Developmentof Novel Pegylated ImidazobenzodiazepinesTargeting α5 GABAA Receptors for Anxiety: A ComparativeProdrug and Metabolite Study

Abstract We report on the development of a highly water-soluble pegylated imidazodiazepine ETK-II-83. The compound binds selectively to GABAA receptors among forty-five other receptors expressed in the brain with a higher affinity for the α5-containing GABAA receptors. The compound is converted in vitro by liver microsomes into ETK-III-9. The metabolic reaction selectively oxidizes the polyethylene glycol groups of ETK-II-83 forming eventually the stable 2-hydroxyethyl amide-bearing ETK-III-9. The conversion is fast with a half-life of 5.33 min. ETK-III-9 strongly binds to α3/α5 containing GABAA receptors. A pharmacokinetic study with ETK-II-83 confirmed metabolic instability resulting in low AUCs for blood and brain. Interestingly, higher brain concentrations than blood concentrations (KP = 5.17) were observed. The in vivo formation of ETK-III-9 inside ETK-II-83-treated animals was rapid and peaked at 40 min. Almost equal amounts of ETK-III-9 were found in blood and brain with half-lives of 219 and 202 min, respectively. We also conducted a pharmacokinetic study with ETK-III-9 at the same dose and observed three times higher AUCs for blood and brain. Importantly, the free brain concentration of ETK-III-9 in ETK-II-83-administrated animals was higher than its affinity for the GABAA receptors. This was confirmed by investigating ETK-II-83 with two anxiety mouse models. For the elevated plus maze, ETK-III-9- and ETK-II-83-treated animals spent significantly more time in the open arm than vehicle-treated animals, without any differences in the overall distance traveled. ETK-III-9- and ETK-II-83-treated animals also buried fewer marbles than vehicle control animals. To demonstrate that these effects were not caused by sedation or inhibition of sensorimotor inhibition, we conducted open field and rotarod tests. For both tests, ETK-III-9- and ETK-II-83-treated animals did not behave differently from vehicle-treated mice in contrast to diazepam-treated animals. The innovation is a highly water-soluble prodrug ETK-II-83 that metabolizes to ETK-III-9, an α3/α5 GABAA receptor-selective compound that reduces anxiety in rodent models.