Ganaxolone Research Articles

Neurosteroids as Novel Anticonvulsants for Refractory Status Epilepticus and Medical Countermeasures for Nerve Agents: A 15-Year Journey to Bring Ganaxolone from Bench to Clinic.

This article describes recent advances in the use of neurosteroids as novel anticonvulsants for refractory status epilepticus (RSE) and as medical countermeasures (MCs) for organophosphates and chemical nerve agents (OPNAs). We highlight a comprehensive 15-year journey to bring the synthetic neurosteroid ganaxolone (GX) from bench to clinic. RSE, including when caused by nerve agents, is associated with devastating morbidity and permanent long-term neurologic dysfunction. Although recent approval of benzodiazepines such as intranasal midazolam and intranasal midazolam offers improved control of acute seizures, novel anticonvulsants are needed to suppress RSE and improve neurologic function outcomes. Currently, few anticonvulsant MCs exist for victims of OPNA exposure and RSE. Standard-of-care MCs for postexposure treatment include benzodiazepines, which do not effectively prevent or mitigate seizures resulting from nerve agent intoxication, leaving an urgent unmet medical need for new anticonvulsants for RSE. Recently, we pioneered neurosteroids as next-generation anticonvulsants that are superior to benzodiazepines for treatment of OPNA intoxication and RSE. Because GX and related neurosteroids that activate extrasynaptic GABA-A receptors rapidly control seizures and offer robust neuroprotection by reducing neuronal damage and neuroinflammation, they effectively improve neurologic outcomes after acute OPNA exposure and RSE. GX has been selected for advanced, Biomedical Advanced Research and Development Authority-supported phase 3 trials of RSE and nerve agent seizures. In addition, in mechanistic studies of neurosteroids at extrasynaptic receptors, we identified novel synthetic analogs with features that are superior to GX for current medical needs. Development of new MCs for RSE is complex, tedious, and uncertain due to scientific and regulatory challenges. Thus, further research will be critical to fill key gaps in evaluating RSE and anticonvulsants in vulnerable (pediatric and geriatric) populations and military persons. SIGNIFICANCE STATEMENT: Following organophosphate and nerve agent intoxication, refractory status epilepticus (RSE) occurs despite benzodiazepine treatment. RSE occurs in 40% of status epilepticus patients, with a 35% mortality rate and significant neurological morbidity in survivors. To treat RSE, neurosteroids are better anticonvulsants than benzodiazepines. Our pioneering use of neurosteroids for RSE and nerve agents led us to develop ganaxolone as a novel anticonvulsant and neuroprotectant with significantly improved neurological outcomes. This article describes the bench-to-bedside journey of bringing neurosteroid therapy to patients, with ganaxolone leading the way.

Long-Term Neuropsychiatric Developmental Defects after Neonatal Organophosphate Exposure: Mitigation by Synthetic Neurosteroids.

