Benzodiazepine Site Of GABAA Receptors Research Articles

Pharmacological Analysis of GABAA Receptor and Sigma1R Chaperone Interaction: Research Report I-Investigation of the Anxiolytic, Anticonvulsant and Hypnotic Effects of Allosteric GABAA Receptors' Ligands.

Two groups of facts have been established in previous drug development studies of the non-benzodiazepine anxiolytic fabomotizole. First, fabomotizole prevents stress-induced decrease in binding ability of the GABAA receptor's benzodiazepine site. Second, fabomotizole is a Sigma1R chaperone agonist, and exposure to Sigma1R antagonists blocks its anxiolytic effect. To prove our main hypothesis of Sigma1R involvement in GABAA receptor-dependent pharmacological effects, we performed a series of experiments on BALB/c and ICR mice using Sigma1R ligands to study anxiolytic effects of benzodiazepine tranquilizers diazepam (1 mg/kg i.p.) and phenazepam (0.1 mg/kg i.p.) in the elevated plus maze test, the anticonvulsant effects of diazepam (1 mg/kg i.p.) in the pentylenetetrazole-induced seizure model, and the hypnotic effects of pentobarbital (50 mg/kg i.p.). Sigma1R antagonists BD-1047 (1, 10, and 20 mg/kg i.p.), NE-100 (1 and 3 mg/kg i.p.), and Sigma1R agonist PRE-084 (1, 5, and 20 mg/kg i.p.) were used in the experiments. Sigma1R antagonists have been found to attenuate while Sigma1R agonists can enhance GABAARs-dependent pharmacological effects.

Synthesis and evaluation of avermectin–imidazo[1,2-a]pyridine hybrids as potent GABAA receptor modulators

The γ-aminobutyric acid type A (GABAA) receptors are pentameric transmembrane protein complexes. They have attracted extensive attention from the scientific community due to their significant pharmacological potential. Here we report the first synthesis of avermectin–imidazo[1,2-a]pyridine hybrids promising as GABAA receptor positive allosteric modulators (PAMs). An efficient multi-step protocol was elaborated for the installation of the 6-methyl-2-(p-tolyl)imidazo[1,2-a]pyridine pendant to the Avermectin B1a and Ivermectin skeletons through a linker. A variety of linkers were used in order to study the effect of disturbances in the hybrid structure on the GABAA receptor affinity. In vitro experiments showed that the lead compounds exhibited high potency (IC50 = 207 and 359 nM) for binding at the benzodiazepine site of GABAA receptors. In silico studies suggest that the hybrids are able to bind at the Ivermectin binding site of the GABAA receptor. The functional properties of the highest-affinity hybrid (compound 15e) as GABAAR PAM were evaluated by patch-clamp electrophysiological recordings of GABA-mediated currents in rat cerebellar Purkinje neurons. The results obtained suggest that the potentiating effect of hybrid compound 15e is due to its interaction both with benzodiazepine- and Ivermectin-binding sites of GABAARs. Drug-induced behavioral responses in adult zebrafish for hybrids correlate with an alternative mode of action of avermectin and imidazo[1,2-a]pyridine pharmacophores. The investigation of avermectin–imidazo[1,2-a]pyridine hybrid molecules with activity as GABAA receptor modulators is important for the discovery of safe and effective drugs for the treatment of neurological disorders and pest control agents.

Mechanism of action involved in the anxiolytic-like effects of Hibalactone isolated from Hydrocotyle umbellata L.

Background and aimHibalactone (HB) is a lignan related to the anxiolytic-like effects of Hydrocotyle umbellata L. However, there is a need to understand better the mechanism of action of this lignan to support the ethnopharmacological uses of the species. This work aimed to evaluate by in vivo and in silico analysis the mechanism of action of HB involved in its anxiolytic-like effects. Experimental procedureThe effects of HB in mice were evaluated on light-dark box (LDB) and elevated plus maze (EPM) tests. The participation of 5-HT1A receptor and the benzodiazepine site of GABAA receptor was evaluated to investigate the possible mechanism of action. In silico tools were used to better elucidate the anxiolytic-like effects of HB. ResultsOral treatment with HB at a dose of 33 mg/kg showed an anxiolytic-like effect in the LDB and EPM tests. Besides that, the treatment altered the ethological parameters, frequency of head dips, and stretched-attend postures (SAP), important to better describe the anxiolytic profile of HB. Pretreatment with flumazenil (2 mg/kg) reverted the anxiolytic-like effect of HB on LDB and EPM tests. On the other hand, pretreatment with NAN-190 (0.5 mg/kg) not reverted the activity observed. In silico predictions revealed the potential of HB to increase GABAergic neurotransmission. Pharmacophore modelling and docking simulations showed that HB might interact with the α1β2γ2 GABAA receptor. ConclusionTogether, the results presented herein suggest that activation of the benzodiazepine site of the GABAA receptor contributes to the anxiolytic-like effect of HB.

