Cannabidiol, Cannabinol and Their Combinations Act As Peripheral Analgesics in a Rat Model of Myofascial Pain T ⁎ Hayes Wong, Brian E

Archives of Oral Biology 104 (2019) 33–39

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Archives of Oral Biology

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Cannabidiol, cannabinol and their combinations act as peripheral analgesics in a rat model of myofascial pain T ⁎ Hayes Wong, Brian E. Cairns

Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC, Canada

ARTICLE INFO ABSTRACT

Keywords: Objective: This study investigated whether local intramuscular injection of non-psychoactive cannabinoids, Cannabidiol cannabidiol (CBD), cannabinol (CBN), cannabichromene (CBC) and their combinations can decrease nerve Cannabinol growth factor (NGF)-induced masticatory muscle sensitization in female rats. Cannabichromene Design: In awake rats, changes in mechanical sensitivity induced by intramuscular injection of NGF and can- Cannabinoid receptors nabinoids were measured by applying an electronic von Frey hair over the masseter muscle to measure the Masseter muscle withdrawal response. The effect of CBD (5 mg/ml) and CBN (1 mg/ml) or their combinations CBD/CBN (1:1 mg/ Nerve growth factor ml or 5:1 mg/ml) were assessed. To confirm a peripheral action, electrophysiological experiments were undertaken in anesthetized rats to examine whether intramuscular injections of CBD (5 mg/ml) and CBN (1 mg/ml) altered the mechanical threshold of masticatory muscle mechanoreceptors. Results: In behavioral experiments, CBD (5 mg/ml) or CBN (1 mg/ml) decreased NGF-induced mechanical sensitization. Combinations of CBD/CBN induced a longer-lasting reduction of mechanical sensitization than either compound alone. No significant change in mechanical withdrawal threshold was observed in the contralateral masseter muscles and no impairment of motor function was found with the inverted screen test after any of the treatments. Consistent with behavioral results, CBD (5 mg/ml), CBN (1 mg/ml) and the combination of CBD/CBN (1:1 mg/ml) increased the mechanical threshold of masseter muscle mechanoreceptors. However, combining CBD/CBN (5:1 mg/ml) at a higher ratio reduced the duration of this effect. This may indicate an inhibitory effect of higher concentrations of CBD on CBN. Conclusions: These results suggest that peripheral application of these non-psychoactive cannabinoids may provide analgesic relief for chronic muscle pain disorders such as temporomandibular disorders and fibromyalgia without central side effects.

1. Introduction properties (Amar, 2006; Izzo, Borrelli, Capasso, Di Marzo, & Mechoulam, 2009; Maione et al., 2013; Pertwee, 2005; Philpott, Cannabis has been used as an analgesic for centuries, but its use is O’Brien, & McDougall, 2017). The only cannabis-based product ap- controversial due to its psychoactive effects (Maione, Costa, & Di proved for pain treatment currently on the market is SATIVEX® (GW Marzo, 2013; Robson, 2014). Advances in our knowledge of the en- Pharma Ltd. Wiltshire, UK), which is formulated as a buccal spray docannabinoid system, in particular the discovery that the psychoactive containing approximate 1-to-1 THC and CBD for the relief of muscle effects of cannabis are primarily due to delta-9-tetrahydrocannabinol spasticity in multiple sclerosis and neuropathic pain. Although it is (THC), renewed interest in cannabis-based analgesics (Maione et al., generally well tolerated, many patients discontinued its use due to 2013; Robson, 2014). There are over 100 chemical compounds in the adverse central nervous system effects, such as dizziness (Etges et al., cannabis plant and the clinical utility of many of these compounds re- 2016; Moreno Torres, Sanchez, & Garcia-Merino, 2014). mains unknown (Ulugol, 2014). For example, another cannabinoid Overproduction of nerve growth factor (NGF) has been proposed to found in the cannabis plant, cannabidiol (CBD), possesses minimal mediate muscle sensitization, while intramuscular injection of NGF has psychotropic effects but has anti-inflammatory and anti-nociceptive been shown to mimic the tender points observed in the craniofacial