Children are much more susceptible to the neurotoxic effects of organophosphate (OP) pesticides and nerve agents than adults. OP poisoning in children leads to acute seizures and neuropsychiatric sequela, including the development of long-term disabilities and cognitive impairments. Despite these risks, there are few chronic rodent models that use pediatric OP exposure for studying neurodevelopmental consequences and interventions. Here, we investigated the protective effect of the neurosteroid ganaxolone (GX) on the long-term developmental impact of neonatal exposure to the OP compound, diisopropyl-fluorophosphate (DFP). Pediatric postnatal day-28 rats were acutely exposed to DFP, and at 3 and 10 months after exposure, they were evaluated using a series of cognitive and behavioral tests with or without the postexposure treatment of GX. Analysis of the neuropathology was performed after 10 months. DFP-exposed animals displayed significant long-term deficits in mood, anxiety, depression, and aggressive traits. In spatial and nonspatial cognitive tests, they displayed striking impairments in learning and memory. Analysis of brain sections showed significant loss of neuronal nuclei antigen(+) principal neurons, parvalbumin(+) inhibitory interneurons, and neurogenesis, along with increased astrogliosis, microglial neuroinflammation, and mossy fiber sprouting. These detrimental neuropathological changes are consistent with behavioral dysfunctions. In the neurosteroid GX-treated cohort, behavioral and cognitive deficits were significantly reduced and were associated with strong protection against long-term neuroinflammation and neurodegeneration. In conclusion, this pediatric model replicates the salient features of children exposed to OPs, and the protective outcomes from neurosteroid intervention support the viability of developing this strategy for mitigating the long-term effects of acute OP exposure in children. SIGNIFICANCE STATEMENT: An estimated 3 million organophosphate exposures occur annually worldwide, with children comprising over 30% of all victims. Our understanding of the neurodevelopmental consequences in children exposed to organophosphates is limited. Here, we investigated the long-term impact of neonatal exposure to diisopropyl-fluorophosphate in pediatric rats. Neurosteroid treatment protected against major deficits in behavior and memory and was well correlated with neuropathological changes. Overall, this pediatric model is helpful to screen novel therapies to mitigate long-term developmental deficits of organophosphate exposure.

Complex Metabolism of the Novel Neurosteroid, Ganaxolone, in Humans: A Unique Challenge for Metabolites in Safety Testing Assessment.

The human pharmacokinetics, metabolism, and excretion of [14C]-ganaxolone (GNX) were characterized in healthy male subjects (n = 8) following a single 300-mg (150 μCi) oral dose. GNX exhibited a short half-life of 4 hours in plasma, whereas total radioactivity had a half-life of 413 hours indicating extensive metabolism to long-lived metabolites. Identification of the major GNX circulating metabolites required extensive isolation and purification for liquid chromatography-tandem mass spectrometry analysis, together with in vitro studies, NMR spectroscopy, and synthetic chemistry support. This revealed that the major routes of GNX metabolism involved hydroxylation at the 16α-hydroxy position, stereoselective reduction of the 20-ketone to afford the corresponding 20α-hydroxysterol, and sulfation of the 3α-hydroxy group. This latter reaction yielded an unstable tertiary sulfate, which eliminated the elements of H2SO4 to introduce a double bond in the A ring. A combination of these pathways, together with oxidation of the 3β-methyl substituent to a carboxylic acid and sulfation at the 20α position, led to the major circulating metabolites in plasma, termed M2 and M17. These studies, which led to the complete or partial identification of no less than 59 metabolites of GNX, demonstrated the high complexity of the metabolic fate of this drug in humans and demonstrated that the major circulating products in plasma can result from multiple sequential processes that may not be easily replicated in animals or with animal or human in vitro systems. SIGNIFICANCE STATEMENT: Studies on the metabolism of [14C]-ganaxolone in humans revealed a complex array of products that circulated in plasma, the two major components of which were formed via an unexpected multi-step pathway. Complete structural characterization of these (disproportionate) human metabolites required extensive in vitro studies, along with contemporary mass spectrometry, NMR spectroscopy, and synthetic chemistry efforts, which served to underscore the limitations of traditional animal studies in predicting major circulating metabolites in man.

Isobolographic Analysis of Antiseizure Activity of the GABA Type A Receptor-Modulating Synthetic Neurosteroids Brexanolone and Ganaxolone with Tiagabine and Midazolam.

