Evaluation of Tetrazine Tracers for Pretargeted Imaging within the Central Nervous System.

The pretargeting approach separates the biological half-life of an antibody from the physical half-life of the radioisotope label, providing a strategy for reducing the radiation burden. A widely explored pretargeting approach makes use of the bioorthogonal click reaction between tetrazines (Tzs) and trans-cyclooctenes (TCOs), combining the targeting specificity of monoclonal antibodies (mAbs) with the rapid clearance and precise reaction of Tzs and TCOs. Such a strategy can allow for the targeting and imaging (e.g., by positron emission tomography (PET)) of molecular markers, which cannot be addressed by solely relying on small molecules. Tz derivatives that undergo inverse electron-demand Diels-Alder (IEDDA) reactions with an antibody bearing TCO moieties have been investigated. This study describes the synthesis and characterization of 11 cold Tz imaging agent candidates. These molecules have the potential to be radiolabeled with 18F or 3H, and with the former label, they could be of use as imaging tracers for positron emission tomography studies. Selection was made using a multiparameter optimization score for the central nervous system (CNS) PET tracers. Novel tetrazines were tested for their pH-dependent chemical stability. Those which turned out to be stable in a pH range of 6.5-8 were further characterized in in vitro assays with regard to their passive permeability, microsomal stability, and P-glycoprotein transport. Furthermore, selected Tzs were examined for their systemic clearance and CNS penetration in a single-dose pharmacokinetic study in rats. Two tetrazines were successfully labeled with 18F, one of which showed brain penetration in a biodistribution study in mice. Another Tz was successfully tritium-labeled and used to demonstrate a bioorthogonal click reaction on a TCO-modified antibody. As a result, we identified one Tz as a potential fluorine-18-labeled CNS-PET agent and a second as a 3H-radioligand for an IEDDA-based reaction with a modified brain-penetrating antibody.

Developing a Healthy Lifestyle Program for Recent Kidney Transplant Recipients.

INTRODUCTION: Many kidney transplant recipients experience weight gain in the first year after transplantation. RESEARCH QUESTION: The objective of this research study was to assess the desires of recent kidney transplant patients about the design features of a healthy lifestyle program to counter unnecessary weight gain. DESIGN: In this descriptive study, recent recipients at 2 transplant centers were invited to participate in an online survey. Survey items included sociodemographic information, current medications, health conditions, weight change posttransplant, diet behaviors, physical activity participation, and desired features of a lifestyle program. RESULTS: Fifty-three participants, mean age 60.5 (11.2) years, primarily males, completed surveys. Forty percent gained weight posttransplantation with many indicating struggling with their diet. Physical activity levels stayed the same (17%) or decreased (40%) posttransplantation. Eighty-seven percent of participants indicated they would participate in an online lifestyle program and 76% wanted online physical activity and nutrition sessions to meet at least once weekly. Suggestions about the type of information and activities, included eating strategies (eg, how to eat healthfully at restaurants, grocery shopping tips, and recipes), resources for at-home physical activities, access to cooking classes, and apps to track both activity and food intake. CONCLUSION: Recent kidney transplant recipients would benefit from and desired to join a lifestyle program featuring tailored nutrition education and physical activity coaching. Gathered information will be used to inform and tailor a lifestyle program. Identifying features for the prevention of unnecessary weight gain with patients' input is essential for promoting and sustaining healthy behaviors.

Transcranial photoacoustic imaging of NMDA-evoked focal circuit dynamics in the rat hippocampus.

OBJECTIVE: We report the transcranial functional photoacoustic (fPA) neuroimaging of N-methyl-D-aspartate (NMDA) evoked neural activity in the rat hippocampus. Concurrent quantitative electroencephalography (qEEG) and microdialysis were used to record real-time circuit dynamics and excitatory neurotransmitter concentrations, respectively. APPROACH: We hypothesized that location-specific fPA voltage-sensitive dye (VSD) contrast would identify neural activity changes in the hippocampus which correlate with NMDA-evoked excitatory neurotransmission. MAIN RESULTS: Transcranial fPA VSD imaging at the contralateral side of the microdialysis probe provided NMDA-evoked VSD responses with positive correlation to extracellular glutamate concentration changes. qEEG validated a wide range of glutamatergic excitation, which culminated in focal seizure activity after a high NMDA dose. We conclude that transcranial fPA VSD imaging can distinguish focal glutamate loads in the rat hippocampus, based on the VSD redistribution mechanism which is sensitive to the electrophysiologic membrane potential. SIGNIFICANCE: Our results suggest the future utility of this emerging technology in both laboratory and clinical sciences as an innovative functional neuroimaging modality.

