DOCSLIB.ORG

Ethanol Potently and Competitively Inhibits Binding of the Alcohol Antagonist Ro15-4513 to ␣4/6␤3␦ GABAA Receptors H

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Ethanol Potently and Competitively Inhibits Binding of the Alcohol Antagonist Ro15-4513 to ␣4/6␤3␦ GABAA Receptors H Ethanol potently and competitively inhibits binding of the alcohol antagonist Ro15-4513 to ␣4/6␤3␦ GABAA receptors H. Jacob Hanchar*†, Panida Chutsrinopkun†‡, Pratap Meera§, Porntip Supavilai‡, Werner Sieghart¶, Martin Wallner*ʈ, and Richard W. Olsen*ʈ Departments of *Molecular and Medical Pharmacology and §Neurobiology, University of California, Los Angeles, CA 90095; ‡Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok 10 400, Thailand; and ¶Center for Brain Research, Division of Biochemistry and Molecular Biology and Section of Biochemical Psychiatry, Medical University of Vienna, A-1090 Vienna, Austria Edited by Floyd E. Bloom, The Scripps Research Institute, La Jolla, CA, and approved February 10, 2006 (received for review November 16, 2005) Although GABAA receptors have long been implicated in mediating (14) concluded that the EtOH-induced increase in tonic current ethanol (EtOH) actions, receptors containing the ‘‘nonsynaptic’’ ␦ is due to increased GABA release rather than a postsynaptic subunit only recently have been shown to be uniquely sensitive to effect. Also, for reasons on which we can only speculate, a recent EtOH. Here, we show that ␦ subunit-containing receptors bind the article by Borghese et al. (15) states that effects of low dose imidazo-benzodiazepines (BZs) flumazenil and Ro15-4513 with EtOH on ␦ subunit-containing receptors could not be observed, high affinity (Kd < 10 nM), contrary to the widely held belief that in particular with human ␣4␤3␦ GABAAR clones expressed in these receptors are insensitive to BZs. In immunopurified native oocytes and cell lines. cerebellar and recombinant ␦ subunit-containing receptors, bind- EtOH pharmacology shares many characteristics with alloste- 3 ing of the alcohol antagonist [ H]Ro15-4513 is inhibited by low ric activators of GABAARs (loosely referred to as GABAAR concentrations of EtOH (Ki Ϸ 8 mM). Also, Ro15-4513 binding is agonists), such as benzodiazepines (BZs) (5). Additional evi- inhibited by BZ-site ligands that have been shown to reverse the dence for a link between EtOH and BZ actions on GABAAR behavioral alcohol antagonism of Ro15-4513 (i.e., flumazenil, comes from the surprising finding that a mutation in the ␣6 ␤ ␤ ␤ PHARMACOLOGY -carbolinecarboxylate ethyl ester ( -CCE), and N-methyl- -carbo- subunit (␣6-R100Q), which was identified in alcohol-nontolerant line-3-carboxamide (FG7142), but not including any classical BZ and -nonpreferring rats in refs. 16 and 17, leads to receptors with agonists like diazepam). Experiments that were designed to dis- increased alcohol sensitivity in recombinant expression (in cer- tinguish between a competitive and allosteric mechanism suggest ebellar granule cell neurons) and to alcohol-hypersensitive an- that EtOH and Ro15-4513 occupy a mutually exclusive binding site. imals in behavioral studies (12). Histidine residues at positions The fact that only Ro15-4513, but not flumazenil, can inhibit the that are homologous to the ␣6-R100 residue (which affects EtOH EtOH effect, and that Ro15-4513 differs from flumazenil by only a sensitivity in ␣6␤3␦ GABAARs) are critical for high-affinity single group in the molecule (an azido group at the C7 position of binding of classical BZ agonists at the interface between ␣ and the BZ ring) suggest that this azido group in Ro15-4513 might be ␥2 subunits. the area that overlaps with the alcohol-binding site. Our findings, It has been thought that the ‘‘extrasynaptic’’ GABAAR ␦ combined with previous observations that Ro15-4513 is a behav- subunit, which presumably takes the position of the ␥2 subunit in ioral alcohol antagonist, suggest that many of the behavioral functional pentameric GABAARs, renders receptors insensitive effects of EtOH at relevant physiological concentrations are me- to BZs (18). Also, most ␦ subunits are