Cannabigerol is a novel, well-tolerated appetite stimulant in pre-satiated rats

Abstract

Rationale

The appetite-stimulating properties of cannabis are well documented and have been predominantly attributed to the hyperphagic activity of the psychoactive phytocannabinoid, ∆9-tetrahydrocannabinol (∆9-THC). However, we have previously shown that a cannabis extract devoid of ∆9-THC still stimulates appetite, indicating that other phytocannabinoids also elicit hyperphagia. One possible candidate is the non-psychoactive phytocannabinoid cannabigerol (CBG), which has affinity for several molecular targets with known involvement in the regulation of feeding behaviour.

Objectives

The objective of the study was to assess the effects of CBG on food intake and feeding pattern microstructure.

Methods

Male Lister hooded rats were administered CBG (30–120 mg/kg, per ora (p.o.)) or placebo and assessed in open field, static beam and grip strength tests to determine a neuromotor tolerability profile for this cannabinoid. Subsequently, CBG (at 30–240 mg/kg, p.o.) or placebo was administered to a further group of pre-satiated rats, and hourly intake and meal pattern data were recorded over 2 h.

Results

CBG produced no adverse effects on any parameter in the neuromotor tolerability test battery. In the feeding assay, 120–240 mg/kg CBG more than doubled total food intake and increased the number of meals consumed, and at 240 mg/kg reduced latency to feed. However, the sizes or durations of individual meals were not significantly increased.

Conclusions

Here, we demonstrate for the first time that CBG elicits hyperphagia, by reducing latency to feed and increasing meal frequency, without producing negative neuromotor side effects. Investigation of the therapeutic potential of CBG for conditions such as cachexia and other disorders of eating and body weight regulation is thus warranted.

Introduction

Cannabis sativa L. has been utilised for medicinal and recreational purposes for millennia and is increasingly being recognised as a valuable source of unique compounds (phytocannabinoids) with a multitude of potential therapeutic applications (Deiana et al. 2012). The main psychoactive constituent of C. sativa, ∆9-tetrahydrocannabinol (∆9-THC), was first isolated and characterised in the 1960s (Mechoulam & Gaoni 1965; Gaoni & Mechoulam 1971), and this has been followed by the discovery of numerous additional phytocannabinoids (pCBs) over the last two decades (see Mechoulam & Hanus 2000 for review). Over 100 pCBs have now been isolated from C. sativa (Elsohly & Slade 2005), but despite their structural similarities, the pCBs show considerable heterogeneity in their pharmacological targets and/or physiological activities (Hill et al. 2012a).

One of the better known properties of C. sativa is its ability to stimulate appetite (hyperphagia), which has been described anecdotally by recreational users and demonstrated under laboratory conditions (Hollister 1971; Mattes et al. 1994). In rodent models, our laboratory has shown that oral administration of ∆9-THC to pre-satiated rats produced significant short-term hyperphagia (Williams et al. 1998), characterised by a marked reduction in latency to begin feeding (Williams and Kirkham 2002a), an effect that was reversed by co-administration of the selective CB1 receptor (CB1R) antagonist SR141716 (Williams and Kirkham 2002b). Similarly, CB1R-mediated hyperphagic effects have also been observed following administration of the endocannabinoids (eCB) anandamide (AEA) (Williams & Kirkham 1999) and 2-arachidonoylglyerol (2-AG) (Kirkham et al. 2002). Importantly, in the latter study, we also demonstrated altered brain levels of AEA and 2-AG during fasting and feeding states, implicating eCBs in the control of appetite.

In the years since these findings, the role of the eCB system in appetite regulation and energy balance has been the subject of intensive research and is now emerging as a major target area for a variety of metabolic disorders (see Di Marzo et al. 2011 for review). Despite promising therapeutic potential, treatments directly targeting CB1R-mediated appetite regulation have, thus far, had limited clinical success. The CB1R antagonist SR141716 was licenced as an anti-obesity treatment in Europe in 2006; however, it was withdrawn 2 years later due to adverse side effects including depression and suicidality (Derosa and Maffioli 2012). Synthetic ∆9-THC (dronabinol) is licenced for treatment of chemotherapy-induced nausea and vomiting; however, it is not recommended as a first-line treatment due to adverse side effects associated with its psychoactivity (Todaro 2012). Two clinical trials conducted on ∆9-THC for appetite stimulation in cancer cachexia patients did not show positive results, possibly due to the low doses required to attain an acceptable tolerability profile (Jatoi et al. 2002; Strasser et al. 2006).