Transcranial Recording of Electrophysiological Neural Activity in the Rodent Brain in vivo Using Functional Photoacoustic Imaging of Near-Infrared Voltage-Sensitive Dye.

Minimally-invasive monitoring of electrophysiological neural activities in real-time-that enables quantification of neural functions without a need for invasive craniotomy and the longer time constants of fMRI and PET-presents a very challenging yet significant task for neuroimaging. In this paper, we present in vivo functional PA (fPA) imaging of chemoconvulsant rat seizure model with intact scalp using a fluorescence quenching-based cyanine voltage-sensitive dye (VSD) characterized by a lipid vesicle model mimicking different levels of membrane potential variation. The framework also involves use of a near-infrared VSD delivered through the blood-brain barrier (BBB), opened by pharmacological modulation of adenosine receptor signaling. Our normalized time-frequency analysis presented in vivo VSD response in the seizure group significantly distinguishable from those of the control groups at sub-mm spatial resolution. Electroencephalogram (EEG) recording confirmed the changes of severity and frequency of brain activities, induced by chemoconvulsant seizures of the rat brain. The findings demonstrate that the near-infrared fPA VSD imaging is a promising tool for in vivo recording of brain activities through intact scalp, which would pave a way to its future translation in real time human brain imaging.

PET imaging of microglia by targeting macrophage colony-stimulating factor 1 receptor (CSF1R).

While neuroinflammation is an evolving concept and the cells involved and their functions are being defined, microglia are understood to be a key cellular mediator of brain injury and repair. The ability to measure microglial activity specifically and noninvasively would be a boon to the study of neuroinflammation, which is involved in a wide variety of neuropsychiatric disorders including traumatic brain injury, demyelinating disease, Alzheimer's disease (AD), and Parkinson's disease, among others. We have developed [11C]CPPC [5-cyano-N-(4-(4-[11C]methylpiperazin-1-yl)-2-(piperidin-1-yl)phenyl)furan-2-carboxamide], a positron-emitting, high-affinity ligand that is specific for the macrophage colony-stimulating factor 1 receptor (CSF1R), the expression of which is essentially restricted to microglia within brain. [11C]CPPC demonstrates high and specific brain uptake in a murine and nonhuman primate lipopolysaccharide model of neuroinflammation. It also shows specific and elevated uptake in a murine model of AD, experimental allergic encephalomyelitis murine model of demyelination and in postmortem brain tissue of patients with AD. Radiation dosimetry in mice indicated [11C]CPPC to be safe for future human studies. [11C]CPPC can be synthesized in sufficient radiochemical yield, purity, and specific radioactivity and possesses binding specificity in relevant models that indicate potential for human PET imaging of CSF1R and the microglial component of neuroinflammation.

Voltage-sensitive dye delivery through the blood brain barrier using adenosine receptor agonist regadenoson.

Optical imaging of brain activity has mostly employed genetically manipulated mice, which cannot be translated to clinical human usage. Observation of brain activity directly is challenging due to the difficulty in delivering dyes and other agents through the blood brain barrier (BBB). Using fluorescence imaging, we have demonstrated the feasibility of delivering the near-infrared voltage-sensitive dye (VSD) IR-780 perchlorate to the brain tissue through pharmacological techniques, via an adenosine agonist (regadenoson). Comparison of VSD fluorescence of mouse brains without and with regadenoson showed significantly increased residence time of the fluorescence signal in the latter case, indicative of VSD diffusion into the brain tissue. Dose and timing of regadenoson were varied to optimize BBB permeability for VSD delivery.

