Pharmacological characterization of SAGE-718, a novel positive allosteric modulator of N-methyl-d-aspartate receptors.

BACKGROUND AND PURPOSE: Select neuroactive steroids tune neural activity by modulating excitatory and inhibitory neurotransmission, including the endogenous cholesterol metabolite 24(S)-hydroxycholesterol (24(S)-HC), which is an N-methyl-d-aspartate (NMDA) receptor positive allosteric modulator (PAM). NMDA receptor PAMs are potentially an effective pharmacotherapeutic strategy to treat conditions associated with NMDA receptor hypofunction. EXPERIMENTAL APPROACH: Using in vitro and in vivo electrophysiological recording experiments and behavioural approaches, we evaluated the effect of SAGE-718, a novel neuroactive steroid NMDA receptor PAM currently in clinical development for the treatment of cognitive impairment, on NMDA receptor function and endpoints that are altered by NMDA receptor hypoactivity and assessed its safety profile. KEY RESULTS: SAGE-718 potentiated GluN1/GluN2A-D NMDA receptors with equipotency and increased NMDA receptor excitatory postsynaptic potential (EPSP) amplitude without affecting decay kinetics in striatal medium spiny neurons. SAGE-718 increased the rate of unblock of the NMDA receptor open channel blocker ketamine on GluN1/GluN2A in vitro and accelerated the rate of return on the ketamine-evoked increase in gamma frequency band power, as measured with electroencephalogram (EEG), suggesting that PAM activity is driven by increased channel open probability. SAGE-718 ameliorated deficits due to NMDA receptor hypofunction, including social deficits induced by subchronic administration of phencyclidine, and behavioural and electrophysiological deficits from cholesterol and 24(S)-HC depletion caused by 7-dehydrocholesterol reductase inhibition. Finally, SAGE-718 did not produce epileptiform activity in a seizure model or neurodegeneration following chronic dosing. CONCLUSIONS AND IMPLICATIONS: These findings provide strong evidence that SAGE-718 is a neuroactive steroid NMDA receptor PAM with a mechanism that is well suited as a treatment for conditions associated with NMDA receptor hypofunction.

Novel neuroactive steroids as positive allosteric modulators of NMDA receptors: mechanism, site of action, and rescue pharmacology on GRIN variants associated with neurological conditions.

N-methyl-D-aspartate receptors (NMDARs) play vital roles in normal brain functions (i.e., learning, memory, and neuronal development) and various neuropathological conditions, such as epilepsy, autism, Parkinson's disease, Alzheimer's disease, and traumatic brain injury. Endogenous neuroactive steroids such as 24(S)-hydroxycholesterol (24(S)-HC) have been shown to influence NMDAR activity, and positive allosteric modulators (PAMs) derived from 24(S)-hydroxycholesterol scaffold can also enhance NMDAR function. This study describes the structural determinants and mechanism of action for 24(S)-hydroxycholesterol and two novel synthetic analogs (SGE-550 and SGE-301) on NMDAR function. We also show that these agents can mitigate the altered function caused by a set of loss-of-function missense variants in NMDAR GluN subunit-encoding GRIN genes associated with neurological and neuropsychiatric disorders. We anticipate that the evaluation of novel neuroactive steroid NMDAR PAMs may catalyze the development of new treatment strategies for GRIN-related neuropsychiatric conditions.

SAGE-718: A First-in-Class N-Methyl-d-Aspartate Receptor Positive Allosteric Modulator for the Potential Treatment of Cognitive Impairment.

N-Methyl-d-aspartate receptor (NMDAR) positive allosteric modulators (PAMs) have received increased interest as a powerful mechanism of action to provide relief as therapies for CNS disorders. Sage Therapeutics has previously published the discovery of endogenous neuroactive steroid 24(S)-hydroxycholesterol as an NMDAR PAM. In this article, we detail the discovery of development candidate SAGE-718 (5), a potent and high intrinsic activity NMDAR PAM with an optimized pharmacokinetic profile for oral dosing. Compound 5 has completed phase 1 single ascending dose and multiple ascending dose clinical trials and is currently undergoing phase 2 clinical trials for treatment of cognitive impairment in Huntington's disease.

