Preclinical Evaluation of the Reversible Monoacylglycerol Lipase PET Tracer (R)-[11C]YH132: Application in Drug Development and Neurodegenerative Diseases.

Monoacylglycerol lipase (MAGL) plays a crucial role in the degradation of 2-arachidonoylglycerol (2-AG), one of the major endocannabinoids in the brain. Inhibiting MAGL could lead to increased levels of 2-AG, which showed beneficial effects on pain management, anxiety, inflammation, and neuroprotection. In the current study, we report the characterization of an enantiomerically pure (R)-[11C]YH132 as a novel MAGL PET tracer. It demonstrates an improved pharmacokinetic profile compared to its racemate. High in vitro MAGL specificity of (R)-[11C]YH132 was confirmed by autoradiography studies using mouse and rat brain sections. In vivo, (R)-[11C]YH132 displayed a high brain penetration, and high specificity and selectivity toward MAGL by dynamic PET imaging using MAGL knockout and wild-type mice. Pretreatment with a MAGL drug candidate revealed a dose-dependent reduction of (R)-[11C]YH132 accumulation in WT mouse brains. This result validates its utility as a PET probe to assist drug development. Moreover, its potential application in neurodegenerative diseases was explored by in vitro autoradiography using brain sections from animal models of Alzheimer's disease and Parkinson's disease.

The impact of tau deposition and hypometabolism on cognitive impairment and longitudinal cognitive decline.

INTRODUCTION: Tau and neurodegeneration strongly correlate with cognitive impairment, as compared to amyloid. However, their contribution in explaining cognition and predicting cognitive decline in memory clinics remains unclarified. METHODS: We included 94 participants with Mini-Mental State Examination (MMSE), tau positron emission tomography (PET), amyloid PET, fluorodeoxyglucose (FDG) PET, and MRI scans from Geneva Memory Center. Linear regression and mediation analyses tested the independent and combined association between biomarkers, cognitive performance, and decline. Linear mixed-effects and Cox proportional hazards models assessed biomarkers' prognostic values. RESULTS: Metabolism had the strongest association with cognition (r = 0.712; p < 0.001), followed by tau (r = -0.682; p < 0.001). Neocortical tau showed the strongest association with cognitive decline (r = -0.677; p < 0.001). Metabolism mediated the association between tau and cognition and marginally mediated the one with decline. Tau positivity represented the strongest risk factor for decline (hazard ratio = 32). DISCUSSION: Tau and neurodegeneration synergistically contribute to global cognitive impairment while tau drives decline. The tau PET superior prognostic value supports its implementation in memory clinics. HIGHLIGHTS: Hypometabolism has the strongest association with concurrent cognitive impairment. Neocortical tau pathology is the main determinant of cognitive decline over time. FDG-PET has a superior value compared to MRI as a measure of neurodegeneration. The prognostic value of tau-PET exceeded all other neuroimaging modalities.

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.

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.

Dual MVK cleavable linkers effectively reduce renal retention of 111In-fibronectin-binding peptides.

