Rest/stress myocardial perfusion imaging by positron emission tomography with 18F-Flurpiridaz: A feasibility study in mice.

BACKGROUND: Myocardial perfusion imaging by positron emission tomography (PET-MPI) is the current gold standard for quantification of myocardial blood flow. 18F-flurpiridaz was recently introduced as a valid alternative to currently used PET-MPI probes. Nonetheless, optimum scan duration and time interval for image analysis are currently unknown. Further, it is unclear whether rest/stress PET-MPI with 18F-flurpiridaz is feasible in mice. METHODS: Rest/stress PET-MPI was performed with 18F-flurpiridaz (0.6-3.0 MBq) in 27 mice aged 7-8 months. Regadenoson (0.1 µg/g) was used for induction of vasodilator stress. Kinetic modeling was performed using a metabolite-corrected arterial input function. Image-derived myocardial 18F-flurpiridaz uptake was assessed for different time intervals by placing a volume of interest in the left ventricular myocardium. RESULTS: Tracer kinetics were best described by a two-tissue compartment model. K1 ranged from 6.7 to 20.0 mL·cm-3·min-1, while myocardial volumes of distribution (VT) were between 34.6 and 83.6 mL·cm-3. Of note, myocardial 18F-flurpiridaz uptake (%ID/g) was significantly correlated with K1 at rest and following pharmacological vasodilation for all time intervals assessed. However, while Spearman's coefficients (rs) ranged between 0.478 and 0.681, R2 values were generally low. In contrast, an excellent correlation of myocardial 18F-flurpiridaz uptake with VT was obtained, particularly when employing the averaged myocardial uptake from 20 to 40 min post tracer injection (R2 ≥ 0.98). Notably, K1 and VT were similarly sensitive to pharmacological vasodilation induction. Further, mean stress-to-rest ratios of K1, VT, and %ID/g 18F-flurpiridaz were virtually identical, suggesting that %ID/g 18F-flurpiridaz can be used to estimate coronary flow reserve (CFR) in mice. CONCLUSION: Our findings suggest that a simplified assessment of relative myocardial perfusion and CFR, based on image-derived tracer uptake, is feasible with 18F-flurpiridaz in mice, enabling high-throughput mechanistic CFR studies in rodents.

Characterization in nonhuman primates of (R)-[18F]OF-Me-NB1 and (S)-[18F]OF-Me-NB1 for imaging the GluN2B subunits of the NMDA receptor.

PURPOSE: GluN2B containing N-methyl-D-aspartate receptors (NMDARs) play an essential role in neurotransmission and are a potential treatment target for multiple neurological and neurodegenerative diseases, including stroke, Alzheimer's disease, and Parkinson's disease. (R)-[18F]OF-Me-NB1 was reported to be more specific and selective than (S)-[18F]OF-Me-NB1 for the GluN2B subunits of the NMDAR based on their binding affinity to GluN2B and sigma-1 receptors. Here we report a comprehensive evaluation of (R)-[18F]OF-Me-NB1 and (S)-[18F]OF-Me-NB1 in nonhuman primates. METHODS: The radiosynthesis of (R)-[18F]OF-Me-NB1 and (S)-[18F]OF-Me-NB1 started from 18F-fluorination of the boronic ester precursor, followed by removal of the acetyl protecting group. PET scans in two rhesus monkeys were conducted on the Focus 220 scanner. Blocking studies were performed after treatment of the animals with the GluN2B antagonist Co101,244 or the sigma-1 receptor antagonist FTC-146. One-tissue compartment (1TC) model and multilinear analysis-1 (MA1) method with arterial input function were used to obtain the regional volume of distribution (VT, mL/cm3). Occupancy values by the two blockers were obtained by the Lassen plot. Regional non-displaceable binding potential (BPND) was calculated from the corresponding baseline VT and the VND derived from the occupancy plot of the Co101,244 blocking scans. RESULTS: (R)- and (S)-[18F]OF-Me-NB1 were produced in > 99% radiochemical and enantiomeric purity, with molar activity of 224.22 ± 161.69 MBq/nmol at the end of synthesis (n = 10). Metabolism was moderate, with ~ 30% parent compound remaining for (R)-[18F]OF-Me-NB1 and 20% for (S)-[18F]OF-Me-NB1 at 30 min postinjection. Plasma free fraction was 1-2%. In brain regions, both (R)- and (S)-[18F]OF-Me-NB1 displayed fast uptake with slower clearance for the (R)- than (S)-enantiomer. For (R)-[18F]OF-Me-NB1, both the 1TC model and MA1 method gave reliable estimates of regional VT values, with MA1 VT (mL/cm3) values ranging from 8.9 in the cerebellum to 12.8 in the cingulate cortex. Blocking with 0.25 mg/kg of Co101,244 greatly reduced the uptake of (R)-[18F]OF-Me-NB1 across all brain regions, resulting in occupancy of 77% and VND of 6.36, while 0.027 mg/kg of FTC-146 reduced specific binding by 30%. Regional BPND, as a measure of specific binding signals, ranged from 0.40 in the cerebellum to 1.01 in the cingulate cortex. CONCLUSIONS: In rhesus monkeys, (R)-[18F]OF-Me-NB1 exhibited fast kinetics and heterogeneous uptake across brain regions, while the (S)-enantiomer displayed a narrower dynamic range of uptake across regions. A Blocking study with a GluN2B antagonist indicated binding specificity. The value of BPND was > 0.5 in most brain regions, suggesting good in vivo specific binding signals. Taken together, results from the current study demonstrated the potential of (R)-[18F]OF-Me-NB1 as a useful radiotracer for imaging the GluN2B receptors.

