An updated synthesis of [11 C]carfentanil for positron emission tomography (PET) imaging of the µ-opioid receptor.

[11 C]Carfentanil ([11 C]CFN) is a selective radiotracer for in vivo positron emission tomography imaging studies of the µ-opioid system that, in our laboratories, is synthesized by methylation of the corresponding carboxylate precursor with [11 C]MeOTf, and purified using a C2 solid-phase extraction cartridge. Changes in the commercial availability of common C2 cartridges have necessitated future proofing the synthesis of [11 C]CFN to maintain reliable delivery of the radiotracer for clinical imaging studies. An updated synthesis of [11 C]CFN is reported that replaces a now obsolete purification cartridge with a new commercially available version and also substitutes the organic solvents used in traditional production methods with ethanol.

PET radioligands for the vesicular transporters for monoamines and acetylcholine.

The vesicular transporters for the monoamine and acetylcholine have been successfully targeted for the development of radioligands for human brain imaging. The vesicular monoamine transporter type 2 ligands are based on the structure of tetrabenazine, a known clinically used drug. In contrast, the radioligands for vesicular acetylcholine transporter are based on vesamicol, a toxic xenobiotic. The similarities and differences in the development of these two classes of radioligands are discussed.

(-)-[(18) F]Flubatine: evaluation in rhesus monkeys and a report of the first fully automated radiosynthesis validated for clinical use.

(-)-[(18) F]Flubatine was selected for clinical imaging of α4 ß2 nicotinic acetylcholine receptors because of its high affinity and appropriate kinetic profile. A fully automated synthesis of (-)-[(18) F]flubatine as a sterile isotonic solution suitable for clinical use is reported, as well as the first evaluation in nonhuman primates (rhesus macaques). (-)-[(18) F]Flubatine was prepared by fluorination of the Boc-protected trimethylammonium iodide precursor with [(18) F]fluoride in an automated synthesis module. Subsequent deprotection of the Boc group with 1-M HCl yielded (-)-[(18) F]flubatine, which was purified by semi-preparative HPLC. (-)-[(18) F]Flubatine was prepared in 25% radiochemical yield (formulated for clinical use at end of synthesis, n = 3), >95% radiochemical purity, and specific activity = 4647 Ci/mmol (171.9 GBq/µmol). Doses met all quality control criteria confirming their suitability for clinical use. Evaluation of (-)-[(18) F]flubatine in rhesus macaques was performed with a Concorde MicroPET P4 scanner (Concorde MicroSystems, Knoxville, TN). The brain was imaged for 90 min, and data were reconstructed using the 3-D maximum a posteriori algorithm. Image analysis revealed higher uptake and slower washout in the thalamus than those in other areas of the brain and peak uptake at 45 min. Injection of 2.5 µg/kg of nifene at 60 min initiated a slow washout of [(18) F]flubatine, with about 25% clearance from the thalamus by the end of imaging at 90 min.

Assessment of mild dementia with amyloid and dopamine terminal positron emission tomography.

We assessed the relationship between consensus clinical diagnostic classification and neurochemical positron emission tomography imaging of striatal vesicular monoamine transporters and cerebrocortical deposition of aß-amyloid in mild dementia. Seventy-five subjects with mild dementia (Mini-Mental State Examination score≥18) underwent a conventional clinical evaluation followed by 11C-dihydrotetrabenazine positron emission tomography imaging of striatal vesicular monoamine transporters and 11C-Pittsburgh compound-B positron emission tomography imaging of cerebrocortical aß-amyloid deposition. Clinical classifications were assigned by consensus of an experienced clinician panel. Neuroimaging classifications were assigned as Alzheimer's disease, frontotemporal dementia or dementia with Lewy bodies on the basis of the combined 11C-dihydrotetrabenazine and 11C-Pittsburgh compound-B results. Thirty-six subjects were classified clinically as having Alzheimer's disease, 25 as having frontotemporal dementia and 14 as having dementia with Lewy bodies. Forty-seven subjects were classified by positron emission tomography neuroimaging as having Alzheimer's disease, 15 as having dementia with Lewy bodies and 13 as having frontotemporal dementia. There was only moderate agreement between clinical consensus and neuroimaging classifications across all dementia subtypes, with discordant classifications in ∼35% of subjects (Cohen's κ=0.39). Discordant classifications were least frequent in clinical consensus Alzheimer's disease (17%), followed by dementia with Lewy bodies (29%) and were most common in frontotemporal dementia (64%). Accurate clinical classification of mild neurodegenerative dementia is challenging. Though additional post-mortem correlations are required, positron emission tomography imaging likely distinguishes subgroups corresponding to neurochemically defined pathologies. Use of these positron emission tomography imaging methods may augment clinical classifications and allow selection of more uniform subject groups in disease-modifying therapeutic trials and other prospective research involving subjects in the early stages of dementia.

