Efficacy of Lacosamide and Rufinamide as Adjuncts to Midazolam-Ketamine Treatment Against Cholinergic-Induced Status Epilepticus in Rats.

Benzodiazepine pharmacoresistance develops when treatment of status epilepticus (SE) is delayed. This response may result from gamma-aminobutyric acid A receptors (GABAAR) internalization that follows prolonged SE; this receptor trafficking results in fewer GABAAR in the synapse to restore inhibition. Increase in synaptic N-methyl-D-aspartate receptors (NMDAR) also occurs in rodent models of SE. Lacosamide, a third-generation antiseizure medication (ASM), acts on the slow inactivation of voltage-gated sodium channels. Another ASM, rufinamide, similarly acts on sodium channels by extending the duration of time spent in the inactivation stage. Combination therapy of the benzodiazepine midazolam, NMDAR antagonist ketamine, and ASMs lacosamide (or rufinamide) was investigated for efficacy against soman (GD)-induced SE and neuropathology. Adult male rats implanted with telemetry transmitters for monitoring electroencephalographic (EEG) activity were exposed to a seizure-inducing dose of GD and treated with an admix of atropine sulfate and HI-6 1 minute later and with midazolam monotherapy or combination therapy 40 minutes after EEG seizure onset. Rats were monitored continuously for seizure activity for two weeks, after which brains were processed for assessment of neurodegeneration, neuronal loss, and neuroinflammatory responses. Simultaneous administration of midazolam, ketamine, and lacosamide (or rufinamide) was more protective against GD-induced SE compared with midazolam monotherapy. In general, lacosamide triple therapy had more positive outcomes on measures of epileptogenesis, EEG power integral, and the number of brain regions protected from neuropathology compared with rats treated with rufinamide triple therapy. Overall, both drugs were well tolerated in these combination models. SIGNIFICANCE STATEMENT: We currently report on improved efficacy of antiseizure medications lacosamide and rufinamide, each administered in combination with ketamine (NMDAR antagonist) and midazolam (benzodiazepine), in combatting soman (GD)-induced seizure, epileptogenesis, and brain pathology over that provided by midazolam monotherapy, or dual therapy of midazolam and lacosamide (or rufinamide) in rats. Administration of lacosamide as adjunct to midazolam and ketamine was particularly effective against GD-induced toxicity. However, protection was incomplete, suggesting the need for further study.

Evaluation of Midazolam-Ketamine-Allopregnanolone Combination Therapy against Cholinergic-Induced Status Epilepticus in Rats.

Status epilepticus (SE) is a life-threatening development of self-sustaining seizures that becomes resistant to benzodiazepines when treatment is delayed. Benzodiazepine pharmacoresistance is thought in part to result from internalization of synaptic GABAA receptors, which are the main target of the drug. The naturally occurring neurosteroid allopregnanolone is a therapy of interest against SE for its ability to modulate all isoforms of GABAA receptors. Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, has been partially effective in combination with benzodiazepines in mitigating SE-associated neurotoxicity. In this study, allopregnanolone as an adjunct to midazolam or midazolam-ketamine combination therapy was evaluated for efficacy against cholinergic-induced SE. Adult male rats implanted with electroencephalographic (EEG) telemetry devices were exposed to the organophosphorus chemical (OP) soman (GD) and treated with an admix of atropine sulfate and HI-6 at 1 minute after exposure followed by midazolam, midazolam-allopregnanolone, or midazolam-ketamine-allopregnanolone 40 minutes after seizure onset. Neurodegeneration, neuronal loss, and neuroinflammation were assessed 2 weeks after GD exposure. Seizure activity, EEG power integral, and epileptogenesis were also compared among groups. Overall, midazolam-ketamine-allopregnanolone combination therapy was effective in reducing cholinergic-induced toxic signs and neuropathology, particularly in the thalamus and hippocampus. Higher dosage of allopregnanolone administered in combination with midazolam and ketamine was also effective in reducing EEG power integral and epileptogenesis. The current study reports that there is a promising potential of neurosteroids in combination with benzodiazepine and ketamine treatments in a GD model of SE. SIGNIFICANCE STATEMENT: Allopregnanolone, a naturally occurring neurosteroid, reduced pathologies associated with soman (GD) exposure such as epileptogenesis, neurodegeneration, and neuroinflammation, and suppressed GD-induced toxic signs when used as an adjunct to midazolam and ketamine in a delayed treatment model of soman-induced status epilepticus (SE) in rats. However, protection was incomplete, suggesting that further studies are needed to identify optimal combinations of antiseizure medications and routes of administration for maximal efficacy against cholinergic-induced SE.