Role of sex hormones in modulating myocardial perfusion and coronary flow reserve.

BACKGROUND: A growing body of evidence highlights sex differences in the diagnostic accuracy of cardiovascular imaging modalities. Nonetheless, the role of sex hormones in modulating myocardial perfusion and coronary flow reserve (CFR) is currently unclear. The aim of our study was to assess the impact of female and male sex hormones on myocardial perfusion and CFR. METHODS: Rest and stress myocardial perfusion imaging (MPI) was conducted by small animal positron emission tomography (PET) with [18F]flurpiridaz in a total of 56 mice (7-8 months old) including gonadectomized (Gx) and sham-operated males and females, respectively. Myocardial [18F]flurpiridaz uptake (% injected dose per mL, % ID/mL) was used as a surrogate for myocardial perfusion at rest and following intravenous regadenoson injection, as previously reported. Apparent coronary flow reserve (CFRApp) was calculated as the ratio of stress and rest myocardial perfusion. Left ventricular (LV) morphology and function were assessed by cardiac magnetic resonance (CMR) imaging. RESULTS: Orchiectomy resulted in a significant decrease of resting myocardial perfusion (Gx vs. sham, 19.4 ± 1.0 vs. 22.2 ± 0.7 % ID/mL, p = 0.034), while myocardial perfusion at stress remained unchanged (Gx vs. sham, 27.5 ± 1.2 vs. 27.3 ± 1.2 % ID/mL, p = 0.896). Accordingly, CFRApp was substantially higher in orchiectomized males (Gx vs. sham, 1.43 ± 0.04 vs. 1.23 ± 0.05, p = 0.004), and low serum testosterone levels were linked to a blunted resting myocardial perfusion (r = 0.438, p = 0.020) as well as an enhanced CFRApp (r = -0.500, p = 0.007). In contrast, oophorectomy did not affect myocardial perfusion in females. Of note, orchiectomized males showed a reduced LV mass, stroke volume, and left ventricular ejection fraction (LVEF) on CMR, while no such effects were observed in oophorectomized females. CONCLUSION: Our experimental data in mice indicate that sex differences in myocardial perfusion are primarily driven by testosterone. Given the diagnostic importance of PET-MPI in clinical routine, further studies are warranted to determine whether testosterone levels affect the interpretation of myocardial perfusion findings in patients.

Regional brain mGlu5 receptor occupancy following single oral doses of mavoglurant as measured by [11C]-ABP688 PET imaging in healthy volunteers.

