Clinical Characterization of [18F]T-008, a Cholesterol 24-Hydroxylase PET Ligand: Dosimetry, Kinetic Modeling, Variability, and Soticlestat Occupancy.

This series of studies characterized [18F]T-008, a PET radiotracer for imaging cholesterol 24-hydroxylase (CH24H), in healthy volunteers (ClinicalTrials.gov identifier NCT02497235). Assessments included radiation dosimetry, kinetic modeling, test-retest variability (TRT) evaluation, and a dose occupancy evaluation using soticlestat, a selective CH24H inhibitor. Soticlestat is currently in phase 3 development for the treatment of seizures in Dravet syndrome and Lennox-Gastaut syndrome. Methods: In the dosimetry study, 5 participants (3 men) underwent serial whole-body scans to estimate organ-absorbed doses and effective doses of [18F]T-008 using OLINDA/EXM 1.1. For the kinetic modeling and TRT study, 6 participants (all men) underwent two 210-min dynamic [18F]T-008 PET scans with arterial blood sampling. The regional total volume of distribution was estimated using a 1-tissue-compartment model, a 2-tissue-compartment model, and Logan graphic analysis. In the dose occupancy study, 11 participants (all men) underwent 120-min scans at baseline and 2 time points (peak and trough) after receiving single oral doses of soticlestat (50-600 mg). The relationship between effect-site soticlestat concentration and brain occupancy was evaluated with a specially developed pharmacokinetic model and a saturable maximal occupancy model. Results: The estimated mean whole-body effective dose was 0.0292 mSv/MBq (SD, 0.00147 mSv/MBq). [18F]T-008 entered the brain rapidly, with a distribution consistent with known CH24H distribution densities. The 2-tissue-compartment model and Logan graphic analysis best described the tracer kinetics. The mean TRT for estimating total volume of distribution was 7%-15%. Single doses of soticlestat in the range 50-600 mg resulted in occupancies of 64%-96% at 2 h and 11%-79% at 24 h. The estimated half-maximal effect-site concentration of soticlestat was 5.52 ng/mL. Conclusion: [18F]T-008 is a suitable PET radiotracer for quantitatively analyzing CH24H in the human brain. Using [18F]T-008 and PET, we demonstrated that soticlestat was brain-penetrant and established target engagement by displacing [18F]T-008 in a dose-dependent manner in the brain.

Design and synthesis of aryl-piperidine derivatives as potent and selective PET tracers for cholesterol 24-hydroxylase (CH24H).

Cholesterol 24-hydroxylase (CH24H, CYP46A1) is a cytochrome P450 family enzyme that maintains the homeostasis of brain cholesterol. Soticlestat, a potent and selective CH24H inhibitor, is in development as a therapeutic agent for Dravet syndrome and Lennox-Gastaut syndrome. Herein, we report the discovery of aryl-piperidine derivatives as potent and selective CH24H positron emission tomography (PET) tracers which can be used for dose guidance of a clinical CH24H inhibitor and as a diagnostic tool for CH24H-related pathology. Starting from compound 1 (IC50 = 16 nM, logD = 1.7), which was reported as a CH24H inhibitor with lower lipophilicity, a18F-labeling site (3-fluoroazetidine) was incorporated by structure-based drug design (SBDD) utilizing the co-crystal structure of a compound 1 analog. Subsequent optimization to adjust key parameters for PET tracers, such as potency, lipophilicity, brain penetration, and unbound plasma protein binding, enabled compounds 3f (IC50 = 8.8 nM) and 3g (IC50 = 8.7 nM) as PET imaging candidates. Selectivity of these compounds for CH24H was validated by a brain distribution study using CH24H-WT and KO mice. In non-human primate PET imaging, [18F]3f and [18F]3g showed similar regional uptake in the brain, indicating that these tracers were specific to the CH24H-expressed regions and validated the expression of CH24H in the living brain by different tracers.

Preclinical characterization of [18F]T-008, a novel PET imaging radioligand for cholesterol 24-hydroxylase.

