptors are the major pharmacological targets for antiemetic compounds. Furthermore, they have become a focus for the treatment of irritable bowel syndrome (IBS) and there is some evidence that pharmacological modulation of 5-HT3 receptors might alleviate symptoms of other neurological disorders. Highly selective, high-affinity antagonists, such as granisetron (Kytril) and palonosetron (Aloxi), belong to a family of drugs (the "setrons") that are well established for clinical use. To enable us to better understand the actions of these drugs in vivo, we report the synthesis of 8-fluoropalonosetron (15) that has a binding affinity (Ki = 0.26 ± 0.05 nM) similar to the parent drug (Ki = 0.21 ± 0.03 nM). We radiolabeled 15 by nucleophilic 18F-fluorination of an unsymmetrical diaryliodonium palonosetron precursor and achieved the radiosynthesis of 1-(methyl-11C)-N-granisetron ([11C]2) through N-alkylation with [11C]CH3I, respectively. Both compounds [18F]15 (chemical and radiochemical purity >95%, specific activity 41 GBq/µmol) and [11C]2 (chemical and radiochemical purity ≥99%, specific activity 170 GBq/µmol) were evaluated for their utility as positron emission tomography (PET) probes. Using mouse and rat brain slices, in vitro autoradiography with both [18F]15 and [11C]2 revealed a heterogeneous and displaceable binding in cortical and hippocampal regions that are known to express 5-HT3 receptors at significant levels. Subsequent PET experiments suggested that [18F]15 and [11C]2 are of limited utility for the PET imaging of brain 5-HT3 receptors in vivo.

Discovery of a fluorinated 4-oxo-quinoline derivative as a potential positron emission tomography radiotracer for imaging cannabinoid receptor type 2.

The cannabinoid receptor type 2 (CB2) is part of the endocannabinoid system and has gained growing attention in recent years because of its important role in neuroinflammatory/neurodegenerative diseases. Recently, we reported on a carbon-11 labeled 4-oxo-quinoline derivative, designated RS-016, as a promising radiotracer for imaging CB2 using PET. In this study, three novel fluorinated analogs of RS-016 were designed, synthesized, and pharmacologically evaluated. The results of our efforts led to the identification of N-(1-adamantyl)-1-(2-(2-fluoroethoxy)ethyl)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide (RS-126) as the most potent candidate for evaluation as a CB2 PET ligand. [(18) F]RS-126 was obtained in ≥ 99% radiochemical purity with an average specific radioactivity of 98 GBq/µmol at the end of the radiosynthesis. [(18) F]RS-126 showed a logD7.4 value of 1.99 and is stable in vitro in rat and human plasma over 120 min, whereas 55% intact parent compound was found in vivo in rat blood plasma at 10 min post injection. In vitro autoradiographic studies with CB2-positive rat spleen tissue revealed high and blockable binding which was confirmed in in vivo displacement experiments with rats by dynamic PET imaging. Ex vivo biodistribution studies confirmed accumulation of [(18) F]RS-126 in rat spleen with a specificity of 79% under blocking conditions. The moderate elevated CB2 levels in LPS-treated mice brain did not permit the detection of CB2 by [(18) F]RS-126 using PET imaging. In summary, [(18) F]RS-126 demonstrated high specificity toward CB2 receptor in vitro and in vivo and is a promising radioligand for imaging CB2 receptor expression. Cannabinoid receptor type 2 (CB2) is an interesting target for PET imaging. Specific binding of [(18) F]RS-126 in CB2-positive spleen tissue (white arrow head) was confirmed in in vivo displacement experiments with rats. Time activity curve of [(18) F]RS-126 in the spleen after the addition of GW405833 (CB2 specific ligand, green) demonstrates faster radiotracer elimination (blue) compared to the tracer only (red).