Mavoglurant binds to same allosteric site on metabotropic glutamate receptor 5 (mGluR5) as [11C]-ABP688, a radioligand. This open-label, single-center pilot study estimates extent of occupancy of mGluR5 receptors following single oral doses of mavoglurant, using [11C]-ABP688 positron emission tomography (PET) imaging, in six healthy males aged 20-40 years. This study comprised three periods and six subjects were divided into two cohorts. On Day 1 (Period 1), baseline clinical data and safety samples were obtained along with PET scan. During Period 2 (1-7 days after Period 1), cohort 1 and 2 received mavoglurant 25 mg and 100 mg, respectively. During Period 3 (7 days after Period 2), cohort 1 and 2 received mavoglurant 200 mg and 400 mg, respectively. Mavoglurant showed the highest distribution volumes in the cingulate region with lower uptake in cerebellum and white matter, possibly because myelinated axonal sheets maybe devoid of mGlu5 receptors. Maximum concentrations of mavoglurant were observed around 2-3.25 h post-dose. Mavoglurant passed the blood-brain barrier and induced dose- and exposure-dependent displacement of [11C]-ABP688 from the mGluR5 receptors, 3-4 h post-administration (27%, 59%, 74%, 85% receptor occupancy for mavoglurant 25 mg, 100 mg, 200 mg, 400 mg dose, respectively). There were no severe adverse effects or clinically significant changes in safety parameters. This is the first human receptor occupancy study completed with Mavoglurant. It served to guide the dosing of mavoglurant in the past and currently ongoing clinical studies. Furthermore, it confirms the utility of [11C]-ABP688 as a unique tool to study drug-induced occupancy of mGlu5 receptors in the living human brain.

A Review of Molecular Imaging of Glutamate Receptors.

Molecular imaging with positron emission tomography (PET) and single photon emission computed tomography (SPECT) is a well-established and important in vivo technique to evaluate fundamental biological processes and unravel the role of neurotransmitter receptors in various neuropsychiatric disorders. Specific ligands are available for PET/SPECT studies of dopamine, serotonin, and opiate receptors, but corresponding development of radiotracers for receptors of glutamate, the main excitatory neurotransmitter in mammalian brain, has lagged behind. This state of affairs has persisted despite the central importance of glutamate neurotransmission in brain physiology and in disorders such as stroke, epilepsy, schizophrenia, and neurodegenerative diseases. Recent years have seen extensive efforts to develop useful ligands for molecular imaging of subtypes of the ionotropic (N-methyl-D-aspartate (NMDA), kainate, and AMPA/quisqualate receptors) and metabotropic glutamate receptors (types I, II, and III mGluRs). We now review the state of development of radioligands for glutamate receptor imaging, placing main emphasis on the suitability of available ligands for reliable in vivo applications. We give a brief account of the radiosynthetic approach for selected molecules. In general, with the exception of ligands for the GluN2B subunit of NMDA receptors, there has been little success in developing radiotracers for imaging ionotropic glutamate receptors; failure of ligands for the PCP/MK801 binding site in vivo doubtless relates their dependence on the open, unblocked state of the ion channel. Many AMPA and kainite receptor ligands with good binding properties in vitro have failed to give measurable specific binding in the living brain. This may reflect the challenge of developing brain-penetrating ligands for amino acid receptors, compounded by conformational differences in vivo. The situation is better with respect to mGluR imaging, particularly for the mGluR5 subtype. Several successful PET ligands serve for investigations of mGluRs in conditions such as schizophrenia, depression, substance abuse and aging. Considering the centrality and diversity of glutamatergic signaling in brain function, we have relatively few selective and sensitive tools for molecular imaging of ionotropic and metabotropic glutamate receptors. Further radiopharmaceutical research targeting specific subtypes and subunits of the glutamate receptors may yet open up new investigational vistas with broad applications in basic and clinical research.

Neuroimaging with Radiopharmaceuticals Targeting the Glutamatergic System.

Radiopharmacy at ETH has worked on the development of novel PET tracers for neuro-, cardiac- and tumor imaging for many years. In this paper, our efforts on targeting the glutamatergic system of the metabotropic glutamate receptor subtype 5 (mGluR5) and the ionotropic N-methyl-D-aspartate (NMDA) receptor are summarized. We briefly described the principles of positron emission tomography (PET) tracer development for the central nervous system (CNS) and the radiolabeling methods used in our laboratory. To assess the radioligands, results of in vitro autoradiography, biodistribution, and metabolite studies as well as PET imaging data are discussed. Furthermore, key PET parameters for kinetic modeling and quantification methods are provided. Two mGluR5 PET tracers, [11C]ABP688 and [18F]PSS232, were translated in our GMP labs and evaluated in human subjects. The newly developed GluN2B PET tracer [11C]Me-NB1 is currently being investigated in a first-in-human PET study and several F-18 labeled tracers are being evaluated in non-human primates in which the first-in-class will be translated for human studies.

Imaging glutamate redistribution after acute N-acetylcysteine administration: A simultaneous PET/MR study.

