The Entourage Effect: A study of Terpene Mechanism of Action and Interaction with Cannabinoid Receptor 1

The cannabis plant has previously been referred to as a ‘pharmacological treasure trove’ due to the hundreds of compounds found within both the flower and other parts of the plant. The main focus in medicinal use has focused on cannabinoids, most notably cannabidiol (CBD) and tetrahydrocannabinol (THC). The reasons for this are obvious as they are available at the greatest concentrations within the plant, whilst also having the greatest evidence for their effects right through from cells in a petri dish to patients. However, there is growing interest in how the other compounds in the cannabis plant may either produce effects on their own, or in combination with one another. This interaction is commonly referred to as the entourage effect, however its existence is continually debated due to mixed levels of evidence.

A key group of compounds which are purported to have their own effects and contribute to any entourage effect are terpenes and terpenoids. These compounds are not unique to the cannabis plant and can be found in many other plants. They are responsible for the characteristic scent of many plants, not least cannabis which contains particularly high concentrations of terpenes and terpenoids.

Studies in animals and human models however have identified pain-relieving, anti-microbial, and anti-inflammatory effects. A recent study in Scientific Reports has sought to identify the effects of four terpenes: α-humulene, geraniol, linalool, and β-pinene with respect to causing effects similar to cannabinoid receptor 1 (CB1R) agonists.

Methods

The study assessed the cannabimimetic (cannabinoid-like) effects for four selected terpenes: α-humulene, geraniol, linalool, and β-pinene. These were assessed in several different models including in vitro cell lines and in vivo mouse models. The responses to each terpene were compared to a CB1R agonist – WIN55,212-2, cannabinoid receptor 2 (CB2R) agonist – β-Caryophyllene and CB1R receptor antagonist – rimonabant.

In vivo models assessed pain-relieving properties, pro-seizure effects, causation of hypothermia, and effects on movement.

Pain relieving properties were assessed using the tail flick thermal latency test where mice were lowered tail-first into a hot water bath (47-52oC) and were timed to see how long it would take for them to remove theirs tail up to maximum of 10 seconds. The terpenes were injected intraperitoneally, and response was assessed. The peak response was then used to guide the dosing used in other tests of cannabimimetic activity.

Pro-seizure effects were tested using the ring test, reduction in movement was tested In the open field test and hypothermia was tested using a rectal thermometer.

Results

Pain-relieving effects

Geraniol and alpha-humulene exhibited 40-50% efficacy in reducing pain in a dose dependent manner. Beta-Pinene showed low efficacy in a non-dose-dependent manner (i.e. partial agonist at high doses). Linalool and β-Caryophyllene demonstrated low efficacy in dose-dependent manner. WIN55,212 demonstrated dose dependent efficacy that was greater than all other tested compounds, whilst rimonabant fully blocked each terpene response. When each terpene was combined with a lower dose of WIN55,212-2 the combined effect was greater compared to terpene or WIN55,212-2 on its own. This suggests that pain-relieving properties tested in this model are CB1R-mediated, and the effect is increased with the addition of CB1R agonists.

Other effects

All terpenes were found to have effects on the other tested models of pro-seizure effects, causation of hypothermia, and effects on movement. Effects on hypothermia were found to be additive with CB1R agonists, but the effect caused by the terpenes is not mediated by the CB1R Effects on pro-seizure behaviour were found to be additive with CB1Rs, but this effect is mostly mediated by A2a adenosine receptors, rather than CB1R. Effects on movement were additive with CB1R agonists and this effect was partially mediated by A2a adenosine receptors.

In vitro models

Studies of in vitro models suggested that terpenes affect downstream extracellular signal-regulated kinase (ERK) signalling in cells that express CB1Rs, therefore they act as CB1R agonist in vitro. In a binding pattern analysis, it was identified that geraniol binds to CB1Rs orthosterically, whilst linalool and β-Pinene bind allosterically. α- Humulene and β-Caryophyllene bind in a combination of orthosteric and allosteric binding.

Each terpene demonstrated inhibition of cAMP but only at high micromolar concentrations >10μM or up to 500μM in some circumstances therefore demonstrating low potency.

Conclusions

Each terpene studied demonstrates additive effects to CB1R agonists which may have implications in clinical studies which use CB1R agonists, such as THC. However, the effect of these clinically has yet to be studied in a robust manner.

Each terpene demonstrated low potency to CB1Rs in this study. However, the doses utilised in the in vivo aspect to this study of 200mg/kg suggest that the clinical effect seen is greater than can be attributed to the low potency at CB1Rs, and this is confirmed by demonstration that the studied effects were secondary to both CB1R-dependent and CB1R-independent mechanisms. Low potency drugs are not necessarily less desirable for therapy (ibuprofen, proglumide and metformin are all low potency for example)

Further evidence is required to assess the potential entourage effect in clinical populations.