BACKGROUND: Previously, we have exploited bacterial adhesins-derived fibronectin-binding peptides (FnBPs) for targeting mechanically altered fibronectin (Fn) fibrils within the cancer-associated extra-cellular matrix (ECM). However, despite the ability of FnBP probes to visualize pathological lesions, when labeled with metallic radionuclides and administered for targeted imaging, they exhibit high and persistent retention of radioactivity within the kidneys. Intending to overcome this issue towards a future translation of FnBPs to the clinic, the goal of the present study was to reduce the renal retention of 111In-labelled FnBPs employing dual renal brush border membrane (BBM) enzyme-sensitive Met-Val-Lys-based linkers, enabling a rapid washout of radioactivity from the kidneys. METHODS: Three maleimide-activated NOTA-conjugated brush border-enzyme cleavable linkers equipped with either single or dual consecutive MVK-based cleavable moieties were designed and synthesized. Their respective NOTA-MVK-based FnBPA5.1 conjugates were obtained by means of maleimide-thiol mediated conjugation at the N-terminus of the Fn-binding sequence, radiolabeled with indium-111, and further evaluated in vitro and in vivo in comparison to the control [111In]In-FnBPA5.1. RESULTS: The linker equipped with two MVK sites displayed a two-fold more effective cleavage rate than the single MVK featuring linker in vitro, as revealed by the quantification of the released Met-containing radiometabolites. SPECT/CT imaging and biodistribution studies of the series of FnBPA5.1 radioconjugates performed at 24 h post-injection (p.i.) confirmed the in vitro results, indicating that the renal retention of 111In-labelled FnBPs can be significantly lowered through the interposition of a single MVK-based sequence between the Fn-targeting moiety and the chelating unit (52.75 ± 9.79 vs 92.88 ± 4.85 % iA/g, P < 0.001), and even further reduced by the addition of a second one (down to 34.82 ± 6.04, P < 0.001), with minor influence on the biodistribution in other organs, such as tumors. CONCLUSIONS: In summary, we report here promising 111In-labelled FnBP radiotracers equipped with dual MVK-based cleavable linkers leading to a more effective reduction of renal retention and improved tumor-to-kidney ratios compared to the single MVK-featuring derivative. Our dual MVK strategy is a crucial step towards the clinical translation of mechano-sensory FnBPs and might as well be adopted for other radiopharmaceuticals suffering from persistent renal retention of radioactivity.

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.

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.

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.

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.

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).

Quantitative positron emission tomography of mGluR5 in rat brain with [(18) F]PSS232 at minimal invasiveness and reduced model complexity.

Imaging the density of metabotropic glutamate receptor 5 (mGluR5) in brain by positron emission tomography (PET) is of interest in relation to several brain disorders. We have recently introduced [(18) F]PSS232, an F-18-labeled analog of the mGluR5-targeting [(11) C]ABP688. Quantitative PET requires kinetic modeling with an input function (IF) or an appropriate reference tissue model. We aimed at minimizing invasiveness of IF recording in rat and employing this protocol for mGluR5 quantitative PET with [(18) F]PSS232. We further aimed at defining models of low complexity for quantitative PET with [(18) F]PSS232. The IF was recorded in an arterio-venous shunt applied by minimally invasive cannulation. PET data were analyzed with a modified two-tissue compartment model including a single variable for radiometabolite correction in brain. We further evaluated a simple reference tissue model. Receptor-dependent accumulation was similar to [(11) C]ABP688 at lower unspecific accumulation of unchanged [(18) F]PSS232, in agreement with its higher plasma protein binding and lower lipophilicity. The minimally invasive protocol revealed similar results as the invasive shunt method and parameters calculated with the modified two-tissue compartment model were similar to those calculated with the standard model. The simple area under the curve ratios agreed with the Logan reference method. [(18) F]PSS232 is a promising radioligand for mGluR5 quantification. Methods were evaluated to quantify mGluR5 in rat brain by PET with [(18) F]PSS232. We present a minimally invasive protocol for input function recording. A two-tissue compartment model correcting for radiometabolites at reduced complexity is compared with the standard model. Finally, we demonstrate and explain why for [(18) F]PSS232 the area-under-the-curve ratio is a valid alternative to the Logan reference tissue analysis.

Preclinical evaluation and test-retest studies of [(18)F]PSS232, a novel radioligand for targeting metabotropic glutamate receptor 5 (mGlu5).

