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PhD Thesis: Rudy Bagnera

Abstract of Dissertation:
Beta1 GABAA Receptor selectivity differentiates sedative and anxiolytic effects of novel GABAergic compounds

By Rudy Eufemio Orlando Bagnera
Doctor of Philosophy in Pharmacology and Toxicology
University of California, Irvine, 2008
Professor Kelvin W. Gee, Chair

Development of novel potential therapeutic agents for the treatment of anxiety includes the designing out of side effects including sedation. Reliable prediction of side effects in the design and testing are not adequately explained by current models of sedative mechanisms found with anxiolytic therapeutics. Chemical structure-activity relationships based upon in vitro electrophysiology, binding, in vivo behavioral and pharmacokinetic-pharmacodynamic studies shaped the design of enaminone positive allosteric modulators of the GABAA receptor.

Newly synthesized compounds were initially screened by [35S]TBPS radioligand binding screen for activity at the GABAA receptor, followed by two-electrode voltage clamp oocyte electrophysiology for further characterization at specific receptor subtypes. Stereospecific effects were seen with enaminone compounds in both screens at nanomolar potency with greater efficacy at beta2 and beta3 containing GABAA receptors over beta1 (b1). The (R)-amide or ester substituted enaminones had greater efficacy at b1 containing receptors which correlated to sedation seen in animal behavior.

Neuroanatomical binding experiments explained the behavioral findings by providing evidence of differential anxiety and arousal-related modulation by the enaminone compounds. Quinolone and enaminone anxiolytics possessing the beta subunit specificity show marked differences in displacement of [35S]TBPS binding in arousal related areas containing higher b1 subunit expression when compared to b2 and b3 expressing regions found in anxiety or non-related brain regions. Stereospecific binding differences were also seen, as the S-enantiomer 2-261 displaced binding in b1-rich regions less than the R-enantiomer 2-262.

A threshold level of activating b1 receptors with known sedatives and novel compounds caused rotarod failure, measured as a sign of sedation. Compounds with b1 activity below that threshold of 80% modulation of GABA EC10 currents in electrophysiology did not cause rotarod deficits. Testing of the non-sedative compounds in mouse light-dark transition test and the rat elevated plus maze anxiety paradigms show these compounds to be non-sedative anxiolytics. This new model of β1 activation is more accurate for predicting sedative liability and could have profound impact in anxiolytic drug discovery methods.