Tolerability and pharmacokinetics of intravenous allopregnanolone with and without midazolam pretreatment in two healthy dogs.

The neurosteroid allopregnanolone (ALLO) is under investigation as a treatment for benzodiazepine-refractory status epilepticus (SE). Here, we assess the cardiopulmonary safety of intravenous ALLO by itself and after a clinically recommended dose of midazolam (MDZ) in two healthy adult beagles. Each dog received ALLO (1 mg/kg, IV), and after a washout period of 2 weeks, each dog was dosed with MDZ (0.2 mg/kg, IV) followed 10 minutes later by ALLO. Behavioral state, vital signs, arterial blood gases, blood chemistries, and plasma ALLO concentrations were monitored for up to 6 hours after dosing. The dogs appeared sleepy but were fully responsive after both treatments. No depression of mean arterial pressure or respiratory rate was noted. Blood gas measurements failed to show evidence of drug-induced acute respiratory acidosis. Estimated maximum plasma ALLO concentrations were in the range of 1500 to 3000 ng/ml. The results indicate that intravenous ALLO can be used safely to treat benzodiazepine-refractory SE, even when administered shortly after a benzodiazepine.

Intravenous and Intramuscular Allopregnanolone for Early Treatment of Status Epilepticus: Pharmacokinetics, Pharmacodynamics, and Safety in Dogs.

Allopregnanolone (ALLO) is a neurosteroid that modulates synaptic and extrasynaptic GABAA receptors. We hypothesize that ALLO may be useful as first-line treatment of status epilepticus (SE). Our objectives were to (1) characterize ALLO pharmacokinetics-pharmacodynamics PK-PD after intravenous (IV) and intramuscular (IM) administration and (2) compare IV and IM ALLO safety and tolerability. Three healthy dogs and two with a history of epilepsy were used. Single ALLO IV doses ranging from 1-6 mg/kg were infused over 5 minutes or injected IM. Blood samples, vital signs, and sedation assessment were collected up to 8 hours postdose. Intracranial EEG (iEEG) was continuously recorded in one dog. IV ALLO exhibited dose-proportional increases in exposure, which were associated with an increase in absolute power spectral density in all iEEG frequency bands. This relationship was best described by an indirect link PK-PD model where concentration-response was described by a sigmoidal maximum response (Emax) equation. Adverse events included site injection pain with higher IM volumes and ataxia and sedation associated with higher doses. IM administration exhibited incomplete absorption and volume-dependent bioavailability. Robust iEEG changes after IM administration were not observed. Based on PK-PD simulations, a 2 mg/kg dose infused over 5 minutes is predicted to achieve plasma concentrations above the EC50, but below those associated with heavy sedation. This study demonstrates that ALLO is safe and well tolerated when administered at 1-4 mg/kg IV and up to 2 mg/kg IM. The rapid onset of effect after IV infusion suggests that ALLO may be useful in the early treatment of SE. SIGNIFICANCE STATEMENT: The characterization of the pharmacokinetics and pharmacodynamics of allopregnanolone is essential in order to design clinical studies evaluating its effectiveness as an early treatment for status epilepticus in dogs and people. This study has proposed a target dose/therapeutic range for a clinical trial in canine status epilepticus.

Strain differences in the extent of brain injury in mice after tetramethylenedisulfotetramine-induced status epilepticus.

Acute intoxication with tetramethylenedisulfotetramine (TETS) can trigger status epilepticus (SE) in humans. Survivors often exhibit long-term neurological effects, including electrographic abnormalities and cognitive deficits, but the pathogenic mechanisms linking the acute toxic effects of TETS to chronic outcomes are not known. Here, we use advanced in vivo imaging techniques to longitudinally monitor the neuropathological consequences of TETS-induced SE in two different mouse strains. Adult male NIH Swiss and C57BL/6J mice were injected with riluzole (10 mg/kg, i.p.), followed 10 min later by an acute dose of TETS (0.2 mg/kg in NIH Swiss; 0.3 mg/kg, i.p. in C57BL/6J) or an equal volume of vehicle (10% DMSO in 0.9% sterile saline). Different TETS doses were administered to trigger comparable seizure behavior between strains. Seizure behavior began within minutes of TETS exposure and rapidly progressed to SE that was terminated after 40 min by administration of midazolam (1.8 mg/kg, i.m.). The brains of vehicle and TETS-exposed mice were imaged using in vivo magnetic resonance (MR) and translocator protein (TSPO) positron emission tomography (PET) at 1, 3, 7, and 14 days post-exposure to monitor brain injury and neuroinflammation, respectively. When the brain scans of TETS mice were compared to those of vehicle controls, subtle and transient neuropathology was observed in both mouse strains, but more extensive and persistent TETS-induced neuropathology was observed in C57BL/6J mice. In addition, one NIH Swiss TETS mouse that did not respond to the midazolam therapy, but remained in SE for more than 2 h, displayed robust neuropathology as determined by in vivo imaging and confirmed by FluoroJade C staining and IBA-1 immunohistochemistry as readouts of neurodegeneration and neuroinflammation, respectively. These findings demonstrate that the extent of injury observed in the mouse brain after TETS-induced SE varied according to strain, dose of TETS and/or the duration of SE. These observations suggest that TETS-intoxicated humans who do not respond to antiseizure medication are at increased risk for brain injury.

