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Electrophysiological shift of GABA by 2-261 and 2-262. GABA concentration-response curves derived from ␣ 1  2 ␥ 2 (A) versus ␣ 1  1 ␥ 2 (B) GABA A Rs Ϯ 0.7 M 2-261. Likewise, the effect of GABA on ␣ 1  2 ␥ 2 (C) versus ␣ 1  1 ␥ 2 (D) GABA A Rs Ϯ 3 M 2-262 is depicted in the bottom panels. GABA responses are expressed as I / I max , with each data point representing the mean Ϯ S.E.M. ( n ϭ 3– 4). The dashed line represents the GABA max
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GABA(A) receptor (R) positive allosteric modulators that selectively modulate GABA(A)Rs containing beta(2)- and/or beta(3)- over beta(1)-subunits have been reported across diverse chemotypes. Examples include loreclezole, mefenamic acid, tracazolate, and etifoxine. In general,"beta(2/3)-selective" GABA(A)R positive allosteric modulators are nonbenz...
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Context 1
... further characterize the -subunit selectivity of 2-261 and 2-262, we obtained GABA concentration-response data in the presence and absence of 2-261 and 2-262 at 1 1 2 and 1 2 2 subtypes. Both enantiomers induced a significant left- ward shift of the GABA concentration-response curve at 1 2 2 , with little or no shift at 1 1 2 (Fig. ...
Citations
... Fenamates pass the blood brain barrier (Bannwarth et al., 1989;Gee et al., 2010;Subaiea et al., 2011), suggesting that their actions at the major inhibitory receptor in the mammalian brain may evoke central nervous system effects. Moreover, their efficacy and ability to potentiate GABA A receptors parallels the sedative-hypnotic and anticonvulsant agents including the benzodiazepines and barbiturates. ...
Repeated and uncontrolled seizures in epilepsy result in brain cell loss and neural inflammation. Current anticonvulsants primarily target ion channels and receptors implicated in seizure activity. Identification of neurotherapeutics that can inhibit epileptiform activity and reduce inflammation in the brain may offer significant benefits in the long-term management of epilepsy. Fenamates are unique because they are both non-steroidal anti-inflammatory drugs (NSAIDs) and highly subunit selective modulators of GABAA receptors. In the current study we have investigated the hypothesis that fenamates have antiseizure properties using mature human stem cell-derived neuro-glia cell cultures, maintained in long-term culture, and previously shown to be sensitive to first, second and third generation antiepileptics. Mefenamic acid, flufenamic acid, meclofenamic acid, niflumic acid, and tolfenamic acid (each tested at 10–100 μM) attenuated 4-aminopyridine (4-AP, 100 μM) evoked epileptiform activity in a dose-dependent fashion. These actions were as effective diazepam (3–30 μM) and up to 200 times more potent than phenobarbital (300–1,000 μM). The low (micromolar) concentrations of fenamates that inhibited 4-AP evoked epileptiform activity correspond to those reported to potentiate GABAA receptor function. In contrast, the fenamates had no effect on neural spike amplitudes, indicating that their antiseizure actions did not result from inhibition of sodium-channels. The antiseizure actions of fenamates were also not replicated by either of the two non-fenamate NSAIDs, ibuprofen (10–100 μM) or indomethacin (10–100 μM), indicating that inhibition of cyclooxygenases is not the mechanism through which fenamates have anticonvulsant properties. This study therefore shows for the first time, using functionally mature human stem cell-derived neuroglial circuits, that fenamate NSAIDs have powerful antiseizure actions independent of, and in addition to their well-established anti-inflammatory properties, suggesting these drugs may provide a novel insight and new approach to the treatment of epilepsy in the future.
... The difference in efficacy between compounds could be due to differences in potency or permeability into zebrafish brain tissue. The EC 50 of compound 2-261 on α1β2γ2 in vitro is reported to be 0.3 µM (Gee et al. 2010), whereas the EC 50 of Zolpidem on α1β3γ2 in vitro is reported to be 0.48 µM (Has et al. 2016). Further in vivo studies are needed to determine the underlying reason the EC 50 values differ between these two PAMs. ...
