Allosteric Modulators Of GABAA Receptors Research Articles

Clinical Sleep-Wake Disorders II: Focus on Insomnia and Circadian Rhythm Sleep Disorders.

Insomnia and circadian rhythm sleep disorders affect large proportions of the population and have pronounced effects on quality of life and daytime performance. While the neurobiology of insomnia is not yet fully understood, circadian rhythm sleep disorders are assumed to be caused by a mismatch between the individual circadian phase position and the desired sleep-wake schedule. Benzodiazepines and non-benzodiazepine positive allosteric GABAA receptor modulators improve sleep onset and maintenance in the short-term treatment of insomnia. However, tolerance and dependence are important side effects. Sedating antidepressants are frequently prescribed for insomnia, however, only few randomised controlled trials have been published so far. Melatonin and melatonin receptor agonists are considered to be an option for the treatment of insomnia especially because of their minimal abuse potential and safety. First data on orexin (aka hypocretin) receptor antagonists are promising, however, the risk-benefit ratio needs to be further evaluated. With respect to circadian rhythm sleep disorders, there is solid evidence from meta-analyses supporting the use of melatonin in jet lag disorder to accelerate entrainment to the new time zone, and in delayed sleep phase disorder to advance sleep-wake rhythms. In addition to that, there is evidence supporting the use of melatonin in patients with shift work disorder in order to promote daytime sleep after night shifts.

Ester to amide substitution improves selectivity, efficacy and kinetic behavior of a benzodiazepine positive modulator of GABAA receptors containing the α5 subunit

We have synthesized and characterized MP-III-022 ((R)−8-ethynyl-6-(2-fluorophenyl)-N,4-dimethyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxamide) in vitro and in vivo as a binding- and efficacy-selective positive allosteric modulator of GABAA receptors containing the α5 subunit (α5GABAARs). By approximation of the electrophysiological responses which the estimated free rat brain concentrations can induce, we demonstrated that convenient systemic administration of MP-III-022 in the dose range 1–10mg/kg may result in a selective potentiation, over a wide range from mild to moderate to strong, of α5βγ2 GABAA receptors. For eliciting a comparable range of potentiation, the widely studied parent ligand SH-053-2′F-R-CH3 containing an ester moiety needs to be administered over a much wider dose range (10–200mg/kg), but at the price of activating non-α5 GABAARs as well as the desired α5GABAARs at the highest dose. At the dose of 10mg/kg, which elicits a strong positive modulation of α5GABAARs, MP-III-022 caused mild, but significant muscle relaxation, while at doses 1–10mg/kg was devoid of ataxia, sedation or an influence on the anxiety level, characteristic for non-selective benzodiazepines. As an amide compound with improved stability and kinetic properties, MP-III-022 may represent an optimized tool to study the influence of α5GABAARs on the neuronal pathways related to CNS disorders such as schizophrenia, Alzheimer's disease, Down syndrome or autism.

Characterization of GABAA receptor ligands with automated patch-clamp using human neurons derived from pluripotent stem cells

IntroductionAutomated patch clamp is a recent but widely used technology to assess pre-clinical drug safety. With the availability of human neurons derived from pluripotent stem cells, this technology can be extended to determine CNS effects of drug candidates, especially those acting on the GABAA receptor. MethodsiCell Neurons (Cellular Dynamics International, A Fujifilm Company) were cultured for ten days and analyzed by patch clamp in the presence of agonist GABA or in combination with positive allosteric GABAA receptor modulators. Both efficacy and affinity were determined. In addition, mRNA of GABAA receptor subunits were quantified by qRT-PCR. ResultsWe have shown that iCell Neurons are compatible with the IonFlux microfluidic system of the automated patch clamp instrument. Resistance ranging from 15 to 25MΩ was achieved for each trap channel of patch clamped cells in a 96-well plate format. GABA induced a robust change of current with an EC50 of 0.43μM. Positive GABAA receptor modulators diazepam, HZ-166, and CW-04-020 exhibited EC50 values of 0.42μM, 1.56μM, and 0.23μM, respectively. The α2/α3/α5 selective compound HZ-166-induced the highest potentiation (efficacy) of 810% of the current induced by 100nM GABA. Quantification of GABAA receptor mRNA in iCell Neurons revealed high levels of α5 and β3 subunits and low levels of α1, which is similar to the configuration in human neonatal brain. DiscussioniCell Neurons represent a new cellular model to characterize GABAergic compounds using automated patch clamp. These cells have excellent representation of cellular GABAA receptor distribution that enable determination of total small molecule efficacy and affinity as measured by cell membrane current change.