Effect of acute and chronic exposure to lovastatin on the anticonvulsant action of classical antiepileptic drugs in the mouse maximal electroshock-induced seizure model.

Numerous studies indicate neuroprotective activity of statins, commonly used cholesterol lowering drugs in epilepsy and several other neurological diseases. Promising anti-convulsant and neuroprotective effects of statins, attributed to their anti-excitotoxic and anti-inflammatory action were reported in several animals' seizure models. To determine the effects of acute (single) and chronic (once daily for 7 consecutive days) administration of lovastatin on the protective activity of four classical antiepileptic drugs such as carbamazepine, phenobarbital, phenytoin and valproate in the mouse maximal electroshock seizure model. Seizure activity (maximal electroconvulsions) in mice were generated by alternating current delivered via ear-clip electrodes. Adverse-effect profile of lovastatin combinations with the tested antiepileptic drugs was assessed in the chimney test (motor performance). Total brain concentrations of antiepileptic drugs were evaluated with the fluorescence polarization immunoassay technique as a measure of the pharmacokinetic interaction between drugs. Lovastatin administered acutely or chronically (5-20 mg/kg) did not significantly affect the threshold for electroconvulsions in mice. Acute lovastatin (10 mg/kg) significantly enhanced the anticonvulsant effect of valproate, which was accompanied with a 34% significant increase in total brain concentration of valproate. Acute lovastatin in combination with phenytoin impaired motor performance by notably decreasing the TD50 value of phenytoin. Chronic lovastatin (10 mg/kg) markedly enhanced the anticonvulsant potential of phenytoin. Acute lovastatin increased anticonvulsant action of valproate but also significantly raised level of valproate in brain after combined administration suggesting pharmacokinetic nature of interaction. The combinations of chronic lovastatin combined with phenytoin can potentially enhance the anticonvulsant potency of phenytoin.

Interactions among Lacosamide and Second-Generation Antiepileptic Drugs in the Tonic-Clonic Seizure Model in Mice.

Combination therapy with two or three antiseizure medications (ASMs) is sometimes a preferred method of treatment in epilepsy patients. (1) Background: To detect the most beneficial combination among three ASMs, a screen test evaluating in vivo interactions with respect to their anticonvulsant properties, was conducted on albino Swiss mice; (2) Methods: Classification of interactions among lacosamide (LCM) and selected second-generation ASMs (lamotrigine (LTG), pregabalin (PGB), oxcarbazepine (OXC), and topiramate (TPM)) was based on the isobolographic analysis in the mouse maximal electroshock-induced seizure (MES) model. Interactions among LCM and second-generation ASMs were visualized using a polygonogram; (3) Results: In the mouse MES model, synergy was observed for the combinations of LCM + TPM + PGB and LCM + OXC + PGB. Additivity was reported for the other combinations tested i.e., LCM + LTG + TPM, LCM + LTG + PGB, LCM + LTG + OXC, and LCM + OXC + TPM in this seizure model. No adverse effects associated with triple ASM combinations, containing LCM and second-generation ASMs were observed in mice; (4) Conclusions: The combination of LCM + TPM + PGB was the most beneficial combination among the tested in this study, offering synergistic suppression of tonic-clonic seizures in mice subjected to the MES model. Both the isobolographic analysis and polygonogram method can be recommended for experimental epileptology when classifying interactions among the ASMs.

Perampanel, a potent AMPA receptor antagonist, protects against tetramethylenedisulfotetramine-induced seizures and lethality in mice: comparison with diazepam.

