Daniel Holden

Imaging synaptic density in the living human brain

Synaptic density in the living human brain was measured with positron emission tomography and a synaptic vesicle glycoprotein 2A tracer. Seeing synapses When synapses “fire,” information is transmitted from one neuron to another. Although many neurological and psychiatric diseases are characterized by misfiring synapses, there is currently no way to visualize healthy or aberrant neuronal connections in the living brain—tissues would need to be sampled, which is an invasive and often unwanted procedure. Finnema and colleagues developed a noninvasive approach to “see” human synapses by using an imaging agent that targets the synaptic vesicle glycoprotein 2A (SV2A). PET imaging allowed the authors to visualize synaptic density in both healthy and epileptic human brains in living patients. In the brains with epilepsy, synaptic density was asymmetric—consistent with damage to certain brain regions. This method opens doors to routine monitoring of the brain in patients with various neurological diseases, where synaptic loss or dynamic changes in density could provide clues to prognosis. Chemical synapses are the predominant neuron-to-neuron contact in the central nervous system. Presynaptic boutons of neurons contain hundreds of vesicles filled with neurotransmitters, the diffusible signaling chemicals. Changes in the number of synapses are associated with numerous brain disorders, including Alzheimer’s disease and epilepsy. However, all current approaches for measuring synaptic density in humans require brain tissue from autopsy or surgical resection. We report the use of the synaptic vesicle glycoprotein 2A (SV2A) radioligand [11C]UCB-J combined with positron emission tomography (PET) to quantify synaptic density in the living human brain. Validation studies in a baboon confirmed that SV2A is an alternative synaptic density marker to synaptophysin. First-in-human PET studies demonstrated that [11C]UCB-J had excellent imaging properties. Finally, we confirmed that PET imaging of SV2A was sensitive to synaptic loss in patients with temporal lobe epilepsy. Thus, [11C]UCB-J PET imaging is a promising approach for in vivo quantification of synaptic density with several potential applications in diagnosis and therapeutic monitoring of neurological and psychiatric disorders.

Brivaracetam, a selective high-affinity synaptic vesicle protein 2A (SV2A) ligand with preclinical evidence of high brain permeability and fast onset of action.

Rapid distribution to the brain is a prerequisite for antiepileptic drugs used for treatment of acute seizures. The preclinical studies described here investigated the high‐affinity synaptic vesicle glycoprotein 2A (SV2A) antiepileptic drug brivara‐cetam (BRV) for its rate of brain penetration and its onset of action. BRV was compared with levetiracetam (LEV).

Synthesis and Preclinical Evaluation of 11C-UCB-J as a PET Tracer for Imaging the Synaptic Vesicle Glycoprotein 2A in the Brain

The synaptic vesicle glycoprotein 2A (SV2A) is found in secretory vesicles in neurons and endocrine cells. PET with a selective SV2A radiotracer will allow characterization of drugs that modulate SV2A (e.g., antiepileptic drugs) and potentially could be a biomarker of synaptic density (e.g., in neurodegenerative disorders). Here we describe the synthesis and characterization of the SV2A PET radiotracer 11C-UCB-J ((R)-1-((3-(11C-methyl-11C)pyridin-4-yl)methyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-2-one) in nonhuman primates, including whole-body biodistribution. Methods: 11C-UCB-J was prepared by C-11C-methylation of the 3-pyridyl trifluoroborate precursor with 11C-methyl iodide via the Suzuki–Miyaura cross-coupling method. Rhesus macaques underwent multiple scans including coinjection with unlabeled UCB-J (17, 50, and 150 μg/kg) or preblocking with the antiepileptic drug levetiracetam at 10 and 30 mg/kg. Scans were acquired for 2 h with arterial sampling and metabolite analysis to measure the input function. Regional volume of distribution (VT) was estimated using the 1-tissue-compartment model. Target occupancy was assessed using the occupancy plot; the dissociation constant (Kd) was determined by fitting self-blocking occupancies to a 1-site model, and the maximum number of receptor binding sites (Bmax) values were derived from baseline VT and from the estimated Kd and the nondisplaceable distribution volume (VND). Results: 11C-UCB-J was synthesized with greater than 98% purity. 11C-UCB-J exhibited high free fraction (0.46 ± 0.02) and metabolized at a moderate rate (39% ± 5% and 24% ± 3% parent remaining at 30 and 90 min) in plasma. In the monkey brain, 11C-UCB-J displayed high uptake and fast kinetics. VT was high (∼25–55 mL/cm3) in all gray matter regions, consistent with the ubiquitous expression of SV2A. Preblocking with 10 and 30 mg/kg of levetiracetam resulted in approximately 60% and 90% occupancy, respectively. Analysis of the self-blocking scans yielded a Kd estimate of 3.4 nM and Bmax of 125–350 nM, in good agreement with the in vitro inhibition constant (Ki) of 6.3 nM and regional Bmax in humans. Whole-body biodistribution revealed that the liver and the brain are the dose-limiting organs for males and females, respectively. Conclusion: 11C-UCB-J exhibited excellent characteristics as an SV2A PET radiotracer in nonhuman primates. The radiotracer is currently undergoing first-in-human evaluation.