found to be associated ͞ diated by EtOH Ro15-4513-sensitive GABAA receptors. with ␣4 and ␣6 subunits that differ from other GABAAR ␣ subunits at a critical position (a histidine in ␣1,2,3,5 replaced by an alcohol receptor ͉ flumazenil ͉ ␤-carbolines ͉ extrasynaptic GABA A arginine in ␣4,6) that makes ␣4/6␤x␥2 receptors insensitive to receptors classical BZ agonists such as diazepam (DZ) and flunitrazepam (19, 20). However, Arg-100 (WT) in ␣4 and ␣6 receptors still lthough many proteins show changes in their function at allows high-affinity binding of the imidazo-BZs Ro15-4513 and Ͼ ␣ ␤ ␥ Avery high alcohol concentrations ( 50 mM), the molecular flumazenil (in x x 2 GABAARs). In functional receptor assays, basis for behavioral alcohol effects at low to moderately intox- Ro15-4513 is a weak partial inverse agonist (i.e., it leads to a icating doses experienced during social alcohol consumption slight reduction in GABAAR activity) on the most abundant remains elusive (1). GABAA receptors (GABAARs) and the GABAAR subtypes in the brain (20). Ro15-4513 is a partial inhibitory GABAergic system have long been suspected to be agonist (i.e., it enhances GABA action, but less than DZ even at targets for acute alcohol effects (2–4). For example, the saturating concentrations) on the ␣4 and ␣6 receptors (with ␤ and GABAAR agonist muscimol potentiates the sedative actions of ␥2 subunits), whereas flumazenil is essentially silent in functional alcohol, whereas the opposite effect, a reduction of ethanol assays. Interestingly, Ro15-4513, but not other inverse agonists (EtOH)-produced sedation, is detected with the GABAAR blockers picrotoxin and bicuculline (5). Although most Conflict of interest statement: M.W., R.W.O., and H.J.H. have filed a U.S. Provisional Patent GABAAR subunit combinations can be activated by high (an- Application, Serial No. 60͞693,844. esthetic) alcohol concentrations (6), only very specific GABAAR This paper was submitted directly (Track II) to the PNAS office. subunit combinations (containing the ␦ as well as the ␤3 subunit) exhibit dose dependencies that mirror blood alcohol levels that Abbreviations: GABAAR, GABAA receptor; HEK, human embryonic kidney; BZ, benzodiaz- epine; EtOH, ethanol; IP, immunoprecipitated͞immunoprecipitation; ␤-CCE, ␤-carboline- are associated with mild to moderate intoxication in humans (7, 3-carboxyethyl ester; DMCM, methyl-6,7-dimethoxy-4-ethyl-␤-carboline-3-carboxylate; 8) (Ϸ3–30 mM, because the legal drinking limit is 17 mM or DZ, diazepam; DZ-IS, DZ-insensitive; FG7142, N-methyl-␤-carboline-3-carboxamide. ␦ 0.08%). GABAARs containing the subunit are located either †H.J.H. and P.C. contributed equally to this work. outside (9) or in the perimeter of (10) synapses but not in the ʈTo whom correspondence may be addressed at: Department of Molecular and Medical subsynaptic membrane, and they give rise to a persistent (tonic) Pharmacology, University of California, Room 23-120 CHS, Charles Young Drive South, Los GABA current (11) that is enhanced by low alcohol concentra- Angeles, CA 90095-1735. E-mail: [email protected] or [email protected]. tions (12–14). However, there is controversy because Carta et al. © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0509903103 PNAS Early Edition ͉ 1of6 Fig. 1. EtOH-sensitive ␣4/6␤3␦ GABAARs have a high-affinity Ro15-4513- binding site. [3H]Ro15-4513 saturation binding in native immuno-purified (␦-IP pellet) and ␦-depleted (␦-IP supernatant) cerebellar GABAAR fractions and to recombinant ␣4␤3␦ and ␣4␤3␥2 GABAARs receptor expressed in the HEK 293T cell line. [like the ␤-carbolines ␤-carboline-3-carboxyethyl ester (␤-CCE) and methyl-6,7-dimethoxy-4-ethyl-␤-carboline-3-carboxylate (DMCM)], has been shown to have fairly dramatic alcohol- antagonist actions, reported in various mammals; in particular, alcohol effects at lower doses can be reversed almost completely by Ro15-4513 (3, 21–25). ␣ ␤ ␦ Fig. 2. Alcohol receptor binding assay. EtOH-displaceable high-affinity Here, we show that alcohol-sensitive 4/6 3 GABAARs bind [3H]Ro15-4513 binding to native and recombinant ␦ subunit-containing 3 