Tetramethylenedisulfotetramine (TETS), a noncompetitive GABAA receptor antagonist, is a potent, highly lethal convulsant that is considered to be a chemical threat agent. Here, we assessed the ability of the AMPA receptor antagonist perampanel to protect against TETS-induced seizures and lethality in mice when administered before or after treatment with the toxicant. For comparison, we conducted parallel testing with diazepam, which is a first-line treatment for chemically induced seizures in humans. Pre-treatment of mice with either perampanel (1-4 mg/kg, i.p.) or diazepam (1-5 mg/kg, i.p.) conferred protection in a dose-dependent fashion against tonic seizures and lethality following a dose of TETS (0.2 mg/kg, i.p.) that rapidly induces seizures and death. The ED50 values for protection against mortality were 1.6 mg/kg for perampanel and 2.1 mg/kg for diazepam. Clonic seizures were unaffected by perampanel and only prevented in a minority of animals by high-dose diazepam. Neither treatment prevented myoclonic body twitches. Perampanel and diazepam also conferred protection against tonic seizures and lethality when administered 15 min following a 0.14 mg/kg, i.p., dose of TETS and 5 min following a 0.2 mg/kg, i.p., dose of TETS. Both posttreatments were highly potent at reducing tonic seizures and lethality in animals exposed to the lower dose of TETS whereas greater doses of both treatments were required in animals exposed to the larger dose of TETS. Neither treatment was as effective suppressing clonic seizures. In an experiment where 0.4 mg/kg TETS was administered by oral gavage and the treatment drugs were administered 5 min later, perampanel only partially protected against lethality whereas diazepam produced nearly complete protection. We conclude that perampanel and diazepam protect against TETS-induced tonic seizures and lethality but have less impact on clonic seizures. Both drugs could have utility in the treatment of TETS intoxication but neither eliminates all seizure activity.

Intranasal Allopregnanolone Confers Rapid Seizure Protection: Evidence for Direct Nose-to-Brain Delivery.

Allopregnanolone, a positive modulator of GABAA receptors with antiseizure activity, has potential in the treatment of seizure emergencies. Instillation of allopregnanolone in 40% sulfobutylether-ß-cyclodextrin into the nose in mice rapidly elevated the seizure threshold in the timed intravenous pentylenetetrazol (ED50, 5.6 mg/kg), picrotoxin (ED50, 5.9 mg/kg), and bicuculline seizure tests. The effect peaked at 15 min, decayed over 1 h, and was still evident in some experiments at 6 h. Intranasal allopregnanolone also delayed the onset of seizures in the maximal PTZ test. At an allopregnanolone dose (16 mg/kg) that conferred comparable effects on seizure threshold as the benzodiazepines midazolam and diazepam (both at doses of 1 mg/kg), allopregnanolone caused minimal sedation or motor toxicity in the horizontal screen test whereas both benzodiazepines produced marked behavioral impairment. In addition, intranasal allopregnanolone failed to cause loss-of-righting reflex in most animals, but when the same dose was administered intramuscularly, all animals became impaired. Intranasal allopregnanolone (10 mg/kg) caused a rapid increase in brain allopregnanolone with a Tmax of ~5 min after initiation of the intranasal delivery. High levels of allopregnanolone were recovered in the olfactory bulb (Cmax, 16,000 ng/mg) whereas much lower levels (Cmax, 670 ng/mg) were present in the remainder of the brain. We conclude that the unique ability of intranasal allopregnanolone to protect against seizures without inducing behavioral adverse effects is due in part to direct nose-to-brain delivery, with preferential transport to brain regions relevant to seizures. Benzodiazepines are commonly administered intranasally for acute seizure therapy, including for the treatment of acute repetitive seizures, but are not transported from nose-to-brain. Intranasal allopregnanolone acts with greater speed, has less propensity for adverse effects, and has the ability to overcome benzodiazepine refractoriness. This is the first study demonstrating rapid functional central nervous system activity of a nose-to-brain-delivered steroid. Intranasal delivery circumvents the poor oral bioavailability of allopregnanolone providing a route of administration permitting its evaluation as a treatment for diverse neuropsychiatric indications.

Polygonogram with isobolographic synergy for three-drug combinations of phenobarbital with second-generation antiepileptic drugs in the tonic-clonic seizure model in mice.

BACKGROUND: Combination therapy consisting of two or more antiepileptic drugs (AEDs) is usually prescribed for patients with refractory epilepsy. The drug-drug interactions, which may occur among currently available AEDs, are the principal criterion taken by physicians when prescribing the AED combination to the patients. Unfortunately, the number of possible three-drug combinations tremendously increases along with the clinical approval of novel AEDs. AIM: To isobolographically characterize three-drug interactions of phenobarbital (PB) with lamotrigine (LTG), oxcarbazepine (OXC), pregabalin (PGB) and topiramate (TPM), the maximal electroshock-induced (MES) seizure model was used in male albino Swiss mice. MATERIALS AND METHOD: The MES-induced seizures in mice were generated by alternating current delivered via auricular electrodes. To classify interactions for 6 various three-drug combinations of AEDs (i.e., PB + TPM + PGB, PB + OXC + TPM, PB + LTG + TPM, PB + OXC + PGB, PB + LTG + PGB and PB + LTG + OXC), the type I isobolographic analysis was used. Total brain concentrations of PB were measured by fluorescent polarization immunoassay technique. RESULTS: The three-drug mixtures of PB + TPM + PGB, PB + OXC + TPM, PB + LTG + TPM, PB + OXC + PGB, PB + LTG + PGB and PB + LTG + OXC protected the male albino Swiss mice from MES-induced seizures. All the observed interactions in this seizure model were supra-additive (synergistic) (p < 0.001), except for the combination of PB + LTG + OXC, which was additive. It was unable to show the impact of the studied second-generation AEDs on total brain content of PB in mice. CONCLUSIONS: The synergistic interactions among PB and LTG, OXC, PGB and TPM in the mouse MES model are worthy of being transferred to clinical trials, especially for the patients with drug resistant epilepsy, who would benefit these treatment options.