Epilepsy is often treated with a combination of antiepileptic drugs. Although neurosteroids are potent anticonvulsants, little is known about their combination potential for the treatment of refractory epilepsy. Here, we investigated the combination efficacy of neurosteroids allopregnanolone (AP, brexanolone) and ganaxolone (GX) with the GABA-reuptake inhibitor tiagabine (TG) or the benzodiazepine midazolam (MDZ) on tonic inhibition in dentate gyrus granule cells and seizure protection in the hippocampus kindling and 6-Hz seizure models. Isobolographic analysis indicated that combinations of GX and TG or AP and TG at three standard ratios (1:1, 3:1, and 1:3) displayed significant synergism in augmenting tonic inhibition. In pharmacological studies, GX, AP, and TG produced dose-dependent antiseizure effects in mice (ED50 = 1.46, 4.20, and 0.20 mg/kg, respectively). The combination of GX and TG at the fixed ratio of 1:1 exerted the greatest combination index (CI = 0.53), indicating strong synergistic interaction in seizure protection. In addition, combination regimens of AP and TG showed robust synergism for seizure protection (CI = 0.4). Finally, combination regimens of GX and MDZ elicited synergistic (CI = 0.6) responses for seizure protection. These results demonstrate striking synergism of neurosteroids and TG combination for seizure protection, likely because of their effects at extrasynaptic GABA type A (GABA-A) receptors from TG-induced elevation in GABA levels. Superadditive antiseizure activity of neurosteroid-MDZ combinations may stem from their actions at both synaptic and extrasynaptic GABA-A receptors. Together, these findings provide a potential mechanistic basis for combination potential of neurosteroids with TG or benzodiazepines for the management of refractory epilepsy, status epilepticus, and seizure disorders. SIGNIFICANCE STATEMENT: This paper investigates for the first time the potential synergistic interactions between two neurosteroids with anticonvulsant properties, allopregnanolone (brexanolone) and the very similar synthetic analog, ganaxolone, and two conventional antiepileptic drugs active at GABA type A receptors: the GABA-reuptake inhibitor tiagabine and a benzodiazepine, midazolam. The results demonstrate a synergistic protective effect of neurosteroid-tiagabine combinations, as well as neurosteroid-midazolam regimens in seizure models.

Genotype Differences in Sensitivity to the Anticonvulsant Effect of the Synthetic Neurosteroid Ganaxolone during Chronic Ethanol Withdrawal

Sensitivity to anticonvulsant effects of the γ-aminobutyric acidA receptor-active neurosteroid allopregnanolone (ALLO) during ethanol withdrawal varies across genotypes, with high sensitivity in genotypes with mild withdrawal and low sensitivity in genotypes with high withdrawal. The present studies determined whether the resistance to ALLO during withdrawal in mouse genotypes with high handling-induced convulsions (HICs) during withdrawal could be overcome with use of ganaxolone (GAN), the metabolically stable derivative of ALLO. In separate studies, male and female Withdrawal Seizure-Prone (WSP-1) and DBA/2J (D2) mice were exposed to air (controls) or 72-h ethanol vapor and then were scored for HICs during withdrawal (hourly for the first 12 h, then at hours 24 and 25). After the HIC scoring at hours 5 and 9, mice were injected with 10 mg/kg GAN or vehicle. Area under the HIC curve (AUC) for hours 5–12 was analyzed. In control WSP-1 mice, GAN significantly reduced AUC by 52% (males) and 63% (females), with effects that were absent or substantially reduced during withdrawal. In contrast, GAN significantly reduced AUC in both control and ethanol-withdrawing male and female D2 mice. AUC was decreased by 81% (males) and 70% (females) in controls and by 35% (males) and 21% (females) during withdrawal. The significant anticonvulsant effect of GAN during withdrawal in D2 but not WSP-1 mice suggests that different mechanisms may contribute to ALLO insensitivity during withdrawal in these two genotypes. Importantly, the results in D2 mice suggest that GAN may be a useful treatment for ethanol withdrawal-induced seizures.