PURPOSE: Cholesterol 24-hydroxylase (CH24H) is a brain-specific enzyme that plays a major role in brain cholesterol homeostasis by converting cholesterol into 24S-hydroxycholesterol. The selective CH24H inhibitor soticlestat (TAK-935) is being pursued as a drug for treatment of seizures in developmental and epileptic encephalopathies. Herein, we describe the successful discovery and the preclinical validation of the novel radiolabeled CH24H ligand (3-[18F]fluoroazetidin-1-yl){1-[4-(4-fluorophenyl)pyrimidin-5-yl]piperidin-4-yl}methanone ([18F]T-008) and its tritiated analog, [3H]T-008. METHODS: In vitro autoradiography (ARG) studies in the CH24H wild-type (WT) and knockout (KO) mouse brain sections were conducted using [3H]T-008. PET imaging was conducted in two adult rhesus macaques using [18F]T-008. Each macaque received two test-retest baseline scans and a series of two blocking doses of soticlestat administered prior to [18F]T-008 to determine the CH24H enzyme occupancy. PET data were analyzed with Logan graphical analysis using plasma input. A Lassen plot was applied to estimate CH24H enzyme occupancy by soticlestat. RESULTS: In ARG studies, binding of [3H]T-008 was specific to CH24H in the mouse brain sections, which was not observed in CH24H KO or in wild-type mice after pretreatment with soticlestat. In rhesus PET studies, the rank order of [18F]T-008 uptake was striatum > cortical regions > cerebellum, which was consistent with CH24H distribution in the brain. Pre-blocking with soticlestat reduced the maximum uptake and increased the washout in all brain regions in a dose-dependent manner. Calculated global occupancy values for soticlestat at a dose of 0.89 mg/kg were 97-98%, indicating maximum occupancy. CONCLUSION: The preclinical in vitro and in vivo evaluation of labeled T-008 demonstrates that [18F]T-008 is suitable for imaging CH24H in the brain and warrants further studies in humans.

Design, Synthesis, and Evaluation of (2-Aminocyclopropyl)phenyl Derivatives as Novel Positron Emission Tomography Imaging Agents for Lysine-Specific Demethylase 1 in the Brain.

Dysregulation of histone H3 lysine 4 (H3K4) methylation is implicated in the pathogenesis of neurodevelopmental disorders. Lysine-specific demethylase 1 (LSD1) determines the methylation status of H3K4 through flavin adenine dinucleotide (FAD)-mediated histone demethylation. Therefore, LSD1 inhibition in the brain can be a novel therapeutic option for treating these disorders. Positron emission tomography (PET) imaging of LSD1 allows for investigating LSD1 expression levels under normal and disease conditions and validating target engagement of therapeutic LSD1 inhibitors. This study designed and synthesized (2-aminocyclopropyl)phenyl derivatives with irreversible binding to LSD1 as PET imaging agents for LSD1 in the brain. We optimized lipophilicity of the lead compound to minimize the risk of nonspecific binding and identified 1e with high selectivity over monoamine oxidase A and B, which are a family of FAD-dependent enzymes homologous to LSD1. PET imaging in a monkey showed a high uptake of [18F]1e to regions enriched with LSD1, indicating its specific binding to LSD1.

Dopamine D1 Receptor Agonist PET Tracer Development: Assessment in Nonhuman Primates.

Non-catechol-based high-affinity selective dopamine D1 receptor (D1R) agonists were recently described, and candidate PET ligands were selected on the basis of favorable properties. The objective of this study was to characterize in vivo in nonhuman primates 2 novel D1R agonist PET radiotracers, racemic 18F-MNI-800 and its more active atropisomeric (-)-enantiomer, 18F-MNI-968. Methods: Ten brain PET experiments were conducted with 18F-MNI-800 on 2 adult rhesus macaques and 2 adult cynomolgus macaques, and 8 brain PET experiments were conducted with 18F-MNI-968 on 2 adult rhesus macaques and 2 adult cynomolgus macaques. PET data were analyzed with both plasma-input-based methods and reference-region-based methods. Whole-body PET images were acquired with 18F-MNI-800 from 2 adult rhesus macaques for radiation dosimetry estimates. Results:18F-MNI-800 and 18F-MNI-968 exhibited regional uptake consistent with D1R distribution. Specificity and selectivity were demonstrated by dose-dependent blocking with the D1 antagonist SCH-23390. 18F-MNI-968 showed a 30% higher specific signal than 18F-MNI-800, with a nondisplaceable binding potential of approximately 0.3 in the cortex and approximately 1.1 in the striatum. Dosimetry radiation exposure was favorable, with an effective dose of about 0.023 mSv/MBq. Conclusion:18F-MNI-968 has significant potential as a D1R agonist PET radiotracer, and further characterization in human subjects is warranted.

The rate of dasotraline brain entry is slow following intravenous administration.