Abbreviations: CBD, cannabidiol; CBN, cannabinol; CBC, cannabichromene; THC, delta-9-tetrahydrocannabinol; GPR, G-protein coupled receptors; NGF, nerve growth factor; TMD, temporomandibular disorders ⁎ Corresponding author at: Faculty of Pharmaceutical Sciences, The University of British Columbia, 2405 Wesbrook, Vancouver, V6T 1Z3, Canada. E-mail address: [email protected] (B.E. Cairns). https://doi.org/10.1016/j.archoralbio.2019.05.028 Received 6 March 2019; Received in revised form 15 May 2019; Accepted 27 May 2019 0003-9969/ © 2019 Elsevier Ltd. All rights reserved. H. Wong and B.E. Cairns Archives of Oral Biology 104 (2019) 33–39 regions of fibromyalgia and myofascial temporomandibular disorders (TMD) patients (Clauw, 2014; Rahman, Underwood, & Carnes, 2014; Wong, Dong, & Cairns, 2014, b). Both disorders show a marked sex- related difference with greater prevalence in women than in men (Cairns, 2007). In a previous study, we investigated whether intramuscularly injected THC reverse NGF-induced mechanical sensitization in female rats. We found that cannabinoid receptor type 1 and 2 (CB1 and CB2) are expressed by trigeminal ganglion neurons that in- Fig. 1. The drawing shows the timeline of experimental interventions. nervate the rat masseter muscle, and that intramuscular injection of THC alleviates NGF-induced mechanical sensitization by activating CB1 et al., 2014, Wong, Kang et al., 2014). The masseter muscle region was receptors (Wong, Hossain, & Cairns, 2017). We also found that in- shaved prior to injection and the injection sites were marked with a tramuscular injection of NGF reduced the expression of CB1 and CB2 permanent marker for subsequent identification. The concentration of receptors on nociceptive trigeminal ganglion neurons, suggesting NGF NGF was selected based on the concentration used in previous human may induce sensitization by a reduction in peripheral inhibitory input and rat experimental studies (Mann, Dong, Svensson, & Cairns, 2006; (Wong et al., 2017). These results suggest that peripheral cannabinoid Svensson, Cairns, Wang, & Arendt-Nielsen, 2003; Svensson, Wang, receptors may be a target for cannabinoid-based analgesics to treat Arendt-Nielsen, & Cairns, 2008; Svensson, Wang, Dong, Kumar, & muscle pain disorders. Cairns, 2010; Wong, Dong et al., 2014) Three days after NGF injection, In the present study, we investigated whether local intramuscular a post-NGF injection baseline (NGF baseline) was assessed. Subse- injection of non-psychoactive cannabinoids including CBD, cannabinol quently, either CBD, CBN, CBC, CBD/CBN or vehicle were injected into (CBN), and cannabichromene (CBC) or their combinations could reduce the left (NGF-injected) masseter muscle of rats under brief isoflurane NGF-induced masseter muscle sensitization in female rats. To in- anesthesia to determine the effects of peripheral application of canna- vestigate whether these cannabinoids were acting peripherally, masti- binoids on NGF-induced sensitization. Vehicle group results have been ff fi catory muscle a erent ber recordings were subsequently undertaken previously published (Wong et al., 2017). This study was part of the ff for each cannabinoid or combination shown to be e ective in the be- same project as the previous study and was performed over the same havioral studies. The overall goal was to determine if there is a po- time period, therefore a separate vehicle group was not tested. The tential for non-psychoactive cannabinoids as novel peripherally active investigator was blinded to the identity of the treatment groups until analgesics. after all data was collected and analyzed.

