Positive Allosteric Modulators Of GABAA Receptors Research Articles

Dorsomedial prefrontal cortex 5-HT6 receptors regulate anxiety-like behavior.

The dorsomedial prefrontal cortex (dmPFC) plays a very important role in decision-related and anxiety-related information processing. It has enriched 5-HT6 receptors; however, the precise role of dmPFC 5-HT6 receptors in anxiety remains to be fully investigated. In this study, we injected dmPFC with the 5-HT6 receptor agonist EMD 386088 and antagonist SB 271046 using stereotactic technology. 5-HT6 receptor activation in mice increased time spent in the center area on the open-field test, increased exploration of the open arms on the elevated plus maze test, and increased ratio on the social interaction test. 5-HT6 receptor inactivation induced the opposite effects. In brain slices, EMD 386088 decreased both spontaneous inhibitory postsynaptic currents (sIPSC) and spontaneous excitatory postsynaptic currents (sEPSC), while SB 271046 only increased sEPSC. These effects of EMD 386088 and SB 271046 could be reversed by the GABAA receptor antagonist bicuculline (BMI) and positive allosteric modulator clonazepam (CLZ), respectively. Our results suggest that neurotransmission in the dmPFC by 5-HT6 receptor activation and inhibition may play an important role in anxiety-like behavior, and may provide new insight into the pathological mechanism and potential target of anxiety disorders.

Functional and molecular plasticity of γ and α1 GABAA receptor subunits in the dorsal motor nucleus of the vagus after experimentally induced diabetes.

Chronic experimentally induced hyperglycemia augments subunit-specific γ-aminobutyric acid A (GABAA) receptor-mediated inhibition of parasympathetic preganglionic motor neurons in the dorsal motor nucleus of the vagus (DMV). However, the contribution of α1 or γ GABAA receptor subunits, which are ubiquitously expressed on central nervous system neurons, to this elevation in inhibitory tone have not been determined. This study investigated the effect of chronic hyperglycemia/hypoinsulinemia on α1- and γ-subunit-specific GABAA receptor-mediated inhibition using electrophysiological recordings in vitro and quantitative RT-PCR. DMV neurons from streptozotocin-treated mice demonstrated enhancement of both phasic and tonic inhibitory currents in response to application of the α1-subunit-selective GABAA receptor-positive allosteric modulator zolpidem. Responses to low concentrations of the GABAA receptor antagonist gabazine suggested an additional increased contribution of γ-subunit-containing receptors to tonic currents in DMV neurons. Consistent with the functional elevation in α1- and γ-subunit-dependent activity, transcription of both the α1- and γ2-subunits was increased in the dorsal vagal complex of streptozotocin-treated mice. Overall, these findings suggest an increased sensitivity to both zolpidem and gabazine after several days of hyperglycemia/hypoinsulinemia, which could contribute to altered parasympathetic output from DMV neurons in diabetes.NEW & NOTEWORTHY Glutamate and GABA signaling in the dorsal vagal complex is elevated after several days of chronic hyperglycemia in a mouse model of type 1 diabetes. We report persistently enhanced GABAA receptor-mediated responses to the somnolescent zolpidem in preganglionic vagal motor neurons. These results imply a broader impact of chronic hyperglycemia on central vagal function than previously appreciated and reinforce the hypothesis that diabetes effects in the brain can impact regulation of metabolic homeostasis.

A randomized double-blind, placebo-controlled trial of ganaxolone in children and adolescents with fragile X syndrome

BackgroundGamma-aminobutyric acid (GABA) system deficits are integral to the pathophysiologic development of fragile X syndrome (FXS). Ganaxolone, a GABAA receptor positive allosteric modulator, is hypothesized to improve symptoms such as anxiety, hyperactivity, and attention deficits in children with FXS.MethodsThis study was a randomized, double-blind, placebo-controlled, crossover trial of ganaxolone in children with FXS, aged 6–17 years.ResultsSixty-one participants were assessed for eligibility, and 59 were randomized to the study. Fifty-five participants completed at least the first arm and were included in the intention-to-treat analysis; 51 participants completed both treatment arms. There were no statistically significant improvements observed on the primary outcome measure (Clinical Global Impression-Improvement), the key secondary outcome measure (Pediatric Anxiety Rating Scale-R), or any other secondary outcome measures in the overall study population. However, post-hoc analyses revealed positive trends in areas of anxiety, attention, and hyperactivity in participants with higher baseline anxiety and low full-scale IQ scores. No serious adverse events (AEs) occurred, although there was a significant increase in the frequency and severity of AEs related to ganaxolone compared to placebo.ConclusionsWhile ganaxolone was found to be safe, there were no significant improvements in the outcome measures in the overall study population. However, ganaxolone in subgroups of children with FXS, including those with higher anxiety or lower cognitive abilities, might have beneficial effects.Trial registrationClinicalTrials.gov, NCT01725152