Zinc reduces antiseizure activity of neurosteroids by selective blockade of extrasynaptic GABA-A receptor-mediated tonic inhibition in the hippocampus

Zinc is an abundant trace metal in the hippocampus nerve terminals. Previous studies demonstrate the ability of zinc to selectively block neurosteroid-sensitive, extrasynaptic GABA-A receptors in the hippocampus (Carver et al, 2016). Here we report that zinc prevents the seizure protective effects of the synthetic neurosteroid ganaxolone (GX) in an experimental model of epilepsy. GABA-gated and tonic currents were recorded from dissociated dentate gyrus granule cells (DGGCs), CA1 pyramidal cells (CA1PCs), and hippocampal slices from adult mice. Antiseizure effects of GX and the reversal of these effects by zinc were evaluated in fully-kindled mice expressing generalized (stage 5) seizures. In electrophysiological studies, zinc blocked the GABA-evoked and GX-potentiated GABA-gated chloride currents in DGGCs and CA1PCs in a concentration-dependent fashion similar to the competitive GABA-A receptor antagonists bicuculline and gabazine. Zinc completely blocked GX potentiation of extrasynaptic tonic currents, but not synaptic phasic currents. In hippocampus kindling studies, systemic administration of GX produced a dose-dependent suppression of behavioral and electrographic seizures in fully-kindled mice with complete seizure protection at the 10 mg/kg dose. However, the antiseizure effects of GX were significantly prevented by intrahippocampal administration of zinc (ED50, 150 μM). The zinc antagonistic response was reversible as animals responded normally to GX administration 24 h post-zinc blockade. These results demonstrate that zinc reduces the antiseizure effects of GX by selectively blocking extrasynaptic δGABA-A receptors in the hippocampus. These pharmacodynamic interactions have clinical implications in neurosteroid therapy for brain conditions associated with zinc fluctuations.

Neurosteroid replacement therapy using the allopregnanolone-analogue ganaxolone following preterm birth in male guinea pigs.

Children born preterm, especially boys, are at increased risk of developing attention deficit hyperactivity disorder (ADHD) and learning difficulties. We propose that neurosteroid-replacement therapy with ganaxolone (GNX) following preterm birth may mitigate preterm-associated neurodevelopmental impairment. Time-mated sows were delivered preterm (d62) or at term (d69). Male preterm pups were randomized to ganaxolone (Prem-GNX; 2.5 mg/kg subcutaneously twice daily until term equivalence), or preterm control (Prem-CON). Surviving male juvenile pups underwent behavioural testing at d25-corrected postnatal age (CPNA). Brain tissue was collected at CPNA28 and mature myelinating oligodendrocytes of the hippocampus and subcortical white matter were quantified by immunostaining of myelin basic protein (MBP). Ganaxolone treatment returned the hyperactive behavioural phenotype of preterm-born juvenile males to a term-born phenotype. Deficits in MBP immunostaining of the preterm hippocampus and subcortical white matter were also ameliorated in animals receiving ganaxolone. However, during the treatment period weight gain was poor, and pups were sedated, ultimately increasing the neonatal mortality rate. Ganaxolone improved neurobehavioural outcomes in males suggesting that neonatal treatment may be an option for reducing preterm-associated neurodevelopmental impairment. However, dosing studies are required to reduce the burden of unwanted side effects.

Effect of nucleus accumbens shell infusions of ganaxolone or gaboxadol on ethanol consumption in mice

Allopregnanolone (ALLO) is an endogenous neuroactive steroid thought to alter the reinforcement value of alcohol (ethanol) due to its actions as a positive modulator of the GABAA receptor (GABAAR). Extrasynaptic GABAARs may be a particularly sensitive target of ethanol and neuroactive steroids. Previous work showed that systemic injections of an ALLO analog, ganaxolone (GAN), or an extrasynaptic GABAAR agonist (gaboxadol; THIP) decreased ethanol intake in male mice with limited access to ethanol. The present studies tested whether activation of GABAARs in the nucleus accumbens (NAc) shell by GAN or THIP was sufficient to reduce ethanol intake. C57BL/6J male mice had 2-h access to 10% ethanol (10E) and water, and 10E intake was measured following site-specific infusions of GAN or THIP. Decreases in limited-access 10E consumption were observed following site-specific bilateral infusions of either drug into the NAc shell. Significant changes in intake were absent when the drugs were infused in a region dorsal to the target site (GAN) or into the lateral ventricle (THIP). Locomotor data confirmed that the decreases in intake were not due to a sedative effect of the drugs. These data demonstrate the sufficiency of GABAAR activation by a positive allosteric modulator or an agonist with selectivity for extrasynaptic GABAARs to decrease ethanol consumption in mice. Importantly, more refined GABAAR-active targets that decrease ethanol intake may enhance our understanding and ability to treat alcohol use disorders.