Glutamate is the most abundant excitatory neurotransmitter in the human brain, but in vivo imaging of acute fluctuations in glutamatergic levels has not been well established. The purpose of this study was to examine acute changes in glutamate after stimulation with N-acetylcysteine (NAC) using a simultaneous positron emission tomography/magnetic resonance spectroscopy (PET/MRS) approach. Ten healthy adult males were examined in two scanning sessions, and 5g NAC was administered 1 h prior to one of the scan sessions. Simultaneous PET/MR data were acquired using an integrated 3T PET/MR scanner. Glutamate (Glu), glutamine (Gln), and glutamate + glutamine (Glx) levels were assessed from MRS data collected from the basal ganglia with PRESS and from the left prefrontal cortex with PRESS and MEGAPRESS, and mGluR5 binding (BPND) was assessed from PET data collected with [18F]PSS232. NAC administration was associated with a significant reduction in Glx and Gln in the basal ganglia spectra, and in Glx in the frontal MEGAPRESS spectra (p < 0.05); no differences in [18F]PSS232 BPND were observed with NAC, although a correlation between pre-/post-treatment Glx and baseline BPnd was found. The MRS-visible Glx signal is sensitive to acute fluctuations in glutamate. The change in Glx was mostly driven by a change in Gln, lending weight to the notion that Gln can provide a proxy marker for neurotransmitter/synaptic glutamate. [18F]PSS232 binding is not sensitive to acute glutamate shifts independently, but was associated with the extent of glutamate liberation upon NAC stimulation.

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.

Modification of the 4-phenylbutyl side chain of potent 3-benzazepine-based GluN2B receptor antagonists.

Excitotoxicity driven by overactivation of NMDA receptors represents a major mechanism of acute and chronic neurological and neurodegenerative disorders. Negative allosteric modulators interacting with the ifenprodil binding site of the NMDA receptor are able to interrupt this ongoing neurodamaging process. Starting from the potent 3-benzazepine-1,7-diol 4a novel NMDA receptor antagonists were designed by modification of the N-(4-phenylbutyl) side chain. With respect to developing novel fluorinated PET tracers, regioisomeric fluoroethoxy derivatives 11, 12, 14, and 15 were synthesized. Analogs 19 and 20 with various heteroaryl moieties at the end of the N-side chain were prepared by Sonogashira reaction and nucleophilic substitution. The fluoroethyl triazole 37 was obtained by 1,3-dipolar cycloaddition. In several new ligands, the flexibility of the (hetero)arylbutyl side chain was restricted by incorporation of a triple bond. The affinity towards the ifenprodil binding site was tested in an established competition assay using [3H]ifenprodil as radioligand. Introduction of a fluoroethoxy moiety at the terminal phenyl ring, replacement of the terminal phenyl ring by a heteroaryl ring and incorporation of a triple bond into the butyl spacer led to considerable reduction of GluN2B affinity. The phenol 15 (Ki = 193 nM) bearing a p-fluoroethoxy moiety at the terminal phenyl ring represents the most promising GluN2B ligand of this series of compounds. With exception of 15 showing moderate σ2 affinity (Ki = 79 nM), the interaction of synthesized 3-benzazepines towards the PCP binding site of the NMDA receptor, σ1 and σ2 receptors was rather low (Ki > 100 nM).

Positron emission tomography of type 2 cannabinoid receptors for detecting inflammation in the central nervous system.

Cannabinoid receptor CB2 (CB2R) is upregulated on activated microglia and astrocytes in the brain under inflammatory conditions and plays important roles in many neurological diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, and ischemic stroke. The advent of positron emission tomography (PET) using CB2R radiotracers has enabled the visualization of CB2R distribution in vivo in animal models of central nervous system inflammation, however translation to humans has been less successful. Several novel CB2R radiotracers have been developed and evaluated to quantify microglial activation. In this review, we summarize the recent preclinical and clinical imaging results of CB2R PET tracers and discuss the prospects of CB2R imaging using PET.

Recent progress in allosteric modulators for GluN2A subunit and development of GluN2A-selective nuclear imaging probes.

