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.

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.

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.

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.

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.

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.

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.

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.

Post-exposure administration of diazepam combined with soluble epoxide hydrolase inhibition stops seizures and modulates neuroinflammation in a murine model of acute TETS intoxication.

Tetramethylenedisulfotetramine (TETS) is a potent convulsant poison for which there is currently no approved antidote. The convulsant action of TETS is thought to be mediated by inhibition of type A gamma-aminobutyric acid receptor (GABAAR) function. We, therefore, investigated the effects of post-exposure administration of diazepam, a GABAAR positive allosteric modulator, on seizure activity, death and neuroinflammation in adult male Swiss mice injected with a lethal dose of TETS (0.15mg/kg, ip). Administration of a high dose of diazepam (5mg/kg, ip) immediately following the second clonic seizure (approximately 20min post-TETS injection) effectively prevented progression to tonic seizures and death. However, this treatment did not prevent persistent reactive astrogliosis and microglial activation, as determined by GFAP and Iba-1 immunoreactivity and microglial cell morphology. Inhibition of soluble epoxide hydrolase (sEH) has been shown to exert potent anti-inflammatory effects and to increase survival in mice intoxicated with other GABAAR antagonists. The sEH inhibitor TUPS (1mg/kg, ip) administered immediately after the second clonic seizure did not protect TETS-intoxicated animals from tonic seizures or death. Combined administration of diazepam (5mg/kg, ip) and TUPS (1mg/kg, ip, starting 1h after diazepam and repeated every 24h) prevented TETS-induced lethality and influenced signs of neuroinflammation in some brain regions. Significantly decreased microglial activation and enhanced reactive astrogliosis were observed in the hippocampus, with no changes in the cortex. Combining an agent that targets specific anti-inflammatory mechanisms with a traditional antiseizure drug may enhance treatment outcome in TETS intoxication.

Neuroactive steroids for the treatment of status epilepticus.

Benzodiazepines are the current first-line standard-of-care treatment for status epilepticus but fail to terminate seizures in about one third of cases. Synaptic GABAA receptors, which mediate phasic inhibition in central circuits, are the molecular target of benzodiazepines. As status epilepticus progresses, these receptors are internalized and become functionally inactivated, conferring benzodiazepine resistance, which is believed to be a major cause of treatment failure. GABAA receptor positive allosteric modulator neuroactive steroids, such as allopregnanolone, also potentiate synaptic GABAA receptors, but in addition they enhance extrasynaptic GABAA receptors that mediate tonic inhibition. Extrasynaptic GABAA receptors are not internalized, and desensitization of these receptors does not occur during continuous seizures in status epilepticus models. Here we review the broad-spectrum antiseizure activity of allopregnanolone in animal seizure models and the evidence for its activity in models of status epilepticus. We also demonstrate that allopregnanolone inhibits ongoing behavioral and electrographic seizures in a model of status epilepticus, even when there is benzodiazepine resistance. Parenteral allopregnanolone may provide an improved treatment for refractory status epilepticus.

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.

Characterization of seizures induced by acute and repeated exposure to tetramethylenedisulfotetramine.

Tetramethylenedisulfotetramine (tetramine; TETS) is a potent convulsant poison that is considered to be a chemical threat agent. To provide a basis for the investigation of antidotes for TETS-induced seizures, we characterized the convulsant activity of TETS in mice and rats when administered by the intraperitoneal, intravenous, oral, and intraventricular routes as a single acute dose and with repeated sublethal doses. In mice, parenteral and oral TETS caused immobility, myoclonic body jerks, clonic seizures of the forelimbs and/or hindlimbs, tonic seizures, and death. The CD50 values for clonic and tonic seizures after oral administration were 0.11 and 0.22 mg/kg, respectively. Intraventricular administration of TETS (5-100 µg) in rats also caused clonic-tonic seizures and death. In mice, repeated sublethal doses of TETS at intervals of 2, 24, and 48 h failed to result in the development of persistent enhanced seizure responsivity ("kindling") as was observed with repeated pentylenetetrazol treatment. In mice, sublethal doses of TETS that produced clonic seizures did not cause observable structural brain damage as assessed with routine histology and Fluoro-Jade B staining 7 days after treatment. However, 1 to 3 days after a single convulsant dose of TETS the expression of glial fibrillary acidic protein, an astrocyte marker, and ionized calcium binding adaptor molecule 1, a microglia marker, were markedly increased in cortex and hippocampus. Although TETS doses that are compatible with survival are not associated with overt evidence of cellular injury or neurodegeneration, there is transient reactive astrocytosis and microglial activation, indicating that brain inflammatory responses are provoked.

