Limiting Activity at 1-Subunit-Containing GABAA Receptor Subtypes Reduces Ataxia

GABAA receptor (R) positive allosteric modulators that selectively modulate GABAARs containing 2and/or 3over 1subunits have been reported across diverse chemotypes. Examples include loreclezole, mefenamic acid, tracazolate, and etifoxine. In general,“ 2/3-selective” GABAAR positive allosteric modulators are nonbenzodiazepines (nonBZs), do not show -subunit isoform selectivity, yet have anxiolytic efficacy with reduced ataxic/sedative effects in animal models and humans. Here, we report on an enantiomeric pair of nonBZ GABAAR positive allosteric modulators that demonstrate differential -subunit isoform selectivity. We have tested this enantiomeric pair along with a series of other 2/3-subunit selective, -subunit isoform-selective, BZ and nonBZ GABAA positive allosteric modulators using electrophysiological, pharmacokinetic, and behavioral assays to test the hypothesis that ataxia may be correlated with the extent of modulation at 1-subunit-containing GABAARs. Our findings provide an alternative strategy for designing anxioselective allosteric modulators of the GABAAR with BZ-like anxiolytic efficacy by reducing or eliminating activity at 1-subunit-containing GABAARs. Positive allosteric modulators of the GABAA receptor (R) such as the benzodiazepines (BZs) continue to be used to treat anxiety, despite the well-known side effect of sedation. Diverse drug discovery efforts over two decades have focused on generating “anxioselective” (i.e., reducing anxiety without sedation) GABAAR positive allosteric modulators. Medicinal chemistry efforts have focused primarily on modifications of the BZ template with limited success in reducing sedative liability (Whiting, 2006). One strategy to generate anxioselective positive allosteric modulators involves creation of positive allosteric modulators that selectively modulate individual GABAAR subtypes involved in anxiety, while avoiding those mediating sedation. Several laboratories have focused on -subunit isoform-selective BZ site agonists that evoke positive modulation of 2and 3but not 1-subunit-containing GABAARs. This “ 2/3-selective” approach is based on pharmacological and genetic data suggesting that 2and 3subunit-containing GABAARs mediate the anxiolytic actions of BZs, whereas those with 1-subunits, especially the 1 2 2 subtype, are thought to mediate their sedative effects (Rudolph et al., 1999; McKernan et al., 2000). Consistent with this general theory, L-838,417 is a 2,3subunit-selective partial agonist BZ receptor ligand reported to be anxioselective in animal models (McKernan et al., 2000). However, recent clinical studies designed to determine whether 2,3-subunit selectivity imparts reduced sedative liability has resulted in equivocal results where BZ-like side effects were observed (de Haas et al., 2008, 2009). Moreover, the 3-subunit-selective BZ site partial agonist adipiplon has potent sedative activity and was in clinical development as a sedative-hypnotic (Sprenger et al., 2007). This work was supported by the National Institutes of Health National Institute of Mental Health [Grant MH082241]; and the University of California Discovery [Grant bio06-10577] (to K.W.G.). Parts of this work were previously presented by Bagnera RE, Johnstone TBC, Tran MB, Whittemore ER, Hogenkamp DJ, and Gee KW (2007) Characterization of a series of novel allosteric modulators of GABAA receptors with differential beta subunit selectivity. The Society for Neuroscience Conference; 2007 Oct 3–Nov 7; San Diego, CA. Society for Neuroscience, Washington, DC. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.161885. □S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: R, receptor; BZ, benzodiazepine; nonBZ, nonbenzodiazepine; DMSO, dimethyl sulfoxide; 5-HT, 5-hydroxytryptamine; nAChR, nicotinic acetylcholine receptor; HPLC, high-performance liquid chromatography; LD, light-dark transition; ANOVA, analysis of variance; RR, Rotarod; AD50, ataxogenic half-maximal dose where half of the mice fail the RR assay; EtOH, ethanol; MED, minimal effective dose; EPM, elevated plus maze; PK, pharmacokinetic; PD, pharmacodynamic; AI, anxioselective index; SAR, structure-activity relationship; LGIC, ligand-gated ion channel. 