Preventing Phosphorylation of the GABA A R ß3 Subunit Compromises the Behavioral Effects of Neuroactive Steroids.

Neuroactive steroids (NASs) have potent anxiolytic, anticonvulsant, sedative, and hypnotic actions, that reflect in part their efficacy as GABA A R positive allosteric modulators (PAM). In addition to this, NAS exert metabotropic effects on GABAergic inhibition via the activation of membrane progesterone receptors (mPRs), which are G-protein coupled receptors. mPR activation enhances the phosphorylation of residues serine 408 and 409 (S408/9) in the ß3 subunit of GABA A Rs, increasing their accumulation in the plasma membrane leading to a sustained increase in tonic inhibition. To explore the significance of NAS-induced phosphorylation of GABA A Rs, we used mice in which S408/9 in the ß3 subunit have been mutated to alanines, mutations that prevent the metabotropic actions of NASs on GABA A R function while preserving NAS allosteric potentiation of GABAergic current. While the sedative actions of NAS were comparable to WT, their anxiolytic actions were reduced in S408/9A mice. Although the induction of hypnosis by NAS were maintained in the mutant mice the duration of the loss of righting reflex was significantly shortened. Finally, ability of NAS to terminate diazepam pharmacoresistant seizures was abolished in S408/9A mice. In conclusion, our results suggest that S408/9 in the GABA A R ß3 subunit contribute to the anxiolytic and anticonvulsant efficacy of NAS, in addition to their ability to regulate the loss of righting reflex.

Oxysterols Modulate the Acute Effects of Ethanol on Hippocampal N-Methyl-d-Aspartate Receptors, Long-Term Potentiation, and Learning.

Ethanol is a noncompetitive inhibitor of N-methyl-d-aspartate receptors (NMDARs) and acutely disrupts hippocampal synaptic plasticity and learning. In the present study, we examined the effects of oxysterol positive allosteric modulators (PAMs) of NMDARs on ethanol-mediated inhibition of NMDARs, block of long-term potentiation (LTP) and long-term depression (LTD) in rat hippocampal slices, and defects in one-trial learning in vivo. We found that 24S-hydroxycholesterol and a synthetic oxysterol analog, SGE-301, overcame effects of ethanol on NMDAR-mediated synaptic responses in the CA1 region but did not alter acute effects of ethanol on LTD; the synthetic oxysterol, however, overcame acute inhibition of LTP. In addition, both oxysterols overcame persistent effects of ethanol on LTP in vitro, and the synthetic analog reversed defects in one-trial inhibitory avoidance learning in vivo. These results indicate that effects of ethanol on both LTP and LTD arise by complex mechanisms beyond NMDAR antagonism and that oxysterol NMDAR PAMS may represent a novel approach for preventing and reversing acute ethanol-mediated changes in cognition. SIGNIFICANCE STATEMENT: Ethanol acutely inhibits hippocampal NMDARs, LTP, and learning. This study found that certain oxysterols that are NMDAR-positive allosteric modulators can overcome the acute effects of ethanol on NMDARs, LTP, and learning. Oxysterols differ in their effects from agents that inhibit integrated cellular stress responses.

Preclinical characterization of zuranolone (SAGE-217), a selective neuroactive steroid GABAA receptor positive allosteric modulator.

Zuranolone (SAGE-217) is a novel, synthetic, clinical stage neuroactive steroid GABAA receptor positive allosteric modulator designed with the pharmacokinetic properties to support oral daily dosing. In vitro, zuranolone enhanced GABAA receptor current at nine unique human recombinant receptor subtypes, including representative receptors for both synaptic (γ subunit-containing) and extrasynaptic (δ subunit-containing) configurations. At a representative synaptic subunit configuration, α1ß2γ2, zuranolone potentiated GABA currents synergistically with the benzodiazepine diazepam, consistent with the non-competitive activity and distinct binding sites of the two classes of compounds at synaptic receptors. In a brain slice preparation, zuranolone produced a sustained increase in GABA currents consistent with metabotropic trafficking of GABAA receptors to the cell surface. In vivo, zuranolone exhibited potent activity, indicating its ability to modulate GABAA receptors in the central nervous system after oral dosing by protecting against chemo-convulsant seizures in a mouse model and enhancing electroencephalogram ß-frequency power in rats. Together, these data establish zuranolone as a potent and efficacious neuroactive steroid GABAA receptor positive allosteric modulator with drug-like properties and CNS exposure in preclinical models. Recent clinical data support the therapeutic promise of neuroactive steroid GABAA receptor positive modulators for treating mood disorders; brexanolone is the first therapeutic approved specifically for the treatment of postpartum depression. Zuranolone is currently under clinical investigation for the treatment of major depressive episodes in major depressive disorder, postpartum depression, and bipolar depression.