Investigation of Proposed Activity of Clarithromycin at GABAA Receptors Using [(11)C]Flumazenil PET.

Clarithromycin is a potential treatment for hypersomnia acting through proposed negative allosteric modulation of GABAA receptors. We were interested whether this therapeutic benefit might extend to Parkinson's disease (PD) patients because GABAergic neurotransmission is implicated in postural control. Prior to initiating clinical studies in PD patients, we wished to better understand clarithromycin's mechanism of action. In this work we investigated whether the proposed activity of clarithromycin at the GABAA receptor is associated with the benzodiazepine binding site using in vivo [(11)C]flumazenil positron emission tomography (PET) in primates and ex vivo [(3)H]flumazenil autoradiography in rat brain. While the studies demonstrate that clarithromycin does not change the K d of FMZ, nor does it competitively displace FMZ, there is preliminary evidence from the primate PET imaging studies that clarithromycin delays dissociation and washout of flumazenil from the primate brain in a dose-dependent fashion. These findings would be consistent with the proposed GABAA allosteric modulator function of clarithromycin. While the results are only preliminary, further investigation of the interaction of clarithromycin with GABA receptors and/or GABAergic medications is warranted, and therapeutic applications of clarithromycin alone or in combination with flumazenil, to treat hyper-GABAergic status in PD at minimally effective doses, should also be pursued.

Novel positive allosteric modulators of GABAA receptors with anesthetic activity.

GABAA receptors are the main inhibitory neurotransmitter receptors in the brain and are targets for numerous clinically important drugs such as benzodiazepines, anxiolytics and anesthetics. We previously identified novel ligands of the classical benzodiazepine binding pocket in α1β2γ2 GABAA receptors using an experiment-guided virtual screening (EGVS) method. This screen also identified novel ligands for intramembrane low affinity diazepam site(s). In the current study we have further characterized compounds 31 and 132 identified with EGVS as well as 4-O-methylhonokiol. We investigated the site of action of these compounds in α1β2γ2 GABAA receptors expressed in Xenopus laevis oocytes using voltage-clamp electrophysiology combined with a benzodiazepine site antagonist and transmembrane domain mutations. All three compounds act mainly through the two β+/α− subunit transmembrane interfaces of the GABAA receptors. We then used concatenated receptors to dissect the involvement of individual β+/α− interfaces. We further demonstrated that these compounds have anesthetic activity in a small aquatic animal model, Xenopus laevis tadpoles. The newly identified compounds may serve as scaffolds for the development of novel anesthetics.

Parkinson’s Disease: New Insights into Pathophysiology and Rehabilitative Approaches