Leucoaraiosis, nigrostriatal denervation and motor symptoms in Parkinson's disease.

Leucoaraiosis is associated with motor symptoms in otherwise normal older adults. Comorbid leucoaraiosis is predicted to contribute also to motor features in Parkinson's disease but previous studies of white matter changes in Parkinson's disease show variable results. No prior studies have compared directly the effects of both leucoaraiosis and the degree of nigrostriatal dopaminergic denervation on motor features. We investigated the effect of leucoaraiosis severity on motor impairment independent of the degree of nigrostriatal dopaminergic denervation in Parkinson's disease. Seventy-three subjects with Parkinson's disease (Hoehn and Yahr stages 1-3) underwent brain magnetic resonance and [(11)C]dihydrotetrabenazine vesicular monoamine transporter type 2 positron emission tomography imaging. Automated assessment of supratentorial fluid-attenuated inversion recovery magnetic resonance hyperintense white matter voxels was performed using cerebellar white matter as the intensity reference. White matter signal hyperintensity burden was log-transformed and normalized for brain volume. Unified Parkinson's Disease Rating Scale total and subscore ratings were assessed to determine motor impairment. Subjects receiving dopaminergic medications were examined in the clinically defined 'OFF' state. Multivariate regression analysis with measures of white matter signal hyperintensity burden and nigrostriatal denervation as independent variables demonstrated a significant overall model for total motor Unified Parkinson's Disease Rating Scale scores (F = 11.4, P < 0.0001) with significant regression effects for both white matter signal hyperintensity burden (t = 2.0, ß = 0.22, P = 0.045) and striatal monoaminergic binding (t = -3.5, ß = -0.38, P = 0.0008). Axial motor impairment demonstrated a robust association with white matter signal hyperintensity burden (t = 4.0, ß = 0.43, P =0.0001) compared with striatal monoaminergic binding (t = -2.1, ß = 0.22, P = 0.043). White matter signal hyperintensity burden regression effects for bradykinesia had borderline significance. No significant white matter signal hyperintensity burden effects were found for rigidity or tremor subscores. White matter signal hyperintensity burden was significantly higher in the subgroup with postural instability and gait difficulties compared with the tremor-predominant subgroup despite no significant differences in age or duration of disease. These findings indicate that increased white matter signal hyperintensity burden is associated with worse motor performance independent of the degree of nigrostriatal dopaminergic denervation in Parkinson's disease. Comorbid white matter disease is a greater determinant of axial motor impairment than nigrostriatal dopaminergic denervation.

11C- and 18F-Radiotracers for In Vivo Imaging of the Dopamine System: Past, Present and Future.

The applications of positron emission tomography (PET) imaging to study brain biochemistry, and in particular the aspects of dopamine neurotransmission, have grown significantly over the 40 years since the first successful in vivo imaging studies in humans. In vivo PET imaging of dopaminergic functions of the central nervous system (CNS) including dopamine synthesis, vesicular storage, synaptic release and receptor binding, and reuptake processes, are now routinely used for studies in neurology, psychiatry, drug abuse and addiction, and drug development. Underlying these advances in PET imaging has been the development of the unique radiotracers labeled with positron-emitting radionuclides such as carbon-11 and fluorine-18. This review focuses on a selection of the more accepted and utilized PET radiotracers currently available, with a look at their past, present and future.

In vitro binding affinity vs. in vivo site occupancy: A PET study of four diastereomers of dihydrotetrabenazine (DTBZ) in monkey brain.

