Positive Modulator Of GABAA Receptors Research Articles

Alcohol use disorder and sleep disturbances: a feed-forward allostatic framework.

The development of alcohol use disorder (AUD) involves binge or heavydrinking to high levels of intoxication that leads to compulsive intake, the loss of control in limiting intake, and a negative emotional state when alcohol is removed. This cascade of events occurs over an extended period within a three-stage cycle: binge/intoxication, withdrawal/negative affect, and preoccupation/anticipation. These threeheuristic stages map onto the dysregulation of functional domains of incentive salience/habits, negative emotional states, and executive function, mediated by the basal ganglia, extended amygdala, and frontal cortex, respectively. Sleep disturbances, alterations of sleep architecture, and the development of insomnia are ubiquitous in AUD and also map onto the three stages of the addiction cycle. During the binge/intoxication stage, alcohol intoxication leads to a faster sleep onset, but sleep quality is poor relative to nights when no alcohol is consumed. The reduction of sleep onset latency and increase in wakefulness later in the night may be related to the acute effects of alcohol on GABAergic systems that are associated with sleep regulation and the effects on brain incentive salience systems, such as dopamine. During the withdrawal/negative affect stage, there is a decrease in slow-wave sleep and some limited recovery in REM sleep when individuals with AUD stop drinking. Limited recovery of sleep disturbances is seen in AUD within the first 30 days of abstinence. The effects of withdrawal on sleep may be related to the loss of alcohol as a positive allosteric modulator of GABAA receptors, a decrease in dopamine function, and the overactivation of stress neuromodulators, including hypocretin/orexin, norepinephrine, corticotropin-releasing factor, and cytokines. During the preoccupation/anticipation stage, individuals with AUD who are abstinent long-term present persistent sleep disturbances, including a longer latency to fall asleep, more time awake during the night, a decrease in slow-wave sleep, decreases in delta electroencephalogram power and evoked delta activity, and an increase in REM sleep. Glutamatergic system dysregulation that is observed in AUD is a likely substrate for some of these persistent sleep disturbances. Sleep pathology contributes to AUD pathology, and vice versa, possibly as a feed-forward drive to an unrecognized allostatic load that drives the addiction process.

Isoallopregnanolone reduces tic-like behaviours in the D1CT-7 mouse model of Tourette syndrome.

Tourette syndrome (TS) is a neuropsychiatric disorder characterised by multiple, persistent tics. These semi-voluntary motor and phonic manifestations are typically aggravated by exposure to acute stress, yet the mechanisms underlying this exacerbation remain unclear. Using a well-characterised animal model of TS, the D1CT-7 mouse, we recently showed that acute stress increases tic-like responses and causes sensorimotor gating deficits, as measured by the prepulse inhibition of the startle. We showed that these effects are promoted by the brain synthesis of the neurosteroid allopregnanolone (AP). In line with this idea, inhibition of AP synthesis by finasteride was found to suppress the tic-exacerbating effects of stress; conversely, AP administration resulted in a marked enhancement of the number of tic-like motor bursts. Given that the primary mechanism of AP is based on the positive allosteric modulation of GABAA receptors, in the present study, we hypothesised that the enhancement in tic-like behaviours induced by either stress or AP may be countered by isoallopregnanolone (isoAP), the natural 3β-epimer of AP that acts as an antagonist to the AP-binding site within GABAA receptors. In agreement with our hypothesis, isoAP (5-10mgkg-1 , s.c.) dose-dependently reduced the number of tic-like behaviours induced by stress in D1CT-7 mice. These effects were comparable to those elicited by both the benchmark TS therapy haloperidol (0.3mgkg-1 , i.p.), as well as finasteride (25mgkg-1 , i.p.). IsoAP also countered the prepulse inhibition deficits secondary to stress in D1CT-7 mice. Finally, isoAP opposed the enhancement of tic-like behaviours induced by AP (15mgkg-1 , i.p.). Given that isoAP is well-tolerated and has an optimal safety profile, these data suggest that this steroid may have therapeutic properties in TS.

