GABAergic Involvement in Selective Attention.

Animals need to cope with abundant sensory information, and one strategy is to selectively direct attention to only the most relevant part of the environment. Although the cortical networks of selective attention have been studied extensively, its underlying neurotransmitter systems, especially the role of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), remain less well understood. Increased GABAA receptor activity because of administration of benzodiazepines such as lorazepam is known to slow reactions in cognitive tasks. However, there is limited knowledge about GABAergic involvement in selective attention. Particularly, it is unknown whether increased GABAA receptor activity slows the build-up of selectivity or generally widens attentional focus. To address this question, participants (n = 29) received 1 mg lorazepam and placebo (within-subjects, double-blind) and performed an extended version of the flanker task. The spatial distribution of selective attention was studied by systematically manipulating number and position of incongruent flankers; the temporal build-up was characterized using delta plots. An online task version was presented to an independent, unmedicated sample (n = 25) to verify task effects. Under placebo and in the unmedicated sample, only the number of incongruent flankers, but not their position, influenced RTs. Incongruent flankers impaired RTs more strongly under lorazepam than placebo, especially when adjacent to the target. Delta plot analyses of RT showed that this effect persisted even when participants reacted slowly, indicating that lorazepam-induced impairments in selective attention do not result from simply slowed down build-up of selectivity. Instead, our data indicate that increased GABAA receptor activity widens the attentional focus.

A neolignan from Connarus tuberosus as an allosteric GABAA receptor modulator at the neurosteroid binding site.

In a screening of a small library of extracts from plants of the Amazonian and Cerrado biomes, a hexane extract of Connarus tuberosus roots was found to significantly potentiate the GABA induced fluorescence in a fluorescence (FLIPR) assay in CHO cells stably expressing the α1ß2γ2 subtype of human GABAA receptors. With the aid of HPLC-based activity profiling the activity was linked to the neolignan connarin. In CHO cells the activity of connarin was not abolished by increasing concentrations of flumazenil, while the effect of diazepam was increased by increasing concentrations of connarin. The effect of connarin was abolished by pregnenolone sulfate (PREGS) in a concentration-dependent manner, and the effect of allopregnanolone was further increased by increasing concentrations of connarin. In a two-microelectrode voltage clamp assay with Xenopus laevis oocytes transiently expressing GABAA receptors composed of human α1ß2γ2S and α1ß2 subunits connarin potentiated the GABA-induced currents, with EC50 values of 1.2 ± 0.3 µM (α1ß2γ2S) and 1.3 ± 0.4 µM (α1ß2), and with a maximum enhancement of currents Emax of 1959 ± 70% (α1ß2γ2S) and 185 ± 48% (α1ß2). The activation induced by connarin was abolished by increasing concentrations of PREGS.

Anxiolytic-like Effect of Quercetin Possibly through GABA Receptor Interaction Pathway: In Vivo and In Silico Studies.

Scientific evidence suggests that quercetin (QUR) has anxiolytic-like effects in experimental animals. However, the mechanism of action responsible for its anxiolytic-like effects is yet to be discovered. The goal of this research is to assess QUR's anxiolytic effects in mouse models to explicate the possible mechanism of action. After acute intraperitoneal (i.p.) treatment with QUR at a dose of 50 mg/kg (i.p.), behavioral models of open-field, hole board, swing box, and light-dark tests were performed. QUR was combined with a GABAergic agonist (diazepam) and/or antagonist (flumazenil) group. Furthermore, in silico analysis was also conducted to observe the interaction of QUR and GABA (α5), GABA (ß1), and GABA (ß2) receptors. In the experimental animal model, QUR had an anxiolytic-like effect. QUR, when combined with diazepam (2 mg/kg, i.p.), drastically potentiated an anxiolytic effect of diazepam. QUR is a more highly competitive ligand for the benzodiazepine recognition site that can displace flumazenil (2.5 mg/kg, i.p.). In all the test models, QUR acted similar to diazepam, with enhanced effects of the standard anxiolytic drug, which were reversed by pre-treatment with flumazenil. QUR showed the best interaction with the GABA (α5) receptor compared to the GABA (ß1) and GABA (ß2) receptors. In conclusion, QUR may exert an anxiolytic-like effect on mice, probably through the GABA-receptor-interacting pathway.