N-methyl-D-aspartate (NMDA) receptors play key roles in physiology by regulating the synaptic plasticity and the cellular mechanism involved in learning and memory. The GluN2A subunit is the most abundant expression of NMDA receptors in mature brain, and its dysfunction has been implicated in various neurological disorders. However, the function of GluN2A subunit in physiological and pathological conditions is not yet completely unveil due to the lack of subunit-selective ligands, including specific positron emission tomography (PET)/single photon emission computed tomography (SPECT) imaging probes. In this review, recent progresses in understanding its pathophysiological role, the structure-activity relationship, and the postulated mechanisms of novel GluN2A ligands as well as status of molecular imaging probes for PET are summarized.

Synthesis and pharmacological evaluation of fluorinated benzo[7]annulen-7-amines as GluN2B-selective NMDA receptor antagonists.

Because of their neuroprotective potential, GluN2B-selective ligands are of great interest for the treatment of various neurological and neurodegenerative disorders. Fluorinated benzo[7]annulen-7-amines, capable for PET, were synthesized by combining fluorinated phenylalkylamines with differently substituted ketones. Relationships between substitution pattern and GluN2B affinity as well as selectivity towards σ1 and σ2 receptors were investigated. Two promising ligands (18a and 20c) were selected for further pharmacological evaluation. Besides a slight serotonin transporter (SERT), norepinephrine transporter (NET), and hERG affinity, they did not show interaction with other targets. Furthermore, the pKa value of a set fluorinated ligands, bearing the fluorine atom in different positions, was determined.

Reduced 18F-Folate Conjugates as a New Class of PET Tracers for Folate Receptor Imaging.

5-Methyltetrahydrofolate (5-MTHF), a reduced folate form, is the biologically active folate involved in many different metabolic processes. To date, there are no studies available in the literature on 18F-labeled 6 S- and 6 R-5-MTHF radiotracers for imaging folate receptor (FR)-α-positive tissues. Therefore, the goal of this study was to synthesize four 18F-labeled 5-MTHF derivatives conjugated at either the α- or γ-carboxylic functionality of glutamate and to assess their suitability for FR-targeting. Organic syntheses of the precursors and the four reference compounds, namely, 6 S-α, 6 S-γ, 6 R-α, and 6 R-γ-click-fluoroethyl-5-MTHF, were carried out in low to moderate overall chemical yields. The radiosyntheses of the α- and γ-conjugated 18F-labeled folate derivatives were accomplished in approximately 100 min, low radiochemical yields (1-7% d.c.) and high molar activities (139-245 GBq/µmol). Radiochemically pure tracers were obtained after the addition of a mixture of antioxidants consisting of sodium ascorbate and l-cysteine. In vitro, all four 5-MTHF conjugates showed similar binding affinities to FR-α (IC50 = 17.7-24.0 nM), whereas folic acid showed a significantly higher binding affinity to the FR-α. Cell uptake and internalization experiments with KB cells demonstrated specific uptake and internalization of the radiofolate conjugates. Metabolite studies in mice revealed high in vivo stability of the radiotracers in mice. Biodistribution and positron emission tomography (PET) imaging studies in FR-positive KB tumor-bearing mice demonstrated that the 6 S- and 6 R-5-MTHF conjugates exhibited a different accumulation pattern in various organs including the kidneys and the liver, whereas no significant differences in radioactivity accumulation in the kidneys and the liver were found for both the α- and γ-conjugated diastereoisomers. Despite the considerably lower binding affinities of the 5-MTHF derivatives compared to the corresponding folic acid conjugates similar high KB tumor uptake was observed for all the folate conjugates investigated (8-11% IA/g). Based on these results, we conclude that 18F-labeled 5-MTHF conjugates are a promising new class of radiotracers for targeting FR-positive tumor tissues.

A first-in-man PET study of [18F]PSS232, a fluorinated ABP688 derivative for imaging metabotropic glutamate receptor subtype 5.

