3ß-Methyl-Neurosteroid Analogs Are Preferential Positive Allosteric Modulators and Direct Activators of Extrasynaptic δ-Subunit γ-Aminobutyric Acid Type A Receptors in the Hippocampus Dentate Gyrus Subfield.

Neurosteroids are powerful modulators of γ-aminobutyric acid (GABA)-A receptors. Ganaxolone (3α-hydroxy-3ß-methyl-5α-pregnan-20-one, GX) and synthetic analogs of the neurosteroid allopregnanolone (AP) are designed to treat epilepsy and related conditions. However, their precise mechanism of action in native neurons remains unclear. Here, we sought to determine the mode of action of GX and its analogs at GABA-A receptors in native hippocampal neurons by analyzing extrasynaptic receptor-mediated tonic currents and synaptic receptor-mediated phasic currents. Concentration-response profiles of GX were determined in two cell types: δ-containing dentate gyrus granule cells (DGGCs) and γ2-containing CA1 pyramidal cells (CA1PCs). GX produced significantly greater potentiation of the GABA-A receptor-activated chloride currents in DGGCs (500%) than CA1PCs (200%). In the absence of GABA, GX evoked 2-fold greater inward currents in DGGCs than CA1PCs, which were 2-fold greater than AP within DGGCs. In hippocampus slices, GX potentiated and directly activated tonic currents in DGGCs. These responses were significantly diminished in DGGCs from δ-subunit knockout (δKO) mice, confirming GX's selectivity for δGABA-A receptors. Like AP, GX potentiation of tonic currents was prevented by protein kinase C inhibition. Furthermore, GX's protection against hippocampus-kindled seizures was significantly diminished in δKO mice. GX analogs exhibited greater potency and efficacy than GX on δGABA-A receptor-mediated tonic inhibition. In summary, these results provide strong evidence that GX and its analogs are preferential allosteric modulators and direct activators of extrasynaptic δGABA-A receptors regulating network inhibition and seizures in the dentate gyrus. Therefore, these findings provide a mechanistic rationale for the clinical use of synthetic neurosteroids in epilepsy and seizure disorders.

Favouring inhibitory synaptic drive mediated by GABA(A) receptors in the basolateral nucleus of the amygdala efficiently reduces pain symptoms in neuropathic mice.

Pain is an emotion and neuropathic pain symptoms are modulated by supraspinal structures such as the amygdala. The central nucleus of the amygdala is often called the 'nociceptive amygdala', but little is known about the role of the basolateral amygdala. Here, we monitored the mechanical nociceptive thresholds in a mouse model of neuropathic pain and infused modulators of the glutamate/GABAergic transmission in the basolateral nucleus of the amygdala (BLA) via chronically-implanted cannulas. We found that an N-methyl-D-aspartate-type glutamate receptor antagonist (MK-801) exerted a potent antiallodynic effect, whereas a transient allodynia was induced after perfusion of bicuculline, a GABA(A) receptor antagonist. Potentiating GABA(A) receptor function using diazepam or etifoxine (a non-benzodiazepine anxiolytic) fully but transiently alleviated mechanical allodynia. Interestingly, the antiallodynic effect of etifoxine disappeared in animals that were incapable of producing 3α-steroids. Diazepam had a similar effect but of shorter duration. As indicated by patch-clamp recordings of BLA neurons, these effects were mediated by a potentiation of GABA(A) receptor-mediated synaptic transmission. Together with a presynaptic elevation of miniature inhibitory postsynaptic current frequency, the duration and amplitude of GABA(A) miniature inhibitory postsynaptic currents were also increased (postsynaptic effect). The analgesic contribution of endogenous neurosteroid seemed to be exclusively postsynaptic. This study highlights the importance of the BLA and the local inhibitory/excitatory neuronal network activity while setting the mechanical nociceptive threshold. Furthermore, it appears that promoting inhibition in this specific nucleus could fully alleviate pain symptoms. Therefore, the BLA could be a novel interesting target for the development of pharmacological or non-pharmacological therapies.

Modulation of Spinal GABAergic Inhibition and Mechanical Hypersensitivity following Chronic Compression of Dorsal Root Ganglion in the Rat.