Structural and dynamic mechanisms of GABAA receptor modulators with opposing activities.

γ-Aminobutyric acid type A (GABAA) receptors are pentameric ligand-gated ion channels abundant in the central nervous system and are prolific drug targets for treating anxiety, sleep disorders and epilepsy. Diverse small molecules exert a spectrum of effects on γ-aminobutyric acid type A (GABAA) receptors by acting at the classical benzodiazepine site. They can potentiate the response to GABA, attenuate channel activity, or counteract modulation by other ligands. Structural mechanisms underlying the actions of these drugs are not fully understood. Here we present two high-resolution structures of GABAA receptors in complex with zolpidem, a positive allosteric modulator and heavily prescribed hypnotic, and DMCM, a negative allosteric modulator with convulsant and anxiogenic properties. These two drugs share the extracellular benzodiazepine site at the α/γ subunit interface and two transmembrane sites at ß/α interfaces. Structural analyses reveal a basis for the subtype selectivity of zolpidem that underlies its clinical success. Molecular dynamics simulations provide insight into how DMCM switches from a negative to a positive modulator as a function of binding site occupancy. Together, these findings expand our understanding of how GABAA receptor allosteric modulators acting through a common site can have diverging activities.

Modulatory effect of long-term treatment with escitalopram and clonazepam on the expression of anxiety-related neuropeptides: neuromedin U, neuropeptide S and their receptors in the rat brain.

BACKGROUND: Newly identified multifunctional peptidergic modulators of stress responses: neuromedin U (NMU) and neuropeptide S (NPS) are involved in the wide spectrum of brain functions. However, there are no reports dealing with potential molecular relationships between the action of diverse anxiolytic or antidepressant drugs and NMU and NPS signaling in the brain. The present work was therefore focused on local expression of the aforementioned stress-related neuropeptides in the rat brain after long-term treatment with escitalopram and clonazepam. METHODS: Studies were carried out on adult, male Sprague-Dawley rats that were divided into 3 groups: animals injected with saline (control) and experimental individuals treated with escitalopram (at single dose 5 mg/kg daily), and clonazepam (at single dose 0.5 mg/kg). All individuals were sacrificed under anaesthesia and the whole brain excised. Total mRNA was isolated from homogenized samples of amygdala, hippocampus, hypothalamus, thalamus, cerebellum and brainstem. Real time-PCR method was used for estimation of related NPS, NPS receptor (NPSR), NMU, NMU and receptor 2 (NMUR2) mRNA expression. The whole brains were also sliced for general immunohistochemical assessment of the neuropeptides expression. RESULTS: Chronic administration of clonazepam resulted in an increase of NMU mRNA expression and formation of NMU-expressing fibers in the amygdala, while escitalopram produced a significant decrease in NPSR mRNA level in hypothalamus. Long-term escitalopram administration affects the local expression of examined neuropeptides mRNA in a varied manner depending on the brain structure. CONCLUSIONS: Pharmacological effects of escitalopram may be connected with local at least partially NPSR-related alterations in the NPS/NMU/NMUR2 gene expression at the level selected rat brain regions. A novel alternative mode of SSRI action can be therefore cautiously proposed.

Clerodane Diterpenes from Casearia corymbosa as Allosteric GABAA Receptor Modulators.