2. Materials and methods 3.2. Mechanical withdrawal threshold 2.1. Animals Mechanical withdrawal threshold was assessed with a rigid elec- Female (225–350 g, n = 54) Sprague–Dawley rats were used for all tronic von Frey hair (IITC Life Science, Woodland Hills, CA). Before experiments. Animals were housed in groups of two with a 12-h light/ NGF injection, rats were habituated to restraint in a towel and me- dark cycle. Food and water were given ad libitum. All animal procedures chanical withdrawal assessments made. The electronic von Frey hair were reviewed and approved by the University of British Columbia was applied perpendicularly to the masseter muscle at the site of NGF Animal Care Committee and complied with the guidelines of the injection previously marked with a permanent marker and the force Canadian Council on Animal Care. was gradually increased until the animal moved its head away from the stimulus (˜ 5 s duration). The mechanical test stimulus was applied at 2.2. Drugs 1 min intervals for 5 min and the average was calculated for further analysis. Mechanical withdrawal threshold was measured daily for 5 CBD, CBN, and CBC were purchased from Cayman Chemicals (Ann days prior to the start of the experiment to determine that measure- Arbor, USA). CBD was dissolved in acetonitrile, while CBN and CBC ments were stable. Mechanical withdrawal threshold readings recorded were dissolved in methanol, all to a concentration of 10 mg/ml in a the day prior to NGF injection were used as naïve baseline. After in- stock solution. Appropriate volumes of each solution were evaporated duction of the NGF-induced sensitization, behavioral testing was per- ff under nitrogen gas, and subsequently re-solubilized with 4% Tween 80 formed to evaluate the antinociceptive e ects of CBD, CBN, CBC and in isotonic saline to final concentrations used for injection. The fol- CBD/CBN, and the vehicle control groups (n = 6/group) at 3 days after lowing treatment groups were tested: CBD (1 and 5 mg/ml), CBN NGF injection. On the test day, a post-NGF injection baseline (NGF (1 mg/ml), CBC (1 mg/ml) and CBD/CBN combination (1:1 or 5:1 mg/ baseline) was recorded before injection of cannabinoids. After treat- ml). A volume of 10 μl was injected intramuscularly for all treatment ment injections, mechanical withdrawal threshold was measured at 10, groups, resulting in a dose of 10 μg (0.030-0.045 mg/kg) cannabinoids 30, 60, and 120 min after injection. injected at a concentration of 1 mg/ml and 50 μg(˜0.145-0.225 mg/kg) cannabinoid injected at a concentration of 5 mg/ml. For the CBD/CBN 3.3. Inverted screen test combination group, 10 or 50 μg CBD and 10 μg CBN were injected together. A modified version of the inverted screen test was performed to evaluate impaired motor function in rats (Coughenour, McLean, & 3. Behavioral experiments Parker, 1977; Maxwell, Brecht, Doctor, & Wolfe, 1993). Rats were trained how to perform the test daily for three days before they were 3.1. Administration of NGF and cannabinoids tested. Rats were placed on a screen (185 mm × 290 mm) with 5 mm diameter holes and the screen was slowly inverted 180 degrees until the Rats received an injection of NGF (25 μg/ml, 10 μl, Sigma, St. Louis, rats were suspended upside down on the bottom of the screen. The MO) or vehicle (phosphate buffered saline, PBS, 10 μ l) into the belly of animals were observed for their ability to climb to the top of the screen the left and right masseter muscles, respectively, under brief isoflurane in the next 60 s and were assigned to a score: (0) animals successfully anesthesia (See Fig. 1 for experimental timeline). NGF has been shown climbed to the top the screen; (1) held on the screen upside down; (2) or to induce local mechanical sensitization at the site of injection which fell from the screen. Baselines were measured prior to treatment and the lasts approximately 5 days after injection in female rats (Wong, Dong animals were re-tested at 10, 30, 60 and 120 min after treatment

34 H. Wong and B.E. Cairns Archives of Oral Biology 104 (2019) 33–39 administration. For the CBD and CBN groups, a separate group of female rats (n = 4/group) were used. For the CBC and the CBD/CBN groups, rats were tested following each behavioral mechanical withdrawal threshold time point (n = 6).