Seizure protection by intrapulmonary delivery of propofol hemisuccinate.

The lung provides a portal of entry for drug delivery that could be used to administer anticonvulsant substances to prevent or abort seizures. Here, we demonstrate that intrapulmonary propofol hemisuccinate (PHS) rapidly confers seizure protection in various rodent chemoconvulsant models. Propofol is a powerful anticonvulsant substance at subanesthetic doses, but it is a viscous, water-immiscible oil that is not suitable for intrapulmonary administration. We found that PHS can be formulated as an aqueous solution that is well tolerated when instilled into the lung. High-dose intraperitoneally administered PHS induced loss-of-righting reflex in rats and mice. The onset of action of PHS was delayed in comparison with propofol, suggesting that conversion to propofol is required for activity. A lower dose of PHS (40 mg/kg i.p.) did not cause general anesthesia but protected against pentylenetetrazol (PTZ)-induced seizures in rats. Intrapulmonary administration of an aqueous PHS solution via a tracheal cannula at lower doses (5 and 10 mg/kg) conferred equivalent seizure protection without acute motor toxicity. In mice, intraperitoneal PHS (60-80 mg/kg) was associated with an elevation in PTZ, bicuculline, picrotoxin, and kainic acid seizure thresholds. Intratracheal PHS was markedly more potent, producing seizure threshold elevations at doses of 10 to 15 mg/kg. In the PTZ threshold model in mice, PHS was active at 5 min, maximally active at 10 min, and no longer active at 20 min. Intratracheal PHS also prolonged the onset of 4-aminopyridine-induced convulsions but did not affect the threshold for N-methyl-D-aspartate-induced convulsions. We conclude that intratracheal administration of an aqueous solution of PHS, a putative propofol prodrug, provides potent seizure protection of rapid onset and brief duration. Intrapulmonary PHS may be useful for preventing the spread of seizures or aborting seizure clusters without causing prolonged sedation.

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.

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.

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.

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.

Evidence for the involvement of dopamine transporters in behavioral stimulant effects of modafinil.

Modafinil is prescribed for numerous medical conditions, but the drug's mechanism of action is unclear. Here, we examined the interaction of modafinil with receptors and transporters in vitro and compared pharmacological effects of the drug with those produced by indirect dopamine (DA) agonists 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR12909) and (+)-methamphetamine (METH). Modafinil was screened at various receptors and transporters using binding assays. Transporter-mediated uptake and release were examined in rat brain synaptosomes. Effects of modafinil on motor activity and neurochemistry were determined in rats undergoing in vivo microdialysis in nucleus accumbens. Of the receptors and transporters assayed, modafinil displayed measurable potency only at DA transporters (DAT), inhibiting [(3)H]DA uptake, with an IC(50) value of 4.0 microM. Accordingly, modafinil pretreatment (10 microM) antagonized METH-induced release of the DAT substrate [(3)H]1-methyl-4-phenylpyridinium. Intravenous modafinil (20 and 60 mg/kg) produced dose-dependent increases in motor activity and extracellular DA, without affecting serotonin (5-HT). Analogous results were observed for GBR12909 (1 and 3 mg/kg), whereas METH (0.3 and 1 mg/kg) increased DA and 5-HT. Locomotor effects of all drugs were positively correlated with dialysate DA (P < 0.001). Interestingly, modafinil pretreatment reduced METH-induced ambulation and DA release. Our data show that modafinil interacts with DAT sites in rat brain, a property shared with agonist medications under investigation for treating cocaine dependence. Nondopaminergic mechanisms may also contribute to the pharmacology of modafinil. Finally, the results suggest that modafinil should be tested as an adjunct for treating METH addiction.

