Status epilepticus (SE) is a neurologic emergency, characterized by prolonged, self-sustaining seizure activity, and is associated with high morbidity and mortality. SE is more prevalent in younger subjects than in adults, with nearly 50% of cases occurring in children <2 years of age. Benzodiazepines (BZDs) are considered the first-line therapy for the treatment of SE. BZDs are positive allosteric modulators of the γ-aminobutyric acid (GABA)A receptor and suppress seizures by enhancing GABAA receptor–mediated inhibition. Unfortunately, clinical experience reveals that SE in at least 35% of patients is refractory to these drugs (Mayer et al., 2002). Benzodiazepine pharmacoresistance can be attributed to a variety of factors, including seizure etiology. Both clinical and animal studies have shown that the likelihood of a patient responding to BZDs decreases as the duration of SE increases. Pharmacoresistance to BZDs develops rapidly after the initiation of SE owing to activity-dependent changes in the subunit composition of the GABAA receptor (Jacob et al., 2008). The ability of BZDs to potentiate current at GABAA receptors requires that these receptors contain γ2 subunits (Mayer et al., 2002). However, following the onset of SE, BZD-sensitive γ2-containing GABAA receptors are rapidly internalized (Naylor et al., 2005; Goodkin et al., 2008), thereby significantly reducing the efficacy of BZDs. In contrast, other BZD-insensitive GABAA receptors, such as those containing α4 and δ subunits, remain functional on the membrane surface (Goodkin et al., 2008). GABAA receptors containing δ subunits are important in mediating tonic inhibition. These observations suggest that agents that potentiate γ2-lacking GABAA receptors may prove beneficial in the treatment of BZD-resistant SE by potentiating synaptic or tonic inhibition.
Stiripentol (STP, Diacomit) is an anticonvulsant drug that has been granted orphan drug status in the European Union as adjunctive therapy for the treatment of severe myoclonic epilepsy in infancy (SMEI, Dravet syndrome). It is a novel compound that is structurally unrelated to other clinically used anticonvulsants. STP is typically coadministered with other antiepileptic drugs, as it has been found to increase the peak concentration and duration of action of these anticonvulsant drugs through inhibition of a variety of hepatic cytochrome P450 (CYP) enzymes, including CYP1A2, CYP3A4, and CYP2C19 (Tran et al., 1997). However, recent studies have shown anticonvulsant effects of STP when it is administered alone. In animal models, STP is anticonvulsant in pentylenetetrazol (Poisson et al., 1984), electrical stimulation (Poisson et al., 1984), and cocaine-induced seizure models (Gasior et al., 1999). STP has been proposed to terminate seizures in these models through enhancement of GABAergic inhibition (Poisson et al., 1984; Quilichini et al., 2006). Recent studies using recombinant receptors support this conclusion by demonstrating that STP is a positive allosteric modulator of GABAA receptors containing any of the α, β, γ, or δ-subunits (Fisher, 2009). STP most strongly potentiates receptors containing α3 or δ subunits, suggesting its ability to potentiate both phasic and tonic inhibition. The subunit selectivity of STP suggests that in established SE, when BZD-sensitive GABAA receptors have been internalized, STP could continue to potentiate GABAergic inhibition. Therefore, STP may hold promise for the treatment of BZD-resistant SE. Furthermore, α3 subunit expression is highest in immature brain (Laurie et al., 1992), suggesting that STP may be particularly effective in terminating SE in children when SE is most prevalent.
The initial goals of this study (Grosenbaugh & Mott, 2013) were to determine whether STP is active in SE and whether its effects were greater in juvenile animals. Behavioral studies were performed on young (15–23 days old) and adult (61–63 days old) male Sprague-Dawley rats. SE was induced using the lithium-pilocarpine model. STP (10–1,000 mg/kg) or the 1,4-benzodiazepine, diazepam (DZP; 0.3–100 mg/kg) was administered at the onset of stage 3 behavioral convulsions (brief SE) and seizures were scored 15 min later. STP and DZP each terminated brief SE in young and adult animals. DZP was equally effective in both age groups. In contrast, STP was significantly more effective in juvenile than adult animals. Although STP acts at GABAA receptors containing any α subunit, it has highest activity at α3-containing GABAA receptors (Fisher, 2009). Accordingly, the reduction in anticonvulsant potency of STP with maturation is likely explained by the developmental loss of α3 subunit expression (Laurie et al., 1992).
To determine whether STP was effective in BZD-resistant SE we compared the ability of STP or DZP to terminate SE when administered at the onset of stage 3 behavioral convulsions (brief SE) or 45 min after the first stage 3 seizure (established SE). As reported previously (Kapur & Macdonald, 1997), in established SE, seizures became pharmacoresistant to DZP. In young animals the dose producing a 50% reduction in seizure score (ED50) for DZP increased 14-fold when DZP was administered during established SE. Similarly, in adult animals the DZP ED50 increased 11-fold during established SE. This decrease in the effectiveness of DZP is thought to be caused by the activity-dependent internalization of γ2-containing GABAA receptors (Naylor et al., 2005; Goodkin et al., 2008). In contrast, STP was equally effective in terminating both brief and established SE in young animals. STP retained effectiveness in established SE in adult animals as well. We suggest that the ability of STP to remain effective in established SE, when seizures have become resistant to BZDs, is due to the unique subunit selectivity profile of STP at GABAA receptors.