Are dopamine receptor and transporter changes in Rett syndrome reflected in Mecp2-deficient mice?

We tested the claim that the dopaminergic dysfunction of Rett Syndrome (RTT) also occurs in Mecp2-deficient mice that serve as a model of the syndrome. We used positron emission tomography (PET) to image dopamine D2 receptors (D2R) and transporters (DAT) in women with RTT and in Mecp2-deficient mice, and D1R and D2R density was measured in postmortem human tissue by autoradiography. Results showed 1) significantly reduced D2R density in the striatum of women with RTT compared to control subjects. 2) PET imaging of mouse striatum similarly demonstrated significant reductions in D2R density of 7-10 week-old hemizygous (Mecp2-null) and heterozygous (HET) mice compared to wild type (WT) mice. With age, the density of D2R declined in WT mice but not HET mice. 3) In contrast, postmortem autoradiography revealed no group differences in the density of D1R and D2R in the caudate and putamen of RTT versus normal control subjects. 4) In humans and in the mouse model, PET revealed only marginal group differences in DAT. The results confirm that dopaminergic dysfunction in RTT is also present in Mecp2-deficient mice and that reductions in D2R more likely explain the impaired ambulation and progressive rigidity observed rather than alterations in DAT.

Synthesis and Evaluation of a New 18F-Labeled Radiotracer for Studying the GABAB Receptor in the Mouse Brain.

New GABAB agonists, fluoropyridyl ether analogues of baclofen, have been synthesized as potential PET radiotracers. The compound with highest inhibition binding affinity as well as greatest agonist response, ( R)-4-amino-3-(4-chloro-3-((2-fluoropyridin-4-yl)methoxy)phenyl)butanoic acid (1b), was radiolabeled with 18F with good radiochemical yield, high radiochemical purity, and high molar radioactivity. The regional brain distribution of the radiolabeled ( R)-4-amino-3-(4-chloro-3-((2-[18F]fluoropyridin-4-yl)methoxy)phenyl)butanoic acid, [18F]1b, was studied in CD-1 male mice. The study demonstrated that [18F]1b enters the mouse brain (1% ID/g tissue). The accumulation of [18F]1b in the mouse brain was inhibited (35%) by preinjection of GABAB agonist 1a, suggesting that the radiotracer brain uptake is partially mediated by GABAB receptors. The presented data demonstrate a feasibility of imaging of GABAB receptors in rodents and justify further development of GABAB PET tracers with improved specific binding and greater blood-brain barrier permeability.

Brain PET Imaging of α7-nAChR with [18F]ASEM: Reproducibility, Occupancy, Receptor Density, and Changes in Schizophrenia.

Background: The α7 nicotinic acetylcholine receptor increasingly has been implicated in normal brain physiology, as well as in neuropsychiatric disorders. The highly cortical distribution of α7 nicotinic acetylcholine receptor suggests a role in cognition. Methods: We expanded the first-in-human PET imaging of α7 nicotinic acetylcholine receptor with [18F]ASEM from 5 to 21 healthy nonsmoking volunteers and added a feasibility study in 6 male patients with schizophrenia. Study aims included: (1) confirmation of test-retest reproducibility of [18F]ASEM binding, (2) demonstration of specificity by competition with DMXB-A, an α7 nicotinic acetylcholine receptor partial agonist, (3) estimation of [18F]ASEM binding potentials and α7 nicotinic acetylcholine receptor density in vivo in humans, and (4) demonstrating the feasibility of studying α7 nicotinic acetylcholine receptor as a target for schizophrenia. Results: Test-retest PET confirmed reproducibility (>90%) (variability ≤7%) of [18F]ASEM volume of distribution (VT) estimates in healthy volunteers. Repeated sessions of PET in 5 healthy subjects included baseline and effect of inhibition after oral administration of 150 mg DMXB-A. From reduction of binding potentials, we estimated the dose-dependent occupancy of α7 nicotinic acetylcholine receptor by DMXB-A at 17% to 49% for plasma concentrations at 60 to 200 nM DMXB-A. In agreement with evidence postmortem, α7 nicotinic acetylcholine receptor density averaged 0.67 to 0.82 nM and inhibitor affinity constant averaged 170 to 385 nM. Median VT in a feasibility study of 6 patients with schizophrenia was lower than in healthy volunteers in cingulate cortex, frontal cortex, and hippocampus (P = 0.02, corrected for multiple comparions, Mann-Whitney test). Conclusions: The current results confirm the reproducibility of [18F]ASEM VT estimates and the specificity of the tracer for α7 nicotinic acetylcholine receptor. Preliminary findings from our feasibility study of [18F]ASEM binding in patients with schizophrenia are suggestive and provide guidance for future studies with more subjects.

