Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2018/05/02/jpet.118.249250.DC1 1521-0103/366/1/145–157$35.00 https://doi.org/10.1124/jpet.118.249250 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 366:145–157, July 2018 Copyright ª 2018 by The American Society for Pharmacology and Experimental Therapeutics Evidence That Sedative Effects of Benzodiazepines Involve Unexpected GABAA Receptor Subtypes: Quantitative Observation Studies in Rhesus Monkeys s Angela N. Duke,1,2 Zhiqiang Meng,1,3 Donna M. Platt, John R. Atack, Gerard R. Dawson, David S. Reynolds, V. V. N. Phani Babu Tiruveedhula, Guanguan Li, Michael Rajesh Stephen, Werner Sieghart, James M. Cook, and James K. Rowlett New England Primate Research Center, Harvard Medical School, Southborough, Massachusetts (A.N.D., Z.M., D.M.P., J.K.R.); Department of Psychiatry and Human Behavior, University of Mississippi Medical Center, Jackson, Mississippi (D.M.P., J.K.R.); Downloaded from Medicines Discovery Institute, Cardiff University, Cardiff, Wales, United Kingdom (J.R.A.); P1Vital, University of Oxford, Warneford Hospital, Headington, Oxford, United Kingdom (G.R.D.); Alzheimer’s Research UK, Granta Park, Great Abington, Cambridge, United Kingdom (D.S.R.); Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin (V.V.N.P.B.T., G.L., M.R.S., J.M.C.); and Division of Biochemistry and Molecular Biology, Center for Brain Research, Medical University of Vienna, Vienna, Austria (W.S.) Received March 19, 2018; accepted April 30, 2018 jpet.aspetjournals.org ABSTRACT In nonhuman primates we tested a new set of behavioral cate- pretreating animals with the a1 subunit-preferring antagonist gories for observable sedative effects using pediatric anesthesiol- b-carboline-3-carboxylate-t-butyl ester (bCCT). Increasing doses ogy classifications as a basis. Using quantitative behavioral of alprazolam and diazepam resulted in the emergence of observation techniques in rhesus monkeys, we examined the observable ataxia, rest/sleep posture, and moderate and deep effects of alprazolam and diazepam (nonselective benzodiaze- sedation. In contrast, zolpidem engendered dose-dependent at ASPET Journals on September 25, 2021 pines), zolpidem (preferential binding to a1 subunit-containing observable ataxia and deep sedation but not rest/sleep posture GABAA receptors), HZ-166 (8-ethynyl-6-(29-pyridine)-4H-2,5,10b- or moderate sedation, and HZ-166 and TPA023 induced primarily triaza-benzo[e]azulene-3-carboxylic acid ethyl ester; function- rest/sleep posture. MRK-696 induced rest/sleep posture and ally selective with relatively high intrinsic efficacy for a2anda3 observable ataxia. Zolpidem, but no other compounds, signifi- subunit-containing GABAA receptors), MRK-696 [7-cyclobutyl-6- cantly increased tactile/oral exploration. The sedative effects (2-methyl-2H-1,2,4-triazol-2-ylmethoxy)-3-(2-flurophenyl)-1,2,4- engendered by alprazolam, diazepam, and zolpidem generally triazolo(4,3-b)pyridazine; no selectivity but partial intrinsic were attenuated by bCCT pretreatments, whereas rest/sleep activity], and TPA023B 6,29-diflouro-59-[3-(1-hydroxy-1-methylethyl) posture and suppression of tactile/oral exploration were insensi- imidazo[1,2-b][1,2,4]triazin-7-yl]biphenyl-2-carbonitrile; partial intrinsic tive to bCCT administration. These data suggest that a2/3- efficacy and selectivity for a2, a3, a5 subunit-containing GABAA containing GABAA receptor subtypes unexpectedly may mediate receptors]. We further examined the role of a1 subunit-containing a mild form of sedation (rest/sleep posture), whereas a1-contain- GABAA receptors in benzodiazepine-induced sedative effects by ing GABAA receptors may play a role in moderate/deep sedation. Introduction This project was funded by the National Institutes of Health National Institute on Drug Abuse [DA011792, DA033795, DA043204 awarded to J.K. Benzodiazepines exert their effects by binding to the R.], National Institute on Alcoholism and Alcohol Abuse [AA016179, awarded g-aminobutyric acid type A (GABAA) receptor. GABAA recep- to D.M.P.], National Institute of Neurologic Disorders and Stroke [NS076517, tors are pentameric chloride ion channels consisting primarily awarded to J.M.C.], National Institute of Mental Health [MH996463, awarded to J.M.C.], as well as National Center for Research Resources [RR000168]. J.M. of two a, two b, and one g subunit(s) with multiple subtypes for C. owns patents on the use of HZ-166 (patent number: US9006233 B2, US each subunit. Accumulating evidence suggests that the be- 8835424 B2). havioral effects of benzodiazepines can be attributed to 1A.N.D. and Z.M. contributed equally to this work. 2 Current affiliation: Wake Forest Baptist Medical Center, Winston-Salem, different