Chronic compression of dorsal root ganglion (CCD) results in neuropathic pain. We investigated the role of spinal GABA in CCD-induced pain using rats with unilateral CCD. A stereological analysis revealed that the proportion of GABA-immunoreactive neurons to total neurons at L4/5 laminae I-III on the injured side decreased in the early phase of CCD (post-CCD week 1) and then returned to the sham-control level in the late phase (post-CCD week 18). In the early phase, the rats showed an increase in both mechanical sensitivity of the hind paw and spinal WDR neuronal excitability on the injured side, and such increase was suppressed by spinally applied muscimol (GABA-A agonist, 5 nmol) and baclofen (GABA-B agonist, 25 nmol), indicating the reduced spinal GABAergic inhibition involved. In the late phase, the CCD-induced increase in mechanical sensitivity and neuronal excitability returned to pre-CCD levels, and such recovered responses were enhanced by spinally applied bicuculline (GABA-A antagonist, 15 nmol) and CGP52432 (GABA-B antagonist, 15 nmol), indicating the regained spinal GABAergic inhibition involved. In conclusion, the alteration of spinal GABAergic inhibition following CCD and leading to a gradual reduction over time of CCD-induced mechanical hypersensitivity is most likely due to changes in GABA content in spinal GABA neurons.

Little evidence of a role for the α1GABAA subunit-containing receptor in a rhesus monkey model of alcohol drinking.

BACKGROUND: Alcohol potentiates GABAergic neurotransmission via action at the GABAA receptor. α1 subunit-containing GABAA receptors have been implicated as mediators, in part, of the behavioral and abuse-related effects of alcohol in rodents. METHODS: We systematically investigated the effects of 1 α1-preferring benzodiazepine agonist, zolpidem, and 2 antagonists, ß-carboline-3-carboxylate-tert-butyl ester (ßCCT) and 3-propoxy-ß-carboline hydrochloride (3-PBC), on oral self-administration of alcohol (2% w/v) or sucrose solution and observable behavior in rhesus macaques. We compared these effects to those of the nonselective benzodiazepine agonist triazolam, antagonist flumazenil, and inverse agonist ß-carboline carboxylate (ßCCE). RESULTS: Alcohol and sucrose solutions maintained reliable baseline drinking behavior across the study. The α1-preferring compounds did not affect intake, number of sipper extensions, or blood alcohol levels (BALs) at any of the doses tested. Zolpidem, ßCCT, and 3-PBC increased latency to first sipper extension in animals self-administering alcohol, but not sucrose, solution. Triazolam exerted biphasic effects on alcohol-drinking behavior, increasing intake at low doses but decreasing BAL and increasing latency at higher doses. At doses higher than those effective in alcohol-drinking animals, triazolam increased sucrose intake and latency. Flumazenil nonsystematically increased number of extensions for alcohol but decreased BAL, with no effects on sucrose drinking. ßCCE decreased sipper extensions for alcohol and increased latency for first sucrose sipper extension, but full dose-effect relationships could not be determined due to seizures at higher doses. CONCLUSIONS: Alcohol-drinking animals appeared more sensitive to the effects of GABAergic compounds on drinking behavior. However, these results do not support a strong contribution of α1GABAA receptors to the reinforcing effects of alcohol in primates.

Seeking potential anticonvulsant agents that target GABAA receptors using experimental and theoretical procedures.

The aim of this study was to identify compounds that possess anticonvulsant activity by using a pentylenetetrazol (PTZ)-induced seizure model. Theoretical studies of a set of ligands, explored the binding affinities of the ligands for the GABA(A) receptor (GABA(A)R), including some benzodiazepines. The ligands satisfy the Lipinski rules and contain a pharmacophore core that has been previously reported to be a GABA(A)R activator. To select the ligands with the best physicochemical properties, all of the compounds were analyzed by quantum mechanics and the energies of the highest occupied molecular orbital and lowest unoccupied molecular orbital were determined. Docking calculations between the ligands and the GABA(A)R were used to identify the complexes with the highest Gibbs binding energies. The identified compound D1 (dibenzo(b,f)(1,4)diazocine-6,11(5H,12H)-dione) was synthesized, experimentally tested, and the GABA(A)R-D1 complex was submitted to 12-ns-long molecular dynamics (MD) simulations to corroborate the binding conformation obtained by docking techniques. MD simulations were also used to analyze the decomposition of the Gibbs binding energy of the residues involved in the stabilization of the complex. To validate our theoretical results, molecular docking and MD simulations were also performed for three reference compounds that are currently in commercial use: clonazepam (CLZ), zolpidem and eszopiclone. The theoretical results show that the GABA(A)R-D1, and GABA(A)R-CLZ complexes bind to the benzodiazepine binding site, share a similar map of binding residues, and have similar Gibbs binding energies and entropic components. Experimental studies using a PTZ-induced seizure model showed that D1 possesses similar activity to CLZ, which corroborates the predicted binding free energy identified by theoretical calculations.