Combination of antiseizure medications phenobarbital, ketamine, and midazolam reduces soman-induced epileptogenesis and brain pathology in rats.

OBJECTIVE: Cholinergic-induced status epilepticus (SE) is associated with a loss of synaptic gamma-aminobutyric acid A receptors (GABAA R) and an increase in N-methyl-D-aspartate receptors (NMDAR) and amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) that may contribute to pharmacoresistance when treatment with benzodiazepine antiseizure medication is delayed. The barbiturate phenobarbital enhances inhibitory neurotransmission by binding to a specific site in the GABAA R to increase the open state of the channel, decrease neuronal excitability, and reduce glutamate-induced currents through AMPA/kainate receptors. We hypothesized that phenobarbital as an adjunct to midazolam would augment the amelioration of soman-induced SE and associated neuropathological changes and that further protection would be provided by the addition of an NMDAR antagonist. METHODS: We investigated the efficacy of combining antiseizure medications to include a benzodiazepine and a barbiturate allosteric GABAA R modulator (midazolam and phenobarbital, respectively) to correct loss of inhibition, and ketamine to reduce excitation caused by increased synaptic localization of NMDAR and AMPAR, which are NMDA-dependent. Rats implanted with transmitters to record electroencephalographic (EEG) activity were exposed to soman and treated with atropine sulfate and HI-6 one min after exposure and with antiseizure medication(s) 40 minutes after seizure onset. RESULTS: The triple therapy combination of phenobarbital, midazolam, and ketamine administered at 40 minutes after seizure onset effectively prevented soman-induced epileptogenesis and reduced neurodegeneration. In addition, dual therapy with phenobarbital and midazolam or ketamine was more effective than monotherapy (midazolam or phenobarbital) in reducing cholinergic-induced toxicity. SIGNIFICANCE: Benzodiazepine efficacy is drastically reduced with time after seizure onset and inversely related to seizure duration. To overcome pharmacoresistance in severe benzodiazepine-refractory cholinergic-induced SE, simultaneous drug combination to include drugs that target both the loss of inhibition (eg, midazolam, phenobarbital) and the increased excitatory response (eg, ketamine) is more effective than benzodiazepine or barbiturate monotherapy.

Novel Genetically Modified Mouse Model to Assess Soman-Induced Toxicity and Medical Countermeasure Efficacy: Human Acetylcholinesterase Knock-in Serum Carboxylesterase Knockout Mice.

The identification of improved medical countermeasures against exposure to chemical warfare nerve agents (CWNAs), a class of organophosphorus compounds, is dependent on the choice of animal model used in preclinical studies. CWNAs bind to acetylcholinesterase and prevent the catalysis of acetylcholine, causing a plethora of peripheral and central physiologic manifestations, including seizure. Rodents are widely used to elucidate the effects of CWNA-induced seizure, albeit with a caveat: they express carboxylesterase activity in plasma. Carboxylesterase, an enzyme involved in the detoxification of some organophosphorus compounds, plays a scavenging role and decreases CWNA availability, thus exerting a protective effect. Furthermore, species-specific amino acid differences in acetylcholinesterase confound studies that use oximes or other compounds to restore its function after inhibition by CWNA. The creation of a human acetylcholinesterase knock-in/serum carboxylesterase knockout (C57BL/6-Ces1ctm1.1LocAChEtm1.1Loc/J; a.k.a KIKO) mouse may facilitate better modeling of CWNA toxicity in a small rodent species. The current studies characterize the effects of exposure to soman, a highly toxic CWNA, and evaluate the efficacy of anti-seizure drugs in this newly developed KIKO mouse model. Data demonstrate that a combination of midazolam and ketamine reduces seizure duration and severity, eliminates the development of spontaneous recurrent seizures, and protects certain brain regions from neuronal damage in a genetically modified model with human relevance to organophosphorus compound toxicity. This new animal model and the results of this study and future studies using it will enhance medical countermeasures development for both defense and homeland security purposes.

Cannabidiol reduces soman-induced lethality and seizure severity in female plasma carboxylesterase knockout mice treated with midazolam.