Инновационный модулятор ГАМК рецепторов — активатор микросомальной цитохром-P450 оксидазной системы печени у больных алкоголизмом

С помощью молекулярного докинга (программа Schrödinger, Glide) показано, что молекула м-Cl-БГМ (мета-хлорбензгидрилмочевина) комплементарна сайту связывания бензодиазепина ГАМКА рецепторов. Энергия связывания низкая (– 11,14 ккал/моль); м-Cl-БГМ взаимодействует с ключевыми аминокислотами на α1γ2 интерфейсе: Tyr159, Tyr209, H101 Phe77 с высокой степенью соответствия модели — dG вставки: 0,741. Связывание [3H]флунитразепама с сайтом бензодиазепина ГАМКА рецепторов головного мозга крыс при экспериментальном алкоголизме, получавших 14 дней м-Cl-БГМ в дозе 100 мг/кг в сутки, увеличивалось. Выявлены изменения фармакокинетических параметров (T1/2, Clt, MRT, МЕТ, AUC) модельного субстрата — антипирина в слюне здоровых добровольцев и больных алкоголизмом мужчин при применении галодифа (м-Cl-БГМ) в дозе 300 мг/сут в течение 21 дня. Элиминация антипирина у больных алкоголизмом была повышена вследствие активации микросомальной цитохром-P450 оксидазной системы печени.

Neuropharmacological assessment in mice and molecular docking of piperazine derivative LQFM212

Piperazine derivatives are an attractive class of chemical compounds for the treatment of various mental illness. Herein, we demonstrated the synthesis of LQFM212, a piperazine derivative, behavioral evaluation in mice and computational studies. In neuropharmacological assessment, LQFM212 treatment at doses of 18, 54 or 162 μmol/kg increased the sleep duration in sodium pentobarbital-induced sleep test. LQFM212 at dose of 162 μmol/kg increased climbing time in the chimney test and decreased the number of squares crossed in the open field test, suggesting that LQFM212 in high doses reduces spontaneous movement. However, LQFM212 treatment at the doses of 18 or 54 μmol/kg increased the preference for the center of field which could be indicative of anxiolytic-like effects. In elevated plus maze and light-dark box tests, LQFM212 treatment altered all parameters observed that demonstrate anxiolytic-like activity. These effects were reversed by flumazenil, mecamylamine, WAY-100635 and PCPA, but not with ketanserin, showing that anxiolytic-like activity involve benzodiazepine site of GABAA receptor, nicotinic and serotonergic pathways. Molecular docking of LQFM212 showed that the ligand has more interactions with GABAA receptor than with 5-HT1A receptor. Despite the involvement of benzodiazepine site on anxiolytic-like effect of LQFM212, treatment with this compound did not alter cognitive function in the step-down avoidance test. In this sense, this piperazine derivative is a good prototype for treating anxiety disorders with putative mechanism of action.

Etizolam: A rapid review on pharmacology, non-medical use and harms.