Hexahydro-1,3,5-trinitro-1,3,5-triazine, or Royal Demolition Explosive (RDX), is a major component of plastic explosives such as C-4. Acute exposures from intentional or accidental ingestion are a documented clinical concern, especially among young male U.S. service members in the armed forces. When ingested in large enough quantity, RDX causes tonic–clonic seizures. Previous in silico and in vitro experiments predict that RDX causes seizures by inhibiting α1β2γ2 γ-aminobutyric acid type A (GABAA) receptor-mediated chloride currents. To determine whether this mechanism translates in vivo, we established a larval zebrafish model of RDX-induced seizures. After a 3 h of exposure to 300 µM RDX, larval zebrafish exhibited a significant increase in motility in comparison to vehicle controls. Researchers blinded to experimental group manually scored a 20-min segment of video starting at 3.5 h post-exposure and found significant seizure behavior that correlated with automated seizure scores. Midazolam (MDZ), an nonselective GABAAR positive allosteric modulator (PAM), and a combination of Zolpidem (α1 selective PAM) and compound 2-261 (β2/3-selective PAM) were effective in mitigating RDX-triggered behavioral and electrographic seizures. These findings confirm that RDX induces seizure activity via inhibition of the α1β2γ2 GABAAR and support the use of GABAAR-targeted anti-seizure drugs for the treatment of RDX-induced seizures.
... GABAARs expressing the α5 subunit were shown to modulate the spatiotemporal memory of benzodiazepines, associated with addiction (Tan et al., 2011;Rudolph and Möhler, 2014;Rudolph and Knoflach, 2011). The beta subunit is associated with sedative, ataxic, and narcotic drug effects (Gee et al., 2010;Sigel and Ernst, 2018). Diazepam is not selective for multiple subunits of the GABAAR and therefore has different effects and side effects. ...
Background: Diazepam is a classic benzodiazepine drug that has been widely used for disorders such as anxiety, sleep disorders, and epilepsy, over the past 59 years. The study of diazepam has always been an important research topic. However, there are few bibliometric analyses or systematic studies in this field. This study undertook bibliometric and visual analysis to ascertain the current status of diazepam research, and to identify research hotspots and trends in the past 10 years, to better understand future developments in basic and clinical research.
Methods: Articles and reviews of diazepam were retrieved from the Web of Science core collection. Using CiteSpace, VOSviewer, and Scimago Graphica software, countries, institutions, authors, journals, references, and keywords in the field were visually analyzed.
Results: A total of 3,870 publications were included. Diazepam-related literature had high volumes of publications and citations. The majority of publications were from the USA and China. The highest number of publications and co-citations, among the authors, was by James M Cook. Epilepsia and the Latin American Journal of Pharmacy were the journals with the most publications on diazepam and Epilepsia was the most frequently cited journal. Through a comprehensive analysis of keywords and references, we found that current research on diazepam has focused on its mechanism of action, application in disease, pharmacokinetics, risk, assessment, and management of use, status epilepticus, gamma-aminobutyric acid receptors (GABAR), intranasal formulation, gephyrin, and that ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) is the current research hotspot.
Conclusion: Research on diazepam is flourishing. We identified research hotspots and trends in diazepam research using bibliometric and visual analytic methods. The clinical applications, mechanisms of action, pharmacokinetics, and assessment and management of the use of diazepam are the focus of current research and the development trend of future research.
... Further significant strong negative correlations were also observed between age and GABA A R β1 and β3 subunit levels in the DG region of males. Reduction or elimination of activity at β1 subunit-containing GABA A Rs has been shown to increase the efficacy of anxiolytic benzodiazepines in rats 55 . Therefore, age-related decreases in β1 subunit expression in older males might be beneficial for the efficacy and reducing potential side-effects of particular benzodiazepine treatments for anxiety and depression. ...
Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the nervous system. The GABA signaling system in the brain is comprised of GABA synthesizing enzymes, transporters, GABAA and GABAB receptors (GABAAR and GABABR). Alterations in the expression of these signaling components have been observed in several brain regions throughout aging and between sexes
in various animal models. The hippocampus is the memory centre of the brain and is impaired in several age-related disorders. It is composed of two main regions: the Cornu Ammonis (CA1-4) and
the Dentate Gyrus (DG), which are interconnected with the Entorhinal Cortex (ECx). The age- and sex-specific changes of GABA signaling components in these regions of the human brain have not
been examined. This study is the first to determine the effect of age and sex on the expression of GABA signaling components-GABAAR α1,2,3,5, β1-3, γ2, GABABR R1 and R2 subunits and the GABA synthesizing enzymes GAD 65/67-in the ECx, and the CA1 and DG regions of the human hippocampus using Western blotting. No significant differences were found in GABAAR α1,2,3,5, β1-3, γ2, GABABR R1 and R2 subunit and GAD65/76 expression levels in the ECx, CA1 and DG regions between the younger and older age groups for both sexes. However, we observed a significant negative correlation between age and GABAAR α1subunit level in the CA1 region for females; significant negative correlation between age and GABAAR β1, β3 and γ2 subunit expression in the DG region for males. In females a significant positive correlation was found between age and GABAAR γ2 subunit expression in the ECx and GABABR R2 subunit expression in the CA1 region. The results indicate that age and sex do not affect the expression of GAD 65/67. In conclusion, our results show age- and sex-related GABAA/BR subunit alterations in the ECx and hippocampus that might significantly influence GABAergic neurotransmission and underlie disease susceptibility and progression.
... The compounds in this study found to be most efficacious in mitigating seizure behavior (SL-651,498, 2-261, and SB-205384) have promising therapeutic potential for treating TETSinduced seizures in mammals. All three compounds (SL-651,498, 2-261, and SB-205384) have previously been used in mammalian models and shown to have anxiolytic properties, with SL-651,498 and 2-261 causing minimal ataxia (Gee et al., 2010;Licata et al., 2005;Navarro et al., 2006). Better understanding of TETS toxicity will allow the direct targeting of necessary subunits, leading to safer and more effective treatments. ...
The chemical threat agent tetramethylenedisulfotetramine (TETS) is a γ-aminobutyric acid type A receptor (GABA AR) antagonist that causes life threatening seizures. Currently, there is no specific antidote for TETS intoxication. TETS-induced seizures are typically treated with benzodiazepines, which function as nonselective positive allosteric modulators (PAMs) of synaptic GABAARs. The major target of TETS was recently identified as the GABAAR α2β3γ2 subtype in electrophysiological studies using recombinantly expressed receptor combinations. Here, we tested whether these in vitro findings translate in vivo by comparing the efficacy of GABAAR subunit-selective PAMs in reducing TETS-induced seizure behavior in larval zebrafish. We tested PAMs targeting α1, α2, α2/3/5, α6, ß2/3, ß1/2/3, and δ subunits and compared their efficacy to the benzodiazepine midazolam (MDZ). The data demonstrate that α2- and α6-selective PAMs (SL-651,498 and SB-205384, respectively) were effective at mitigating TETS-induced seizure-like behavior. Combinations of SB-205384 and MDZ or SL-651,498 and 2–261 (ß2/3-selective) mitigated TETS-induced seizure-like behavior at concentrations that did not elicit sedating effects in a photomotor behavioral assay, whereas MDZ alone caused sedation at the concentration required to stop seizure behavior. Isobologram analyses suggested that SB-205384 and MDZ interacted in an antagonistic fashion, while the effects of SL-651,498 and 2–261 were additive. These results further elucidate the molecular mechanism by which TETS induces seizures and provide mechanistic insight regarding specific countermeasures against this chemical convulsant.
... The differential influence of the β subunit appeared selective for α2, but not the α1 subunit-containing GABA A R (Table 1). This reduced activity at the β1-subunit subtype may diminish the propensity to cause sedative/ataxic side effects in vivo at therapeutically relevant doses (Gee et al., 2010;Hogenkamp et al., 2014), although note the sedative effects of etomidate are mediated by GABA A Rs incorporating the β2-subunit (Reynolds et al., 2003). ...