GABAA receptor subtypes (Rowlett et al., 2005; North Carolina. Rudolph and Knoflach, 2011; Tan et al., 2011). Studies have 3Current affiliation: Brandeis University, Waltham, Massachusetts. https://doi.org/10.1124/jpet.118.249250. shown that GABAA receptors containing the a1 subunit (i.e., s This article has supplemental material available at jpet.aspetjournals.org. a1GABAA receptors) play a role in the sedative and motor- ABBREVIATIONS: a1GABAA receptors, GABAA receptors containing the a1 subunit; a2GABAA receptors, GABAA receptors containing the a2 subunit; a3GABAA receptors, GABAA receptors containing the a3 subunit; a5GABAA receptors, GABAA receptors containing the a5 subunit; ANOVA, analysis of variance; bCCT, b-carboline-3-carboxylate-t-butyl ester; HZ-166, 8-ethynyl-6-(29-pyridine)-4H-2,5,10b-triaza-benzo[e]azulene- 3-carboxylic acid ethyl ester; MRK-696, 7-cyclobutyl-6-(2-methyl-2H-1,2,4-triazol-2-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo(4,3-b)pyridazine; TPA023B, 6,29-diflouro-59-[3-(1-hydroxy-1-methylethyl)imidazo[1,2-b][1,2,4]triazin-7-yl]biphenyl-2-carbonitrile. 145 146 Duke et al. impairing effects of benzodiazepines (e.g., Rudolph et al., in loss of benzodiazepine sensitivity for all but a3GABAA 1999; McKernan et al., 2000). In contrast, GABAA receptors receptors showed significant inhibition of locomotor activity containing a2 and/or a3 subunits (i.e., a2GABAA and/or when administered diazepam (Behlke et al., 2016). These a3GABAA receptors) are believed to be involved in the results raise the possibility that subtypes other than anxiolytic properties of benzodiazepines (e.g., Löw et al., a1GABAA subtypes may play a role in sedative and motoric 2000; McKernan et al., 2000; Rowlett et al., 2005), and recent effects of benzodiazepines. evidence suggests a role for the a5 subunit-containing GABAA The incongruent results with respect to GABAA receptor receptors (a5GABAA receptors; Behlke et al., 2016). subtypes and sedative effects of benzodiazepine-type com- Although the idea that a1GABAA receptors mediate the pounds may reflect, in part, differences across both laborato- sedative and motoric effects of benzodiazepines has gained ries and species in definitions of sedation. These definitions considerable acceptance, there are findings in the literature vary widely across laboratories using nonhuman animal that suggest some aspects of the sedative actions of benzodi- species, ranging from measures of locomotor activity to azepines might involve other GABAA receptor subtypes. For operant behavior to observational measures. The extent to example, administration of zolpidem, a benzodiazepine-type which these preclinical measures are reliable measures of the ligand that binds preferentially to a1GABAA receptors, was typical sedative metrics used in human clinical studies (e.g., associated with procumbent posture (essentially a measure of self-report of “dizziness, light headedness, tiredness, drowsi- robust sedation and/or anesthesia) in squirrel monkeys and ness,” and so on) is unclear at present. To begin to address this was insensitive to pretreatments with b-carboline-3-carboxylate- issue, we adapted definitions of sedation that used as a basis Downloaded from t-butyl ester (bCCT), an a1GABAA-preferring antagonist levels of arousal as defined by the American Society of (Platt et al., 2002). Moreover, in a review of the clinical Anesthesiologists, which has published standard defini- literature, Skolnick (2012) noted several instances of com- tions for drug-induced levels of sedation in pediatric patients pounds lacking activity at a1GABAA receptors but having (see Table 1, footnote a; American Society of Anesthesiolo- demonstrable sedative effects in human subjects (see also gists Task Force on Sedation and Analgesia by Non- Atack, 2011; Nickolls et al., 2018). A recent study using Anesthesiologists, 2002). Arousal was measured by trained jpet.aspetjournals.org transgenic mouse technology may provide a clue to these observers who assessed the ability of the animal to attend to effects in human subjects: Mice with point mutations resulting external stimuli (e.g., the observer, other animals in the room) TABLE 1 Behavioral categories, abbreviations, and definitions at ASPET Journals on September 25, 2021 Behavior Abbreviation Brief Description Species-typical behaviors Passive visual VIS Animal is standing or sitting motionless with eyes open Locomotion LOC At least two directed steps in the horizontal and/or vertical plane Self-groom GRM Picking, scraping, spreading, or licking of an animal’s own hair Tactile/oral exploration TAC Any tactile or oral manipulation of the cage or environment Scratch SCR Vigorous strokes of the hair