An EtOAc extract of Casearia corymbosa leaves led to an allosteric potentiation of the GABA signal in a fluorometric imaging plate reader (FLIPR) assay on Chinese hamster ovary (CHO) cells stably expressing GABAA receptors with an α1ß2γ2 subunit composition. The activity was tracked by HPLC-based activity profiling, and four known (2, 3, 4, and 8) and five new clerodane-type diterpenoids (1, 5-7, and 9) were isolated. Compounds 1-8 were obtained from the active time window. The absolute configuration of all compounds was established by ECD. Compounds 3, 7, and 8 exhibited EC50 values of 0.5, 4.6, and 1.4 µM, respectively. To explore possible binding sites at the receptor, the most abundant diterpenoid 8 was tested in combination with diazepam, etazolate, and allopregnanolone. An additive potentiation of the GABA signal was observed with these compounds, while the effect of 8 was not inhibited by flumazenil, a negative allosteric modulator at the benzodiazepine binding site. Finally, the activity was validated in voltage clamp studies on Xenopus laevis oocytes transiently expressing GABAA receptors of the α1ß2γ2S and α1ß2 subtypes. Compound 8 potentiated GABA-induced currents with both receptor subunit compositions [EC50 (α1ß2γ2S) = 43.6 µM; Emax = 809% and EC50 (α1ß2) = 57.6 µM; Emax = 534%]. The positive modulation of GABA-induced currents was not inhibited by flumazenil, thereby confirming an allosteric modulation independent of the benzodiazepine binding site.

Phenolics as GABAA Receptor Ligands: An Updated Review.

Natural products can act as potential GABA modulators, avoiding the undesirable effects of traditional pharmacology used for the inhibition of the central nervous system such as benzodiazepines (BZD). Phenolics, especially flavonoids and phlorotannins, have been considered as modulators of the BZD-site of GABAA receptors (GABAARs), with sedative, anxiolytic or anticonvulsant effects. However, the wide chemical structural variability of flavonoids shows their potential action at more than one additional binding site on GABAARs, which may act either negatively, positively, by neutralizing GABAARs, or directly as allosteric agonists. Therefore, the aim of the present review is to compile and discuss an update of the role of phenolics, namely as pharmacological targets involving dysfunctions of the GABA system, analyzing both their different compounds and their mechanism as GABAergic modulators. We focus this review on articles written in English since the year 2010 until the present. Of course, although more research would be necessary to fully establish the type specificity of phenolics and their pharmacological activity, the evidence supports their potential as GABAAR modulators, thereby favoring their inclusion in the development of new therapeutic targets based on natural products. Specifically, the data compiled in this review allows for the directing of future research towards ortho-dihydroxy diterpene galdosol, the flavonoids isoliquiritigenin (chalcone), rhusflavone and agathisflavone (biflavonoids), as well as the phlorotannins, dieckol and triphlorethol A. Clinically, flavonoids are the most interesting phenolics due to their potential as anticonvulsant and anxiolytic drugs, and phlorotannins are also of interest as sedative agents.

Dynamic Recovery: GABA Agonism Restores Neural Variability in Older, Poorer Performing Adults.

Aging is associated with cognitive impairment, but there are large individual differences in these declines. One neural measure that is lower in older adults and predicts these individual differences is moment-to-moment brain signal variability. Testing the assumption that GABA should heighten neural variability, we examined whether reduced brain signal variability in older, poorer performing adults could be boosted by increasing GABA pharmacologically. Brain signal variability was estimated using fMRI in 20 young and 24 older healthy human adults during placebo and GABA agonist sessions. As expected, older adults exhibited lower signal variability at placebo, and, crucially, GABA agonism boosted older adults' variability to the levels of young adults. Furthermore, poorer performing older adults experienced a greater increase in variability on drug, suggesting that those with more to gain benefit the most from GABA system potentiation. GABA may thus serve as a core neurochemical target in future work on aging- and cognition-related human brain dynamics.SIGNIFICANCE STATEMENT Prior research indicates that moment-to-moment brain signal variability is lower in older, poorer performing adults. We found that this reduced brain signal variability could be boosted through GABA agonism in older adults to the levels of young adults and that this boost was largest in the poorer performing older adults. These results provide the first evidence that brain signal variability can be restored by increasing GABAergic activity and suggest the promise of developing interventions targeting inhibitory systems to help slow cognitive declines in healthy aging.

Novel Therapeutics for Neonatal Seizures.

Neonatal seizures are a common neurologic emergency for which therapies have not significantly changed in decades. Improvements in diagnosis and pathophysiologic understanding of the distinct features of acute symptomatic seizures and neonatal-onset epilepsies present exceptional opportunities for development of precision therapies with potential to improve outcomes. Herein, we discuss the pathophysiology of neonatal seizures and review the evidence for currently available treatment. We present emerging therapies in clinical and preclinical development for the treatment of acute symptomatic neonatal seizures. Lastly, we discuss the role of precision therapies for genetic neonatal-onset epilepsies and address barriers and goals for developing new therapies for clinical care.