25-Hydroxycholesterol amplifies microglial IL-1ß production in an apoE isoform-dependent manner.

BACKGROUND: Genome-wide association studies of Alzheimer's disease (AD) have implicated pathways related to lipid homeostasis and innate immunity in AD pathophysiology. However, the exact cellular and chemical mediators of neuroinflammation in AD remain poorly understood. The oxysterol 25-hydroxycholesterol (25-HC) is an important immunomodulator produced by peripheral macrophages with wide-ranging effects on cell signaling and innate immunity. Cholesterol 25-hydroxylase (CH25H), the enzyme responsible for 25-HC production, has also been found to be one of the disease-associated microglial (DAM) genes that are upregulated in the brain of AD and AD transgenic mouse models. METHODS: We used real-time PCR and immunoblotting to examine CH25H expression in human AD brain tissue and in transgenic mouse brain tissue-bearing amyloid-ß plaques or tau pathology. The innate immune response of primary mouse microglia under different treatment conditions or bearing different genetic backgrounds was analyzed using ELISA, western blotting, or immunocytochemistry. RESULTS: We found that CH25H expression is upregulated in human AD brain tissue and in transgenic mouse brain tissue-bearing amyloid-ß plaques or tau pathology. Treatment with the toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS) markedly upregulates CH25H expression in the mouse brain and stimulates CH25H expression and 25-HC secretion in mouse primary microglia. We found that LPS-induced microglial production of the pro-inflammatory cytokine IL-1ß is markedly potentiated by 25-HC and attenuated by the deletion of CH25H. Microglia expressing apolipoprotein E4 (apoE4), a genetic risk factor for AD, produce greater amounts of 25-HC than apoE3-expressing microglia following treatment with LPS. Remarkably, 25-HC treatment results in a greater level of IL-1ß secretion in LPS-activated apoE4-expressing microglia than in apoE2- or apoE3-expressing microglia. Blocking potassium efflux or inhibiting caspase-1 prevents 25-HC-potentiated IL-1ß release in apoE4-expressing microglia, indicating the involvement of caspase-1 inflammasome activity. CONCLUSION: 25-HC may function as a microglial-secreted inflammatory mediator in the brain, promoting IL-1ß-mediated neuroinflammation in an apoE isoform-dependent manner (E4>>E2/E3) and thus may be an important mediator of neuroinflammation in AD.

Trial of SAGE-217 in Patients with Major Depressive Disorder.

BACKGROUND: Altered neurotransmission of γ-aminobutyric acid (GABA) has been implicated in the pathogenesis of depression. Whether SAGE-217, an oral, positive allosteric modulator of GABA type A receptors, is effective and safe for the treatment of major depressive disorder is unknown. METHODS: In this double-blind, phase 2 trial, we enrolled patients with major depression and randomly assigned them in a 1:1 ratio to receive 30 mg of SAGE-217 or placebo once daily. The primary end point was the change from baseline to day 15 in the score on the 17-item Hamilton Depression Rating Scale (HAM-D; scores range from 0 to 52, with higher scores indicating more severe depression). Secondary efficacy end points, which were assessed on days 2 through 8 and on days 15, 21, 28, 35, and 42, included changes from baseline in scores on additional depression and anxiety scales, a reduction from baseline of more than 50% in the HAM-D score, a HAM-D score of 7 or lower, and a Clinical Global Impression of Improvement score of 1 (very much improved) or 2 (much improved) (on a scale of 1 to 7, with a score of 7 indicating that symptoms are very much worse). RESULTS: A total of 89 patients underwent randomization: 45 patients were assigned to the SAGE-217 group, and 44 to the placebo group. The mean baseline HAM-D score was 25.2 in the SAGE-217 group and 25.7 in the placebo group. The least-squares mean (±SE) change in the HAM-D score from baseline to day 15 was -17.4±1.3 points in the SAGE-217 group and -10.3±1.3 points in the placebo group (least-squares mean difference in change, -7.0 points; 95% confidence interval, -10.2 to -3.9; P<0.001). The differences in secondary end points were generally in the same direction as those of the primary end point. There were no serious adverse events. The most common adverse events in the SAGE-217 group were headache, dizziness, nausea, and somnolence. CONCLUSIONS: Administration of SAGE-217 daily for 14 days resulted in a reduction in depressive symptoms at day 15. Adverse events were more common in the SAGE-217 group than in the placebo group. Further trials are needed to determine the durability and safety of SAGE-217 in major depressive disorder and to compare SAGE-217 with available treatments. (Funded by Sage Therapeutics; ClinicalTrials.gov number, NCT03000530.).