Nectandra grandiflora essential oil and its isolated sesquiterpenoids minimize anxiety-related behaviors in mice through GABAergic mechanisms

Nectandra grandiflora Ness (Lauraceae) essential oil (EO) main constituent, the sesquiterpenoid (+)-dehydrofukinone (DHF), has sedative and anticonvulsant effects through GABAergic mechanisms. Other DHF-related sesquiterpenoids have been identified in the EO, such as, dehydrofukinone epoxide (DFX), eremophil-11-en-10-ol (ERM) and selin-11-en-4-α-ol (SEL). However, the neuronal effects of these compounds in mammals remain unknown. Therefore, the aim of this study was to evaluate the anxiolytic potential of the N. grandiflora EO and the isolated compounds in in mice. For this purpose, mice were administered orally with vehicle, 10, 30 or 100 mg/kg EO, DHF, DFX, ERM or SEL or 1 mg/kg diazepam. Locomotion and ethological parameters in the open field (OF) and elevated plus maze (EPM) were recorded. We also examined the effect of DFX, ERM and SEL on the membrane potential and calcium influx in synaptosomes, and the presence of the compounds in the cortical tissue using gas chromatography. EOs and isolated compounds reduced anxiety-related parameters in the EPM (open arms time and entries, end activity, head dipping) and OF (center time and entries, total rearing, unprotected rearing, sniffing, grooming) without alter ambulation or induce sedation. Flumazenil (2 mg/kg, i.p.) altered the anxiolytic-like effect of all treatments and vanished the DFX, ERM and SEL-induced changes in membrane potential. However, FMZ did not blocked the DFX-, ERM- and SEL-induced inhibition of calcium influx. Therefore, our results suggest that N. grandiflora EO and isolated compounds induced anxiolytic-like effect in mice due to positive modulation of GABAa receptors and/or inhibition of neuronal calcium influx.

T117. Pro-Cognitive Properties of a Novel GABA-A Receptor Positive Modulator in Animal Models of Depression and Aging

T117. Pro-Cognitive Properties of a Novel GABA-A Receptor Positive Modulator in Animal Models of Depression and Aging

Kami-shoyo-san ameliorates sociability deficits in ovariectomized mice, a putative female model of autism spectrum disorder, via facilitating dopamine D1 and GABAA receptor functions

Ethnopharmacological relevanceKami-shoyo-san (KSS) is a Kampo formula used clinically for menopause-related symptoms in Japan. However, the effect of KSS on autism spectrum disorder (ASD), a developmental disorder with a higher prevalence in males than in females, has not been reported yet. Aim of the studyIt is accepted generally that dysfunction in the GABAergic system is associated with pathogenesis of ASD. In our previous study, a decrease in brain allopregnanolone (ALLO), a positive allosteric GABAA receptor modulator, induced ASD-like symptoms such as impaired sociability-related performance and increased repetitive self-grooming behavior in male mice, and that KSS ameliorated these behavioral abnormalities via GABAA receptor- and dopamine D1 receptor-mediated mechanisms. In this study, to better understand a gender difference in the prevalence of ASD, we examined whether dissection of ovary (OVX), a major organ secreting progesterone in females, causes ASD-like behaviors in a manner dependent on brain ALLO levels, and if so, how KSS affects the behaviors. Materials and methodsSix-week-old ICR female mice received ovariectomy, and KSS (74 mg/kg and 222 mg/kg, p.o.) were treated before 1 h starting each behavioral test. The sociability, social anxiety-like behavior, and self-grooming behavior were analyzed by the resident-intruder test, mirror chamber test, and open field test, respectively. After finishing the behavioral experiment, the ALLO content in the brain was measured by ELISA. Furthermore, we examined the effects of OVX on the neuro-signaling pathways in the prefrontal cortex and striatum by Western blotting. ResultsThe results revealed that OVX induced sociability deficits and social anxiety-related behaviors, but not repetitive self-grooming behavior, and that these behavioral changes were accompanied not only by a decrease of brain ALLO levels, but also by impairment of CREB- and CaMKIIα-mediated neuro-signaling in the prefrontal cortex. Moreover, the administration of KSS had no effect on the brain ALLO level, but significantly ameliorated the OVX-induced behavioral and neurochemical changes via facilitation of GABAA receptor and dopamine D1 receptor-mediated neurotransmission. ConclusionsThese findings suggest that a decrease in gonadal hormone-derived ALLO plays a major role in ASD-like behaviors in female mice and that KSS is beneficial for the treatment of ASD in females.