PURPOSE: Non-invasive imaging of metabotropic glutamate receptor 5 (mGlu5) in the brain using PET is of interest in e.g., anxiety, depression, and Parkinson's disease. Widespread application of the most widely used mGlu5 tracer, [11C]ABP688, is limited by the short physical half-life of carbon-11. [18F]PSS232 is a fluorinated analog with promising preclinical properties and high selectivity and specificity for mGlu5. In this first-in-man study, we evaluated the brain uptake pattern and kinetics of [18F]PSS232 in healthy volunteers. METHODS: [18F]PSS232 PET was performed with ten healthy male volunteers aged 20-40 years. Seven of the subjects received a bolus injection and the remainder a bolus/infusion protocol. Cerebral blood flow was determined in seven subjects using [15O]water PET. Arterial blood activity was measured using an online blood counter. Tracer kinetics were evaluated by compartment modeling and parametric maps were generated for both tracers. RESULTS: At 90 min post-injection, 59.2 ± 11.1% of total radioactivity in plasma corresponded to intact tracer. The regional first pass extraction fraction of [18F]PSS232 ranged from 0.41 ± 0.06 to 0.55 ± 0.03 and brain distribution pattern matched that of [11C]ABP688. Uptake kinetics followed a simple two-tissue compartment model. The volume of distribution of total tracer (V T, ml/cm3) ranged from 1.18 ± 0.20 for white matter to 2.91 ± 0.51 for putamen. The respective mean distribution volume ratios (DVR) with cerebellum as the reference tissue were 0.88 ± 0.06 and 2.12 ± 0.10, respectively. The tissue/cerebellum ratios of a bolus/infusion protocol (30/70 dose ratio) were close to the DVR values. CONCLUSIONS: Brain uptake of [18F]PSS232 matched the distribution of mGlu5 and followed a two-tissue compartment model. The well-defined kinetics and the possibility to use reference tissue models, obviating the need for arterial blood sampling, make [18F]PSS232 a promising fluorine-18 labeled radioligand for measuring mGlu5 density in humans.

Radioligands for positron emission tomography imaging of cannabinoid type 2 receptor.

The cannabinoid type 2 (CB2) receptor is an immunomodulatory receptor mainly expressed in peripheral cells and organs of the immune system. The expression level of CB2 in the central nervous system under physiological conditions is negligible, however under neuroinflammatory conditions an upregulation of CB2 protein or mRNA mainly colocalized with activated microglial cells has been reported. Consequently, CB2 agonists have been confirmed to play a role in neuroprotective and anti-inflammatory processes. A suitable positron emission tomography radioligand for imaging CB2 would provide an invaluable research tool to explore the role of CB2 receptor expression in inflammatory disorders. In this review, we provide a summary of so far published CB2 radioligands as well as their in vitro and in vivo binding characteristics.

Automated cGMP-compliant radiosynthesis of [18 F]-(E)-PSS232 for brain PET imaging of metabotropic glutamate receptor subtype 5.

(E)-3-(Pyridin-2-yl ethynyl)cyclohex-2-enone O-(3-(2-[18 F]-fluoroethoxy)propyl) oxime ([18 F]-(E)-PSS232, [18 F]2a) is a recently developed radiotracer that can be used to visualize metabotropic glutamate receptor subtype 5 (mGlu5 ) in vivo. The mGlu5 has become an attractive therapeutic and diagnostic target owing to its role in many neuropsychiatric disorders. Several carbon-11-labeled and fluorine-18-labeled radiotracers have been developed to measure mGlu5 receptor occupancy in the human brain. The radiotracer [18 F]2a, which is used as an analogue for [11 C]ABP688 ([11 C]1) and has a longer physical half-life, is a selective radiotracer that exhibits high binding affinity for mGlu5 . Herein, we report the fully automated radiosynthesis of [18 F]2a using a commercial GE TRACERlab™ FX-FN synthesizer for routine production and distribution to nearby satellite clinics. Nucleophilic substitution of the corresponding mesylate precursor with cyclotron-produced [18 F]fluoride ion at 100°C in dimethyl sulfoxide (DMSO), followed by high-performance liquid chromatography (HPLC) purification and formulation, readily provided [18 F]2a with a radiochemical yield of 40 ± 2% (decay corrected, n = 5) at the end of synthesis. Radiochemical purity for the [18 F]-(E)-conformer was greater than 95%. Molar activity was determined to be 63.6 ± 9.6 GBq/µmol (n = 5), and the overall synthesis time was 70 minutes.

Imaging the glutamate receptor subtypes-Much achieved, and still much to do.

Functional imaging of glutamate receptors using PET imaging modality can be used to study numerous CNS disorders and also to select appropriate doses of clinically relevant glutamate-receptor-targeting candidate drugs. Great strides have been made in developing PET imaging probes for the non-invasive detection of glutamate receptors in the brain. This review highlights recent progress made towards the development of glutamatergic PET imaging agents. Focus is placed on PET imaging probes that have been labelled with either carbon-11 or fluorine-18.

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).

Comparative Studies of Three Pairs of α- and γ-Conjugated Folic Acid Derivatives Labeled with Fluorine-18.