INTRODUCTION: In vivo imaging methods such as Positron Emission Tomography (PET) can be used to examine the relationship between in vitro binding affinity and in vivo occupancy of binding sites in the brain for new drug candidates. In this study, PET imaging in monkey brain was used to evaluate that correlation for a set of four diastereomers of the compound dihydrotetrabenazine (DTBZ), the pharmacologically active metabolite of the drug tetrabenazine. METHODS: PET studies of DTBZ diastereomers were completed in a single monkey brain. In vivo occupancies (ED50) were estimated using multiple drug doses and the vesicular monoamine transporter 2 specific radioligand (+)-α-[11C] DTBZ, employing a test-retest sequence of control PET scan, drug administration and a second PET scan completed on a single day. RESULTS: DTBZ has three chiral carbon centers and eight possible stereoisomers, and in vivo occupancy of the target site VMAT2 was observed only for the four diastereomers of DTBZ having the 11bR absolute configuration. The estimated in vivo occupancies (ED50 values from 0.023 to >3.15 mg/kg) correlated well (R2 = 0.95) with the in vitro binding affinities (Ki values of 4 to 600 nM for the VMAT2), and an even better correlation (R2 = 0.99) was found for the three isomers with in vitro binding affinities <100 nM. CONCLUSIONS: If the physiochemical (MW, log P, pKa) or physiological (metabolism, transport, protein binding) properties of a set of drug stereoisomers are considered similar, the binding affinities determined from in vitro assays may predict the in vivo occupancies of the target binding site in the monkey brain.

Classics in Neuroimaging: Imaging the Cholinergic System with Positron Emission Tomography.

Since the earliest days of nuclear medicine, there has been interest in using PET and SPECT imaging to interrogate and quantify the cholinergic system. In this Viewpoint we highlight key milestones in the development of cholinergic imaging agents, including identification of radiopharmaceuticals targeting the receptors, transporters, and enzymes of the cholinergic synapse, as well as fundamental developments in the radiopharmaceutical sciences (e.g., cyclotron targetry, radiochemistry) that have enabled translation of the most promising agents into clinical use. We also provide an overview of the current state-of-the-art in cholinergic PET imaging, with an emphasis on radiotracers that are in human studies at PET centers around the world.

Classics in Neuroimaging: Shedding Light on Opioid Receptors with Positron Emission Tomography Imaging.

Imaging of the opioid system was one of the earliest applications of Positron Emission Tomography (PET) imaging in neuroscience that remains in widespread use today and in the age of the opioid crisis the technique is as important as ever. In this viewpoint the rich history of opioid imaging using PET is highlighted, including discussion of the preferred radiotracers for imaging of µ, δ, κ and ORL-1 receptors in clinical applications. We also draw attention to key innovations that were essential to development of radiotracers for imaging opioid receptors including production of high molar activity PET radionuclides and new approaches to radiochemistry.

Improved Synthesis of [11C]COU and [11C]PHXY, Evaluation of Neurotoxicity, and Imaging of MAOs in Rodent Heart.

The radiotracers [11C]COU and [11C]PHXY are potential PET imaging agents for in vivo studies of monoamine oxidases (MAOs), as previously shown in rodent and primate brain. One-pot, automated methods for the radiosynthesis of [11C]PHXY and [11C]COU were developed to provide reliable and improved radiochemical yields. Although derived from the structure of the neurotoxin MPTP, COU did not exhibit in vivo neurotoxicity to dopaminergic nerve terminals in the mouse brain as assayed by losses of VMAT2 radioligand binding. PET imaging studies in rats demonstrated that both [11C]COU and [11C]PHXY exhibit retention in cardiac tissues that can be blocked by pretreatment with the MAO inhibitors deprenyl (MAO-B) and pargyline (MAO-A and -B). In addition to prior neuroimaging applications, [11C]COU and [11C]PHXY are thus also of interest for studies of MAO enzymatic activity and imaging of sympathetic nerve density in heart.

Classics in Neuroimaging: Development of Positron Emission Tomography Tracers for Imaging the GABAergic Pathway.

