Background Parkinson's disease (PD) and multiple system atrophy (MSA) are considered α-synucleinopathies, characterized by the presence of pathological α-synuclein (α-syn) aggregates. A positron emission tomography (PET) tracer for imaging α-syn aggregates in vivo is highly sought after, as disease progression correlates with the accumulation of aggregated α-syn. We recently reported [ 18 F]asyn-44 as a radiotracer for α-syn, worthy of evaluation in higher species, based on in vitro binding data from human brain tissues and in vivo PET imaging studies in rodents. Objective [ 3 H]ACI-12589 is a promising α-syn PET tracer which recently showed binding in MSA patients but appears to have limited utility in other α-synucleinopathies. Objective 1) compare the in vitro binding properties of our lead, [ 3 H]asyn-44, to [ 3 H]ACI-12589; Objective 2) evaluate [ 18 F]asyn-44 and [ 18 F]ACI-12589 kinetics by in vivo PET imaging in normal rodents; Objective 3) assess pharmacokinetic properties and metabolism of [ 18 F]asyn-44 in normal pig and non-human primate (NHP). Methods In vitro autoradiography with [ 3 H]asyn-44 and [ 3 H]ACI-12589 was performed to compare radiotracer binding in PD, MSA, Alzheimer's disease and healthy control post-mortem brain tissue. Additionally, preclinical PET imaging was performed in rats with [ 18 F]ACI-12589 to compare with our previously reported [ 18 F]asyn-44 data. Further evaluation of [ 18 F]asyn-44 in higher species was carried out by preclinical PET imaging in pig and NHP with metabolite analysis. Liver microsome assays and mass spectrometry were performed to identify the metabolites formed in NHP. Results [ 3 H]Asyn-44 and [ 3 H]ACI-12589 displayed different binding properties in both PD and MSA tissue, suggesting that the tracers target different binding sites and asyn-44 might therefore be more suited for PD imaging. In the pig, [ 18 F]asyn-44 readily entered the brain and no brain penetrant metabolites were observed in arterial blood samples. In the NHP, [ 18 F]asyn-44 readily entered the brain but was rapidly metabolized. Radiolabeled metabolites of asyn-44 were proposed and will be considered in the design of future derivatives. Conclusions Species differences in metabolism of [ 18 F]asyn-44 are observed between pig and NHP, and do not support the further translation of [ 18 F]asyn-44. Additionally, autoradiography with [ 3 H]asyn-44 revealed low signal specificity and high non-displaceable binding. We report evidence for off-target binding of [ 3 H]ACI-12589 to amyloid-β plaques. The limitations of both [ 3 H]asyn-44 and [ 3 H]ACI-12589 reported here support the development of additional derivatives and structural scaffolds of asyn-44 with the potential to improve radiotracer specificity and selectivity towards α-syn.

The endocannabinoid system is a critical brain signaling pathway that is dysregulated in various brain disorders, including Alzheimer's disease (AD). Cannabinoid-targeted therapies and imaging approaches have gained increasing interest; however, the biological impact of the endocannabinoid system in disease needs further validation. We aimed to study changes in cannabinoid receptor 1 (CB1) and monoacylglycerol lipase (MAGL), components of endocannabinoid signaling and degradation, in a mouse model of AD by PET imaging. Methods: [18F]FMPEP-d 2 and [18F]MAGL-2102 were produced on a commercial radiosynthesis module. PET-CT images with both tracers were acquired in a knock-in mouse model of AD bearing mutated human amyloid precursor protein (AppNL-G-F ) at 3 ages, and compared to wild-type mice. Excised brains were used for in vitro autoradiography with [18F]FMPEP-d 2 and [18F]MAGL-2102, immunofluorescence, and western blotting. Male wild-type and 5xFAD mice were chronically treated with MAGL inhibitor JZL184 and imaged with [18F]MAGL-2102 two days after ending treatment. Results: PET imaging showed sex-, age- and genotype-dependent changes in CB1 and MAGL availability. At 4-months (early-stage β-amyloid pathology), female AppNL-G-F mice had lower CB1 availability, and MAGL availability was increased in male AppNL-G-F , compared to wild-types. At 8-months, no genotype differences in CB1 were observed, yet MAGL availability was reduced in AppNL-G-F frontal cortex, and male AppNL-G-F mice exhibited higher MAGL than transgenic females brain-wide. At 12-months (late-stage β-amyloid pathology), significantly lower uptake of [18F]FMPEP-d 2 was observed in AppNL-G-F compared to wild-type, with no changes in [18F]MAGL-2102 binding. AppNL-G-F plaque staging was confirmed by Thioflavin-S staining. Imaging findings were supplemented by autoradiography, immunofluorescence, and western blots. [18F]MAGL-2102 availability was responsive to target engagement of the MAGL inhibitor JZL184 in wild-type and 5xFAD mice. Conclusions: The present study showed dynamic age-, sex- and pathology-related changes in CB1 and MAGL availability from early-stage β-amyloid pathology, suggesting that the endocannabinoid system is a useful target for diagnostics and treatment of AD. Finally, these results highlight that endocannabinoid sex differences should be considered in diagnostics and drug development.