Feasibility Evaluation of Myocardial Cannabinoid Type 1 Receptor Imaging in Obesity: A Translational Approach.

OBJECTIVES: The aim of this study was to evaluate the feasibility of targeted imaging of myocardial cannabinoid type 1 receptor (CB1-R) and its potential up-regulation in obese mice with translation to humans using [11C]-OMAR and positron emission tomography (PET)/computed tomography (CT). BACKGROUND: Activation of myocardial CB1-R by endocannabinoids has been implicated in cardiac dysfunction in diabetic mice. Obesity may lead to an up-regulation of myocardial CB1-R, potentially providing a mechanistic link between obesity and the initiation and/or progression of cardiomyopathy. METHODS: Binding specificity of [11C]-OMAR to CB1-R was investigated by blocking studies with rimonabant in mice. The heart was harvested from each mouse, and its radioactivity was determined by γ-counter. Furthermore, [11C]-OMAR dynamic micro-PET/CT was carried out in obese and normal-weight mice. Ex vivo validation was performed by droplet digital polymerase chain reaction (absolute quantification) and RNAscope Technology (an in situ ribonucleic acid analysis platform). Subsequently, myocardial CB1-R expression was probed noninvasively with intravenous injection of CB1-R ligand [11C]-OMAR and PET/CT in humans with advanced obesity and normal-weight human control subjects, respectively. RESULTS: Rimonabant significantly blocked OMAR uptake in the heart muscle compared with vehicle, signifying specific binding of OMAR to the CB1-R in the myocardium. The myocardial OMAR retention quantified by micro-PET/CT in mice was significantly higher in obese compared with normal-weight mice. Absolute quantification of CB1-R gene expression with droplet digital polymerase chain reaction and in situ hybridization confirmed CB1-R up-regulation in all major myocardial cell types (e.g., cardiomyocytes, endothelium, vascular smooth muscle cells, and fibroblasts) of obese mice. Obese mice also had elevated myocardial levels of endocannabinoids anandamide and 2-arachidonoylglycerol compared with lean mice. Translation to humans revealed higher myocardial OMAR retention in advanced obesity compared with normal-weight subjects. CONCLUSIONS: Noninvasive imaging of cardiac CB1-R expression in obesity is feasible applying [11C]-OMAR and PET/CT. These results may provide a rationale for further clinical testing of CB1-R-targeted molecular imaging in cardiometabolic diseases.

Preclinical Evaluation of 18F-RO6958948, 11C-RO6931643, and 11C-RO6924963 as Novel PET Radiotracers for Imaging Tau Aggregates in Alzheimer Disease.

