A sleepy cannabis constituent: cannabinol and its active metabolite influence sleep architecture in rats

Abstract

Medicinal cannabis is being used worldwide and there is increasing use of novel cannabis products in the community. Cannabis contains the major cannabinoids, Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and cannabidiol (CBD), but also an array of minor cannabinoids that have undergone much less pharmacological characterization. Cannabinol (CBN) is a minor cannabinoid used in the community in "isolate' products and is claimed to have pro-sleep effects comparable to conventional sleep medications. However, no study has yet examined whether it impacts sleep architecture using objective sleep measures. The effects of CBN on sleep in rats using polysomnography were therefore examined. CBN increased total sleep time, although there was evidence of biphasic effects with initial sleep suppression before a dramatic increase in sleep. CBN increased both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. The magnitude of the effect of CBN on NREM was comparable to the sleep aid zolpidem, although, unlike CBN, zolpidem did not influence REM sleep. Following CBN dosing, 11-hydroxy-CBN, a primary metabolite of CBN surprisingly attained equivalently high brain concentrations to CBN. 11-hydroxy-CBN was active at cannabinoid CB₁ receptors with comparable potency and efficacy to Δ⁹-THC, however, CBN had much lower activity. We then discovered that the metabolite 11-hydroxy-CBN also influenced sleep architecture, albeit with some subtle differences from CBN itself. This study shows CBN affects sleep using objective sleep measures and suggests an active metabolite may contribute to its hypnotic action.

Fig. 1. Effects of acute CBN and zolpidem on sleep and wake in rats.

A Study design. B CBN increased total sleep time (χ²₃ = 10.732, P = 0.013), but had biphasic effects on C cumulative sleep time (CBN main effect, χ²₃ = 19.908, P = 0.013; CBN and time interaction, χ²₃₀ = 85.65, P < 0.0001). Zolpidem increased D total sleep time and E cumulative total sleep time (CBN treatment effects, χ²₁ = 6.057, P = 0.014; χ²₁ = 62.428, P < 0.0001 respectively). CBN did not affect F NREM sleep onset latency but increased G % NREM sleep (CBN and time interaction, χ²₃₀ = 74.4289, P < 0.0001₎, and had a biphasic effect on H cumulative NREM time (CBN treatment, χ²₃ = 66.926, P < 0.0001; CBN and time interaction, χ²₃₀ = 73.541, P < 0.0001). Zolpidem decreased I NREM sleep onset latency and increased J % NREM (zolpidem main effect, χ²₁ = 72.941, P < 0.0001; zolpidem by time interaction, χ²₁₀ = 83.479, P < 0.0001). CBN increased K REM sleep onset latency (main effect CBN, χ²₃ = 14.172, P = 0.003). CBN initially suppressed L % REM sleep, which was followed by a transient increase in % REM at 10 mg/kg only (main effect of CBN, χ²₃ = 13.655, P = 0.003; CBN and time interaction, χ²₃₀ = 76.089, P < 0.0001). CBN similarly influenced M cumulative REM time (CBN treatment, χ²₃ = 17.332, P < 0.0001; CBN and time interaction, χ²₃₀ = 91.312, P < 0.0001). Zolpidem did not affect N REM sleep onset latency, or O % REM sleep. CBN decreased P total wake time and decreased Q % active wake (CBN and time interaction, χ²₃₀ = 62.048, P = 0.001), and affected R % quiet wake (CBN main effect; χ²₃ = 10.340, P = 0.016). Zolpidem decreased S total wake time and decreased T % active wake (zolpidem main effect, χ²₁ = 105.464, P < 0.0001, zolpidem by time interaction, χ²₁₀ = 117.882, P < 0.0001). Time is expressed relative to lights on (ZT). Dunn–Šidák corrected multiple comparisons test *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars display ± SEM, n = 8 per group. CBN cannabinol, WSD within-subjects design (Latin square), EEG electroencephalography, EMG electromyography, IP intraperitoneal, NREM non-rapid eye movement sleep, REM rapid eye movement sleep, ZT zeitgeber time. Created with BioRender.com.

Fig. 2. Effects of repeated CBN exposure on sleep and wake in rats.