Neuroactive Steroids and GABAergic Involvement in the Neuroendocrine Dysfunction Associated With Major Depressive Disorder and Postpartum Depression

Stress and previous adverse life events are well-established risk factors for depression. Further, neuroendocrine disruptions are associated with both major depressive disorder (MDD) and postpartum depression (PPD). However, the mechanisms whereby stress contributes to the underlying neurobiology of depression remains poorly understood. The hypothalamic-pituitary-adrenal (HPA) axis, which mediates the body’s neuroendocrine response to stress, is tightly controlled by GABAergic signaling and there is accumulating evidence that GABAergic dysfunction contributes to the impact of stress on depression. GABAergic signaling plays a critical role in the neurobiological effects of stress, not only by tightly controlling the activity of the HPA axis, but also mediating stress effects in stress-related brain regions. Deficits in neuroactive steroids and neurosteroids, some of which are positive allosteric modulators of GABAA receptors (GABAARs), such as allopregnanolone and THDOC, have also been implicated in MDD and PPD, further supporting a role for GABAergic signaling in depression. Alterations in neurosteroid levels and GABAergic signaling are implicated as potential contributing factors to neuroendocrine dysfunction and vulnerability to MDD and PPD. Further, potential novel treatment strategies targeting these proposed underlying neurobiological mechanisms are discussed. The evidence summarized in the current review supports the notion that MDD and PPD are stress-related psychiatric disorders involving neurosteroids and GABAergic dysfunction.

The importance of being specific

In this issue, you will find a paper by Vasović et al., entitled “Trigeminal neuropathic pain development and maintenance in rats are suppressed by a positive modulator of α6GABAA receptors” (Vasović et al., 2019). Loss of GABA-mediated inhibition within pain circuits is one of the mechanisms commonly invoked to explain allodynia and hyperalgesia following injury. Intrathecal administration of picrotoxin produces pain like signs. Positive allosteric modulators of GABAA receptors like benzodiazepines are known to produce analgesia although their main effects are sedative, hypnotic and anxiolytic, and therefore, their use as analgesics is curtailed (Schliessbach et al., 2017). GABAA receptors mediate fast neuronal inhibition, and they are widely expressed in the CNS modulating all sorts of functions. However, their structure is heterogeneous. They are formed by the ensemble of typically 5 protein subunits out of 19 possible with certain rules for combination. Structural heterogeneity opens up the door to designing new specific compounds targeting single receptor subunits thus binding to a subset of GABAA receptors and modulating a reduced range of functions or ideally a single function, like pain signalling. The strategy of developing specific pharmacological tools to interact with single GABA receptor subunits has been explored before (Munro, Hansen, & Mirza, 2013). Alpha5 preferring compounds have been shown to have potential for analgesia using animal models of inflammatory and neuropathic pain while having relatively low impact on other functions. However, the alpha 6 subunit does not contain a benzodiazepine site and it is insensitive to this class of compounds. The PAM activity of pyrazoloquinolinones at diazepam insensitive GABAA receptors has been studied since the 90s, and recently, it has been shown that certain drugs of this class can interact specifically with alpha6-containing receptors to potentiate chloride flow upon receptor activation (Treven et al., 2018). The commented article uses two pyrazoloquinolinones, one of which is active at modulating alpha6-containing GABA receptors (DK-I-56-1) and the other is inactive and serves as a control. The authors present electrophysiological proof for the PAM activity of DK-I-56-1 on receptors containing the alpha6 subunit. They also perform pharmacokinetic studies to show that this compound crosses the blood–brain barrier and has a good enough metabolic stability to ensure pharmacologically relevant levels of the compound during daily IP injections of the compound. Finally, they show that subchronic treatment with this compound produces clear signs of analgesia in rats that have undergone a lesion to the infraorbital nerve (a model of trigeminal neuropathic pain). Interestingly, these analgesic effects occur in the absence of ethologic signs of sedation and are not produced by the inactive compound. As a proof of mechanism, the results of this paper are very promising. This is a great job done! Unfortunately, this is only the beginning of a long and complicated road to success. GABA receptors containing alpha6 subunits are not exclusive of trigeminal ganglia. They are present in cerebellar granule cells and spinal motor neurons were they may mediate sensorimotor processing, in hippocampal interneurons perhaps modulating certain forms of depression and in other nuclei of the CNS mediating a variety of functions. Therefore, the potential use of this chemical family of compounds to produce analgesics for specific pain conditions, such as trigeminal neuropathy, is just starting to be analysed. On rational grounds, the strategy of developing new psychoactive compounds with highly specific profiles is definitively good, probably the best. The questions open to the future are how specific can we get and how specific is the linkage between receptor structure and body function. *In this issue, you will find a paper by Vasović et al.: ‘Trigeminal neuropathic pain development and maintenance in rats are suppressed by a positive modulator of α6GABAA receptors’. Eur J Pain, 23, 973-984

The α2,3-selective potentiator of GABAA receptors, KRM-II-81, reduces nociceptive-associated behaviors induced by formalin and spinal nerve ligation in rats