Tau aggregates and amyloid-ß (Aß) plaques are key histopathologic features in Alzheimer disease (AD) and are considered targets for therapeutic intervention as well as biomarkers for diagnostic in vivo imaging agents. This article describes the preclinical in vitro and in vivo characterization of 3 novel compounds-RO6958948, RO6931643, and RO6924963-that bind specifically to tau aggregates and have the potential to become PET tracers for future human use. Methods: RO6958948, RO6931643, and RO6924963 were identified as high-affinity competitors at the 3H-T808 binding site on native tau aggregates in human late-stage AD brain tissue. Binding of tritiated compounds to brain tissue sections of AD patients and healthy controls was analyzed by macro- and microautoradiography and by costaining of tau aggregates and Aß plaques on the same tissue section using specific antibodies. All 3 tracer candidates were radiolabeled with a PET nuclide and tested in vivo in tau-naïve baboons to assess brain uptake, distribution, clearance, and metabolism. Results:3H-RO6958948, 3H-RO6931643, and 3H-RO6924963 bound with high affinity and specificity to tau aggregates, clearly lacking affinity for concomitant Aß plaques in human AD Braak V tissue sections. The specificity of all 3 radioligands for tau aggregates was supported, first, by binding patterns in AD sections comparable to the tau-specific radioligand 3H-T808; second, by very low nonspecific binding in brain tissue devoid of tau pathology, excluding significant radioligand binding to any other central nervous system target; and third, by macroscopic and microscopic colocalization and quantitative correlation of radioligand binding and tau antibody staining on the same tissue section. RO6958948, RO6931643, and RO6924963 were successfully radiolabeled with a PET nuclide at high specific activity, radiochemical purity, and yield. After intravenous administration of 18F-RO6958948, 11C-RO6931643, and 11C-RO6924963 to baboons, PET scans indicated good brain entry, rapid washout, and a favorable metabolism pattern. Conclusion:18F-RO6958948, 11C-RO6931643, and 11C-RO6924963 are promising PET tracers for visualization of tau aggregates in AD. Head-to-head comparison and validation of these tracer candidates in AD patients and healthy controls will be reported in due course.

Development of a radioligand for imaging V1a vasopressin receptors with PET.

A series of vasopressin receptor V1a ligands have been synthesized for positron emission tomography (PET) imaging. The lead compound (1S,5R)-1 ((4-(1H-indol-3-yl)-3-methoxyphenyl) ((1S,5R)-1,3,3-trimethyl-6-azabicyclo[3.2.1]octan-6-yl)methanone) and its F-ethyl analog 6c exhibited the best combination of high binding affinity and optimal lipophilicity within the series. (1S,5R)-1 was radiolabeled with 11C for PET studies. [11CH3](1S,5R)-1 readily entered the mouse (4.7% ID/g tissue) and prairie vole brains (∼2% ID/g tissue) and specifically (30-34%) labeled V1a receptor. The common animal anesthetic Propofol significantly blocked the brain uptake of [11CH3](1S,5R)-1 in the mouse brain, whereas anesthetics Ketamine and Saffan increased the uptake variability. Future PET imaging studies with V1a radiotracers in non-human primates should be performed in awake animals or using anesthetics that do not affect the V1a receptor.

Identification of Three Novel Radiotracers for Imaging Aggregated Tau in Alzheimer's Disease with Positron Emission Tomography.

Aggregates of tau and beta amyloid (Aß) plaques constitute the histopathological hallmarks of Alzheimer's disease and are prominent targets for novel therapeutics as well as for biomarkers for diagnostic in vivo imaging. In recent years much attention has been devoted to the discovery and development of new PET tracers to image tau aggregates in the living human brain. Access to a selective PET tracer to image and quantify tau aggregates represents a unique tool to support the development of any novel therapeutic agent targeting pathological forms of tau. The objective of the study described herein was to identify such a novel radiotracer. As a result of this work, we discovered three novel PET tracers (2-(4-[11C]methoxyphenyl)imidazo[1,2-a]pyridin-7-amine 7 ([11C]RO6924963), N-[11C]methyl-2-(3-methylphenyl)imidazo[1,2-a]pyrimidin-7-amine 8 ([11C]RO6931643), and [18F]2-(6-fluoropyridin-3-yl)pyrrolo[2,3-b:4,5-c']dipyridine 9 ([18F]RO6958948)) with high affinity for tau neurofibrillary tangles, excellent selectivity against Aß plaques, and appropriate pharmacokinetic and metabolic properties in mice and non-human primates.

Development of a High-Affinity PET Radioligand for Imaging Cannabinoid Subtype 2 Receptor.

