Pharmacological Targeting the REV-ERBs in Sleep/Wake Regulation

Abstract

The circadian clock maintains appropriate timing for a wide range of behaviors and physiological processes. Circadian behaviors such as sleep and wakefulness are intrinsically dependent on the precise oscillation of the endogenous molecular machinery that regulates the circadian clock. The identical core clock machinery regulates myriad endocrine and metabolic functions providing a link between sleep and metabolic health. The REV-ERBs (REV-ERBα and REV-ERBβ) are nuclear receptors that are key regulators of the molecular clock and have been successfully targeted using small molecule ligands. Recent studies in mice suggest that REV-ERB-specific synthetic agonists modulate metabolic activity as well as alter sleep architecture, inducing wakefulness during the light period. Therefore, these small molecules represent unique tools to extensively study REV-ERB regulation of sleep and wakefulness. In these studies, our aim was to further investigate the therapeutic potential of targeting the REV-ERBs for regulation of sleep by characterizing efficacy, and optimal dosing time of the REV-ERB agonist SR9009 using electroencephalographic (EEG) recordings. Applying different experimental paradigms in mice, our studies establish that SR9009 does not lose efficacy when administered more than once a day, nor does tolerance develop when administered once a day over a three-day dosing regimen. Moreover, through use of a time response paradigm, we determined that although there is an optimal time for administration of SR9009 in terms of maximal efficacy, there is a 12-hour window in which SR9009 elicited a response. Our studies indicate that the REV-ERBs are potential therapeutic targets for treating sleep problems as those encountered as a consequence of shift work or jet lag.

Fig 1. SR9009 remains effective at three-hour interval administration and causes sleep rebound.

(A) Schematic of experimental paradigm to assess efficacy of the REV-ERB agonist SR9009. Mice were administered with SR9009 at ZT6, ZT9 and ZT12 consecutively in order to assess efficacy of the drug when administered at three-hour intervals. (B) SR9009 increases wakefulness when dosed at ZT6 (F(1,14) = 17.3, p<0.005, ZT7 p = 0.002, ZT8 p = 0.03) and ZT9 (F(1,14) = 4.7, p<0.05, ZT10 p = 0.005, ZT11 N.S., but not ZT12 (F(1,14) = 0.54, N.S.). SR9009 inhibits SWS at ZT6 (F(1,14) = 11.0, p<0.01, ZT7 p = 0.001, ZT8 p = 0.004); but not ZT9 (F(1,14) = 1.5, N.S.) and ZT12 (F(1,14) = 0.95, N.S.); and SR9009 inhibits REM sleep at ZT6 (F(1,14) = 16.0, p<0.005, ZT8 p = 0.0002, ZT9 p = 0.001), ZT9 (F(1,14) = 13.9, p<0.005, ZT10 p = 0.03, ZT11 p = 0.006, ZT12 p = 0.007), and ZT12 (F(1,14) = 7.7, p < 0.05, ZT13 p = 0.04, ZT15 P = 0.007). There was no rebound in wakefulness (F(1,14) = 2.7, N.S.) or REM (F(1,14) = 8.9, N.S.) during the following dark phase, but there was a significant SWS rebound (F(1,14) = 13.7, p<0.005, ZT16 p = 0.004, ZT17 p = 0.001, ZT18 p = 0.01, and ZT24 p = 0.007). (C) SR9009 significantly increased REM sleep latency (F(1,8) = 6.8, p<0.05) at ZT6 (p = 0.003) and ZT12 (p = 0.002). *P<0.05, ** P<0.01, *** P<0.005.

Fig 2

Acute daily administration of SR9009 does not induce short-term tolerance (A) Schematic of experimental paradigm to assess short-term tolerance in response to the REV-ERB agonist SR9009. Mice were injected with SR9009 (i.p. 100mg kg^-1) at ZT6 on three consecutive days in order to assess tolerance. (B) SR9009 increased wakefulness, but only reached significance on day 3 (p<0.05), decreased SWS on days 2 and 3 (p<0.05), and decreased REM on day 3 (C) Overall, SR9009 increased SWS latency (F(1,5) = 7.7, p<0.05, only significant on Day 2 p = 0.02). SR9009 increased REM sleep latency, reaching significance on all days tested (F(1,5) = 183.4, p<0.0001, Day 1 p = 0.0004, Day 2 p<0.0001, and Day 3 p<0.0001). *P<0.05, *** P<0.005.

Fig 3. Time-Response EEG activity of SR9009 during the light phase.

(A) Schematic of experimental paradigm to assess SR9009 time-dependent behavioral activity of mice EEG. Mice were dosed with SR9009 at different time points (ZT3, ZT6, and ZT9) on separate days in order to generate a Time-Response Curve. (B) Injections with SR9009 (i.p. 100mg kg^-1) induced wakefulness at ZT6 (p<0.05), but not ZT3 and ZT9, reduced SWS at ZT6 (p<0.05), but not ZT3 and ZT9, and decreased significantly REM sleep at ZT3 (p<0.05), and ZT6 (p<0.05), but not at ZT9. The maximal effect on wakefulness was achieved when the animals were dosed at ZT6 compared to ZT3 and ZT9. *P<0.05

Fig 4. Time-Response EEG activity of SR9009 during the dark phase Mice were dosed with SR9009 at different time points (ZT15, ZT18, ZT21) on separate days in order to generate a Time-Response Curve.

Injections with SR9009 (i.p. 100mg kg^-1) failed to induce wakefulness at ZT15 and ZT18. but rather decreased wakefulness at ZT21 (p<0.05). SR9009 increased SWS at ZT21 (p<0.05) and had no effect on REM at any of these time points. *P<0.05.

Fig 5. SR9009 effect during transitions from light-to-dark and dark-to-light.

There was no effect of SR9009 on wakefulness or SWS at ZT0/24; however REM was significantly decreased at this time (p<0.05). There were trends toward increased wakefulness and decreased SWS and REM by SR9009 at ZT12 (all p’s = 0.06), but did not reach statistical significance. *P<0.05.

Fig 6. SR9009 SWS and REM sleep latency during a 24h period.

SR9009 injections increased the latency to enter REM sleep (F(1,4) = 16.9, p<0.05, ZT6 p = 0.003, ZT15, p = 0.005) in a circadian manner. The effects on SWS were not significant in the overall ANOVA. Latencies to enter SWS and REM following the acute injections at the three-hour intervals presented in Figs 3–6 are graphed across a 24h period. ** P<0.01.