3 [ H]Ro15-4513 with high affinity, and that this binding is inhib- GABAAR. (a)[H]Ro15-4513 binding is inhibited by low concentrations of ited dose-dependently by low doses of EtOH. This result is EtOH. Receptors were equilibrated with 10 nM [3H]Ro15-4513 and varying consistent with the observation that Ro15-4513 potently inhibits EtOH concentrations. We tested native immunopurified (␦-IP pellet) and ␦ ␦ EtOH enhancement of functional ␣ ␤ ␦ GABA Rs (45). The -depleted ( -IP supernatant) cerebellar GABAAR fractions, and recombinant 4/6 3 A ␣ ␤ ␦ ␣ ␤ ␥ Ro15-4513͞EtOH antagonism appears to be competitive. Im- 4 3 and 4 3 2 GABAARs expressed in the HEK 293T cell line. (b) Approxi- mately one-third of the DZ-IS [3H]Ro15-4513 binding to cow cerebellar portantly, our data explain the puzzling observation that Ro15- GABAARs is antagonized by low EtOH concentrations. (Inset) Total, 10 ␮M 4513 is a behavioral alcohol antagonist and suggest that EtOH͞ DZ-sensitive, and DZ-IS 30 nM [3H]Ro15-4513 binding to cow cerebellar Ro15-4513-sensitive GABAARs are important mediators of membranes. alcohol actions that are experienced during moderate social drinking. EtOH Inhibits [3H]Ro15-4513 Binding to ␦ Subunit-Containing Results GABAARs. Inspired by the finding that these alcohol-sensitive receptors bind the alcohol antagonist Ro15-4513 with high Alcohol Antagonist Ro15-4513 Binds to ␦ Subunit-Containing affinity, we investigated whether [3H]Ro15-4513 binding to ␦ GABAARs. Based on the observations that ␦ subunit-containing subunit-containing GABAAR can be inhibited by EtOH. Fig. 2a receptors are uniquely sensitive to EtOH (7), and that a BZ-site 3 ␦ ␣ shows that [ H]Ro15-4513 binding to native cerebellar subunit single-nucleotide polymorphism ( 6-R100Q) increases alcohol ␦ ␣ ␤ ␦ immunopurified receptors ( -IP pellet), as well as to recombi- sensitivity in 6 3 receptors (12), we decided to investigate ␣ ␤ ␦ ␦ nant 4 3 receptors, was dose-dependently inhibited by 3–300 whether native and recombinant subunit-containing receptors mM EtOH (IC Ϸ 12 mM). In marked contrast, [3H]Ro15-4513 could bind the behavioral alcohol antagonist Ro15-4513, which 50 binding to ␣4␤3␥2 and the ␦ subunit immunodepleted superna- is commercially available as a tritiated radioligand.
Load more

Recommended publications
  • NIH Public Access Author Manuscript Synapse
    NIH Public Access Author Manuscript Synapse. Author manuscript; available in PMC 2010 May 4. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Synapse. 2006 November ; 60(6): 411±419. doi:10.1002/syn.20314. GABAA Receptor Regulation of Voluntary Ethanol Drinking Requires PKCε Joyce Besheer, Veronique Lepoutre, Beth Mole, and Clyde W. Hodge* Bowles Center for Alcohol Studies, Department of Psychiatry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Abstract Protein kinase C (PKC) regulates a variety of neural functions, including ion channel activity, neurotransmitter release, receptor desensitization and differentiation. We have shown previously that mice lacking the ε-isoform of PKC (PKCε) self-administer 75% less ethanol and exhibit supersensitivity to acute ethanol and allosteric positive modulators of GABAA receptors when compared with wild-type controls. The purpose of the present study was to examine involvement of PKCε in GABAA receptor regulation of voluntary ethanol drinking. To address this question, PKCε null-mutant and wild-type control mice were allowed to drink ethanol (10% v/v) vs. water on a two-bottle continuous access protocol. The effects of diazepam (nonselective GABAA BZ positive modulator), zolpidem (GABAA α1 agonist), L-655,708 (BZ-sensitive GABAA α5 inverse agonist), and flumazenil (BZ antagonist) were then tested on ethanol drinking. Ethanol intake (grams/kg/day) by wild-type mice decreased significantly after diazepam or zolpidem but increased after L-655,708 administration. Flumazenil antagonized diazepam-induced reductions in ethanol drinking in wild- type mice. However, ethanol intake by PKCε null mice was not altered by any of the GABAergic compounds even though effects were seen on water drinking in these mice.