Persistent behavior deficits, neuroinflammation, and oxidative stress in a rat model of acute organophosphate intoxication.

Current medical countermeasures for organophosphate (OP)-induced status epilepticus (SE) are not effective in preventing long-term morbidity and there is an urgent need for improved therapies. Rat models of acute intoxication with the OP, diisopropylfluorophosphate (DFP), are increasingly being used to evaluate therapeutic candidates for efficacy in mitigating the long-term neurologic effects associated with OP-induced SE. Many of these therapeutic candidates target neuroinflammation and oxidative stress because of their implication in the pathogenesis of persistent neurologic deficits associated with OP-induced SE. Critical to these efforts is the rigorous characterization of the rat DFP model with respect to outcomes associated with acute OP intoxication in humans, which include long-term electroencephalographic, neurobehavioral, and neuropathologic effects, and their temporal relationship to neuroinflammation and oxidative stress. To address these needs, we examined a range of outcomes at later times post-exposure than have previously been reported for this model. Adult male Sprague-Dawley rats were given pyridostigmine bromide (0.1 mg/kg, im) 30 min prior to administration of DFP (4 mg/kg, sc), which was immediately followed by atropine sulfate (2 mg/kg, im) and pralidoxime (25 mg/kg, im). This exposure paradigm triggered robust electroencephalographic and behavioral seizures that rapidly progressed to SE lasting several hours in 90% of exposed animals. Animals that survived DFP-induced SE (~70%) exhibited spontaneous recurrent seizures and hyperreactive responses to tactile stimuli over the first 2 months post-exposure. Performance in the elevated plus maze, open field, and Pavlovian fear conditioning tests indicated that acute DFP intoxication reduced anxiety-like behavior and impaired learning and memory at 1 and 2 months post-exposure in the absence of effects on general locomotor behavior. Immunohistochemical analyses revealed significantly increased expression of biomarkers of reactive astrogliosis, microglial activation and oxidative stress in multiple brain regions at 1 and 2 months post-DFP, although there was significant spatiotemporal heterogeneity across these endpoints. Collectively, these data largely support the relevance of the rat model of acute DFP intoxication as a model for acute OP intoxication in the human, and support the hypothesis that neuroinflammation and/or oxidative stress represent potential therapeutic targets for mitigating the long-term neurologic sequelae of acute OP intoxication.

Combination of phenobarbital with phenytoin and pregabalin produces synergy in the mouse tonic-clonic seizure model: An isobolographic analysis.

AIMS: Despite many antiepileptic drugs (AEDs) are available to treat epilepsy, there is still about 30% of epilepsy patients inadequately treated with these AEDs. For these patients, polytherapy with two or three AEDs to fully control their seizure attacks is recommended. Unfortunately, polytherapy is always associated with drug interactions, whose nature may be beneficial, neutral or unfavorable. To determine a type of interaction for the combination of three AEDs (i.e., phenobarbital [PB], phenytoin [PHT] and pregabalin [PGB]) at the fixed-ratio of 1:1:1, we used a model of tonic-clonic seizures in male albino Swiss mice. MATERIALS AND METHOD: Tonic-clonic seizures in mice were evoked by a current (sine-wave, 25 mA, 500 V, 0.2 s stimulus duration) delivered via auricular electrodes. The anticonvulsant effects of the three-drug combination (PB, PHT and PGB) in terms of suppression of tonic-clonic seizures in mice were assessed with type I isobolographic analysis. Potential acute side effects for the mixture of PB, PHT and PGB along with total brain concentrations of the AEDs were determined to confirm pharmacodynamic nature of observed interaction. RESULTS: The three-drug combination of PB, PHT and PGB (at the fixed-ratio of 1:1:1) exerted synergistic interaction (at P < 0.01) in the mouse model of tonic-clonic seizures. The combination of PB, PHT and PGB did not produce any side effects in experimental animals, when measuring long-term memory, muscular strength and motor coordination. The measurement of total brain concentrations of PB, PHT and PGB was conducted to confirm that none of the three AEDs significantly influenced total brain concentrations (pharmacokinetic profiles) of the other co-administered AEDs in mice. CONCLUSIONS: The synergistic pharmacodynamic interaction for the combination of PB, PHT and PGB observed in this preclinical study can be translated into clinical settings and this favorable AED combination is worthy of being recommended to some patients with refractory epilepsy.

