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The sphingosine-1-phosphate receptor 1 (S1PR1) radiotracer [ 11 C] CS1P1 has shown promise in proof-of-concept PET imaging of neuroinflammation in multiple sclerosis (MS). Our HPLC radiometabolite analysis of human plasma samples collected during PET scans with [ 11 C] CS1P1 detected a radiometabolite peak that is more lipophilic than [ 11 C] CS1P1 . Radiolabeled metabolites that cross the blood-brain barrier complicate quantitative modeling of neuroimaging tracers; thus, characterizing such radiometabolites is important. Here, we report our detailed investigation of the metabolite profile of [ 11 C] CS1P1 in rats, nonhuman primates, and humans. CS1P1 is a fluorine-containing ligand that we labeled with C-11 or F-18 for preclinical studies; the brain uptake was similar for both radiotracers. The same lipophilic radiometabolite found in human studies also was observed in plasma samples of rats and NHPs for CS1P1 labeled with either C-11 or F-18. We characterized the metabolite in detail using rats after injection of the nonradioactive CS1P1 . To authenticate the molecular structure of this radiometabolite, we injected rats with 8 mg/kg of CS1P1 to collect plasma for solvent extraction and HPLC injection, followed by LC/MS analysis of the same metabolite. The LC/MS data indicated in vivo mono-oxidation of CS1P1 produces the metabolite. Subsequently, we synthesized three different mono-oxidized derivatives of CS1P1 for further investigation. Comparing the retention times of the mono-oxidized derivatives with the metabolite observed in rats injected with CS1P1 identified the metabolite as N -oxide 1 , also named TZ82121 . The MS fragmentation pattern of N -oxide 1 also matched that of the major metabolite in rat plasma. To confirm that metabolite TZ82121 does not enter the brain, we radiosynthesized [ 18 F] TZ82121 by the oxidation of [ 18 F] FS1P1 . Radio-HPLC analysis confirmed that [ 18 F] TZ82121 matched the radiometabolite observed in rat plasma post injection of [ 18 F] FS1P1 . Furthermore, the acute biodistribution study in SD rats and PET brain imaging in a nonhuman primate showed that [ 18 F] TZ82121 does not enter the rat or nonhuman primate brain. Consequently, we concluded that the major lipophilic radiometabolite N -oxide [ 11 C] TZ82121 , detected in human plasma post injection of [ 11 C] CS1P1 , does not enter the brain to confound quantitative PET data analysis. [ 11 C] CS1P1 is a promising S1PR1 radiotracer for detecting S1PR1 expression in the CNS.

Sphingosine-1-phosphate receptor 1 (S1PR1) is recognized as a novel therapeutic and diagnostic target in neurological disorders. We recently transferred the S1PR1 radioligand [ 11 C] CS1P1 into clinical investigation for multiple sclerosis. Herein, we reported the design, synthesis and evaluation of novel F-18 S1PR1 radioligands. We combined the structural advantages of our two lead S1PR1 radioligands and synthesized 14 new S1PR1 compounds, then performed F-18 radiochemistry on the most promising compounds. Compound 6h is potent (IC 50 = 8.7 nM) and selective for S1PR1. [ 18 F] 6h exhibited a high uptake in macaque brain (SUV > 3.0) and favorable brain washout pharmacokinetics in positron emission tomography (PET) study. PET blocking and displacement studies confirmed the specificity of [ 18 F] 6h in vivo . Radiometabolite analysis confirmed no radiometabolite of [ 18 entered into the brain to confound the PET measurement. In summary, [ 6h entered into the brain to confound the PET measurement. In summary, [ 18 is a promising radioligand to image S1PR1 and worth translational clinical investigation for humans with brain disorders.6h is a promising radioligand to image S1PR1 and worth translational clinical investigation for humans with brain disorders.

Purpose: TRPC5 belongs to the mammalian superfamily of transient receptor potential (TRP) Ca 2+ -permeable cationic channels and it has been implicated in various CNS disorders. As part of our ongoing interest in the development of a PET radiotracer for imaging TRPC5, herein, we explored the radiosynthesis, and in vitro and in vivo evaluation of a new C-11 radiotracer [ 11 C]HC070 in rodents and nonhuman primates., Procedures: [ 11 C]HC070 was radiolabeled utilizing the corresponding precursor and [ 11 C]CH 3 I via N-methylation protocol. Ex vivo biodistribution study of [ 11 C]HC070 was performed in Sprague-Dawley rats. In vitro autoradiography study was conducted for the rat brain sections to characterize the radiotracer distribution in the brain regionals. MicroPET brain imaging studies of [ 11 C]HC070 were done for 129S1/SvImJ wild-type mice and 129S1/SvImJ TRPC5 knockout mice for 0-60-min dynamic data acquisition after intravenous administration of the radiotracer. Dynamic PET scans (0-120 min) for the brain of cynomolgus male macaques were performed after the radiotracer injection., Results: [ 11 C]HC070 was efficiently prepared with good radiochemical yield (45 ± 5%, n = 15), high chemical and radiochemical purity (> 99%), and high molar activity (320.6 ± 7.4 GBq/μmol, 8.6 ± 0.2 Ci/μmol) at the end of bombardment (EOB). Radiotracer [ 11 C]HC070 has good solubility in the aqueous dose solution. The ex vivo biodistribution study showed that [ 11 C]HC070 had a quick rat brain clearance. Autoradiography demonstrated that [ 11 C]HC070 specifically binds to TRPC5-enriched regions in rat brain. MicroPET study showed the peak brain uptake (SUV value) was 0.63 in 129S1/SvImJ TRPC5 knockout mice compared to 1.13 in 129S1/SvImJ wild-type mice. PET study showed that [ 11 C]HC070 has good brain uptake with maximum SUV of ~ 2.2 in the macaque brain, followed by rapid clearance., Conclusions: Our data showed that [ 11 C]HC070 is a TRPC5-specific radiotracer with high brain uptake and good brain washout pharmacokinetics in both rodents and nonhuman primates. The radiotracer is worth further investigating of its suitability to be a PET radiotracer for imaging TRPC5 in animals and human subjects in vivo., (© 2022. The Author(s), under exclusive licence to World Molecular Imaging Society.)