Differences in the reinstatement of ethanol seeking with ganaxolone and gaboxadol

The endogenous neuroactive steroid allopregnanolone (ALLO) has previously been shown to induce reinstatement of ethanol seeking in rodents. ALLO is a positive allosteric modulator at both synaptic and extrasynaptic GABAA receptors. The contribution of each class of GABAA receptors in mediating reinstatement of ethanol seeking is unknown. The first aim of the present study was to determine whether ganaxolone (GAN), a longer-acting synthetic analog of ALLO, also promotes reinstatement of ethanol seeking. The second aim was to examine whether preferentially activating extrasynaptic GABAA receptors with the selective agonist gaboxadol (THIP) was sufficient to reinstate responding for ethanol in mice. Male C57BL/6J mice were trained to lever press for access to a 10% ethanol (v/v) solution (10E), using a sucrose-fading procedure. Following extinction of the lever-pressing behavior, systemic THIP (0, 4 and 6mg/kg) and GAN (0, 10, and 15mg/kg) were tested for their ability to reinstate ethanol-appropriate responding in the absence of 10E access. GAN significantly increased lever pressing on the previously active lever, while THIP did not alter lever-pressing behavior. The results of this study suggest that direct activation of extrasynaptic GABAA receptors at the GABA site is not sufficient to induce ethanol seeking in the reinstatement procedure. Future studies are necessary to elucidate the mechanisms and brain areas by which differences in the pharmacological activity of GAN and THIP at the GABAA receptor contribute to the dissimilarity in their effect on the reinstatement of ethanol seeking. Nonetheless, based on the increased use of these drugs in clinical trials across multiple disease states, the effects of GAN or THIP on alcohol seeking may be an important consideration if these drugs are to be used clinically in a population with a co-occurring alcohol use disorder.

GABA transport and neuroinflammation are coupled in multiple sclerosis: Regulation of the GABA transporter-2 by ganaxolone

Interactions between neurotransmitters and the immune system represent new prospects for understanding neuroinflammation and associated neurological disease. GABA is the chief inhibitory neurotransmitter but its actions on immune pathways in the brain are unclear. In the present study, we investigated GABAergic transport in conjunction with neuroinflammation in models of multiple sclerosis (MS). Protein and mRNA levels of γ-amino butyric acid transporter 2 (GAT-2) were examined in cerebral white matter from MS and control (Non-MS) patients, in cultured human macrophages, microglia and astrocytes, and in spinal cords from mice with and without experimental autoimmune encephalomyelitis (EAE) using western blotting, immunocytochemistry and quantitative real-time polymerase chain reaction (qRT-PCR). GABA levels were measured by HPLC. The GAT-2′s expression was increased in MS patients’ (n=6) white matter, particularly in macrophage lineage cells, compared to Non-MS patients (n=6) (p<0.05). Interferon-γ (IFN-γ) stimulation of human macrophage lineage cells induced GAT-2 expression and reduced extracellular GABA levels (p<0.05) but soluble GABA treatment suppressed HLA-DRα, GAT-2 and XBP-1/s expression in stimulated macrophage lineage cells (p<0.05). Similarly, the synthetic allopregnanolone analog, ganaxolone (GNX), repressed GAT-2, JAK-1 and STAT-1 expression in activated macrophage lineage cells (p<0.05). In vivo GNX treatment reduced Gat-2, Cd3ε, MhcII, and Xbp-1/s expression in spinal cords following EAE induction (p<0.05), which was correlated with improved neurobehavioral outcomes and reduced neuroinflammation, demyelination and axonal injury. These findings highlight altered GABAergic transport through GAT-2 induction during neuroinflammation. GABA transport and neuroinflammation are closely coupled but regulated by GNX, pointing to GABAergic pathways as therapeutic targets in neuroinflammatory diseases.