Aggregated alpha-synuclein (alpha-syn) protein is a pathological hallmark of Parkinson's disease (PD) and Lewy body dementia (LBD). Development of positron emission tomography (PET) radiotracers to image alpha-syn aggregates has been a longstanding goal. This work explores the suitability of a pyridothiophene scaffold for alpha-syn PET radiotracers, where 47 derivatives of a potent pyridothiophene (asyn-44; K-d=1.85 nM) were synthesized and screened against [H-3]asyn-44 in competitive binding assays using post-mortem PD brain homogenates. Equilibrium inhibition constant (K-i) values of the most potent compounds were determined, of which three had K-i ' s in the lower nanomolar range (12-15 nM). An autoradiography study confirmed that [H-3]asyn-44 is promising for imaging brain sections from multiple system atrophy and PD donors. Fluorine-18 labelled asyn-44 was synthesized in 6 +/- 2 % radiochemical yield (decay-corrected, n=5) with a molar activity of 263 +/- 121 GBq/mu mol. Preliminary PET imaging of [F-18]asyn-44 in rats showed high initial brain uptake (>1.5 standardized uptake value (SUV)), moderate washout (similar to 0.4 SUV at 60 min), and low variability. Radiometabolite analysis showed 60-80 % parent tracer in the brain after 30 and 60 mins. While [F-18]asyn-44 displayed good in vitro properties and acceptable brain uptake, troublesome radiometabolites precluded further PET imaging studies. The synthesis and in vitro evaluation of additional pyridothiophene derivatives are underway, with the goal of attaining improved affinity and metabolic stability.

Positron emission tomography (PET) is a powerful imaging tool for drug discovery, clinical diagnosis, and monitoring of disease progression. Fluorine-18 is the most common radionuclide used for PET, but advances in radiotracer development have been limited by the historical lack of methodologies and precursors amenable to radiolabeling with fluorine-18. Radiolabeling of electron-rich (hetero)aromatic rings remains a long-standing challenge in the production of PET radiopharmaceuticals. In this personal account, we discuss the history of spirocyclic iodonium ylide precursors, from inception to applications in clinical research, for the incorporation of fluorine-18 into complex non-activated (hetero)aromatic rings.

Positron emission tomography (PET) is a powerful tool for imaging biological processes in the central nervous system (CNS). Designing PET radiotracers capable of crossing the blood-brain barrier (BBB) remains a major challenge. In addition to being brain-penetrant, a quantifiable CNS PET radiotracer must have high target affinity and selectivity, appropriate pharmacokinetics, minimal non-specific binding, negligible radiometabolites in the brain, and generally must be amenable to labeling with carbon-11 (11 C) or fluorine-18 (18 F). This review aims to give an overview of some of the critical physicochemical and biochemical contributors specific for CNS PET radiotracer design and how they can differ from pharmaceutical drug development, including in vitro assays, in silico predictions, and in vivo studies, with examples for how such methods can be implemented to optimize brain uptake of radiotracers based on experiences from our neuroimaging program.

Positron emission tomography (PET) is a molecular imaging technique that makes use of radiolabelled molecules for in vivo evaluation. Carbon-11 is a frequently used radionuclide for the labelling of small molecule PET tracers and can be incorporated into organic molecules without changing their physicochemical properties. While the short half-life of carbon-11 (11C; t½ = 20.4 min) offers other advantages for imaging including multiple PET scans in the same subject on the same day, its use is limited to facilities that have an on-site cyclotron, and the radiochemical transformations are consequently more restrictive. Many researchers have embraced this challenge by discovering novel carbon-11 radiolabelling methodologies to broaden the synthetic versatility of this radionuclide. This review presents new carbon-11 building blocks and radiochemical transformations as well as PET tracers that have advanced to first-in-human studies over the past five years.

