Radioactivity In Brain Research Articles

Imaging Mutant Huntingtin Aggregates using [18F]CHDI-650 PET in a preclinical model of Huntington’s disease

Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene (HTT) that encodes pathogenic mutant huntingtin (mHTT) protein. Therapeutic strategies aimed at lowering mHTT would benefit from non-invasive PET-based methods to evaluate treatment response. The CHDI foundation developed the first-generation PET ligands labelled with 11C, which advanced to clinical evaluation. Further optimization generated an 18F radioligand with improved metabolic stability, brain exposure and specificity, shown by PET imaging in wild-type mice and autoradiography in HD brain samples1,2. Here, we characterised the PET radioligand [18F]CHDI-650 in the R6/1 mouse model of HD to assess its sensitivity for monitoring treatment response of mHTT lowering therapies. [18F]CHDI-650 was produced according to an established method3 using a TRACERlab system. Radiochemical purity/molar activity of the final [18F]CHDI-650 product was detected by reverse-phase HPLC analysis. Dynamic PET imaging was conducted using R6/1 mice and wild-type littermates at various disease stages. Image processing and kinetic modelling (2TCM) analysis were performed using PMOD software. Ex vivo brain radioactivity was measured using gamma counting. Brains were cryo-sectioned and processed for immunofluorescence to identify HTT load. [18F]CHDI-650 was produced (1.3 ± 0.2 GBq) with an average radiochemical purity of 99.9 ± 0.1 % and a molar activity of 32.4 ± 5.2 GBq/µmol (n = 9). The radioligand was able to discriminate R6/1 HET from WT mice at an early presymptomatic age for both dynamic PET imaging (striatal) and ex vivo whole brain gamma. A well-defined age associated increase was also evident using both measures. Dual immunofluorescence spatially defined the level of aggregated HTT in striatal and hippocampal regions across the age groups. [18F]CHDI-650 PET shows great potential as a non-invasive tool for visualising the development of mHTT pathology and as a biomarker for preclinical efficacy studies. Please click on the 'PDF' for the full abstract!

A novel PET probe to selectively image heat shock protein 90α/β isoforms in the brain

BackgroundHeat shock proteins (HSPs) are present throughout the brain. They function as molecular chaperones, meaning they help with the folding and unfolding of large protein complexes. These chaperones are vital in the development of neuropathological conditions such as Alzheimer’s disease and Lewy body disease, with HSP90, a specific subtype of HSP, playing a key role. Many studies have shown that drugs that inhibit HSP90 activity have beneficial effects in the neurodegenerative diseases. Therefore, HSP90 PET imaging ligand can be used effectively to study HSP90 in neurodegenerative diseases. Among four HSP90 isoforms, two cytosolic isoforms (HSP90α and HSP90β) thought to be involved in the structural homeostasis of the proteins related to the neurodegenerative diseases. Currently, no useful PET imaging ligands selectively targeting the two cytosolic isoforms of HSP90 have been available yet.ResultsIn this study, we developed a novel positron emission tomography (PET) imaging ligand, [11C]BIIB021, by 11C-radiolabeling (a positron emitter with a half-life of 20.4 min) 6-Chloro-9-[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]-9H-purin-2-amine (BIIB021), an inhibitor with a high affinity for and selectivity to HSP90α and HSP90β. [11C]BIIB021 was synthesized with a high yield, molar activity and radiochemical purity. [11C]BIIB021 showed a high binding affinity for rat brain homogenate as well as human recombinant HSP90α and HSP90β proteins. Radioactivity was well detected in the rat brain (SUV 1.4). It showed clear specific binding in PET imaging of healthy rats and autoradiography of healthy rat and human brain sections. Radiometabolite was detected in the brain, however, total distribution volume was well quantified using dual-input graphical model. Inhibition of p-glycoprotein increased brain radioactivity concentrations. However, total distribution volume values with and without p-glycoprotein inhibition were nearly the same.ConclusionsWe have developed a new PET imaging agent, [11C]BIIB021, specifically targeting HSP90α/β. We have been successful in synthesizing [11C]BIIB021 and in vitro and in vivo imaging HSP90α/β. However, the quantification of HSP90α/β is complicated by the presence of radiometabolites in the brain and the potential to be a substrate for p-glycoprotein. Further efforts are needed to develop radioligand suitable for imaging of HSP90α/β.