Cannabinoid receptors type 2 (CB2) represent a target with increasing importance for neuroimaging due to its upregulation under various pathological conditions. Encouraged by preliminary results obtained with [(11)C](Z)-N-(3-(2-methoxyethyl)-4,5-dimethylthiazol-2(3H)-ylidene)-2,2,3,3-tetramethyl-cyclopropanecarboxamide ([(11)C]A-836339, [(11)C]1) in a mouse model of acute neuroinflammation (induced by lipopolysaccharide, LPS), we designed a library of fluorinated analogues aiming for an [(18)F]-labeled radiotracer with improved CB2 binding affinity and selectivity. Compound (Z)-N-(3-(4-fluorobutyl)-4,5-dimethylthiazol-2(3H)-ylidene)-2,2,3,3-tetramethyl-cyclopropanecarboxamide (29) was selected as the ligand with the highest CB2 affinity (Ki = 0.39 nM) and selectivity over those of CB1 (factor of 1000). [(18)F]29 was prepared starting from the bromo precursor (53). Specific binding was shown in vitro, whereas fast metabolism was observed in vivo in CD-1 mice. Animal PET revealed a brain uptake comparable to that of [(11)C]1. In the LPS-treated mice, a 20-30% higher uptake in brain was found in comparison to that in nontreated mice (n = 3, P < 0.05).

Quantitative Multi-modal Brain Autoradiography of Glutamatergic, Dopaminergic, Cannabinoid, and Nicotinic Receptors in Mutant Disrupted-In-Schizophrenia-1 (DISC1) Mice.

PURPOSE: Disrupted-in-schizophrenia-1 (DISC1) is a promising genetic susceptibility factor for major psychiatric conditions, such as schizophrenia. We hypothesized that the mutant DISC1 alters the homeostasis of multi-receptor interactions between dopaminergic [dopamine 2/3 (D(2/3)R)], glutamatergic [metabotropic glutamate 5 (mGluR5)], cannabinoid 1 (CB(1)R), and nicotinic acetylcholine (α4ß2-nAChR) receptors in the brains of mice with inducible forebrain neuronal expression of dominant-negative mutant DISC1. PROCEDURES: The quantitative in vitro autoradiography was performed with positron emission tomography (PET) ligands using [(11)C]raclopride (D2/3R), [(11)C]ABP688 (mGluR5), [(11)C]OMAR (CB(1)R), and [(18)F]AZAN (nAChR). Total binding (pmol/cc) from standard and binding index, defined as [(region of interest - reference) / reference], was analyzed in the parasagittal sections. The cerebellum was used as a reference for D(2/3)R, mGluR5, and α4ß2-nAChR, while the midbrain was the reference tissue for CB(1)R, because of the high density of CB(1)R in the cerebellum. RESULTS: We observed a significant positive correlation between mGluR5 and D2/3R in the nucleus accumbens (NAc) in mutant DISC1 (rho = 0.6, p = 0.04; y = 0.02 x + 6.7) and a trend of negative correlation between those receptors in the dorsal striatum (DS) in control animals (rho = -0.5, p = 0.09; y = -0.03 x + 23), suggesting a co-release of dopamine (DA) and glutamate (Glu) in the NAc, but not in the DS. There were trends of an inverse relationship between striatal CB(1)R and D(2/3)R (rho = -0.7, p = 0.07) as well as between dorsal thalamic nAChR and striatal D2/3R (rho = -0.5, p = 0.08). There was no statistically significant difference of the individual receptor density in the majority of brain regions. CONCLUSIONS: The mutant DISC1 altered the homeostasis of multi-receptor interactions of coincident signaling of DA and Glu in the NAc, but not in the DS, and mutually negative control of striatal CB(1)R and D2/3R. Multi-receptor mapping with PET ligands in relevant animal models could be a valuable translational approach for psychiatric drug development.

Human brain imaging of α7 nAChR with [(18)F]ASEM: a new PET radiotracer for neuropsychiatry and determination of drug occupancy.