Evaluation of 11C-BU99008, a PET Ligand for the Imidazoline2 Binding Sites in Rhesus Brain

The development of a PET radioligand selective for I2-imidazoline binding sites (I2BS) would enable, for the first time, specific, measurable in vivo imaging of this target protein, along with assessment of alterations in expression patterns of this protein in disease pathophysiology. Methods: BU99008 was identified as the most promising I2BS radioligand candidate and radiolabeled with 11C via methylation. The in vivo binding properties of 11C-BU99008 were assessed in rhesus monkeys to determine brain penetration, brain distribution, binding specificity and selectivity (via the use of the unlabeled blockers), and the most appropriate kinetic model for analyzing data generated with this PET radioligand. Results: 11C-BU99008 was demonstrated to readily enter the brain, resulting in a heterogeneous distribution (globus pallidus > cortical regions > cerebellum) consistent with the reported regional I2BS densities as determined by human tissue section autoradiography and preclinical in vivo PET studies in the pig. In vivo competition studies revealed that 11C-BU99008 displayed reversible kinetics specific for the I2BS. The multilinear analysis (MA1) model was the most appropriate analysis method for this PET radioligand in this species. The selective I2BS blocker BU224 was shown to cause a saturable, dose-dependent decrease in 11C-BU99008 binding in all regions of the brain assessed, further demonstrating the heterogeneous distribution of I2BS protein in the rhesus brain and binding specificity for this radioligand. Conclusion: These data demonstrate that 11C-BU99008 represents a specific and selective PET radioligand for imaging and quantifying the I2BS, in vivo, in the rhesus monkey. Further work is under way to translate the use of 11C-BU99008 to the clinic.

Further evaluation of [11C]MP-10 as a radiotracer for phosphodiesterase 10A: PET imaging study in rhesus monkeys and brain tissue metabolite analysis.

[11C]MP‐10 is a potent and specific PET tracer previously shown to be suitable for imaging the phosphodiesterase 10A (PDE10A) in baboons with reversible kinetics and high specific binding. However, another report indicated that [11C]MP‐10 displayed seemingly irreversible kinetics in rhesus monkeys, potentially due to the presence of a radiolabeled metabolite capable of penetrating the blood‐brain‐barrier (BBB) into the brain. This study was designed to address the discrepancies between the species by re‐evaluating [11C]MP‐10 in vivo in rhesus monkey with baseline scans to assess tissue uptake kinetics and self‐blocking scans with unlabeled MP‐10 to determine binding specificity. Ex vivo studies with one rhesus monkey and 4 Sprague‐Dawley rats were also performed to investigate the presence of radiolabeled metabolites in the brain. Our results indicated that [11C]MP‐10 displayed reversible uptake kinetics in rhesus monkeys, albeit slower than in baboons. Administration of unlabeled MP‐10 reduced the binding of [11C]MP‐10 in a dose‐dependent manner in all brain regions including the cerebellum. Consequently, the cerebellum appeared not to be a suitable reference tissue in rhesus monkeys. Regional volume of distribution (VT) was mostly reliably derived with the multilinear analysis (MA1) method. In ex vivo studies in the monkey and rats only negligible amount of radiometabolites was seen in the brain of either species. In summary, results from the present study strongly support the suitability of [11C]MP‐10 as a radiotracer for PET imaging and quantification of PDE10A in nonhuman primates. Synapse 69:86–95, 2015.

A click-chemistry based strategy for labeling an anti-phosphatidylserine (PS) antibody with copper-64 via a cross-bridged tetraazamacrocyclic chelator scaffold