GABAA receptor positive allosteric modulators modify the abuse-related behavioral and neurochemical effects of methamphetamine in rhesus monkeys

GABAA receptor positive allosteric modulators (GABAA receptor modulators) are commonly used for the treatment of insomnia. Nevertheless, the effects of these compounds on psychostimulant-induced sleep impairment are poorly understood. Because GABAA receptor modulators have been shown to decrease the abuse-related effects of psychostimulants, the aim of the present study was to evaluate the effects of temazepam (0.3, 1.0 or 3.0 mg/kg) and eszopiclone (0.3, 1.0 or 3.0 mg/kg), two GABAA receptor modulators, on the behavioral neuropharmacology of methamphetamine in adult rhesus macaques (n = 5). Sleep-like measures and general daytime activity were evaluated with Actiwatch monitors. Methamphetamine self-administration (0.03 mg/kg/inf) was evaluated during morning sessions. Methamphetamine-induced dopamine overflow was assessed through in vivo microdialysis targeting the nucleus accumbens. Nighttime treatment with either temazepam or eszopiclone was ineffective in improving sleep-like measures disrupted by methamphetamine self-administration. Acute pretreatment with a low dose of temazepam before self-administration sessions increased methamphetamine self-administration without affecting normal daytime home-cage activity. At a high dose, acute temazepam pretreatment decreased methamphetamine self-administration and attenuated methamphetamine-induced increases in dopamine in the nucleus accumbens, without decreasing general daytime activity. Acute eszopiclone treatment exerted no effects on methamphetamine intake or drug-induced increases in dopamine. Our study suggests that treatments based on GABAA receptor modulators are not effective for the treatment of sleep disruption in the context of psychostimulant use. In addition, distinct GABAA receptor modulators differentially modulated the abuse-related effects of methamphetamine, with acute treatment with the high efficacy GABAA receptor modulator temazepam decreasing the behavioral and neurochemical effects of methamphetamine.

Abuse-related effects of subtype-selective GABAA receptor positive allosteric modulators in an assay of intracranial self-stimulation in rats.

GABAA positive allosteric modulators (GABAA PAMs), such as diazepam and zolpidem, are used clinically for anxiety and insomnia, but abuse liability is a concern. Novel GABAA PAMS may have lower abuse liability while retaining clinical utility. The present study compared abuse-related effects of the non-selective GABAA PAM diazepam, the α1-selective GABAA PAM zolpidem, and three novel GABAA PAMs (JY-XHe-053, XHe-II-053, and HZ-166) using intracranial self-stimulation (ICSS) in rats. These novel compounds have relatively low efficacy at α1-, α2-, and α3-containing GABAA receptors, putative in vivo selectivity at α2/α3-containing GABAA receptors, and produce anxiolytic-like effects with limited sedation in non-human primates. Adult, male Sprague-Dawley rats (n=17) were each implanted with a bipolar electrode in the medial forebrain bundle and trained to respond under a fixed-ratio 1 schedule of reinforcement for electrical brain stimulation. The potency and time course of effects were compared for diazepam (0.1-10mg/kg), zolpidem (0.032-3.2mg/kg), and the three novel compounds (JY-XHe-053, XHe-II-053, and HZ-166; all 3.2-32mg/kg). Zolpidem and diazepam produced transient facilitation of ICSS at small doses and more sustained rate-decreasing effects at larger doses. JY-XHe-053 and HZ-166 produced weak and inconsistent ICSS facilitation, whereas XHe-II-053 had no effect on ICSS. These results support a key role for α1-containing GABAA receptors in mediating GABAA PAM-induced ICSS facilitation. These results are concordant with drug self-administration studies in monkeys in suggesting that GABAA PAMs with low α1 efficacy and putative α2/α3 selectivity have lower abuse liability than high-efficacy non-selective or α1-selective GABAA PAMs.