Etizolam is a thienodiazepine derivative, with high affinity for the benzodiazepine site of GABAA receptors. It is often referred to as a new (or novel) psychoactive substance, a 'designer' benzodiazepine or a 'street benzodiazepine'. Increasing reports of non-medical use, identification of etizolam as an ingredient in counterfeit medications and the common identification of etizolam in drug-related deaths, highlight the need for a greater understanding of etizolam. A rapid narrative review was conducted using PubMed and Google Scholar to synthesise what is known about etizolam to answer two research questions: (i) Does the pharmacological or toxicological profile of etizolam differ from other benzodiazepines?; and (ii) What is the nature and context of non-medical use and harms related to etizolam? Etizolam has a higher potency as an anxiolytic but lower lethality compared with diazepam. Few harms are documented with the therapeutic use of pharmaceutical products. Harms appear to be predominantly related to the use of etizolam in illicitly manufactured pills and occur almost exclusively in the context of mixed-drug toxicity. In therapeutic doses, there is little to suggest that etizolam is more harmful than other benzodiazepines. Most harms with etizolam appear to be related to the wide availability of illicitly manufactured pills, which are taken in unknown doses and combined with other substances. Current harm reduction advice, including avoiding combining opioids and benzodiazepines, remains relevant and increasingly important within an emerging culture of non-medical use.

Pharmacological Profile of the Novel Antiepileptic Drug Candidate Padsevonil: Characterization in Rodent Seizure and Epilepsy Models.

The antiepileptic drug (AED) candidate, (4R)-4-(2-chloro-2,2-difluoroethyl)-1-{[2-(methoxymethyl)-6-(trifluoromethyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl]methyl}pyrrolidin-2-one (padsevonil), is the first in a novel class of drugs that bind to synaptic vesicle protein 2 (SV2) proteins and the GABAA receptor benzodiazepine site, allowing for pre- and postsynaptic activity, respectively. In acute seizure models, padsevonil provided potent, dose-dependent protection against seizures induced by administration of pilocarpine or 11-deoxycortisol, and those induced acoustically or through 6 Hz stimulation; it was less potent in the pentylenetetrazol, bicuculline, and maximal electroshock models. Padsevonil displayed dose-dependent protective effects in chronic epilepsy models, including the intrahippocampal kainate and Genetic Absence Epilepsy Rats from Strasbourg models, which represent human mesial temporal lobe and absence epilepsy, respectively. In the amygdala kindling model, which is predictive of efficacy against focal to bilateral tonic-clonic seizures, padsevonil provided significant protection in kindled rodents; in mice specifically, it was the most potent AED compared with nine others with different mechanisms of action. Its therapeutic index was also the highest, potentially translating into a favorable efficacy and tolerability profile in humans. Importantly, in contrast to diazepam, tolerance to padsevonil's antiseizure effects was not observed in the pentylenetetrazol-induced clonic seizure threshold test. Further results in the 6 Hz model showed that padsevonil provided significantly greater protection than the combination of diazepam with either 2S-(2-oxo-1-pyrrolidinyl)butanamide (levetiracetam) or 2S-2-[(4R)-2-oxo-4-propylpyrrolidin-1-yl] butanamide (brivaracetam), both selective SV2A ligands. This observation suggests that padsevonil's unique mechanism of action confers antiseizure properties beyond the combination of compounds targeting SV2A and the benzodiazepine site. Overall, padsevonil displayed robust efficacy across validated seizure and epilepsy models, including those considered to represent drug-resistant epilepsy. SIGNIFICANCE STATEMENT: Padsevonil, a first-in-class antiepileptic drug candidate, targets SV2 proteins and the benzodiazepine site of GABAA receptors. It demonstrated robust efficacy across a broad range of rodent seizure and epilepsy models, several representing drug-resistant epilepsy. Furthermore, in one rodent model, its efficacy extended beyond the combination of drugs interacting separately with SV2 or the benzodiazepine site. Padsevonil displayed a high therapeutic index, potentially translating into a favorable safety profile in humans; tolerance to antiseizure effects was not observed.

Neuroprotective action of diazepam at very low and moderate doses in Alzheimer's disease model rats