Stigmasterol can be new steroidal drug for neurological disorders: Evidence of the GABAergic mechanism via receptor modulation.

BACKGROUND: Gamma-aminobutyric acid A (GABAA) receptors have been implicated in anxiety and epileptic disorders. HYPOTHESIS/PURPOSE: This study aimed to investigate the effects of stigmasterol, a plant sterol (phytosterol) isolated from Artemisia indica Linn on neurological disorders. METHODS: Stigmasterol was evaluated on various recombinant GABAA receptor subtypes expressed in Xenopus laevis oocytes and its anxiolytic and anticonvulsant potential was assessed using the elevated plus maze (EPM), light-dark box (LDB) test, and pentylenetetrazole- (PTZ-) induced seizure paradigms. Furthermore, computational modeling of α2ß2γ2L, α4ß3δ, and α4ß3 subtypes was performed to gain insights into the GABAergic mechanism of stigmasterol. For the first time, a model of GABAδ subtype was generated. Stigmasterol was targeted to all the binding sites (neurotransmitters, positive and negative modulator binding sites) of GABAA α2ß2γ2L, α4ß3, and α4ß3δ complexes by in silico docking. RESULTS: Stigmasterol enhanced GABA-induced currents at ternary α2ß2γ2L, α4ß3δ, and binary α4ß3 GABAAR subtypes. The potentiation of GABA-induced currents at extrasynaptic α4ß3δ was significantly higher compared to the binary α4ß3 subtype, indicating that the δ subunit is important for efficacy. Stigmasterol was found to be a potent positive modulator of the extrasynaptic α4ß3δ subtype, which was also confirmed by computational analysis. The computational analysis reveals that stigmasterol preferentially binds at the transmembrane region shared by positive modulators or a binding site constituted by the M2-M3 region of α4 and M1-M2 of ß3 at α4ß3δ complex. In in vivo studies, Stigmasterol (0.5-3.0 mg/kg, i.p.) exerted significant anxiolytic and anticonvulsant effects in an identical manner of allopregnanolone, indicating the involvement of a GABAergic mechanism. CONCLUSION: To our knowledge, this is the first study reporting the positive modulation of GABAA receptors, anxiolytic and anticonvulsant potential of stigmasterol. Thus, stigmasterol is considered to be a candidate steroidal drug for the treatment of neurological disorders due to its positive modulation of GABA receptors.

The efficacy of γ-aminobutyric acid type A receptor (GABA AR) subtype-selective positive allosteric modulators in blocking tetramethylenedisulfotetramine (TETS)-induced seizure-like behavior in larval zebrafish with minimal sedation.

The chemical threat agent tetramethylenedisulfotetramine (TETS) is a γ-aminobutyric acid type A receptor (GABA AR) antagonist that causes life threatening seizures. Currently, there is no specific antidote for TETS intoxication. TETS-induced seizures are typically treated with benzodiazepines, which function as nonselective positive allosteric modulators (PAMs) of synaptic GABAARs. The major target of TETS was recently identified as the GABAAR α2ß3γ2 subtype in electrophysiological studies using recombinantly expressed receptor combinations. Here, we tested whether these in vitro findings translate in vivo by comparing the efficacy of GABAAR subunit-selective PAMs in reducing TETS-induced seizure behavior in larval zebrafish. We tested PAMs targeting α1, α2, α2/3/5, α6, ß2/3, ß1/2/3, and δ subunits and compared their efficacy to the benzodiazepine midazolam (MDZ). The data demonstrate that α2- and α6-selective PAMs (SL-651,498 and SB-205384, respectively) were effective at mitigating TETS-induced seizure-like behavior. Combinations of SB-205384 and MDZ or SL-651,498 and 2-261 (ß2/3-selective) mitigated TETS-induced seizure-like behavior at concentrations that did not elicit sedating effects in a photomotor behavioral assay, whereas MDZ alone caused sedation at the concentration required to stop seizure behavior. Isobologram analyses suggested that SB-205384 and MDZ interacted in an antagonistic fashion, while the effects of SL-651,498 and 2-261 were additive. These results further elucidate the molecular mechanism by which TETS induces seizures and provide mechanistic insight regarding specific countermeasures against this chemical convulsant.

