[11C]UCB-A, a novel PET tracer for synaptic vesicle protein 2 A
Introduction
Epilepsy, with an incidence estimated of 1.5 to 5% of the population, is still a clinical challenge and at the same time one of the most common neurological diseases [1]. Although a number of drugs with different mechanisms of action are available for the treatment of seizures, around 30% of patients do not have seizure control on their current medication and these patients with refractory epilepsy represent a severe unmet clinical need [2], [3]. Pharmacotherapy is directed towards maintaining the patient seizure-free by controlling the pathological electric activity in the brain which is the typical hallmark of epilepsy. There are several hypotheses for the reason of pharmacoresistant epilepsy but so far, both the etiology and mechanism are unknown, although efflux systems such as P-glycoprotein (Pgp), a member of the ATP-binding cassette transporters limiting drug entry into brain, have been the focus for many research studies [3]. There is, however, an ongoing debate as to whether Pgp plays a role in treatment resistant epilepsy. One potential explanation for the controversy could be that local, rather than, global up-regulation of Pgp occurs, however, current diagnostic imaging tools have not been able to quantify this. Other efflux systems than Pgp could also be involved. One argument against the Pgp hypothesis is the fact that many antiepileptic drugs (AEDs) are not Pgp substrates or inhibitors [4]. Another hypothesis of AED therapy resistance involves the expression of specific drug-related molecular targets.
One such drug-related molecular target is synaptic vesicle protein 2 A (SV2A) which is a 12-transmembrane glycoprotein present on synaptic vesicles of all neural cells [5]. Although its exact molecular function is still unclear, the current hypothesis is that SV2A plays a role in the exocytosis of neurotransmitters and acts as a modulator of vesicle fusion [6]. The importance of SV2A has been demonstrated in knockout mice which suffer from spontaneous seizures from birth and typically die within 3 weeks of age [7]. It has been speculated that SV2A is associated with action potential-dependent synaptic transmission and therefore reduced SV2A expression could lead to both increased excitability and higher propensity for seizures. The phenotype of homozygous SV2A knock-out mice, being characterized by uncontrolled spontaneous seizures leading to premature death, suggests an agonistic or modulating action of SV2A-binding antiepileptic drugs, such as levetiracetam. Studies in brain tissue from patients that died from status epilepticus or from pharmacoresistant temporal lobe epilepsy patients showed that they had a reduced SV2A expression in hippocampus [8] compared to levels measured in the brain from nonepileptic patients.
SV2A has been identified as the unique brain-specific protein binding site of the antiepileptic drug levetiracetam, therefore suggesting a major role played by SV2A in its mechanism of action [9], [10], [11]. Even though SV2A has been identified as the brain binding site of levetiracetam, the precise molecular mechanism(s) by which this binding protects against seizures is not well understood at present. In order to further investigate the role of SV2A in epilepsy, a non-invasive imaging method such as positron emission tomography (PET) and an SV2A-selective tracer would thus be of significant value. PET is a powerful nuclear imaging technique allowing the in vivo quantification of a radioligand labeled with a short-lived positron-emitting isotope, typically carbon-11 or fluorine-18. The opportunity of quantifying the SV2A expression in patients with focal epilepsy, for example temporal lobe epilepsy, and to measure the extent of SV2A occupancy resulting in a therapeutic effect would give further insight into the mechanism of action of levetiracetam and other SV2A modulating antiepileptic drugs. In a recent publication Mercier et al. presented three potential PET tracer candidates developed from the pharmacophore of levetiracetam, coined UCB-A, UCB-H and UCB-J [12]. Recently two radioligands for the study of SV2A were presented, [18F]UCB-H and [11C]levetiracetam [13], [14], [15]. Here, we present the development and preclinical evaluation, including tracer kinetic modeling, of [11C]UCB-A, as a selective SV2A PET tracer.
Following development of a labeling synthesis the potential of [11C]UCB-A as an in vivo biomarker for quantification of regional SV2A expression was investigated in rats and in pigs using PET. Receptor kinetics were studied in an in vivo displacement study in rats using PET. Dosimetry was determined by ex vivo biodistribution studies in rats and extrapolated to give estimates of effective radiation doses in humans. Binding potential and specific binding was assessed in pigs by pre-treatment with brivaracetam, a selective SV2A ligand with antiepileptic properties. A tracer kinetic model, based on a metabolite-corrected input function from arterial blood, was applied to the data to create values of brain SV2A binding potential.
Section snippets
Equipment
The radiosynthesis was carried out on an in-house built Synthia robot system based upon a Gilson ASPEC module. The semi-preparative HPLC purification of the tracer was carried out on a Phenomenex Gemini NX C18 5 μm 150 × 10 mm column or a Reprosil PUR Basic C18 5 μm 150 × 10 mm using a mixture of 8 mM aqueous ammonium carbonate buffer and acetonitrile as the mobile phase with a flow rate of 10 mL/min. The product identification and purity check was done by analytical reversed-phase HPLC utilizing a
Synthesis of [11C]UCB-A
[11C]UCB-A was synthesized in a two-step reaction giving 3–5 GBq of the finished product. It was obtained with a radiochemical purity higher than 98% and with a specific radioactivity of 63 ± 30 GBq/μmol (n = 19, average ± SD). The chemical purity, defined as absence of precursor or other UV absorbing substances, was high, with no traces of the corresponding detritylated UCB-A precursor 6.
Metabolite analysis
The developed metabolite analysis method indicated a relatively slow metabolism of [11C]UCB-A, with 93% and 42%
Discussion
The purpose of the present work was to evaluate [11C]UCB-A as a PET tracer for SV2A in rats and pigs. [11C]UCB-A has a high brain uptake as measured in rats and pigs using PET. In the latter an SUV exceeding 6 was measured at 90 min p.i. In both species, the apparent brain kinetics suggested irreversible binding of [11C]UCB-A during the time course of the PET investigation. It could, however, be demonstrated by a displacement study in rat using the selective SV2A drug brivaracetam, that the
Conclusions
We have developed the novel PET tracer, [11C]UCB-A, that can be used to measure intra-individual heterogeneity in SV2A expression and potentially to measure absolute SV2A expression in vivo. 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 dosimetry allows up to 5 administrations of 400 MBq
Acknowledgements
This work was performed as a collaboration between Uppsala University and UCB Biopharma, which financially supported the study. The authors want to thank the staff at the Pre-clinical PET platform, Uppsala University, for their assistance in this study.
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- 1
Both authors contributed equally.
- 2
Currently employed by Sanofi-Aventis Deutschland GmbH, Frankfurt am Main, Germany.