3.4. In vivo electrophysiology

In vivo electrophysiology recordings of single ganglion neurons that innervate the craniofacial muscles were performed to investigate the mechanism of action of peripherally injected cannabinoids. Rats (n = 6/group except CBD/CBN (1:1) with n = 4 and CBD/CBN (5:1) with n = 5)) from the behavioral experiments were used for subsequent in vivo electrophysiological experiments. Recordings were performed at least 7 days after the behavioral experiments and experiments were conducted on the PBS-injected masseter muscles (right side) of the rats to minimize potential residual effects of the previous cannabinoid treatments. Rats were anesthetized with isoflurane (2–2.5% in oxygen 97–98%; AErrane; Baxter) and surgically prepared for electrophysiological recording. Heart rate, blood pressure and body temperature were mon- itored and a trachea tube was inserted for ventilation throughout the experiment. The hair of the face was shaved and the animal’s head was positioned in a stereotaxic frame. A parylene-coated tungsten microelectrode (0.10”,2MΩ,A–M Systems Inc.) was lowered into the trigeminal ganglion through the brain via a small trephination in the skull. An incision was made over the neck to expose the brain stem, and the dura was removed to allow access for a stimulating electrode to contact the caudal brain stem. Mechanoreceptors innervating craniofacial muscles (masseter and temporalis) were identified by mechanical probing using a fine-tipped cotton swab. Antidromic collisions were performed to confirm projection of the muscle primary afferent nerve fiber to the caudal brain stem (Cairns, 2007; Wong, Dong et al., 2014). A stimulating electrode (parylene-coated tungsten microelectrode, 0.10”,2MΩ,A–M Systems Inc.) was lowered into the ipsilateral caudal brainstem and constant-current electrical stimuli (100 μs biphasic pulse, 10–90 μA, 0.5 Hz) were applied to evoke antidromic action potentials. Orthodromic action potentials were evoked by mechanical stimulation of the tissue. Collision was demonstrated by disappearance of the antidromic spike. The straight line distance between the stimulating and recording electrodes was divided by the latency of the antidromic action potential to estimate conduction velocity. Mechanical threshold was assessed with an electronic von Frey hair (IITC Life Sci- ence) by applying mechanical stimuli at 1 min intervals for 10 min to obtain baseline threshold. After baseline measurement, treatment was administered and mechanical threshold was reassessed at 10, 30, 60, and 120 min thereafter. At the end of the experiments, animals were euthanized with pentobarbital (Nembutal 100 mg/kg, Abbott Labora- tories, Chicago, IL).

3.5. Data analysis

For the behavioral experiments, mechanical withdrawal threshold from NGF and vehicle-injected sides was analyzed with a two-way repeated measures analysis of variance (ANOVA) with time and treatment as factors. For electrophysiology experiments, mechanical withdrawal threshold was also assessed with a two-way repeated measures analysis of variance (ANOVA) with time and treatment as factors. Relative Fig. 2. The line and scatter plots show the mean ( ± SE) relative mechanical mechanical threshold was calculated as post-treatment threshold withdrawal threshold of 6 female rats per treatment group following in- /baseline threshold. Post hoc Holm Sidak’s multiple comparison tests tramuscular injections of (A) CBD (1 and 5 mg/ml), (B) CBN (1 mg/ml) and (C) CBC (1 mg/ml) in behavioral experiments. Significant differences were ob- were used to compare post injection mechanical thresholds between served for CBD (5 mg/ml) and CBN compared to vehicle by 2-way repeated treatment groups at each time point. One-way repeated measures measures ANOVA. The asterisks (*) indicate significant differences compared ANOVA on ranks was used to analyze test scores in the inverted screen with the vehicle group and pound signs (#) indicate a significant difference fi tests. A probability level of 0.05 was considered signi cant for all tests. compared to the NGF baseline within the treatment group (Holm Sidak multiple Error bars represented standard error of the mean. comparison test, p < 0.05).

35 H. Wong and B.E. Cairns Archives of Oral Biology 104 (2019) 33–39

Fig. 3. The line and scatter plots show the mean ( ± SE) relative mechanical withdrawal threshold of 6 female rats per treatment group following in- Fig. 4. The line and scatter plots show the mean ( ± SE) relative mechanical tramuscular injections of CBD/CBN combinations (A) 1:1 mg/ml and (B) withdrawal threshold of 6 female rats per treatment group in the contralateral 5:1 mg/ml in behavioral experiments. Significant differences were observed by masseter muscles after intramuscular cannabinoid injections in behavioral ex- 2-way repeated measures ANOVA. The asterisks (*) indicate significant differ- periments (n = 6/group). No significant differences between treatment groups ences compared with the vehicle group and pound signs (#) indicate a sig- were found by 2-way repeated measures ANOVA. nificant difference compared to the NGF baseline within the treatment group (Holm Sidak multiple comparison test, p < 0.05). score for all treatments was 0).