Abstract ID 129967Poster Board 537Background: Several conditions, including schizophrenia (SCZ), are believed to be related to dysregulation in dopamine (DA) signalling. Furthermore, the endocannabinoid system (ECS) is suggested to be dysregulated in DA pathologies: biosynthesis pathway of 2-arachidnoylglycerol (2-AG), a major endocannabinoid neurotransmitter, shown to be altered in SCZ; DAGL (2-AG synthesis) levels decreased; MAGL (primary 2-AG metabolism) expression levels significantly lower; elevated 2-AG observed. Despite mixed findings, elevation of 2-AG is coveted in certain contexts; clinical trials of MAGL inhibitors (MAGLi) currently underway for PTSD and Tourette syndrome. Evidence suggests that increasing 2-AG might be detrimental in hyperDA pathologies. It’s imperative to understand MAGLi in vulnerable populations, and whether decreasing 2-AG is therapeutic. Therefore, we assessed pre-clinical effects of modulating 2-AG in two models of hyperDA; based on well-established associations between psychopathologies and increased subcortical dopamine.Methods: Genetic (adult DAT-knockout (DATKO) and pharmacological (C57Bl/6J with amphetamine) models of hyperDA were treated acutely with a MAGLi (MJN110, 5mg/kg) or DAGLi (DO34, 30mg/kg), and tested on several behavioural assays. Lipidomic and molecular analyses were completed (striatal brain samples), and partial correlation networks were generated. Using the novel positron emission tomography (PET) radiotracer for MAGL, [18F]MAGL-2102, and an established radiotracer for imaging the cannabinoid receptor type-1 (CB1), [18F]FMPEP-d2, we interrogated the status of MAGL and CB1 in vivo in hyperDA states, comparing DATKO vs WT littermate controls. Data analyzed using three-way ANOVA (behaviour), Student’s t-test (lipidomics), and repeated measures ANOVA for PET quantification (with appropriate post hoc analyses for all tests).Results: DATKO show exploratory hyperactivity, impaired sensorimotor gating, blunted response to psychostimulants, and disrupted lipid profiles. Brain uptake of [18F]MAGL-2102 (male > female) was similarly and significantly decreased in both sexes in DATKO (whole brain AUC: -21% and -17% in female and male DATKO, respectively). [18F]FMPEP-d2 (CB1), on the other hand, showed the opposite sex-dependent binding in WT (female > male), with a sex-dependent significant decrease in tracer uptake in female DATKO (-27% in whole brain AUC), but not males (-11%, non-significant). When treated with a MAGLi, DATKO showed exacerbation of hyperlocomotion, sensorimotor deficits, and further disruption of lipid networks. MAGLi increased reward association in DATKO, but not WT, suggesting an addiction liability in certain populations. MAGLi effects weren’t limited to DATKO; it exacerbated psychostimulant responses in C57BL/6J. Data suggests that increasing 2-AG via MAGLi exacerbates states of hyperdopaminergia, mediated by CB1. Interestingly, decreasing 2-AG synthesis (via DAGLi) presented opposite effects on all measured hyperdopaminergic behavioural outputs in both DATKO and C57BL/6J.Conclusion: Present study demonstrates profound brain-region specific remodelling of 2-AG in hyperDA states. The work highlights hitherto unrecognized potential for detrimental effects of MAGLi in certain disease states. It also revealed a potential therapeutic for hyperDA pathologies by reducing 2-AG synthesis via DAGLi.

(3R,5R)-5-(3-([F-18]fluoromethoxy-d(2))phenyl)-3-(((R)-1-phenylethyl)amino)-1-(4-(trifluoromethyl)phenyl)pyrrolidin-2-one ([F-18]FMPEP-d(2)) is a promising positron emission tomography (PET) radiopharmaceutical for the imaging of cannabinoid type 1 receptors in human studies. To facilitate widespread use of [F-18]FMPEP-d(2) we herein report a simplified one-pot synthesis procedure that is broadly applicable for F-18-fluoromethylation of phenols and can be applied for routine clinical production using a commercial radiofluorination module (GE TRACERlab FX2 N). The present method overcomes previous challenges in the [F-18]FMPEP-d(2) synthesis related to intermediate purification of a [F-18]fluoromethyl building block by using ditosylmethane-d(2) and reacting it directly with (3R,5R)-5-(3-hydroxyphenyl)-3-[(R)-1-phenylethylamino]-1-(4-trifluoromethylphenyl)pyrrolidine-2-one in a one-pot nucleophilic reaction with [F-18]fluoride (K2CO3, K-222, CH3CN, 80 degrees C, 10 min). After purification of the product by semi-preparative HPLC under isocratic conditions and formulation, [F-18]FMPEP-d(2) was obtained in a decay-corrected radiochemical yield of 8 +/- 1 %, a radiochemical purity >95 % and a molar activity of 322 +/- 101 GBq/mu mol in a synthesis time of 70 +/- 5 min (n = 8). Validation and regulatory submission of [F-18]FMPEP-d(2) is underway with the new methodology and will facilitate widespread human use as well as multi-center clinical trials.