    [Show full text]
  • GABA Receptors
    D Reviews • BIOTREND Reviews • BIOTREND Reviews • BIOTREND Reviews • BIOTREND Reviews Review No.7 / 1-2011 GABA receptors Wolfgang Froestl , CNS & Chemistry Expert, AC Immune SA, PSE Building B - EPFL, CH-1015 Lausanne, Phone: +41 21 693 91 43, FAX: +41 21 693 91 20, E-mail: [email protected] GABA Activation of the GABA A receptor leads to an influx of chloride GABA ( -aminobutyric acid; Figure 1) is the most important and ions and to a hyperpolarization of the membrane. 16 subunits with γ most abundant inhibitory neurotransmitter in the mammalian molecular weights between 50 and 65 kD have been identified brain 1,2 , where it was first discovered in 1950 3-5 . It is a small achiral so far, 6 subunits, 3 subunits, 3 subunits, and the , , α β γ δ ε θ molecule with molecular weight of 103 g/mol and high water solu - and subunits 8,9 . π bility. At 25°C one gram of water can dissolve 1.3 grams of GABA. 2 Such a hydrophilic molecule (log P = -2.13, PSA = 63.3 Å ) cannot In the meantime all GABA A receptor binding sites have been eluci - cross the blood brain barrier. It is produced in the brain by decarb- dated in great detail. The GABA site is located at the interface oxylation of L-glutamic acid by the enzyme glutamic acid decarb- between and subunits. Benzodiazepines interact with subunit α β oxylase (GAD, EC 4.1.1.15). It is a neutral amino acid with pK = combinations ( ) ( ) , which is the most abundant combi - 1 α1 2 β2 2 γ2 4.23 and pK = 10.43.
    [Show full text]
  • Benzodiazepine Actions Mediated by Specificg-Aminobutyric Acida
    letters to nature ..................................................................................................................................... erratum Benzodiazepine actions mediated by speci®c g-aminobutyric acidA receptor subtypes Uwe Rudolph, Florence Crestani, Dietmar Benke, Ina BruÈnig, Jack A. Benson, Jean-Marc Fritschy, James R. Martin, Horst Bluethmann & Hanns MoÈhler Nature 401, 796±800 (1999) .......................................................................................................................................................................................................................................................................... The quality of Fig. 2 was unsatisfactory as published. The ®gure is reproduced again here. M 3 3 a d [ H]Ro15-4513 [ H]Ro15-4513, Ki (nM) + diazepam 1(H101R) 1(H101R) α α Wild-type Wild-type α1 α5 Wild-type α2 β2/3 α3 γ2 1(H101R) α α b Wild-type 1(H101R) e Wild-type 3 µM GABA+Dz +Ro15 3 µM GABA α1 α1(H101R) 3 µ µ c Displacement of [ H]Ro15-4513, Ki (nM) 3 M GABA+Dz +Ro15 3 M GABA Wild-type α1(H101R) Clonazepam 3 ± 2 > 10,000 Diazepam 28 ± 15 > 10,000 Zolpidem 13 ± 5 > 10,000 500 pA 2 s ................................................................. suggest that pairing occurs primarily on the electrons on the g- correction sheet of the Fermi surface under the conditions of the experiment. However, the orientation of the FLL, interpreted within Agterberg's two-component Ginzburg±Landau (TCGL) theory1,2, is no longer consistent with g-band pairing if we take the values of Fermi surface Observation of a square ¯ux-line anisotropy used in refs 1, 2, and obtained by recent band structure calculations3. However, other calculations (T. Oguchi, private lattice in the unconventional communication) give a different sign of Fermi surface anisotropy. TCGL theory predictions may be modi®ed by extra anisotropy of superconductor Sr2RuO4 the superconducting energy gap3, by substantial pairing on the a- and/or b-sheets4 or by anisotropy in the electron mass enhance- T.
