Allosteric Modulators Of GABAA Receptors Research Articles

Antidepressant with novel mechanism of action shows promise in Phase 2 trial

Daily oral administration of SAGE‐217, a synthetic neurosteroid and positive allosteric GABAA receptor modulator, reduced depressive symptoms relative to placebo in a Phase 2 study involving 89 patients with major depression. The promising results were seen both among patients taking and not taking an antidepressant, researchers reported. Study results were published Sept. 5 in the New England Journal of Medicine.

The allopregnanolone to progesterone ratio across the menstrual cycle and in menopause

The neuroactive steroid 3α-5α-tetrahydroprogesterone (allopregnanolone), a metabolite of progesterone, is a positive allosteric modulator of GABAA receptors, and low levels have been implicated in the etiology of mood disorders. However, it is not known whether metabolism of progesterone to allopregnanolone varies across the menstrual cycle or is low after menopause. We hypothesized that the allopregnanolone/progesterone ratio would decrease from the follicular to luteal phase. We also hypothesized that postmenopausal women would have lower levels of progesterone and allopregnanolone but similar allopregnanolone/progesterone ratios as premenopausal women in the follicular phase.Serum fasting allopregnanolone and progesterone levels were measured by gas chromatography-mass spectrometry in ten premenopausal women at the follicular, mid-cycle, and luteal phases of the menstrual cycle and in twenty-four postmenopausal women. Although allopregnanolone and progesterone levels increased from the follicular to luteal phase, the allopregnanolone/progesterone ratio decreased 8-fold [0.33 ± 0.08 (follicular) vs 0.16 ± 0.09 (mid-cycle) vs 0.04 ± 0.007 (luteal), p = 0.0003]. Mean allopregnanolone and progesterone levels were lower in postmenopausal than premenopausal women at all menstrual cycle phases (p < 0.01). The mean allopregnanolone/progesterone ratio was similar in postmenopausal and premenopausal women in the follicular phase (0.39 ± 0.08 vs 0.33 ± 0.08, p = 0.94) but was significantly lower at mid-cycle and in the luteal phase than in postmenopausal women (p < 0.01). In conclusion, the serum allopregnanolone/progesterone ratio decreases 8-fold from the follicular to luteal phase and is lower at mid-cycle and the luteal phase than in postmenopausal women. Whether these data have implications for luteal phase and other mood disorders merits further study.

Mechanisms Responsible for Genetic Hypertension in Schlager BPH/2 Mice.

It has been 45 years since Gunther Schlager used a cross breeding program in mice to develop inbred strains with high, normal, and low blood pressure (BPH/2, BPN/3, and BPL/1 respectively). Thus, it is timely to gather together the studies that have characterized and explored the mechanisms associated with the hypertension to take stock of exactly what is known and what remains to be determined. Growing evidence supports the notion that the mechanism of hypertension in BPH/2 mice is predominantly neurogenic with some of the early studies showing aberrant brain noradrenaline levels in BPH/2 compared with BPN/3. Analysis of the adrenal gland using microarray suggested an association with the activity of the sympathetic nervous system. Indeed, in support of this, there is a larger depressor response to ganglion blockade, which reduced blood pressure in BPH/2 mice to the same level as BPN/3 mice. Greater renal tyrosine hydroxylase staining and greater renal noradrenaline levels in BPH/2 mice suggest sympathetic hyperinnervation of the kidney. Renal denervation markedly reduced the blood pressure in BPH/2 but not BPN/3 mice, confirming the importance of renal sympathetic nervous activity contributing to the hypertension. Further, there is an important contribution to the hypertension from miR-181a and renal renin in this strain. BPH/2 mice also display greater neuronal activity of amygdalo-hypothalamic cardiovascular regulatory regions. Lesions of the medial nucleus of the amygdala reduced the hypertension in BPH/2 mice and abolished the strain difference in the effect of ganglion blockade, suggesting a sympathetic mechanism. Further studies suggest that aberrant GABAergic inhibition may play a role since BPH/2 mice have low GABAA receptor δ, α4 and β2 subunit mRNA expression in the hypothalamus, which are predominantly involved in promoting tonic neuronal inhibition. Allopregnanolone, an allosteric modulator of GABAA receptors, which increase the expression of these subunits in the amygdala and hypothalamus, is shown to reduce the hypertension and sympathetic nervous system contribution in BPH/2 mice. Thus far, evidence suggests that BPH/2 mice have aberrant GABAergic inhibition, which drives neuronal overactivity within amygdalo-hypothalamic brain regions. This overactivity is responsible for the greater sympathetic contribution to the hypertension in BPH/2 mice, thus making this an ideal model of neurogenic hypertension.