Emergency drug kits: pharmacological and technical considerations.

The risk for complications while providing dental procedures is greatest when caring for patients having significant medical compromise. It is comforting that significant adverse events can generally be prevented by careful preoperative assessment, along with attentive intraoperative monitoring and support. Nevertheless, the office team must be prepared to manage untoward events should they arise. This continuing education article will address basic emergency drugs that should be available in all dental practices and additional agents that become essential for those practices providing various levels of procedural sedation or general anesthesia.

Superior colliculus mediates cervical dystonia evoked by inhibition of the substantia nigra pars reticulata.

Cervical dystonia (CD; spasmodic torticollis) can be evoked by inhibition of substantia nigra pars reticulata (SNpr) in the nonhuman primate (Burbaud et al., 1998; Dybdal et al., 2012). Suppression of GABAergic neurons that project from SNpr results in the disinhibition of the targets to which these neurons project. It therefore should be possible to prevent CD by inhibition of the appropriate nigral target region(s). Here we tested the hypothesis that the deep and intermediate layers of the superior colliculus (DLSC), a key target of nigral projections, are required for the emergence of CD. To test this hypothesis, we pretreated the DLSC of four macaques with the GABA(A) agonist muscimol to determine whether this treatment would prevent CD evoked by muscimol infusions in SNpr. Our data supported this hypothesis: inhibition of DLSC attenuated CD evoked by muscimol in SNpr in all four animals. In two of the four subjects, quadrupedal rotations were evoked by muscimol application into SNpr sites that were distinct from those that induced dystonia. We found that inhibition of DLSC did not significantly alter quadrupedal rotations, suggesting that this response is dissociable from the SNpr-evoked CD. Our results are the first to demonstrate a role of DLSC in mediating the expression of CD. Furthermore, these data reveal a functional relationship between SNpr and DLSC in regulating posture and movement in the nonhuman primate, raising the possibility that the nigrotectal pathway has potential as a target for therapeutic interventions for CD.

Inhibition of α5 γ-Aminobutyric acid type A receptors restores recognition memory after general anesthesia.