... However, the activity of δ-GABA A R PAMs are governed in part by physiological firing patterns (Section 2), whereas this is presumably less so for direct agonists such as gaboxadol Weir et al., 2017). The non-steroidal small molecules 2-261 and 2-329 (Table 1), act as GABA A R PAMs via a binding site distinct from that of neurosteroids, or benzodiazepines (Gee et al., 2010). They compare favorably to the neurosteroids, including those currently in clinical development, in terms of potency, efficacy and selectivity for GABA A R subtypes including the α4β3δ isoform (Table 1). ...
... They display unique pharmacological properties, which can be readily manipulated through their novel SAR to produce desirable side effect/safety profiles based on GABA A R receptor subunit-selectivity. For example, the orally bioavailable prototype, 2-261, retains anxiolytic activity without ataxia, cognitive impairment, ethanol potentiation, rewarding effects (linked to addiction), tolerance, or withdrawal (Gee et al., 2010;Yoshimura et al., 2014). Furthermore, in rodents 2-261 has a neurosteroid-like profile in blocking electrical-and chemical-kindled seizures and organophosphate-induced SE, and exhibits activity in chronic pain (Reddy et al., 2018;Johnstone et al., 2019a, b). ...
In the 1980s particular endogenous metabolites of progesterone and of deoxycorticosterone were revealed to be potent, efficacious, positive allosteric modulators (PAMs) of the GABAA receptor (GABAAR). These reports were followed by the discovery that such steroids may be synthesised not only in peripheral endocrine glands, but locally in the central nervous system (CNS), to potentially act as paracrine, or autocrine “neurosteroid” messengers, thereby fine tuning neuronal inhibition. These discoveries triggered enthusiasm to elucidate the physiological role of such neurosteroids and explore whether their levels may be perturbed in particular psychiatric and neurological disorders. In preclinical studies the GABAAR-active steroids were shown to exhibit anxiolytic, anticonvulsant, analgesic and sedative properties and at relatively high doses to induce a state of general anaesthesia. Collectively, these findings encouraged efforts to investigate the therapeutic potential of neurosteroids and related synthetic analogues. However, following over 30 years of investigation, realising their possible medical potential has proved challenging. The recent FDA approval for the natural neurosteroid allopregnanolone (brexanolone) to treat postpartum depression (PPD) should trigger renewed enthusiasm for neurosteroid research. Here we focus on the influence of neuroactive steroids on GABA-ergic signalling and on the challenges faced in developing such steroids as anaesthetics, sedatives, analgesics, anticonvulsants, antidepressants and as treatments for neurodegenerative disorders.
... We recently reported 2-261 as a prototype non-steroid small molecule with neurosteroid-like activity at extrasynaptic α 4 β 3 δ GABA A Rs similar to allopregnanolone or ganaxolone (unpublished observations). 2-261 and related enaminone modulators are non-steroidal small molecules that modulate synaptic GABA A Rs similar to neurosteroids (Hogenkamp et al., 2007;Gee et al., 2010;Yoshimura et al., 2014). 2-261 binds to a distinct site on GABA A Rs and is functionally coupled to neurosteroid and benzodiazepine sites by positive heterotropic cooperativity ( Hogenkamp et al., 2007). ...
... The topological separation of drug binding sites on the receptor offers the potential for additive or synergistic effects when combined with benzodiazepines due to allosteric changes in receptor conformation. 2-261 and related enaminones display unique pharmacological properties that can be manipulated to produce desirable side effect/safety profiles based on receptor subunitselectivity not possible with other classes of GABA A R modulators (Gee et al., 2010). For example, 2-261 retains anxiolytic activity without ataxia, sedation, cognitive impairment, rewarding effects (linked to addiction), tolerance or withdrawal ( Yoshimura et al., 2014). ...
