DOI: 10.5061/dryad.zkh1893j6
title: A sleepy cannabis constituent: cannabinol and its active metabolite influence sleep architecture in rats

description: Products containing purified minor cannabinoids are being consumed without
 sufficient scientific evidence. Cannabinol (CBN) is claimed to be a
 “sleepy cannabinoid”; however,  no study has examined whether it
 impacts sleep architecture using objective measures. We therefore examined
 the effects of CBN on sleep in rats using polysomnography. We found CBN
 increased total sleep time, although there was evidence of biphasic
 effects. Following CBN dosing, a primary metabolite of CBN attained
 equivalently high brain concentrations to CBN. The primary metabolite of
 CBN was active at cannabinoid receptors, but CBN had lower activity. We
 then discovered that the primary metabolite also influenced sleep
 architecture. This study shows CBN affects sleep and suggests an active
 metabolite may contribute to its hypnotic action.
description: Sleep data was collected from EEG/EMG recordings in
 rats. Rat tissue concentrations of CBN and its
 metabolites data were collected using LC-MS/MS.
 Molecular pharmacology data was collected from the CB1/CB2
 receptor assay using FLIPR and membrane potential assay.
description: # A sleepy cannabis constituent: cannabinol and its active metabolite
 influence sleep architecture in rats
 [https://doi.org/10.5061/dryad.zkh1893j6](https://doi.org/10.5061/dryad.zkh1893j6) ## Description of the data and file structure The data from each of the figures in the main paper and supplementary data section are in separate Excel files. The data is listed as Fig 1B etc which corresponds to the summary data presented in the manuscript. **Fig. 1.** Effects of acute CBN and zolpidem on sleep and wake in rats. (A) Study design. (B) CBN increased total sleep time (min), but had biphasic effects on (C) cumulative sleep time (min). Zolpidem increased (D) total sleep time (min) and (E) cumulative total sleep time (min). CBN did not affect (F) NREM sleep onset latency (min) but increased (G) % NREM sleep, and had a biphasic effect on (H) cumulative NREM time (min). Zolpidem decreased (I) NREM sleep onset latency (min), and increased (J) % NREM. CBN increased (K) REM sleep onset latency (min). CBN initially suppressed (L) % REM sleep, which was followed by a transient increase in % REM at 10 mg/kg only. CBN similarly influenced (M) cumulative REM time (min). Zolpidem did not affect (N) REM sleep onset latency (min), or (O) % REM sleep. CBN decreased (P) total wake time (min) and decreased (Q) % active wake, and affected (R) % quiet wake. Zolpidem decreased (S) total wake time (min) and decreased (T) % active wake. Time is expressed relative to lights on (ZT). n/a refers to not applicable/there was no data collected here. **Fig. 2.** Effects of repeated CBN exposure on sleep and wake in rats. (A) Study design. CBN increased (B) total sleep time over days (min). CBN increased (C) NREM sleep onset latency (min). The effects of repeated CBN on % NREM on days (D) 1, (E) 8, and (F) 15 were subject to a degree of tolerance. CBN increased (G) REM sleep onset latency (min). CBN affected % REM over days (H) 1, (I) 8 and (J) 15. The effects of repeated CBN on % REM sleep were different over days. Repeated CBN decreased (K) total wake time (min). CBN decreased % active wake over days (L) 1, (M) 8, and (N) 15, which was subject to tolerance. Time is expressed relative to lights on (ZT). n/a refers to not applicable/there was no data collected here. **Fig. 3.** Total synthesis of CBN metabolites and their pharmacological characterization. **(A**) Synthetic route of primary metabolite 11-OH-CBN and terminal metabolite 11-COOH-CBN. (**B**) Brain (pg/mg) and (**C**) plasma (ng/ml) pharmacokinetic profile of CBN, 11-OH-CBN, and 11-COOH-CBN following administration of 10 mg/kg CBN IP to rats. 11-OH-CBN attained similar brain exposures to CBN. n = 4 per group. (**D**) Overview of pharmacological characterization of CBN and its major metabolites. Assessment of CBN and its metabolites at human cannabinoid (**E**) CB1 and (**F**) CB2 receptors expressed in AtT20 cells using membrane potential assay (change in fluorescence, % CP55,940). CBN showed low activity at CB1 receptors, whilst 11-OH-CBN behaved as a modestly potent partial agonist. Responses as depicted as a percentage of the response 1 µM CP 55,940, a potent, non-selective CB1/CB2 receptor agonist. n = 5-11 per group, performed in technical duplicate. n/a refers to not applicable/there was no data collected here. x data are missing data due to experimental error. **Fig. 4.