RATIONALE: Drugs that rapidly increase dopamine levels have an increased risk of abuse. Dasotraline (DAS) is a dopamine and norepinephrine reuptake inhibitor characterized by slow oral absorption with low potential for abuse. However, it remains unclear whether intravenous (i.v.) administration would facilitate the rapid elevation of dopamine levels associated with stimulant drugs. OBJECTIVE: To assess the kinetics of DAS across the blood-brain barrier and time to onset of dopamine transporters (DAT) inhibition. METHODS: We compared the onset of DAT occupancy and the associated elevation of synaptic dopamine levels in rhesus monkey following i.v. administration of DAS or methylphenidate (MPH) using positron emission tomography (PET). Brain entry times were estimated by reductions in [18F]-FE-PE2I binding to DAT in rhesus monkeys. Elevations of synaptic dopamine were estimated by reductions in [11C]-Raclopride binding to D2 receptors. RESULTS: Intravenous administration of DAS (0.1 and 0.2 mg/kg) resulted in striatal DAT occupancies of 54% and 68%, respectively; i.v. administered MPH (0.1 and 0.5 mg/kg) achieved occupancies of 69% and 88% respectively. Brain entry times of DAS (22 and 15 min, respectively) were longer than for MPH (3 and 2 min). Elevations in synaptic dopamine were similar for both DAS and MPH however the time for half-maximal displacement by MPH (t = 23 min) was 4-fold more rapid than for DAS (t = 88 min). CONCLUSIONS: These results demonstrate that the pharmacodynamics effects of DAS on DAT occupancy and synaptic dopamine levels are more gradual in onset than those of MPH even with i.v. administration that is favored by recreational drug abusers.

Clinical evaluation of [18F] JNJ-64326067, a novel candidate PET tracer for the detection of tau pathology in Alzheimer's disease.

PURPOSE: The accumulation of misfolded tau is a common feature of several neurodegenerative disorders, with Alzheimer's disease (AD) being the most common. Earlier we identified JNJ-64326067, a novel isoquinoline derivative with high affinity and selectivity for tau aggregates from human AD brain. We report the dosimetry of [18F] JNJ-64326067 and results of a proof-of-concept study comparing subjects with probable Alzheimer's disease to age-matched healthy controls. METHODS: [18F] JNJ-64326067 PET scans were acquired for 90 min and then from 120 to 180 min in 5 participants with [18F]-florbetapir PET amyloid positive probable AD (73 ± 9 years) and 5 [18F]-florbetapir PET amyloid negative healthy controls (71 ± 7 years). Whole-body [18F] JNJ-64326067 PET CT scans were acquired in six healthy subjects for 5.5 h in 3 scanning sessions. Brain PET scans were visually reviewed. Regional quantification included kinetic analysis of distribution volume ration (DVR) estimated by Logan graphical analysis over the entire scan and static analysis of SUVr in late frames. Both methods used ventral cerebellar cortex as a reference region. RESULTS: One of the healthy controls had focal areas of PET signal in occipital and parietal cortex underlying the site of a gunshot injury as an adolescent; the other four healthy subjects had no tau brain signal. Four of the 5 AD participants had visually apparent retention of [18F] JNJ-64326067 in relevant cortical regions. One of the AD subjects was visually negative. Cortical signal in visually positive subjects approached steady state by 120 min. Temporal and frontal cortical SUVr/DVR values in visually positive AD subjects ranged from 1.21 to 3.09/1.2 to 2.18 and from 0.92 to 1.28/0.91 to 1.16 in healthy controls. Whole-body effective dose was estimated to be 0.0257 mSv/MBq for females and 0.0254 mSv/MBq for males. CONCLUSIONS: [18F] JNJ-64326067 could be useful for detection and quantitation of tau aggregates.

Evaluation of Dosimetry, Quantitative Methods, and Test-Retest Variability of 18F-PI-2620 PET for the Assessment of Tau Deposits in the Human Brain.

18F-PI-2620 is a next-generation tau PET tracer that has demonstrated ability to image the spatial distribution of suspected tau pathology. The objective of this study was to assess the tracer biodistribution, dosimetry, and quantitative methods of 18F-PI-2620 in the human brain. Full kinetic modeling to quantify tau load was investigated. Noninvasive kinetic modeling and semiquantitative methods were evaluated against the full tracer kinetics. Finally, the reproducibility of PET measurements from test and retest scans was assessed. Methods: Three healthy controls (HCs) and 4 Alzheimer disease (AD) subjects underwent 2 dynamic PET scans, including arterial sampling. Distribution volume ratio (DVR) was estimated using full tracer kinetics (reversible 2-tissue-compartment [2TC] model and Logan graphical analysis [LGA]) and noninvasive kinetic models (noninvasive LGA [NI-LGA] and the multilinear reference tissue model [MRTM2]). SUV ratio (SUVR) was determined at different imaging windows after injection. The correlation between DVR and SUVR, effect size (Cohen's d), and test-retest variability (TRV) were evaluated. Additionally, 6 HCs received 1 tracer administration and underwent whole-body PET for dosimetry calculation. Organ doses and the whole-body effective dose were calculated using OLINDA 2.0. Results: A strong correlation was found across different kinetic models (R2 > 0.97) and between DVR(2TC) and SUVR between 30 and 90 min, with an R2 of more than 0.95. Secular equilibrium was reached at around 40 min after injection in most regions and subjects. TRV and effect size for SUVR across different regions were similar at 30-60 min (TRV, 3.8%; Cohen's d, 3.80), 45-75 min (TRV, 4.3%; Cohen's d, 3.77) and 60-90 min (TRV, 4.9%; Cohen's d, 3.73) and increased at later time points. Elimination was via the hepatobiliary and urinary systems. The whole-body effective dose was 33.3 ± 2.1 µSv/MBq for an adult female and 33.1 ± 1.4 µSv/MBq for an adult male, with a 1.5-h urinary bladder voiding interval. Conclusion:18F-PI-2620 exhibits fast kinetics, suitable dosimetry, and low TRV. DVR measured using the 2TC model with arterial sampling correlated strongly with DVR measured by NI-LGA, MRTM2, and SUVR. SUVR can be used for 18F-PI-2620 PET quantification of tau deposits, avoiding arterial blood sampling. Static 18F-PI-2620 PET scans between 45 and 75 min after injection provide excellent quantification accuracy, a large effect size, and low TRV.