Manipulation of Dietary DHA does not Alter DHA Brain Uptake Kinetics in Mice Despite Changing Brain and Plasma DHA Levelsvs

AbstractBackgroundIncreased brain docosahexaenoic acid (DHA) uptake reflects a compensatory mechanism for maintaining brain DHA homeostasis and can be captured through PET‐MRI scan uptake studies using radiolabeled DHA. It has been previously shown that APOE4 status and alcohol intake alter brain DHA uptake in mice. Whether a change in DHA intake affects the brain DHA incorporation coefficient (K*) however is not known.MethodFour‐month‐old C57BL/6J mice were fed either a diet rich in DHA (15 kcal% Fat, 1.7 g/kg ALA, and 7g/kg DHA) or deficient in DHA (15 kcal% Fat and 0.9 mg/kg ALA) for 4 months. At 8 months, anesthetized mice were subjected to dynamic PET‐MRI for 30 min following i.v. injection of 22‐[18F]fluorodocosahexaenoic acid ([18F]FDHA), prepared in‐house through radiosynthesis. Using an image‐derived cardiac input function, brain uptake was assessed by estimating [18F]FDHA K* values using both the Patlak model approach and the irreversible two‐tissue compartmental model (Irr2TCM) with and without correction of the brain radioactivity for radioactive spillover from the skull. Mice were then sacrificed, and the plasma and brains were analyzed using liquid chromatography‐mass spectrometry (LC‐MS) to evaluate differences in the unlabeled concentrations of fatty acids into phospholipids, triglycerides, and other lipids.ResultThere was no significant difference in the [18F]FDHA K* estimates (with either Patlak or Irr2TCM) between mice fed a DHA‐rich or a DHA‐deficient diet. LC‐MS based lipidomic analysis showed that the DHA‐rich diet significantly increased DHA concentration in triglycerides in plasma and in phosphatidylethanolamine in the brain.ConclusionThese results suggest that under physiological conditions, increased dietary intake of DHA in wild‐type mice increases DHA in brain phosphatidylethanolamine and in plasma triglycerides without changing the DHA K*. The net effect likely is increased brain DHA uptake, equal to the product of unesterified DHA plasma concentration and K*. Imaging brain DHA with dynamic [18F]FDHA PET‐MRI is a potentially useful tool not only to investigate the APOE4 phenotype, which promotes DHA dysregulation, but also to assess dietary and pharmacological interventions that affect brain DHA homeostasis as well as to guide future treatments.

11C]Paraoxon: Radiosynthesis, Biodistribution and In Vivo Positron Emission Tomography Imaging in Rat.

Synthesis of the acetylcholinesterase inhibitor paraoxon (POX) as a carbon-11 positron emission tomography tracer ([11C]POX) and profiling in live rats is reported. Naïve rats intravenously injected with [11C]POX showed a rapid decrease in parent tracer to ∼1%, with an increase in radiolabeled serum proteins to 87% and red blood cells (RBCs) to 9%. Protein and RBC leveled over 60 minutes, reflecting covalent modification of proteins by [11C]POX. Ex vivo biodistribution and imaging profiles in naïve rats had the highest radioactivity levels in lung followed by heart and kidney, and brain and liver the lowest. Brain radioactivity levels were low but observed immediately after injection and persisted over the 60-minute experiment. This showed for the first time that even low POX exposures (∼200 ng tracer) can rapidly enter brain. Rats given an LD50 dose of nonradioactive paraoxon at the LD50 20 or 60 minutes prior to [11C]POX tracer revealed that protein pools were blocked. Blood radioactivity at 20 minutes was markedly lower than naïve levels due to rapid protein modification by nonradioactive POX; however, by 60 minutes the blood radioactivity returned to near naïve levels. Live rat tissue imaging-derived radioactivity values were 10%-37% of naïve levels in nonradioactive POX pretreated rats at 20 minutes, but by 60 minutes the area under the curve (AUC) values had recovered to 25%-80% of naïve. The live rat imaging supported blockade by nonradioactive POX pretreatment at 20 minutes and recovery of proteins by 60 minutes. SIGNIFICANCE STATEMENT: Paraoxon (POX) is an organophosphorus (OP) compound and a powerful prototype and substitute for OP chemical warfare agents (CWAs) such as sarin, VX, etc. To study the distribution and penetration of POX into the central nervous system (CNS) and other tissues, a positron emission tomography (PET) tracer analog, carbon-11-labeled paraoxon ([11C]POX), was prepared. Blood and tissue radioactivity levels in live rats demonstrated immediate penetration into the CNS and persistent radioactivity levels in tissues indicative of covalent target modification.

Application of In vitro transcytosis models to brain targeted biologics.

