Introduction

The majority of available drugs that target GPCRs act at the receptor's orthosteric site – the site at which the endogenous ligand binds (Christopoulos and Kenakin, 2002). The cannabinoid CB 1 receptor is the most abundant GPCR in the central nervous system and is expressed throughout the periphery (reviewed in Ross, 2007; Pertwee, 2008). Orthosteric ligands of CB 1 receptors have been proposed as possible treatments for anxiety and depression, epilepsy, neurodegenerative diseases such as Huntington disease and Parkinson disease, and chronic pain (Pertwee, 2008; Piscitelli et al., 2012) and have been tested in the treatment of addiction, obesity and diabetes (Pertwee, 2008; Piscitelli et al., 2012). Despite their therapeutic potential, orthosteric agonists of CB 1 receptors are limited by their potential psychomimetic effects while orthosteric antagonists of CB 1 receptors are limited by their depressant effects (Ross, 2007).

An allosteric binding site is a distinct domain from the orthosteric site that can bind to small molecules or other proteins in order to modulate receptor activity (Wootten et al., 2013). All class A, B and C GPCRs investigated to date possess allosteric binding sites (Wootten et al., 2013). Ligands that bind to receptor allosteric sites may be classified as allosteric agonists that can activate a receptor independent of other ligands, allosteric modulators that alter the potency and efficacy of the orthosteric ligand but cannot activate the receptor alone and mixed agonist/modulator ligands. As therapeutic agents, allosteric modulators, unlike allosteric agonists and mixed agonist/modulator ligands, are attractive because they lack intrinsic efficacy. Therefore, the effect ceiling of an allosteric modulator is determined by the endogenous or exogenous orthosteric ligand (Wootten et al., 2013). In contrast, exogenous orthosteric ligands may produce adverse effects through supraphysiological over‐activation or down‐regulation of a receptor (Wootten et al., 2013). Unlike orthosteric ligands, allosteric modulators of CB 1 receptors may not produce these undesirable side effects because their efficacy depends on the presence of orthosteric ligands, such as the two major endocannabinoids, anandamide and 2‐arachidonylglycerol (2‐AG) (Ross, 2007; Wootten et al., 2013).

To date, the best‐characterized allosteric modulators of CB 1 receptors are the positive allosteric modulator lipoxin A 4 (Pamplona et al., 2012) and the negative allosteric modulators (NAMs) ORG27569 and PSNCBAM‐1 (Price et al., 2005; Horswill et al., 2007; Wang et al., 2011; Ahn et al., 2013). ORG27569 and PSNCBAM‐1 reduce the efficacy and potency of the CB 1 receptor agonists WIN55,212‐2 and CP55,940 to stimulate GTPγS35, enhance Gα i/o ‐dependent signalling and arrestin recruitment and inhibit CB 1 receptor internalization and cAMP accumulation at submicromolar concentrations (Price et al., 2005; Horswill et al., 2007; Wang et al., 2011; Ahn et al., 2013; Cawston et al., 2013). The well‐characterized NAM activities of ORG27569 and PSNCBAM‐1 are the standards against which new possible CB 1 receptor NAMs can be assessed.

The phytocannabinoid, cannabidiol (CBD) is known to modulate the activity of many cellular effectors, including CB 1 and CB 2 receptors (Hayakawa et al., 2008), 5HT 1A receptors (Russo et al., 2005), GPR55 (Ryberg et al., 2007), the μ‐ and δ‐opioid receptors (Kathmann et al., 2006), the TRPV1 cation channels (Bisogno et al., 2001), PPARγ (Campos et al., 2012) and fatty acid amide hydrolase (FAAH) (Bisogno et al., 2001). With regard to cannabinoid receptor‐specific effects, several in vitro and in vivo studies have reported that CBD acts as an antagonist of cannabinoid agonists at CB 1 receptors at concentrations well below the reported affinity (K i ) for CBD to the orthosteric agonist site of these receptors (Pertwee et al., 2002; Ryan et al., 2007; Thomas et al., 2007; McPartland et al., 2014). We recently reported that the effects of CBD on intracellular signalling were largely independent of CB 1 receptors (Laprairie et al., 2014a). However, CBD inhibited internalization of CB 1 receptors in vitro at submicromolar concentrations where no other CB 1 receptor‐dependent effect on signalling was observed (Laprairie et al., 2014a). Because ORG27569 and PSNCBAM‐1 are also known to inhibit CB 1 receptor internalization and taking into account earlier in vivo data suggesting that CBD can act as a potent antagonist at CB 1 receptors, we hypothesized that CBD could have NAM activity at CB 1 receptors.