PURPOSE: A novel, (18)F-labelled metabotropic glutamate receptor subtype 5 (mGlu5) derivative of [(11)C]ABP688 ([(11)C]1), [(18)F]PSS232 ([(18)F] ]5), was evaluated in vitro and in vivo for its potential as a PET agent and was used in test-retest reliability studies METHODS: The radiosynthesis of [(18)F]5 was accomplished via a one-step reaction using a mesylate precursor. In vitro stability was determined in PBS and plasma, and with liver microsomal enzymes. Metabolite studies were performed using rat brain extracts, blood and urine. In vitro autoradiography was performed on horizontal slices of rat brain using 1 and 8, antagonists for mGlu5 and mGlu1, respectively. Small-animal PET, biodistribution, and test-retest studies were performed in Wistar rats. In vivo, dose-dependent displacement studies were performed using 6 and blocking studies with 7. RESULTS: [(18)F]5 was obtained in decay-corrected maximal radiochemical yield of 37 % with a specific activity of 80 - 400 GBq/µmol. Treatment with rat and human microsomal enzymes in vitro for 60 min resulted in 20 % and 4 % of hydrophilic radiometabolites, respectively. No hydrophilic decomposition products or radiometabolites were found in PBS or plasma. In vitro autoradiography on rat brain slices showed a heterogeneous distribution consistent with the known distribution of mGlu5 with high binding to hippocampal and cortical regions, and negligible radioactivity in the cerebellum. Similar distribution of radioactivity was found in PET images. Under displacement conditions with 6, reduced [(18)F]5 binding was found in all brain regions except the cerebellum. 7 reduced binding in the striatum by 84 % on average. Test-retest studies were reproducible with a variability ranging from 6.8 % to 8.2 %. An extended single-dose toxicity study in Wistar rats showed no compound-related adverse effects. CONCLUSION: The new mGlu5 radiotracer, [(18)F]5, showed specific and selective in vitro and in vivo properties and is a promising radioligand for PET imaging of mGlu5 in humans.

Synthesis and preliminary biological evaluation of O-2((2-[(18)F]fluoroethyl)methylamino)ethyltyrosine ([(18)F]FEMAET) as a potential cationic amino acid PET tracer for tumor imaging.

Amino acid transport is an attractive target for oncologic imaging. Despite a high demand of cancer cells for cationic amino acids, their potential as PET probes remains unexplored. Arginine, in particular, is involved in a number of biosynthetic pathways that significantly influence carcinogenesis and tumor biology. Cationic amino acids are transported by several cationic transport systems including, ATB(0,+) (SLC6A14), which is upregulated in certain human cancers including cervical, colorectal and estrogen receptor-positive breast cancer. In this work, we report the synthesis and preliminary biological evaluation of a new cationic analog of the clinically used PET tumor imaging agent O-(2-[(18)F]fluroethyl)-L-tyrosine ([(18)F]FET), namely O-2((2-[(18)F]fluoroethyl)methylamino)ethyltyrosine ([(18)F]FEMAET). Reference compound and precursor were prepared by multi-step approaches. Radiosynthesis was achieved by no-carrier-added nucleophilic [(18)F]fluorination in 16-20% decay-corrected yields with radiochemical purity >99%. The new tracer showed good stability in vitro and in vivo. Cell uptake assays demonstrated that FEMAET and [(18)F]FEMAET accumulate in prostate cancer (PC-3) and small cell lung cancer cells (NCI-H69), with an energy-dependent mechanism. Small animal PET imaging with NCI-H69 xenograft-bearing mice revealed good tumor visualization comparable to [(18)F]FET and low brain uptake, indicating negligible transport across the blood-brain barrier. In conclusion, the non-natural cationic amino acid PET probe [(18)F]FEMAET accumulates in cancer cells in vitro and in vivo with possible involvement of ATB(0,+).

Synthesis and biological evaluation of (18)F-labeled Fluoroethoxy tryptophan analogues as potential PET tumor imaging agents.