Pharmacological Potential and Synthetic Approaches of Imidazo[4,5-b]pyridine and Imidazo[4,5-c]pyridine Derivatives.

The structural resemblance between the fused imidazopyridine heterocyclic ring system and purines has prompted biological investigations to assess their potential therapeutic significance. They are known to play a crucial role in numerous disease conditions. The discovery of their first bioactivity as GABAA receptor positive allosteric modulators divulged their medicinal potential. Proton pump inhibitors, aromatase inhibitors, and NSAIDs were also found in this chemical group. Imidazopyridines have the ability to influence many cellular pathways necessary for the proper functioning of cancerous cells, pathogens, components of the immune system, enzymes involved in carbohydrate metabolism, etc. The collective results of biochemical and biophysical properties foregrounded their medicinal significance in central nervous system, digestive system, cancer, inflammation, etc. In recent years, new preparative methods for the synthesis of imidazopyridines using various catalysts have been described. The present manuscript to the best of our knowledge is the complete compilation on the synthesis and medicinal aspects of imidazo[4,5-b]pyridines and imidazo[4,5-c]pyridines reported from the year 2000 to date, including structure–activity relationships.

Antinociceptive Effects of a Novel α2/α3-Subtype Selective GABAA Receptor Positive Allosteric Modulator.

Pain remains a challenging clinical condition and spinal GABAA receptors are crucial modulators of pain processing. α2/α3-subtype GABAA receptors mediate the analgesic actions of benzodiazepines. Positive allosteric modulators (PAMs) at α2/α3-subtype GABAA receptors may have analgesic potential. Here we report a new selective α2/α3-subtype GABAA receptor PAM in in vitro and in vivo pain assays. KRM-II-81 demonstrated similar efficacy at α1/α2/α3 GABAA receptors and negligible efficacy at α4/α5/α6 GABAA receptors, with α2 and α3-subtypes being 17- and 28-fold more potent than α1 subtypes in HEK-293T cells expressing GABAA receptors with different α subunits. In contrast, KRM-II-18B showed significant efficacy at α1/α2/α3/ α5 subtypes, with similar potency at α1/α2/α3 subtypes. Both PAMs and morphine dose-dependently decreased 0.6% acetic acid- and 0.32% lactic acid-induced writhing. The effects of both PAMs were reversed by the benzodiazepine receptor antagonist flumazenil, confirming their action at the benzodiazepine binding site of GABAA receptors. Both PAMS and morphine all dose-dependently reversed 0.32% lactic acid (but not 0.6% acetic acid) induced suppression of nesting behavior. Acetaminophen, but not the PAMs, reversed acid-depressed locomotor activity. Combined, these findings suggest that KRM-II-81 is a selective α2/α3 subtype GABAA PAM with significant antinociceptive effects in chemical stimulation-induced pain in mice.

The synthetic neuroactive steroid SGE-516 reduces status epilepticus and neuronal cell death in a rat model of soman intoxication.

Organophosphorus nerve agents (OPNAs) are irreversible inhibitors of acetylcholinesterase that pose a serious threat to public health because of their use as chemical weapons. Exposure to high doses of OPNAs can dramatically potentiate cholinergic synaptic activity and cause status epilepticus (SE). Current standard of care for OPNA exposure involves treatment with cholinergic antagonists, oxime cholinesterase reactivators, and benzodiazepines. However, data from pre-clinical models suggest that OPNA-induced SE rapidly becomes refractory to benzodiazepines. Neuroactive steroids (NAS), such as allopregnanolone, retain anticonvulsant activity in rodent models of benzodiazepine-resistant SE, perhaps because they modulate a broader variety of GABAA receptor subtypes. SGE-516 is a novel, next generation NAS and a potent and selective GABAA receptor positive allosteric modulator (PAM). The present study first established that SGE-516 reduced electrographic seizures in the rat lithium-pilocarpine model of pharmacoresistant SE. Then the anticonvulsant activity of SGE-516 was investigated in the soman-intoxication model of OPNA-induced SE. SGE-516 (5.6, 7.5, and 10mg/kg, IP) significantly reduced electrographic seizure activity compared to control when administered 20min after SE onset. When 10mg/kg SGE-516 was administered 40min after SE onset, seizure activity was still significantly reduced compared to control. In addition, all cohorts of rats treated with SGE-516 exhibited significantly reduced neuronal cell death as measured by FluoroJade B immunohistochemistry. These data suggest synthetic NASs that positively modulate both synaptic and extrasynaptic GABAA receptors may be candidates for further study in the treatment of OPNA-induced SE.