The blood brain barrier (BBB) efficiently limits the penetration of biologics drugs from blood to brain. Establishment of an in vitro BBB model can facilitate screening of central nervous system (CNS) drug candidates and accelerate CNS drug development. Despite many established in vitro models, their application to biologics drug selection has been limited. Here, we report the evaluation of in vitro transcytosis of anti-human transferrin receptor (TfR) antibodies across human, cynomolgus and mouse species. We first evaluated human models including human cerebral microvascular endothelial cell line hCMEC/D3 and human colon epithelial cell line Caco-2 models. hCMEC/D3 model displayed low trans-epithelial electrical resistance (TEER), strong paracellular transport, and similar transcytosis of anti-TfR and control antibodies. In contrast, the Caco-2 model displayed high TEER value and low paracellular transport. Anti-hTfR antibodies demonstrated up to 70-fold better transcytosis compared to control IgG. Transcytosis of anti-hTfR.B1 antibody in Caco-2 model was dose-dependent and saturated at 3 μg/mL. Enhanced transcytosis of anti-hTfR.B1 was also observed in a monkey brain endothelial cell based (MBT) model. Importantly, anti-hTfR.B1 showed relatively high brain radioactivity concentration in a non-human primate positron emission tomography study indicating that the in vitro transcytosis from both Caco-2 and MBT models aligns with in vivo brain exposure. Typically, brain exposure of CNS targeted biologics is evaluated in mice. However, antibodies, such as the anti-human TfR antibodies, do not cross-react with the mouse target. Therefore, validation of a mouse in vitro transcytosis model is needed to better understand the in vitro in vivo correlation. Here, we performed transcytosis of anti-mouse TfR antibodies in mouse brain endothelial cell-based models, bEnd3 and the murine intestinal epithelial cell line mIEC. There is a good correlation between in vitro transcytosis of anti-mTfR antibodies and bispecifics in mIEC model and their mouse brain uptake. These data strengthen our confidence in the predictive power of the in vitro transcytosis models. Both mouse and human in vitro models will serve as important screening assays for brain targeted biologics selection in CNS drug development.

Development of a Novel [11C]CO-Labeled Positron Emission Tomography Radioligand [11C]BIO-1819578 for the Detection of O-GlcNAcase Enzyme Activity.

Imaging O-GlcNAcase OGA by positron emission tomography (PET) could provide information on the pathophysiological pathway of neurodegenerative diseases and important information on drug-target engagement and be helpful in dose selection of therapeutic drugs. Our aim was to develop an efficient synthetic method for labeling BIO-1819578 with carbon-11 using 11CO for evaluation of its potential to measure levels of OGA enzyme in non-human primate (NHP) brain using PET. Radiolabeling was achieved in one-pot via a carbon-11 carbonylation reaction using [11C]CO. The detailed regional brain distribution of [11C]BIO-1819578 binding was evaluated using PET measurements in NHPs. Brain radioactivity was measured for 93 min using a high-resolution PET system, and radiometabolites were measured in monkey plasma using gradient radio HPLC. Radiolabeling of [11C]BIO-1819578 was successfully accomplished, and the product was found to be stable at 1 h after formulation. [11C]BIO-1819578 was characterized in the cynomolgus monkey brain where a high brain uptake was found (7 SUV at 4 min). A pronounced pretreatment effect was found, indicating specific binding to OGA enzyme. Radiolabeling of [11C]BIO-1819578 with [11C]CO was successfully accomplished. [11C]BIO-1819578 binds specifically to OGA enzyme. The results suggest that [11C]BIO-1819578 is a potential radioligand for imaging and for measuring target engagement of OGA in the human brain.

In vivo evaluation of a novel 18F-labeled PET radioligand for translocator protein 18 kDa (TSPO) in monkey brain

Purpose[18F]SF51 was previously found to have high binding affinity and selectivity for 18 kDa translocator protein (TSPO) in mouse brain. This study sought to assess the ability of [18F]SF51 to quantify TSPO in rhesus monkey brain.MethodsPositron emission tomography (PET) imaging was performed in monkey brain (n = 3) at baseline and after pre-blockade with the TSPO ligands PK11195 and PBR28. TSPO binding was calculated as total distribution volume corrected for free parent fraction in plasma (VT/fP) using a two-tissue compartment model. Receptor occupancy and nondisplaceable uptake were determined via Lassen plot. Binding potential (BPND) was calculated as the ratio of specific binding to nondisplaceable uptake. Time stability of VT was used as an indirect probe to detect radiometabolite accumulation in the brain. In vivo and ex vivo experiments were performed in mice to determine the distribution of the radioligand.ResultsAfter [18F]SF51 injection, the concentration of brain radioactivity peaked at 2.0 standardized uptake value (SUV) at ~ 10 min and declined to 30% of the peak at 180 min. VT/fP at baseline was generally high (203 ± 15 mL· cm−3) and decreased by ~ 90% after blockade with PK11195. BPND of the whole brain was 7.6 ± 4.3. VT values reached levels similar to terminal 180-min values by 100 min and remained relatively stable thereafter with excellent identifiability (standard errors < 5%), suggesting that no significant radiometabolites accumulated in the brain. Ex vivo experiments in mouse brain showed that 96% of radioactivity was parent. No significant uptake was observed in the skull, suggesting a lack of defluorination in vivo.ConclusionThe results demonstrate that [18F]SF51 is an excellent radioligand that can quantify TSPO with a good ratio of specific to nondisplaceable uptake and has minimal radiometabolite accumulation in brain. Collectively, the results suggest that [18F]SF51 warrants further evaluation in humans.