Recently, we have started investigating the effects of the non-∆9-THC pCBs on feeding behaviour (Farrimond et al. 2010a; Farrimond et al. 2010b; Farrimond et al. 2012a; Farrimond et al. 2012b; Brierley et al. 2016). This work suggests that some pCBs may offer potential for therapeutic appetite regulation without the psychoactive side effect profile of ∆9-THC-containing preparations. In the present study, we investigated the effects of one such pCB, cannabigerol (CBG), which does not produce ‘cannabimimetic’ psychoactive side effects and is hence typically described as non-psychoactive (Mechoulam et al. 1970). CBG is a relatively little-studied biosynthetic precursor (in C. sativa) of the major pCBs ∆9-THC and cannabidiol (CBD) (Hill et al. 2012a). Pharmacodynamic studies of CBG in vitro have determined that it acts as a potent α2-adrenoceptor agonist (EC50 = 0.2 nM) and a modest 5-HT1AR competitive antagonist (at 10 μM) and can weakly bind, but not activate, CB1R and CB2R (K i  = 81 and 2600 nM, respectively) (Cascio et al. 2010; Pertwee et al. 2010). Furthermore, it has been shown to inhibit the reuptake of the endocannabinoid AEA (IC50 = 11.3 μM) and interacts with a number of transient receptor potential (TRP) channels, acting as an agonist at TRPA1, TRPV1 and TRPV2 (EC50 = 0.7, 1.3 and 1.7 μM, respectively) and as a potent antagonist at TRPM8 (IC50 = 0.16 μM) (De Petrocellis et al. 2011). CBG has also recently been shown to block voltage-gated sodium channels Nav 1.1, 1.2 and 1.5 (IC50 = 88, 79 and 36 μM, respectively) (Hill et al. 2014). Given that CBG readily penetrates the blood brain barrier (Deiana et al. 2012) and interacts with a number of eCB and non-eCB targets with known involvement in the control of feeding and energy balance (Halford et al. 2004; Di Marzo and Matias 2005; Lee et al. 2015), the in vitro data available suggest this that pCB could conceivably elicit a centrally mediated stimulation of feeding behaviour. Although there is a paucity of in vivo studies of CBG in general, and feeding studies in particular, it has been reported that an acute low dose (2.5 mg/kg, i.p.) administered to rats enhanced saccharin palatability in a taste reactivity test (O’Brien et al. 2013). As part of one of our previous studies, we investigated the effects on feeding behaviour of low doses of CBG (0.176–17.6 mg/kg, p.o.), which were scaled to the concentrations found in a low-∆9-THC cannabis extract which induced hyperphagia (Farrimond et al. 2012b). In that study however, despite a suggestion of increased food intake over 2 h, no significant effects of CBG on appetite were found.

The present study was thus conducted with the aim of investigating whether CBG was able to stimulate appetite, at higher doses than previously tested. To ensure that any potential therapeutic utility of CBG would not be compromised by detrimental neuromotor side effects, our first experiment comprised a neuromotor tolerability test battery to assess the pCB’s effects on locomotor activity, balance, fine motor control and muscular strength. As CBG has previously been found to have no cannabimimetic effects in the mouse tetrad assay up to a maximal tested dose of 80 mg/kg (El-Alfy et al. 2010), had minimal behavioural effects (at 3–100 mg/kg) in a mouse Irwin assay (Duncan et al. 2014) and no signs of acute toxicity were reported in a pharmacokinetic study of 120-mg/kg doses (Deiana et al. 2012), a dose range of 30–120 mg/kg was used in this tolerability experiment. Subsequently, a second experiment was conducted using a pre-feed satiation paradigm to assess the acute hyperphagic effects of CBG. Results from a pilot of this experiment suggested that CBG may elicit dose-dependent hyperphagia, which did not appear to have a ceiling effect up to 120 mg/kg, and so an additional 240-mg/kg dose group was included in the design of the experiment. Infrared activity monitoring was performed concurrently throughout the feeding experiment to corroborate the effects of CBG on locomotor activity determined during the tolerability battery and to extend this investigation over a longer time frame and higher dose range.