Preclinical Evaluation and Nonhuman Primate Receptor Occupancy Study of 18F-JNJ-64413739, a PET Radioligand for P2X7 Receptors.

The P2X7 receptor is an adenosine triphosphate-gated ion channel, which is abundantly expressed in glial cells within the central nervous system and in the periphery. P2X7 receptor activation leads to the release of the proinflammatory cytokine IL-1ß in the brain, and antagonism of the P2X7 receptor is a novel therapeutic strategy to dampen adenosine triphosphate-dependent IL-1ß signaling. PET ligands for the P2X7 receptor will not only be valuable to assess central target engagement of drug candidates but also hold promise as surrogate markers of central neuroinflammation. Herein we describe the in vitro and in vivo evaluation of 18F-JNJ-64413739, an 18F-labeled PET ligand for imaging the P2X7 receptor in the brain. Methods: P2X7 receptor affinity and specificity, pharmacokinetics, metabolic stability, blood-brain barrier permeability, and off-target binding of JNJ-64413739 were evaluated in a series of in vitro, ex vivo, and in vivo assays. 18F-JNJ-64413739 was radiolabeled via a one-step nucleophilic aromatic substitution. The tracer was also studied in rhesus macaques, and PET images were analyzed with an arterial plasma input function-based Logan graphical analysis. Results: The potency (half-maximal inhibitory concentration) of the P2X7 receptor antagonist JNJ-64413739 is 1.0 ± 0.2 nM and 2.0 ± 0.6 nM at the recombinant human and rat P2X7 receptor, respectively, and the binding affinity is 2.7 nM (rat cortex binding assay) and 15.9 nM (human P2X7 receptor). In nonhuman primate PET imaging studies, dose-dependent receptor occupancy of JNJ-54175446 was observed in 2 rhesus monkeys. At a 0.1 mg/kg dose (intravenous) of JNJ-54175446, the receptor occupancy was calculated to be 17% by Logan graphical analysis, whereas a dose of 2.5 mg/kg yielded a receptor occupancy of 60%. Conclusion: The preclinical evaluation of 18F-JNJ-64413739 demonstrates that the tracer engages the P2X7 receptor. Reproducible and dose-dependent receptor occupancy studies with the P2X7 receptor antagonist JNJ-54175446 were obtained in rhesus monkeys. This novel PET tracer exhibits in vitro and in vivo characteristics suitable for imaging the P2X7 receptor in the brain and warrants further studies in humans.

Evaluation of [18 F]2FP3 in pigs and non-human primates.

So far, no suitable 5-HT7 R radioligand exists for clinical positron emission tomography (PET) imaging. [18 F]2FP3 was first tested in vivo in cats, and the results were promising for further evaluations. Here, we evaluate the radioligand in pigs and non-human primates (NHPs). Furthermore, we investigate species differences in 5-HT7 R binding with [3 H]SB-269970 autoradiography in post-mortem pig, NHP, and human brain tissue. Specific binding of [18 F]2FP3 was investigated by intravenous administration of the 5-HT7 R specific antagonist SB-269970. [3 H]SB-269970 autoradiography was performed as previously described. [18 F]2FP3 was synthesized in an overall yield of 35% to 45%. High brain uptake of the tracer was found in both pigs and NHPs; however, pretreatment with SB-269970 only resulted in decreased binding of 20% in the thalamus, a 5-HT7 R-rich region. Autoradiography on post-mortem pig, NHP, and human tissues revealed that specific binding of [3 H]SB-269970 was comparable in the thalamus of pig and NHP. Despite the high uptake of [18 F]2FP3 in both species, the binding could only be blocked to a limited degree with the 5-HT7 R antagonists. We speculate that the affinity of the radioligand is too low for imaging the 5-HT7 Rs in vivo and that part of the PET signal arises from targets other than the 5-HT7 R.