Clinical evidence indicates that positive allosteric modulators (PAMs) of GABAA receptors have analgesic benefit in addition to efficacy in anxiety disorders. However, the utility of GABAA receptor PAMs as analgesics is compromised by the central nervous system side effects of non-selective potentiators. A selective potentiator of GABAA receptors associated with α2/3 subunits, KRM-II-81(5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepin-3-yl)oxazole), has demonstrated anxiolytic, anticonvulsant, and antinociceptive effects in rodents with reduced motoric side effects. The present study evaluated the potential of KRM-II-81 as a novel analgesic. Oral administration of KRM-II-81 attenuated formalin-induced flinching; in contrast, diazepam was not active. KRM-II-81 attenuated nociceptive-associated behaviors engendered by chronic spinal nerve ligation (L5/L6). Diazepam decreased locomotion of rats at the dose tested in the formalin assay (10 mg/kg) whereas KRM-II-81 produced small decreases that were not dose-dependent (10–100 mg/kg). Plasma and brain levels of KRM-II-81 were used to demonstrate selectivity for α2/3- over α1-associated GABAA receptors and to define the degree of engagement of these receptors. Plasma and brain concentrations of KRM-II-81 were positively-associated with analgesic efficacy. GABA currents from isolated rat dorsal-root ganglion cultures were potentiated by KRM-II-81 with an ED50 of 32 nM. Measures of respiratory depression were reduced by alprazolam whereas KRM-II-81 was either inactive or produced effects with lower potency and efficacy. These findings add to the growing body of data supporting the idea that α2/3-selective GABAA receptor PAMs will have efficacy and tolerability as pain medications including those for neuropathic pain. Given their predicted anxiolytic effects, α2/3-selective GABAA receptor PAMs offer an additional inroad into the management of pain.

Positive allosteric modulation of native and recombinant GABAA receptors by hops prenylflavonoids

Hops are a major component of beer that is added during brewing. In addition to its wide range of bioactivity, it exhibits neuroactive properties as a sedative and sleeping aid. The compounds responsible for this activity are yet to be revealed and understood in terms of their pharmacological properties. Here we evaluated the potential of several hops flavonoids in modulating the GABAergic activity and assessed their selectivity to GABAA receptors subtypes. GABA-potentiating effects were measured using [3H]ethynylbicycloorthobenzoate (EBOB) radioligand binding assay in native and recombinant α1β3γ2, α2β3γ2 and α6β3δ receptors expressed in HEK293 cells. Flumazenil sensitivity of GABA-potentiating effects and [3H]Ro 15-4513 binding assay were used to examine the flavonoids binding to benzodiazepine site. The prenylflavonoids xanthohumol (XN), isoxanthohumol (IXN) and 8-prenylnaringenin (8PN) potentiated GABA-induced displacement of [3H]EBOB binding in a concentration-dependent manner. The IC50 for this potentiation in native GABAA receptors were 29.7 µM, 11.6 µM, 7.3 µM, respectively. In recombinant receptors, the sensitivity to prenylflavonoid potentiation of GABA-induced displacement of [3H]EBOB binding followed the order α6β3δ > α2β3γ2 > α1β3γ2 with the strongest inhibition observed by 8PN in α6β3δ (IC50 = 3.6 μM). Flumazenil had no significant effect on the prenylflavonoid-induced displacement of [3H]EBOB binding and [3H]Ro 15-4513 displacement from native GABAA receptors was only detected at high micromolar concentrations (100 µM). We identified potent prenylflavonoids in hops that positively modulate GABA-induced responses in native and αβγ/δ recombinant GABAA receptors at low micromolar concentrations. These GABAergic modulatory effects were not mediated via the high-affinity benzodiazepine binding site.

Zolpidem Activation of Alpha 1-Containing GABAA Receptors Selectively Inhibits High Frequency Action Potential Firing of Cortical Neurons.

Introduction: High frequency neuronal activity in the cerebral cortex can be induced by noxious stimulation during surgery, brain injury or poisoning. In this scenario, it is essential to block cortical hyperactivity to protect the brain against damage, e.g., by using drugs that act as positive allosteric modulators at GABAA receptors. Yet, cortical neurons express multiple, functionally distinct GABAA receptor subtypes. Currently there is a lack of knowledge which GABAA receptor subtypes would be a good pharmacological target to reduce extensive cortical activity.Methods: Spontaneous action potential activity was monitored by performing extracellular recordings from organotypic neocortical slice cultures of wild type and GABAAR-α1(H101R) mutant mice. Phases of high neuronal activity were characterized using peri-event time histograms. Drug effects on within-up state firing rates were quantified via Hedges’ g.Results: We quantified the effects of zolpidem, a positive modulator of GABAA receptors harboring α1-subunits, and the experimental benzodiazepine SH-053-2′F-S-CH3, which preferably acts at α2/3/5- but spares α1-subunits. Both agents decreased spontaneous action potential activity but altered the firing patterns in different ways. Zolpidem reduced action potential firing during highly active network states. This action was abolished by flumazenil, suggesting that it was mediated by benzodiazepine-sensitive GABAA receptors. SH-053-2′F-S-CH3 also attenuated neuronal activity, but unlike zolpidem, failed to reduce high frequency firing. To confirm that zolpidem actions were indeed mediated via α1-dependent actions, it was evaluated in slices from wild type and α(H101R) knock-in mice. Inhibition of high frequency action potential firing was observed in slices from wild type but not mutant mice.Conclusion: Our results suggest that during episodes of scarce and high neuronal activity action potential firing of cortical neurons is controlled by different GABAA receptor subtypes. Exaggerated firing of cortical neurons is reduced by positive modulation of α1-, but not α2/3/5-subunit containing GABAA receptors.