Intramuscular allopregnanolone and ganaxolone in a mouse model of treatment-resistant status epilepticus.

Allopregnanolone (5α-pregnan-3α-ol-20-one) and its synthetic 3ß-methyl analog, ganaxolone, are positive allosteric modulators of synaptic and extrasynaptic γ-aminobutyric acid (GABA)A receptors that exhibit antiseizure activity in diverse animal seizure models, including models of status epilepticus (SE). The 2 neuroactive steroids are being investigated as treatments for SE, including as a treatment for SE induced by chemical threat agents. Intramuscular injection is the preferred route of administration in the prehospital treatment of SE. The objective of this study was to assess the efficacy of intramuscular allopregnanolone and ganaxolone in the treatment of SE induced by the chemical threat agent tetramethylenedisulfotetramine (TETS). The test agents were administered 40 minutes after the onset of SE when mice are refractory to treatment. Allopregnanolone and ganaxolone (each at 3 mg/kg) terminated SE in, respectively, 92% and 75% of animals, and prevented mortality in 85% and 50% of animals; the mean times to termination of behavioral seizures were, respectively, 172 ± 16 and 447 ± 52 seconds. In a separate series of experiments, mice were dosed with the neuroactive steroids by intramuscular injection, and plasma and brain levels were sampled at various time points following injection to estimate pharmacokinetic parameters. Plasma Cmax (maximum concentration) values for allopregnanolone and ganaxolone were 645 and 550 ng/mL, respectively. Brain exposure of both steroids was approximately 3-fold the plasma exposure. Two-compartment pharmacokinetic analysis revealed that the central compartment Vd (volume of distribution), CL (clearance), t½ (terminal half-life), and F (intramuscular bioavailability) values for allopregnanolone and ganaxolone were, respectively, 4.95 L/kg 12.88 L/kg/h,16 minutes, 97%, and 5.07 L/kg, 8.35 L/kg/h, 25 minutes, 95%. Allopregnanolone and ganaxolone are effective in the treatment of TETS-induced SE when administered by the intramuscular route. Allopregnanolone is more rapidly acting and modestly more effective, possibly because it has greater potency on GABAA receptors.

Neuronal overexpression of Ube3a isoform 2 causes behavioral impairments and neuroanatomical pathology relevant to 15q11.2-q13.3 duplication syndrome.

Maternally derived copy number gains of human chromosome 15q11.2-q13.3 (Dup15q syndrome or Dup15q) cause intellectual disability, epilepsy, developmental delay, hypotonia, speech impairments, and minor dysmorphic features. Dup15q syndrome is one of the most common and penetrant chromosomal abnormalities observed in individuals with autism spectrum disorder (ASD). Although ∼40 genes are located in the 15q11.2-q13.3 region, overexpression of the ubiquitin-protein E3A ligase (UBE3A) gene is thought to be the predominant molecular cause of the phenotypes observed in Dup15q syndrome. The UBE3A gene demonstrates maternal-specific expression in neurons and loss of maternal UBE3A causes Angelman syndrome, a neurodevelopmental disorder with some overlapping neurological features to Dup15q. To directly test the hypothesis that overexpression of UBE3A is an important underlying molecular cause of neurodevelopmental dysfunction, we developed and characterized a mouse overexpressing Ube3a isoform 2 in excitatory neurons. Ube3a isoform 2 is conserved between mouse and human and known to play key roles in neuronal function. Transgenic mice overexpressing Ube3a isoform 2 in excitatory forebrain neurons exhibited increased anxiety-like behaviors, learning impairments, and reduced seizure thresholds. However, these transgenic mice displayed normal social approach, social interactions, and repetitive motor stereotypies that are relevant to ASD. Reduced forebrain, hippocampus, striatum, amygdala, and cortical volume were also observed. Altogether, these findings show neuronal overexpression of Ube3a isoform 2 causes phenotypes translatable to neurodevelopmental disorders.