As a continuation of our research efforts toward the development of tryptophan-based radiotracers for tumor imaging with positron emission tomography (PET), three new fluoroethoxy tryptophan analogues were synthesized and evaluated in vivo. These new tracers (namely, 4-(2-[(18)F]fluoroethoxy)-dl-tryptophan ([(18)F]4-FEHTP), 6-(2-[(18)F]fluoroethoxy)-dl-tryptophan ([(18)F]6-FEHTP), and 7-(2-[(18)F]fluoroethoxy)-dl-tryptophan ([(18)F]7-FEHTP) carry the fluoroethoxy side chain either at positions 4-, 6-, or 7- of the indole core. Reference compounds and precursors were synthesized by multistep approaches. Radiosynthesis was accomplished by no-carrier-added nucleophilic (18)F-fluorination following either an indirect approach (O-alkylation of the corresponding hydroxytryptophan with [(18)F]fluoroethyltosylate) or a direct approach (nucleophilic [(18)F] fluorination using a protected mesyl precursor). Radiochemical yields (decay corrected) for both methods were in the range of 10-18%. Small animal PET imaging with xenograft-bearing mice revealed the highest tumor/background ratio for [(18)F]6-FEHTP which, in a direct comparison, outperformed the other two tryptophan tracers and also the well-established tyrosine analogue O-(2-[(18)F]fluoroethyl)-l-tyrosine ([(18)F]l-FET). Investigation of the transport mechanism of [(18)F]6-FEHTP in small cell lung cancer cells (NCI-H69) revealed that it is most probably taken up exclusively via the large neutral amino acid transporter(s) (LAT).

Development and evaluation of novel PET tracers for imaging cannabinoid receptor type 2 in brain.

The cannabinoid receptor type 2 (CB2) has a very low expression level in brain tissue under basal conditions, but it is up-regulated in diverse pathological conditions. Two promising lead structures from the literature, N-((3S,5S,7S)-adamantan-1-yl)-8-methoxy-4-oxo-1-pentyl-1,4-dihydroquinoline-3-carboxamide and 8-butoxy-N-(2-fluoro-2-phenylethyl)-7-methoxy-2-oxo-1,2-dihydroquinoline-3-carboxamide - designated KD2 and KP23, respectively - were evaluated as potential PET ligands for imaging CB2. Both KD2 and KP23 were synthesized and labeled with carbon-11. In vitro autoradiographic studies on rodent spleen tissues showed that [(11)C]KD2 exhibits superior properties. A pilot study using [(11)C]KD2 on human post mortem ALS spinal cord slices indicated high CB2 expression level and specific binding, a very exciting finding if considering the future diagnostic application of CB2 ligands and their utility in therapy monitoring. In vivo blocking studies in rats with [(11)C]KD2 showed also high specific uptake in spleen tissue. Although the protein-bound fraction is relatively high, KD2 or KD2 derivatives could be very useful tools for the non-invasive investigation of CB2 levels under various neuroinflammatory conditions.

Metabotropic glutamate receptor 5 (mGluR5) is associated with neurodegeneration and amyloid deposition in Alzheimer's disease: A [18F]PSS232 PET/MRI study.

BACKGROUND: Metabotropic glutamate receptor 5 (mGluR5) is involved in regulating integrative brain function and synaptic transmission. Aberrant mGluR5 signaling and relevant synaptic failure play a key role in the initial pathophysiological mechanism of Alzheimer's disease (AD). The study aims to investigate the association between mGluR5 availability and AD's biomarkers and cognitive function. METHODS: We examined 35 individuals with mGluR5 tracer [18F]PSS232 to assess mGluR5 availability, and with [18F]Florbetapir PET to assess global amyloid deposition, and [18F]FDG PET to assess glucose metabolism. The plasma neurofilament light (NfL) and p-tau181 levels in a subset of individuals were measured (n = 27). The difference in mGluR5 availability between the AD and normal control (NC) groups was explored. The associations of mGluR5 availability with amyloid deposition, glucose metabolism, gray matter volume (GMV), neuropsychological assessment scores, and plasma biomarkers were analyzed. RESULTS: The mGluR5 availability was significantly reduced in AD patients' hippocampus and parahippocampal gyrus compared to NCs. Global amyloid deposition was positively associated with mGluR5 availability in the AD group and reversely associated in the NC group. The mGluR5 availability was positively correlated with regional glucose metabolism in the overall and stratified analyses. The availability of mGluR5 in the hippocampus and parahippocampal gyrus demonstrated a strong relationship with the GMV of the medial temporal lobe, plasma p-tau181 or NfL levels, and global cognitive performance. CONCLUSIONS: [18F]PSS232 PET can quantify the changes of mGluR5 availability in the progression of AD. mGluR5 availability correlated not only with neuropathological biomarkers of AD but also with neurodegenerative biomarkers and cognitive performance. mGluR5 may be a novel neurodegenerative biomarker, and whether mGluR5 could be a potential therapeutic target for AD needs to be further studied.

