## Terpenoids From Cannabis Do Not Mediate an Entourage Effect by Acting at Cannabinoid Receptors

David B. Finlay, Kathleen J. Sircombe, Mhairi Nimick, Callum Jones and Michelle Glass

Frontiers in Pharmacology, 2020, Volume 11, Article 359, 1-9.

doi : 10.3389/fphar.2020.00359

The entourage effect was a proposed explanation for biological observations that endocannabinoid ligand activities can be modified by other lipids released from cells at the same time. An increasing volume of anecdotal reports and interest in the plant have provoked research into the activity of minor chemical constituents of the plant—including volatile terpenoids such as myrcene, a- and b- pinene, b-caryophyllene, and limonene. However, to date, no clear interaction has been identified. The current study was designed to determine whether terpenes in the cannabis plant have detectable receptor-mediated activity, or modify the activity of D9-tetrahydrocannabinol, cannabidiol, or the endocannabinoid 2-arachidonylglycerol at the cannabinoid receptors. In addition, we have utilized a standard radioligand binding paradigm with ability to detect orthosteric and allosteric interactions of test compounds. With the possible exception of a weak interaction of b-caryophyllene with CB2, no data were produced to support the hypothesis that any of the five terpenes tested (either alone or in mixtures) have direct interactions with CB1 or CB2, as the binding of radioligand ([3H]-CP55,940), D9- tetra-hydrocannabinol, and cannabidiol were unaltered by the presence of terpenes. Similarly, terpene functional effects were also not detected, either alone or in combination with D9-tetrahydrocannabinol, cannabidiol, or 2-arachidonoylglycerol. This study adds to the evidence that the putative entourage effect cannot be explained by direct effects at CB1 or CB2.

Keywords : cannabis, cannabinoid, terpenoid, terpene, entourage effect, signaling, binding

INTRODUCTION

Cannabinol (CBN) was the first cannabinoid from the cannabis plant for which a structure was
identified (Cahn, 1933). Cannabidiol (CBD) was identified a few years later (Adams et al., 1940a),
and the same research group later came close to identifying the structure of tetrahydrocannabinols, in a study involving isomerization of CBD (Adams et al., 1940b). Shortly after, tetrahydrocannabinols were isolated from cannabis resin (Wollner et al., 1942)—though it was more than 20 years before chemical analytical methods were adequate for resolving the final structure of the main psychoactive component of cannabis, (−)D9-tetrahydrocannabinol (D9-THC; Gaoni and Mechoulam, 1964).

Meanwhile, Loewe was also the first to observe pharmacological differences between cannabinoids (Loewe, 1946), in a study differentiating D9-THC and a synthetic hexyl analog, from CBD: the former, but not the latter, caused catalepsy and central excitation (with some additional species differences). In the years since, at least 489 different compounds (ElSohly and Slade, 2005), including at least 113 cannabinoids (Aizpurua-Olaizola et al., 2016), have been identified from cannabis. The most abundant of these are D9-THC and CBD (Aizpurua-Olaizola et al., 2016; Scherma et al., 2018). D9-THC acts as a partial agonist at type 1 cannabinoid receptors (CB1), which are found mostly in the central and peripheral nervous system and mediate the intoxicating effects for which cannabis is well known (reviewed in Pertwee, 2008a; Pertwee, 2008b). It also acts at type 2 cannabinoid receptors (CB2), which are most highly expressed in immune cells (reviewed in Turcotte et al., 2016). In general, many of the effects of CBD are thought to occur through non-cannabinoid receptor mechanisms (Turner et al., 2017). However, CBD has been demonstrated to bind to CB2 at high (micromolar) concentrations (Pertwee, 2008b)— although this is also controversial, as some evidence suggests that at much lower concentrations than this, CBD may behave as an inverse agonist at CB2 and an antagonist (Thomas et al., 2007) or allosteric modulator (Laprairie et al., 2015) of CB1.

More recently, interest has also turned to the biological activity of the less abundant, “minor” phytocannabinoids and phytoterpenoids, and their ability to produce an “entourage effect”. This phenomenon was first described for endogenous glycerol esters (Ben-Shabat et al., 1998), when 2-linoleoylglycerol and 2-palmitoylglycerol were found to increase the on-target affinity and efficacy of the endogenous cannabinoid 2- arachidonoylglycerol (2-AG), with which they co-occur, in spleen—yet without detectable direct interaction with the cannabinoid receptors themselves (though these data were not shown). Similar observations have been described for Npalmitoylethanolamide and N-oleoylethanol-amide (which are co-synthesized with anandamide) and may potentiate anandamide-induced relaxation of arteries (Ho et al., 2008). Since the publication of the Ben-Shabat et al. study, the term “entourage effect” has been co-opted to refer to the idea that whole cannabis possesses greater therapeutic potential than its individual components (Russo, 2011; Worth, 2019), with many websites suggesting that terpenes can modify the high produced by D9-THC (e.g., https://www.heylocannabis.com/post/whatare- terpenes). Terpenoids are commonly found in plants (Gershenzon and Dudareva, 2007), and at least 120 have been found in cannabis (ElSohly and Slade, 2005)—of which some of the most commonly referenced appear to include linalool, myrcene, limonene, b-caryophyllene, and a- and b-pinene. Previous work has suggested that b-caryophyllene may act as a CB2 agonist (Gertsch et al., 2008), though subsequent studies have questioned this (Santiago et al., 2019). Evidence for cannabis-derived terpenoids having entourage activity is also sparse. A very recent study has attempted to examine the six terpenoids referred to above for potential entourage activity at cannabinoid receptors. When used either alone or in combination to stimulate AtT-20 cells expressing CB1 or CB2, D9-THC-induced hyperpolarization was unaffected (Santiago et al., 2019)—indeed no GIRK channel-related modulatory effects were detected in this molecular study for any of the terpenes. In a related GIRK assay paradigm, receptor desensitization was also unaffected (Santiago et al., 2019).

The current study aimed to clarify the putative molecular activity of five terpenoids of interest acting specifically (ontarget) through CB1/CB2, in a canonical activity pathway (cAMP) which can capture receptor effects with high sensitivity. Effects on orthosteric ligand binding were also included in the study design, as in addition to detecting orthosteric interactions this assay has been shown to be very sensitive to allosteric modulation of CB1 (Ahn et al., 2012; Ignatowska-Jankowska et al., 2015).

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