Neuroactive Steroid N-Methyl-d-aspartate Receptor Positive Allosteric Modulators: Synthesis, SAR, and Pharmacological Activity.

Neuroactive steroids (NASs) play a pivotal role in maintaining homeostasis is the CNS. We have discovered that one NAS in particular, 24(S)-hydroxycholesterol (24(S)-HC), is a positive allosteric modulator (PAM) of NMDA receptors. Using 24(S)-HC as a chemical starting point, we have identified other NASs that have good in vitro potency and efficacy. Herein, we describe the structure activity relationship and pharmacokinetic optimization of this series that ultimately led to SGE-301 (42). We demonstrate that SGE-301 enhances long-term potentiation (LTP) in rat hippocampal slices and, in a dose-dependent manner, improves cognition in a rat social recognition study.

Metabotropic, but not allosteric, effects of neurosteroids on GABAergic inhibition depend on the phosphorylation of GABAA receptors.

Neuroactive steroids (NASs) are synthesized within the brain and exert profound effects on behavior. These effects are primarily believed to arise from the activities of NASs as positive allosteric modulators (PAMs) of the GABA-type A receptor (GABAAR). NASs also activate a family of G protein-coupled receptors known as membrane progesterone receptors (mPRs). Here, using surface-biotinylation assays and electrophysiology techniques, we examined mPRs' role in mediating the effects of NAS on the efficacy of GABAergic inhibition. Selective mPR activation enhanced phosphorylation of Ser-408 and Ser-409 (Ser-408/9) within the GABAAR ß3 subunit, which depended on the activity of cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC). mPR activation did not directly modify GABAAR activity and had no acute effects on phasic or tonic inhibition. Instead, mPR activation induced a sustained elevation in tonic current, which was blocked by PKA and PKC inhibition. Substitution of Ser-408/9 to alanine residues also prevented the effects of mPR activation on tonic current. Furthermore, this substitution abolished the effects of sustained NAS exposure on tonic inhibition. Interestingly, the allosteric effects of NAS on GABAergic inhibition were independent of Ser-408/9 in the ß3 subunit. Additionally, although allosteric effects of NAS on GABAergic inhibition were sensitive to a recently developed "NAS antagonist," the sustained effects of NAS on tonic inhibition were not. We conclude that metabotropic effects of NAS on GABAergic inhibition are mediated by mPR-dependent modulation of GABAAR phosphorylation. We propose that this mechanism may contribute to the varying behavioral effects of NAS.