Cannabidiol, approved for treatment of pediatric refractory epilepsy, has anti-seizure effects in various animal seizure models. Chemical warfare nerve agents, including soman, are organophosphorus chemicals that can induce seizure and death if untreated or if treatment is delayed. Our objective was to evaluate whether cannabidiol would ameliorate soman-induced toxicity using a mouse model that similar to humans lacks plasma carboxylesterase. In the present study, adult female plasma carboxylesterase knockout (Es1-/-) mice were pre-treated with cannabidiol (20-150 mg/kg) or vehicle 1 h prior to exposure to a seizure-inducing dose of soman and evaluated for survival and seizure activity. The muscarinic antagonist atropine sulfate and the oxime HI-6 were administered at 1 min after exposure, and the benzodiazepine midazolam was administered at 30 min after seizure onset. Cannabidiol (150 mg/kg) pre-treatment led to a robust increase in survival rate and attenuated body weight loss in soman-exposed mice treated with medical countermeasures, compared to mice pre-treated with vehicle. In addition, mice pretreated with cannabidiol (150 mg/kg) had a modest reduction in seizure severity after midazolam treatment compared to vehicle-pretreated. These findings of improved outcome with cannabidiol administration in a severe seizure model of soman exposure provide additional pre-clinical support for the benefits of cannabidiol against exposure to seizure-inducing chemical agents and suggest cannabidiol may augment the anti-seizure effects of midazolam.

Ketamine as adjunct to midazolam treatment following soman-induced status epilepticus reduces seizure severity, epileptogenesis, and brain pathology in plasma carboxylesterase knockout mice.

Delayed treatment of cholinergic seizure results in benzodiazepine-refractory status epilepticus (SE) that is thought, at least in part, to result from maladaptive trafficking of N-methyl-d-aspartate (NMDA) and gamma-aminobutyric acid type A (GABAA) receptors, the effects of which may be ameliorated by combination therapy with the NMDA receptor antagonist ketamine. Our objective was to establish whether ketamine and midazolam dual therapy would improve outcome over midazolam monotherapy following soman (GD) exposure when evaluated in a mouse model that, similar to humans, lacks plasma carboxylesterase, greatly reducing endogenous scavenging of GD. In the current study, continuous cortical electroencephalographic activity was evaluated in male and female plasma carboxylesterase knockout mice exposed to a seizure-inducing dose of GD and treated with midazolam or with midazolam and ketamine combination at 40 min after seizure onset. Ketamine and midazolam combination reduced GD-induced lethality, seizure severity, and the number of mice that developed spontaneous recurrent seizure (SRS) compared with midazolam monotherapy. In addition, ketamine-midazolam combination treatment reduced GD-induced neuronal degeneration and microgliosis. These results support that combination of antiepileptic drug therapies aimed at correcting the maladaptive GABAA and NMDA receptor trafficking reduces the detrimental effects of GD exposure. Ketamine may be a beneficial adjunct to midazolam in reducing the epileptogenesis and neuroanatomical damage that follows nerve agent exposure and pharmacoresistant SE.

Dataset of EEG power integral, spontaneous recurrent seizure and behavioral responses following combination drug therapy in soman-exposed rats.

This article investigated the efficacy of the combination of antiepileptic drug therapy in protecting against soman-induced seizure severity, epileptogenesis and performance deficits. Adult male rats with implanted telemetry transmitters for continuous recording of electroencephalographic (EEG) activity were exposed to soman and treated with atropine sulfate and the oxime HI-6 one minute after soman exposure and with midazolam, ketamine and/or valproic acid 40 min after seizure onset. Rats exposed to soman and treated with medical countermeasures were evaluated for survival, seizure severity, the development of spontaneous recurrent seizure and performance deficits; combination anti-epileptic drug therapy was compared with midazolam monotherapy. Telemetry transmitters were used to record EEG activity, and a customized MATLAB algorithm was used to analyze the telemetry data. Survival data, EEG power integral data, spontaneous recurrent seizure data and behavioral data are illustrated in figures and included as raw data. In addition, edf files of one month telemetry recordings from soman-exposed rats treated with delayed midazolam are provided as supplementary materials. Data presented in this article are related to research articles "Rational Polytherapy in the Treatment of Cholinergic Seizures" [1] and "Early polytherapy for benzodiazepine-refractory status epilepticus [4].

Soman-induced status epilepticus, epileptogenesis, and neuropathology in carboxylesterase knockout mice treated with midazolam.