Clonazepam attenuates neurobehavioral abnormalities in offspring exposed to maternal immune activation by enhancing GABAergic neurotransmission.

Ample evidence indicates that maternal immune activation (MIA) during gestation is linked to an increased risk for neurodevelopmental and psychiatric disorders, such as autism spectrum disorder (ASD), anxiety and depression, in offspring. However, the underlying mechanism for such a link remains largely elusive. Here, we performed RNA sequencing (RNA-seq) to examine the transcriptional profiles changes in mice in response to MIA and identified that the expression of Scn1a gene, encoding the pore-forming α-subunit of the brain voltage-gated sodium channel type-1 (NaV1.1) primarily in fast-spiking inhibitory interneurons, was significantly decreased in the medial prefrontal cortex (mPFC) of juvenile offspring after MIA. Moreover, diminished excitatory drive onto interneurons causes reduction of spontaneous gamma-aminobutyric acid (GABA)ergic neurotransmission in the mPFC of MIA offspring, leading to hyperactivity in this brain region. Remarkably, treatment with low-dose benzodiazepines clonazepam, an agonist of GABAA receptors, completely prevented the behavioral abnormalities, including stereotypies, social deficits, anxiety- and depression-like behavior, via increasing inhibitory neurotransmission as well as decreasing neural activity in the mPFC of MIA offspring. Our results demonstrate that decreased expression of NaV1.1 in the mPFC leads to abnormalities in maternal inflammation-related behaviors and provides a potential therapeutic strategy for the abnormal behavioral phenotypes observed in the offspring exposed to MIA.

Effect of Zuranolone vs Placebo in Postpartum Depression: A Randomized Clinical Trial.

Importance: Postpartum depression (PPD) is one of the most common medical complications during and after pregnancy, negatively affecting both mother and child. Objective: To demonstrate the efficacy and safety of zuranolone, a neuroactive steroid γ-aminobutyric acid receptor-positive allosteric modulator, in PPD. Design, Setting, and Participants: This phase 3, double-blind, randomized, outpatient, placebo-controlled clinical trial was conducted between January 2017 and December 2018 in 27 enrolling US sites. Participant were women aged 18 to 45 years, 6 months or fewer post partum, with PPD (major depressive episode beginning third trimester or ≤4 weeks postdelivery), and baseline 17-item Hamilton Rating Scale for Depression (HAMD-17) score of 26 or higher. Analysis was intention to treat and began December 2018 and ended March 2019. Interventions: Randomization 1:1 to placebo:zuranolone, 30 mg, administered orally each evening for 2 weeks. Main Outcomes and Measures: Primary end point was change from baseline in HAMD-17 score for zuranolone vs placebo at day 15. Secondary end points included changes from baseline in HAMD-17 total score at other time points, HAMD-17 response (≥50% score reduction) and remission (score ≤7) rates, Montgomery-Åsberg Depression Rating Scale score, and Hamilton Rating Scale for Anxiety score. Safety was assessed by adverse events and clinical assessments. Results: Of 153 randomized patients, the efficacy set comprised 150 patients (mean [SD] age, 28.3 [5.4] years), and 148 (98.7%) completed treatment. A total of 76 patients were randomized to placebo, and 77 were randomized to zuranolone, 30 mg. Zuranolone demonstrated significant day 15 HAMD-17 score improvements from baseline vs placebo (-17.8 vs -13.6; difference, -4.2; 95% CI, -6.9 to -1.5; P = .003). Sustained differences in HAMD-17 scores favoring zuranolone were observed from day 3 (difference, -2.7; 95% CI, -5.1 to -0.3; P = .03) through day 45 (difference, -4.1; 95% CI, -6.7 to -1.4; P = .003). Sustained differences at day 15 favoring zuranolone were observed in HAMD-17 response (odds ratio, 2.63; 95% CI, 1.34-5.16; P = .005), HAMD-17 score remission (odds ratio, 2.53; 95% CI, 1.24-5.17; P = .01), change from baseline for Montgomery-Åsberg Depression Rating Scale score (difference, -4.6; 95% CI, -8.3 to -0.8; P = .02), and Hamilton Rating Scale for Anxiety score (difference, -3.9; 95% CI, -6.7 to -1.1; P = .006). One patient per group experienced a serious adverse event (confusional state in the zuranolone group and pancreatitis in the placebo group). One patient in the zuranolone group discontinued because of an adverse event vs none for placebo. Conclusions and Relevance: In this randomized clinical trial, zuranolone improved the core symptoms of depression as measured by HAMD-17 scores in women with PPD and was generally well tolerated, supporting further development of zuranolone in the treatment of PPD. Trial Registration: ClinicalTrials.gov Identifier: NCT02978326.