Parkinson's disease (PD) is a multifactorial neurodegenerative disorder, but up till now, its etiology remains largely unknown. Progressive impairment of voluntary motor control, which represents the primary clinical feature of the disease, is caused by a loss of midbrain substantia nigra dopamine (DA) neurons. PD prevalence increases steadily with age. Therefore, with the aging population, we will see an increase in social and health impact of this disease. Currently, the drugs available improve physical performances and lengthen life expectancy and autonomy in the activities of daily living by reducing adverse events; it follows that they improve the quality of life of patients. However, these drugs are symptomatic and not curative. When the patient reaches the maximum allowable therapeutic dosage of levodopa, we have to face great difficulties and impotence in disease's management. Recently, the role of rehabilitation in the field of neurodegenerative diseases is becoming more and more important as it results from several studies. In this special issue, we wanted to focus our attention on the “holistic view” of the disease because we believe that only a multidisciplinary approach could help to face it in the best way. PD involves miscellaneous systems (physical, mental, and behavioral structures) affecting also the autonomy in self-care management. Furthermore, in this special issue we wanted to draw the attention to the importance of rehabilitation in PD. An amount of epidemiological evidence stressed the importance of the relationships between genetic and environmental factors. L. Polito et al. summarize evidence on gene-environment interplay in the development of PD focusing on susceptibility factors and causal genetic mutation. To the best of our knowledge, this review is the first attempt to analyze all those genetic factors that modify the impact of environmental exposure. Brilliantly, F. Magrinelli et al. contributed to examining the neuropathology, neuropharmacology, and neurophysiology of motor dysfunction of PD, with particular attention to those clinical features that impair quality of life and functional ability of patients. Starting from anatomic basis of the main circuit involved in PD, subsequently they analyzed the pathophysiology of PD motor symptoms (bradykinesia and tremor rigidity) and signs (postural balance, gait impairment, fatigue, and pain). Currently, pharmacological treatment of motor symptoms of PD depends mainly on the administration of dopaminergic drugs (levodopa and dopamine agonists) and other drugs involved in levodopa metabolism (monoamine oxidase-B inhibitors and catechol-O-methyltransferase inhibitors). Usually, in the late disease stages, PD patients treated by dopaminergic drugs display various complications, such as on-off fluctuations of Parkinsonian symptoms and dyskinesia. Unfortunately, most patients with advanced PD are unsatisfactorily controlled by the usual therapeutic approach. A. Daniele et al. examine other pharmacological targets regardless of the dopaminergic pathway. In particular, they focus on potential beneficial effects of zolpidem, a positive allosteric modulator of GABAA receptors, on the motor symptoms of PD. In fact, some studies have previously suggested that there is a high density of zolpidem binding sites in the globus pallidus and in the substantia nigra, which are the output structures of the basal ganglia. Two experimental works complete this brief special issue. The first is a case control study carried out by M. Tramontano et al. with the aim of investigating the efficacy of a blindfolded balance training in the improvement of gait parameters in people with PD compared to patients who underwent a standard physical therapy program. The second is a prospective open-label feasibility study evaluating the impact of 8-week action observation training (video-therapy) for the treatment of postural instability and balance impairment in PD patients. Although in this latter study A. Santamato et al. did not find positive results, they underline one of the most important shortcomings in PD rehabilitation field. In fact, absence of standard procedures to improve balance and posture in PD subjects makes this area a virgin field to explore. We hope that the contents of this special issue may help clinicians better understand the rationale of current pharmacological and rehabilitation strategies for PD and give viability to further studies that integrate current knowledge with innovations in the field of rehabilitation. Vincenza Frisardi Andrea Santamato Binith Cheeran

The role of neurosteroids in the pathogenesis of hepatic encephalopathy

Hepatic Encephalopathy (HE) represents a neuropsychiatric syndrome caused by acute or chronic liver failure. Hyperammonemia plays a pivotal role in the development of HE through modulation of neurotransmission, oxidative stress, neuroinflammation, mitochondrial dysfunction, and energy deficit. Neurosteroids contribute significantly to increased GABAergic tone in HE. Ammonia, in combination with manganese and proinflammatory cytokines, stimulate neurosteroid synthesis by up-regulation of translocator protein, a component of multiprotein complex that stimulate cholesterol transport into astrocytic mitochondria. Cholesterol serves as a substrate for the synthesis of neurosteroids allopregnanolone and tetrahydro-deoxycorticosterone. After release from astrocytes, allopregnanolone and tetrahydro-deoxycorticosterone potentiate GABAergic transmission by positive allosteric modulation of GABAA receptor, thus contributing to cognitive deficit and alterations in sleep-wake cycle. Additional potential mechanisms of neurosteroid action in HE include modulation of serotoninergic, cholinergic, glutamatergic, glycinergic, and opioid receptor activities, as well as modulation of gene expression. This review aimed to summarize current knowledge of the role of neurosteroids in the pathogenesis of HE.