4. Results 4.2. Effect of intramuscular injections of cannabinoids on masseter muscle mechanoreceptors 4.1. Effect of intramuscular injections of cannabinoids on NGF-induced sensitization Recordings from 33 masticatory muscle mechanoreceptors were undertaken. The median conduction velocity was 9.9 m/s and the In the behavioral studies, intramuscular injection of NGF decreased median mechanical threshold was 30.6 g. The population consisted of masseter muscle relative mechanical threshold at 3 days after injection 79% Aδ fibers (2–12 m/s) and 21% Aβ fibers (> 12 m/s). The results by 31%, consistent with earlier studies (Fig. 2;(Wong, Dong et al., from the electrophysiology experiments for CBD and CBN paralleled the 2014, Wong, Kang et al., 2014). A dose dependent effect was observed results from the behavioral experiments. CBD 5 mg/ml, but not 1 mg/ with CBD on NGF-induced mechanical sensitization. CBD (1 mg/ml) ml, significantly increased relative mechanical threshold, 30 min after had no effect, but a higher concentration of CBD (5 mg/ml) significantly injection (Fig. 5A). CBN 1 mg/ml significantly increased relative me- reversed NGF-induced mechanical sensitization at 10 and 30 min after chanical threshold at 30 and 60 min after its injection (Fig. 5B). CBC injection (Fig. 2A). CBN (1 mg/ml) significantly reversed NGF-induced had no effect on behavioral mechanical withdrawal threshold, therefore mechanical sensitization at 10 min post injection (Fig. 2B). No effect on its effect on individual mechanoreceptors was not assessed. In- mechanical withdrawal threshold was observed following intramuscular injection of CBD/CBN (1:1) significantly increased the re- tramuscular injection of CBC (1 mg/ml) (Fig. 2C). Intramuscular in- lative mechanical threshold of masseter muscle mechanoreceptors at jection of CBD/CBN (1:1) had a longer lasting effect when compared to 10, 30 and 60 min after injection (Fig. 6A) while CBD/CBN (5:1) sig- the individual cannabinoids at the same concentration with significant nificantly increased relative mechanical threshold at 10 min after in- increases in mechanical withdrawal threshold at 10 and 30 min after jection, however, the effect was shorter lasting with relative mechanical injection (Fig. 3A). CBD/CBN (5:1) had a similar effect on mechanical threshold returning to baseline values at 30 and 60 min after injection sensitization as CBD/CBN (1:1; Fig. 3B). No effect of any injection was (Fig. 6B). observed on the mechanical withdrawal threshold of contralateral masseter muscles for all treatment groups (Fig. 4). No impairment of 5. Discussion motor functions was found in the inverted screen tests after intramuscular injections of CBD, CBN and CBD/CBN treatments (median In a previous study, a single intramuscular injection of THC, the

36 H. Wong and B.E. Cairns Archives of Oral Biology 104 (2019) 33–39

Fig. 6. The line and scatter plots show the mean relative mechanical threshold ( ± SE) of masseter muscle mechanoreceptors following intramuscular injections of CBD/CBN combinations (A) 1:1 mg/ml (n = 4) and (B) 5:1 mg/ml (n = 5) in electrophysiological experiments. Significant differences were observed by 2-way repeated measures ANOVA. Asterisks indicate significant differences compared with the vehicle group (Holm Sidak multiple comparison test, p < 0.05).