GABAA Receptors and Maternally Derived Taurine Regulate the Temporal Specification of Progenitors of Excitatory Glutamatergic Neurons in the Mouse Developing Cortex.

Temporal specification of the neural progenitors (NPs) producing excitatory glutamatergic neurons is essential for histogenesis of the cerebral cortex. Neuroepithelial cells, the primary NPs, transit to radial glia (RG). To coincide with the transition, NPs start to differentiate into neurons, undergoing a switch from symmetric to asymmetric cell division. After the onset of neurogenesis, NPs produce layer-specific neurons in a defined order with precise timing. Here, we show that GABAA receptors (GABAARs) and taurine are involved in this regulatory mechanism. Foetal exposure to GABAAR-antagonists suppressed the transition to RG, switch to asymmetric division, and differentiation into upper-layer neurons. Foetal exposure to GABAAR-agonists caused the opposite effects. Mammalian foetuses are dependent on taurine derived from the mothers. GABA and taurine function as endogenous ligands for GABAARs. Ca2+ imaging showed that NPs principally responded to taurine but not GABA before E13. The histological phenotypes of the taurine transporter knockout mice resembled those of the mice foetally exposed to GABAAR-antagonists. Foetal exposure to GABAAR-modulators resulted in considerable alterations in offspring behavior like core symptoms of autism. These results show that taurine regulates the temporal specification of NPs and that disrupting the taurine-receptor interaction possibly leads to neurodevelopmental disorders.

A honokiol-enriched Magnolia officinalis Rehder & E.H. Wilson. bark extract possesses anxiolytic-like activity with neuroprotective effect through the modulation of CB1 receptor.

OBJECTIVES: The exposure of neurons to an excessive excitatory stimulation induces the alteration of the normal neuronal function. Mood disorders are among the first signs of alterations in the central nervous system function. Magnolia officinalis bark extract has been extensively used in the traditional medicine systems of several countries, showing several pharmacological activities. Honokiol, the main constituent of M. officinalis, is a GABA modulator and a CB1 agonist, which is deeply investigated for its role in modulating mood disorders. METHODS: Thus, we evaluated the possible neuroprotective effect of a standardized M. officinalis bark extract (MOE), enriched in honokiol, and its effect on animal mood behavioural tests and in an in vitro model of excitotoxicity. KEY FINDINGS: MOE showed neuroprotective effect using SH-SY5Y cells, by normalizing brain-derived neurotrophic factor release. Then, we tested the effect of MOE in different behavioural tests evaluating anxiety and depression and we observed a selective anxiolytic-like effect. Finally, we confirmed the involvement of CB1 in the final effect of MOE by the co-administration of the CB1 antagonist, AM251. CONCLUSION: These results suggest that MOE could be considered an effective and safe anxiolytic candidate with neuroprotective activity.

COR758, a negative allosteric modulator of GABAB receptors.