Can a Positive Allosteric Modulation of GABAergic Receptors Improve Motor Symptoms in Patients with Parkinson’s Disease? The Potential Role of Zolpidem in the Treatment of Parkinson’s Disease

At present, patients with advanced Parkinson's disease (PD) are unsatisfactorily controlled by currently used anti-Parkinsonian dopaminergic drugs. Various studies suggest that therapeutic strategies based on nondopaminergic drugs might be helpful in PD. Zolpidem, an imidazopyridine widely used as sleep inducer, shows high affinity only for GABAA receptors containing the α-1 subunit and facilitates GABAergic neurotransmission through a positive allosteric modulation of GABAA receptors. Various observations, although preliminary, consistently suggest that in PD patients zolpidem may induce beneficial (and sometimes remarkable) effects on motor symptoms even after single doses and may also improve dyskinesias. Since a high density of zolpidem binding sites is in the two main output structures of the basal ganglia which are abnormally overactive in PD (internal globus pallidus, GPi, and substantia nigra pars reticulata, SNr), it was hypothesized that in PD patients zolpidem may induce through GABAA receptors an inhibition of GPi and SNr (and, possibly, of the subthalamic nucleus also), resulting in an increased activity of motor cortical areas (such as supplementary motor area), which may give rise to improvement of motor symptoms of PD. Randomized clinical trials are needed in order to assess the efficacy, safety, and tolerability of zolpidem in treating motor symptoms of PD.

Inhibitory respiratory responses to progesterone and allopregnanolone in newborn rats chronically treated with caffeine.

In premature newborns, recurrent apnoea is systematically treated with caffeine to prevent long-term neurocognitive disorders, but a substantial percentage of apnoea persists particularly in neonates born before 28 weeks of gestation. Progesterone has been proposed as a respiratory stimulant potentially suitable for the treatment of newborn apnoea persistent to caffeine. Accordingly we asked whether acute progesterone administration reduces apnoea frequency in newborn rats treated with caffeine. Surprisingly our results show that in newborn rats treated with caffeine, administration of progesterone inhibits breathing and increases apnoea frequency. Additional experiments showed an enhanced GABAergic inhibitory drive on breathing after caffeine treatment, and that progesterone is converted to allopregnanolone (an allosteric modulator of GABAA receptors) to inhibit breathing. We conclude that combining progesterone and chronic caffeine is not an option in preterm neonates, unless the effects of allopregnanolone can be counteracted. Caffeine is the main treatment for apnoea in preterm neonates, but its interactions with other respiratory stimulants like progesterone are unknown. We tested the hypothesis that the addition of progesterone to caffeine treatments further stimulates ventilation. Newborn rats were treated with water (control) or caffeine (15 mg kg(-1)) by daily gavage between postnatal day (P)3 and P12. At P4 and P12, we measured apnoea frequency, ventilatory responses and metabolic parameters under both normoxia and hypoxia (12% O2, 20 min) following an acute administration of either saline or progesterone (4 mg kg(-1); i.p.). Progesterone injection increased the serum levels of both progesterone and its neuroactive metabolite allopregnanolone. Progesterone had no effect on ventilation in control rats under normoxia. Progesterone depressed ventilation in P12 caffeine-treated rats under normoxia and hypoxia and increased apnoea frequency in both P4 and P12 rats. Because allopregnanolone is an allosteric modulator of GABAA receptors and caffeine may enhance GABAergic inhibition in newborns, we studied the effects of the GABAA receptor antagonist bicuculline at 0, 1, 2 and 3 mg kg(-1) doses and allopregnanolone (10 mg kg(-1) dose) in P12 rats. In caffeine-treated rats, bicuculline enhanced ventilation, while allopregnanolone decreased ventilation and increased total apnoea time. Progesterone had no effect on ventilation and apnoea frequency in caffeine-treated rats injected with finasteride, which blocks the conversion of progesterone to allopregnanolone. We conclude that combining progesterone and chronic caffeine therapy is not an option for the treatment of persistent apnoea in preterm neonates, unless the effects of allopregnanolone can be counteracted.