Death by Opioids: Are there non-addictive scientific solutions?

In the face of the current Opioid crisis in America killing close to 800,000 people since 2004, we are proposing a novel approach to assist in at least attenuating these unwanted premature deaths. While we applaud the wonderful efforts of our governmental institutes and professional societies (NIDA, NIAAA, ASAM, ABAM ) in their extraordinary efforts in combating this continued dilemma, the current approach is failing, and other alternative approaches should at least be tested. These truths present a serious ethical dilemma to scientists, clinicians and counselors in the Reward Deficiency Syndrome (RDS) treatment community. It is important to realize that the current DSM-5 does not actually accurately display the natural brain reward process. The human brain has not been designed to carve out specific drugs like opioids, alcohol, nicotine, cocaine, benzodiazepines or cannabis and process addictions such as gambling as distinct endophenotypes. This is true in spite of natural ligands for cannabinoids, endorphins, or even benzodiazepines. The most accurate endophenotype is indeed reward dysfunction (e.g hypodopaminergic or hyperdopaminergic). With this mind, we are hereby proposing that the current Medication Assisted Treatment (i.e. 'MAT') expands to needed individuals as an initial "Band-Aid" to reduce harm avoidance, with the long-term goal of prophylaxis. So, to be clear, there may be other promising modalities other than MAT such as repetitive transcranial magnetic stimulation (rTMS), exercise and even new medications with positive allosteric modulators of GABA-A receptors, as well as the highly researched Genetic Addiction Risk Score (GARS) coupled with precision KB220Z. This will induce "dopamine homeostasis" to effectively rebalance and restore healthier brain function by promoting the cross talk between various brain regions (e.g. Nucleus accumbens, cingulate gyrus, hippocampus etc.) resulting in dopamine homeostasis. Our laudable goal is to not only save lives, but to redeem joy and improve the quality of life in the recovery community through scientifically sound natural non-addicting alternatives.

86: Brexanolone iv efficacy in postpartum depression in three pivotal trials: montgomery-åsberg depression rating scale assessment

The investigational, positive allosteric modulator of ɣ-aminobutyric acid A (GABAA) receptors, brexanolone iv (BRX), was evaluated for efficacy and safety in women with postpartum depression (PPD) in 3 double-blind, placebo-controlled, randomized trials. BRX met the primary efficacy endpoint with a significantly greater change from baseline in Hamilton Rating Scale for Depression (HAM-D) total score at Hour 60 than placebo (PBO). This abstract presents the results of a secondary measure, the Montgomery-Åsberg Depression Rating Scale (MADRS). An umbrella protocol for the 3 studies allowed integrated dataset analysis. Women aged 18-45 years, ≤ 6 months postpartum, with PPD (i.e. major depressive episode no earlier than third trimester and no later than 4 weeks post-delivery) and a qualifying 17-item HAM-D total score (≥ 26 in Studies A and B; 20-25 in Study C) were enrolled. Randomized subjects received a single, continuous 60-hour infusion of PBO, BRX 60 μg/kg/hour (BRX60), or 90 μg/kg/hour (BRX90) and were followed through Day 30. Change from baseline in MADRS total score (MADRSts) was a secondary endpoint, analyzed by a mixed effects model for repeated measures (BRX90). Safety and tolerability were assessed by adverse event (AE) reporting and clinical laboratory testing (BRX60 and BRX90). Least squares mean (SE) change from baseline in MADRSts at Hour 60 in the PBO (N=107) or BRX90 (N=102) groups was -17.7 (1.2) or -23.5 (1.2) respectively. A significantly greater mean reduction versus PBO was achieved with BRX90 starting at Hour 24 and maintained through Day 7 (Figure). At Day 30, the mean reduction of BRX90 versus PBO was numerically greater. The most common AEs (≥ 10%) reported in all BRX subjects included headache, dizziness, and somnolence. Brexanolone iv was generally well tolerated and showed statistically significant greater reductions of depressive symptoms in women with PPD compared to PBO, as measured by MADRSts. The proposed mechanism of action of brexanolone iv (i.e., positive allosteric modulation of GABAA receptors) is a novel approach in the development of a therapeutic agent for PPD and, if regulatory approval is obtained, has the potential to provide meaningful new treatment options for women with PPD.