Development of 1,3-thiazole analogues of imidazopyridines as potent positive allosteric modulators of GABAA receptors.

Structure-activity relationship studies were conducted in the search for 1,3-thiazole isosteric analogs of imidazopyridine drugs (Zolpidem, Alpidem). Three series of novel γ-aminobutyric acid receptor (GABAAR) ligands belonging to imidazo[2,1-b]thiazoles, imidazo[2,1-b][1,3,4]thiadiazoles, and benzo[d]imidazo[2,1-b]thiazoles were synthesized and characterized as active agents against GABAAR benzodiazepine-binding site. In each of these series, potent compounds were discovered using a radioligand competition binding assay. The functional properties of highest-affinity compounds 28 and 37 as GABAAR positive allosteric modulators (PAMs) were determined by electrophysiological measurements. In vivo studies on zebrafish demonstrated their potential for the further development of anxiolytics. Using the OECD "Fish, Acute Toxicity Test" active compounds were found safe and non-toxic. Structural bases for activity of benzo[d]imidazo[2,1-b]thiazoles were proposed using molecular docking studies. The isosteric replacement of the pyridine nuclei by 1,3-thiazole, 1,3,4-thiadiazole, or 1,3-benzothiazole in the ring-fused imidazole class of GABAAR PAMs was shown to be promising for the development of novel hypnotics, anxiolytics, anticonvulsants, and sedatives drug-candidates.

Neurosteroids as novel antidepressants and anxiolytics: GABA-A receptors and beyond

The recent FDA approval of the neurosteroid, brexanolone (allopregnanolone), as a treatment for women with postpartum depression, and successful trials of a related neuroactive steroid, SGE-217, for men and women with major depressive disorder offer the hope of a new era in treating mood and anxiety disorders based on the potential of neurosteroids as modulators of brain function. This review considers potential mechanisms contributing to antidepressant and anxiolytic effects of allopregnanolone and other GABAergic neurosteroids focusing on their actions as positive allosteric modulators of GABAA receptors. We also consider their roles as endogenous “stress” modulators and possible additional mechanisms contributing to their therapeutic effects. We argue that further understanding of the molecular, cellular, network and psychiatric effects of neurosteroids offers the hope of further advances in the treatment of mood and anxiety disorders.

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.

A FLIPR Assay for Discovery of GABAA Receptor Modulators of Natural Origin.

A fluorometric imaging plate reader (FLIPR) assay utilizing Chinese hamster ovary (CHO) cells stably transfected with GABAA receptors of α 1 β 2 γ 2 subunit composition was evaluated and validated for rapid screening of plant extract libraries and efficient localization of active compounds in extracts. Validation was performed with pure compounds and extracts known to contain allosteric GABAA receptor modulators. Plants extracts that had been previously reported as active in an assay using Xenopus laevis oocytes transiently expressing GABAA receptors of α 1 β 2 γ 2 subunit composition were also active in the FLIPR assay. A protocol for HPLC-based activity profiling was developed, whereby separations of 0.4 - 1.2 mg of extracts on an analytical HPLC column were found to be sufficient for the sensitivity of the bioassay. The protocol successfully localized the activity of known GABAergic natural products, such as magnolol in Magnolia officinalis, valerenic acid in Valeriana officinalis, and piperine in Piper nigrum extract. EC50 values of compounds (magnolol: 4.81 ± 1.0 µM, valerenic acid: 12.56 ± 1.2 µM, and piperine: 5.76 ± 0.7 µM) were found to be comparable or lower than those reported using Xenopus oocyte assays.

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

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.

Engineered Flumazenil Recognition Site Provides Mechanistic Insight Governing Benzodiazepine Modulation in GABAA Receptors.