Identification of (R)-[18F]YH134 for Monoacylglycerol Lipase Neuroimaging and Exploration of Its Use for Central Nervous System and Peripheral Drug Development.

This study aimed to evaluate (R)-[18F]YH134 as a novel PET tracer for imaging monoacylglycerol lipase (MAGL). Considering the ubiquitous expression of MAGL throughout the whole body, the impact of various MAGL inhibitors on (R)-[18F]YH134 brain uptake and its application in brain-periphery crosstalk were explored. Methods: MAGL knockout and wild-type mice were used to evaluate (R)-[18F]YH134 in in vitro autoradiography and PET experiments. To explore the impact of peripheral MAGL occupancy on (R)-[18F]YH134 brain uptake, PET kinetics with an arterial input function were studied in male Wistar rats under baseline and blocking conditions. Results: In in vitro autoradiography, (R)-[18F]YH134 revealed a heterogeneous distribution pattern with high binding to MAGL-rich brain regions in wild-type mouse brain slices, whereas the radioactive signal was negligible in MAGL knockout mouse brain slices. The in vivo brain PET images of (R)-[18F]YH134 in wild-type and MAGL knockout mice demonstrated its high specificity and selectivity in mouse brain. A Logan plot with plasma input function was applied to estimate the distribution volume (V T) of (R)-[18F]YH134. V T was significantly reduced by a brain-penetrant MAGL inhibitor but was unchanged by a peripherally restricted MAGL inhibitor. The MAGL target occupancy in the periphery was estimated using (R)-[18F]YH134 PET imaging data from the brain. Conclusion: (R)-[18F]YH134 is a highly specific and selective PET tracer with favorable kinetic properties for imaging MAGL in rodent brain. Our results showed that blocking of the peripheral target influences brain uptake but not the V T of (R)-[18F]YH134. (R)-[18F]YH134 can be used for estimating the dose of MAGL inhibitor at half-maximal peripheral target occupancy.

Radiolabeling and in vitro /in vivo evaluation of N-(1-adamantyl)-8-methoxy-4-oxo-1-phenyl-1,4-dihydroquinoline-3-carboxamide as a PET probe for imaging cannabinoid type 2 receptor.

The cannabinoid type 2 (CB2) receptor plays an important role in neuroinflammatory and neurodegenerative diseases such as multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimer's disease and is therefore a very promising target for therapeutic approaches as well as for imaging. Based on the literature, we identified one 4-oxoquinoline derivative(designated KD2) as the lead structure. It was synthesized, radiolabeled and evaluated as a potential imaging tracer for CB2. [11C]KD2 was obtained in 99% radiochemical purity.Moderate blood­brain barrier (BBB) passage was predicted for KD2 from an in vitro transport assay with P-glycoprotein-transfected Madin Darby canine kidney cells. No efflux of KD2 by P-glycoprotein was detected. In vitro autoradiography of rat and mouse spleen slices demonstrated that [11C]KD2 exhibits high specific binding towards CB2. High spleen uptake of [11C]KD2 was observed in dynamic positron emission tomography(PET) studies with Wistar rats and its specificity was confirmed by displacement study with a selective CB2 agonist, GW405833. A pilot autoradiography study with post-mortem spinal cord slices from amyotrophic lateral sclerosis (ALS)patients with [11C]KD2 suggested the presence of CB2 receptors under disease conditions. Specificity of [11C]KD2 binding could also be demonstrated on these human tissues. In conclusion, [11C]KD2 shows good in vitro and in vivo properties as a potential PET tracer for CB2.