Ivabradine attenuates the anticonvulsant potency of lamotrigine, but not that of lacosamide, pregabalin and topiramate in the tonic-clonic seizure model in mice.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are involved not only in synaptic transmission and neuronal excitability under physiological conditions, but also in seizure activity. To determine the influence of ivabradine (an HCN channel inhibitor) on the anticonvulsant potency of four novel antiepileptic drugs (AEDs: lacosamide, lamotrigine, pregabalin and topiramate) in the mouse maximal electroshock-induced seizure (MES) model. Adult male albino Swiss mice were challenged with maximal electroconvulsions (electric current of 25mA delivered via auricular electrodes). Total brain concentrations of AEDs were measured with high-pressure liquid chromatography. Ivabradine (10mg/kg, i.p.) significantly reduced the anticonvulsant potency of lamotrigine by elevating the ED50 value of the AED from 7.48 (6.15-9.11) to 10.07 (8.85-11.45) mg/kg (P<0.05) in the mouse MES model. In contrast, ivabradine (10mg/kg, i.p.) did not significantly affect the anticonvulsant potency of lacosamide, pregabalin or topiramate in the mouse MES model. Additionally, ivabradine had no impact on total brain concentrations of all the studied AEDs in mice. A special caution is advised when combining ivabradine with lamotrigine in epilepsy patients due to the possible pharmacodynamic reduction of the anticonvulsant action of the later drug. The combinations of ivabradine with lacosamide, pregabalin and topiramate seem to be pharmacodynamic and neutral from a preclinical viewpoint.

Influence of Ivabradine on the Anticonvulsant Action of Four Classical Antiepileptic Drugs Against Maximal Electroshock-Induced Seizures in Mice.

Although the role of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in neuronal excitability and synaptic transmission is still unclear, it is postulated that the HCN channels may be involved in seizure activity. The aim of this study was to assess the effects of ivabradine (an HCN channel inhibitor) on the protective action of four classical antiepileptic drugs (carbamazepine, phenobarbital, phenytoin and valproate) against maximal electroshock-induced seizures in mice. Tonic seizures (maximal electroconvulsions) were evoked in adult male albino Swiss mice by an electric current (sine-wave, 25 mA, 0.2 s stimulus duration) delivered via auricular electrodes. Acute adverse-effect profiles of the combinations of ivabradine with classical antiepileptic drugs were measured in mice along with total brain antiepileptic drug concentrations. Results indicate that ivabradine (10 mg/kg, i.p.) significantly enhanced the anticonvulsant activity of valproate and considerably reduced that of phenytoin in the mouse maximal electroshock-induced seizure model. Ivabradine (10 mg/kg) had no impact on the anticonvulsant potency of carbamazepine and phenobarbital in the maximal electroshock-induced seizure test in mice. Ivabradine (10 mg/kg) significantly diminished total brain concentration of phenytoin and had no effect on total brain valproate concentration in mice. In conclusion, the enhanced anticonvulsant action of valproate by ivabradine in the mouse maximal electroshock-induced seizure model was pharmacodynamic in nature. A special attention is required when combining ivabradine with phenytoin due to a pharmacokinetic interaction and reduction of the anticonvulsant action of phenytoin in mice. The combinations of ivabradine with carbamazepine and phenobarbital were neutral from a preclinical viewpoint.

Cytisine inhibits the protective activity of various classical and novel antiepileptic drugs against 6 Hz-induced psychomotor seizures in mice.

BACKGROUND: Cytisine (CYT) is a partial agonist of brain α4ß2 nicotinic acetylcholine receptors widely used in Central/Eastern Europe for smoking cessation. OBJECTIVES: This study evaluated the effect of CYT on the ability of classical and novel antiepileptic drugs to prevent seizures evoked by the 6-Hz test, a model of psychomotor seizures in mice thought as a model of drug-resistant seizures. RESULTS: CYT administered intraperitoneally (i.p.) in a dose of 2 mg kg-1 significantly inhibited the anticonvulsant activity of lacosamide, levetiracetam, and pregabalin, increasing their median effective doses 50 (ED50) values from 6.88 to 10.52 mg kg-1 (P < 0.05) for lacosamide, from 22.08 to 38.26 mg kg-1 (P < 0.05) for levetiracetam, and from 40.48 to 64.61 mg kg-1 (P < 0.01) for pregabalin, respectively. There were no significant changes in total brain concentrations of lacosamide, levetiracetam, and pregabalin following CYT i.p. administration. CYT administered in a dose of 2 mg kg-1 failed to change the protective action of clobazam, clonazepam, phenobarbital, tiagabine, and valproate in the 6-Hz test. Neither CYT (2 mg kg-1) alone nor its combination with the anticonvulsant drugs (at their ED50 values from the 6-Hz test) affected motor coordination; skeletal muscular strength and long-term memory, as determined in the chimney; and grip strength and passive avoidance tests, respectively. CONCLUSION: CYT-evoked alterations in the protection provided by some antiepileptic drugs against seizures can be of serious concern for epileptic smokers, who might demonstrate therapeutic failure to lacosamide, levetiracetam, and pregabalin, resulting in possible breakthrough seizure attacks.

