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Purpose [18F]-labeled positron emission tomography (PET) radioligands permit in vivo assessment of Alzheimer’s disease biomarkers, including aggregated neurofibrillary tau (NFT) with [18F]flortaucipir. Due to structural similarities of flortaucipir with some monoamine oxidase A (MAO-A) inhibitors, this study aimed to evaluate flortaucipir binding to MAO-A and MAO-B and any potential impact on PET interpretation. Methods [18F]Flortaucipir autoradiography was performed on frozen human brain tissue slices, and PET imaging was conducted in rats. Dissociation constants were determined by saturation binding, association and dissociation rates were measured by kinetic binding experiments, and IC50 values were determined by competition binding. Results Under stringent wash conditions, specific [18F]flortaucipir binding was observed on tau NFT-rich Alzheimer’s disease tissue and not control tissue. In vivo PET experiments in rats revealed no evidence of [18F]flortaucipir binding to MAO-A; pre-treatment with MAO inhibitor pargyline did not impact uptake or wash-out of [18F]flortaucipir. [18F]Flortaucipir bound with low nanomolar affinity to human MAO-A in a microsomal preparation in vitro but with a fast dissociation rate relative to MAO-A ligand fluoroethyl-harmol, consistent with no observed in vivo binding in rats of [18F]flortaucipir to MAO-A. Direct binding of flortaucipir to human MAO-B was not detected in a microsomal preparation. A high concentration of flortaucipir (IC50 of 1.3 μM) was found to block binding of the MAO-B ligand safinamide to MAO-B on microsomes suggesting that, at micromolar concentrations, flortaucipir weakly binds to MAO-B in vitro. Conclusion These data suggest neither MAO-A nor MAO-B binding will contribute significantly to the PET signal in cortical target areas relevant to the interpretation of [18F]flortaucipir.

INTRODUCTION: Sphingosine-1-phosphate receptor 2 (S1PR2) activation exerts a critical role in biological abnormalities and diseases. A suitable radiotracer will advance our understanding of S1PR2 pathophysiology of diseases. The objective of this study is to evaluate the potential of iodine-125 labeled [(125)I]TZ6544 to be used for screening new compounds binding toward S1PR2, and assessing the changes of S1PR2 expression in the kidney of streptozotocin-induced diabetic rats. METHODS: [(125)I]TZ6544 was synthesized from borate precursor by copper (II)-catalyzed iodization reaction with [(125)I]NaI. [(125)I]TZ6544 was characterized using human recombinant S1PR2 cell membrane and biodistribution studies of [(125)]TZ6544 were performed on Wistar rats that were euthanized at 5 and 30 minutes post-injection. A rat model of diabetes was induced by IV injection of streptozotocin (55 mg/kg). In vitro autoradiography studies, immunostaining, and enzyme-linked immunosorbent assay (ELISA) analysis were performed in both diabetic and control rats. RESULTS: Radiosynthesis of [(125)I]TZ6544 was achieved successfully with good radiochemical yields of ~47% and high radiochemical purity of >99%. [(125)I]TZ6544 is a potent ligand in vitro for S1PR2 with K(d) value of 4.31 nM. [(125)I]TZ6544 and [(32)P]-labelled endogenous S1P provided comparable IC(50) values in radioactive competitive binding assays against known S1PR2 ligands. Compared to control, the kidney of diabetic rats had increased uptake of [(125)I]TZ6544, which could be reduced by a S1PR2 antagonist, JTE-013. Immunostaining and ELISA analysis confirmed that the diabetic rat had increased S1PR2 expression in the kidney. CONCLUSIONS: [(125)I]TZ6544 was synthesized successfully in high yields, and in vitro evaluation suggested [(125)I]TZ6544 has high potential to be used for screening new S1PR2 compounds and investigating the pathophysiology of S1PR2 functions. The available of [(125)I]TZ6544 may facilitate the development of therapeutics and imaging agents targeting S1PR2. ADVANCES IN KNOWLEDGE: [(125)I]TZ6544 showed increased expression of S1PR2 in diabetic rat kidney and can be used to determine binding potency of S1PR2 compounds.