Candidate 3-benzazepine-1-ol type GluN2B receptor radioligands (11C-NR2B-Me enantiomers) have high binding in cerebellum but not to σ1 receptors

IntroductionWe recently reported 11C-NR2B-SMe ([S-methyl-11C](R,S)-7-thiomethoxy-3-(4-(4-methyl-phenyl)butyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepin-1-ol) and its enantiomers as candidate radioligands for imaging the GluN2B subunit within rat N-methyl-D-aspartate receptors. However, these radioligands gave unexpectedly high and displaceable binding in rat cerebellum, possibly due to cross-reactivity with sigma-1 (σ1) receptors. This study investigated 11C-labeled enantiomers of a close analogue (7-methoxy-3-(4-(p-tolyl)butyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepin-1-ol; NR2B-Me) of 11C-NR2B-SMe as new candidate GluN2B radioligands. PET was used to evaluate these radioligands in rats and to assess potential cross-reactivity to σ1 receptors.MethodsNR2B-Me was assayed for binding affinity and selectivity to GluN2B in vitro. 11C-NR2B-Me and its enantiomers were prepared by Pd-mediated treatment of boronic ester precursors with 11C-iodomethane. Brain PET scans were conducted after radioligand intravenous injection into rats. Various ligands for GluN2B receptors or σ1 receptors were administered at set doses in pre-blocking or displacement experiments to assess their impact on imaging data. 18F-FTC146 and enantiomers of 11C-NR2B-SMe were used for comparison. Radiometabolites from brain and plasma were measured ex vivo and in vitro.ResultsNR2B-Me enantiomers showed high GluN2B affinity and selectivity in vitro. 11C-NR2B-Me enantiomers gave high early whole rat brain uptake of radioactivity, including high uptake in cerebellum, followed by slower decline. Radioactivity in brain at 30 min ex vivo was virtually all unchanged radioligand. Only less lipophilic radiometabolites appeared in plasma. When 11C-(R)-NR2B-Me was used, three high-affinity GluN2B ligands—NR2B-SMe, Ro25-6981, and CO101,244—showed increasing pre-block of whole brain radioactivity retention with increasing dose. Two σ1 receptor antagonists, FTC146 and BD1407, were ineffective pre-blocking agents. Together, these results strongly resemble those obtained with 11C-NR2B-SMe enantiomers, except that 11C-NR2B-Me enantiomers showed faster reversibility of binding. When 18F-FTC146 was used as a radioligand, FTC146 and BD1407 showed strong pre-blocking effects whereas GluN2B ligands showed only weak blocking effects.Conclusion11C-NR2B-Me enantiomers showed specific binding to GluN2B receptors in rat brain in vivo. High unexpected specific binding in cerebellum was not due to σ1 receptors. Additional investigation is needed to identify the source of the high specific binding.Graphical

Synthesis and Preclinical Evaluation of [11C]AZ11895530 for PET Imaging of the Serotonin 1A Receptor

The serotonin 1A (5-HT1A) receptor is a G-protein-coupled receptor implicated in the pathophysiology of several neuropsychiatric and neurodegenerative disorders. We here report the preparation of two candidate 5-HT1A radioligands, [11C]AZ11132132 ([11C]3) and [11C]AZ11895530 ([11C]4), and their subsequent evaluation in vitro using autoradiography and in vivo using positron emission tomography (PET). Compounds 3 and 4 were radiolabeled at high radiochemical purity (>99%) and high molar activity (>38 GBq/μmol) by heteroatom methylation with [11C]methyl iodide. Autoradiography on whole hemispheres from post-mortem human brain revealed substantial nonspecific binding of [11C]3, while the binding of [11C]4 to brain tissue was consistent with the distribution of 5-HT1A receptors and sensitive to co-incubation with the reference 5-HT1A antagonist WAY-100635 (10 μM). Following intravenous injection of [11C]4 into a cynomolgus monkey, brain radioactivity concentration (Cmax ∼ 2.2 SUV) was high whereafter it decreased rapidly. The regional binding potential (BPND) values were calculated using the simplified reference tissue model with cerebellum as reference region. The values varied between 0.2 and 1.0 for temporal cortex, raphe nuclei, frontal cortex, and hippocampus which is consistent with the known 5-HT1A expression pattern. After pretreatment with WAY100635 (0.5 mg/kg), a homogeneous distribution of radioactivity was observed in non-human primate (NHP) brain. Although [11C]4 fulfilled important criteria for successful in vivo neuroimaging, including good blood-brain-barrier permeability and high specific binding in vitro to human brain tissue, the regional BPND values for [11C]4 in NHP brain were low when compared to those obtained with existing radioligands and thus do not merit further investigation of [11C]4. Evaluation of structurally related analogues is underway in our laboratory to identify improved candidates for clinical imaging.