850: Evaluation of breast milk concentrations following brexanolone iv administration to healthy lactating women

Brexanolone iv, a proprietary, intravenous formulation of allopregnanolone, is an investigational positive allosteric modulator of GABAA receptors and is under FDA review for the treatment of postpartum depression. The brexanolone iv dose regimen yields plasma concentrations of allopregnanolone that approximate endogenous levels at the end of pregnancy. We evaluated concentrations of allopregnanolone in breast milk and plasma for 7 days after the start of brexanolone iv infusion. This open-label, Phase 1b trial enrolled 12 healthy, lactating women, who were ≤6 months postpartum. Subjects received a 60-hour brexanolone iv infusion, titrated to a maximum of 90 μg/kg/h, as inpatients and were followed through Day 7. Breast milk was collected ad libitum at least every 12 hours through Day 7. Milk samples were retained for analysis. Expressed milk was not given to infants during the study. Plasma and breast milk were analyzed for allopregnanolone concentrations. Brexanolone iv safety and tolerability were monitored throughout the study. Plasma concentrations of allopregnanolone increased during dose titration and declined during dose taper, with an average steady state concentration (Cavg) of 70.56 ng/mL (95% CI 61.8-80.4), a t1/2 of 11.3 hours (95% CI 9.8- 13.1), and an area under the curve to infinity (AUC∞) of 3736.5 ng·h/mL (95% CI 3309.1-4219.1). Breast milk concentrations of allopregnanolone followed a similar pattern and fell below the limit of detection (5 ng/mL) in the majority of the women by approximately 3 days after infusion cessation. Overall pharmacokinetics results showed a rapid equilibrium between milk and plasma. The milk to plasma ratio appeared to be constant over a large range of plasma concentrations, with varying volumes of milk expressed and across subjects. There were no deaths, serious or severe adverse events, or clinically significant changes in safety tests, including laboratory results. Allopregnanolone concentrations were detected in breast milk of women receiving brexanolone iv. Concentrations in milk followed plasma concentrations closely, with rapid decline after the end of the 60-hour infusion to undetectable levels 3 days after the end of the infusion. There was no apparent accumulation of allopregnanolone in plasma or breast milk.

The role of loops B and C in determining the potentiation of GABAA receptors by midazolam.

Many benzodiazepines are positive allosteric modulators (PAMs) of GABAA receptors that cause sedation, hypnosis, and anxiolysis. Benzodiazepines bind GABAA receptors at the extracellular interface of the α and γ subunits. Within the α subunit, the benzodiazepine binding site is defined by three highly conserved structural loops, loops A‐C. Although previous mutagenesis studies have identified His102 in Loop A as important for benzodiazepine modulation of GABAA receptors, the functional roles of many of the other conserved residues in loops A‐C remain incompletely understood. In this study, we made single mutations in loops A‐C of the benzodiazepine binding‐site across all six α subunits. We used whole‐cell patch clamp recording to measure the functional effects of these mutations on midazolam potentiation. The results showed that mutating the threonine in loop B and serine in loop C (Thr163 and S206 in human α1) did not abolish the receptors’ responsiveness to midazolam, as the α1(H102R) mutation did. The loop C mutations exhibited a novel array of α‐isoform specific effects on midazolam potentiation. The α3(S230I) and α5(S209I) mutations had the largest effect on midazolam potentiation, increasing the efficacy of midazolam. Novel benzodiazepines targeting loop C may represent a future direction for designing new drugs that specifically alter the activity of α3‐ and α5‐containing GABAA receptors.

2-(4-methyl-thiazol-5-yl) ethyl nitrate maleate-potentiated GABAA receptor response in hippocampal neurons.

