Treatment of cholinergic-induced status epilepticus with polytherapy targeting GABA and glutamate receptors.

Despite new antiseizure medications, the development of cholinergic-induced refractory status epilepticus (RSE) continues to be a therapeutic challenge as pharmacoresistance to benzodiazepines and other antiseizure medications quickly develops. Studies conducted by Epilepsia. 2005;46:142 demonstrated that the initiation and maintenance of cholinergic-induced RSE are associated with trafficking and inactivation of gamma-aminobutyric acid A receptors (GABAA R) thought to contribute to the development of benzodiazepine pharmacoresistance. In addition, Dr. Wasterlain's laboratory reported that increased N-methyl-d-aspartate receptors (NMDAR) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) contribute to enhanced glutamatergic excitation (Neurobiol Dis. 2013;54:225; Epilepsia. 2013;54:78). Thus, Dr. Wasterlain postulated that targeting both maladaptive responses of reduced inhibition and increased excitation that is associated with cholinergic-induced RSE should improve therapeutic outcome. We currently review studies in several animal models of cholinergic-induced RSE that demonstrate that benzodiazepine monotherapy has reduced efficacy when treatment is delayed and that polytherapy with drugs that include a benzodiazepine (eg midazolam and diazepam) to counter loss of inhibition, concurrent with an NMDA antagonist (eg ketamine) to reduce excitation provide improved efficacy. Improved efficacy with polytherapy against cholinergic-induced seizure is demonstrated by reduction in (1) seizure severity, (2) epileptogenesis, and (3) neurodegeneration compared with monotherapy. Animal models reviewed include pilocarpine-induced seizure in rats, organophosphorus nerve agent (OPNA)-induced seizure in rats, and OPNA-induced seizure in two mouse models: (1) carboxylesterase knockout (Es1-/- ) mice which, similarly to humans, lack plasma carboxylesterase and (2) human acetylcholinesterase knock-in carboxylesterase knockout (KIKO) mice. We also review studies showing that supplementing midazolam and ketamine with a third antiseizure medication (valproate or phenobarbital) that targets a nonbenzodiazepine site rapidly terminates RSE and provides further protection against cholinergic-induced SE. Finally, we review studies on the benefits of simultaneous compared with sequential drug treatments and the clinical implications that lead us to predict improved efficacy of early combination drug therapies. The data generated from seminal rodent studies of efficacious treatment of cholinergic-induced RSE conducted under Dr. Wasterlain's guidance suggest that future clinical trials should treat the inadequate inhibition and temper the excess excitation that characterize RSE and that early combination therapies may provide improved outcome over benzodiazepine monotherapy.

Rational polytherapy in the treatment of cholinergic seizures.

The initiation and maintenance phases of cholinergic status epilepticus (SE) are associated with maladaptive trafficking of synaptic GABAA and glutamate receptors. The resulting pharmacoresistance reflects a decrease in synaptic GABAA receptors and increase in NMDA and AMPA receptors, which tilt the balance between inhibition and excitation in favor of the latter. If these changes are important to the pathophysiology of SE, both should be treated, and blocking their consequences should have therapeutic potential. We used a model of benzodiazepine-refractory SE (RSE) (Tetz et al., 2006) and a model of soman-induced SE to test this hypothesis. Treatment of RSE with combinations of the GABAAR agonists midazolam or diazepam and the NMDAR antagonists MK-801 or ketamine terminated RSE unresponsive to high-dose monotherapy with benzodiazepines, ketamine or other antiepileptic drugs (AEDs). It also reduced RSE-associated neuronal injury, spatial memory deficits and the occurrence of spontaneous recurrent seizures (SRS), tested several weeks after SE. Treatment of sc soman-induced SE similarly showed much greater reduction of EEG power by a combination of midazolam with ketamine, compared to midazolam monotherapy. When treating late (40 min after seizure onset), there may not be enough synaptic GABAAR left to be able to restore inhibition with maximal GABAAR stimulation, and further benefit is derived from the addition of an AED which increases inhibition or reduces excitation by a non-GABAergic mechanism. The midazolam-ketamine-valproate combination is effective in terminating RSE. 3-D isobolograms demonstrate positive cooperativity between midazolam, ketamine and valproate, without any interaction between the toxicity of these drugs, so that the therapeutic index is increased by combination therapy between GABAAR agonist, NMDAR antagonist and selective AEDs. We compared this drug combination based on the receptor trafficking hypothesis to treatments based on clinical practice. The midazolam-ketamine-valproate combination is far more effective in stopping RSE than the midazolam-fosphenytoin-valproate combination inspired from clinical guidelines. Furthermore, sequential administration of midazolam, ketamine and valproate is far less effective than simultaneous treatment with the same drugs at the same dose. These data suggest that we should re-evaluate our traditional treatment of RSE, and that treatment should be based on pathophysiology. The search for a better drug has to deal with the fact that most monotherapy leaves half the problem untreated. The search for a better benzodiazepine should acknowledge the main cause of pharmacoresistance, which is loss of synaptic GABAAR. Future clinical trials should consider treating both the failure of inhibition and the runaway excitation which characterize RSE, and should include an early polytherapy arm.

