Joël Mercier

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).

Crystal structure of the adenosine A2A receptor bound to an antagonist reveals a potential allosteric pocket.

Significance The A2AR is a G protein-coupled receptor (GPCR) that plays important roles in cardiovascular physiology and immune function. The A2AR is also a target for the treatment of Parkinson’s disease, where A2AR antagonists have been shown to enhance signaling through the D2 dopamine receptor. Here we present the crystal structure of the A2AR bound to a novel bitopic antagonist. As a result of structural changes needed to accommodate the bound antagonist, crystals could not be grown in lipidic cubic phase. Instead, crystals were grown in detergent with a type II packing rarely observed in GPCR crystals. The structure revealed a potential allosteric pocket that that can be exploited to develop subtype-selective allosteric modulators. The adenosine A2A receptor (A2AR) has long been implicated in cardiovascular disorders. As more selective A2AR ligands are being identified, its roles in other disorders, such as Parkinson’s disease, are starting to emerge, and A2AR antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A2A receptor bound to compound 1 (Cmpd-1), a novel A2AR/N-methyl d-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A2A receptor with a cytochrome b562-RIL (BRIL) fusion (A2AR–BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phase diffusion. Whereas A2AR–BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1–bound A2AR–BRIL prevented formation of the lattice observed with the ZM241385–bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A2AR structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr91.35 and Tyr2717.36, which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A2AR structures, highlighting flexibility in the binding pocket that may facilitate the development of A2AR-selective compounds for the treatment of Parkinson’s disease.

T807, a reported selective tau tracer, binds with nanomolar affinity to monoamine oxidase a

to determine the legal implications of biomarkers for insurance or employment purposes. Federal law prohibiting employment discrimination based on disability offered potential protections for individuals. Comparatively, federal law explicitly delegates insurance regulation to states. Regulations vary between states on issues regarding underwriting, eligibility, and benefits. Additionally, while the Affordable Care Act provides protections regarding health insurance by prohibiting discrimination based on pre-existing conditions, similar protections are not extended for life, disability, or long-term care insurance. Conclusions: Current laws are largely insufficient to protect individuals from employment or insurance discrimination based on preclinical Alzheimer’s disease status. This supports exploring the potential routes for advocacy to improve protections and research regarding other routes for mitigating the potential risks related to insurance and employment. This work was supported by a grant from the Alzheimer’s Association (MNIRGD-14-319284).

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 18F-UCB-H as a Novel PET Tracer for Synaptic Vesicle Protein 2A in the Brain

Synaptic vesicle protein 2 isoforms are critical for proper nervous system function and are involved in vesicle trafficking. The synaptic vesicle protein 2A (SV2A) isoform has been identified as the binding site of the antiepileptic levetiracetam (LEV), making it an interesting therapeutic target for epilepsy. 18F-UCB-H is a novel PET imaging agent with a nanomolar affinity for human SV2A. Methods: Preclinical PET studies were performed with isoflurane-anesthetized rats. The arterial input function was measured with an arteriovenous shunt and a β-microprobe system. 18F-UCB-H was injected intravenously (bolus of 140 ± 20 MBq). Results: Brain uptake of 18F-UCB-H was high, matching the expected homogeneous distribution of SV2A. The distribution volume (Vt) for 18F-UCB-H was calculated with Logan graphic analysis, and the effect of LEV pretreatment on Vt was measured. In control animals the whole-brain Vt was 9.76 ± 0.52 mL/cm3 (mean ± SD; n = 4; test–retest), and the reproducibility in test–retest studies was 10.4% ± 6.5% (mean ± SD). The uptake of 18F-UCB-H was dose dependently blocked by pretreatment with LEV (0.1–100 mg/kg intravenously). Conclusion: Our results indicated that 18F-UCB-H is a suitable radiotracer for the imaging of SV2A in vivo. To our knowledge, this is the first PET tracer for the in vivo quantification of SV2A. The necessary steps for the implementation of 18F-UCB-H production under good manufacturing practice conditions and the first human studies are being planned.

Discovery of Heterocyclic Nonacetamide Synaptic Vesicle Protein 2A (SV2A) Ligands with Single-Digit Nanomolar Potency: Opening Avenues towards the First SV2A Positron Emission Tomography (PET) Ligands

The role of the synaptic vesicle protein 2A (SV2A) protein, target of the antiepileptic drug levetiracetam, is still mostly unknown. Considering its potential to provide in vivo functional insights into the role of SV2A in epileptic patients, the development of an SV2A positron emission tomography (PET) tracer has been undertaken. Using a 3D pharmacophore model based on close analogues of levetiracetam, we report the rationale design of three heterocyclic non‐acetamide lead compounds, UCB‐A, UCB‐H and UCB‐J, the first single‐digit nanomolar SV2A ligands with suitable properties for development as PET tracers.

