Limited evidence has suggested that terpenes found in Cannabis sativa are analgesic, and could produce an “entourage effect” whereby they modulate cannabinoids to result in improved outcomes. However this hypothesis is controversial, with limited evidence. We thus investigated Cannabis sativa terpenes alone and with the cannabinoid agonist WIN55,212 using in vitro and in vivo approaches. We found that the terpenes α-humulene, geraniol, linalool, and β-pinene produced cannabinoid tetrad behaviors in mice, suggesting cannabimimetic activity. Some behaviors could be blocked by cannabinoid or adenosine receptor antagonists, suggesting a mixed mechanism of action. These behavioral effects were selectively additive with WIN55,212, suggesting terpenes can boost cannabinoid activity. In vitro experiments showed that all terpenes activated the CB1R, while some activated other targets. Our findings suggest that these Cannabis terpenes are multifunctional cannabimimetic ligands that provide conceptual support for the entourage effect hypothesis and could be used to enhance the therapeutic properties of cannabinoids.

The opioid receptors are important regulators of pain, reward, and addiction. Limited evidence suggests the mu and delta opioid receptors form a heterodimer (MDOR), which may act as a negative feedback brake on opioid-induced analgesia. However, evidence for the MDOR in vivo is indirect and limited, and there are few selective tools available. We recently published the first MDOR-selective antagonist, D24M, allowing us to test the role of the MDOR in mice. We thus cotreated CD-1 mice with D24M and opioids in tail flick, paw incision, and chemotherapy-induced peripheral neuropathy pain models. D24M treatment enhanced oxymorphone antinociception in all models by 54.7% to 628%. This enhancement could not be replicated with the mu and delta selective antagonists CTAP, naltrindole, and naloxonazine, and D24M had a mild transient effect in the rotarod test, suggesting this increase is selective to the MDOR. However, D24M had no effect on morphine or buprenorphine, suggesting that only specific opioids interact with the MDOR. To find a mechanism, we performed phosphoproteomic analysis on brainstems of mice. We found that the kinases Src and CaMKII were repressed by oxymorphone, which was restored by D24M. We were able to confirm the role of Src and CaMKII in D24M-enhanced antinociception using small molecule inhibitors (KN93 and Src-I1). Together, these results provide direct in vivo evidence that the MDOR acts as an opioid negative feedback brake, which occurs through the repression of Src and CaMKII signal transduction. These results further suggest that MDOR antagonism could be a means to improve clinical opioid therapy.

Terpenes are small hydrocarbon compounds that impart aroma and taste to many plants, includingCannabis sativa. A number of studies have shown that terpenes can produce pain relief in various pain states in both humans and animals. However, these studies were methodologically limited and few established mechanisms of action. In our previous work, we showed that the terpenes geraniol, linalool, β-pinene, α-humulene, and β-caryophyllene produced cannabimimetic behavioral effects via multiple receptor targets. We thus expanded this work to explore the efficacy and mechanism of theseCannabisterpenes in relieving chronic pain. We first tested for antinociceptive efficacy by injecting terpenes (200 mg/kg, IP) into male and female CD-1 mice with chemotherapy-induced peripheral neuropathy (CIPN) or lipopolysaccharide-induced inflammatory pain, finding that the terpenes produced roughly equal efficacy to 10 mg/kg morphine or 3.2 mg/kg WIN55,212. We further found that none of the terpenes produced reward as measured by conditioned place preference, while low doses of terpene (100 mg/kg) combined with morphine (3.2 mg/kg) produced enhanced antinociception vs. either alone. We then used the adenosine A2A receptor (A2AR) selective antagonist istradefylline (3.2 mg/kg, IP) and spinal cord-specific CRISPR knockdown of the A2AR to identify this receptor as the mechanism for terpene antinociception in CIPN. In vitro cAMP and binding studies and in silico modeling studies further suggested that the terpenes act as A2AR agonists. Together these studies identifyCannabisterpenes as potential therapeutics for chronic neuropathic pain, and identify a receptor mechanism in the spinal cord for this activity.