Early polytherapy for benzodiazepine-refractory status epilepticus.

The transition from single seizures to status epilepticus (SE) is associated with malaptive trafficking of synaptic gamma-aminobutyric acid (GABAA) and glutamate receptors. The receptor trafficking hypothesis proposes that these changes are key events in the development of pharmacoresistance to antiepileptic drugs (AEDs) during SE, and that blocking their expression will help control drug-refractory SE (RSE). We tested this hypothesis in a model of SE induced by very high-dose lithium and pilocarpine (RSE), and in a model of SE induced by sc soman. Both models are refractory to benzodiazepines when treated 40 min after seizure onset. Our treatments aimed to correct the loss of inhibition because of SE-associated internalization of synaptic GABAA receptors (GABAAR), using an allosteric GABAAR modulator, sometimes supplemented by an AED acting at a nonbenzodiazepine site. At the same time, we reduced excitation because of increased synaptic localization of NMDA and AMPA (?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate) receptors (NMDAR, AMPAR (?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, N-methyl-D-aspartate receptors)) with an NMDAR channel blocker, since AMPAR changes are NMDAR-dependent. Treatment of RSE with combinations of the GABAAR allosteric modulators midazolam or diazepam and the NMDAR antagonists dizocilpine or ketamine terminated RSE unresponsive to high-dose monotherapy. It also reduced RSE-associated neuronal injury, spatial memory deficits, and the occurrence of spontaneous recurrent seizures (SRS), tested several weeks after SE. Treatment of soman-induced SE also reduced seizures, behavioral deficits, and epileptogenesis. Addition of an AED further improved seizure outcome in both models. Three-dimensional isobolograms demonstrated positive cooperativity between midazolam, ketamine, and valproate, without any interaction between the toxicity of these drugs, so that the therapeutic index was increased by combination therapy. The midazolam-ketamine-valproate combination based on the receptor trafficking hypothesis was far more effective in stopping RSE than the midazolam-fosphenytoin-valproate combination inspired from clinical guidelines for the treatment of SE. Furthermore, sequential administration of midazolam, ketamine, and valproate was far less effective than simultaneous treatment with the same drugs at the same dose. These data suggest that treatment of RSE should be based at least in part on its pathophysiology. The search for a better treatment should focus on the cause of pharmacoresistance, which is loss of synaptic GABAAR and gain of synaptic glutamate receptors. Both need to be treated. Monotherapy addresses only half the problem. Improved pharmacokinetics will not help pharmacoresistance because of loss of receptors. Waiting for one drug to fail before giving the second drugs gives pharmacoresistance time to develop. Future clinical trials should consider treating both the failure of inhibition and the runaway excitation which characterize RSE, and should include an early polytherapy arm. This article is part of the Special Issue "Proceedings of the 7th London-Innsbruck Colloquium on Status Epilepticus and Acute Seizures".

Simultaneous triple therapy for the treatment of status epilepticus.