    [Show full text]
  • Pharmacological Properties of GABAA- Receptors Containing Gamma1
    Molecular Pharmacology Fast Forward. Published on November 4, 2005 as DOI: 10.1124/mol.105.017236 Molecular PharmacologyThis article hasFast not Forward.been copyedited Published and formatted. on The November final version 7, may 2005 differ as from doi:10.1124/mol.105.017236 this version. MOLPHARM/2005/017236 Pharmacological properties of GABAA- receptors containing gamma1- subunits Khom S.1, Baburin I.1, Timin EN, Hohaus A., Sieghart W., Hering S. Downloaded from Department of Pharmacology and Toxicology, University of Vienna Center of Brain Research , Medical University of Vienna, Division of Biochemistry and molpharm.aspetjournals.org Molecular Biology at ASPET Journals on September 27, 2021 1 Copyright 2005 by the American Society for Pharmacology and Experimental Therapeutics. Molecular Pharmacology Fast Forward. Published on November 4, 2005 as DOI: 10.1124/mol.105.017236 This article has not been copyedited and formatted. The final version may differ from this version. MOLPHARM/2005/017236 Running Title: GABAA- receptors containing gamma1- subunits Corresponding author: Steffen Hering Department of Pharmacology and Toxicology University of Vienna Althanstrasse 14 Downloaded from A-1090 Vienna Telephone number: +43-1-4277-55301 Fax number: +43-1-4277-9553 molpharm.aspetjournals.org [email protected] Text pages: 29 at ASPET Journals on September 27, 2021 Tables: 2 Figures: 7 References: 26 Abstract: 236 words Introduction:575 words Discussion:1383 words 2 Molecular Pharmacology Fast Forward. Published on November 4, 2005 as DOI: 10.1124/mol.105.017236 This article has not been copyedited and formatted. The final version may differ from this version. MOLPHARM/2005/017236 Abstract GABAA receptors composed of α1, β2, γ1-subunits are expressed in only a few areas of the brain and thus represent interesting drug targets.
    [Show full text]
  • Total Intravenous Anesthesia with Midazolam, Ketamine
    Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2010 Total invtravenous anesthesia with midazolam, ketamine, and xylazine or detomidine following induction with tiletamine, zolazepam, and xylazine in red deer (Cervus elaphus hippelaphus) undergoing surgery Auer, U ; Wenger, S ; Beigelböck, C ; Zenker, W ; Mosing, M Abstract: Sixteen captive female red deer were successfully anesthetized to surgically implant a telemetry system. The deer were immobilized with (mean±SD) 1.79±0.29 mg/kg xylazine and 1.79±0.29 mg/kg tiletamine/zolazepam given intramuscularly with a dart gun. Anesthesia was maintained for 69±2 min using a total intravenous protocol with a catheter placed in the jugular vein. Group X received xylazine (0.5±0.055 mg/kg/hr) and group D, detomidine (2±0.22 µg/kg/hr), both in combination with ketamine (2±0.02 mg/kg/hr) and midazolam (0.03±0.0033 mg/kg/hr), as a constant rate infusion. Anesthesia was reversed with 0.09±0.01 mg/kg atipamezole and 8.7±1.21 µg/kg sarmazenil given intravenously in both groups. These drug combinations provided smooth induction, stable anesthesia for surgery, and rapid recovery. Respiratory depression and mild hypoxemia were seen, and we, therefore, recommend using supplemental intranasal oxygen. DOI: https://doi.org/10.7589/0090-3558-46.4.1196 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-36361 Journal Article Originally published at: Auer, U; Wenger, S; Beigelböck, C; Zenker, W; Mosing, M (2010).