Neuroactive Steroids Reverse Tonic Inhibitory Deficits in Fragile X Syndrome Mouse Model.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. A reduction in neuronal inhibition mediated by γ-aminobutyric acid type A receptors (GABAARs) has been implicated in the pathophysiology of FXS. Neuroactive steroids (NASs) are known allosteric modulators of GABAAR channel function, but recent studies from our laboratory have revealed that NASs also exert persistent metabotropic effects on the efficacy of tonic inhibition by increasing the protein kinase C (PKC)-mediated phosphorylation of the α4 and ß3 subunits which increase the membrane expression and boosts tonic inhibition. We have assessed the GABAergic signaling in the hippocampus of fragile X mental retardation protein (FMRP) knock-out (Fmr1 KO) mouse. The GABAergic tonic current in dentate gyrus granule cells (DGGCs) from 3- to 5-week-old (p21-35) Fmr1 KO mice was significantly reduced compared to WT mice. Additionally, spontaneous inhibitory post synaptic inhibitory current (sIPSC) amplitudes were increased in DGGCs from Fmr1 KO mice. While sIPSCs decay in both genotypes was prolonged by the prototypic benzodiazepine diazepam, those in Frm1-KO mice were selectively potentiated by RO15-4513. Consistent with this altered pharmacology, modifications in the expression levels and phosphorylation of receptor GABAAR subtypes that mediate tonic inhibition were seen in Fmr1 KO mice. Significantly, exposure to NASs induced a sustained elevation in tonic current in Fmr1 KO mice which was prevented with PKC inhibition. Likewise, exposure reduced elevated membrane excitability seen in the mutant mice. Collectively, our results suggest that NAS act to reverse the deficits of tonic inhibition seen in FXS, and thereby reduce aberrant neuronal hyperexcitability seen in this disorder.

The synthetic neuroactive steroid SGE-516 reduces seizure burden and improves survival in a Dravet syndrome mouse model.

Dravet syndrome is an infant-onset epileptic encephalopathy with multiple seizure types that are often refractory to conventional therapies. Treatment with standard benzodiazepines like clobazam, in combination with valproate and stiripentol, provides only modest seizure control. While benzodiazepines are a first-line therapy for Dravet syndrome, they are limited by their ability to only modulate synaptic receptors. Unlike benzodiazepines, neuroactive steroids potentiate a wider-range of GABAA receptors. The synthetic neuroactive steroid SGE-516 is a potent positive allosteric modulator of both synaptic and extrasynaptic GABAA receptors. Prior work demonstrated anticonvulsant activity of SGE-516 in acute seizure assays in rodents. In this study, we evaluated activity of SGE-516 on epilepsy phenotypes in the Scn1a +/- mouse model that recapitulates many features of Dravet syndrome, including spontaneous seizures, premature death and seizures triggered by hyperthermia. To evaluate SGE-516 in Scn1a +/- mice, we determined the effect of treatment on hyperthermia-induced seizures, spontaneous seizure frequency and survival. SGE-516 treatment protected against hyperthermia-induced seizures, reduced spontaneous seizure frequency and prolonged survival in the Scn1a +/- mice. This provides the first evidence of SGE-516 activity in a mouse model of Dravet syndrome, and supports further investigation of neuroactive steroids as potential anticonvulsant compounds for refractory epilepsies.

Brexanolone as adjunctive therapy in super-refractory status epilepticus.

OBJECTIVE: Super-refractory status epilepticus (SRSE) is a life-threatening form of status epilepticus that continues or recurs despite 24 hours or more of anesthetic treatment. We conducted a multicenter, phase 1/2 study in SRSE patients to evaluate the safety and tolerability of brexanolone (USAN; formerly SAGE-547 Injection), a proprietary, aqueous formulation of the neuroactive steroid, allopregnanolone. Secondary objectives included pharmacokinetic assessment and open-label evaluation of brexanolone response during and after anesthetic third-line agent (TLA) weaning. METHODS: Patients receiving TLAs for SRSE control were eligible for open-label, 1-hour brexanolone loading infusions, followed by maintenance infusion. After 48 hours of brexanolone infusion, TLAs were weaned during brexanolone maintenance. After 4 days, the brexanolone dose was tapered. Safety and functional status were assessed over 3 weeks of follow-up. RESULTS: Twenty-five patients received open-label study drug. No serious adverse events (SAEs) were attributable to study drug, as determined by the Safety Review Committee. Sixteen patients (64%) experienced ≥1 SAE. Six patient deaths occurred, all deemed related to underlying medical conditions. Twenty-two patients underwent ≥1 TLA wean attempt. Seventeen (77%) met the response endpoint of weaning successfully off TLAs before tapering brexanolone. Sixteen (73%) were successfully weaned off TLAs within 5 days of initiating brexanolone infusion without anesthetic agent reinstatement in the following 24 hours. INTERPRETATION: In an open-label cohort of limited size, brexanolone demonstrated tolerability among SRSE patients of heterogeneous etiologies and was associated with a high rate of successful TLA weaning. The results suggest the possible development of brexanolone as an adjunctive therapy for SRSE requiring pharmacological coma for seizure control. Ann Neurol 2017;82:342-352.