Negative allosteric modulation of GABAA receptors inhibits facilitation of brain stimulation reward by drugs of abuse in C57BL6/J mice.

There is an emerging body of evidence that implicates a crucial role of γ-aminobutyric acid subtype A (GABAA) receptors in modulating the rewarding effects of a number of abused drugs. Modulation of GABAA receptors may therefore represent a novel drug-class independent mechanism for the development of abuse treatment pharmacotherapeutics. We tested the hypothesis that the GABAA receptor benzodiazepine-site (BDZ) negative modulator Ro15-4513 would reduce the reward-related effects of three pharmacologically dissimilar drugs; toluene vapor, d-methamphetamine, and diazepam using intracranial self-stimulation (ICSS) in mice. We also examined whether Ro15-4513 attenuated dopamine release produced by d-methamphetamine in an in vivo microdialysis procedure. Ro15-4513 abolished ICSS reward facilitation produced by all three abused drugs at Ro15-4513 doses which had no effect on ICSS when administered alone. In contrast, the BDZ antagonist flumazenil only attenuated the ICSS-facilitating effects of diazepam. Administration of the same dose of Ro15-4513 which abolished drug-facilitated ICSS produced a 58% decrease in d-methamphetamine-stimulated dopamine in the nucleus accumbens of mice relative to d-methamphetamine alone. These results demonstrate that negative modulation of GABAA receptors can produce profound reductions in reward-related effects of a diverse group of drugs that activate the mesolimbic reward pathway through different mechanisms. These data suggest that pharmacological modulation of GABAA receptors may represent a viable pathway for the development of drug abuse pharmacotherapies.

Clarithromycin in γ-aminobutyric acid-Related hypersomnolence: A randomized, crossover trial.

Some central hypersomnolence syndromes are associated with a positive allosteric modulator of γ-aminobutyric acid (GABA)-A receptors in cerebrospinal fluid. Negative allosteric modulators of GABA-A receptors, including clarithromycin, have been reported to reduce sleepiness in these patients. We sought to systematically assess the effects of clarithromycin on objective vigilance and subjective sleepiness. This was a 5-week, randomized, placebo-controlled, double-blind, crossover trial of clarithromycin 500mg with breakfast and lunch, in patients with hypersomnolence syndromes (excluding narcolepsy with cataplexy) and evidence for abnormal cerebrospinal fluid potentiation of GABA-A receptors. The study occurred at a university-affiliated medical center. The primary outcome measure was median reaction time on the psychomotor vigilance task (PVT) at week 2 in each condition. Secondary outcomes included the Epworth Sleepiness Scale, Stanford Sleepiness Scale, Functional Outcomes of Sleep Questionnaire, Pittsburgh Sleep Quality Index, SF-36, and additional PVT measures. Twenty-three patients began treatment. Three patients dropped out, and final analyses were performed on 20 complete cases. Median reaction time was not significantly different between clarithromycin and placebo. Subjective measures of sleepiness were significantly improved on clarithromycin versus placebo. Altered taste perception occurred, but was the only side effect more common on clarithromycin than placebo. No serious adverse events occurred. Subjective sleepiness, but not psychomotor vigilance, improved during a 2-week course of clarithromycin. Although additional studies are needed, this suggests that clarithromycin may be a reasonable treatment option in patients with treatment-refractory hypersomnolence. This trial was registered at ClinicalTrials.gov (NCT01146600) and supported by the American Sleep Medicine Foundation.

Rapid Antidepressant Action and Restoration of Excitatory Synaptic Strength After Chronic Stress by Negative Modulators of Alpha5-Containing GABAA Receptors.