OBJECTIVE: Exposure to chemical warfare nerve agents (CWNAs), such as soman (GD), can induce status epilepticus (SE) that becomes refractory to benzodiazepines when treatment is delayed, leading to increased risk of epileptogenesis, severe neuropathology, and long-term behavioral and cognitive deficits. Rodent models, widely used to evaluate novel medical countermeasures (MCMs) against CWNA exposure, normally express plasma carboxylesterase, an enzyme involved in the metabolism of certain organophosphorus compounds. To better predict the efficacy of novel MCMs against CWNA exposure in human casualties, it is crucial to use appropriate animal models that mirror the human condition. We present a comprehensive characterization of the seizurogenic, epileptogenic, and neuropathologic effects of GD exposure with delayed anticonvulsant treatment in the plasma carboxylesterase knockout (ES1-/-) mouse. METHODS: Electroencephalography (EEG) electrode-implanted ES1-/- and wild-type (C57BL/6) mice were exposed to various seizure-inducing doses of GD, treated with atropine sulfate and the oxime HI-6 at 1 minute after exposure, and administered midazolam at 15-30 minutes following the onset of seizure activity. The latency of acute seizure onset and spontaneous recurrent seizures (SRS) was assessed, as were changes in EEG power spectra. At 2 weeks after GD exposure, neurodegeneration and neuroinflammation were assessed. RESULTS: GD-exposed ES1-/- mice displayed a dose-dependent response in seizure severity. Only ES1-/- mice exposed to the highest tested dose of GD developed SE, subchronic alterations in EEG power spectra, and SRS. Degree of neuronal cell loss and neuroinflammation were dose-dependent; no significant neuropathology was observed in C57BL/6 mice or ES1-/- mice exposed to lower GD doses. SIGNIFICANCE: The US Food and Drug Administration (FDA) animal rule requires the use of relevant animal models for the advancement of MCMs against CWNAs. We present evidence that argues for the use of the ES1-/- mouse model to screen anticonvulsant, antiepileptic, and/or neuroprotective drugs against GD-induced toxicity, as well as to identify mechanisms of GD-induced epileptogenesis.

Poisoning with Soman, an Organophosphorus Nerve Agent, Alters Fecal Bacterial Biota and Urine Metabolites: a Case for Novel Signatures for Asymptomatic Nerve Agent Exposure.

The experimental pathophysiology of organophosphorus (OP) chemical exposure has been extensively reported. Here, we describe an altered fecal bacterial biota and urine metabolome following intoxication with soman, a lipophilic G class chemical warfare nerve agent. Nonanesthetized Sprague-Dawley male rats were subcutaneously administered soman at 0.8 (subseizurogenic) or 1.0 (seizurogenic) of the 50% lethal dose (LD50) and evaluated for signs of toxicity. Animals were stratified based on seizing activity to evaluate effects of soman exposure on fecal bacterial biota and urine metabolites. Soman exposure reshaped fecal bacterial biota by altering Facklamia, Rhizobium, Bilophila, Enterobacter, and Morganella genera of the Firmicutes and Proteobacteria phyla, some of which are known to hydrolyze OP chemicals. However, analogous changes were not observed in the bacterial biota of the ileum, which remained the same irrespective of dose or seizing status of animals after soman intoxication. However, at 75 days after soman exposure, the bacterial biota stabilized and no differences were observed between groups. Interestingly, in considering just the seizing status of animals, we found that the urine metabolomes were markedly different. Leukotriene C4, kynurenic acid, 5-hydroxyindoleacetic acid, norepinephrine, and aldosterone were excreted at much higher rates at 72 h in seizing animals, consistent with early multiorgan involvement during soman poisoning. These findings demonstrate the feasibility of using the dysbiosis of fecal bacterial biota in combination with urine metabolome alterations as forensic evidence for presymptomatic OP exposure temporally to enable administration of neuroprotective therapies of the future.IMPORTANCE The paucity of assays to determine physiologically relevant OP exposure presents an opportunity to explore the use of fecal bacteria as sentinels in combination with urine to assess changes in the exposed host. Recent advances in sequencing technologies and computational approaches have enabled researchers to survey large community-level changes of gut bacterial biota and metabolomic changes in various biospecimens. Here, we profiled changes in fecal bacterial biota and urine metabolome following a chemical warfare nerve agent exposure. The significance of this work is a proof of concept that the fecal bacterial biota and urine metabolites are two separate biospecimens rich in surrogate indicators suitable for monitoring OP exposure. The larger value of such an approach is that assays developed on the basis of these observations can be deployed in any setting with moderate clinical chemistry and microbiology capability. This can enable estimation of the affected radius as well as screening, triage, or ruling out of suspected cases of exposures in mass casualty scenarios, transportation accidents involving hazardous materials, refugee movements, humanitarian missions, and training settings when coupled to an established and validated decision tree with clinical features.