Neurosteroid Agonist at GABAA Receptor Induces Persistent Neuroplasticity in VTA Dopamine Neurons

The main fast-acting inhibitory receptors in the mammalian brain are γ-aminobutyric acid type-A (GABAA) receptors for which neurosteroids, a subclass of steroids synthesized de novo in the brain, constitute a group of endogenous ligands with the most potent positive modulatory actions known. Neurosteroids can act on all subtypes of GABAA receptors, with a preference for δ-subunit-containing receptors that mediate extrasynaptic tonic inhibition. Pathological conditions characterized by emotional and motivational disturbances are often associated with perturbation in the levels of endogenous neurosteroids. We studied the effects of ganaxolone (GAN)-a synthetic analog of endogenous allopregnanolone that lacks activity on nuclear steroid receptors-on the mesolimbic dopamine (DA) system involved in emotions and motivation. A single dose of GAN in young mice induced a dose-dependent, long-lasting neuroplasticity of glutamate synapses of DA neurons ex vivo in the ventral tegmental area (VTA). Increased α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/N-methyl-D-aspartate ratio and rectification of AMPA receptor responses even at 6 days after GAN administration suggested persistent synaptic targeting of GluA2-lacking AMPA receptors. This glutamate neuroplasticity was not observed in GABAA receptor δ-subunit-knockout (δ-KO) mice. GAN (500 nM) applied locally to VTA selectively increased tonic inhibition of GABA interneurons and triggered potentiation of DA neurons within 4 h in vitro. Place-conditioning experiments in adult wild-type C57BL/6J and δ-KO mice revealed aversive properties of repeated GAN administration that were dependent on the δ-subunits. Prolonged neuroadaptation to neurosteroids in the VTA might contribute to both the physiology and pathophysiology underlying processes and changes in motivation, mood, cognition, and drug addiction.

English

BACKGROUND: Presently, treatment of epilepsy with standard anti-epileptic drugs is associated with a number of shortcomings inviting us to study newer agents that would overcome these problems or search for the drugs or substances, which would enhance the efficacy or reduce the dose or toxicity of these standard anti-epileptic drugs. Ganaxolone (GNX) (3α-hydroxy-3β-methyl-5α-pregnan-20-one), a synthetic analog of the endogenous neurosteroid allopregnanolone and a positive allosteric modulator of GABA A receptors, may represent a new treatment approach for epilepsy. AIM: To evaluate the effect of Ganaxolone on maximal electroshock (MES) induced and Pentylenetetrazol (PTZ) induced convulsions and also their effect in combination with conventional antiepileptic drugs (CAEDs). MATERIAL AND METHODS: Wister strain albino rats weighing 200-250 gm were used. Effects of Ganaxolone (5 &10 mg/kg) alone and in combination with standard drugs were studied in MES and PTZ induced seizure models. Abolition of tonic hind limb extension was an index of anticonvulsant activity in MES, while for PTZ seizures; failure to observe clonus for 5sec duration for 30min was the index. Following that, percentage inhibition was calculated. STATISTICS: ANOVA followed by Newman-Keuls Multiple Comparison Test was used for analysis of data between the groups. RESULTS : In MES seizures, significant antiepileptic activity was observed with 10mg of Ganaxolone compared to control group but has less activity when compared to that of Phenobarbitone. In PTZ induced convulsions, the anti-epileptic effect of Sodium Valproate was higher than Ganaxolone, but both were statistically significant as compared to control. In PTZ-induced seizures, augmented effects were obtained when Ganaxolone was combined with sodium valproate i.e. 55%. CONCLUSIONS: The results provide a lead for potential benefit of adding Ganaxolone to Sodium Valproate in the treatment of epilepsy, which needs to be explored further.