The κ-opioid receptors (KORs) are implicated in several neuropsychiatric diseases and addictive disorders. PET with radioligands provides a means to image the KOR in vivo and investigate its function in health and disease. The purpose of this study was to develop the selective KOR antagonist 11C-LY2459989 as a PET radioligand and characterize its imaging performance in nonhuman primates. Methods: LY2459989 was synthesized and assayed for in vitro binding to opioid receptors. Ex vivo studies in rodents were conducted to assess its potential as a tracer candidate. 11C-LY2459989 was synthesized by reaction of its iodophenyl precursor with 11C-cyanide, followed by partial hydrolysis of the resulting 11C-cyanophenyl intermediate. Imaging experiments with 11C-LY2459989 were performed in rhesus monkeys with arterial input function measurement. Imaging data were analyzed with kinetic models to derive in vivo binding parameters. Results: LY2459989 is a full antagonist with high binding affinity and selectivity for KOR (0.18, 7.68, and 91.3 nM, respectively, for κ, μ, and δ receptors). Ex vivo studies in rats indicated LY2459989 as an appropriate tracer candidate with high specific binding signals and confirmed its KOR binding selectivity in vivo. 11C-LY2459989 was synthesized in high radiochemical purity and good specific activity. In rhesus monkeys, 11C-LY2459989 displayed a fast rate of peripheral metabolism. Similarly, 11C-LY2459989 displayed fast uptake kinetics in the brain and an uptake pattern consistent with the distribution of KOR in primates. Pretreatment with naloxone (1 mg/kg, intravenously) resulted in a uniform distribution of radioactivity in the brain. Further, specific binding of 11C-LY2459989 was dose-dependently reduced by the selective KOR antagonist LY2456302 and the unlabeled LY2459989. Regional binding potential values derived from the multilinear analysis-1 (MA1) method, as a measure of in vivo specific binding signal, were 2.18, 1.39, 1.08, 1.04, 1.03, 0.59, 0.51, and 0.50, respectively, for the globus pallidus, cingulate cortex, insula, caudate, putamen, frontal cortex, temporal cortex, and thalamus. Conclusion: The novel PET radioligand 11C-LY2459989 displayed favorable pharmacokinetic properties, a specific and KOR-selective binding profile, and high specific binding signals in vivo, thus making it a promising PET imaging agent for KOR.

In Vivo Evaluation of 18F-MNI698: An 18F-Labeled Radiotracer for Imaging of Serotonin 4 Receptors in Brain

Serotonin 4 receptors (5-hydroxytryptamine receptor 4 [5HT4R]) hold promise as a novel therapeutic approach to multiple brain disorders, including Alzheimer and Huntington disease. In vivo imaging of these receptors with selective 5HT4R radiotracers and PET would be valuable to investigate alterations in 5HT4R in different brain disorders and to assist drug discovery. In this study, 18F-MNI698 was evaluated as a potential PET radiotracer for imaging of 5HT4R in the brain. Methods: Eighteen PET studies were performed in 3 adult rhesus monkeys. The radiotracer was administered as a bolus intravenous injection or bolus plus constant infusion (time that would be required to inject the bolus at the infusion rate = 60 min), and arterial blood was collected for data quantification. Kinetic models were used to estimate distribution volumes and binding potentials, for which the cerebellum was used as a reference region. 18F-MNI698 test–retest variability and upper mass dose limits were determined. Preblocking studies using several doses of SB204070, a selective 5HT4R antagonist, were performed. Results: 18F-MNI698 avidly entered the monkey brain (peak percentage injected dose of ∼6.6%), and its brain distribution was consistent with known 5HT4R densities. At 120 min after bolus injection and after the start of radiotracer infusion, only less than 5% and approximately 10% parent compound was present in blood, respectively. Measured binding potentials were underestimated by 22%–36% when noninvasive methods were used for data quantification in comparison with invasive methods. A good agreement was found between test–retest measurements. The radiotracer upper mass dose limit (<5% occupancy) was determined to be 13.1 μg per 70 kg of body weight. SB204070 blocked the radiotracer binding in a dose-dependent manner. Conclusion: Data indicate that 18F-MNI698 is a promising PET radiotracer for imaging of 5HT4R in the brain, and human studies are warranted based on these study results.

PET Imaging Evaluation of Four Novel Sigma-1 Radiotracers in Nonhuman Primates

The σ1 receptors (S1Rs) are implicated in a variety of diseases including Alzheimer disease and cancer. Previous PET S1R radiotracers are characterized by slow kinetics or off-target binding that impedes their use in humans. Here, we report the first PET imaging evaluation in rhesus monkeys of 4 18F-labeled spirocyclic piperidine-based PET radiotracers (18F-1 to 18F-4). Methods: Baseline scans for the 4 radiotracers were obtained on an adult male rhesus monkey. Blocking scans were obtained with the S1R-selective agonist SA4503 to assess binding specificity of 18F-2 and 18F-4. Arterial input functions were measured, and binding parameters were determined with kinetic modeling analysis. Results: In the rhesus brain, all 4 radiotracers showed high and fast uptake. Tissue activity washout was rapid for 18F-2 and 18F-4, and much slower for 18F-1 and 18F-3, in line with their respective in vitro S1R-binding affinities. Both the 1-tissue-compartment and multilinear analysis-1 kinetic models provided good fits of time–activity curves and reliable estimates of distribution volume. Regional distribution volume values were highest in the cingulate cortex and lowest in the thalamus for all radiotracers. 18F-4 showed greater differential uptake across brain regions and 3-fold-higher binding potential than 18F-2. SA4503 at the dose of 0.5 mg/kg blocked approximately 85% (18F-2) and 95% (18F-4) of radiotracer binding. Conclusion: Tracers 18F-2 and 18F-4 displayed high brain uptake and fast tissue kinetics, with 18F-4 having higher specific binding signals than 18F-2 in the same monkey. Taken together, these data indicate that both 18F-2 and 18F-4 possess the requisite kinetic and imaging properties as viable PET tracers for imaging S1R in the human brain.