Receptor-interacting protein kinase 1 (RIPK1) has emerged as a crucial regulator of necroptosis and the inflammatory response by activating a group of downstream immune receptors. It has been recognized as a pivotal contributor to cell death and inflammation in various physiological and pathological processes. RIPK1 deficiency or dysregulation in humans can cause severe immunodeficiency and neurodegenerative diseases such as multiple sclerosis and amyotrophic lateral sclerosis. Recently, diverse structures of RIPK1 inhibitors have been developed as potential therapeutics for neurodegenerative diseases and other pathological inflammatory processes. 7-oxo-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine (Compound 5 or TZ7774) was reported as a novel RIPK1 inhibitor with a KSynthesis of the desmethyl precursor TZ7790 was performed and optimized. The radiosynthesis of [The radiosynthesis of [

[[Under stringent wash conditions, specific [These data suggest neither MAO-A nor MAO-B binding will contribute significantly to the PET signal in cortical target areas relevant to the interpretation of [

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.

The nonselective Ca2+ -permeable transient receptor potential channel subfamily member 5 (TRPC5) belongs to the transient receptor potential canonical (TRPC) superfamily and is widely expressed in the brain. Compelling evidence reveals that TRPC5 plays crucial roles in depression and other psychiatric disorders. To develop a TRPC5 radioligand, following up on our previous effort, we synthesized the iodine compound TZ66127 and its iodine-125-labeled counterpart [125 I]TZ66127. The synthesis of TZ66127 was achieved by replacing chloride with iodide in the structure of HC608, and the [125 I]TZ66127 was radiosynthesized using its corresponding tributylstannylated precursor. We established a stable human TRPC5-overexpressed HEK293-hTRPC5 cell line and performed Ca2+ imaging and a cell-binding assay study of TZ66127; these indicated that TZ66127 had good inhibition activity for TRPC5, and the inhibitory efficiency of TZ66127 toward TRPC5 presented in a dose-dependent manner. An in vitro autoradiography and immunohistochemistry study of rat brain sections suggested that [125 I]TZ66127 had binding specificity toward TRPC5. Altogether, [125 I]TZ66127 has high potential to serve as a radioligand for screening the binding activity of other new compounds toward TRPC5. The availability of [125 I]TZ66127 might facilitate the development of therapeutic drugs and PET imaging agents that target TRPC5.

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 [18F]VAT to target VAChT in vivo with high affinity and selectivity. Here we report in vitro characterization of [3H]VAT, a tritiated counterpart of [18F]VAT. Using human VAChT-rich cell membrane extracts, a saturated binding curve was obtained for [3H]VAT with Kd = 6.5 nM and Bmax = 22.89 pmol/mg protein. In the [3H]VAT competition-binding assay with a panel of CNS ligands, binding inhibition of [3H]VAT was observed using VAChT ligands, the Ki values ranged from 5.41 to 33.3 nM. No inhibition was detected using a panel of other CNS ligands. In vitro [3H]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 [3H]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 [3H]VAT in caudate and putamen were blocked significantly by either VAChT ligand TZ659 or (−)-vesamicol, but not by the σ1 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 [18F]VAT as a suitable PET radiotracer for quantitative assessment of VAChT levels in vivo.

The transient receptor potential channels 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) combining 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 [(11)C]methyl iodide. Under optimized conditions, [(11)C]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). In vitro autoradiography study reveals that [(11)C]HC608 specifically binds to TRPC5. Moreover, initial in vivo evaluation of [(11)C]HC608 performed in rodents and microPET study in the brain of non-human primates further deomonstrate that [(11)C]HC608 was able to penetrate the blood brain barrier and sufficiently accumulate in the brain. These results suggest that [(11)C]HC608 has potential to be a PET tracer for imaging TRPC5 in vivo.

Nine new compounds targeting on transient receptor potential vanilloid-4 (TRPV4) were synthesized and their biological activities toward TRPV4 were determined using freshly isolated mouse skin macrophages through live cell Ca(2+) imaging assay. Three compounds 4b, 4c, and 4i exhibit higher percentages of activating TRPV4 in vitro as 48.1%, 59.3% and 33.5%, comparable with 56.4 % activation response of a reported TRPV4 agonist GSK1016790A (3). The compound 4i was chosen for (11)C-radiosynthesis using its phenol precursor 4g to react with [(11)C]methyl iodide. The radiosynthesis was achieved with good radiochemical yield (16 ± 5%), high chemical and radiochemical purity (> 95%), and high molar activity (16-21 GBq/μmol, decay corrected to the end of bombardment, EOB n ≥ 4). Furthermore, the initial ex vivo biodistribution study showed that [(11)C]4i had relative higher uptakes the kidney, liver and small intestine compared to other tissues and quick washout from the animal body.