BACKGROUND: General anesthetics cause cognitive deficits that persist much longer than would be expected on the basis of their pharmacokinetics. The cellular mechanisms underlying these postanesthetic cognitive deficits remain unknown. γ-Aminobutyric acid type A (GABA(A)) receptors are principal targets for most anesthetics. In particular, the α5GABA(A) receptor subtype has been implicated in acute memory blockade during anesthesia and memory deficits in the early postoperative period. We first sought to determine whether working memory and short-term recognition memory are impaired after isoflurane anesthesia. The second aim of the study was to determine whether memory deficits after isoflurane can be reversed by inhibiting α5GABA(A) receptors. We also sought to determine whether the expression of α5GABA(A) receptors is necessary for the development of memory dysfunction after isoflurane. Lastly, the effect of sevoflurane on memory was studied. METHODS: Wild-type and α5GABA(A) receptor null-mutant (Gabra5-/-) mice were treated with isoflurane (1.3%; 1 minimum alveolar concentration [MAC]) or sevoflurane (2.3%; 1 MAC) or vehicle gas for 1 hour. Memory performance was assessed with a novel object recognition task. Mice were trained on the recognition task either 24 hours or 72 hours after isoflurane anesthesia. Working memory and short-term memory were tested 1 minute and 1 hour after training, respectively. To determine whether inhibition of α5GABA(A) receptors reverses memory deficits, we treated a subset of mice with L-655,708 (0.35 mg/kg or 0.7 mg/kg) 23.5 hours after isoflurane and 30 minutes before behavioral training. RESULTS: Short-term memory was impaired in wild-type mice 24 hours after isoflurane as evidenced by a decrease in the discrimination ratio (control 0.66 ± 0.03 vs isoflurane 0.51 ± 0.03, P = 0.0005). In contrast, working memory was not impaired by isoflurane (control 0.68 ± 0.05 vs isoflurane 0.67 ± 0.04, P = 0.979). The deficit in short-term memory was fully reversed by L-655,708 (effect of isoflurane × L-655,708, F(2,102) = 3.59, P = 0.032; isoflurane 0.51 ± 0.03 vs isoflurane + L-655,708 at 0.35 mg/kg 0.67 ± 0.03, P < 0.05). By 72 hours, the deficits in short-term memory resolved spontaneously (control 0.65 ± 0.05 vs isoflurane 0.60 ± 0.04, P = 0.441). Gabra5-/- mice showed no short-term memory deficits 24 hours after isoflurane (effect of isoflurane F(1,47) = 0.375, P = 0.544). Sevoflurane also caused memory deficits 24 hours after anesthesia, as evidence by a reduction in the discrimination ratio (control 0.63 ± 0.02 vs sevoflurane 0.53 ± 0.03, P = 0.039). CONCLUSIONS: Inhalational anesthetics cause deficits in anterograde recognition memory. This proof-of-concept study shows that α5GABA(A) receptors are necessary for the development of postanesthetic deficits in recognition memory and that these receptors can be targeted to restore memory even after the anesthetic has been eliminated.

A double-blind randomised crossover trial of low-dose flumazenil for benzodiazepine withdrawal: A proof of concept.

INTRODUCTION: Benzodiazepines (BZD) are a class of anxiolytics with varying uses, which primarily act on the GABAA receptor resulting in hyperpolarisation. BZDs are often a difficult drug class to cease once neuroadaptation has occurred; recommendations usually involve gradual dose reductions at variable rates. A growing body of evidence has suggested that low-dose flumazenil, a GABAA receptor antagonist, may be a useful agent to allow for rapid detoxification. AIM: To collect pilot data on the safety and efficacy of low-dose subcutaneous flumazenil to reduce BZD use, withdrawal symptoms, and craving in participants taking above and below the therapeutic maximum diazepam equivalent of 30 mg to inform on sample size for future trials. METHOD: In a randomised double-blinded crossover study design, participants received low-dose flumazenil first (4 mg/24 h for approximately eight days) or placebo first. Groups were divided into those taking < 30 mg diazepam equivalent and ≥ 30 mg diazepam equivalent at baseline. Main outcome measures were percentage reduction in daily diazepam use, withdrawal symptoms, and craving scores from baseline, difference in diazepam use across the placebo first group, and flumazenil related adverse events. RESULTS: Twenty-eight participants were recruited and randomised to flumazenil first (n = 14) and placebo first (n = 14). In participants taking ≥ 30 mg diazepam equivalent at baseline (n = 15), flumazenil significantly reduced diazepam use by 30.5% (p = 0.024) compared to placebo. CONCLUSION: Low-dose flumazenil may aid in BZD detoxification in participants taking daily diazepam equivalent doses greater than or equal to the therapeutic maximum (≥30 mg) by reducing the need for diazepam.

The role of spinal cord extrasynaptic α5 GABAA receptors in chronic pain.

Chronic pain is an incapacitating condition that affects a large population worldwide. Until now, there is no drug treatment to relieve it. The impairment of GABAergic inhibition mediated by GABAA receptors (GABAA R) is considered a relevant factor in mediating chronic pain. Even though both synaptic and extrasynaptic GABAA inhibition are present in neurons that process nociceptive information, the latter is not considered relevant as a target for the development of pain treatments. In particular, the extrasynaptic α5 GABAA Rs are expressed in laminae I-II of the spinal cord neurons, sensory neurons, and motoneurons. In this review, we discuss evidence showing that blockade of the extrasynaptic α5 GABAA Rs reduces mechanical allodynia in various models of chronic pain and restores the associated loss of rate-dependent depression of the Hoffmann reflex. Furthermore, in healthy animals, extrasynaptic α5 GABAA R blockade induces both allodynia and hyperalgesia. These results indicate that this receptor may have an antinociceptive and pronociceptive role in healthy and chronic pain-affected animals, respectively. We propose a hypothesis to explain the relevant role of the extrasynaptic α5 GABAA Rs in the processing of nociceptive information. The data discussed here strongly suggest that this receptor could be a valid pharmacological target to treat chronic pain states.