Development and In Vivo Preclinical Imaging of Fluorine-18-Labeled Synaptic Vesicle Protein 2A (SV2A) PET Tracers.

PURPOSE: Synaptic vesicle protein 2A (SV2A) serves as a biomarker of synaptic density and positron emission tomography (PET) imaging of SV2A could provide a tool to assess progression of neurodegenerative diseases. Two tracers have primarily been reported and characterized in vivo: [11C]UCB-J and [18F]UCB-H. In early human studies, [11C]UCB-J showed promising results, while its F-18-labeled analogue [18F]UCB-H showed suboptimal specific signal in comparison to [11C]UCB-J. Considering the limited use of [11C]UCB-J to facilities with a cyclotron, having a F-18 variant would facilitate large, multicenter imaging trials. We have screened several F-18 derivatives of UCB-J in non-human primates and identified a promising F-18 PET candidate, [18F]MNI-1126, with additional investigations of the racemate [18F]MNI-1038, affording a signal comparable to [11C]UCB-J. PROCEDURES: F-18 derivatives of UCB-J and UCB-H were synthesized and administered to non-human primates for microPET imaging. Following screenings, [18F]MNI-1038 (racemate) and [18F]MNI-1126 (R-enantiomer) were identified with the highest signal and favorable kinetics and were selected for further imaging. Kinetic modeling with one- and two-tissue compartmental models, and linear methods were applied to PET data using metabolite-corrected arterial input function. Pre-block scans with levetiracetam (LEV, 10, 30 mg/kg, iv) were performed to determine the tracers' in vivo specificity for SV2A. Two whole-body PET studies were performed with [18F]MNI-1038 in one male and one female rhesus, and radiation absorbed dose estimates and effective dose (ED, ICRP-103) were estimated with OLINDA/EXM 2.0. RESULTS: All compounds screened displayed very good brain penetration, with a plasma-free fraction of ~ 40 %. [18F]MNI-1126 and [18F]MNI-1038 showed uptake and distribution the most consistent with UCB-J, while the other derivatives showed suboptimal results, with similar or lower uptake than [18F]UCB-H. VT of [18F]MNI-1126 and [18F]MNI-1038 was high in all gray matter regions (within animal averages ~ 30 ml/cm3) and highly correlated with [11C]UCB-J (r > 0.99). Pre-blocking of [18F]MNI-1126 or [18F]MNI-1038 with LEV showed robust occupancy across all gray matter regions, similar to that reported with [11C]UCB-J (~ 85 % at 30 mg/kg, ~ 65 % at 10 mg/kg). Using the centrum semiovale as a reference region, BPND of [18F]MNI-1126 reached values of up to ~ 30 to 40 % higher than those reported for [11C]UCB-J. From whole-body imaging average ED of [18F]MNI-1038 was estimated to be 22.3 µSv/MBq, with tracer being eliminated via both urinary and hepatobiliary pathways. CONCLUSIONS: We have identified a F-18-labeled tracer ([18F]MNI-1126) that exhibits comparable in vivo characteristics and specificity for SV2A to [11C]UCB-J in non-human primates, which makes [18F]MNI-1126 a promising PET radiotracer for imaging SV2A in human trials.

18F-F13640 preclinical evaluation in rodent, cat and primate as a 5-HT1A receptor agonist for PET neuroimaging.

Serotonin 1A receptors are known to play an important role in many psychiatric and neurodegenerative disorders. Currently, all available 5-HT1A receptor PET radiopharmaceuticals that are radiolabeled with fluorine-18 are antagonists. As agonists bind preferentially to the high-affinity state of receptors, it would be of great interest to develop agonist radioligands which could provide a measure of the functional 5-HT1A receptors in pathophysiological processes. The 5-HT1A receptor agonist candidates we recently proposed had promising in vitro properties but were not optimal in terms of PET imaging. F13640, a.k.a befiradol or NLX-112, is a 5-HT1A receptor agonist with a high affinity (Ki = 1 nM) and a high selectivity that would be suitable for a potential PET radiopharmaceutical. With propose here the first preclinical evaluation of 18F-F13640. 18F-F13640's nitro-precursor was synthesized and radiolabeled via a fluoro-nucleophilic substitution. Its radiopharmacological characterization included autoradiographic studies, metabolic studies, and in vivo PET scans in rat, cat and non-human primate. Some of the results were compared with the radiotracer 18F-MPPF, a 5-HT1A receptor antagonist. The radiochemical purity of 18F-F13640 was > 98%. In vitro binding pattern was consistent with the 5-HT1A receptor distribution. Metabolic studies revealed that the radiotracer rapidly entered the brain and led to few brain radiometabolites. Although 18F-F13640 in vivo binding was blocked by the 5-HT1A antagonist WAY-100635 and the 5-HT1A agonist 8-OH-DPAT, the distribution pattern was markedly different from antagonist radiotracers in the three species, suggesting it provides novel information on 5-HT1A receptors. Preliminary studies also suggest a high sensitivity of 18F-F13640 to endogenous serotonin release. 18F-F13640 has suitable characteristics for probing in vitro and in vivo the 5-HT1A receptors in high-affinity state. Quantification analyses with kinetic modeling are in progress to prepare the first-in-man study of 18F-F13640.