Antiepileptic potential of ganaxolone

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Expression of the K+/Cl− cotransporter, KCC2, in cerebellar Purkinje cells is regulated by group-I metabotropic glutamate receptors

The neuronal K+/Cl− symporter, KCC2, shapes synaptic responses mediated by Cl−-permeant GABAA receptors. Moving from the evidence that excitatory neurotransmission drives changes in KCC2 expression in cerebellar neurons, we studied the regulation of KCC2 expression by group-I metabotropic glutamate (mGlu) receptors in the cerebellum of adult mice. Mice lacking mGlu5 receptors showed a large reduction in cerebellar KCC2 protein levels and a loss of KCC2 immunoreactivity in Purkinje cells. Similar changes were seen in mice treated with the mGlu5 receptor antagonist, MPEP, whereas treatment with the mGlu5 receptor positive allosteric modulator (PAM), VU0360172, increased KCC2 expression. In contrast, pharmacological inhibition of mGlu1 receptors with JNJ16259685 enhanced cerebellar KCC2 protein levels and KCC2 immunoreactivity in Purkinje cells, whereas treatment with the mGlu1 receptor PAM, RO0711401, reduced KCC2 expression. To examine whether the reduction in KCC2 expression caused by the absence or the inhibition of mGlu5 receptors could affect GABAergic transmission, we performed electrophysiological and behavioral studies. Recording of extracellular action potentials in Purkinje cells showed that the inhibitory effect of the GABAA receptor agonist, muscimol, was lost in cerebellar slices prepared from mGlu5−/− mice or from mice treated systemically with MPEP, in line with the reduction in KCC2 expression. Similarly, motor impairment caused by the GABAA receptor PAM, diazepam, was attenuated in mice pre-treated with MPEP. These findings disclose a novel function of mGlu5 receptors in the cerebellum and suggest that mGlu5 receptor ligands might influence GABAergic transmission in the cerebellum and affect motor responses to GABA-mimetic drugs.This article is part of the Special Issue entitled ‘Metabotropic Glutamate Receptors, 5 years on’.

Early postnatal GABAA receptor modulation reverses deficits in neuronal maturation in a conditional neurodevelopmental mouse model of DISC1.

Exploring drug targets based on disease-associated molecular mechanisms during development is crucial for the generation of novel prevention and treatment strategies for neurodevelopmental psychiatric conditions. We report that prefrontal cortex-specific postnatal knockdown of DISC1 via in utero electroporation combined with an inducible knockdown expression system drives deficits in synaptic GABAA function and dendritic development in pyramidal neurons, as well as abnormalities in sensorimotor gating, albeit without profound memory deficits. We show for the first time that DISC1 is specifically involved in regulating cell surface expression of α2 subunit-containing GABAA receptors in immature developing neurons, but not after full maturation. Notably, pharmacological intervention with α2/3 subtype-selective GABAA receptor positive allosteric modulators during the early postnatal period ameliorates dendritic deficits and behavioral abnormalities induced by knockdown of DISC1. These findings highlight a critical role of DISC1-mediated disruption of postnatal GABA signaling in aberrant prefrontal cortex maturation and function.