Early manifestations of Alzheimer's disease (AD) include neuroinflammation, disrupted neurotransmission and cognitive deficits. Impairment of the GABAergic system is essentially involved in the pathogenesis of AD. Traditionally, agonists of GABAA receptors at doses above 1 mg/kg are known to possess memory impairing effects. However, we have previously found that GABAA receptor GABA site ligand muscimol at very low doses acted contrary – enhanced spatial learning/memory, as well as prevented neuroinflammation and augmented neurotransmission in AD model rats. Therefore, in the present study we focused on the assessment of the effects of non-sedative – very low (0.05 mg/kg) and moderate (1 mg/kg) – doses of diazepam, a positive allosteric modulator of benzodiazepine site of GABAA receptors. Its effects on spatial learning/memory and brain proteins related to neuroinflammation (GFAP and Iba-1), synaptic plasticity (SYP1), as well as acetylcholine breakdown and GABA biosynthesis were studied. Non-transgenic AD model rats (intracerebroventricular streptozocin injection) were used with the aim to mimic the pre-dementia stage of AD in humans. The obtained data showed that diazepam at both doses protected against streptozocin induced detrimental effects by enhancing spatial learning/memory, preventing neuroinflammation, preserving synaptic plasticity, as well as normalizing the hippocampal and cortical protein expression related to acetylcholine breakdown and GABA biosynthesis. One may suggest that at low and moderate doses diazepam is targeting non-specific, probably allosteric GABAA receptor sites, thus leading to stimulatory effects that can be beneficial for diazepam use in early pre-dementia stages of AD.

Behavioral Effects of α2/α3 Subtype‐Selective GABAA Receptor Positive Allosteric Modulators

Millions of individuals suffer from chronic pain. Opioids are effective for treating chronic pain but their use is limited due to undesirable side‐effects. Previous studies have shown that different subunits of the GABAA receptor are associated with different actions of the benzodiazepine site. While α2/α3 subunit‐containing GABAA receptors mediate antinociceptive effects, α1 subunit‐containing GABAA receptors mediate sedative and abuse‐related effects. This study sought to examine the behavioral effects of two α2/α3‐subtype selective PAMs: KRM‐II‐81 and NS16085. In a rat model of inflammatory pain (complete Freund's adjuvant) and a rat model of neuropathic pain (chronic constriction injury), both PAMs and the classical benzodiazepine midazolam attenuated mechanical hyperalgesia (as measured by von Frey test). In the procedure of food‐maintained operant responding, KRM‐II‐81 and NS16085 did not significantly decrease the response rate at doses that produced maximal antinociception. Contrastingly, midazolam significantly reduced the response rate at doses that attenuated mechanical hyperalgesia. In a horizontal wire test aimed to measure muscle relaxation, within the dose range that produced antinociception, only midazolam dose‐dependently increased the percentage of rats unable to grasp the wire, indicating muscle‐relaxant activity. These behavioral effects can be attenuated by benzodiazepine receptor antagonist flumazenil, confirming that the behavioral effects of these subtype‐selective GABAA PAMs are mediated through the benzodiazepine site of GABAA receptors. Taken together, while midazolam produced antinociceptive, rate‐suppressing, and muscle‐relaxant activity at similar doses, both subtype‐selective GABAA receptor PAMs selectively produced antinocicpetive effects. Collectively, these data support the notion of α2/α3‐subtype selective GABAA PAMs as novel analgesics.Support or Funding InformationR01DA034806This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Involvement of 5-HT1A Receptors in the Anxiolytic-Like Effects of Quercitrin and Evidence of the Involvement of the Monoaminergic System.

Quercitrin is a well-known flavonoid that is contained in Flos Albiziae, which has been used for the treatment of anxiety. The present study investigated the anxiolytic-like effects of quercitrin in experimental models of anxiety. Compared with the control group, repeated treatment with quercitrin (5.0 and 10.0 mg/kg/day, p.o.) for seven days significantly increased the percentage of entries into and time spent on the open arms of the elevated plus maze. In the light/dark box test, quercitrin exerted an anxiolytic-like effect at 5 and 10 mg/kg. In the marble-burying test, quercitrin (5.0 and 10.0 mg/kg) also exerted an anxiolytic-like effect. Furthermore, quercitrin did not affect spontaneous locomotor activity. The anxiolytic-like effects of quercitrin in the elevated plus maze and light/dark box test were blocked by the serotonin-1A (5-hydroxytryptamine-1A (5-HT1A)) receptor antagonist WAY-100635 (3.0 mg/kg, i.p.) but not by the γ-aminobutyric acid-A (GABAA) receptor antagonist flumazenil (0.5 mg/kg, i.p.). The levels of brain monoamines (5-HT and dopamine) and their metabolites (5-hydroxy-3-indoleacetic acid, 3,4-dihydroxyphenylacetic acid, and homovanillic acid) were decreased after quercitrin treatment. These data suggest that the anxiolytic-like effects of quercitrin might be mediated by 5-HT1A receptors but not by benzodiazepine site of GABAA receptors. The results of the neurochemical studies suggest that these effects are mediated by modulation of the levels of monoamine neurotransmitters.