Biocompatible mucoadhesive nanoparticles for brain targeting of ropinirole hydrochloride: Formulations, radiolabeling and biodistribution.

Two nanoformulations with mucoadhesive properties and brain-targeting mechanisms were designed to deliver the anti-Parkinson's drug, ropinirole hydrochloride (RH). In the first formulation, RH and the amphiphilic block copolymer methoxy poly(ethylene glycol)-b-poly(caprolactone) were assembled in a core-shell morphology followed by coating with a mucoadhesive chitosan outer layer producing a multilayer vehicle (MLV). In the second formulation, RH was encapsulated during the polyelectrolyte complexation of two natural polymers, chitosan and alginate producing RH-loaded chitosan-alginate polyelectrolyte (PEC) nanocomplex. Conditions of each formulation were adopted for optimal drug loading. Physico-chemical characterization of the prepared formulations (particle size, polydispersity index and zeta-potential) exhibited stable monodispersed nanoparticles. RH was radiolabeled by I-131 radiotracer in a high-radiochemical yield. Biodistribution and brain targeting of RH from the prepared formulations were studied after administration of 131 I-RH-loaded nanoparticles to albino mice via intranasal and intravenous routs. Elevated brain radioactivity was detected post IN administration of (131 I-RH/PCL-PEG/CS) nanoparticles and (131 I-RH/CS-ALG) nanoparticles comparing with the IN administrated RH solutions (Cmax =2.8 ± 0.3, 2 ± 0.3, 0.93 ± 0.03% radioactivity/g, 1h post administration, respectively). This demonstrated that a relatively high-brain targeting could be achieved via intranasal route of administration of RH-loaded nanoparticles. The proposed models are further potential for application to deliver many other brain-targeting therapeutics.

Visualization of Receptor-Interacting Protein Kinase 1 (RIPK1) by Brain Imaging with Positron Emission Tomography.

We report the development of the first positron emission tomography (PET) radiotracer, [18F]CNY-07, based on a highly specific and potent RIPK1 inhibitor, Nec-1s, for RIPK1/necroptosis brain imaging in rodents. [18F]CNY-07 was synthesized through copper-mediated 18F-radiolabeling from an aryl boronic ester precursor and studied in vivo PET imaging in rodents. PET imaging results showed that [18F]CNY-07 can penetrate the blood-brain barrier with a maximum percent injected dose per unit volume of 3 at 10 min postinjection in the brain in vivo. Self-blocking studies of [18F]CNY-07 by pretreating with unlabeled molecules in rodents showed reduced radioactivity in animal brains (30% radioactivity decreased), indicating the binding specificity of our radiotracer. Our studies demonstrate that [18F]CNY-07 has provided a useful PET radioligand enabling brain RIPK1 imaging, which could be a valuable research tool in studying RIPK1-related neurological disorders in animals and potentially humans.

Brain pharmacokinetics of two BBB penetrating bispecific antibodies of different size

BackgroundTransferrin receptor (TfR1) mediated enhanced brain delivery of antibodies have been studied extensively in preclinical settings. However, the brain pharmacokinetics, i.e. brain entry, distribution and elimination are still not fully understood for this class of antibodies. The overall aim of the study was to compare the brain pharmacokinetics of two BBB-penetrating bispecific antibodies of different size (210 vs 58 kDa). Specifically, we wanted to investigate if the faster systemic clearance of the smaller non-IgG antibody di-scFv3D6-8D3, in comparison with the IgG-based bispecific antibody mAb3D6-scFv8D3, was also reflected in the brain.MethodsWild-type (C57/Bl6) mice were injected with 125I-iodinated ([125I]) mAb3D6-scFv8D3 (n = 46) or [125I]di-scFv3D6-8D3 (n = 32) and euthanized 2, 4, 6, 8, 10, 12, 16, or 24 h post injection. Ex vivo radioactivity in whole blood, peripheral organs and brain was measured by γ-counting. Ex vivo autoradiography and nuclear track emulsion were performed on brain sections to investigate brain and parenchymal distribution. Capillary depletion was carried out at 2, 6, and 24 h after injection of [125I]mAb3D6-scFv8D3 (n = 12) or [125I]di-scFv3D6-8D3 (n = 12), to estimate the relative levels of radiolabelled antibody in brain capillaries versus brain parenchyma. In vitro binding kinetics for [125I]mAb3D6-scFv8D3 or [125I]di-scFv3D6-8D3 to murine TfR were determined by LigandTracer.Results[125I]di-scFv3D6-8D3 showed faster elimination from blood, lower brain Cmax, and Tmax, a larger parenchymal-to-capillary concentration ratio, and a net elimination from brain at an earlier time point after injection compared with the larger [125I]mAb3D6-scFv8D3. However, the elimination rate from brain did not differ between the antibodies. The study also indicated that [125I]di-scFv3D6-8D3 displayed lower avidity than [125I]mAb3D6-scFv8D3 towards TfR1 in vitro and potentially in vivo, at least at the BBB.ConclusionA smaller size and lower TfR1 avidity are likely important for fast parenchymal delivery, while elimination of brain-associated bispecific antibodies may not be dependent on these characteristics.