2-(4-methyl-thiazol-5-yl) ethyl nitrate maleate (NMZM), a derivative of clomethiazole (CMZ), had been investigated for the treatment of Alzheimer's disease (AD). The beneficial effects of NMZM in AD included reversing cognitive deficit, improving learning and memory as well as neuroprotection. The pharmacological effects of NMZM on GABAA receptors were reported previously; however, the mechanisms were unclear and were explored therefore. In this study, we demonstrated that NMZM improved learning and memory by alleviating scopolamine-induced long-term potentiation (LTP) suppression in the dentate gyrus of rats, indicating that NMZM had protective effects against scopolamine-induced depression of LTP. Next, we investigated the action of NMZM on GABAA receptors in hippocampal neurons and the binding site of NMZM on GABAA receptors. NMZM directly activated GABAA receptors in hippocampal neurons in a weak manner. However, NMZM could potentiate the response of GABAA receptors to GABA and NMZM positively modulated GABAA receptors with an EC50 value of 465μmol/L at 3μmol/L GABA while this potentiation at low concentration of GABA (1, 3μmol/L) was more significant than that at high concentration (10, 30μmol/L). In addition, NMZM could enhance GABA currents after using diazepam and pentobarbital, the positive modulators of GABAA receptors. NMZM could not affect the etomidate-potentiated GABAA current. It suggested that the binding site of NMZM on GABAA receptors is the same as etomidate. These results provided support for the neuroprotective effect of NMZM, which was partly dependent on the potentiation of GABAA receptors. The etomidate binding site might be a new target for neuronal protection and for drug development.

Attaining in vivo selectivity of positive modulation of α3βγ2 GABAA receptors in rats: A hard task!

It is unclear whether GABAA receptors (GABAARs) that contain the α3-subunit are substantially involved in the anxiolytic effects of benzodiazepines (BDZs). In the present study, we tested YT-III-31, a newer BDZ ligand with functional preference for α3βγ2 GABAARs, in two paradigms of unconditioned anxiety, the open field and elevated plus maze in rats. The effective dose of YT-III-31 (2 mg/kg) displayed a clear anxiolytic-like profile, unhampered by sedative action, in both tests. At a higher dose (10 mg/kg), YT-III-31 induced ataxia in the rotarod and sedation in spontaneous locomotor activity test. The latter effect was preventable by flumazenil and βCCt, the non-selective and α1βγ2 GABAAR affinity-selective antagonist, respectively, demonstrating that sedative properties of YT-III-31, when attained, are mediated by the α1γ2 site. To elucidate the receptor substrate of subtle behavioral differences between YT-III-31 and diazepam, we approximated in vivo receptor potentiation for both ligands, based on estimated unbound concentrations in rat brains. Far different from diazepam, YT-III-31 has significantly lower affinity for the α1γ2 over other BDZ-sensitive sites, and at lower doses (1–2 mg/kg) was devoid of potentiation at α1βγ2 GABAARs. The approximation approach revealed a modest selectivity of YT-III-31 for α3γ2- in comparison to α2γ2 and α5γ2 binding sites, suggesting that its anxiolytic-like activity may not necessarily or predominantly reflect potentiation at α3βγ2 GABAARs. Nonetheless, as the anxiolytic effects are achievable at a dose devoid of any sedative potential, and having favorable safety (cytotoxicity) and metabolic stability profile, YT-III-31 represents a valuable candidate for further translational research.

Simultaneous quantification of EVT201, a novel partial positive allosteric GABAA receptor modulator, and its two metabolites in human plasma by UHPLC/MS/MS

A sensitive, accurate and rapid UHPLC–MS/MS method was developed and validated for the simultaneous determination of EVT201 and its two metabolites, Ro46-1927 and Ro18-5528, in human plasma. D6-EVT201 was used as the internal standard (IS). Plasma samples were extracted using ethyl acetate after being alkalized with saturated sodium carbonate solution. Chromatographic separation was carried out on an Acquity BEH C18 column (2.1 × 50 mm, 1.7 μm) with a gradient mobile phase at a flow-rate of 0.50 mL/min. The analytical run time was 5.5 min. For mass spectrometric detection, multiple reaction monitoring (MRM) was used. The MRM ion transitions were m/z 373.2 → 58.0 for EVT201, m/z 359.2 → 316.1 for Ro46-1927, m/z 291.2 → 274.1 for Ro18-5528 and m/z 379.3 → 64.0 for the IS. The linear range was 0.2–200 ng/mL for each analyte, with a correlation coefficient (r) over 0.9900. The intra-/inter- precision was within 7.9% and 4.5% for EVT201, 13.2% and 6.3% for Ro46-1927, 3.7% and 4.1% for Ro18-5528. For the accuracy, the relative bias of intra-/inter- run was no more than 6.4% and 4.6% for EVT201, 9.4% and 7.9% for Ro46-1927, −6.9% and −7.5% for Ro18-5528. The validated method was successfully applied to the analysis of more than 1500 samples from a Phase I clinical trial. The incurred sample reanalysis (ISR) of 146 samples from the study was evaluated and the results met the acceptance criteria. It indicated that the method could be used for the pharmacokinetic study of EVT201.