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.

Sh-I-048A, an in vitro non-selective super-agonist at the benzodiazepine site of GABAA receptors: The approximated activation of receptor subtypes may explain behavioral effects

Enormous progress in understanding the role of four populations of benzodiazepine-sensitive GABAA receptors was paralleled by the puzzling findings suggesting that substantial separation of behavioral effects may be accomplished by apparently non-selective modulators. We report on SH-I-048A, a newly synthesized chiral positive modulator of GABAA receptors characterized by exceptional subnanomolar affinity, high efficacy and non-selectivity. Its influence on behavior was assessed in Wistar rats and contrasted to that obtained with 2mg/kg diazepam. SH-I-048A reached micromolar concentrations in brain tissue, while the unbound fraction in brain homogenate was around 1.5%. The approximated electrophysiological responses, which estimated free concentrations of SH-I-048A or diazepam are able to elicit, suggested a similarity between the 10mg/kg dose of the novel ligand and 2mg/kg diazepam; however, SH-I-048A was relatively more active at α1- and α5-containing GABAA receptors. Behaviorally, SH-I-048A induced sedative, muscle relaxant and ataxic effects, reversed mechanical hyperalgesia 24h after injury, while it was devoid of clear anxiolytic actions and did not affect water-maze performance. While lack of clear anxiolytic actions may be connected with an enhanced potentiation at α1-containing GABAA receptors, the observed behavior in the rotarod, water maze and peripheral nerve injury tests was possibly affected by its prominent action at receptors containing the α5 subunit. The current results encourage further innovative approaches aimed at linking in vitro and in vivo data in order to help define fine-tuning mechanisms at four sensitive receptor populations that underlie subtle differences in behavioral profiles of benzodiazepine site ligands.

Sh-I-048A, an in vitro non-selective super-agonist at the benzodiazepine site of GABAA receptors: The approximated activation of receptor subtypes may explain behavioral effects

Sh-I-048A, an in vitro non-selective super-agonist at the benzodiazepine site of GABAA receptors: The approximated activation of receptor subtypes may explain behavioral effects

Effects of Phenazepam on the Behavior of C57Bl/6 and BALB/c Mice in the Open Field Test after Naloxone Pretreatment

We studied the effects of phenazepam (0.075 mg/kg) after pretreatment (5 minutes before) with naloxone (10 mg/kg) on open-field behavior of C57Bl/6 and BALB/c mice. In ex vivo experiments, we studied the effects of naloxone (1 and 10 mg/kg) on receptor binding of [(3)H]-flunitrazepam by membranes of brain fraction (P1+P2) of C57Bl/6 and BALB/c mice. It was shown that naloxone increased motor activity in the open field in BALB/c mice and decreased this parameter in C57Bl/6 mice. During combined treatment, naloxone potentiated the activating effects of phenazepam on the open-field behavior of BALB/c mice and slightly increased the sedative effect of this drug in C57Bl/6 mice. Naloxone stimulated reception of [(3)H]-flunitrazepam in BALB/c mice and slightly increased radioligand binding in C57Bl/6 mice. These data attest to enhanced reception in benzodiazepine site of GABAA-receptor under conditions of opioid receptor blockade, the presence of anxiolytic or sedative (depending on the phenotype of the response to emotional stress) effect of naloxone, and co-directed effects of naloxone and benzodiazepine tranquilizer on open-field behavior of C57Bl/6 and BALB/c mice.