Selective serotonin reuptake inhibitors (SSRIs) are the primary pharmacological treatment for depression, but SSRIs are effective in only half of the patients and typically take several weeks to relieve symptoms. The NMDA receptor antagonist ketamine exerts a rapid antidepressant action, but has troubling side effects. We hypothesized that negative allosteric modulators of GABAA receptors would exert similar effects on brain activity as ketamine, but would not exert as many side effects if targeted only to GABAA receptors containing α5 subunits, which are enriched in the hippocampus and prefrontal cortex. Here, we show that the α5-selective negative modulator L-655,708 reversed the alterations in hedonic behavior in the sucrose preference and social interaction tests produced by two different chronic stress paradigms in rats within 24 h of systemic administration. Similar effects were observed with another α5-selective negative modulator, MRK-016. L-655,708 had no effect on hedonic or open-field behavior in unstressed animals. Within 24 h, L-655,708 injection also restored the strength of pathologically weakened excitatory synaptic transmission at the stress-sensitive temporoammonic-CA1 synapse, measured electrophysiologically, and increased levels of the GluA1 subunit of the AMPA receptor, measured with western blotting. We suggest that the ability of L-655,708 to restore excitatory synaptic strength rapidly may underlie its ability to restore stress-induced behavioral alterations rapidly, supporting evidence that dysfunction of multiple excitatory synapses in cortico-mesolimbic reward pathways contributes, in part, to the genesis of depression. Negative allosteric modulators of α5 subunit-containing GABAA receptors represent a promising novel class of fast-acting and clinically viable antidepressant compounds.

Combined treatment with diazepam and allopregnanolone reverses tetramethylenedisulfotetramine (TETS)-induced calcium dysregulation in cultured neurons and protects TETS-intoxicated mice against lethal seizures

Tetramethylenedisulfotetramine (TETS) is a potent convulsant GABAA receptor blocker. Mice receiving a lethal dose of TETS (0.15 mg/kg i.p.) are rescued from death by a high dose of diazepam (5 mg/kg i.p.) administered shortly after the second clonic seizure (∼20 min post-TETS). However, this high dose of diazepam significantly impairs blood pressure and mobility, and does not prevent TETS-induced neuroinflammation in the brain. We previously demonstrated that TETS alters synchronous Ca2+ oscillations in primary mouse hippocampal neuronal cell cultures and that pretreatment with the combination of diazepam and allopregnanolone at concentrations having negligible effects individually prevents TETS effects on intracellular Ca2+ dynamics. Here, we show that treatment with diazepam and allopregnanolone (0.1 μM) 20 min after TETS challenge normalizes synchronous Ca2+ oscillations when added in combination but not when added singly. Similarly, doses (0.03–0.1 mg/kg i.p.) of diazepam and allopregnanolone that provide minimal protection when administered singly to TETS intoxicated mice increase survival from 10% to 90% when given in combination either 10 min prior to TETS or following the second clonic seizure. This therapeutic combination has negligible effects on blood pressure or mobility. Combined treatment with diazepam and allopregnanolone also decreases TETS-induced microglial activation. Diazepam and allopregnanolone have distinct actions as positive allosteric modulators of GABAA receptors that in combination enhance survival and mitigate neuropathology following TETS intoxication without the adverse side effects associated with high dose benzodiazepines. Combination therapy with a benzodiazepine and neurosteroid represents a novel neurotherapeutic strategy with potentially broad application.

The effects of central delivery of a positive allosteric modulator of GABAA receptors upon stress and hypertension in Schlager hypertensive mice

Hypertensive Schlager mice (BPH/2J) have neurogenic hypertension associated with abnormal reactivity of neurons in the forebrain integrating the response to aversive stress. We found that they also have functional and molecular differences in GABAA receptors compared to their normotensive counterparts (BPN/3J). Allopregnanolone is an endogenous neurosteroid reduced by chronic stress and when administered, decreases anxiety by positive allosteric modulation of GABAA receptors. We therefore compared the effect of chronic intracerebroventricular allopregnanolone and its vehicle on blood pressure and stress reactivity in BPH/2J and BPH/3J mice. Implantation of telemetric probes enabled continuous recordings of blood pressure (BP) at rest and during aversive stressors (restraint and dirty cage switch stress). Two weeks of allopregnanolone reduced MAP (‐9.8±4.3mmHg) and the depressor response to ganglionic blockade (‐10.5mmHg) in BPH/2J but had little effect in BPN/3J. Furthermore, allopregnanolone selectively reduced the pressor response to restraint stress by 27.4% in BPH/2J mice. The selective antihypertensive and stress inhibitory effects of allopregnanolone in BPH/2J mice suggests that allosteric modulation of GABAA receptors has therapeutic potential to treat stress related hypertension.