PET radiotracer development for imaging high-affinity state of dopamine D2 and D3 receptors: Binding studies of fluorine-18 labeled aminotetralins in rodents.

Imaging the high-affinity, functional state (HA) of dopamine D2 and D3 receptors has been pursued in PET imaging studies of various brain functions. We report further evaluation of 18 F-5-OH-FPPAT, and the newer 18 F-5-OH-FHXPAT and 18 F-7-OH-FHXPAT. Syntheses of 18 F-5-OH-FHXPAT and 18 F-7-OH-FHXPAT were improved by modifications of our previously reported procedures. Brain slices and brain homogenates from male Sprague-Dawley rats were used with the 3 radiotracers (74-111 kBq/cc). Competition with dopamine (1-100 nM) and Gpp(NH)p (10-50 µM) were carried out to demonstrate binding to dopamine D2 and D3 HA-states and binding kinetics of 18 F-5-OH-FPPAT measured. Ex vivo brain slice autoradiography was carried out on rats administered with 18 F-5-OH-FHXPAT to ascertain HA-state binding. PET/CT imaging in rats and wild type (WT) and D2 knock-out mice were carried out using 18 F-7-OH-FHXPAT (2-37 MBq). Striatum was clearly visualized by the three radiotracers in brain slices and dopamine displaced more than 80% of binding, with dissociation rate in homogenates of 2.2 × 10-2 min-1 for 18 F-5-OH-FPPAT. Treatment with Gpp(NH)p significantly reduced 50-80% striatal binding with faster dissociation rates (5.0 × 10-2 min-1 ), suggesting HA-state binding of 18 F-5-OH-FPPAT and 18 F-5-OH-FHXPAT. Striatal binding of 18 F-5-OH-FHXPAT in ex vivo brain slices were sensitive to Gpp(NH)p, suggesting HA-state binding in vivo. PET binding ratios of 18 F-7-OH-FHXPAT in rat brain were ventral striatum/cerebellum = 2.09 and dorsal striatum/cerebellum = 1.65; similar binding ratios were found in the D2 WT mice. These results suggest that in vivo PET measures of agonists in the brain at least in part reflect binding to the membrane-bound HA-state of the dopamine receptor.

Synthesis and in†vivo Evaluation of Fluorine-18 and Iodine-123 Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine Derivatives as PET and SPECT Radiotracers for Mapping A2A Receptors.

Imaging agents that target adenosine type 2A (A2A ) receptors play an important role in evaluating new pharmaceuticals targeting these receptors, such as those currently being developed for the treatment of movement disorders like Parkinson's disease. They are also useful for monitoring progression and treatment efficacy by providing a noninvasive tool to map changes in A2A receptor density and function in neurodegenerative diseases. We previously described the successful evaluation of two A2A -specific radiotracers in both nonhuman primates and in subsequent human clinical trials: [(123) I]MNI-420 and [(18) F]MNI-444. Herein we describe the development of both of these radiotracers by selection from a series of A2A ligands, based on the pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine core of preladenant. Each of this series of 16 ligands was found to bind to recombinant human A2A receptor in the low nanomolar range, and of these 16, six were radiolabeled with either fluorine-18 or iodine-123 and evaluated in nonhuman primates. These initial in vivo results resulted in the identification of 7-(2-(4-(4-(2-[(18) F]fluoroethoxy)phenyl)piperazin-1-yl)ethyl)-2-(furan-2-yl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine ([(18) F]MNI-444) and 7-(2-(4-(2-fluoro-4-[(123) I]iodophenyl)piperazin-1-yl)ethyl)-2-(furan-2-yl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-amine ([(123) I]MNI-420) as PET and SPECT radiopharmaceuticals for mapping A2A receptors in brain.

Identification and in vivo evaluation of a fluorine-18 rolipram analogue, [(18) F]MNI-617, as a radioligand for PDE4 imaging in mammalian brain.

