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A series of novel carbazole-containing amides and ureas were synthesized. A structure-activity relationship study of these compounds led to the identification of potent cryptochrome modulators. Based on the desired pharmacokinetic/pharmacodynamic parameters and the results of efficacy studies in db/db mice, compound 50 was selected for further profiling.

A series of novel carbazole-containing sulfonamides and sulfamides were synthesized. A structure-activity relationship study of these compounds led to the identification of potent cryptochrome modulators. Based on the results of efficacy studies in diet-induced obese (DIO) mice, and the desired pharmacokinetic parameters, compound 41 was selected for further profiling.

Narcolepsy type 1 (Na-1) and 2 (Na-2) are characterized by an inability to sustain wakefulness and are likely caused by degeneration of orexin neurons. Near complete orexin neurodegeneration depletes orexin-A from the cerebrospinal fluid and produces Na-1. The pathophysiology of Na-2 is less understood, but has been hypothesized to be due to less extensive loss of orexin neurotransmission. The orexin-tTA; TetO diphtheria toxin A mouse allows conditional control over the extent and timing of orexin neurodegeneration. To evaluate partial ablation of the orexin field as a model of Na-2, orexin-A positive cell counts and sleep/wake phenotypes (determined by piezoelectric monitoring) were correlated within individual mice after different protocols of diet-controlled neurodegeneration. Partial ablations that began during the first 8 days of study were 14% larger than partial ablations induced during the last 8 days of study, six weeks later and prior to sacrifice of all mice, suggesting orexin-A positive cell death continued despite the resumption of conditions intended to keep orexin neurons intact. Sleep/wake of mice with 71.0% orexin-A positive cell loss, initiated at the beginning of study, resembled that of orexin-intact controls more than mice with near complete neurodegeneration. Conversely, mice with 56.6% orexin-A positive cell loss, created at the end of study, had sleep/wake phenotypes that were similar to those of mice with near complete orexin-A positive cell loss. Collectively, these results suggest that compensatory wake-promotion develops in mice that have some critical portion of their orexinergic system remaining after partial ablation.Statement of significanceThe pathophysiology of narcolepsy type 2 is poorly understood but has been hypothesized to be due, at least in part, to degeneration of a smaller proportion of the orexin neuronal field than occurs in narcolepsy type 1. To evaluate a transgenic mouse model of narcolepsy type 2, we correlated the sleep/wake phenotypes of individual, male and female adult mice that received diet-induced conditional ablations of orexin neurons with their orexin cell counts. Using a translatable measure of narcolepsy sleepiness severity, we demonstrated that compensatory wake-promoting responses developed in mice concurrent with progressive orexin neurodegeneration. These results provide important details necessary for preclinical drug discovery for therapeutic areas characterized by orexin insufficiency, such as narcolepsy, Parkinson’s disease, and other neurodegenerative disorders.

BackgroundAssessment of sleep/wake by electroencephalography (EEG) and electromyography (EMG) is invasive, resource intensive, and not amenable to rapid screening at scale for drug discovery. In the preclinical development of therapeutics for narcolepsy, efficacy tests are hindered by the lack of a non-EEG/EMG based translational test of symptom severity. The current methods study offers proof-of-principle that PiezoSleep (noninvasive, unsupervised piezoelectric monitoring of gross body movement, together with respiration patterns during behavioral quiescence), can be used to determine sleep/wake as applicable to the development of wake-promoting therapeutics. First, the translational wake-maintenance score (WMS, the ratio of time during the first half of the dark period spent in long wake bouts to short sleep bouts) of the PiezoSleep narcolepsy screen was introduced as a means by which to rank narcoleptic orexin/ataxin-3 mice and wild type mice by sleep/wake fragmentation severity. Accuracy of the WMS to detect narcoleptic phenotypes were determined in genotype-confirmed orexin/ataxin-3 mice and wild type colony mates. The WMS was used to identify the most highly symptomatic mice for resource-intensive EEG/EMG studies for further analysis of specific arousal states. Second, PiezoSleep was demonstrated for use in high-throughput screening of wake-promoting compounds using modafinil in orexin/ataxin-3 and wild type mice.ResultsThe WMS detected a narcoleptic phenotype with 89% sensitivity, 92% specificity and 98% positive predictive value. A 15-fold difference in WMS differentiated wild type littermates from the most severely affected orexin/ataxin-3 mice. Follow-up EEG/EMG study indicated 82% of the orexin/ataxin-3 mice with the lowest wake-maintenance scores met or exceeded the cataplexy-occurrence threshold (≥ 3 bouts) for inclusion in therapeutic efficacy studies. In the PiezoSleep dose-response study, the ED50 for wake-promotion by modafinil was approximately 50 mg/kg in both genotypes. Using unsupervised piezoelectric monitoring, the efficacy of wake-promoting compounds can be determined in a 5-arm study with 60 mice in less than one week—a fraction of the time compared to EEG/EMG studies.ConclusionsThe WMS on the PiezoSleep narcolepsy screen quantifies the inability to sustain wakefulness and provides an accurate measure of the narcoleptic phenotype in mice. PiezoSleep offers rapid, scalable assessment of sleep/wake for high-throughput screening in drug discovery.