    [Show full text]
  • Xyrem (Sodium Oxybate) Is a CNS Depressant
    HIGHLIGHTS OF PRESCRIBING INFORMATION These highlights do not include all the information needed to use Important Administration Information for All Patients XYREM safely and effectively. See full prescribing information for • Take each dose while in bed and lie down after dosing (2.3). XYREM. • Allow 2 hours after eating before dosing (2.3). • Prepare both doses prior to bedtime; dilute each dose with approximately ¼ XYREM® (sodium oxybate) oral solution, CIII cup of water in pharmacy-provided containers (2.3). Initial U.S. Approval: 2002 • Patients with Hepatic Impairment: starting dose is one-half of the original dosage per night, administered orally divided into two doses (2.4). • WARNING: CENTRAL NERVOUS SYSTEM (CNS) DEPRESSION Concomitant use with Divalproex Sodium: an initial reduction in Xyrem dose and ABUSE AND MISUSE. of at least 20% is recommended (2.5, 7.2). --------------------DOSAGE FORMS AND STRENGTHS------------------- See full prescribing information for complete boxed warning. Oral solution, 0.5 g per mL (3) Central Nervous System Depression ----------------------------CONTRAINDICATIONS------------------------------ • Xyrem is a CNS depressant, and respiratory depression can occur with • In combination with sedative hypnotics or alcohol (4) Xyrem use (5.1, 5.4) • Succinic semialdehyde dehydrogenase deficiency (4) Abuse and Misuse • Xyrem is the sodium salt of gamma-hydroxybutyrate (GHB). Abuse or misuse of illicit GHB is associated with CNS adverse reactions, ---------------------WARNINGS AND PRECAUTIONS----------------------
    [Show full text]
  • Romazicon®) in an Animal Model
    Scientific Article Comparative pharmacokinetics of submucosal vs. intravenous flumazenil (Romazicon®) in an animal model Faith M. Oliver, DDS Trevor W. Sweatman, PhD John H. Unkel, DDS, MPA Michael A. Kahn, DDS Mildred M. Randolph, DVM Kristopher L. Arheart, EdD Timothy D. Mandrell, DVM Dr. Oliver is a resident, Pediatric Dentistry; Dr. Sweatman is an associate professor, Pharmacology; Dr. Unkel is an associate professor and postgraduate program director, Pediatric Dentistry; Dr. Kahn is a professor, Biologic & Diagnostic Sciences; Dr. Randolph is a veterinarian, Comparative Medicine; Dr. Arheart is an assistant professor, Preventive Medicine; and Dr. Mandrell is an associate professor, Comparative Medicine, they are all at the University of Tennessee. Correspond with Dr. Unkel at [email protected] Abstract Purpose: This study was performed to determine the in post-synaptic membrane hyperpolarization and reduced bioavailability and local tissue toxicological safety of flumazenil cortical stimulation via the lower brain centers. Traditional (Romazicon®) when administered by oral submucosal (SM) as endpoints for benzodiazepine-induced conscious sedation (pto- opposed to intravenous (IV) injection. sis, dysarthria, and drowsiness) are very close to an hypnotic Methods: Six dogs each received SM flumazenil (0.2 mg), and state in which the patient is unresponsive to verbal command.1,2 their serum was collected at predetermined time intervals (0-2 h) More recently, anxiolysis, amnesia, and patient cooperation and frozen (-70°C). Seven days later, the dogs received an iden- have been used as measures of drug effectiveness.3 With re- tical dose of IV flumazenil, and serum samples were again collected, gard to dentistry, the intranasal (IN: 0.2-0.4 mg/kg) or oral as above.
    [Show full text]
  • Characterisation of the Contribution of the GABA-Benzodiazepine &Alpha
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central Journal of Cerebral Blood Flow & Metabolism (2012) 32, 731–744 & 2012 ISCBFM All rights reserved 0271-678X/12 www.jcbfm.com Characterisation of the contribution of the GABA-benzodiazepine a1 receptor subtype to [11C]Ro15-4513 PET images James FM Myers1, Lula Rosso2, Ben J Watson1, Sue J Wilson1,3, Nicola J Kalk3, Nicoletta Clementi1, David J Brooks2, David J Nutt3, Federico E Turkheimer2 and Anne R Lingford-Hughes3 1Psychopharmacology Unit, University of Bristol, Bristol, UK; 2Department of Medicine, Centre for Neuroscience, Imperial College London, London, UK; 3Division of Experimental Medicine, Neuropsychopharmacology Unit, Imperial College London, London, UK This positron emission tomography (PET) study aimed to further define selectivity of [11C]Ro15-4513 binding to the GABARa5 relative to the GABARa1 benzodiazepine receptor subtype. The impact of zolpidem, a GABARa1-selective agonist, on [11C]Ro15-4513, which shows selectivity for GABARa5, and the nonselective benzodiazepine ligand [11C]flumazenil binding was assessed in humans. Compartmental modelling of the kinetics of [11C]Ro15-4513 time-activity curves was used to describe distribution volume (VT) differences in regions populated by different GABA receptor subtypes. Those with low a5 were best fitted by one-tissue compartment models; and those with high a5 required a more complex model. The heterogeneity between brain regions suggested spectral analysis as a more appropriate method to quantify binding as it does not a priori specify compartments. Spectral analysis revealed that zolpidem caused a significant VT decrease (B10%) in [11C]flumazenil, but no decrease in [11C]Ro15-4513 binding.
