Absence of Entourage: Terpenoids Commonly Found in Cannabis sativa Do Not Modulate the Functional Activity of Δ9-THC at Human CB1 and CB2 Receptors.

Introduction: Compounds present in Cannabis sativa such as phytocannabinoids and terpenoids may act in concert to elicit therapeutic effects. Cannabinoids such as Δ⁹-tetrahydrocannabinol (Δ⁹-THC) directly activate cannabinoid receptor 1 (CB₁) and cannabinoid receptor 2 (CB₂); however, it is not known if terpenoids present in Cannabis also affect cannabinoid receptor signaling. Therefore, we examined six common terpenoids alone, and in combination with cannabinoid receptor agonists, on CB₁ and CB₂ signaling in vitro. Materials and Methods: Potassium channel activity in AtT20 FlpIn cells transfected with human CB₁ or CB₂ receptors was measured in real time using FLIPR^® membrane potential dye in a FlexStation 3 plate reader. Terpenoids were tested individually and in combination for periods up to 30 min. Endogenous somatostatin receptors served as a control for direct effects of drugs on potassium channels. Results: α-Pinene, β-pinene, β-caryophyllene, linalool, limonene, and β-myrcene (up to 30-100 μM) did not change membrane potential in AtT20 cells expressing CB₁ or CB₂, or affect the response to a maximally effective concentration of the synthetic cannabinoid CP55,940. The presence of individual or a combination of terpenoids did not affect the hyperpolarization produced by Δ⁹-THC (10 μM): (CB₁: control, 59%±7%; with terpenoids (10 μM each) 55%±4%; CB₂: Δ⁹-THC 16%±5%, with terpenoids (10 μM each) 17%±4%). To investigate possible effect on desensitization of CB₁ responses, all six terpenoids were added together with Δ⁹-THC and signaling measured continuously over 30 min. Terpenoids did not affect desensitization, after 30 min the control hyperpolarization recovered by 63%±6% in the presence of the terpenoids recovery was 61%±5%. Discussion: None of the six of the most common terpenoids in Cannabis directly activated CB₁ or CB₂, or modulated the signaling of the phytocannabinoid agonist Δ⁹-THC. These results suggest that if a phytocannabinoid-terpenoid entourage effect exists, it is not at the CB₁ or CB₂ receptor level. It remains possible that terpenoids activate CB₁ and CB₂ signaling pathways that do not involve potassium channels; however, it seems more likely that they may act at different molecular target(s) in the neuronal circuits important for the behavioral effect of Cannabis.