combination of THC and CBD (Etges et al., 2016; Moreno Torres et al., 2014; Romero-Sandoval, Asbill, Paige, & Byrd-Glover, 2015). There- fore, in this study, we investigated whether intramuscular injections of other non-psychoactive cannabinoids alone (CBD, CBN, CBC) and in combination could provide similar relief in the rat NGF-induced muscle sensitization model while further minimizing the potential for limiting central adverse effects. The inverted screen test was used to evaluate Fig. 5. The line and scatter plots show the mean relative mechanical threshold effects on central motor function. While no impairments were found in ( ± SE) of masseter muscle mechanoreceptors following intramuscular injec- ff tions of (A) CBD (1 and 5 mg/ml) and (B) CBN (1 mg/ml) in electro- any of the treatment groups, which suggests minimal motor e ects, a physiological experiments. Six mechanoreceptors are recorded for each treat- more comprehensive battery of tests may be needed to evaluate the full ment group. Significant differences were observed for CBD (5 mg/ml) and CBN effects of CBD, CBN and CBC on central functions. compared to vehicle by 2-way repeated measures ANOVA. Asterisks indicate It was found that the non-psychoactive cannabinoids CBD, CBN and significant differences compared with the vehicle group (Holm Sidak multiple CBC were less efficacious than THC at the same concentration (1 mg/ comparison test, p < 0.05). ml) in reducing NGF-induced muscle sensitization in both behavioral and electrophysiological studies (Wong et al., 2017). This is not sur- primary psychoactive substance in the cannabis plant, was found to prising as these cannabinoids have weaker binding affinity for the CB1 th th alleviate NGF-induced sensitization in the masseter muscle by acti- receptors when compared to THC: CBN (˜1/10 ), CBC (˜1/20 ) and th vating CB1 receptors expressed by nociceptive fibers innervating this CBD (˜1/100 )(Pertwee, 2008; Rosenthaler et al., 2014), and we have muscle (Wong et al., 2017). This effect was not accompanied by evi- previously demonstrated that CB1 receptor activation is responsible for dence of central nervous system effects. It was proposed that peripheral the local analgesic effect of THC (Wong et al., 2017). This result is also cannabinoid receptors may be a potential target for analgesia without consistent with earlier studies showing similar ranking in effectiveness central adverse effects. Although this result was encouraging, repeated of these cannabinoids when administered systemically in different pain peripheral applications of THC may still induce significant undesirable models (Sofia, Vassar, & Knobloch, 1975; Welburn, Starmer, Chesher, & central effects that limit the use of many of the current cannabinoid Jackson, 1976). Interestingly, CBD at a higher concentration of 5 mg/ preparations for chronic pain treatment including Sativex, which is a ml significantly reduced NGF-induced mechanical sensitization in both

37 H. Wong and B.E. Cairns Archives of Oral Biology 104 (2019) 33–39 behavioral and electrophysiological experiments. This concentration is Funding much less than would be expected based on the binding affinity of these two compounds for the CB1 receptor, where CBD is ˜100x less potent This project was jointly funded by MITACS Canada and InMed than THC (Pertwee, 2008). It is, therefore, possible that CBD may have Pharmaceuticals Inc. Hayes Wong was the recipient of a MITACS effects on receptors other than CB1 when administered peripherally at Elevate Postdoctoral Fellowship. higher concentrations (Petrosino, Ligresti, & Di Marzo, 2009). For example, recent studies suggest that CBD may have effects on the orphan Declarations of interest G-protein coupled receptors (GPR)3, GPR6, GPR12 and GPR55 (Laun, Shrader, Brown, & Song, 2019; Ryberg et al., 2007). These receptors None. have no confirmed endogenous ligands and are phylogenetically closely related to the cannabinoid receptors (Laun et al., 2019; Ryberg et al., Acknowledgements 2007). GPR55 is predominantly expressed in the brain but is also found in many tissues and organs (Shore & Reggio, 2015). Lysophophatidy- Special thanks to Dr. Sazzad Hussein for his valuable comments on linositol has been proposed as its endogenous ligand, but many can- the initial design of the study. nabinoids have also been found to bind to it (Ryberg et al., 2007; Shore & Reggio, 2015). The physiological function of GPR55 is unclear but it References has been proposed to play a role in regulation of energy intake, bone resorption, cancer and pain (Marichal-Cancino, Fajardo-Valdez, Ruiz- Amar, B. (2006). Cannabinoids in medicine: A review of their therapeutic potential. Contreras, Mendez-Diaz, & Prospero-Garcia, 2017; Shore & Reggio, Journal of Ethnopharmacology, 105,1–25. Cairns, B. E. (2007). 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