Progesterone modulates neuronal excitability bidirectionally.

Progesterone acts on neurons directly by activating its receptor and through metabolic conversion to neurosteroids. There is emerging evidence that progesterone exerts excitatory effects by activating its cognate receptors (progesterone receptors, PRs) through enhanced expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs). Progesterone metabolite 5α,3α-tetrahydro-progesterone (allopregnanolone, THP) mediates its anxiolytic and sedative actions through the potentiation of synaptic and extrasynaptic γ-aminobutyric acid type-A receptors (GABAARs). Here, we review progesterone's neuromodulatory actions exerted through PRs and THP and their opposing role in regulating seizures, catamenial epilepsy, and seizure exacerbation associated with progesterone withdrawal.

Bumetanide prevents diazepam-modified anxiety-like behavior in lipopolysaccharide-treated mice.

Benzodiazepine receptor agonists are widely prescribed therapeutic agents that alter gamma-aminobutyric acid (GABA)A receptor activity and have anxiolytic effects. Post-operative use of benzodiazepines is a risk factor of delirium. Inflammatory conditions alter the anxiolytic effects of benzodiazepine. We investigated the effect of diazepam, a typical benzodiazepine anxiolytic, on changes in the emotional behavior of mice in a hole-board test after lipopolysaccharide (LPS) treatment. Diazepam dose-dependently increased the number of head-dips at doses that did not alter locomotor activity; however, diazepam dose-dependently significantly decreased the number of head-dips at doses that did not alter locomotor activity in LPS-treated mice. Flumazenil, a benzodiazepine receptor antagonist, normalized the decrease in head-dipping behavior caused by diazepam treatment in normal and LPS-treated mice. The decrease of the head-dipping effect caused by diazepam was attenuated by minocycline in LPS-treated mice. We further found that the decrease in head-dipping behavior caused by diazepam was blocked by bumetanide, a Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) antagonist, in LPS-treated mice. These findings suggest that diazepam induces the anxiety-like behavior under inflammation conditions, and may cause the GABAA receptor dysfunction associated with the chloride plasticity mediated by NKCC1, which contributes to benzodiazepine-induced delirium after surgery.

Design, synthesis, and subtype selectivity of 3,6-disubstituted ß-carbolines at Bz/GABA(A)ergic receptors. SAR and studies directed toward agents for treatment of alcohol abuse.

A series of 3,6-disubstituted ß-carbolines was synthesized and evaluated for their in vitro affinities at α(x)ß(3)γ(2) GABA(A)/benzodiazepine receptor subtypes by radioligand binding assays in search of α(1) subtype selective ligands to treat alcohol abuse. Analogues of ß-carboline-3-carboxylate-t-butyl ester (ßCCt, 1) were synthesized via a CDI-mediated process and the related 6-substituted ß-carboline-3-carboxylates 6 including WYS8 (7) were synthesized via a Sonogashira or Stille coupling processes from 6-iodo-ßCCt (5). The bivalent ligands of ßCCt (32 and 33) were also designed and prepared via a palladium-catalyzed homocoupling process to expand the structure-activity relationships (SAR) to larger ligands. Based on the pharmacophore/receptor model, a preliminary SAR study on 34 analogues illustrated that large substituents at position-6 of the ß-carbolines were well tolerated. As expected, these groups are proposed to project into the extracellular domain (L(Di) region) of GABA(A)/Bz receptors (see 32 and 33). Moreover, substituents located at position-3 of the ß-carboline nucleus exhibited a conserved stereo interaction in lipophilic pocket L(1), while N(2) presumably underwent a hydrogen bonding interaction with H(1). Three novel ß-carboline ligands (ßCCt, 3PBC and WYS8), which preferentially bound to α1 BzR subtypes permitted a comparison of the pharmacological efficacies with a range of classical BzR antagonists (flumazenil, ZK93426) from several different structural groups and indicated these ß-carbolines were 'near GABA neutral antagonists'. Based on the SAR, the most potent (in vitro) α(1) selective ligand was the 6-substituted acetylenyl ßCCt (WYS8, 7). Earlier both ßCCt and 3PBC had been shown to reduce alcohol self-administration in alcohol preferring (P) and high alcohol drinking (HAD) rats but had little or no effect on sucrose self-administration.(1-3) Moreover, these two ß-carbolines were orally active, and in addition, were anxiolytic in P rats but were only weakly anxiolytic in rodents. These data prompted the synthesis of the ß-carbolines presented here.