Methods

Experiment 1: effects of CBG in a neuromotor tolerability test battery

Drugs

CBG (GW Pharmaceuticals, UK) was dissolved directly into sesame seed oil (by magnetic stirring at 57 °C) to produce a maximal working concentration of 120 mg/ml. Working solutions of 60 and 30 mg/ml were then prepared by serial dilution. CBG solutions were prepared freshly on each test day and protected from light until administration.

Doses of CBG or sesame seed oil vehicle alone were administered using a within-subject design, with all experimental units (individual animals) receiving 0, 30, 60 and 120 mg/kg CBG according to a pseudo-random, counterbalanced, Latin square protocol. All animals received doses separated by a minimum 48-h washout period. On test days, animals were administered CBG or vehicle 60 min prior to commencement of testing. CBG or sesame seed oil vehicle was administered per ora (p.o.) via a syringe placed into the cheek pouch at 1-ml/kg dosing volume.

Animals

Twelve young adult male Lister Hooded rats (Harlan, UK), weighing 200–225 g on delivery, were housed in pairs in temperature and humidity-controlled rooms with reversed light cycles (dim red light 12:00–24:00), with standard laboratory chow and water available ad libitum.

Procedure

Prior to testing, animals were subjected to a 5-day habituation process, consisting of daily handling, vehicle drug administration, habituation to open field and static beam test procedures. On test days, all procedures were conducted during the first half of the dark period (12:00–18:00) in the same room as the animals were housed. All test equipment was cleaned with 70 % ethanol and allowed to dry completely between animals. All tasks were presented in the order below with animals having a 5-min rest period in their home cage between tasks.

Open field

Consisting of a 1.1 × 1.1 × 0.4-m black acrylic-lined box, delineated into a 5 × 5 square grid and comprising a 3 × 3 central sector and a single square-wide peripheral sector, the open field was illuminated by dim red light (∼10 lx). Animals were placed in a consistent corner of the open field, and behaviour was video recorded for 5 min. Videos were analysed offline using Observer XT software (Noldus, Netherlands). Locomotor activity was quantified based on the number of times animals crossed the lines on the open field floor, with time spent in the central area of the field and latency to first entry used to quantify anxiety-like behaviour (i.e. degree of thigmotaxis). It should be noted that the habituation period animals received to the open field component of the test battery is necessary for within-subject assessment of drug-induced changes of locomotor activity. However, as a consequence, the aversive/novel nature of the environment is attenuated in comparison to the non-habituated version of this task, which is primarily used to assess anxiety-like behaviour.

Static beam

The apparatus consisted of a 3.2-cm-diameter cylindrical beam, 1 m in length and suspended 0.5 m above floor level. A bright light was positioned at the start of the beam and an enclosed goal box at the end. Animals were placed at the start of the beam and allowed a maximum of 5 min to successfully traverse its length to reach the goal box. Animals were then given a 2-min rest period in home cages prior to repeating the test. Tests were video recorded for off-line coding using Observer XT software (Noldus, Netherlands). In the static beam test, performance generated four outcome measures, based on successful completion or length of beam traversed prior to falling (pass rate and distance travelled), number of times paws were fully extended past the beam (foot slips) and time taken to traverse the middle 50 cm of beam (speed).

Forelimb grip strength

Animals completed two repeats of the forelimb grip strength test, separated by a 30-s rest period. Animals were placed with forelimbs gripping a trapeze bar connected to a digital force gauge (FH50, Sauter GmbH, Germany), then uniformly pulled by the tail base away from bar along the horizontal plane until grip was released and peak force recorded.

Forelimb grip strength

Analysis All behavioural coding was conducted by an experimenter blinded to treatment allocation. For static beam and forelimb grip strength outcome measures, where animals were subjected to two tests during the battery, data represent the mean of the two technical repeats, with the exception of pass rate on static beam in which a score of 0–2 was allocated based on number of successfully completed tests. All continuous data were analysed using SPSS 18 (IBM, UK) by one-way repeated measures ANOVA (ordinal pass rate data were analysed by Friedman’s ANOVA), with degrees of freedom and p values corrected, where assumptions of sphericity were violated (using Greenhouse-Geisser correction). When significant overall dose effects were observed, planned comparisons of all dose groups vs vehicle group were conducted to reveal any significant pairwise comparisons. Results were considered significant if p < 0.05.

 

https://link.springer.com/article/10.1007/s00213-016-4397-4

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