Advances in drug discovery and diverse radiochemical methodologies have led to the discovery of novel positron emission tomography (PET) radiotracers used to image the GABAergic system, shaping our fundamental understanding of a variety of brain health illnesses, including epilepsy, stroke, cerebral palsy, schizophrenia, autism, Alzheimer's disease, and addictions. In this Viewpoint, we review the state-of-the art of PET imaging with radiotracers that target the GABAA-benzodiazepine receptor complex, challenges and opportunities for imaging GABAB receptors and GABA transporters, and highlight an ongoing need to develop more sensitive radiotracers for imaging GABA release in the central nervous system.

Use of 55 PET radiotracers under approval of a Radioactive Drug Research Committee (RDRC).

BACKGROUND: In the US, EU and elsewhere, basic clinical research studies with positron emission tomography (PET) radiotracers that are generally recognized as safe and effective (GRASE) can often be conducted under institutional approval. For example, in the United States, such research is conducted under the oversight of a Radioactive Drug Research Committee (RDRC) as long as certain requirements are met. Firstly, the research must be for basic science and cannot be intended for immediate therapeutic or diagnostic purposes, or to determine the safety and effectiveness of the PET radiotracer. Secondly, the PET radiotracer must be generally recognized as safe and effective. Specifically, the mass dose to be administered must not cause any clinically detectable pharmacological effect in humans, and the radiation dose to be administered must be the smallest dose practical to perform the study and not exceed regulatory dose limits within a 1-year period. In our experience, the main barrier to using a PET radiotracer under RDRC approval is accessing the required information about mass and radioactive dosing. RESULTS: The University of Michigan (UM) has a long history of using PET radiotracers in clinical research studies. Herein we provide dosing information for 55 radiotracers that will enable other PET Centers to use them under the approval of their own RDRC committees. CONCLUSIONS: The data provided herein will streamline future RDRC approval, and facilitate further basic science investigation of 55 PET radiotracers that target functionally relevant biomarkers in high impact disease states.

Classics in Neuroimaging: Radioligands for the Vesicular Monoamine Transporter 2.

Positron emission tomography (PET) studies of the monoamine neurotransmitter systems in the human brain employ a variety of radiotracers targeting the many receptors, transporters, and enzymes present in monoaminergic neurons. One of these is the vesicular monoamine transporter 2 (VMAT2), the protein responsible for the energy-dependent accumulation of monoamines into synaptic vesicles. The development of in vivo imaging radiotracers for VMAT2 is a story of starting with a well-characterized clinically used drug (tetrabenazine) which had a pharmacologically active metabolite: that metabolite that was in stepwise fashion refined and modified to provide both carbon-11 and fluorine-18 labeled VMAT2 radiotracers that are now used for human PET studies of neurodegenerative and psychiatric diseases. The design approach taken, which involved understanding the metabolism of the radiotracers and identification of the optimal ligand stereochemistry, are representative of important steps in the general concepts behind successful in vivo radiotracer design for brain imaging agents.

Identification of AV-1451 as a Weak, Nonselective Inhibitor of Monoamine Oxidase.

[18F]AV-1451 is one of the most widely used radiotracers for positron emission tomography (PET) imaging of tau protein aggregates in neurodegenerative disorders. While the radiotracer binds with high affinity to tau neurofibrillary tangles, extensive clinical studies have simultaneously revealed off-target tracer accumulation in areas of low tau burden such as the basal ganglia and choroid plexus. Though there are a number of possible reasons for this accumulation, it is often attributed to off-target binding to monoamine oxidase (MAO). In this paper, we investigate the association between [18F]AV-1451 and MAO through (i) enzyme inhibition assays, (ii) autoradiography with postmortem tissue samples, and (iii) nonhuman primate PET imaging. We confirm that [18F]AV-1451 is a weak inhibitor of MAO-A and -B and that MAO inhibitors can alter binding of [18F]AV-1451 in autoradiography and in vivo PET imaging.

Classics in Neuroimaging: Development of PET Tracers for Imaging Monoamine Oxidases.