Early maladaptive internalization of synaptic GABAA receptors (GABAAR) and externalization of NMDA receptors (NMDAR) may explain the time-dependent loss of potency of standard anti-epileptic drugs (AED) in refractory status epilepticus (SE). We hypothesized that correcting the effects of changes in GABAAR and NMDAR would terminate SE, even when treatment is delayed 40 minutes. SE was induced in adult Sprague-Dawley rats with a high dose of lithium and pilocarpine. The GABAAR agonist midazolam, the NMDAR antagonist ketamine and the AED valproate were injected 40 min after SE onset in combination or as monotherapy. The midazolam-ketamine-valproate combination was more efficient than triple-dose midazolam, ketamine or valproate monotherapy or higher-dose dual therapy in reducing several parameters of SE severity. Triple therapy also reduced SE-induced acute neuronal injury and spatial memory deficits. In addition, simultaneous triple therapy was more efficient than sequential triple therapy: giving the three drugs simultaneously was more efficient at stopping seizures than the standard practice of giving them sequentially. Furthermore, midazolam-ketamine-valproate therapy suppressed seizures far better than the midazolam-fosphenytoin-valproate therapy, which follows evidence-based AES guidelines. These results show that a treatment aimed at correcting maladaptive GABAAR and NMDAR trafficking can reduce the severity of SE and its long-term consequences.

Midazolam-ketamine dual therapy stops cholinergic status epilepticus and reduces Morris water maze deficits.

OBJECTIVE: Pharmacoresistance remains an unsolved therapeutic challenge in status epilepticus (SE) and in cholinergic SE induced by nerve agent intoxication. SE triggers a rapid internalization of synaptic γ-aminobutyric acid A (GABAA ) receptors and externalization of N-methyl-d-aspartate (NMDA) receptors that may explain the loss of potency of standard antiepileptic drugs (AEDs). We hypothesized that a drug combination aimed at correcting the consequences of receptor trafficking would reduce SE severity and its long-term consequences. METHODS: A severe model of SE was induced in adult Sprague-Dawley rats with a high dose of lithium and pilocarpine. The GABAA receptor agonist midazolam, the NMDA receptor antagonist ketamine, and/or the AED valproate were injected 40 min after SE onset in combination or as monotherapy. Measures of SE severity were the primary outcome. Secondary outcomes were acute neuronal injury, spontaneous recurrent seizures (SRS), and Morris water maze (MWM) deficits. RESULTS: Midazolam-ketamine dual therapy was more efficient than double-dose midazolam or ketamine monotherapy or than valproate-midazolam or valproate-ketamine dual therapy in reducing several parameters of SE severity, suggesting a synergistic mechanism. In addition, midazolam-ketamine dual therapy reduced SE-induced acute neuronal injury, epileptogenesis, and MWM deficits. SIGNIFICANCE: This study showed that a treatment aimed at correcting maladaptive GABAA receptor and NMDA receptor trafficking can stop SE and reduce its long-term consequences. Early midazolam-ketamine dual therapy may be superior to monotherapy in the treatment of benzodiazepine-refractory SE.

Transcriptional responses of the nerve agent-sensitive brain regions amygdala, hippocampus, piriform cortex, septum, and thalamus following exposure to the organophosphonate anticholinesterase sarin.

