P2X7 receptor antagonism reduces the severity of spontaneous seizures in a chronic model of temporal lobe epilepsy

Highlights

• P2X7 receptor antagonism caused a shift in the severity of chronic seizures.

• A decrease in severe seizures was visible after one week of treatment.

• P2X7 receptor antagonism was unsuccessful in decreasing the number of seizures.

• P2X7 receptor antagonism did not alter microglial activation or astrogliosis.

Abstract

Background

The available pharmacotherapy for patients with epilepsy primarily address the symptoms and are ineffective in about 40% of patients. Brain inflammation gained support as potential target for developing new therapies, especially the P2X7 receptor (P2X7R), involved in processing of IL-1β, might be an interesting candidate. This study was designed to investigate the effect of a novel P2X7R antagonist on the severity and on the number of chronic spontaneous recurrent seizures (SRS), which was unexplored until now.

Methods

After one-week of vehicle treatment (20% HP-β-cyclodextrin), JNJ-42253432 was administered subcutaneously for another week under continuous video-electroencephalography monitoring (n = 17) in Sprague Dawley rats 3 months after kainic acid-induced status epilepticus. The proportion of different seizure classes, as well as the number of SRS/day were calculated for the vehicle and treatment period. In addition, post-mortem microglial activation and astrogliosis were assessed.

Results

A significant decrease of the proportion of type 4–5 SRS (p < 0.05), while an increase of type 1–3 was demonstrated (p < 0.05) from the vehicle to the treatment period. There was no effect of the P2X7R antagonist on the number of SRS/day or the glial markers.

Conclusions

The P2X7R antagonist gave rise to a less severe profile of the chronic seizure burden without suppressing the SRS frequency. More studies are needed to unravel the underlying mechanisms of the beneficial effect on seizure severity and whether the administration of the compound during early epileptogenesis could induce long-term disease-modifying effects.

Introduction

Epilepsy is a chronic neurological disorder characterised by the occurrence of spontaneous recurrent seizures (SRS) due to aberrant neuronal discharges. Currently available anti-epileptic drugs (AEDs) are merely symptomatic treatments and cannot prevent or cure epilepsy. There are more than 20 AEDs in clinical use and newer generations of AEDs have been developed with a better side effect profile. However, the number of patients being medically intractable has not changed, remaining at 30–40% (Kwan and Brodie, 2000, Wiebe and Jette, 2012). Inadequate control of seizures and the exposure to inefficient therapy regimens concomitantly leads to significant comorbidities such as intellectual disability and psychiatric problems, poor quality of life and even sudden unexpected death in epilepsy (Pugliatti et al., 2007, Weaver and Pohlmann-Eden, 2013). Therefore, there remains a tremendous need for the development of medicinal therapies targeting alternative mechanisms important in disease ontogenesis.

Epileptogenesis – the process in which a normal neuronal network develops into a hyperexcitable one – is characterised by different processes such as excitotoxicity, neurodegeneration, gliosis and metabolic changes (Goffin et al., 2008). A plethora of evidence supports an important contribution of brain inflammation in the disease ontogenesis. It has been shown that different cytokines, including IL-1β and TNF-α, can cause hyperexcitability (Hu et al., 2000, Dube et al., 2005, Stellwagen et al., 2005, Ravizza et al., 2006, Galic et al., 2012). Only recently, Bogdanovic et al. (2014) showed a positive relationship between hippocampal brain inflammation and seizure frequency in an epilepsy rat model by means of [¹¹C]-PK11195 translocator protein (TSPO) positron emission tomography imaging. The P2X7 receptor (P2X7R), an ATP-gated, non-selective cation channel, has gained a lot of interest as a target to modulate brain inflammation owing to its important role in the processing and release of IL-1β (Solle et al., 2001), which in turn is a potent pro-convulsive mediator (Ravizza et al., 2006). In the brain, this receptor is preferably, but not solely, expressed on the cell-surface of immune-competent cells such as microglia. An upregulation of the P2X7R has been shown during different stages of epileptogenesis in several epilepsy models in the limbic regions, including hippocampus, amygdala and piriform cortex, as well as in the neocortex (Vianna et al., 2002, Rappold et al., 2006, Avignone et al., 2008, Dona et al., 2009, Kim et al., 2009, Jimenez-Pacheco et al., 2013). An increased expression has also been shown in the neocortex in surgical specimen of patients with temporal lobe epilepsy (TLE), which is the most common focal form of acquired epilepsy (Jimenez-Pacheco et al., 2013). All together, these facts make the P2X7R an attractive and potential alternative drug target for epilepsy (Henshall and Engel, 2015).

Several preclinical studies have already demonstrated a seizure suppressive and neuroprotective effect of P2X7R antagonists on acutely provoked status epilepticus (SE) and the consequent pathology such as neuronal death (Engel et al., 2012, Jimenez-Pacheco et al., 2013, Mesuret et al., 2014). However, the effects of P2X7R antagonists on spontaneous seizures in the chronic epilepsy phase, remain unexplored. This study was designed to investigate the effects of the novel P2X7R antagonist JNJ-42253432 [2-methyl-N-([1-(4-phenylpiperazin-1-yl)cyclohexyl]methyl)-1,2,3,4-tetrahydroisoquinoline-5-carboxamide] (Lord et al., 2014) on the expression of SRS, in the established chronic epilepsy phase, after kainic acid-induced status epilepticus (KASE), a very common model for TLE. Most of the other available P2X7R antagonists have several limitations such as a lower affinity in rodents, lower specificity to the receptors and limited blood brain barrier permeability (Bartlett et al., 2014). On the contrary, the novel compound JNJ-42253432 has excellent pharmacokinetic and –dynamic properties, and has been demonstrated to be a potent P2X7R antagonist with a brain to plasma ratio equal to one (Lord et al., 2014).

With this study, we firstly aimed to investigate whether the P2X7R antagonist JNJ-42253432 is able to change the severity of SRS, using a modified scale of Racine (Racine, 1972), during the chronic phase by using a within group treatment paradigm. Secondly, we assessed the effect of JNJ-42253432 on the number of SRS. Finally, we determined whether JNJ-42253432 had an influence on the pathological hallmarks of the KASE model, more specifically microgliosis (OX-42 and TSPO) and astrogliosis (GFAP), during the chronic period.