To further examine the effect of STP on GABAergic inhibition, we characterized the effect of the drug on GABAA receptor–mediated currents in dentate granule cells. As previously reported (Quilichini et al., 2006), we found that STP prolonged the decay time of the GABAA receptor–mediated inhibitory postsynaptic current (IPSC). However, this IPSC prolongation was significantly greater in young animals (5–8 days old) than in animals from older age groups (20–26 days old or 59–65 days old). It is important to note that the age-dependence of IPSC potentiation by STP was similar to the observed age-dependence of the anticonvulsant effect of the drug.
For treatment of Dravet syndrome STP is often combined with the 1,5-benzodiazepine, clobazam. To determine whether these drugs act at a common site on the receptor we first assessed whether their actions were additive. We found that IPSC potentiation by STP summed with the potentiation produced by a maximal concentration of clobazam, suggesting that the drugs acted at separate sites. In addition, we found that the benzodiazepine-site antagonist flumazenil blocked potentiation by both DZP and clobazam, but not STP. These findings demonstrate that STP acts on the GABAA receptor at a site independent from the BZD binding site, as has been previously proposed (Quilichini et al., 2006).
The ability of STP to retain anticonvulsant potency in established SE could be explained by the continued ability of the drug to potentiate the IPSC during SE. To investigate this possibility, we compared the effect of STP on IPSCs in dentate granule cells from naive animals and from animals that had experienced 45 min of continuous seizure activity. STP potentiated the IPSC in granule cells from both groups of animals to a similar extent. In contrast, potentiation of the IPSC by DZP was significantly reduced in animals that had experienced prolonged seizure activity. These findings demonstrate that STP, but not DZP, retains potency in established SE and suggest that the continued ability of STP to potentiate the IPSC underlies its ability to remain effective in established SE.
Tonic inhibition in the dentate gyrus is mediated by GABAA receptors containing α4 and δ subunits as well as those containing α5 and γ2 subunits (Zhang et al., 2007). The ability of STP to act at receptors containing these subunits suggests that potentiation of tonic GABA currents may contribute to the anticonvulsant effect of STP. We compared the effect of STP on tonic GABA current in dentate granule cells in young (21–23 day old) naive animals to that in animals with established SE. STP significantly and similarly potentiated the tonic current in both groups of animals. The continued effectiveness of STP on tonic inhibition in established SE was likely due to the persistence of δ-containing GABAA receptors on the membrane surface (Goodkin et al., 2008). In contrast, DZP does not act at δ-containing receptors. However, DZP still potentiated the tonic GABA current in granule cells in naive animals, possibly by acting on extrasynaptic α5/γ2-containing GABAA receptors (Zhang et al., 2007). This potentiation by DZP was lost in established SE, likely due to internalization of these γ2-containing receptors (Naylor et al., 2005; Goodkin et al., 2008).
STP can increase GABA release by acting presynaptically on inhibitory terminals (Quilichini et al., 2006). This action could contribute to the effects of STP on IPSC decay and tonic inhibition as well as the anticonvulsant effect of the compound. In naive animals, we found that STP did not alter the amplitude of spontaneous miniature IPSCs (mIPSCs), but significantly increased the frequency and decay time of the events. These findings point to both presynaptic and postsynaptic actions of STP on GABAergic inhibition. The effects of STP on mIPSCs persisted in established SE. In contrast, in naive animals DZP significantly potentiated only the decay time of mIPSCs, without affecting mIPSC frequency. Therefore, DZP acted entirely postsynaptically on GABAA receptors. As predicted, in established SE, the effect of DZP on mIPSC decay was significantly reduced. Therefore, STP, but not DZP acts presynaptically to facilitate GABAergic inhibition, and this effect remains in established SE. The presynaptic effect of STP to enhance GABA release differentiates it from both barbiturates and BZDs, further demonstrating the unique pharmacologic profile of this drug.
In summary, these findings suggest that at doses that yield therapeutically relevant concentrations, STP is anticonvulsant by potentiating GABAergic inhibition. The subunit selectivity profile of STP enables it to remain effective despite GABAA receptor subunit changes in established SE. The need for novel therapies for established SE is underscored by clinical studies showing that the majority of patients undergoing SE do not receive treatment within 30 min of seizure onset (Pellock et al., 2004). The increased duration of these seizures decreases their likelihood of being successfully treated with BZDs. The additive effects of BZDs and STP observed in the present study suggest that the combination of STP and BZDs would produce stronger enhancement of GABAergic inhibition and potentiate a greater variety of GABAA receptors than would BZDs alone. These findings point to the potential utility of STP, either alone or as add-on therapy, for treatment of established and BZD-resistant SE. Furthermore, the age dependence of its anticonvulsant effect suggests that STP would be most effective in treating SE in children where it is most prevalent.