Neuroactive Steroids. 2. 3α-Hydroxy-3ß-methyl-21-(4-cyano-1H-pyrazol-1'-yl)-19-nor-5ß-pregnan-20-one (SAGE-217): A Clinical Next Generation Neuroactive Steroid Positive Allosteric Modulator of the (γ-Aminobutyric Acid)A Receptor.

Certain classes of neuroactive steroids (NASs) are positive allosteric modulators (PAM) of synaptic and extrasynaptic GABAA receptors. Herein, we report new SAR insights in a series of 5ß-nor-19-pregnan-20-one analogues bearing substituted pyrazoles and triazoles at C-21, culminating in the discovery of 3α-hydroxy-3ß-methyl-21-(4-cyano-1H-pyrazol-1'-yl)-19-nor-5ß-pregnan-20-one (SAGE-217, 3), a potent GABAA receptor modulator at both synaptic and extrasynaptic receptor subtypes, with excellent oral DMPK properties. Compound 3 has completed a phase 1 single ascending dose (SAD) and multiple ascending dose (MAD) clinical trial and is currently being studied in parallel phase 2 clinical trials for the treatment of postpartum depression (PPD), major depressive disorder (MDD), and essential tremor (ET).

Anticonvulsant profile of the neuroactive steroid, SGE-516, in animal models.

Despite the availability of multiple antiepileptic drugs (AED), failure to adequately control seizures is a challenge for approximately one third of epilepsy patients, and new therapies with a differentiated mechanism of action are needed. The neuroactive steroid, SGE-516, is a positive allosteric modulator of both gamma- and delta-containing GABAA receptors. This broad GABAA receptor activity differentiates neuroactive steroids like SGE-516 from benzodiazepines, a class of anticonvulsants which have been shown in vitro to selectively target gamma-subunit containing GABAA receptors. As a neuroactive steroid, SGE-516 has pharmacokinetic properties that are intended to allow for chronic oral dosing. We investigated the anticonvulsant activity of SGE-516 across numerous in vitro and in vivo models of seizure activity. SGE-516 dose-dependently reduced neuronal firing rates and epileptiform activity in vitro. In mice, SGE-516 protected against acute seizures in the PTZ-induced chemo-convulsant seizure model and the 6Hz psychomotor seizure model. In addition, SGE-516 demonstrated anticonvulsant activity in the mouse corneal kindling model. These data suggest that SGE-516 may have potential for development as a novel oral AED for the treatment of refractory seizures.

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

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

Endogenous and synthetic neuroactive steroids evoke sustained increases in the efficacy of GABAergic inhibition via a protein kinase C-dependent mechanism.

The neuroactive steroid (NAS) tetrahydrodeoxycorticosterone (THDOC) increases protein kinase C (PKC) mediated phosphorylation of extrasynaptic GABAA receptor (GABAAR) subunits leading to increased surface expression of α4/ß3 subunit-containing extrasynaptic GABAARs, leading to a sustained increase in GABAAR tonic current density. Whether other naturally occurring and synthetic NASs share both an allosteric and metabotropic action on GABAARs is unknown. Here, we examine the allosteric and metabotropic properties of allopregnanolone (ALLO), and synthetic NASs SGE-516 and ganaxolone. ALLO, SGE-516, and ganaxolone all allosterically enhanced prototypical synaptic and extrasynaptic recombinant GABAARs. In dentate gyrus granule cells (DGGCs) all three NASs, when applied acutely, allosterically enhanced tonic and phasic GABAergic currents. In separate experiments, slices were exposed to NASs for 15 min, and then transferred to a steroid naïve recording chamber followed by ≥ 30 min wash before tonic currents were measured. A sustained increase in tonic current was observed following exposure to ALLO, or SGE-516 and was prevented by inhibiting PKC with GF 109203X. No increase in tonic current was observed with exposure to ganaxolone. In agreement with the observations of an increased tonic current, the NASs ALLO and SGE-516 increased the phosphorylation and surface expression of the ß3 subunit-containing GABAARs. Our studies demonstrate that neuroactive steroids have differential abilities to induce sustained increases in the efficacy of tonic inhibition by promoting GABAAR phosphorylation and membrane trafficking dependent on PKC activity.