Evaluation of Anti - Anxiety, Sedative and Motor Co-Ordination Properties of Ganaxolone in Comparison with Diazepam in Rodent Models

Presently, treatment of anxiety with standard anti-anxiety drugs is associated with a number of shortcomings inviting us to study newer agents that would overcome these problems or search for the drugs or substances, which would enhance the efficacy or reduce the dose or toxicity of this standard anti-anxiety drug diazepam. Ganaxolone (GNX) a positive allosteric modulator of GABAA receptors, may represent a new treatment approach for anxiety. The present study was undertaken to evaluate the anti - anxiety effect of ganaxolone in comparison with diazepam and also to study their effect on sleep and motor co-ordination using elevated plus maze, thiopentone sodium induced sleeping and rotarod model in rodents. Wistar strain albino rats weighing 200-250 gm were used. Ganaxolone produced significant anti-anxiety effect compared to control group but the activity was less when compared to diazepam. The sedative and muscle relaxant properties of ganaxolone were weak as compared to diazepam. Ganaxolone had significant synergistic effect with diazepam by potentiating its actions, thereby providing a lead for exploring potential benefit of adding ganaxolone to decrease the dose of diazepam in the treatment of anxiety.

Alteration of Ethanol Drinking in Mice via Modulation of the GABAA Receptor with Ganaxolone, Finasteride, and Gaboxadol

Neurosteroids and other γ-aminobutyric acid(A) (GABA(A) ) receptor-modulating compounds have been shown to affect ethanol intake, although their mechanism remains unclear. This study examined how patterns of 24-hour ethanol drinking in mice were altered with the synthetic GABAergic neurosteroid ganaxolone (GAN), with an inhibitor of neurosteroid synthesis (finasteride [FIN]), or a GABA(A) receptor agonist with some selectivity at extrasynaptic receptors (gaboxadol HCL [THIP]). Male C57BL/6J mice had continuous access to a 10% v/v ethanol solution (10E) or water. Using lickometer chambers, drinking patterns were analyzed among mice treated in succession to GAN (0, 5, and 10 mg/kg), FIN (0 or 100 mg/kg), and THIP (0, 2, 4, 8, and 16 mg/kg). GAN shifted drinking in a similar but extended manner to previous reports using low doses of the neurosteroid allopregnanolone (ALLO); drinking was increased in hour 1, decreased in hours 2 and 3, and increased in hours 4 and 5 postinjection. THIP (8 mg/kg) and FIN both decreased 10E drinking during the first 5 hours postinjection by 30 and 53%, respectively, while having no effect on or increasing water drinking, respectively. All 3 drugs altered the initiation of drinking sessions in a dose-dependent fashion. FIN increased and GAN decreased time to first lick and first bout. THIP (8 mg/kg) decreased time to first lick but increased time to first bout and attenuated first bout size. The present findings support a role for the modulation of ethanol intake by neurosteroids and GABA(A) receptor-acting compounds and provide hints as to how drinking patterns are shifted. The ability of THIP to alter 10E drinking suggests that extrasynaptic GABA(A) receptors may be involved in the modulation of ethanol intake. Further, the consistent results with THIP to that seen previously with high doses of ALLO suggest that future studies should further examine the relationship between neurosteroids and extrasynaptic GABA(A) receptors, which could provide a better understanding of the mechanism by which neurosteroids influence ethanol intake.

S.03.03 Paracapsular cells of the amygdala: an inhibitory gate modulated by D1 dopamine receptors