Subtype selectivity of α+β− site ligands of GABAA receptors: identification of the first highly specific positive modulators at α6β2/3γ2 receptors

GABAA receptors are the major inhibitory neurotransmitter receptors in the mammalian brain and the target of many clinically important drugs interacting with different binding sites. Recently, we demonstrated that CGS 9895 (2-(4-methoxyphenyl)-2H-pyrazolo[4,3-c]quinolin-3(5H)-one) elicits a strong and subtype-dependent enhancement of GABA-induced currents via a novel drug-binding site at extracellular αx+βy- (x = 1-6, y = 1-3) interfaces. Here, we investigated 16 structural analogues of CGS 9895 for their ability to modulate GABA-induced currents of various GABAA receptor subtypes. Recombinant GABAA receptor subtypes were expressed in Xenopus laevis oocytes and investigated by the two-electrode voltage clamp method. Most of the compounds investigated were able to modulate GABA-induced currents of αβ and αβγ receptors to a comparable extent, suggesting that the effect of these drugs is not dependent on the benzodiazepine site of GABAA receptors. Steric hindrance experiments demonstrated that these compounds exert their action predominantly via the αx+βy- (x = 1-6, y = 1-3) interfaces. Whereas some compounds are unselectively modulating a broad range of receptor subtypes, other compounds feature remarkable functional selectivity for the α6β3γ2 receptor, or behave as null modulators at some receptor subtypes investigated. Pyrazoloquinolinones and pyrazolopyridinones represent the first prototypes of drugs exerting benzodiazepine-like modulatory effects via the α+β- interface of GABAA receptors. The discovery of modulators with functional subtype selectivity at this class of binding sites provides a highly useful tool for the investigation of α6β2/3γ2 receptor function, and may lead to novel therapeutic principles.

Pharmacophore, 3D‐QSAR, and Bayesian Model Analysis for Ligands Binding at the Benzodiazepine Site of GABAA Receptors: the Key Roles of Amino Group and Hydrophobic Sites

Ligands binding at the benzodiazepine site of GABAA receptor play important pharmacological roles in clinical application. In this study, ligand-based pharmacophore modeling, 3D-QSAR analysis, and Bayesian model studies have been performed on a set of 84 diverse ligands binding at the benzodiazepine site. The results showed the best pharmacophore hypothesis AADHR.4, which included two hydrogen acceptors (A), one hydrogen donor (D), one hydrophobic group (H), and one aromatic ring (R). Atom-based 3D-QSAR model was built, and it showed good statistical significance (R(2) =0.936) and excellent predictive ability (Q(2) =0.821). Moreover, Bayesian model was developed and used to identify the key molecular features which are good or bad for the ligand binding activity. All the results from the pharmacophore, 3D-QSAR, and Bayesian modeling studies revealed that a hydrogen-bond donor (e.g., N-H) and a hydrophobic group (e.g., Br) are critical structural features for the ligands binding at the benzodiazepine site.

Magnolol, a major bioactive constituent of the bark of Magnolia officinalis, induces sleep via the benzodiazepine site of GABAA receptor in mice

Magnolol (6,6′,7,12-tetramethoxy-2,2′-dimethyl-1-beta-berbaman, C18H18O2), an active ingredient of the bark of Magnolia officinalis, has been reported to exert potent anti-epileptic effects via the GABAA receptor. The receptor also mediates sleep in humans and animals. The aim of this study was to determine whether magnolol could modulate sleep behaviors by recording EEG and electromyogram in mice. The results showed that magnolol administered i.p. at a dose of 5 or 25 mg/kg could significantly shorten the sleep latency, increase the amount of non-rapid eye movement (non-REM, NREM) and rapid eye movement (REM) sleep for 3 h after administration with an increase in the number of NREM and REM sleep episodes. Magnolol at doses of 5 and 25 mg/kg increased the number of bouts of wakefulness but decreased their duration. On the other hand, magnolol increased the number of state transitions from wakefulness to NREM sleep and subsequently from NREM sleep to wakefulness. Immunohistochemical study showed that magnolol increased c-Fos expression in the neurons of ventrolateral preoptic area, a sleep center in the anterior hypothalamus, and decreased c-Fos expression in the arousal tuberomammillary nucleus, which was located in the caudolateral hypothalamus. The sleep-promoting effects and changes in c-Fos induced by magnolol were reversed by flumazenil, an antagonist at the benzodiazepine site of the GABAA receptor. These results indicate that magnolol increased NREM and REM sleep via the GABAA receptor.