    [Show full text]
  • Human Pharmacology of Positive GABA-A Subtype-Selective Receptor Modulators for the Treatment of Anxiety
    www.nature.com/aps REVIEW ARTICLE Human pharmacology of positive GABA-A subtype-selective receptor modulators for the treatment of anxiety Xia Chen1,2,3,4, Joop van Gerven4,5, Adam Cohen4 and Gabriel Jacobs4 Anxiety disorders arise from disruptions among the highly interconnected circuits that normally serve to process the streams of potentially threatening stimuli. The resulting imbalance among these circuits can cause a fundamental misinterpretation of neural sensory information as threatening and can lead to the inappropriate emotional and behavioral responses observed in anxiety disorders. There is considerable preclinical evidence that the GABAergic system, in general, and its α2- and/or α5-subunit- containing GABA(A) receptor subtypes, in particular, are involved in the pathophysiology of anxiety disorders. However, the clinical efficacy of GABA-A α2-selective agonists for the treatment of anxiety disorders has not been unequivocally demonstrated. In this review, we present several human pharmacological studies that have been performed with the aim of identifying the pharmacologically active doses/exposure levels of several GABA-A subtype-selective novel compounds with potential anxiolytic effects. The pharmacological selectivity of novel α2-subtype-selective GABA(A) receptor partial agonists has been demonstrated by their distinct effect profiles on the neurophysiological and neuropsychological measurements that reflect the functions of multiple CNS domains compared with those of benzodiazepines, which are nonselective, full GABA(A) agonists. Normalizing the undesired pharmacodynamic side effects against the desired on-target effects on the saccadic peak velocity is a useful approach for presenting the pharmacological features of GABA(A)-ergic modulators. Moreover, combining the anxiogenic symptom provocation paradigm with validated neurophysiological and neuropsychological biomarkers may provide further construct validity for the clinical effects of novel anxiolytic agents.
    [Show full text]
  • DBL Flumazenil Data Sheet
    NEW ZEALAND DATA SHEET 1. PRODUCT NAME DBL™ Flumazenil Injection 2. QUALITATIVE AND QUANTITATIVE COMPOSITION Ampoules contain 0.1 mg/mL flumazenil in aqueous solution (for intravenous administration) and also the following ingredients: disodium edetate, acetic acid, sodium chloride, sodium hydroxide in water for injections adjusted to pH 4.0. DBL Flumazenil Injection is available as 0.5 mg/5 mL and 1 mg/10 mL ampoules. For the full list of excipients, see section 6.1. 3. PHARMACEUTICAL FORM DBL Flumazenil Injection is a colourless to almost colourless clear liquid, adjusted to pH 4.0. Solution for injection. 4. CLINICAL PARTICULARS 4.1 Therapeutic indications DBL Flumazenil Injection is indicated for use in hospitalised patients for the reversal of acute benzodiazepine effects (overdose or therapeutic). Hospitalised patients are patients admitted to hospital, inpatient care and under continued professional observation while under the influence of flumazenil. Not to be used in outpatients or short stay patients. Not to be used as a diagnostic. 4.2 Dose and method of administration DBL Flumazenil Injection should be administered intravenously by an anaesthetist or experienced physician. Dosage should be titrated for the intended effect. Since the duration of action of some benzodiazepines may exceed that of DBL Flumazenil Injection, repeated doses may be required if sedation recurs following awakening. The use of DBL Flumazenil Injection should be balanced against the risk of precipitating withdrawal symptoms (see section 4.4). The desirability of retaining a degree of sedation in the early postoperative period should be considered. DBL Flumazenil Injection may be diluted in glucose 5% in water or 0.9% NaCl for infusion and may also be used concurrently with other resuscitative procedures.