Dose Formulation, Biodistribution and PET Imaging Studies of a First-in-Class Fluorine-18 Organophosphorus Cholinesterase Inhibitor Tracer in Rat

Background:: To investigate dynamic live tissue organophosphorus nerve agent uptake and distribution fates resulting in acetylcholinesterase inhibition, we recently reported the first-in-class fluorine-18 [18F] radiolabeled Positron Emission Tomography (PET) imaging tracer known as [18F]O-(2-fluoroethyl)-O-(p-nitrophenyl)methylphosphonate. This tracer has been initially studied in live rats with PET imaging. Objective.: We sought to evaluate the PET tracer in vivo using a new dose formulation of saline, ethanol and L-ascorbic acid, and compare the influence of this formulation on in vivo tracer performance to previous data collected using a CH3CN:PBS formulation. Methods:: A high molar activity [18F]tracer radiosynthesis was used. Doses were formulated as saline, ethanol (≤ 1%) and L-ascorbic acid (0.1%), pH 4.0-4.5. Stability was evaluated to 6 h. Dose injection (i.v.) into male rats was followed by either ex vivo biodistribution profiling at 5, 30, 90 min, or dynamic 90 min PET imaging. Rat biodistribution and PET imaging data were compared. Results and Discussion:: An optimized radiosynthesis (8 ± 2 % RCY) resulted in stable doses for 6 h (&gt;99%). Arterial blood included a tracer and a single metabolite. The ex vivo biodistribution and live tissue PET imaging data revealed rapid radioactivity uptake and distributed tissue levels: heart and lung, highest; liver, moderate; and brain, lowest. Conclusions:: Imaging and biodistribution data were highly correlated with expected radioactivity tissue uptake and distribution in target organs. Lower brain radioactivity levels by PET imaging were found for the new formulation (saline, 1% L-ascorbic acid, &lt; 1% ethanol) as compared to the established CH3CN:PBS formulation. Overall, we found that the i.v. dose formulation changed the in vivo profile of an organophosphorus PET tracer that is considered an important finding for future organophosphorus PET tracer studies.

Positron emission tomography evaluation of oxime countermeasures in live rats using the tracer O-(2-[18 F]fluoroethyl)-O-(p-nitrophenyl)methylphosphonate [18 F]-VXS.

Oxime antidotes regenerate organophosphate-inhibited acetylcholinesterase (AChE). Although they share a common mechanism of AChE reactivation, the rate and amount of oxime that enters the brain are critical to the efficacy, a process linked to the oxime structure and charge. Using a platform based on the organophosphate [18 F]-VXS as a positron emission tomography tracer for active AChE, the in vivo distribution of [18 F]-VXS was evaluated after an LD50 dose (250 μg/kg) of the organophosphate paraoxon (POX) and following oximes as antidotes. Rats given [18 F]-VXS tracer alone had significantly higher radioactivity (two- to threefold) in the heart and lung than rats given LD50 POX at 20 or 60 min prior to [18 F]-VXS. When rats were given LD50 POX followed by 2-PAM (cationic), RS194b (ionizable), or monoisonitrosoacetone (MINA) (neutral), central nervous system (CNS) radioactivity returned to levels at or above untreated naive rats (no POX), whereas CNS radioactivity did not increase in rats given the dication oximes HI-6 or MMB-4. MINA showed a significant, pairwise increase in CNS brain radioactivity compared with POX-treated rats. This new in vivo dynamic platform using [18 F]-VXS tracer measures and quantifies peripheral and CNS relative changes in AChE availability after POX exposure and is suitable for comparing oxime delivery and AChE reactivation in rats.

Synthesis and Preclinical Evaluation of 6-[18F]Fluorine-α-methyl-l-tryptophan, a Novel PET Tracer for Measuring Tryptophan Uptake.