PURPOSE: Using the α7-nAChR radiotracer, [(18)F]ASEM, we present the first successful human positron emission tomography (PET) studies. Rodent occupancy with three clinically employed α7-nAChR drugs confirms the specificity of the radiotracer. PROCEDURES: Five healthy male subjects were imaged for 90 min following IV [(18)F]ASEM. Two subjects were scanned for the second time (test/retest; TRV). Mouse biodistribution of [(18)F]ASEM was carried out in CD1 mice injected with using human equivalent doses of DMXB-A, EVP-6124, and varenicline to block specific binding. RESULTS: [(18)F]ASEM readily entered the brain and peaked at 15 min post-injection with reversible kinetics and a peak %SUV of about 400 %. The regional human brain distribution of [(18)F]ASEM matched previous in vitro data and baboon PET results. The precuneus, parietal, occipital, cingulate cortexes, putamen, and thalamus showed high values of distribution volume (>20 ml/ml) and binding potentials >1 with TRV averaged 10.8 ± 5.1 %. In mouse distribution studies, there was significant dose-dependent blockade in the mouse brain with DMXB-A as well as the other two α7-nAChR drugs. CONCLUSIONS: The characteristics of [(18)F]ASEM are consistent with the ability to quantify α7-nAChR in the human brain. [(18)F]ASEM is suitable for imaging neuropsychiatric disorders and target engagement (receptor occupancy) of potential α7-nAChR drugs.

Dose-dependent, saturable occupancy of the metabotropic glutamate subtype 5 receptor by fenobam as measured with [11 C]ABP688 PET imaging.

Fenobam is a negative allosteric modulator of the metabotropic glutamate receptor subtype 5 (mGluR5) with inverse agonist activity and is expected to contribute to the treatment of neuropsychiatric disorders involving dysfunction of mGluR5 including Fragile X syndrome. This study examined whether [11 C]ABP688, an antagonist PET radioligand, competes with fenobam for the same binding site in the nonhuman primate brain and would allow examination of occupancy-plasma concentration relationships in the evaluation of the drug for target disorders in the human brain. Four paired PET studies with [11 C]ABP688 were performed in baboons at a baseline condition and after intravenous treatment with fenobam at different dose levels (0.3-1.33 mg/kg). Total distribution volume (VT ) and binding potential (BPND ) using the cerebellum as a reference region were obtained by the plasma reference graphical method. Then it was examined whether occupancy follows a dose-dependent, saturating pattern that was predicted by a modified first-order Hill equation in individual regions. Baseline regional VT and BPND values agreed with previously published data. Occupancy showed dose-dependent and saturating patterns in individual regions, reaching >90% occupancy at 1.33 mg/kg dose of fenobam in the majority of regions. To our knowledge, this is the first use of PET to characterize the mGluR5 therapeutic drug fenobam. This study demonstrates a proof of principle for determining the in vivo occupancy of fenobam in primates. The results indicate that [11 C]ABP688 and PET may be useful for examination of occupancy of mGluR5 by fenobam, which should prove to be useful for designing future studies and treatment of human disease states. Synapse 68:565-573, 2014. © 2014 Wiley Periodicals, Inc.

PET imaging of high-affinity α4ß2 nicotinic acetylcholine receptors in humans with 18F-AZAN, a radioligand with optimal brain kinetics.

UNLABELLED: We evaluated (-)-2-(6-[(18)F]fluoro-2,3'-bipyridin-5'-yl)-7-methyl-7-aza-bicyclo[2.2.1]heptane ((18)F-AZAN), a novel radiotracer that binds to α4ß2 nicotinic acetylcholine receptors (α4ß2-nAChRs) and shows high specific binding and rapid and reversible kinetics in the baboon and human brain. METHODS: We tested safety tolerability and test-retest reliability (n = 5) and proposed initial quantification of (18)F-AZAN receptors in 3 healthy human subjects who had nicotine exposure and 9 who did not. We also present a receptor blocking study in a nicotine subject dosed with the α4ß2-nAChR-selective partial agonist varenicline. RESULTS: Radiation dosimetry PET/CT experiments indicated that most human organs received doses between 0.008 and 0.015 mSv/MBq, with an effective dose of approximately 0.014 mSv/MBq. The tracer rapidly entered the brain, and the peak was reached before 20 min, even for thalamus. Ninety-minute scans were sufficient for (18)F-AZAN to obtain the ratio at equilibrium of specifically bound radioligand to nondisplaceable radioligand in tissue (BPND) using plasma reference graphical analysis, which showed excellent reproducibility of BPND (test-retest variability < 10%) in the nAChR-rich brain regions. Regional plasma reference graphical analysis BP(ND) values exceeded 2 in the midbrain tegmental nuclei, lateral geniculate body, and thalamus for nonsmokers (n = 9) but were less than 1 in the nAChR-poor brain regions. There was a dramatic reduction of (18)F-AZAN brain uptake in smokers and varenicline-treated subjects. CONCLUSION: (18)F-AZAN is a highly specific, safe, and effective PET radioligand for human subjects that requires only 90 min of PET scanning to estimate high-affinity α4ß2-nAChR in the living human brain.