0022-3565/10/3323-1040–1053$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 332, No. 3 Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 161885/3557798 JPET 332:1040–1053, 2010 Printed in U.S.A. 1040 http://jpet.aspetjournals.org/content/suppl/2009/11/25/jpet.109.161885.DC1 Supplemental material to this article can be found at: at A PE T Jornals on A uust 0, 2017 jpet.asjournals.org D ow nladed from Selective activity at different -subunit isoforms does not fully explain the sedative effects of all BZ site agonists. A prominent example that argues against the theory is the BZ site ligand ocinaplon. It has robust full agonist-like activity at 1-subunit-containing GABAARs in vitro, yet it has reduced sedative liability in preclinical and clinical studies (Basile et al., 2004; Lippa et al., 2005; Popik et al., 2006). Furthermore, there have been no reports of compounds with 2,3-subunit selectivity achieving clinical proof-of-concept (i.e., anxioselectivity), despite the passage of almost a decade since the initial proposal of the 2,3subunit selectivity hypothesis. Apart from the intense focus on GABAAR subtypeselective BZs, other ways to elicit anxioselectivity via the GABAAR are relatively unexplored, especially as it relates to nonBZ site positive allosteric modulators with receptor subtype selectivity (Whiting, 2006). One interesting approach focuses on GABAAR positive allosteric modulators that are selective modulators of 2or 3over 1-subunitcontaining GABAARs. This type of selective modulation has been reported for compounds across diverse chemotypes (e.g., loreclezole, mefenamic acid, tracazolate, and etifoxine) (Halliwell et al., 1999; Thompson et al., 2002; Hamon et al., 2003; Groves et al., 2006). Empirical observations of these positive allosteric modulators in both animals and humans provide anecdotal evidence that suggests that the degree of activation of 1-subunit-containing GABAARs may contribute to their sedative/ataxic potential. Based on this correlative evidence, we propose the hypothesis that among nonBZ site positive allosteric modulators the continuum of sedative, ataxic and hypnotic effects elicited by GABAAR activation may also depend on -subunit isoform selectivity. Thus, limiting or eliminating activity at 1-subunit-containing GABAARs will reduce or abolish these effects, respectively. We recently identified a series of nonBZ site enaminone positive allosteric modulators with a range of potencies and efficacies, thus providing us with a template to create tools to test our hypothesis (Hogenkamp et al., 2007). Here, we report the characterization of an enantiomeric pair of enaminones, 2-261 and 2-262, that have differential -subunit isoform selectivity. We have tested this pair and 15 other -subunit isoform-selective and -nonselective positive allosteric modulators for GABAAR potency/efficacy, pharmacokinetic profiles, and behavior in anxiety models. Our studies provide correlative pharmacological evidence that selective modulation of -subunit-containing GABAAR subtypes may be another mechanism to separate anxiolytic from ataxic effects among nonBZ site GABAAR positive allosteric modulators. Materials and Methods Drugs. Loreclezole (Heeres, 1985; Astleford et al., 1989), tracazolate (Bare et al., 1989), etifoxine (Putman et al., 2007), and ocinaplon (Skolnick and Epstein, 2005) were synthesized in our lab using methods reported in the literature. The enaminones were synthesized as described previously (Hogenkamp et al., 2007; Table 1). Diazepam, bretazenil, etomidate, and mefenamic acid were purchased from Sigma-Aldrich (St. Louis, MO), whereas L-838,417 (McKernan et al., 2000) was from Tocris Biosciences (Ellisville, MO). For electrophysiology experiments, drugs were first dissolved in dimethyl sulfoxide (DMSO) to 10 mM and diluted in Ringer’s salt solution (0.1% total DMSO final solution). Drugs for per os administration were dissolved in polyethylene glycol400 and administered at 2 ml/kg. Drugs administered by intraperitoneal injection were dissolved in DMSO and administered at