Zolpidem for persistent vegetative state--a placebo-controlled trial in pediatrics.

OBJECTIVE: The aim of this study was to determine if zolpidem is associated with improved responsiveness or regional cerebral perfusion in patients with persistent vegetative states. METHODS: Following ethics approval, children with persistent vegetative state were enrolled in a prospective, double-blind, placebo-controlled randomised trial. Patients underwent 2 treatments of 4 days, separated by 10 days. Each child received either a daily dose of zolpidem or placebo with a dosage of 0.14-0.2 mg/kg. Responsiveness and regional cerebral perfusion were the outcomes of interest. These were assessed using the Rancho levels of cognitive functioning scale, the coma/near-coma scale and F (18)-FDG positron emission tomography. These were conducted at baseline and after completion of the treatments. RESULTS: 3 children were enrolled. The Rancho assessment scales showed no change with treatment. The coma/near-coma scale showed a tendency to increase with zolpidem, suggesting reduced responsiveness - when compared to baseline or placebo. The positron emission tomography scans showed no significant changes between treatments. CONCLUSION: Zolpidem was associated with a tendency towards reduced responsiveness in patients with persistent vegetative states. There were no objective changes on positron emission tomography suggestive of an associated increase in cerebral blood flow with zolpidem. It would appear that zolpidem does not offer a beneficial effect in this setting.

Safety and efficacy of GABAA α5 antagonist S44819 in patients with ischaemic stroke: a multicentre, double-blind, randomised, placebo-controlled trial.