Influence of xanthotoxin (8-methoxypsoralen) on the anticonvulsant activity of various novel antiepileptic drugs against maximal electroshock-induced seizures in mice.

The aim of this study was to determine the effects of xanthotoxin (8-methoxypsoralen) on the protective action of 5 various second- and third-generation antiepileptic drugs (i.e., lacosamide, lamotrigine, oxcarbazepine, pregabalin and topiramate) in the mouse maximal electroshock-induced seizure model. Seizure activity was evoked in adult male albino Swiss mice by a current (25mA, 500V, 0.2s stimulus duration) delivered via auricular electrodes. Drug-related adverse effects were determined in the chimney, grip-strength and passive avoidance tests. Total brain antiepileptic drug concentrations were measured to confirm pharmacodynamic nature of observed interactions with xanthotoxin. Results indicate that xanthotoxin (100mg/kg, i.p.) significantly enhanced the anticonvulsant action of lacosamide (P<0.01), oxcarbazepine (P<0.05), pregabalin (P<0.01), and topiramate (P<0.001), but not that of lamotrigine in the maximal electroshock-induced seizure test. Moreover, xanthotoxin (50mg/kg) still significantly potentiated the anticonvulsant action of lacosamide (P<0.05), pregabalin (P<0.05), and topiramate (P<0.001) in this seizure test. Xanthotoxin had no significant impact on total brain concentrations of the studied antiepileptic drugs in mice. Furthermore, combinations of xanthotoxin with oxcarbazepine or topiramate produced no adverse effects. However, xanthotoxin in combination with lacosamide, lamotrigine or pregabalin significantly reduced muscular strength in mice in the grip-strength test. In the chimney test, only the combinations of xanthotoxin with pregabalin significantly impaired motor coordination in mice. In conclusion, the combinations of xanthotoxin with oxcarbazepine and topiramate produce beneficial anticonvulsant pharmacodynamic interactions in the maximal electroshock-induced seizure test. A special caution is advised when combining xanthotoxin with pregabalin due to appearance of acute adverse effects.

Models to identify treatments for the acute and persistent effects of seizure-inducing chemical threat agents.

Exposures to seizure-inducing chemical threat agents are a major public health concern. Of particular need is improved treatment to terminate convulsions and to prevent the long-term neurological sequelae in survivors. We are studying the organophosphorus cholinesterase inhibitor diisopropyl fluorophosphate (DFP) and the GABA receptor inhibitor tetramethylenedisulfotetramine (TETS), which arguably encompass the mechanistic spectrum of seizure-inducing chemical threats, with the goal of identifying therapeutic approaches with broad-spectrum efficacy. Research efforts have focused on developing translational models and translational diagnostic approaches, including (1) in vivo models of DFP- and TETS-induced seizures for studying neuropathological mechanisms and identifying treatment approaches; (2) in vivo imaging modalities for noninvasive longitudinal monitoring of neurological damage and response to therapeutic candidates; and (3) higher-throughput in vitro platforms for rapid screening of compounds to identify potential antiseizure and neuroprotective agents, as well as mechanistically relevant novel drug targets. This review summarizes our progress toward realizing these goals and discusses best practices and mechanistic insights derived from our modeling efforts.

Evaluation of the neuroactive steroid ganaxolone on social and repetitive behaviors in the BTBR mouse model of autism.

RATIONALE: Abnormalities in excitatory/inhibitory neurotransmission are hypothesized to contribute to autism spectrum disorder (ASD) etiology. BTBR T (+) Itpr3 (tf) /J (BTBR), an inbred mouse strain, displays social deficits and repetitive self-grooming, offering face validity to ASD diagnostic symptoms. Reduced GABAergic neurotransmission in BTBR suggests that GABAA receptor positive allosteric modulators (PAMs) could improve ASD-relevant BTBR phenotypes. The neuroactive steroid ganaxolone acts as a PAM, displaying anticonvulsant properties in rodent epilepsy models and an anxiolytic-like profile in the elevated plus-maze. OBJECTIVES: We evaluated ganaxolone in BTBR and C57BL/6J mice in standardized assays for sociability and repetitive behaviors. Open field and anxiety-related behaviors were tested as internal controls and for comparison with the existing neuroactive steroid literature. RESULTS: Ganaxolone improved aspects of social approach and reciprocal social interactions in BTBR, with no effect on repetitive self-grooming, and no detrimental effects in C57BL/6J. Ganaxolone increased overall exploratory activity in BTBR and C57BL/6J in the open field, social approach, and elevated plus-maze, introducing a confound for the interpretation of social improvements. Allopregnanolone and diazepam similarly increased total entries in the elevated plus-maze, indicating that behavioral activation may be a general property of GABAA receptor PAMs in these strains. CONCLUSIONS: Ganaxolone shows promise for improving sociability. In addition, ganaxolone, as well as other GABAA receptor PAMs, enhanced overall BTBR activity. The translational implications of specific sociability improvements and nonspecific behavioral activation by ganaxolone in the BTBR model remain to be determined. Future studies to explore whether PAMs provide a novel profile with unique benefits for ASD treatment will be worthwhile.