Can a Positive Allosteric Modulation of GABAergic Receptors Improve Motor Symptoms in Patients with Parkinson’s Disease? The Potential Role of Zolpidem in the Treatment of Parkinson’s Disease

At present, patients with advanced Parkinson's disease (PD) are unsatisfactorily controlled by currently used anti-Parkinsonian dopaminergic drugs. Various studies suggest that therapeutic strategies based on nondopaminergic drugs might be helpful in PD. Zolpidem, an imidazopyridine widely used as sleep inducer, shows high affinity only for GABAA receptors containing the α-1 subunit and facilitates GABAergic neurotransmission through a positive allosteric modulation of GABAA receptors. Various observations, although preliminary, consistently suggest that in PD patients zolpidem may induce beneficial (and sometimes remarkable) effects on motor symptoms even after single doses and may also improve dyskinesias. Since a high density of zolpidem binding sites is in the two main output structures of the basal ganglia which are abnormally overactive in PD (internal globus pallidus, GPi, and substantia nigra pars reticulata, SNr), it was hypothesized that in PD patients zolpidem may induce through GABAA receptors an inhibition of GPi and SNr (and, possibly, of the subthalamic nucleus also), resulting in an increased activity of motor cortical areas (such as supplementary motor area), which may give rise to improvement of motor symptoms of PD. Randomized clinical trials are needed in order to assess the efficacy, safety, and tolerability of zolpidem in treating motor symptoms of PD.

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

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. 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. 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. 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.

GABAA α5 subunit-containing receptors do not contribute to reversal of inflammatory-induced spinal sensitization as indicated by the unique selectivity profile of the GABAA receptor allosteric modulator NS16085

GABAA receptor positive allosteric modulators (PAMs) mediate robust analgesia in animal models of pathological pain. Restoration of diminished spinal GABAA-α2 and -α3 subunit-containing receptor function is a principal contributor to this analgesia, albeit involvement of GABAA-α5-receptors has not been excluded. Thus, we compared NS11394 and TPA023 (PAMs with selectivity/efficacy at GABAA-α2/α3/α5 receptors) with TP003 (a reportedly GABAA-α3 selective PAM) against spinal sensitization. However, in-house electrophysiology studies designed to confirm the selectivity of TPA023 and TP003 for human GABAA receptors did not corroborate published data, with TP003 displaying considerable GABAA-α5 receptor efficacy. Therefore, we identified a novel PAM, NS16085, which possesses negligible efficacy at GABAA-α5 receptors, but with GABAA-α2/α3 efficacy equivalent to NS11394. At the GABAA-α1 receptor the compound gives low level of negative modulation further separating it from the other compounds. Rat pups with carrageenan-induced hindpaw inflammatory hyperalgesia were used to make ex vivo spinal dorsal root-evoked ventral root recordings. Some spontaneous activity and large numbers of spikes to repetitive stimulation of dorsal roots at C-fibre intensity, indicative of wind-up and sensitization were observed. Equimolar concentrations of NS11394, TP003 and NS16085 all attenuated wind-up to a similar degree; TPA023 was clearly less effective. In adult rats, NS16085 (3–30mg/kg, p.o.) dose-dependently reduced formalin-induced hindpaw flinching with efficacy comparable to NS11394. Thus, potentiation of GABAA-α2 and-α3 receptors is sufficient to depress spinal sensitization and mediate analgesia after inflammatory injury. Positive modulation at GABAA-α5-receptors is apparently dispensable for this process, an important consideration given the role of this receptor subtype in cognitive function.

Involvement of glutamatergic and GABAergic transmission in MK-801-increased gamma band oscillation power in rat cortical electroencephalograms

Hypofunction of the N-methyl-d-aspartic acid receptor (NMDAr) has been considered to play a crucial role in the pathophysiology of schizophrenia. In rodent electroencephalogram (EEG) studies, non-competitive NMDAr antagonists have been reported to produce aberrant basal gamma band oscillation (GBO), as observed in schizophrenia. Aberrations in GBO power have attracted attention as a translational biomarker for the development of novel antipsychotic drugs. However, the neuronal mechanisms as well as the pharmacological significance of NMDAr antagonist-induced aberrant GBO power have not been fully investigated. In the present study, to address the above questions, we examined the pharmacological properties of MK-801 (0.1mg/kg)-increased basal GBO power in rat cortical EEG. Riluzole (3–10mg/kg), a glutamate release inhibitor, reduced the MK-801-increased basal GBO power. In contrast, L-838,417 (1–3mg/kg), an α2/3/5 subunit-selective GABAA receptor-positive allosteric modulator, enhanced the GBO increase. Antipsychotics such as haloperidol (0.05–0.3mg/kg) and clozapine (1–10mg/kg) dose-dependently attenuated the MK-801-increased GBO power. Likewise, LY379268 (0.3–3mg/kg), an metabotropic glutamate 2/3 receptor (mGlu2/3 receptor) agonist, reduced the GBO increase in a dose-dependent manner, which was antagonized by an mGlu2/3 receptor antagonist LY341495. These results suggest that an increase in cortical GBO power induced by NMDAr hypofunction can be attributed to the aberrant activities of both excitatory pyramidal neurons and inhibitory interneurons in local circuits. The aberrant cortical GBO power reflecting cortical network dysfunction observed in schizophrenia might be a useful biomarker for the discovery of novel antipsychotic drugs.