The trifluoromethyl group is a prominent motif in biologically active compounds and therefore of great interest for the labeling with the positron emitter fluorine-18 for positron emission tomography (PET) imaging. Multiple labeling strategies have been explored in the past; however, most of them suffer from low molar activity due to precursor degradation. In this study, the potential of 1-(difluoromethyl)-3-methyl-4-phenyl-1H-1,2,3-triazol-3-ium triflate as precursor for the synthesis of the [F-18]trifluoromethylation building block [F-18]fluoroform with high molar activity was investigated. The triazolium precursor was reacted under various conditions with [F-18]fluoride, providing [F-18]fluoroform with radiochemical yields (RCY) and molar activities (A(m)) comparable and even superior with already existing methods. Highest molar activities (A(m) = 153 +/- 14 GBq/mu mol, dc, EOS) were observed for the automated procedure on the Neptis (R) perform module. Due to its easy handling and good RCY and A(m) in the [F-18]fluoroform synthesis, the triazolium precursor is a valuable alternative to already known precursors.

Introduction: Parathyroid hyperplasia is a disease characterized by overactive parathyroid glands secreting increased levels of parathyroid hormone. Surgical removal of the parathyroid glands is the standard treatment but requires precise pre-operative localization of the glands. However, currently available imaging modalities show limited sensitivity. Since positron emission tomography (PET) is a molecular imaging technique with high accuracy and sensitivity, our aim was to develop a new PET tracer for overactive parathyroid glands imaging by radiolabelling cinacalcet, a drug binding to the calcium-sensing receptor of the parathyroid glands. Methods: [F-18]Cinacalcet was synthesized by copper-catalysed [F-18]trifluoromethylation of a boronic acid precursor using high molar activity [F-18]fluoroform. Ex vivo biodistribution and metabolism were evaluated in 12 healthy male Wistar rats at 5, 15, 45 and 90 min. PET scans were performed at baseline and after blocking with NPS R-568. Results: [F-18]Cinacalcet was obtained in an overall radiosynthesis time of 1 h with a radiochemical purity of 98 +/- 1%, a radiochemical yield of 8 +/- 4% (overall, n=7, corrected for decay) and a molar activity of 40 +/- 11 GBq/mu mol (n = 7, at EOS). The ex vivo biodistribution showed uptake in the thyroid and parathyroid glands as well as in other glands such as adrenals, salivary glands and pancreas. The tracer was rapidly cleared from the blood via liver and kidneys and showed fast metabolism. PET images confirmed uptake in the target organ. However, in a blocking study with NPS R-568 specific binding of [F-18]cinacalcet to the CaSR could not be confirmed. Conclusions: [F-18]Cinacalcet was successfully synthesized. First in vivo experiments in healthy rats showed uptake of the tracer in the target organ and fast metabolism, encouraging further in vivo evaluation of this tracer. (c) 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

This article describes the first synthesis and application of fluorine-18 labelled Ruppert-Prakash reagent [18F]Me3SiCF3. [18F]Me3SiCF3 was synthesized from [18F]fluoroform with radiochemical yields of 85-95% and radiochemical purities of >95% within 20 minutes. 18F-trifluoromethylated compounds were successfully prepared by reaction of [18F]Me3SiCF3 with benzaldehydes, acetophenones and benzophenones.

Fluoroform is an interesting motif for the isotopologue labelling of biologically active compounds with fluorine‐18 for PET imaging. However, so far the building block [ 18 F]fluoroform and consequently the [ 18 F]trifluoromethylated products suffered from low molar activities ranging from 0.1 to 30 GBq/µmol due to isotopic dilution under the strongly basic standard radiofluorination conditions. In this article the synthesis of high molar activity [ 18 F]fluoroform is described. By implementing a recently reported novel radiofluorination reagent, [ 18 F]triflyl fluoride, the concentration of base‐cryptand complex in the reaction could be reduced 100‐fold compared to standard radiofluorination conditions and molar activities close to 100 GBq/µmol (at end of [ 18 F]fluoroform synthesis) could be obtained, enabling the imaging of low density receptors. Furthermore, an automated procedure was developed on the commercially available NEPTIS® perform synthesizer to provide access of high molar activity [ 18 F]fluoroform to other PET centres.