The folate receptor (FR) is upregulated in various epithelial cancer types (FR α-isoform), while healthy tissues show only restricted expression. FR-targeted imaging using folate radiopharmaceuticals is therefore a promising approach for the detection of FR-positive cancer tissue. Almost all folate-based radiopharmaceuticals have been prepared by conjugation at the γ-carboxylic functionality of the glutamate moiety of folic acid. In this work, three pairs of fluorinated α- and γ-conjugated folate derivatives were synthesized and their in vitro and in vivo properties compared. The syntheses of all six regioisomers were obtained in good chemical yields using a multistep synthetic approach including the highly selective Cu(I)-catalyzed 1,3-dipolar cycloaddition. The radiosyntheses of the α- and γ-conjugated (18)F-labeled folate derivatives were accomplished in moderate to good radiochemical yields, high radiochemical purities (>95%), and specific activities ranging from 25 to 196 GBq/µmol. In vitro, all folate derivatives showed high binding affinity to the FR-α (IC50 = 1.4-2.2 nM). In vivo PET imaging and biodistribution studies in FR-positive KB tumor-bearing mice demonstrated similar FR-specific tumor uptake for both regioisomers of each pair of compounds. However, FR-unspecific liver uptake was significantly lower for the α-regioisomers compared to the corresponding γ-regioisomers. In contrast, kidney uptake was up to 50% lower for the γ-regioisomers than for the α-regioisomers. These results show that the site of conjugation in the glutamyl moiety of folic acid has a significant impact on the in vivo behavior of (18)F-based radiofolates, but not on their in vitro FR-binding affinity. These findings may potentially stimulate new directions for the design of novel (18)F-labeled folate-based radiotracers.

(64)Cu- and (68)Ga-Based PET Imaging of Folate Receptor-Positive Tumors: Development and Evaluation of an Albumin-Binding NODAGA-Folate.

A number of folate-based radioconjugates have been synthesized and evaluated for nuclear imaging purposes of folate receptor (FR)-positive tumors and potential therapeutic application. A common shortcoming of radiofolates is, however, a significant accumulation of radioactivity in the kidneys. This situation has been faced by modifying the folate conjugate with an albumin-binding entity to increase the circulation time of the radiofolate, which led to significantly improved tumor-to-kidney ratios. The aim of this study was to develop an albumin-binding folate conjugate with a NODAGA-chelator (rf42) for labeling with (64)Cu and (68)Ga, allowing application for PET imaging. The folate conjugate rf42 was synthesized in 8 steps, with an overall yield of 5%. Radiolabeling with (64)Cu and (68)Ga was carried out at room temperature within 10 min resulting in (64)Cu-rf42 and (68)Ga-rf42 with >95% radiochemical purity. (64)Cu-rf42 and (68)Ga-rf42 were stable (>95% intact) in phosphate-buffered saline over more than 4 half-lives of the corresponding radionuclide. In vitro, the plasma protein-bound fraction of (64)Cu-rf42 and (68)Ga-rf42 was determined to be >96%. Cell experiments proved FR-specific uptake of both radiofolates, as it was reduced to <1% when KB tumor cells were coincubated with excess folic acid. In vivo, high accumulation of (64)Cu-rf42 and (68)Ga-rf42 was found in KB tumors of mice (14.52 ± 0.99% IA/g and 11.92 ± 1.68% IA/g, respectively) at 4 h after injection. The tumor-to-kidney ratios were in the range of 0.43-0.55 over the first 4 h of investigation. At later time points (up to 72 h p.i. of (64)Cu-rf42) the tumor-to-kidney ratio increased to 0.73. High-quality PET/CT images were obtained 2 h after injection of (64)Cu-rf42 and (68)Ga-rf42, respectively, allowing distinct visualization of tumors and kidneys. Comparison of PET/CT images obtained with (64)Cu-rf42 and a (64)Cu-labeled DOTA-folate conjugate (cm10) clearly proved the superiority of NODAGA for stable coordination of (64)Cu. (64)Cu-cm10 showed high liver uptake, most probably as a consequence of released (64)Cu(2+). The data reported in this study clearly proved the promising features of (64)Cu-rf42, particularly in terms of favorable tumor-to-kidney ratios. The relatively long half-life of (64)Cu (T1/2 = 12.7 h) matches well with the enhanced circulation time of the albumin-binding NODAGA-folate, allowing PET imaging at longer time points after injection than is possible when using (68)Ga (T1/2 = 68 min).