BACKGROUND: S44819, a selective GABAA α5 receptor antagonist, reduces tonic post-ischaemic inhibition of the peri-infarct cortex. S44819 improved stroke recovery in rodents and increased cortical excitability in a transcranial magnetic stimulation study in healthy volunteers. The Randomized Efficacy and Safety Trial of Oral GABAA α5 antagonist S44819 after Recent ischemic Event (RESTORE BRAIN) aimed to evaluate the safety and efficacy of S44819 for enhancing clinical recovery of patients with ischaemic stroke. METHODS: RESTORE BRAIN was an international, randomised, double-blind, parallel-group, placebo-controlled, multicentre phase 2 trial that evaluated the safety and efficacy of oral S44189 in patients with recent ischaemic stroke. The study was done in specialised stroke units in 92 actively recruiting centres in 14 countries: ten were European countries (Belgium, Czech Republic, France, Germany, Hungary, Italy, Netherlands, Poland, Spain, and the UK) and four were non-European countries (Australia, Brazil, Canada, and South Korea). Patients aged 18-85 years with acute ischaemic stroke involving cerebral cortex (National Institute of Health Stroke Scale [NIHSS] score 7-20) without previous disability were eligible for inclusion. Participants were randomly assigned to receive 150 mg S44819 twice a day, 300 mg S44819 twice a day, or placebo twice a day by a balanced, non-adaptive randomisation method with a 1:1:1 ratio. Treatment randomisation and allocation were centralised via the interactive web response system using computer-generated random sequences with a block size of 3. Blinding of treatment was achieved by identical appearance and taste of all sachets. Patients, investigators and individuals involved in the analysis of the trial were masked to group assignment. The primary endpoint was the modified Rankin Scale (mRS) score 90 days from onset of treatment, evaluated by shift analysis (predefined main analysis) or by dichotomised analyses using 0-1 versus 2-6 and 0-2 versus 3-6 cutoffs (predefined secondary analysis). Secondary endpoints were the effects of S44819 on the NIHSS and Montreal Cognitive Assessment (MoCA) scores, time needed to complete parts A and B of the Trail Making Test, and the Barthel index. Efficacy analyses were done on all patients who received at least one dose of treatment and had at least one mRS score taken after day 5 (specifically, on or after day 30). Safety was compared across treatment groups for all patients who received at least one dose of treatment. The study was registered at ClinicalTrials.gov, NCT02877615. FINDINGS: Between Dec 19, 2016, and Nov 16, 2018, 585 patients were enrolled in the study. Of these, 197 (34%) were randomly assigned to receive 150 mg S44819 twice a day, 195 (33%) to receive 300 mg S44819 twice a day, and 193 (33%) to receive placebo twice a day. 189 (96%) of 197 patients in the 150 mg S44819 group, 188 (96%) of 195 patients in the 300 mg S44819 group, and 191 (99%) patients in the placebo group received at least one dose of treatment and had at least one mRS score taken after day 5, and were included in efficacy analyses. 195 (99%) of 197 patients in the 150 mg S44819 group, 194 (99%) of 195 patients in the 300 mg S44819 group, and 193 (100%) patients in the placebo group received at least one dose of treatment, and were included in safety analyses. The primary endpoint of mRS at day 90 did not differ between each of the two S44819 groups and the placebo group (OR 0·91 [95% CI 0·64-1·31]; p=0·80 for 150 mg S44819 compared with placebo and OR 1·17 [95% CI 0·81-1·67]; p=0·80 for 300 mg S44819 compared with placebo). Likewise, dichotomised mRS scores at day 90 (mRS 0-2 vs 3-6 or mRS 0-1 vs 2-6) did not differ between groups. Secondary endpoints did not reveal any significant group differences. The median NIHSS score at day 90 did not differ between groups (4 [IQR 2-8] in 150 mg S44819 group, 4 [2-7] in 300 mg S44819 group, and 4 [2-6] in placebo group), nor did the number of patients at day 90 with an NIHSS score of up to 5 (95 [61%] of 156 in 150 mg S44819 group, 106 [66%] of 161 in 300 mg S44819 group, and 104 [66%] of 157 in placebo group) versus more than 5 (61 [39%] in 150 mg S44819 group, 55 [34%] in 300 mg S44819 group, and 53 [34%] in placebo group). Likewise, the median MoCA score (22·0 [IQR 17·0-26·0] in 150 mg S44819 group, 23·0 [19·0-26·5] in 300 mg S44819 group, and 22·0 [17·0-26·0] in placebo group), time needed to complete parts A (50 s [IQR 42-68] in 150 mg S44819 group, 49 s [36-63] in 300 mg S44819 group, and 50 s [38-68] in placebo group) and B (107 s [81-144] in 150 mg S44819 group, 121 s [76-159] in 300 mg S44819 group, and 130 s [86-175] in placebo group) of the Trail Making Test, and the Barthel index (90 [IQR 60-100] in 150 mg S44819 group, 90 [70-100] in 300 mg S44819 group, and 90 [70-100] in placebo group) were similar in all groups. Number and type of adverse events were similar between the three groups. There were no drug-related adverse events and no drug-related deaths. INTERPRETATION: There was no evidence that S44819 improved clinical outcome in patients after ischaemic stroke, and thus S44819 cannot be recommended for stroke therapy. The concept of tonic inhibition after stroke should be re-evaluated in humans. FUNDING: Servier.

Subtype Selective γ-Aminobutyric Acid Type A Receptor (GABAAR) Modulators Acting at the Benzodiazepine Binding Site: An Update.

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter within the central nervous system (CNS) with fast, transsynaptic, and modulatory extrasynaptic effects being mediated by the ionotropic GABA type A receptors (GABAARs). These receptors are of particular interest because they are the molecular target of a number of pharmacological agents, of which the benzodiazepines (BZDs), such as diazepam, are the best described. The anxiolytic, sedating, and myorelaxant effects of BZDs are mediated by separate populations of GABAARs containing either α1, α2, α3, or α5 subunits and the molecular dissection of the pharmacology of BZDs indicates that subtype-selective GABAAR modulators will have novel pharmacological profiles. This is best exemplified by α2/α3-GABAAR positive allosteric modulators (PAMs) and α5-GABAAR negative allosteric modulators (NAMs), which were originally developed as nonsedating anxiolytics and cognition enhancers, respectively. This review aims to summarize the current state of the field of subtype-selective GABAAR modulators acting via the BZD binding site and their potential clinical indications.