The endogenous neuroactive steroid allopregnanolone (ALLO) has previously been shown to induce reinstatement of ethanol seeking in rodents. ALLO is a positive allosteric modulator at both synaptic and extrasynaptic GABAA receptors. The contribution of each class of GABAA receptors in mediating reinstatement of ethanol seeking is unknown. The first aim of the present study was to determine whether ganaxolone (GAN), a longer-acting synthetic analog of ALLO, also promotes reinstatement of ethanol seeking. The second aim was to examine whether preferentially activating extrasynaptic GABAA receptors with the selective agonist gaboxadol (THIP) was sufficient to reinstate responding for ethanol in mice. Male C57BL/6J mice were trained to lever press for access to a 10% ethanol (v/v) solution (10E), using a sucrose-fading procedure. Following extinction of the lever-pressing behavior, systemic THIP (0, 4 and 6 mg/kg) and GAN (0, 10, and 15 mg/kg) were tested for their ability to reinstate ethanol-appropriate responding in the absence of 10E access. GAN significantly increased lever pressing on the previously active lever, while THIP did not alter lever-pressing behavior. The results of this study suggest that direct activation of extrasynaptic GABAA receptors at the GABA site is not sufficient to induce ethanol seeking in the reinstatement procedure. Future studies are necessary to elucidate the mechanisms and brain areas by which differences in the pharmacological activity of GAN and THIP at the GABAA receptor contribute to the dissimilarity in their effect on the reinstatement of ethanol seeking. Nonetheless, based on the increased use of these drugs in clinical trials across multiple disease states, the effects of GAN or THIP on alcohol seeking may be an important consideration if these drugs are to be used clinically in a population with a co-occurring alcohol use disorder.

Ganaxolone for treating intractable infantile spasms: a multicenter, open-label, add-on trial

This is a multicenter, open-label, add-on trial, investigating the safety and efficacy of ganaxolone (GNX) in a population of children with refractory infantile spasms, or with continuing seizures after a prior history of infantile spasms. A total of 20 children aged 7 months to 7 years were enrolled in this dose-escalation study, after baseline seizure frequencies were established. Concomitant antiepilepsy drugs were maintained throughout the study period. The dose of GNX was progressively increased to 36 mg/kg/d (or to the maximally tolerated dose) over a period of 4 weeks, then maintained for 8 weeks before tapering and discontinuation. Seizure diaries were maintained by the families, and spasm frequency was compared with the baseline period. The occurrence of adverse events was clinically monitored, and global evaluations of seizure severity and response to treatment were obtained. A total of 16 of the 20 subjects completed the study, 15 of whom had refractory infantile spasms at the time of study enrollment. Spasm frequency was reduced by at least 50% in 33% of these subjects, with an additional 33% experiencing some improvement (25–50% reduction in spasm frequency). Ganaxolone was well tolerated, and adverse events attributed to GNX were generally mild. Ganaxolone was safe and effective in treating this group of refractory infantile spasms patients in an open-label, add-on trial. Further investigation with randomized, controlled study design is warranted.

Effect of ganaxolone on flurothyl seizures in developing rats.

To determine the effects of a newly synthesized epalon, ganaxolone (GNX), on primarily generalized seizures in rats of various ages during development. Epalons are classified as neuroactive steroids that interact at unique site of the GABAA receptor-Cl- channel complex in the central nervous system. Sprague-Dawley male rats were used at 9, 15, 30, and 60 postnatal days (PN). GNX dissolved in 2-hydroxypropylbeta-cyclodextrine was administered intraperitoneally in different doses at various time points before flurothyl testing. The incidence and threshold of clonic and tonic-clonic flurothyl seizures were evaluated. Behavioral changes were also assessed. In all age groups, the effects of GNX were dose dependent and more prominent 10 min after its administration. In PN 60 and PN 30 rats, GNX had dose-dependent anticonvulsant effects; tonic-clonic seizures were more sensitive to GNX treatment than clonic seizures. In PN 15 and PN 9 rats, GNX demonstrated dose-and time-dependent anticonvulsant effects against both types of flurothyl-induced seizures. GNX was more effective in PN 15 rats than in other age groups, but at doses that altered motor behavior. GNX has anticonvulsant effects against flurothylinduced seizures in all age groups tested. Its effects are more prominent in the two younger age groups, especially in PN 15 rats, but are associated with motor side effects.

Ganaxolone: potential in epilepsy and migraine

Ganaxolone: potential in epilepsy and migraine