Analysis of γ-aminobutyric acid (GABA) type A receptor subtypes using isosteric and allosteric ligands.

The GABAA receptors (GABAARs) play an important role in inhibitory transmission in the brain. The GABAARs could be identified using a medicinal chemistry approach to characterize with a series of chemical structural analogues, some identified in nature, some synthesized, to control the structural conformational rigidity/flexibility so as to define the 'receptor-specific' GABA agonist ligand structure. In addition to the isosteric site ligands, these ligand-gated chloride ion channel proteins exhibited modulation by several chemotypes of allosteric ligands, that help define structure and function. The channel blocker picrotoxin identified a noncompetitive channel blocker site in GABAARs. This ligand site is located in the transmembrane channel pore, whereas the GABA agonist site is in the extracellular domain at subunit interfaces, a site useful for low energy coupled conformational changes of the functional channel domain. Also in the trans-membrane domain are allosteric modulatory ligand sites, mostly positive, for diverse chemotypes with general anesthetic efficacy, namely, the volatile and intravenous agents: barbiturates, etomidate, propofol, long-chain alcohols, and neurosteroids. The last are apparent endogenous positive allosteric modulators of GABAARs. These binding sites depend on the GABAAR heteropentameric subunit composition, i.e., subtypes. Two classes of pharmacologically very important allosteric modulatory ligand binding site reside in the extracellular domain at modified agonist sites at other subunit interfaces: the benzodiazepine site, and the low-dose ethanol site. The benzodiazepine site is specific for certain subunit combination subtypes, mainly synaptically localized. In contrast, the low-dose (high affinity) ethanol site(s) is found at a modified benzodiazepine site on different, extrasynaptic, subtypes.

Anticonvulsant potencies of the enantiomers of the neurosteroids androsterone and etiocholanolone exceed those of the natural forms

Androsterone [(3α,5α)-3-hydroxyandrostan-17-one; 5α,3α-A] and its 5β-epimer etiocholanolone [(3α,5β)-3-hydroxyandrostan-17-one; 5β,3α-A)], the major excreted metabolites of testosterone, are neurosteroid positive modulators of GABAA receptors. Such neurosteroids typically show enantioselectivity in which the natural form is more potent than the corresponding unnatural enantiomer. For 5α,3α-A and 5β,3α-A, the unnatural enantiomers are more potent at GABAA receptors than the natural forms. The aim of this study was to compare the anticonvulsant potencies and time courses of 5α,3α-A and 5β,3α-A with their enantiomers in mouse seizure models. Steroids were administered intraperitoneally to male NIH Swiss mice 15 min (or up to 6 h in time course experiments) prior to administration of an electrical stimulus in the 6-Hz or maximal electroshock (MES) seizure tests or the convulsant pentylenetetrazol (PTZ). In the 6-Hz test, the ED50 values of ent-5α,3α-A was 5.0 mg/kg whereas the value for 5α,3α-A was 12.1 mg/kg; the corresponding values in the PTZ seizure test were 22.8 and 51.8 mg/kg. Neurosteroid GABAA receptor-positive allosteric modulators are generally weak in the MES seizure test and this was confirmed in the present study. However, the atypical relative potency relationship was maintained with ED50 values of 140 and 223 mg/kg for ent-5α,3α-A and 5α,3α-A, respectively. Similar relationships were obtained for the 5β-isomers, except that the enantioselectivity was accentuated. In the 6-Hz and PTZ tests, the ED50 values of ent-5β,3α-A were 11.8 and 20.4 mg/kg whereas the values for 5β,3α-A were 57.6 and 109.1 mg/kg. Protective activity in the 6-Hz test of ent-5α,3α-A persisted for somewhat longer (~5 h) than for 5α,3α-A (~4 h); protection by ent-5β,3α-A also persisted longer (~3 h) than for 5β,3α-A (~2 h). The unnatural enantiomers of 17-keto androgen class neurosteroids have greater in vivo potency and a longer duration of action than their natural counterparts. The more prolonged duration of action of the unnatural enantiomers could reflect reduced susceptibility to metabolism. Unnatural enantiomers of androgen class neurosteroids could have therapeutic utility and may provide advantages over the corresponding natural isomers due to enhanced potency and improved pharmacokinetic characteristics.