A Study design. CBN increased B total sleep time over days (CBN main effect, χ²₁ = 5.291, P = 0.021; there was no CBN treatment by day interaction, inconsistent with tolerance). CBN increased C NREM sleep onset latency (CBN main effect, χ²₁ = 5.632, P = 0.018). The effects of repeated CBN on % NREM on days D 1, E 8, and F 15 were subject to a degree of tolerance (CBN main effect, χ²₁ = 7.946, P = 0.005, CBN and day interaction, χ²₂ = 8.48, P = 0.014). CBN increased G REM sleep onset latency (CBN main effect, χ²₁ = 4.073, P = 0.044). CBN affected % REM over days H 1, I 8, and J 15 (CBN main effect, χ²₁ = 14.822, P = 0.0001; CBN and time interaction, χ²₁₀ = 100.778, P < 0.0001). The effects of repeated CBN on % REM sleep were different over days (CBN, time and day interaction, χ²₂₀ = 47.475, P = 0.001). Repeated CBN decreased K total wake time (CBN main effect, χ²₁ = 5.467, P = 0.019, but no CBN and day interaction). CBN decreased % active wake over days L 1, M 8, and N 15 (CBN and time interaction, χ²₁₀ = 40.168, P < 0.0001), which was subject to tolerance (CBN, time and day interaction, χ²₂₀ = 32.219, P = 0.041). Time is expressed relative to lights on (ZT). Dunn–Šidák corrected multiple comparisons test *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars display ± SEM, n = 8 per group. CBN cannabinol, BSD between-subjects design, EEG electroencephalography, EMG electromyography, IP intraperitoneal, NREM non-rapid eye movement sleep, REM rapid eye movement sleep, ZT zeitgeber time. Created with BioRender.com.

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 and C plasma 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 that reflects activation of G-protein-coupled inwardly rectifying potassium channels (GIRK). 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 = 5–8 per group, performed in technical duplicate. CBN cannabinol, 11-OH-CBN 11-hydroxy-cannabinol; 11-COOH-CBN 11-carboxy-cannabinol, IP intraperitoneal. Created with BioRender.com.

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, although it did increase C cumulative total sleep time, but in a biphasic manner with early-phase sleep suppression before subsequent enhancement (main effect 11-OH-CBN, χ²₃ = 34.612, P < 0.0001, 11-OH-CBN and time interaction, χ²₃₀ = 99.18, P < 0.0001). 11-OH-CBN significantly increased D NREM sleep onset latency (main effect 11-OH-CBN, χ²₃ = 13.679, P = 0.003). E 11-OH-CBN increased % NREM (main effect 11-OH-CBN, χ²₃ = 55.64, P < 0.0001, 11-OH-CBN and time interaction, χ²₃₀ = 113.816, P < 0.0001). F CBN biphasically influenced cumulative total NREM sleep (main effect 11-OH-CBN, χ²₃ = 36.884, P < 0.0001, 11-OH-CBN and time interaction, χ²₃₀ = 107.499, P < 0.0001). 11-OH-CBN increased G REM sleep onset latency (main effect 11-OH-CBN, χ²₃ = 26.885, P < 0.0001). 11-OH-CBN affected H % REM sleep (11-OH-CBN main effect, χ²₃ = 9.676, P = 0.022; 11-OH CB and time interaction, χ²₃₀ = 146.099, P < 0.0001). 11-OH-CBN suppressed I cumulative REM at the highest dose (11-OH-CBN and time interaction, χ²₃₀ = 79.744, P < 0.0001). 11-OH-CBN did not affect J total wake, but decreased K % active wake (11-OH-CBN and time interaction, χ²₃₀ = 94.753, P < 0.0001), and L % quiet wake (11-OH-CBN and time interaction χ²₃₀ = 120.4587, P < 0.0001). Time is expressed relative to lights on (ZT). Dunn–Šidák corrected multiple comparisons test *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars display ± SEM, n = 8 per group. 11-OH-CBN 11-hydroxy-cannabinol, WSD within-subjects design (Latin square), EEG electroencephalography, EMG electromyography, IP intraperitoneal, NREM non-rapid eye movement sleep, REM rapid eye movement sleep, ZT zeitgeber time. Created with BioRender.