In this Viewpoint, we highlight the history of positron emission tomography (PET) radiotracer development to quantify changes in monoamine oxidase (MAO)-A and -B enzyme expression or activity. MAO-A and MAO-B are critical for understanding monoaminergic pathways in psychiatric addiction disorders, and more recently in neurodegenerative disorders with MAO-B expression in astrogliosis. Unique radiochemical innovations have been shown for neuroimaging of MAOs including the clinical translation of irreversible propargylamine-based suicide inhibitors, application of deuterium-substitution to slow down metabolism, development of trapped metabolite imaging agents, and unique 11C-carbonylation chemistry toward novel high-affinity reversibly binding inhibitors.

In vivo imaging of beta-cell mass in rats using 18F-FP-(+)-DTBZ: a potential PET ligand for studying diabetes mellitus.

UNLABELLED: Recent studies on gene expression of beta-cell mass (BCM) in the pancreas showed that vesicular monoamine transporter 2 (VMAT2) is highly expressed in the BCM (mainly in the islets of Langerhans). Imaging pancreatic BCM may provide an important tool for understanding the relationship between loss of insulin-secreting beta-cells and onset of diabetes mellitus. In this article, 9-fluoropropyl-(+)-dihydrotetrabenazine (FP-(+)-DTBZ), which is a VMAT2 imaging agent, was evaluated as a PET agent for estimating BCM in vivo. METHODS: Organ biodistribution after an intravenous injection of (18)F-FP-(+)-DTBZ (active isomer) and (18)F-FP-(-)-DTBZ (inactive isomer) was evaluated in normal rats. The specificity of uptake of (18)F-FP-(+)-DTBZ was assessed by a pretreatment (3.8 mg of (+)-DTBZ per kilogram and 3.5 mg of FP-(+)-DTBZ per kilogram, intravenously, 5 min prior) or coadministration (2 mg of (+)-DTBZ per kilogram). PET studies were performed in normal rats. RESULTS: The in vivo biodistribution of (18)F-FP-(+)-DTBZ in rats showed the highest uptake in the pancreas (5% dose/g at 30 min after injection), whereas (18)F-FP-(-)-DTBZ showed a very low pancreas uptake. Rats pretreated with FP-(+)-DTBZ displayed a 78% blockade of pancreas uptake. PET studies in normal rats demonstrated an avid pancreas uptake of (18)F-FP-(+)-DTBZ. CONCLUSION: The preliminary data obtained with (18)F-FP-(+)-DTBZ suggest that this fluorinated derivative of DTBZ shows good pancreas specificity and has the potential to be useful for quantitative measurement of VMAT2 binding sites reflecting BCM in the pancreas.

5-tert-Butyl-2-(4'-[18F]fluoropropynylphenyl)-1,3-dithiane oxides: potential new GABA A receptor radioligands.

As potential new ligands targeting the binding site of gamma-aminobutyric acid (GABA) receptor ionophore, trans-5-tert-butyl-2-(4'-fluoropropynylphenyl)-2-methyl-1,1-dioxo-1,3-dithiane (1) and cis/trans-5-tert-butyl-2-(4'-fluoropropynylphenyl)-2-methyl-1,1,3,3-tetroxo-1,3-dithiane (2) were selected for radiolabeling and initial evaluation as in vivo imaging agents for positron emission tomography (PET). Both compounds exhibited identical high in vitro binding affinities (K(i)=6.5 nM). Appropriate tosylate-substituted ethynyl precursors were prepared by multistep syntheses involving stepwise sulfur oxidation and chromatographic isolation of desired trans isomers. Radiolabeling was accomplished in one step using nucleophilic [(18)F]fluorination. In vivo biodistribution studies with trans-[(18)F]1 and trans-[(18)F]2 showed significant initial uptake into mouse brain and gradual washout, with heterogeneous regional brain distributions and higher retention in the cerebral cortex and cerebellum and lower retention in the striatum and pons-medulla. These regional distributions of the new radioligands correlated with in vitro and ex vivo measures of standard radioligands binding to the ionophore- and benzodiazepine-binding sites of GABA(A) receptor in rodent brain. A comparison of these results with previously prepared radiotracers for other neurochemical targets, including successes and failures as in vivo radioligands, suggests that higher-affinity compounds with increased retention in target brain tissues will likely be needed before a successful radiopharmaceutical for human PET imaging can be identified.

Evaluation of Enzyme Substrate Radiotracers as PET/MRS Hybrid Imaging Agents.