[11C]UCB-A, a novel PET tracer for synaptic vesicle protein 2 A

INTRODUCTION Development of a selective and specific high affinity PET tracer, [(11)C]UCB-A, for the in vivo study of SV2A expression in humans. Radiochemistry and preclinical studies in rats and pigs including development of a tracer kinetic model to determine VT. A method for the measurement of percent intact tracer in plasma was developed and the radiation dosimetry was determined in rats. RESULTS 3-5GBq of [(11)C]UCB-A could be produced with radiochemical purity exceeding 98% with a specific radioactivity of around 65GBq/μmol. In vitro binding showed high selective binding towards SV2A. [(11)C]UCB-A displayed a dose-dependent and reversible binding to SV2A as measured with PET in rats and pigs and the VT could be determined by Logan analysis. The dosimetry was favorable and low enough to allow multiple administrations of [(11)C]UCB-A to healthy volunteers, and the metabolite analysis showed no sign of labeled metabolites in brain. CONCLUSIONS We have developed the novel PET tracer, [(11)C]UCB-A, that can be used to measure SV2A expression in vivo. The dosimetry allows up to 5 administrations of 400MBq of [(11)C]UCB-A in humans. Apart from measuring drug occupancy, as we have shown, the tracer can potentially be used to compare SV2A expression between individuals because of the rather narrow range of baseline VT values. This will have to be further validated in human studies.

The tau positron-emission tomography tracer AV-1451 binds with similar affinities to tau fibrils and monoamine oxidases: AV-1451 Tau Pet Tracer Binds Mao-a and -B

Background: Lilly/Avids AV‐1451 is one of the most advanced tau PET tracers in the clinic. Although results obtained in Alzheimers disease patients are compelling, discrimination of tracer uptake in healthy individuals and patients with supranuclear palsy (PSP) is less clear as there is substantial overlap of signal in multiple brain regions. Moreover, accurate quantification of [18F]AV‐1451 uptake in Alzheimers disease may not be possible.

Discovery of Indolone Acetamides as Novel SV2A Ligands with Improved Potency Toward Seizure Suppression

A major goal in epilepsy research is to develop new antiepileptic drugs (AEDs) that combine improved seizure control with enhanced tolerability while avoiding drug–drug interactions. Although the prognosis for seizure control is acceptable in up to 70 % of patients, approximately 30 % suffer from intractable pharmaco-resistant epilepsy. 3] Furthermore, the clinical use of most older AEDs is hampered by their limited tolerability; this most commonly consists of CNS-related adverse effects, idiosyncratic reactions such as skin rashes, and the potential for unfavorable drug–drug interactions. It was previously reported that the AED levetiracetam (2 ; Keppra ) has a unique brainspecific binding site. This site was recently identified as the synaptic vesicle protein 2A (SV2A). A strong correlation between the affinity of levetiracetam analogues for SV2A and their anti-seizure potency in the audiogenic mouse model of epilepsy has been reported. This suggests that levetiracetam’s interaction with SV2A has a major functional role in its anticonvulsant mechanism of action. SV2, a protein specific to synaptic vesicles, is a 12-transmembrane region glycoprotein present in all neural cells, and it is present in three isoforms: SV2A, SV2B, and SV2C. SV2A is the most widely distributed isoform and is ubiquitous in the CNS, but is also present in endocrine cells. Although the exact molecular role of SV2A is still unknown, it is believed to play a role in the exocytosis of neurotransmitters and to act as a modulator of vesicle fusion. Given the proven clinical efficacy of levetiracetam as an AED, its unique mechanism of action, and its excellent tolerability, we decided to initiate a drug discovery program focused on SV2A as a novel molecular target. The aim was to identify a new generation of SV2A ligands with an equal or better tolerability profile than 2 and an improved potency toward seizure suppression in animal models. At the outset of our work in the SV2A field, we systematically investigated the various positions of the pyrrolidone acetamide scaffold of 2. Among others, we discovered the importance of the carboxamide moiety on 2 and the preferred substitution position a to the carboxamide. This research lead to the identification of two compounds currently in clinical development: brivaracetam (3) and seletracetam (4), both of which are more potent than 2 in vitro toward SV2A and in vivo as anticonvulsant agents in audiogenic seizure-prone mice. (Figure 1)