Vesicular acetylcholine transporter plays a crucial role in the cholinergic system, and its alterations is implicated in several neurodegenerative disorders. We recently developed a PET imaging tracer [ 18 F]VAT to target VAChT in vivo with high affinity and selectivity. Here we report in vitro characterization of [ 3 H]VAT, a tritiated counterpart of [ 18 F]VAT. Using human VAChT-rich cell membrane extracts, a saturated binding curve was obtained for [ 3 H]VAT with K d  = 6.5 nM and B max  = 22.89 pmol/mg protein. In the [ 3 H]VAT competition-binding assay with a panel of CNS ligands, binding inhibition of [ 3 H]VAT was observed using VAChT ligands, the K i values ranged from 5.41 to 33.3 nM. No inhibition was detected using a panel of other CNS ligands. In vitro [ 3 H]VAT autoradiography of rat brain sections showed strong signals in the striatum, moderate to high signals in vermis, thalamus, cortex, and hippocampus, and weak signals in cerebellum. Strong [ 3 H]VAT ARG signals were also observed from striatal sections of normal nonhuman primates and human brains. Competitive ARG study with human striatal sections demonstrated strong ARG signals of [ 3 H]VAT in caudate and putamen were blocked significantly by either VAChT ligand TZ659 or (-)-vesamicol, but not by the σ 1 receptor ligand Yun-122. ARG study also indicated that signal in the striatal sections from PSP human brains was lower than normal human brains. These data provide solid evidence supporting [ 18 F]VAT as a suitable PET radiotracer for quantitative assessment of VAChT levels in vivo., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The transient receptor potential channel subfamily member 5 (TRPC5) is a calcium permeable cation channel widely expressed in the brain. Accumulating evidence indicates that it plays a crucial role in psychiatric disorders including depression and anxiety. Positron emission tomography (PET) combined with a TRPC5 specific radioligand may provide a unique tool to investigate the functions of TRPC5 in animal disease models to guide drug development targeting TRPC5. To develop a TRPC5 PET radiotracer, the potent TRPC5 inhibitor HC608 was chosen for C-11 radiosynthesis through the N-demethyl amide precursor 7 reacting with [11C]methyl iodide. Under optimized conditions, [11C]HC608 was achieved with good radiochemical yield (25 ± 5%), high chemical and radiochemical purity (>99%), and high specific activity (204-377 GBq μmol-1, decay corrected to the end of bombardment, EOB). The in vitro autoradiography study revealed that [11C]HC608 specifically binds to TRPC5. Moreover, initial in vivo evaluation of [11C]HC608 performed in rodents and the microPET study in the brain of non-human primates further demonstrated that [11C]HC608 was able to penetrate the blood brain barrier and sufficiently accumulate in the brain. These results suggest that [11C]HC608 has the potential to be a PET tracer for imaging TRPC5 in vivo.

Sixteen new sulfur-containing compounds targeting the vesicular acetylcholine transporter (VAChT) were synthesized and assessed for in vitro binding affinities. Enantiomers (-)-(1-(3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(4-(methylthio)phenyl)methanone [(-)-8] and (-)-(4-((2-fluoroethyl)thio)phenyl)(1-(3-hydroxy-1,2,3,4-tetrahydronaph-thalen-2-yl)piperidin-4-yl)methanone [(-)-14 a] displayed high binding affinities, with respective K i values of 1.4 and 2.2 nm for human VAChT, moderate and high selectivity for human VAChT over σ 1 (≈13-fold) and σ 2 receptors (>420-fold). Radiosyntheses of (-)-[ 11 C]8 and (-)-[ 18 F]14 a were achieved using conventional methods. Ex vivo autoradiography and biodistribution studies in Sprague-Dawley rats indicated that both radiotracers have the capacity to penetrate the blood-brain barrier, with high initial brain uptake at 5 min and rapid washout. The striatal region had the highest accumulation for both radiotracers. Pretreating the rats with the VAChT ligand (-)-vesamicol decreased brain uptake for both radiotracers. Pretreating the rats with the σ 1 ligand YUN-122 (N-(4-benzylcyclohexyl)-2-(2-fluorophenyl)acetamide) also decreased brain uptake, suggesting these two radiotracers also bind to the σ 1 receptor in vivo. The microPET study of (-)-[ 11 C]8 in the brain of a non-human primate showed high striatal accumulation that peaked quickly and washed out rapidly. Although preliminary results indicated these two sulfur-containing radiotracers have high binding affinities for VAChT with rapid washout kinetics from the striatum, their σ 1 receptor binding properties limit their potential as radiotracers for quantifying VAChT in vivo., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)