The development of a positron emission tomography (PET)/magnetic resonance spectroscopy (MRS) hybrid imaging agent allows for functional imaging by both methods with a single imaging agent. Enzyme substrates that are cleaved to form two metabolites present an interesting opportunity, as the unique metabolites generated might each be detected by a different modality. To be successful, such enzyme substrates would require administration of doses that (a) reach the in vivo target tissue at concentrations necessary for MRS imaging, (b) do not show substrate inhibition of tissue uptake or enzymatic activity, and (c) provide PET images that still reflect the action of the enzyme. We report in vitro and in vivo proof-of-concept studies of a carbon-11 small molecule substrate for brain monoamine oxidases that, upon enzyme-mediated cleavage, produces two metabolites, one detectable by PET and the other by MRS.

Deuterium Kinetic Isotope Effect Studies of a Potential in Vivo Metabolic Trapping Agent for Monoamine Oxidase B.

Visualizing the in vivo activity of monoamine oxidase B (MAO-B) is a valuable tool in the ongoing investigation of astrogliosis in neurodegeneration. Existing strategies for imaging changes in MAO enzyme expression or activity have utilized the irreversible suicide inhibitors or high-affinity reversibly binding inhibitors as positron emission tomography (PET) ligands. As an alternative approach, we developed 4-methyl-7-[(1-[11C]methyl-1,2,3,6-tetrahydropyridin-4-yl)oxy]-2 H-chromen-2-one ([11C]Cou) as a metabolic trapping agent for MAO-B. Trapping of [11C]Cou in rhesus monkey brain demonstrated MAO-B selectivity. In this work, we have attempted to improve on the in vivo pharmacokinetics of [11C]Cou by using the deuterium kinetic isotope effect (KIE) to slow the MAO-B-mediated oxidation step and thus reduce the rate of trapping in brain tissues. However, in vitro assays of enzyme kinetics and in vivo PET imaging of pharmacokinetics in primate brain showed no effects of deuterium substitution on the tetrahydropyridine ring of [11C]Cou. The results are possibly due to masking of the KIE by a second step in the overall metabolism of the new imaging agent.

Development of Positron Emission Tomography Radiotracers for the GABA Transporter 1.

In vivo positron emission tomography (PET) imaging of the γ-aminobutyric acid (GABA) receptor complex has been accomplished using radiolabeled benzodiazepine derivatives, but development of specific presynaptic radioligands targeting the neuronal membrane GABA transporter type 1 (GAT-1) has been less successful. The availability of new structure-activity studies of GAT-1 inhibitors and the introduction of a GAT-1 inhibitor (tiagabine, Gabatril) into clinical use prompted us to reinvestigate the syntheses of PET ligands for this transporter. Initial synthesis and rodent PET studies of N-[11C]methylnipecotic acid confirmed the low brain uptake of that small and polar molecule. The common design approach to improve blood-brain barrier permeability of GAT-1 inhibitors is the attachment of a large lipophilic substituent. We selected an unsymmetrical bis-aromatic residue attached to the ring nitrogen by a vinyl ether spacer from a series recently reported by Wanner and coworkers. Nucleophilic aromatic substitution of an aryl chloride precursor with [18F]fluoride was used to prepare the desired candidate radiotracer ( R, E/ Z)-1-(2-((4-fluoro-2-(4-[18F]fluorobenzoyl)styryl)oxy)ethyl)piperidine-3-carboxylic acid (( R, E/ Z)-[18F]10). PET studies in rats showed no brain uptake, which was not altered by pretreatment of animals with the P-glycoprotein inhibitor cyclosporine A, indicating efflux by Pgp was not responsible. Subsequent PET imaging studies of ( R, E/ Z)-[18F]10 in rhesus monkey brain showed very low brain uptake. Finally, to test if the free carboxylic acid group was the likely cause of poor brain uptake, PET studies were done using the ethyl ester derivative of ( R, E/ Z)-[18F]10. Rapid and significant monkey brain uptake of the ester was observed, followed by a slow washout over 90 min. The blood-brain barrier permeability of the ester supports a hypothesis that the free acid function limits brain uptake of nipecotic acid-based GAT-1 radioligands, and future radiotracer efforts should investigate the use of carboxylic acid bioisosteres.