... While this sedative/hypnotic state will likely reduce gamma power or spike rate and halt seizure progression in a hospital setting for certain periods of time, there is questionable benefit to soldiers on the battlefield who need to remain at least conscious enough to be ambulatory for extraction to a hospital setting. Therefore, we tested whether a non-sedating enaminone, 2-261 ( Gee et al., 2010;Yoshimura et al., 2017), could be an alternative to other extrasynaptic GABA A Rs modulators to reverse organophosphosphorus nerve agent-induced SE. ...
Seizures induced by organophosphorus nerve agent exposure become refractory to treatment with benzodiazepines because these drugs engage synaptic γ-aminobutyric acid-A receptors (GABAARs) that rapidly internalize during status epilepticus (SE). Extrasynaptic GABAARs, such as those containing α4β3δ subunits, are a putative pharmacological target to comprehensively manage nerve agent-induced seizures since they do not internalize during SE and are continuously available for activation. Neurosteroids related to allopregnanolone have been tested as a possible replacement for benzodiazepines because they target both synaptic and extrasynaptic GABAARs receptors. A longer effective treatment window, extended treatment efficacy, and enhanced neuroprotection represent significant advantages of neurosteroids over benzodiazepines. However, neurosteroid use is limited by poor physicochemical properties arising from the intrinsic requirement of the pregnane steroid core structure for efficacy rendering drug formulation problematic. We tested a non-steroidal enaminone GABAAR modulator that interacts with both synaptic and extrasynaptic GABAARs on a binding site distinct from neurosteroids or benzodiazepines for efficacy to control electrographic SE induced by diisopropyl fluorophosphate or soman intoxication in rats. Animals were treated with standard antidotes, and experimental therapeutic treatment was given following 1 h (diisopropyl fluorophosphate model) or 20 min (soman model) after SE onset. We found that the enaminone 2-261 had an extended duration of seizure termination (>10 h) in the diisopropyl fluorophosphate intoxication model in the presence or absence of midazolam (MDZ). 2-261 also moderately potentiated MDZ in the soman-induced seizure model but had limited efficacy as a stand-alone anticonvulsant treatment due to slow onset of action. 2-261 significantly reduced neuronal death in brain areas associated with either diisopropyl fluorophosphate- or soman-induced SE. 2-261 represents an alternate chemical template from neurosteroids for enhancing extrasynaptic α4β3δ GABAAR activity to reverse SE from organophosphorous intoxication.
... subtype-selective GABAA receptor PAMs have been shown to produce reduced motor-impacting effects[86][87][88].Finally, while lower sedative effects are desirable, their elimination is not necessarily required for an improved therapeutic agent. The benzodiazepine, alprazolam, is highly prescribed for anxiety despite sedative properties. ...
Enhancement of GABAA receptor inhibition has long been used in the treatment of
anxiety beginning with meprobamate, diazepam, chlordiazepoxide, and alprazolam
in present times. Positive allosteric modulation of GABAA receptors has thus proven its
place in medical practice. Subsequent work focused on the design of compounds with
reduced sedative liabilities. Several non-benzodiazepine GABAA-positive allosteric
modulator (PAM) compounds (MRK-409, TPA-023, TPA-023B, NS11821, AZD7325
and PF-06372865) were tested in early clinical trials but suffered from signs of
sedation and motor impairment and only three compounds progressed to proof of
concept studies (TPA-023, AZD7325 and PF-06372865). TPA-023 was terminated
due to toxicity in preclinical species while AZD7325 and PF-06372865 did not
achieve efficacy endpoints in clinical trials. All compounds tested in Phase-II trials
produced some signs of sedation at the minimum effective dose. We highlight a new
compound, KRM-II-81, that is an imidazodiazepine selective for GABAA receptors
containing 2/3 proteins. KRM-II-81 has demonstrated a reduced liability for motorimpairing and respiratory effects compared to non-selective agents. KRM-II-81 has
shown efficacy in animal models of epilepsy and is active in models for which other
standard-of-care antiepileptics are not active. KRM-II-81 also produces anxiolyticlike effects but with minimal sedation. In contrast to benzodiazepines like diazepam,
KRM-II-81 also produces anti-nociceptive effects including reduction in pain responses
in models of neuropathic pain. Unlike diazepam, KRM-II-81 displays antidepressantlike effects. KRM-II-81 dampens cortical excitability in mice with traumatic brain
injury. Thus, KRM-II-81 is a newly discovered, non-benzodiazepine compound, which
targets a selective population of GABAA receptors for improved therapeutic gain and
reduced side effects.