Allosteric modulators of G protein coupled receptors (GPCRs), including GABABRs (GABABRs), are promising therapeutic candidates. While several positive allosteric modulators (PAM) of GABABRs have been characterized, only recently the first negative allosteric modulator (NAM) has been described. In the present study, we report the characterization of COR758, which acts as GABABR NAM in rat cortical membranes and CHO cells stably expressing GABABRs (CHO-GABAB). COR758 failed to displace the antagonist [3H]CGP54626 from the orthosteric binding site of GABABRs showing that it acts through an allosteric binding site. Docking studies revealed a possible new allosteric binding site for COR758 in the intrahelical pocket of the GABAB1 monomer. COR758 inhibited basal and GABABR-stimulated O-(3-[35Sthio)-triphosphate ([35S]GTPγS) binding in brain membranes and blocked the enhancement of GABABR-stimulated [35S]GTPγS binding by the PAM GS39783. Bioluminescent resonance energy transfer (BRET) measurements in CHO-GABAB cells showed that COR758 inhibited G protein activation by GABA and altered GABABR subunit rearrangements. Additionally, the compound altered GABABR-mediated signaling such as baclofen-induced inhibition of cAMP production in transfected HEK293 cells, agonist-induced Ca2+ mobilization as well as baclofen and the ago-PAM CGP7930 induced phosphorylation of extracellular signal-regulated kinases (ERK1/2) in CHO-GABAB cells. COR758 also prevented baclofen-induced outward currents recorded from rat dopamine neurons, substantiating its property as a NAM for GABABRs. Altogether, these data indicate that COR758 inhibits G protein signaling by GABABRs, likely by interacting with an allosteric binding-site. Therefore, COR758 might serve as a scaffold to develop additional NAMs for therapeutic intervention.

Tertiary Oxidation of Deoxycholate Is Predictive of CYP3A Activity in Dogs.

Deoxycholic acid (DCA, 3α, 12α-dihydroxy-5ß-cholan-24-oic acid) is the major circulating secondary bile acid, which is synthesized by gut flora in the lower gut and selectively oxidized by CYP3A into tertiary metabolites, including 1ß,3α,12α-trihydroxy-5ß-cholan-24-oic acid (DCA-1ß-ol) and 3α,5ß,12α-trihydroxy-5ß-cholan-24-oic acid (DCA-5ß-ol) in humans. Since DCA has the similar exogenous nature and disposition mechanisms as xenobiotics, this work aimed to investigate whether the tertiary oxidations of DCA are predictive of in vivo CYP3A activities in beagle dogs. In vitro metabolism of midazolam (MDZ) and DCA in recombinant canine CYP1A1, 1A2, 2B11, 2C21, 2C41, 2D15, 3A12, and 3A26 enzymes clarified that CYP3A12 was primarily responsible for either the oxidation elimination of MDZ or the regioselective oxidation metabolism of DCA into DCA-1ß-ol and DCA-5ß-ol in dog liver microsomes. Six male dogs completed the CYP3A intervention studies including phases of baseline, inhibition (ketoconazole treatments), recovery, and induction (rifampicin treatments). The oral MDZ clearance after a single dose was determined on the last day of the baseline, inhibition, and induction phases, and subjected to correlation analysis with the tertiary oxidation ratios of DCA detected in serum and urine samples. The results confirmed that the predosing serum ratios of DCA oxidation, DCA-5ß-ol/DCA, and DCA-1ß-ol/DCA were significantly and positively correlated both intraindividually and interindividually with oral MDZ clearance. It was therefore concluded that the tertiary oxidation of DCA is predictive of CYP3A activity in beagle dogs. Clinical transitional studies following the preclinical evidence are promising to provide novel biomarkers of the enterohepatic CYP3A activities. SIGNIFICANCE STATEMENT: Drug development, clinical pharmacology, and therapeutics are under insistent demands of endogenous CYP3A biomarkers that avoid unnecessary drug exposure and invasive sampling. This work has provided the first proof-of-concept preclinical evidence that the CYP3A catalyzed tertiary oxidation of deoxycholate, the major circulating secondary bile acid synthesized in the lower gut by bacteria, may be developed as novel in vivo biomarkers of the enterohepatic CYP3A activities.

Anxiolytic-like effect of brominated compounds from the marine sponge Aplysina fulva on adult zebrafish (Danio rerio): Involvement of the GABAergic system.