Effects of dose and route of administration on pharmacokinetics of (+ or -)-3,4-methylenedioxymethamphetamine in the rat.

Based on animal data, there is speculation that (+ or -)-3,4-methylenedioxymethamphetamine (MDMA) is neurotoxic to humans. Extrapolation of MDMA findings from animals to humans requires assessment of pharmacokinetics in various species, and low-dose administration data from rats are lacking. In this study, we examine MDMA pharmacokinetics in rats given low (2 mg/kg) and high (10 mg/kg) doses of racemic MDMA via intraperitoneal, subcutaneous, and oral routes. Repeated blood specimens were collected from venous catheters, and plasma was assayed for MDMA and its metabolites, 4-hydroxy-3-methoxymethamphetamine (HMMA) and 3,4-methylenedioxyamphetamine (MDA), by gas chromatography-mass spectrometry. After 2 mg/kg, maximum MDMA concentrations (C(max)) were approximately 200 ng/ml for intraperitoneal and subcutaneous routes, but less for the oral route. MDMA plasma half-lives were <1 h for low-dose groups, whereas HMMA and MDA half-lives were >2 h. After 10 mg/kg, MDMA areas under the curve (AUCs) were 21-fold (intraperitoneal), 10-fold (subcutaneous), and 36-fold (oral) greater than those at 2 mg/kg. In contrast, HMMA AUC values in high-dose groups were <3-fold above those at 2 mg/kg. Several new findings emerge from this report of low-dose MDMA pharmacokinetics in rats. First, 2 mg/kg MDMA in rats can produce MDMA C(max) values similar to those in humans, perhaps explaining why both species discriminate 1.5 mg/kg MDMA in laboratory paradigms. Second, our data provide additional support for nonlinear kinetics of MDMA in rats, and, analogous to humans, this phenomenon appears to involve impaired drug metabolism. Finally, given key similarities between MDMA pharmacokinetics in rats and humans, data from rats may be clinically relevant when appropriate dosing conditions are used.

Chronic fenfluramine administration increases plasma serotonin (5-hydroxytryptamine) to nontoxic levels.

Large elevations in blood serotonin (5-hydroxytryptamine; 5-HT) can produce valvular heart disease in humans and laboratory animals. In accordance, one prevailing hypothesis (i.e., the "5-HT hypothesis") suggests that 5-HT transporter substrates like fenfluramine increase the risk for valvular heart disease by elevating plasma 5-HT, secondary to the release of 5-HT from platelets. The main purpose of this study was to determine whether chronic administration of fenfluramine increases plasma 5-HT to concentrations that are associated with the development of valvular heart disease. To the best of our knowledge, this is the first study to address this issue using an in vivo microdialysis method that measures plasma 5-HT in nonhypoxic rats. We examined the effects of chronic (+/-)-fenfluramine and fluoxetine on plasma levels of 5-HT and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), in blood samples from conscious catheterized rats. Plasma indoles were measured by high-performance liquid chromatography with electrochemical detection in the dialysates of whole blood. The baseline plasma 5-HT level was <1.0 nM. Chronic fenfluramine (14-day minipump infusion) produced small increases in baseline plasma 5-HT ( approximately 2-4-fold), whereas chronic fluoxetine had no effect. Chronic fenfluramine and fluoxetine markedly decreased whole-blood 5-HT and reduced the ability of acute fenfluramine to evoke 5-HT release. Elevations in baseline plasma 5-HT produced by chronic fenfluramine are far below the micromolar levels necessary to produce valvular heart disease. Furthermore, chronic fenfluramine reduces the ability of acute fenfluramine to increase plasma 5-HT, suggesting that the 5-HT hypothesis cannot explain the increased risk of valvular heart disease in patients treated with fenfluramine.