** Effects of acute 11-OH-CBN on sleep and wake in rats. (A) Study design. 11-OH did not significantly increase (B) total sleep time (min), although it did increase (C) cumulative total sleep time (min), but in a biphasic manner with early phase sleep suppression before subsequent enhancement. 11-OH-CBN significantly increased (D) NREM sleep onset latency (min). (E) 11-OH-CBN increased % NREM. (F) CBN biphasically influenced cumulative total NREM sleep (min). 11-OH-CBN increased (G) REM sleep onset latency (min). 11-OH-CBN affected (H) % REM sleep. 11-OH-CBN suppressed (I) cumulative REM (min) at the highest dose. 11-OH-CBN did not affect (J) total wake (min) but decreased (K) % active wake, and (L) % quiet wake. Time is expressed relative to lights on (ZT). n/a refers to not applicable/there was no data collected here. **Fig. S1.** Effects of CBN and zolpidem on duration and number of NREM and REM bouts in rats. (A) CBN and (B) zolpidem increased the mean duration of NREM bouts per hour (s). (C) CBN but not (D) zolpidem decreased the number of NREM bouts per hour (n). (E) CBN initially decreased REM bout duration (s) and then increased the duration of REM bouts (n). (F) Zolpidem did not affect the duration of REM bouts (s). (G) CBN biphasically influenced REM bout number (n). (H) Zolpidem did not affect the number of REM bouts nor (I) % quiet wake. Time is expressed relative to lights on (ZT). n/a refers to not applicable/there was no data collected here. **Fig. S2.** Effects of CBN and zolpidem on EEG power spectra, locomotor activity, and body temperature. **A** CBN nor **B** zolpidem increased NREM delta power (nV`2`), although there were increases at specific time points for both compounds. **C** CBN and **D** zolpidem increased REM theta power (CBN by time interaction (nV`2`). **E** CBN and F zolpidem decreased locomotor motor activity counts. G CBN and **H** zolpidem did not robustly affect body temperature (no treatment or treatment by time interactions) (degrees Celsius). Time is expressed relative to lights on (ZT). n/a refers to not applicable/there was no data collected here. **Fig. S3**. Effects of repeated CBN exposure on duration and number of NREM and REM sleep bouts in rats. CBN increased NREM bout duration over the days 1 **A**, 8 **B**, and 15 **C** (s), which was not subject to tolerance. CBN decreased NREM bout number over the days 1 **D**, 8 **E**, and 15 **F **(n), which was not subject to tolerance. CBN affected REM bout duration over the days 1 **G**, 8 **H**, and 15 **I (s)**. CBN affected REM bout number over the days 1 **J**, 8 **K**, and 15 **L **(n), which differed across days. CBN affected % quiet wake over days 1 **M**, 8 **N**, and 15 **O**, which was not subject to tolerance. Time is expressed relative to lights on (ZT). n/a refers to not applicable/there was no data collected here. **Fig. S4.** Effects of 11-OH-CBN on duration and number of NREM and REM sleep bouts in rats. 11-OH-CBN significantly decreased **A** duration of NREM bouts (s) and increased **B** NREM bout number (n). 11-OH-CBN did not affect duration of **C** REM bouts (s), however affected **D** REM bout number (n). Time is expressed relative to lights on (ZT). n/a refers to not applicable/there was no data collected here. **Fig. S5.** Effects of 11-OH-CBN on EEG power spectra, locomotor activity, and body temperature. 11-OH-CBN significantly increased **A** NREM delta power and **B** REM theta power (nV`2`). **C** 11-OH-CBN had a biphasic effect on locomotor activity (counts). **D** 11-OH-CBN decreased body temperature (degrees Celsius). Time is expressed relative to lights on (ZT). n/a refers to not applicable/there was no data collected here.