Identification and Validation of a Novel Biologics Target in Triple Negative Breast Cancer.

The goal of this study was to identify a novel target for antibody-drug conjugate (ADC) development in triple negative breast cancer (TNBC), which has limited treatment options, using gene expression datasets and in vitro siRNA/CRISPR and in vivo functional assays. We analyzed 4467 breast cancers and identified GABRP as top expressed gene in TNBC with low expression in most normal tissues. GABRP protein was localized to cell membrane with broad range of receptors/cell (815-53,714) and expressed by nearly half of breast cancers tissues. GABRP gene knockdown inhibited TNBC cell growth and colony formation in vitro and growth of MDA-MB-468 xenografts in nude mice. Commercially available anti-GABRP antibody (5-100 µg/ml) or de novo generated Fabs (20 µg/ml) inhibited TNBC cell growth in vitro. The same antibody conjugated to mertansine (DM1) also showed significant anticancer activity at nanomolar concentrations. Our results indicate that GABRP is a potential novel therapeutic target for ADC development.

Update on treatment for idiopathic hypersomnia.

INTRODUCTION: Idiopathic hypersomnia (IH) is a poorly characterized orphan central disorder of hypersomnolence responsible for excessive daytime sleepiness (EDS), prolonged nighttime sleep and sleep inertia that often require long-term symptomatic stimulant medication. To date, no drug has currently the authorization for the treatment of IH patients worldwide. Areas covered: The authors reviewed data on pharmacological treatment of IH obtained from published literature (Medline/PubMed/Web of Science) and Clinicaltrial.gov database from 1997 to 2017. Most of data on treatment of IH derived from observational studies and case series with only three well-designed clinical trials available. Expert opinion: In two recent randomized, double-blind, placebo-controlled trials, modafinil improves EDS in IH. Most of other wakefulness-promoting agents labeled for narcolepsy have similar efficacy in cases series of IH patients. Pitolisant and sodium oxybate show promising results in two retrospective studies. The efficacy of γ-aminobutyric acid-A receptor antagonists on objective EDS needs to be clarified. All these medications are used off-label for the management of EDS in IH. Specific clinical instruments and objective tests are required in IH to better evaluate the severity of EDS and responsiveness to medications, but also prolonged sleep and sleep inertia, to optimize the whole management of IH patients.

Pharmacotherapy for Refractory and Super-Refractory Status Epilepticus in Adults.

Patients with prolonged seizures that do not respond to intravenous benzodiazepines and a second-line anticonvulsant suffer from refractory status epilepticus and those with seizures that do not respond to continuous intravenous anesthetic anticonvulsants suffer from super-refractory status epilepticus. Both conditions are associated with significant morbidity and mortality. A strict pharmacological treatment regimen is urgently required, but the level of evidence for the available drugs is very low. Refractory complex focal status epilepticus generally does not require anesthetics, but all intravenous non-anesthetizing anticonvulsants may be used. Most descriptive data are available for levetiracetam, phenytoin and valproate. Refractory generalized convulsive status epilepticus is a life-threatening emergency, and long-term clinical consequences are eminent. Administration of intravenous anesthetics is mandatory, and drugs acting at the inhibitory gamma-aminobutyric acid (GABA)A receptor such as midazolam, propofol and thiopental/pentobarbital are recommended without preference for one of those. One in five patients with anesthetic treatment does not respond and has super-refractory status epilepticus. With sustained seizure activity, excitatory N-methyl-d-aspartate (NMDA) receptors are increasingly expressed post-synaptically. Ketamine is an antagonist at this receptor and may prove efficient in some patients at later stages. Neurosteroids such as allopregnanolone increase sensitivity at GABAA receptors; a Phase 1/2 trial demonstrated safety and tolerability, but randomized controlled data failed to demonstrate efficacy. Adjunct ketogenic diet may contribute to termination of difficult-to-treat status epilepticus. Randomized controlled trials are needed to increase evidence for treatment of refractory and super-refractory status epilepticus, but there are multiple obstacles for realization. Hitherto, prospective multicenter registries for pharmacological treatment may help to improve our knowledge.