GABAergic modulation in central sensitization in humans: a randomized placebo-controlled pharmacokinetic-pharmacodynamic study comparing clobazam with clonazepam in healthy volunteers.

Positive allosteric modulators of GABAA receptors (GAMs) acting at specific subtypes of GABAA receptors effectively restore compromised spinal pain control in rodents. Studies addressing a similar antihyperalgesic effect in humans are sparse and are hampered by sedative effects of nonselective GAMs available for use in humans. We present results from a randomized controlled double-blind crossover study in 25 healthy volunteers, which addressed potential antihyperalgesic actions of clobazam (CBZ) and clonazepam (CLN) at mildly sedating equianticonvulsive doses. Clobazam was chosen because of its relatively low sedative properties and CLN because of its use in neuropathic pain. Tolterodine (TLT) was used as an active placebo. The primary outcome parameter was a change in the area of cutaneous UVB irradiation-induced secondary hyperalgesia (ASH), which was monitored for 8 hours after drug application. Sedative effects were assessed in parallel to antihyperalgesia. Compared with TLT, recovery from hyperalgesia was significantly faster in the CBZ and CLN groups (P = 0.009). At the time point of maximum effect, the rate of recovery from hyperalgesia was accelerated by CBZ and CLN, relative to placebo by 15.7% (95% confidence interval [CI] 0.8-30.5), P = 0.040, and 28.6% (95% CI 4.5-52.6), P = 0.022, respectively. Active compounds induced stronger sedation than placebo, but these differences disappeared 8 hours after drug application. We demonstrate here that GAMs effectively reduce central sensitization in healthy volunteers. These results provide proof-of-principle evidence supporting efficacy of GAMs as antihyperalgesic agents in humans and should stimulate further research on compounds with improved subtype specificity.

The legacy of the benzodiazepine receptor: from flumazenil to enhancing cognition in Down syndrome and social interaction in autism.

The study of the psychopharmacology of benzodiazepines continues to provide new insights into diverse brain functions related to vigilance, anxiety, mood, epileptiform activity, schizophrenia, cognitive performance, and autism-related social behavior. In this endeavor, the discovery of the benzodiazepine receptor was a key event, as it supplied the primary benzodiazepine drug-target site, provided the molecular link to the allosteric modulation of GABAA receptors and, following the recognition of GABAA receptor subtypes, furnished the platform for future, more selective drug actions. This review has two parts. In a retrospective first part, it acknowledges the contributions to the field made by my collaborators over the years, initially at Hoffmann-La Roche in Basle and later, in academia, at the University and the ETH of Zurich. In the second part, the new frontier of GABA pharmacology, targeting GABAA receptor subtypes, is reviewed with special focus on nonsedative anxiolytics, antidepressants, analgesics, as well as enhancers of cognition in Down syndrome and attenuators of symptoms of autism spectrum disorders. It is encouraging that a clinical trial has been initiated with a partial inverse agonist acting on α5 GABAA receptors in an attempt to alleviate the cognitive deficits in Down syndrome.

Allosteric Modulation of GABAA Receptors by an Anilino Enaminone in an Olfactory Center of the Mouse Brain.