Limited central side effects of a β-subunit subtype-selective GABAA receptor allosteric modulator

GABAergic anxiolytics have well-documented centrally mediated side effects including sedation, potentiation of ethanol, tolerance, abuse liability and memory impairment. Most research directed towards identifying an anxioselective GABAergic therapeutic has been based upon the theory that these side effects could be mitigated by avoiding α1/5-subunit GABAA receptors while specifically targeting those with the α2/3-subunit. Unfortunately, there are prominent exceptions to this theory and it has yet to be translated into clinical success. We previously demonstrated that β2/3-subunit-selective GABAA receptor-positive allosteric modulators act as anxiolytics with reduced sedation and ethanol potentiation regardless of their activity at α1-subunit GABAA receptors. The prototypical β2/3-subunit-selective positive allosteric modulator, 2-261, is further characterized here for additional side effects commonly associated with central GABAA receptor activation. In mice, 10 times the anxiolytic dose (10 mg/kg) of 2-261 does not induce behavioral tolerance in the elevated plus maze following a 2 week subchronic treatment. In rats, an anxiolytic dose (10 mg/kg) of 2-261 is inactive in conditioned place preference, suggesting a reduced abuse liability. In rats, 10 times the anxiolytic dose (100 mg/kg) of 2-261 does not have a significant amnestic effect in the radial arm maze, suggesting a greater therapeutic index for memory impairment. These results suggest that β2/3-subunit subtype-selective GABAA receptor-positive allosteric modulators not only have reduced sedative liability, but also a reduction in other central side effects commonly associated with broader GABAA receptor activation. β2/3-subunit-selective compounds may represent a novel design template for anxiolytics with benzodiazepine-like efficacy and mitigated side effects.

P.1.g.020 Pretreatment with selective I2-imidazoline receptor ligands prevents kainic acid-induced neurotoxic signaling in the mouse hippocampus

Hypofunction of the N-methyl-d-aspartic acid receptor (NMDAr) has been considered to play a crucial role in the pathophysiology of schizophrenia. In rodent electroencephalogram (EEG) studies, non-competitive NMDAr antagonists have been reported to produce aberrant basal gamma band oscillation (GBO), as observed in schizophrenia. Aberrations in GBO power have attracted attention as a translational biomarker for the development of novel antipsychotic drugs. However, the neuronal mechanisms as well as the pharmacological significance of NMDAr antagonist-induced aberrant GBO power have not been fully investigated. In the present study, to address the above questions, we examined the pharmacological properties of MK-801 (0.1 mg/kg)-increased basal GBO power in rat cortical EEG. Riluzole (3–10 mg/kg), a glutamate release inhibitor, reduced the MK-801-increased basal GBO power. In contrast, L-838,417 (1–3 mg/kg), an α2/3/5 subunit-selective GABAA receptor-positive allosteric modulator, enhanced the GBO increase. Antipsychotics such as haloperidol (0.05–0.3 mg/kg) and clozapine (1–10 mg/kg) dose-dependently attenuated the MK-801-increased GBO power. Likewise, LY379268 (0.3–3 mg/kg), an metabotropic glutamate 2/3 receptor (mGlu2/3 receptor) agonist, reduced the GBO increase in a dose-dependent manner, which was antagonized by an mGlu2/3 receptor antagonist LY341495. These results suggest that an increase in cortical GBO power induced by NMDAr hypofunction can be attributed to the aberrant activities of both excitatory pyramidal neurons and inhibitory interneurons in local circuits. The aberrant cortical GBO power reflecting cortical network dysfunction observed in schizophrenia might be a useful biomarker for the discovery of novel antipsychotic drugs.

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.