Development of new carbon-11 labelled radiotracers for imaging GABAA- and GABAB-benzodiazepine receptors

Two quinolines identified as positive allosteric modulators of γ-aminobutyric acid (GABA)A receptors containing the α2 subunit, 9-amino-2-cyclobutyl-5-(6-methoxy-2-methylpyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one (4) and 9-amino-2-cyclobutyl-5-(2-methoxypyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one (5), were radiolabelled at the methoxy position with carbon-11 (half-life=20.4min). These quinolines represent a new class of potential radiotracers for imaging the benzodiazepine site of GABAA receptors with positron emission tomography (PET). Both radiotracers were reliably isolated following reaction of their respective pyridinone/pyridinol tautomeric precursors with [11C]CH3I in clinically useful, formulated quantities (2.9% and 2.7% uncorrected radiochemical yield, respectively, relative to [11C]CO2) with high specific activities (>70GBqμmol−1; >2Ciμmol–1) and high radiochemical purities (>95%). The radiosyntheses reported herein represent rare examples of selectively isolating radiolabelled compounds bearing [11C]2-methoxypyridine moieties. Although both radiotracers demonstrated promising imaging characteristics based on preliminary ex vivo biodistribution studies in conscious rodents, higher brain uptake was observed with [11C]5 and therefore this radiotracer was further evaluated. Carbon-11 labelled 5 readily penetrated the brain (>1 standard uptake value in cortical regions at 15min post-injection of the radiotracer), had an appropriate regional brain distribution for GABAA receptors that appeared to be reversible, and did not show any appreciable radiometabolites in rat brain homogenates up to 15min post-injection. Preadministration of flumazenil (1, 10mgkg−1) or 5 (5mgkg−1) effectively blocked >50% of [11C]5 binding to the GABAA receptor-rich regions, thereby suggesting that this radiotracer is worthy of further evaluation for imaging GABAA receptors. Additionally (R,S)-N-(1-(3-chloro-4-methoxyphenyl)ethyl)-3,3-diphenylpropan-1-amine, 6, an allosteric modulator of GABAB receptors, was efficiently labelled in one step using [11C]methyl iodide. Ex vivo biodistribution studies in conscious rats showed low brain uptake, therefore, efforts are underway to discover alternative radiotracers to image GABAB. In conclusion, [11C]5 is worthy of further evaluation in higher species for imaging GABAA receptors in the central nervous system.

Unravelling the role of GABAAreceptor subtypes in distinct neurons and behaviour

Unravelling the role of GABA_Areceptor subtypes in distinct neurons and behaviour

Involvement of 5-HT1A in the anxiolytic-like effect of dichloromethane fraction of Pimenta pseudocaryophyllus

Ethnopharmacological relevanceMedicinal applications of Pimenta pseudocaryophyllus infusion as a diuretic and aphrodisiac agent as well as tranquilizer in the form of tea for the treatment of emotional tension in Brazilian folk medicine has been in practice since time immemorial. Despite its popular therapeutic acceptance and claims, there are scanty scientific reports to corroborate its central biological activities. AimTo characterize anxiolytic-like effect of the dichloromethane fraction (DF) obtained from ethanolic leaf extract of the Pimenta pseudocaryophyllus and identify mechanisms of action involved while seeking to support its popular use as a soothing agent. Material and methodsMice (25–35g) were treated orally with DF obtained from ethanolic leaf extract of Pimenta pseudocaryophyllus and were submitted to light-dark box (LDB) and elevated plus maze (EPM) tests. Different groups of mice were treated with flumazenil and NAN-190 to identify mechanisms of action involved in the anxiolytic-like effect of DF. ResultsTreatment with DF increased number of transitions and time spent in the light compartment of the LDB while the time spent and numbers of entries in the open arm of the LCE were significantly increased. Pre-treatment of the animal with flumazenil (2mg/kg, i.p. – competitive antagonist of benzodiazepine site of GABAA receptor) did not block this effect, thereby excluding participation of benzodiazepine site of the GABAA receptor. However, anxiolytic-like effect of DF was reversed by pre-treatment with NAN-190 (0.5mg/kg, i.p. – an antagonist of the 5-HT1A receptor) thereby suggesting involvement of 5-HT1A receptor. The thin layer chromatography and high-performance liquid chromatography analysis indicated the predominance of (E)-methyl isoeugenol and oleanolic acid in DF. ConclusionThese results support the popular use of Pimenta pseudocaryophyllus as a calming agent and suggest the involvement of 5-HT1A receptor.