... But actually they are not b2/3 selective at all, and only preferentially modulate b2/3-containing GABA A receptors. In addition, they also modulate b1-containing receptors and sometimes to a quite significant extent (Gee et al., 2010). ...
... 3. b2/3-Selective Enaminones (Compound 2-261). Empirical observations of the b2/3 selective compounds loreclezole, mefenamic acid, tracazolate, and etifoxine (section V.J.1) in both animals and humans provided anecdotal evidence for the possibility that the degree of activation of b1 subunit-containing GABA A receptors may contribute to their sedative/ataxic properties (Gee et al., 2010). Based on this hypothesis, several enaminones were investigated for their positive allosteric modulation of a1b1g2 or a1b2g2 GABA A receptors. ...
... Based on this hypothesis, several enaminones were investigated for their positive allosteric modulation of a1b1g2 or a1b2g2 GABA A receptors. These compounds allosterically modulate GABA A receptors via a so far unidentified binding site and compared with other b2/3-selective compounds, such as loreclezole, tracazolate, or etomidate, exert a dramatically reduced maximal efficacy at a1b1g2 over a1b2g2 GABA A receptors (Gee et al., 2010). GABA-dependent modulation by the prototypic compound 2-261 (Fig. 38) at b2/3containing receptors did not appear to be strongly dependent on the type of a subunits. ...
GABAA receptors are the major inhibitory transmitter receptors in the brain. They are ligand-gated chloride channels and the site of action of benzodiazepines, barbiturates, neuroactive steroids, anesthetics, and convulsants. GABAA receptors are composed of five subunits that can belong to different subunit classes. The existence of 19 homologous subunits and their distinct regional, cellular, and subcellular distribution gives rise to a large number of GABAA receptor subtypes with distinct pharmacology, which modulate different functions of the brain. A variety of compounds have been identified that were claimed to modulate selectively individual GABAA receptor subtypes. However, many of these compounds have only incompletely been investigated or, in addition to a preferential modulation of a receptor subtype, also modulate other subtypes at similar concentrations. Although their differential efficacy at distinct receptor subtypes reduced side effects in behavioral experiments in rodents, the exact receptor subtypes mediating their behavioral effects cannot be unequivocally delineated. In addition, the discrepant in vivo effects of some of these compounds in rodents and man raised doubts on the applicability of the concept of receptor subtype selectivity as a guide for the development of clinically useful drugs. Here, we provide an up-to-date review on the currently available GABAA receptor subtype-selective ligands. We present data on their actual activity at GABAA receptor subtypes, discuss the translational aspect of subtype-selective drugs, and make proposals for the future development of ligands with better anxioselectivity in humans. Finally, we discuss possible ways to strengthen the conclusions of behavioral studies with the currently available drugs.
... It has been pointed out, however, that non-sedating anxiolytics are not generally a1-sparing in functional assays using recombinant systems [46]. Other lines of research in fact correlated b isoforms with sedative-like, ataxic, or narcosisinducing drug efficacy [47,48]. ...
Everyday activity is based on a subtle equilibrium of excitatory and inhibitory neuronal systems. The most prominent players in neuronal inhibition are synaptic and extrasynaptic GABAA receptors. Benzodiazepines are popular drugs that act as positive allosteric modulators of a subset of these receptors. Benzodiazepines have sedative, hypnotic, muscle-relaxant, and anticonvulsive effects, and are of outstandingly low overdose risk. The discovery of a large number of subtypes of GABAA receptors has raised hopes for a clear separation of this spectrum of actions. We discuss here how far this separation has been achieved, and outline recent progress towards the discovery of novel ligands for canonical and non-canonical binding sites.