The Search for a Subtype-Selective PET Imaging Agent for the GABAA Receptor Complex: Evaluation of the Radiotracer [11C]ADO in Nonhuman Primates

The myriad physiological functions of γ-amino butyric acid (GABA) are mediated by the GABA-benzodiazepine receptor complex comprising of the GABAA, GABAB, and GABAC groups. The various GABAA subunits with region-specific distributions in the brain subserve different functional and physiological roles. For example, the sedative and anticonvulsive effects of classical benzodiazepines are attributed to the α1 subunit, and the α2 and α3 subunits mediate the anxiolytic effect. To optimize pharmacotherapies with improved efficacy and devoid of undesirable side effects for the treatment of anxiety disorders, subtype-selective imaging radiotracers are required to assess target engagement at GABA sites and determine the dose–receptor occupancy relationships. The goal of this work was to characterize, in nonhuman primates, the in vivo binding profile of a novel positron emission tomography (PET) radiotracer, [11C]ADO, which has been indicated to have functional selectivity for the GABAA α2/α3 subunits. High specific activity [11C]ADO was administrated to 3 rhesus monkeys, and PET scans of 120-minute duration were performed on the Focus-220 scanner. In the blood, [11C]ADO metabolized at a fairly rapid rate, with ∼36% of the parent tracer remaining at 30 minutes postinjection. Uptake levels of [11C]ADO in the brain were high (peak standardized uptake value of ∼3.0) and consistent with GABAA distribution, with highest activity levels in cortical areas, intermediate levels in cerebellum and thalamus, and lowest uptake in striatal regions and amygdala. Tissue kinetics was fast, with peak uptake in all brain regions within 20 minutes of tracer injection. The one-tissue compartment model provided good fits to regional time–activity curves and reliable measurement of kinetic parameters. The absolute test–retest variability of regional distribution volumes (V T) was low, ranging from 4.5% to 8.7%. Pretreatment with flumazenil (a subtype nonselective ligand, 0.2 mg/kg, intravenous [IV], n = 1), Ro15-4513 (an α5-selective ligand, 0.03 mg/kg, IV, n = 2), and zolpidem (an α1-selective ligand, 1.7 mg/kg, IV, n = 1) led to blockade of [11C]ADO binding by 96.5%, 52.5%, and 76.5%, respectively, indicating the in vivo binding specificity of the radiotracer. Using the nondisplaceable volume of distribution (V ND) determined from the blocking studies, specific binding signals, as measured by values of regional binding potential (BP ND), ranged from 0.6 to 4.4, which are comparable to those of [11C]flumazenil. In conclusion, [11C]ADO was demonstrated to be a specific radiotracer for the GABAA receptors with several favorable properties: high brain uptake, fast tissue kinetics, and high levels of specific binding in nonhuman primates. However, subtype selectivity in vivo is not obvious for the radiotracer, and thus, the search for subtype-selective GABAA radiotracers continues.

Measurement of Bmax and Kd with the glycine transporter 1 radiotracer 18F-MK6577 using a novel multi-infusion paradigm

Glycine is a co-agonist of glutamate at the NMDA receptor. Glycine transporter 1 (GlyT1) inhibitors are reported to be potential therapeutic agents for schizophrenia. 18F-MK6577 is a new positron emission tomography (PET) radiotracer useful for imaging brain GlyT1 and its occupancy in humans. We devised a novel multi-infusion paradigm of radiolabeled and unlabeled compound and an iterative linear/nonlinear alternating fitting method to allow for the determination of in vivo affinity (Kd) and target concentration (Bmax) images, constraining Kd to be uniform across the brain. This paradigm was tested with 18F-MK6577 in baboons. Voxel-based analysis produced high quality Bmax images and reliable Kd estimates, and also suggested that the nondisplaceable distribution volume (VND) is not uniform throughout the brain. In vivo GlyT1 Kd was estimated to be 1.87 nmol/L for 18F-MK6577, and the rank order of GlyT1 distribution measured in the baboon brain was: high in the brainstem (133 nmol/L), medium in the cerebellum (83 nmol/L), and low in the cortex (30 nmol/L). These in vivo Kd and Bmax values agreed well with those determined in vitro, thus validating our novel multi-infusion approach.