AZD8529, a positive allosteric modulator at the mGluR2 receptor, does not improve symptoms in schizophrenia: A proof of principle study.

INTRODUCTION: Activation of metabotropic glutamate (mGluR2/3) receptors has been proposed as an alternative mechanism to dopaminergic-based antipsychotics to correct glutamatergic deficits hypothesized to underlie schizophrenia symptoms. This study investigates the efficacy and safety of AZD8529, a selective positive allosteric modulator (PAM) at the mGlu2 receptor, in symptomatic patients with schizophrenia. METHODS: Patients were randomized to receive AZD8529 40 mg, risperidone 4 mg, or placebo as monotherapy. Treatment lasted for 28 days, and clinical efficacy was assessed using Positive and Negative Syndrome Scale (PANSS) and Clinical Global Impression (CGI) scores. RESULTS: There were no significant differences between patients treated with AZD8529 versus placebo in change from baseline to endpoint in PANSS total, negative and positive symptom subscale, or CGI-S scores. In contrast, risperidone demonstrated significant efficacy relative to placebo. CONCLUSION: These results do not support a role for the mGluR-2 PAM AZD8529 as an antipsychotic and indicate that positive modulation of mGluR type 2 receptors alone is not sufficient for antipsychotic effects in acutely ill schizophrenia patients.

Rescue of deficient amygdala tonic γ-aminobutyric acidergic currents in the Fmr-/y mouse model of fragile X syndrome by a novel γ-aminobutyric acid type A receptor-positive allosteric modulator.

Alterations in the ratio of excitatory to inhibitory transmission are emerging as a common component of many nervous system disorders, including autism spectrum disorders (ASDs). Tonic γ-aminobutyric acidergic (GABAergic) transmission provided by peri- and extrasynaptic GABA type A (GABAA ) receptors powerfully controls neuronal excitability and plasticity and, therefore, provides a rational therapeutic target for normalizing hyperexcitable networks across a variety of disorders, including ASDs. Our previous studies revealed tonic GABAergic deficits in principal excitatory neurons in the basolateral amygdala (BLA) in the Fmr1(-/y) knockout (KO) mouse model fragile X syndrome. To correct amygdala deficits in tonic GABAergic neurotransmission in Fmr1(-/y) KO mice, we developed a novel positive allosteric modulator of GABAA receptors, SGE-872, based on endogenously active neurosteroids. This study shows that SGE-872 is nearly as potent and twice as efficacious for positively modulating GABAA receptors as its parent molecule, allopregnanolone. Furthermore, at submicromolar concentrations (≤1 µM), SGE-872 is selective for tonic, extrasynaptic α4ß3δ-containing GABAA receptors over typical synaptic α1ß2γ2 receptors. We further find that SGE-872 strikingly rescues the tonic GABAergic transmission deficit in principal excitatory neurons in the Fmr1(-/y) KO BLA, a structure heavily implicated in the neuropathology of ASDs. Therefore, the potent and selective action of SGE-872 on tonic GABAA receptors containing α4 subunits may represent a novel and highly useful therapeutic avenue for ASDs and related disorders involving hyperexcitability of neuronal networks.

Neuroactive Steroids. 1. Positive Allosteric Modulators of the (γ-Aminobutyric Acid)A Receptor: Structure-Activity Relationships of Heterocyclic Substitution at C-21.

Neuroactive steroids (NASs) have been shown to impact central nervous system (CNS) function through positive allosteric modulation of the GABA(A) receptor (GABA(A)-R). Herein we report the effects on the activity and pharmacokinetic properties of a series of nor-19 pregnanolone analogues bearing a heterocyclic substituent at C-21. These efforts resulted in the identification of SGE-516, a balanced synaptic/extrasynaptic GABA(A) receptor modulator, and SGE-872, a selective extrasynaptic GABA(A) receptor modulator. Both molecules possess excellent druglike properties, making them advanced leads for oral delivery of GABA(A) receptor modulators.