Synthesis and evaluation of new radioligands [(11)C]A-833834 and [(11)C]A-752274 for positron-emission tomography of α7-nicotinic acetylcholine receptors.

INTRODUCTION: α7-nicotinic acetylcholine receptor (α7-nAChR) is one of the major neuronal nAChR subtypes. α7-nAChR is involved in variety of neuronal processes and disorders including schizophrenia and Alzheimer's disease. A number of α7-nAChR PET radioligands have been developed, but a quality radiotracer remains to be discovered. METHODS: High binding affinity α7-nAChR ligands A-833834 and A-752274 were radiolabeled with (11)C. Baseline and blockade biodistribution studies in the mouse brain of [(11)C]A-833834 (5-(6-(5-[(11)C]methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyridazin-3-yl)-1H-indole) and [(11)C]A-752274 (2-(6-[(11)C]methyl-3,6-diazabicyclo[3.2.0]heptan-3-yl)-7-(6-methyl-3,6-diazabicyclo[3.2.0]heptan-3-yl)-9H-fluoren-9-one) were performed. [(11)C]A-752274 was evaluated in a baseline baboon PET study. RESULTS: [(11)C]A-833834 and [(11)C]A-752274 were synthesized by radiomethylation of corresponding des-methyl precursors. The radioligands were prepared with radiochemical yield of 12%-32%, high specific radioactivity (330-403GBq/µmol) and radiochemical purity>95%. Dissection studies with [(11)C]A-833834 demonstrated low specific α7-nAChR binding in the mouse brain. [(11)C]A-752274 specifically (~50%) labeled α7-nAChR in the mouse thalamus. However, [(11)CA-752274 exhibited low brain uptake in baboon (%SUV<100). CONCLUSION: Two novel α7-nAChR ligands radioligands were synthesized and studied in animals. Specific binding of [(11)C]A-833834 in the mouse brain is low due to the insufficient binding affinity of the radioligand. The very high binding affinity [(11)C]A-752274 exhibited good specific binding in the α7-nAChR-rich mouse brain regions. The low uptake of [(11)C]A-752274 in the baboon brain is due to its high hydrophilicity, rapid metabolism or other properties. Future development of α7-nAChR PET radioligands will be based on compounds with high binding affinities and good blood-brain barrier permeability.

5-(5-(6-[(11)C]methyl-3,6-diazabicyclo[3.2.0]heptan-3-yl)pyridin-2-yl)-1H-indole as a potential PET radioligand for imaging cerebral α7-nAChR in mice.

The radiosynthesis and in vivo evaluation of 5-(5-(6-[(11)C]methyl-3,6-diazabicyclo[3.2.0]heptan-3-yl)pyridin-2-yl)-1H-indole [(11)C]rac-(1), a potential PET tracer for α7 nicotinic acetylcholine receptors (α7-nAChR), are described. Syntheses of the nonradioactive standard rac-1 and corresponding desmethyl precursor 7 were achieved in several reaction steps. Radiomethylation of 7 with [(11)C]CH(3)I afforded [(11)C]rac-1 in an average radiochemical yield of 30 ± 5% (n=5) with high radiochemical purity and an average specific radioactivity of 444 ± 74 GBq/µmol (n=5). The total synthesis time was 30 min from end-of-bombardment. Biodistribution studies in mice showed that [(11)C]rac-1 penetrates the blood-brain barrier and specifically labels neuronal α7-nAChRs.