Phosphodiesterase (PDE) 4 is the most prevalent PDE in the central nervous system (CNS) and catalyzes hydrolysis of intracellular cAMP, a secondary messenger. By therapeutic inhibition of PDE4, intracellular cAMP levels can be stabilized, and the symptoms of psychiatric and neurodegenerative disorders including depression, memory loss and Parkinson's disease can be ameliorated. Radiotracers targeting PDE4 can be used to study PDE4 density and function, and evaluate new PDE4 therapeutics, in vivo in a non-invasive way, as has been shown using the carbon-11 labeled PDE4 inhibitor R-(-)-rolipram. Herein we describe a small series of rolipram analogs that contain fluoro- or iodo-substituents that could be used as fluorine-18 PET or iodine-123 SPECT PDE4 radiotracers. This series was evaluated with an in vitro binding assay and a 4-(fluoromethyl) derivative of rolipram, MNI-617, was identified, with a five-fold increase in affinity for PDE4 (Kd = 0.26 nM) over R-(-)-rolipram (Kd = 1.6 nM). A deutero-analogue d2 -[(18) F]MNI-617 was radiolabeled and produced in 23% yield with high (>5 Ci/µmol) specific activity and evaluated in non-human primate, where it rapidly entered the brain, with SUVs between 4 and 5, and with a distribution pattern consistent with that of PDE4.

Evaluation of [11C]TAZA for amyloid ß plaque imaging in postmortem human Alzheimer's disease brain region and whole body distribution in rodent PET/CT.

OBJECTIVE: Alzheimer's disease (AD) is a neurodegenerative disease characterized by Aß plaques in the brain. The aim of this study was to evaluate the effectiveness of a novel radiotracer, 4-[(11) C]methylamino-4'-N,N-dimethylaminoazobenzene ([(11)C]TAZA), for binding to Aß plaques in postmortem human brain (AD and normal control (NC)). METHODS: Radiosyntheses of [(11)C]TAZA, related [(11)C]Dalene ((11)C-methylamino-4'-dimethylaminostyrylbenzene), and reference [(11)C]PIB were carried out using [(11)C]methyltriflate prepared from [(11) C]CO(2) and purified using HPLC. In vitro binding affinities were carried out in human AD brain homogenate with Aß plaques labeled with [(3) H]PIB. In vitro autoradiography studies with the three radiotracers were performed on hippocampus of AD and NC brains. PET/CT studies were carried out in normal rats to study brain and whole body distribution. RESULTS: The three radiotracers were produced in high radiochemical yields (>40%) and had specific activities >37 GBq/µmol. TAZA had an affinity, K(i) = 0.84 nM and was five times more potent than PIB. [(11)C]TAZA bound specifically to Aß plaques present in AD brains with gray matter to white matter ratios >20. [(11)C]TAZA was displaced by PIB (>90%), suggesting similar binding site for [(11)C]TAZA and [(11)C]PIB. [(11)C]TAZA exhibited slow kinetics of uptake in the rat brain and whole body images showed uptake in interscapular brown adipose tissue (IBAT). Binding in brain and IBAT were affected by preinjection of atomoxetine, a norepinephrine transporter blocker. CONCLUSION: [(11)C]TAZA exhibited high binding to Aß plaques in human AD hippocampus. Rat brain kinetics was slow and peripheral binding to IBAT needs to be further evaluated.

Dopamine D3 receptor binding of (18)F-fallypride: Evaluation using in vitro and in vivo PET imaging studies.

Identification of dopamine D3 receptors (D3R) in vivo is important to understand several brain functions related to addiction. The goal of this work was to identify D3R binding of the dopamine D2 receptor (D2R)/D3R imaging agent, (18)F-fallypride. Brain slices from male Sprague-Dawley rats (n = 6) and New Zealand White rabbits (n = 6) were incubated with (18)F-fallypride and D3R selective agonist (R)-7-OH-DPAT (98-fold D3R selective). Rat slices were also treated with BP 897 (68-fold D3R selective partial agonist) and NGB 2904 (56-fold D3R selective antagonist). In vivo rat studies (n = 6) were done on Inveon PET using 18-37 MBq (18)F-fallypride and drug-induced displacement by (R)-7-OH-DPAT, BP 897 and NGB 2904. PET/CT imaging of wild type (WT, n = 2) and D2R knock-out (KO, n = 2) mice were carried out with (18)F-fallypride. (R)-7-OH-DPAT displaced binding of (18)F-fallypride, both in vitro and in vivo. In vitro, at 10 nM (R)-7-OH-DPAT, (18)F-fallypride binding in the rat ventral striatum (VST) and dorsal striatum (DST) and rabbit nucleus accumbens were reduced by ∼10-15%. At 10 µM (R)-7-OH-DPAT all regions in rat and rabbit were reduced by ≥85%. In vivo reductions for DST and VST before and after (R)-7-OH-DPAT were: low-dose (0.015 mg kg(-1)) DST -22%, VST -29%; high-dose (1.88 mg kg(-1)) DST -58%, VST -77%, suggesting D3R/D2R displacement. BP 897 and NGB 2904 competed with (18)F-fallypride in vitro, but unlike BP 897, NGB 2904 did not displace (18)F-fallypride in vivo. The D2R KO mice lacked (18)F-fallypride binding in the DST. In summary, our findings suggest that up to 20% of (18)F-fallypride may be bound to D3R sites in vivo.