The anxiolytic, anticonvulsant, muscle-relaxant, and sedative-hypnotic effects of benzodiazepine site ligands are mainly elicited by allosteric modulation of GABAA receptors via their extracellular αx+/γ2- ( x = 1, 2, 3, 5) interfaces. In addition, a low affinity binding site at the homologous α+/β- interfaces was reported for some benzodiazepine site ligands. Classical benzodiazepines and pyrazoloquinolinones have been used as molecular probes to develop structure-activity relationship models for benzodiazepine site activity. Considering all possible α+/β- and α+/γ- interfaces, such ligands potentially interact with as many as 36 interfaces, giving rise to undesired side effects. Understanding the binding modes at their binding sites will enable rational strategies to design ligands with desired selectivity profiles. Here, we compared benzodiazepine site ligand interactions in the high affinity α1+/γ2- site with the homologous α1+/β3- site using a successive mutational approach. We incorporated key amino acids known to contribute to high affinity benzodiazepine binding of the γ2- subunit into the β3- subunit, resulting in a quadruple mutant β3(4mut) with high affinity flumazenil (Ro 15-1788) binding properties. Intriguingly, some benzodiazepine site ligands displayed positive allosteric modulation in the tested recombinant α1β3(4mut) constructs while diazepam remained inactive. Consequently, we performed in silico molecular docking in the wildtype receptor and the quadruple mutant. The results led to the conclusion that different benzodiazepine site ligands seem to use distinct binding modes, rather than a common binding mode. These findings provide structural hypotheses for the future optimization of both benzodiazepine site ligands, and ligands that interact with the homologous α+/β- sites.

Pharmacological characterization of the neurotrophic sesquiterpene jiadifenolide reveals a non-convulsant signature and potential for progression in neurodegenerative disease studies

The ‘neurotrophic sesquiterpenes’ refer to a group of molecules derived from the Illicium genus of flowering plant. They display neurotrophic effects in cultured neuron preparations and have been suggested to be cognitive enhancers and potential therapeutics for neurodegenerative disorders and dementias. Recent synthetic advances generated sufficient quantities of jiadifenolide for in vivo investigation into its biological effects. Jiadifenolide did not induce convulsions in mice nor did it enhance or diminish convulsions induced by pentylenetetrazole. Other negative allosteric modulators of GABAA receptors, picrotoxin, tetramethylenedisulfotetramine (TETS), and bilobalide all induced convulsions. Either i.p. or i.c.v. dosing generated micromolar plasma and brain levels of jiadifenolide but only small effects on locomotion of mice. However, jiadifenolide decreased d-amphetamine-induced hyperlocomotion in mice, an antipsychotic-like drug effect. Jiadifenolide did not significantly alter body temperature or behavior in the forced-swim test in mice. Molecular simulation data suggested a potential site in the pore/M2 helix region that is at an overlapping, yet lower position than those observed for other ‘cage convulsant’ compounds such as TETS and picrotoxin. We hypothesize that a position nearer to the entrance of the pore channel may allow for easier displacement of jiadifenolide from its blocking location leading to lower potency and lower side-effect liability. Like jiadifenolide, memantine (Namenda), one of the few drugs used in the symptomatic treatment of dementias, occupies a unique site on the NMDA receptor complex that creates low binding affinity that is associated with its reduced side-effect profile. Given the potential therapeutic applications of jiadifenolide and its relatively inert effects on overt behavior, the possibility of clinical utility for jiadifenolide and related compounds becomes intriguing.

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.

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).

Peripheral GABAA receptor-mediated signaling facilitates persistent inflammatory hypersensitivity

Unlike in the central nervous system (CNS), in the adult peripheral nervous system (PNS), activation of GABAA receptors (GABAAR) is excitatory because of the relatively high concentration of intracellular chloride in these neurons. Indeed, exogenous GABA and muscimol, a GABAAR agonist, exacerbate acute inflammatory hypersensitivity in rodents. However, it remains unclear whether peripheral GABAAR and the endogenous GABA play an important role in persistent inflammatory hypersensitivity. In this study, we thus investigated how peripheral GABAAR affects pain hypersensitivity by using the complete Freund's adjuvant (CFA)-induced persistent inflammatory pain mouse model. We found that intraplantar (i.pl.) administration of GABAAR antagonists, picrotoxin, and 1(S),9(R)-(−)-bicuculline methiodide significantly inhibited both spontaneous nociceptive (paw licking and flinching) behavior and mechanical hypersensitivity in CFA-injected mice at day 3 (D3), but not in naïve mice. Interestingly, CFA-induced mechanical hypersensitivity was significantly reversed by anti-GABA antibody (anti-GABA, i.pl.). In addition, RT-qPCR revealed that glutamate decarboxylase Gad1 (GAD 67) and Gad2 (GAD 65) mRNA expression was also upregulated in the ipsilateral hind paw of CFA-injected mice at D3. Finally, 5α-pregnan-3α-ol-20-one (3α,5α-THP), a selective positive allosteric modulator of GABAAR, produced mechanical hypersensitivity in naïve mice in a dose-dependent manner. Taken together, our results indicate that peripheral GABAAR and endogenous GABA, possibly produced by the inflamed tissue, potentiate CFA-induced persistent inflammatory hypersensitivity, suggesting that they can be used as a therapeutic target for alleviating inflammatory pain.