Cerebellar α6 -subunit-containing GABAA receptors: a novel therapeutic target for disrupted prepulse inhibition in neuropsychiatric disorders.

The pathophysiological role of α6 -subunit-containing GABAA receptors, which are mainly expressed in cerebellar granule cells, remains unclear. Recently, we demonstrated that hispidulin, a flavonoid isolated from a local herb that remitted a patient's intractable motor tics, attenuated methamphetamine-induced hyperlocomotion in mice as a positive allosteric modulator (PAM) of cerebellar α6 GABAA receptors. Here, using hispidulin and a selective α6 GABAA receptor PAM, the pyrazoloquinolinone Compound 6, we revealed an unprecedented role of cerebellar α6 GABAA receptors in disrupted prepulse inhibition of the startle response (PPI), which reflects sensorimotor gating deficits manifested in several neuropsychiatric disorders. PPI disruptions were induced by methamphetamine and NMDA receptor antagonists in mice. Effects of the tested compounds were measured in Xenopus oocytes expressing recombinant α6 β3 γ2S GABAA receptors. Hispidulin given i.p. or by bilateral intracerebellar (i.cb.) injection rescued PPI disruptions induced by methamphetamine, ketamine, MK-801 and phencyclidine. Intracerebellar effects of hispidulin were mimicked by Ro15-4513 and loreclezole (two α6 GABAA receptor PAMs), but not by diazepam (an α6 GABAA receptor-inactive benzodiazepine) and were antagonized by furosemide (i.cb.), an α6 GABAA receptor antagonist. Importantly, Compound 6 (i.p.) also rescued methamphetamine-induced PPI disruption, an effect prevented by furosemide (i.cb.). Both hispidulin and Compound 6 potentiated α6 β3 γ2S GABAA receptor-mediated GABA currents. Positive allosteric modulation of cerebellar α6 GABAA receptors rescued disrupted PPI by attenuating granule cell activity. α6 GABAA receptor-selective PAMs are potential medicines for treating sensorimotor gating deficits in neuropsychiatric disorders. A mechanistic hypothesis is based on evidence for cerebellar contributions to cognitive functioning including sensorimotor gating.

Phase 3 Study Evaluating Brexanolone, a GABAA Receptor Modulator, in Moderate Postpartum Depression [25H

INTRODUCTION: A double-blind, placebo-controlled Phase 2 study of brexanolone (USAN; formerly SAGE-547 Injection), a positive allosteric modulator of GABAA receptors, demonstrated rapid and durable improvements in postpartum depression (PPD). The current study (NCT 02942017) is one of two randomized, multi-center, double-blind, placebo-controlled Phase 3 studies that examined the efficacy and safety of brexanolone in women with PPD. METHODS: Approximately 100 women with moderate PPD (Hamilton Rating Scale for Depression [HAM-D] total score ≥20 and ≤25) were randomized 1:1 to a 60-hour infusion of blinded brexanolone (90 µg/kg/h) or placebo. The primary endpoint was the change from baseline in the mean HAM-D total score at Hour 60 following brexanolone compared with placebo. Durability of effect and safety were assessed through Day 30. Safety and tolerability were assessed by adverse events, vital signs, laboratory evaluations, electrocardiogram, and the Columbia Suicide Severity Rating Scale. Secondary efficacy endpoints included change from baseline in the Montgomery-Åsberg Depression Rating Scale, HAM-D response, HAM-D remission, and Clinical Global Impression-Improvement. RESULTS: This Phase 3 study will be completed in 2017. This presentation will be the first comprehensive reporting of primary and secondary endpoints. These data seek to validate and expand upon the Phase 2 results, which showed a rapid reduction in mean HAM-D score (Hour 60, brexanolone -21.0 vs. placebo -8.8, p=0.008) and durability through Day 30 (20.8 vs. -8.8, p=0.010). CONCLUSION: This study, along with a companion Phase 3 study with brexanolone in subjects with severe PPD, seeks to validate and expand upon the earlier Phase 2 data.

O46. SAGE-217 a First in Class GABAA Receptor Positive Allosteric Modulator in Major Depressive Disorder: A Multicenter, Randomized, Double-Blind, Phase 2 Placebo-Controlled Trial

This randomized double-blind, placebo-controlled clinical trial evaluated the anti-depressant efficacy and safety of SAGE-217, a positive allosteric modulator of GABAA receptors, in subjects with moderate-to-severe Major Depressive Disorder (MDD).