Enhancement of 18F-fluorodeoxyglucose metabolism in rat brain frontal cortex using a ß3 adrenoceptor agonist.

We report the use of ß3 adrenergic receptor mediated activation of rat brain frontal cortex using mirabegron (a selective ß3 adrenoceptor agonist), measured by (18)F-FDG PET/CT. Another ß3 agonist, CL 316,243, did not have this effect due to impermeability through the blood brain barrier (BBB), while atomoxetine, a norepinephrine transporter blocker, did increase (18)F-FDG uptake in the frontal cortex. Mirabegron exhibited a dose-dependent increase in frontal cortex (18)F-FDG uptake. These findings suggest a possible use of selective ß3 adrenoceptor agonists in reversing regional glucose hypometabolism in the brain.

Spinal cord dopamine D2/D3 receptors: in vivo and ex vivo imaging in the rat using (18)F/(11)C-fallypride.

OBJECTIVES: The spinal cord is known to be innervated with dopaminergic cells with catecholaminergic projections arising from the medulla and pons and dopaminergic transmission in the spinal cord is vital for sensory and motor function. Our goal was to evaluate and compare the imaging capability of dopamine D2/D3 receptors in the rat spinal cord using PET ligands (18)F-fallypride and (11)C-fallypride. METHODS: Male Sprague-Dawley rats were used in all in vitro and in vivo studies. Spinal cord and brain sections were used for in vitro autoradiography and ex vivo autoradiography. For in vivo studies animals received a (18)F-fallypride scan or a (11)C-fallypride PET scan. The spinal cord and the brain were then harvested, flash-frozen and imaged ex vivo. For in vivo analysis Logan plots with cerebellum as a reference was used to evaluate binding potentials (BP). Tissue ratios were used for ex vivo analysis. Drug effects were evaluated using clozapine, haloperidol and dopamine were evaluated on spinal cord sections in vitro. RESULTS: In vitro studies showed (18)F-fallypride binding to superficial dorsal horn (SDH), dorsal horn (DH), ventral horn (VH) and the pars centralis (PC). In the cervical section, the greatest amount of binding appeared to be in the SDH. Ex vivo studies showed approximately 6% of (18)F-fallypride in SDH compared to that observed in the striatum. In vivo analysis of both (18)F-fallypride and (11)C-fallypride in the spinal cord were comparable to that in the extrastriatal regions. Haloperidol and clozapine displaced more than 75% of the (18)F-fallypride in spinal cord sections. CONCLUSIONS: Our studies showed (18)F-fallypride and (11)C-fallypride binding in the spinal cord in vitro and in vivo. The binding pattern correlates well with the known distribution of dopamine D2/D3 receptors in the spinal cord.

2-deoxy-2-[18F]fluoro-D-mannose positron emission tomography imaging in atherosclerosis.

Progressive inflammation in atherosclerotic plaques is associated with increasing risk of plaque rupture. Molecular imaging of activated macrophages with 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) has been proposed for identification of patients at higher risk for acute vascular events. Because mannose is an isomer of glucose that is taken up by macrophages through glucose transporters and because mannose receptors are expressed on a subset of the macrophage population in high-risk plaques, we applied (18)F-labeled mannose (2-deoxy-2-[(18)F]fluoro-D-mannose, [(18)F]FDM) for targeting of plaque inflammation. Here, we describe comparable uptake of [(18)F]FDM and [(18)F]FDG in atherosclerotic lesions in a rabbit model; [(18)F]FDM uptake was proportional to the plaque macrophage population. Our FDM competition studies in cultured cells with 2-deoxy-2-[(14)C]carbon-D-glucose ([(14)C]2DG) support at least 35% higher [(18)F]FDM uptake by macrophages in cell experiments. We also demonstrate that FDM restricts binding of anti-mannose receptor antibody to macrophages by approximately 35% and that mannose receptor targeting may provide an additional avenue for imaging of plaque inflammation.