Influence of MPEP (a selective mGluR5 antagonist) on the anticonvulsant action of novel antiepileptic drugs against maximal electroshock-induced seizures in mice.

The aim of this study was to determine the effects of 2-methyl-6-(phenylethynyl)pyridine (MPEP - a selective antagonist for the glutamate metabotropic receptor subtype mGluR5) on the protective action of some novel antiepileptic drugs (lamotrigine, oxcarbazepine, pregabalin and topiramate) against maximal electroshock-induced seizures in mice. Brain concentrations of antiepileptic drugs were measured to determine whether MPEP altered pharmacokinetics of antiepileptic drugs. Intraperitoneal injection of 1.5 and 2mg/kg of MPEP significantly elevated the threshold for electroconvulsions in mice, whereas MPEP at a dose of 1mg/kg considerably enhanced the anticonvulsant activity of pregabalin and topiramate, but not that of lamotrigine or oxcarbazepine in the maximal electroshock-induced seizures in mice. Pharmacokinetic results revealed that MPEP (1mg/kg) did not alter total brain concentrations of pregabalin and topiramate, and the observed effect in the mouse maximal electroshock seizure model was pharmacodynamic in nature. Collectively, our preclinical data suggest that MPEP may be a safe and beneficial adjunct to the therapeutic effects of antiepileptic drugs in human patients.

Contrasting actions of a convulsant barbiturate and its anticonvulsant enantiomer on the α1 ß3 γ2L GABAA receptor account for their in vivo effects.

KEY POINTS: Most barbiturates are anaesthetics but unexpectedly a few are convulsants whose mechanism of action is poorly understood. We synthesized and characterized a novel pair of chiral barbiturates that are capable of photolabelling their binding sites on GABAA receptors. In mice the S-enantiomer is a convulsant, but the R-enantiomer is an anticonvulsant. The convulsant S-enantiomer binds solely at an inhibitory site. It is both an open state inhibitor and a resting state inhibitor. Its action is pH independent, suggesting the pyrimidine ring plays little part in binding. The inhibitory site is not enantioselective because the R-enantiomer inhibits with equal affinity. In contrast, only the anticonvulsant R-enantiomer binds to the enhancing site on open channels, causing them to stay open longer. The enhancing site is enantioselective. The in vivo actions of the convulsant S-enantiomer are accounted for by its interactions with GABAA receptors. ABSTRACT: Most barbiturates are anaesthetics but a few unexpectedly are convulsants. We recently located the anaesthetic sites on GABAA receptors (GABAA Rs) by photolabelling with an anaesthetic barbiturate. To apply the same strategy to locate the convulsant sites requires the creation and mechanistic characterization of a suitable agent. We synthesized enantiomers of a novel, photoactivable barbiturate, 1-methyl-5-propyly-5-(m-trifluoromethyldiazirinyl) phenyl barbituric acid (mTFD-MPPB). In mice, S-mTFD-MPPB acted as a convulsant, whereas R-mTFD-MPPB acted as an anticonvulsant. Using patch clamp electrophysiology and fast solution exchange on recombinant human α1 ß3 γ2L GABAA Rs expressed in HEK cells, we found that S-mTFD-MPPB inhibited GABA-induced currents, whereas R-mTFD-MPPB enhanced them. S-mTFD-MPPB caused inhibition by binding to either of two inhibitory sites on open channels with bimolecular kinetics. It also inhibited closed, resting state receptors at similar concentrations, decreasing the channel opening rate and shifting the GABA concentration-response curve to the right. R-mTFD-MPPB, like most anaesthetics, enhanced receptor gating by rapidly binding to allosteric sites on open channels, initiating a rate-limiting conformation change to stabilized open channel states. These states had slower closing rates, thus shifting the GABA concentration-response curve to the left. Under conditions when most GABAA Rs were open, an inhibitory action of R-mTFD-MPPB was revealed that had a similar IC50 to that of S-mTFD-MPPB. Thus, the inhibitory sites are not enantioselective, and the convulsant action of S-mTFD-MPPB results from its negligible affinity for the enhancing, anaesthetic sites. Interactions with these two classes of barbiturate binding sites on GABAA Rs underlie the enantiomers' different pharmacological activities in mice.