Cannabis sativa, or marijuana, is the most commonly used illicit drug in the United States. Recreational and medical use has increased over the past decade due to loosening laws and legalization in 11 states. Anecdotal evidence from users suggests the therapeutic value of C. sativa is greater when the whole plant is consumed versus individual constituents such as D9‐tetrahydrocannabinol (D9‐THC). This observation has been termed the “entourage effect” and is hypothesized to derive from the combinatorial effect of phytocannabinoids and terpenes; the sum of the constituents found in the whole plant. C. sativa varieties contain ~150 phytocannabinoids and over 100 terpenoids. Prevalent monoterpenes include a‐pinene, linalool, limonene, and b‐myrcene, while prevalent sesquiterpenes include b‐caryophyllene (a known cannabinoid receptor 2 (CB2) agonist) and a‐humulene. These terpenoid compounds dictate the specific aromas and flavors of cannabis. Although terpenes can be lost during heat extractions, cold extraction using CO2 can increase terpene content upwards of 10‐fold. With increased terpene concentrations, a stronger case for the “entourage effect” can be made. Recent evidence has been published against the “entourage effect”, however, terpenes in these studies were used at very low doses or were studied in a single in vitro model. We sought to test the question: is the “entourage effect” a possible physiological phenomenon? We thus tested individual terpenes of C. sativa for cannabinoid receptor 1 (CB1R) activity in comparison with the synthetic cannabinoid WIN55,212. Four terpenes, varying in amounts in C. sativa, were chosen for screening: linalool and a‐humulene (med‐high), b‐pinene (low‐med), and geraniol (low). Preliminary in vitro results demonstrated that each of these terpenes were agonists at the CB1R. In mice, activation of CB1Rs by CB1 agonists induces four distinct physiological responses known as the CB1R tetrad: antinociception, hypolocomotion, catalepsy, and hypothermia. Each terpene was subsequently tested for activity in the tetrad. Intraperitoneal injection (£ 200 mg/kg) of a‐humulene, b‐pinene, and geraniol resulted in a dose‐dependent increase in tail flick thermal latency, an indication of antinociception; linalool had no effect. Interestingly, at 200 mg/kg a‐humulene, b‐pinene, and geraniol were able to induce an effect greater than or equivalent to 5.6 mg/kg WIN55,212. These effects were at least partially mediated by the CB1R, as 3 mg/kg rimonabant (CB1R antagonist) caused partial reversal. At 200 mg/kg, each terpene caused a significant decrease in hypolocomotion, an increase in catalepsy, and decrease in core temperature. These preliminary results suggest these terpenes may induce CB1R‐dependent behaviors in mice, thus promoting the possibly of an “entourage effect”. Ongoing studies are examining the interaction between known CB1R ligands and these terpenes to identify whether their interactions at the CB1R are competitive or allosteric in nature, and whether these ligands interact behav...

Marijuana has been understudied for decades, primarily due to social stigma and legal restrictions. However, as legal restrictions begin to loosen among states, the potential medical benefits and pharmacological properties of marijuana are beginning to be explored. Terpenes, an expansive group of organic chemicals that impart odor and taste, are found in the Cannabis sativa plant and may work synergistically with cannabinoids, such as THC and CBD, in a term deemed the “entourage effect”. Anecdotally among the recreational and medical use community, terpenes have been reported to enhance the potency and physiological effects of marijuana. However, scientific evidence for the “entourage effect” is very limited. To evaluate this hypothesis, we obtained the C. sativa‐relevant terpenes: β‐pinene, α‐humulene, geraniol, and linalool. Utilizing Chinese hamster ovary cells (CHO) expressing the human cannabinoid receptor type 1 (CB1, CB1‐CHO) we screened these terpenes for CB1‐dependent phosphorylation of extracellular signal‐regulated kinase 1/2 (ERK1/2), a well‐known downstream target of CB1 activation, using Western blot. We observed that pERK levels were efficaciously stimulated by all four terpenes when compared to positive control, the selective CB1 agonist WIN 55,212‐2. These results appeared to be CB1‐dependent, as pre‐treatment of these cells with a selective CB1 antagonist, rimonabant (SRI141716), blocked ERK phosphorylation by each of the terpenes. We further verified the CB1‐dependent nature of these effects by examining ERK phosphorylation by the terpenes in wild type CHO (WT CHO) cells, which do not express the CB1 receptor. In these cells, β‐pinene and α‐humulene treatment resulted in ERK phosphorylation while linalool or geraniol treatment did not. In WT CHO cells, the ERK phosphorylation induced by β‐pinene and α‐humulene was not CB1‐dependent, as rimonabant pre‐treatment did not block it. These results thus suggest that geraniol and linalool could be CB1‐selective agonists, whereas β‐pinene and α‐humulene are non‐selective and may also activate one or more receptors besides CB1. Follow‐up studies will examine other measures of CB1 activity (binding, cAMP signaling, GTPgS coupling) to characterize the binding and functional properties of these terpenes at the CB1 receptor, as well as identifying the other targets of β‐pinene and α‐humulene. Once we’ve characterized these terpenes individually, we aim to investigate their role in the “entourage effect”, by testing their modulation of typical cannabinoid (THC, etc.) pharmacology. Translationally, these findings could have implications in marijuana cultivar breeding and could help produce strains optimized for specific terpene profiles, which could be more efficacious for chronic pain management and other therapeutic uses. Support or Funding Information This study was supported by institutional funds from the University of Arizona. JMS has received research funding from Botanical Results, LLC, a company that develops cannabidiol products (not related to cur...