BACKGROUND: Although the acute toxicity of organophosphorus nerve agents is known to result from acetylcholinesterase inhibition, the molecular mechanisms involved in the development of neuropathology following nerve agent-induced seizure are not well understood. To help determine these pathways, we previously used microarray analysis to identify gene expression changes in the rat piriform cortex, a region of the rat brain sensitive to nerve agent exposure, over a 24-h time period following sarin-induced seizure. We found significant differences in gene expression profiles and identified secondary responses that potentially lead to brain injury and cell death. To advance our understanding of the molecular mechanisms involved in sarin-induced toxicity, we analyzed gene expression changes in four other areas of the rat brain known to be affected by nerve agent-induced seizure (amygdala, hippocampus, septum, and thalamus). METHODS: We compared the transcriptional response of these four brain regions to sarin-induced seizure with the response previously characterized in the piriform cortex. In this study, rats were challenged with 1.0 × LD50 sarin and subsequently treated with atropine sulfate, 2-pyridine aldoxime methylchloride, and diazepam. The four brain regions were collected at 0.25, 1, 3, 6, and 24 h after seizure onset, and total RNA was processed for microarray analysis. RESULTS: Principal component analysis identified brain region and time following seizure onset as major sources of variability within the dataset. Analysis of variance identified genes significantly changed following sarin-induced seizure, and gene ontology analysis identified biological pathways, functions, and networks of genes significantly affected by sarin-induced seizure over the 24-h time course. Many of the molecular functions and pathways identified as being most significant across all of the brain regions were indicative of an inflammatory response. There were also a number of molecular responses that were unique for each brain region, with the thalamus having the most distinct response to nerve agent-induced seizure. CONCLUSIONS: Identifying the molecular mechanisms involved in sarin-induced neurotoxicity in these sensitive brain regions will facilitate the development of novel therapeutics that can potentially provide broad-spectrum protection in five areas of the central nervous system known to be damaged by nerve agent-induced seizure.

Impaired auditory and contextual fear conditioning in soman-exposed rats.

Exposure to soman (GD) can result in prolonged seizures and subsequent neuropathology in a variety of brain regions including the amygdala and hippocampus. Both regions are believed to play important roles in the development and expression of fear conditioning. The purpose of this experiment was to test these conditioning tasks as a possible behavioral correlate of the observed neuropathology. Male rats were exposed to GD (1.0 or 1.2×LD50) or saline followed with injections of atropine sulfate, the oxime HI-6 and diazepam. Fear conditioning was conducted on post-exposure day (PED) 8 followed by measuring freezing to contextual and auditory conditioned stimuli on PED 9 and 10 respectively. Contextual and auditory fear conditioning was severely impaired in both the 1.0×LD50 and 1.2×LD50 GD groups. Both GD groups spent less time freezing than controls when returned to the context in which conditioning occurred. The 1.0×LD50 and 1.2×LD50 groups had very low levels of freezing following presentation of the auditory conditioned stimulus. Neuronal fiber degeneration was present in the piriform cortex, thalamus, and amygdala in GD-exposed animals regardless of dose. The present study suggests that contextual and auditory fear conditioning is impaired in GD-exposed rats possibly due to neuropathology observed in the hippocampus, amygdala and thalamus.

Spontaneous recurrent seizures after status epilepticus induced by soman in Sprague-Dawley rats.

PURPOSE: Exposure to toxic levels of organophosphorus (OP) nerve agents can lead to seizures, respiratory failure, and, if untreated, death. The cholinesterase inhibitor soman belongs to the class of OP nerve agents and can cause status epilepticus (SE) and brain damage due to neuroexcitotoxicity. In the present study, electroencephalographic seizures are characterized through telemetry implants in rats exposed to soman, followed by treatment with therapeutics similar to those administered after nerve agent exposure. METHODS: Cortical electroencephalography (EEG), motor activity and body temperature were recorded continuously for 2 days preexposure and 15 days postexposure to verify the occurrence of spontaneous recurrent seizures (SRS) after soman exposure. RESULTS: Behavioral seizures were monitored and the latency to SE was 7.8 ± 4.0 min after exposure. Among the rats that showed SE, approximately 90% had prolonged seizures within the initial 3 days after soman exposure. Five percent of the rats developed stage 1 seizures, 16% stage 2, 23% stage 3, 18% stage 4, and 38% stage 5. Seventy-nine percent of the rats presented SE and epileptiform-like discharges several days after SE, and 28.9% of those with SE experienced electrographic SRS. The latency to the appearance of SRS ranged from 5-10 days. Fiber degeneration evaluated through silver staining revealed damage in cortical and subcortical areas directly correlated with SE. DISCUSSION: The presence of SRS after seizures induced by soman highlights the importance of quantifying SRS in studies where the objective is to find new therapeutics against soman-induced seizures.