    [Show full text]
  • Requirement Ofα5-GABAA Receptors for the Development of Tolerance To
    The Journal of Neuroscience, July 28, 2004 • 24(30):6785–6790 • 6785 Behavioral/Systems/Cognitive ␣ Requirement of 5-GABAA Receptors for the Development of Tolerance to the Sedative Action of Diazepam in Mice Carolien van Rijnsoever,1* Marcus Ta¨uber,1* Mohamed Khaled Choulli,1 Ruth Keist,1 Uwe Rudolph,1 Hanns Mohler,1,2 Jean Marc Fritschy,1 and Florence Crestani1 1Institute of Pharmacology and Toxicology, University of Zu¨rich, 8057 Zu¨rich, Switzerland, and 2Department of Applied Biosciences, Swiss Federal Institute of Technology, 8057 Zu¨rich, Switzerland Despite its pharmacological relevance, the mechanism of the development of tolerance to the action of benzodiazepines is essentially ␣ unknown. The acute sedative action of diazepam is mediated via 1-GABAA receptors. Therefore, we tested whether chronic activation of ␣ ␣ ␣ ␣ these receptors by diazepam is sufficient to induce tolerance to its sedative action. Knock-in mice, in which the 1-, 2-, 3-, or 5-GABAA receptors had been rendered insensitive to diazepam by histidine–arginine point mutation, were chronically treated with diazepam (8 d; ⅐ Ϫ1 ⅐ Ϫ1 ␣ ␣ 15 mg kg d ) and tested for motor activity. Wild-type, 2(H101R), and 3(H126R) mice showed a robust diminution of the ␣ ␣ motor-depressant drug action. In contrast, 5(H105R) mice failed to display any sedative tolerance. 1(H101R) mice showed no alter- ation of motor activity with chronic diazepam treatment. Autoradiography with [ 3H]flumazenil revealed no change in benzodiazepine ␣ binding sites. However, a decrease in 5-subunit radioligand binding was detected selectively in the dentate gyrus with specific ligands. This alteration was observed only in diazepam-tolerant animals, indicating that the manifestation of tolerance to the sedative action of ␣ ␣ diazepam is associated with a downregulation of 5-GABAA receptors in the dentate gyrus.
    [Show full text]
  • An Investigation of Flumazenil to Antagonize Gamma-Hydroxybutyrate Intoxication in a Murine Model
    Toxicology Investigations An Investigation of Flumazenil to Antagonize Gamma-Hydroxybutyrate Intoxication in a Murine Model David C. Lee, MDab, Wayne A. Satz, MDac, Timothy Dougherty, MDa, Tucker Greene, MDa a Department of Emergency Medicine, Drexel University/Medical College of Pennsylvania b Department of Emergency Medicine, North Shore University Hospital c Department of Emergency Medicine, Temple University Hospital Abstract Objective: Prior animal studies have suggested that flumazenil may blunt GHB’s sedative-hypnotic affects. We hypothesized that flumazenil would decrease the affects of GHB in a murine model of intoxication. Methods: We performed a controlled, pilot experiment using 32 mice divided into 3 groups. All mice received intraperitoneal injections of GHB (1.5 g/kg). Group I received sham injections at time 0, and then GHB at 5 minutes. Group II received flumazenil (0.3 mg/kg) at time 0, and then GHB at 5 minutes. Group III received sham injection at time 0, then GHB at 5 minutes, and then 4 escalating flumazenil doses administered at 3-minute intervals (0.003 to 1 mg/kg). We measured certain functions: time to loss/recovery of righting reflexes (RR), time to sprawl/recovery of sprawl (postural tone [PT]), and death. Results: There were statistically significant delays in the loss of PT and shortened recovery time to RR in pre-treated mice (group II) versus controls (group I). There were no differences in group III versus group I for any outcome parameters. Conclusions: In this model, pre-dosing flumazenil prior to GHB administration delayed clinical intoxication. INTRODUCTION a toxic dose of GHB4. There are no reported human studies de- scribing the use and clinical effect of flumazenil in GHB over- Gamma-hydroxybutyrate (GHB) is a sedative-hypnotic agent doses.
    [Show full text]