The positron emission tomography (PET) radioligand α-[11C]methyl-l-tryptophan ([11C]AMT) has been used to assess tryptophan metabolism in cancer, epilepsy, migraine, and autism. Despite its extensive application, the utility of this tracer is currently hampered by the short half-life of the radionuclide used for its labeling (11C, t1/2 = 20.4 min). We herein report the design, synthesis, radiolabeling, and initial in vivo evaluation of a fluorine-18 (18F, t1/2 = 109.7 min) labeled analogue that is fluorinated in the 6-position of the aromatic ring ([18F]6-F-AMTr). In a head-to-head comparison between [18F]6-F-AMTr and [11C]AMT in mice using PET, peak brain radioactivity, regional brain distribution, and kinetic profiles were similar between the two tracers. [18F]6-F-AMTr was however not a substrate for IDO1 or TPH as determined in in vitro enzymatic assays. The brain uptake of the tracer is thus more likely related to LAT1 transport over the blood-brain barrier than metabolism along the serotonin or kynurenine pathways.

Evaluation of 11C-NR2B-SMe and Its Enantiomers as PET Radioligands for Imaging the NR2B Subunit Within the NMDA Receptor Complex in Rats.

[S-methyl-11C](±)-7-methoxy-3-(4-(4-(methylthio)phenyl)butyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepin-1-ol (11C-NR2B-SMe) and its enantiomers were synthesized as candidates for imaging the NR2B subunit within the N-methyl-d-aspartate receptor with PET. Methods: Brains were scanned with PET for 90 min after intravenous injection of one of the candidate radioligands into rats. To detect any NR2B-specific binding of radioligand in brain, various preblocking or displacing agents were evaluated for their impact on the PET brain imaging data. Radiometabolites from brain and other tissues were measured ex vivo and in vitro. Results: Each radioligand gave high early whole-brain uptake of radioactivity, followed by a brief fast decline and then a slow final decline. 11C-(S)-NR2B-SMe was studied extensively. Ex vivo measurements showed that radioactivity in rat brain at 30 min after radioligand injection was virtually unchanged radioligand. Only less lipophilic radiometabolites appeared in plasma. High-affinity NR2B ligands, Ro-25-6981, ifenprodil, and CO101244, showed increasing preblocking of whole-brain radioactivity retention with increasing dose (0.01-3.00 mg/kg, intravenously). Five σ1 antagonists (FTC146, BD1407, F3, F4, and NE100) and 4 σ1 agonists ((+)-pentazocine, (±)-PPCC, PRE-084, and (+)-SKF10047) were ineffective preblocking agents, except FTC146 and F4 at a high dose. Two potent σ1 receptor agonists, TC1 and SA4503, showed dose-dependent preblocking effects in the presence or absence of pharmacologic σ1 receptor blockade with FTC146. Conclusion:11C-(S)-NR2B-SMe has adequate NR2B-specific PET signal in rat brain to warrant further evaluation in higher species. TC1 and SA4503 likely have off-target binding to NR2B in vivo.

Synthesis and characterization of novel radiofluorinated probes for positron emission tomography imaging of monoamine oxidase B.

Monoamine oxidase B (MAO-B), predominantly expressed in glial cells, plays an important role in neurotransmitter regulation, and MAO-B activity relates to several neuronal diseases. Here, we aimed to develop a radiofluorinated MAO-B imaging probe based on the structure of a selective MAO-B inhibitor, MD-230254. We synthesized and evaluated a series of compounds in vitro and in vivo. A series of fluorinated analogs of MD-230254 were synthesized and evaluated for inhibitory potency and selectivity toward MAO-B. 5-[4-(2-[18 F]Fluorobenzyloxy)phenyl]-3-(2-cyanoethyl)-1,3,4-oxadiazol-2(3H)-one (2-[18 F]FBPO) was synthesized from a corresponding tributylstannyl precursor and [18 F]CH3 COOF. Biodistribution after intravenous injection of 2-[18 F]FBPO was evaluated in male ddY mice with or without pretreatment by inhibitors. Among the compounds synthesized and evaluated, 2-FBPO showed high inhibitory potency and selectivity toward MAO-B comparable with MD-230254. 2-[18 F]FBPO was successfully synthesized by an electrophilic reaction with a high radiochemical purity of more than 99%. 2-[18 F]FBPO was efficiently taken up by the brain and showed rapid blood clearance, which provided a brain/blood radioactivity ratio of 3.7 at 90 minutes postinjection. The brain radioactivity was significantly decreased by pretreatment with an MAO-B selective inhibitor. The great potential of 2-[18 F]FBPO as an MAO-B imaging probe, applicable to a variety of diseases, is indicated.

Synthesis and preclinical evaluation of [18F]FSL25.1188, a reversible PET radioligand for monoamine oxidase-B.