Benzodiazepines are commonly used to treat disorders of the central nervous system, including anxiety. However, due to their adverse effects, there is a continuing interest in discovering new safe and effective drugs. Marine natural products have emerged as a prolific source of bioactive nitrogenated compounds. Aiming to discover new biologically active natural compounds, the marine sponge Aplysina fulva, a nitrogen-bearing heterocyst producer, was investigated. The main isolated compounds (4, 6, and 9) were evaluated on adult zebrafish (Danio rerio). A group of fishes (n = 6) was preliminarily subjected to acute toxicity, and open field tests using 0.1, 0.5, and 1.0 mg/mL (v. o.) of those compounds was performed. The anxiolytic effect was further investigated in the light/dark assay based on the locomotor response at zebrafish. Interactions through the GABAergic system were investigated using flumazenil, a silent modulator of GABA receptors. To improve the results, a study of molecular docking using the GABAA receptor also was performed. Based on the results, the bromotyrosine derivative compounds 4, 6, and 9 exhibited anxiolytic-like effects mediated by the GABAergic system.

Vasodilatory effects of a variety of positive allosteric modulators of GABAA receptors on rat thoracic aorta.

Different subtypes of GABAA (gamma-aminobutyric acid A) receptors, through their specific regional and cellular localization, are involved in the manifestation of various functions, both at the central and peripheral levels. We hypothesized that various non-neuronal GABAA receptors are expressed on blood vessels, through which positive allosteric modulators of GABAA receptors exhibit vasodilatory effects. This study involved two parts: one to determine the presence of α1-6 subunit GABAA receptor mRNAs in the rat thoracic aorta, and the other to determine the vasoactivity of the various selective and non-selective positive GABAA receptor modulators: zolpidem (α1-selective), XHe-III-074 (α4-selective), MP-III-022 (α5-selective), DK-I-56-1 (α6-selective), SH-I-048A and diazepam (non-selective). Reverse transcription-polymerase chain reaction (RT-PCR) analysis data demonstrated for the first time the expression of α1, α2, α3, α4 and α5 subunits in the rat thoracic aorta tissue. Tissue bath assays on isolated rat aortic rings revealed significant vasodilatory effects of diazepam, SH-I-048A, XHe-III-074, MP-III-022 and DK-I-56-1, all in terms of achieved relaxations (over 50% of relative tension decrease), as well as in terms of preventive effects on phenylephrine (PE) contraction. Diazepam was the most efficient ligand in the present study, while zolpidem showed the weakest vascular effects. In addition, diazepam-induced relaxations in the presence of antagonists PK11195 or bicuculline were significantly reduced (P < 0.001 and P < 0.05, respectively) at lower concentrations of diazepam (10-7 M and 3 × 10-7 M). The present work suggests that the observed vasoactivity is due to modulation of "vascular" GABAA receptors, which after further detailed research may provide a therapeutic target.

Pharmacological uses of flumazenil in benzodiazepine use disorders: a systematic review of limited data.

BACKGROUND: The estimated annual prevalence of drug use disorders is as high as 3%, underpinning the need to continually develop more effective treatments. Central nervous system dysregulation, contributing to acute and post-withdrawal syndromes, has traditionally been managed with benzodiazepines; however, a small but growing body of data indicate that the GABAA receptor antagonist, flumazenil, may offer some advantages over traditional management. AIM: To review the literature on the safety and efficacy of flumazenil in benzodiazepine use disorders and identify gaps in the literature. METHOD: A systematic method was used to identify randomised control trials. Where randomised control trials existed, non-randomised control trials were included to supplement findings. RESULTS: Eleven flumazenil trials were included with varying doses, frequencies and routes of administration. The evidence for flumazenil alone showed generally a reduction in withdrawal symptoms with the exception of one study where withdrawal symptoms initially increased. Flumazenil plus benzodiazepine tapering was assessed in one randomised control trial and a series of non-randomised control trials. Randomised control trial results showed that flumazenil plus benzodiazepine tapering was superior at reducing withdrawal symptoms compared to benzodiazepine tapering alone and placebo. Flumazenil was associated with no serious adverse events; however there remains a risk of seizures. CONCLUSION: Although flumazenil shows promising efficacy in the management of benzodiazepine use disorders and withdrawal, more randomized control trials are required before a definitive recommendation can be made around its use.