In an ongoing effort to identify novel drugs that can be used as neurotherapeutic compounds, we have focused on anilino enaminones as potential anticonvulsant agents. Enaminones are organic compounds containing a conjugated system of an amine, an alkene and a ketone. Here, we review the effects of a small library of anilino enaminones on neuronal activity. Our experimental approach employs an olfactory bulb brain slice preparation using whole-cell patch-clamp recording from mitral cells in the main olfactory bulb. The main olfactory bulb is a key integrative center in the olfactory pathway. Mitral cells are the principal output neurons of the main olfactory bulb, receiving olfactory receptor neuron input at their dendrites within glomeruli, and projecting glutamatergic axons through the lateral olfactory tract to the olfactory cortex. The compounds tested are known to be effective in attenuating pentylenetetrazol (PTZ) induced convulsions in rodent models. One compound in particular, KRS-5Me-4-OCF3, evokes potent inhibition of mitral cell activity. Experiments aimed at understanding the cellular mechanism underlying the inhibitory effect revealed that KRS-5Me-4-OCF3 shifts the concentration-response curve for GABA to the left. KRS-5Me-4-OCF3 enhances GABA affinity and acts as a positive allosteric modulator of GABAA receptors. Application of a benzodiazepine site antagonist blocks the effect of KRS-5Me-4-OCF3 indicating that KRS-5Me-4-OCF3 binds at the classical benzodiazepine site to exert its pharmacological action. This anilino enaminone KRS-5Me-4-OCF3 emerges as a candidate for clinical use as an anticonvulsant agent in the battle against epileptic seizures.

Allosteric modulation of GABAA receptors via multiple drug-binding sites.

GABAA receptors are ligand-gated ion channels composed of five subunits that can be opened by GABA and be modulated by multiple pharmacologically and clinically important drugs. Over the time, hundreds of compounds from different structural classes have been demonstrated to modulate, directly activate, or inhibit GABAA receptors, and most of these compounds interact with more than one binding site at these receptors. Crystal structures of proteins and receptors homologous to GABAA receptors as well as homology modeling studies have provided insights into the possible location of ligand interaction sites. Some of these sites have been identified by mutagenesis, photolabeling, and docking studies. For most of these ligands, however, binding sites are not known. Due to the high flexibility of GABAA receptors and the existence of multiple drug-binding sites, the unequivocal identification of interaction sites for individual drugs is extremely difficult. The existence of multiple GABAA receptor subtypes with distinct subunit composition, the contribution of distinct subunit sequences to binding sites of different receptor subtypes, as well as the observation that even subunits not directly contributing to a binding site are able to influence affinity and efficacy of drugs, contribute to a unique pharmacology of each GABAA receptor subtype. Thus, each receptor subtype has to be investigated to identify a possible subtype selectivity of a compound. Although multiple binding sites make GABAA receptor pharmacology even more complicated, the exploitation of ligand interaction with novel-binding sites also offers additional possibilities for a subtype-selective modulation of GABAA receptors.

Profile of anticonvulsant action of levetiracetam, tiagabine and phenobarbital against seizures evoked by DMCM (methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate) in neonatal rats

Levetiracetam (LEV) and tiagabine (TGB) are utilized for the treatment of seizures, including neonatal seizures. However, relatively little is known about the preclinical therapeutic profile of these drugs during brain development. The relative paucity of information regarding these drugs in neonatal animals may be due to their unusual profile of anticonvulsant action in experimental models. LEV and TGB are without effect against seizures in several common screening models (e.g., the maximal electroshock test, maximal pentylenetetrazole seizures), instead showing preferential efficacy against models of partial seizures. We have recently described a method for reliably evoking partial seizures in neonatal animals by systemic administration of the chemoconvulsant, DMCM (Kulick et al., 2014, Eur. J. Pharmacol., doi:10.1016/j.ejphar.2014.06.012). DMCM is a negative allosteric modulator of GABAA receptors, and offers a wide separation between doses required to evoke complex partial as compared to tonic–clonic seizures. Here we used DMCM to evaluate the effect of LEV and TGB against seizures in postnatal day (P) 10 rat pups. We compared the profile of LEV and TGB to that of phenobarbital (PB), the most widely utilized anticonvulsant in neonates. We found that LEV significantly protected against DMCM seizures when administered in doses of 10mg/kg and greater. TGB protected against DMCM-evoked seizures when administered in doses of 1mg/kg or greater. PB protected against DMCM-evoked seizures when administered in doses of 5mg/kg or greater. These data provide preclinical evidence for the efficacy of LEV and TGB in neonates and underscore the utility of DMCM for screening anticonvulsant action in neonatal animals.

New perspectives in neurosteroid action: open questions for future research.

New perspectives in neurosteroid action: open questions for future research.