Carbon-11 labeled SL25.1188 is a promising reversible monoamine oxidase-B (MAO-B) radioligand that was recently translated for human positron emission tomography (PET) imaging. Herein, we report the development of a novel fluorinated derivative, namely, [18F](S)-3-(6-(3-fluoropropoxy)benzo[d]isoxazol-3-yl)-5-(methoxymethyl)oxazolidin-2-one ([18F]FSL25.1188; [18F]6), as a candidate 18F-labeled MAO-B radioligand, and, its subsequent preclinical evaluation in non-human primates (NHP). [18F]6 was produced and isolated (>6 GBq) with high radiochemical purity (>99%), and molar activity (>100 GBq/µmol at time of injection). Autoradiography studies conducted in post-mortem human brain sections revealed [18F]6 binding in MAO-B rich regions. PET imaging study of [18F]6 in NHP showed high brain uptake (SUV > 2.5) as well as a regional brain radioactivity distribution in accordance with MAO-B expression. [18F]6 displayed favorable in vivo kinetics, with an early peak in the time-activity curve followed by progressive wash-out from the NHP brain. Specificity of [18F]6 was investigated in a pre-treatment study with l-deprenyl (1.0 mg/kg) wherein reduced radioligand uptake was observed in all MAO-B rich regions. Results from the current preclinical investigation suggests [18F]6 is a promising MAO-B PET radioligand. Further evaluation of [18F]6 and structurally related 18F-analogs are underway to identify an optimized candidate for clinical research studies.

Synthesis and evaluation of two new candidate high-affinity full agonist PET radioligands for imaging 5-HT1B receptors

IntroductionThe serotonin 1B receptor subtype is of interest in the pathophysiology and treatment of depression, anxiety, and migraine. Over recent years 5-HT1B receptor binding in human brain has been examined with PET using radioligands that are partial but not full agonists. To explore how the intrinsic activity of a PET radioligand may affect imaging performance, two high-affinity full 5-HT1B receptor agonists (AZ11136118, 4; and AZ11895987, 5) were selected from a large compound library and radiolabeled for PET examination in non-human primates. Methods[11C]4 was obtained through Pd(0)-mediated insertion of [11C]carbon monoxide between prepared iodoarene and homochiral amine precursors. [11C]5 was obtained through N-11C-methylation of N-desmethyl precursor 6 with [11C]methyl triflate. [11C]4 and [11C]5 were studied with PET in rhesus or cynomolgus monkey. [11C]4 was studied with PET in mice and rats to measure brain uptake and specific binding. Ex-vivo experiments in rats were performed to identify whether there were radiometabolites in brain. Physiochemical parameters for [11C]4 (pKa, logD and conformational energetics) were evaluated. ResultsBoth [11C]4 and [11C]5 were successfully produced in high radiochemical purity and in adequate amounts for PET experiments. After intravenous injection of [11C]4, brain radioactivity peaked at a low level (0.2 SUV). Pretreatment with tariquidar, an inhibitor of the brain P-gp efflux transporter, increased brain exposure four-fold whereas pretreatment with a high pharmacological dose of the 5-HT1B antagonist, AR-A000002, had no effect on the binding. Ex-vivo experiments in rats showed no radiometabolites entering brain. [11C]5 also failed to enter monkey brain under baseline conditions. Conclusions[11C]4 and [11C]5 show too low brain uptake and specific binding to be useful PET radioligands. Low brain uptake is partly ascribed to efflux transporter action as well as unfavorable conformations.

Radiosynthesis and evaluation of [11C]CMP, a high affinity GSK3 ligand

Dysfunction of GSK3 is implicated in the etiology of many brain, inflammatory, cardiac diseases, and cancer. PET imaging would enable in vivo detection and quantification of GSK3 and can impact the choice of therapy, allow non-invasive monitoring of disease progression and treatment effects. In this report, the synthesis and evaluation of a high affinity GSK3 ligand, [11C]2-(cyclopropanecarboxamido)-N-(4-methoxypyridin-3-yl)isonicotinamide, ([11C]CMP, (3), (IC50 = 3.4 nM, LogP = 1.1) is described. [11C]CMP was synthesized in 25 ± 5% yield by radiomethylating the corresponding phenolate using [11C]CH3I. The radioligand exhibited modest uptake in U251 human glioblastoma cell lines with ∼50% specific binding. MicroPET studies in rats indicated negligible blood–brain barrier (BBB) penetration of [11C]CMP, despite its high affinity and suitable logP value for BBB penetration. However, administration of cyclosporine prior to [11C]CMP injection showed significant improvement in brain radioactivity uptake and the tracer binding. This finding indicates that [11C]CMP might be a P-gp efflux substrate and therefore has some limitations for routine in vivo PET evaluations in brain.