In vitro and in vivo pharmacological activity of minor cannabinoids isolated from Cannabis sativa, Ayat Zagzoog et al., 2020

In vitro and in vivo pharmacological activity of minor cannabinoids isolated from Cannabis sativa

Ayat Zagzoog, Kawthar A. Mohamed, Hye Ji J. Kim, Eunhyun D. Kim, Connor S. Frank,
Tallan Black, Pramodkumar D. Jadhav, Larry A. Holbrook & Robert B. Laprairie

Scientific Reports,, 2020, 10, 20405, 1-13.

Doi : 10.1038/s41598-020-77175-y


The Cannabis sativa plant contains more than 120 cannabinoids. With the exceptions of Δ9-tetrahydro-cannabinol (Δ9-THC) and cannabidiol (CBD), comparatively little is known about the pharmacology of the less-abundant plant-derived (phyto) cannabinoids. The best-studied transducers of cannabinoid-dependent effects are type 1 and type 2 cannabinoid receptors (CB1R, CB2R). Partial agonism of CB1R by Δ9-THC is known to bring about the ‘high’ associated with Cannabis use, as well as the pain-, appetite-, and anxiety-modulating effects that are potentially therapeutic. CB2R activation by certain cannabinoids has been associated with anti-inflammatory activities. We assessed the activity of 8 phytocannabinoids at human CB1R, and CB2R in Chinese hamster ovary (CHO) cells stably expressing these receptors and in C57BL/6 mice in an attempt to better understand their pharmacodynamics. Specifically, Δ9-THC, Δ9-tetrahydrocannabinolic acid (Δ9- THCa), Δ9-tetrahydrocannabivarin (THCV), CBD, cannabidiolic acid (CBDa), cannabidivarin (CBDV), cannabigerol (CBG), and cannabichromene (CBC) were evaluated. Compounds were assessed for their affinity to receptors, ability to inhibit cAMP accumulation, βarrestin2 recruitment, receptor selectivity, and ligand bias in cell culture; and cataleptic, hypothermic, anti-nociceptive, hypolocomotive, and anxiolytic effects in mice. Our data reveal partial agonist activity for many phytocannabinoids tested at CB1R and/or CB2R, as well as in vivo responses often associated with activation of CB1R. These data build on the growing body of literature showing cannabinoid receptor-dependent pharmacology for these less-abundant phytocannabinoids and are critical in understanding the complex and interactive pharmacology of Cannabis-derived molecules.


Biologically active compounds derived from the Cannabis sativa plant are referred to as ‘phytocannabinoids’. The two most-thoroughly studied phytocannabinoids are Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). Both of these compounds are being intensively studied for their utility in treating chronic and acute pain, epilepsy, anxiety, and modulating appetite, and their potential toxicities and side effects1. The effects of phytocannabinoids in the human body are thought to be mediated by many different receptors including the type 1 and type 2 cannabinoid receptors (CB1R and CB2R, respectively); as well as other G protein-coupled receptors (GPCRs) such as serotonin (5HT) receptors, orphan GPCRs (e.g. GPR18, GPR55, and GPR119) and ligand-gated ion channels. The pharmacology of Δ9-THC is relatively well-established: THC is a CB1R and CB2R partial agonist1–5. The pharmacology of CBD is far-less clear. CBD has been described as a CB1R negative allosteric modulator, CB2R antagonist, GPR18 agonist, GPR55 antagonist, among other effects6–16. Beyond Δ9-THC and CBD, there are thought to be at least 120 other phytocannabinoids found in Cannabis for which the
receptor-mediated mechanisms are still actively being investigated2–5. Examples of these lesser-known phytocannabinoids include Δ9-tetrahydrocannabidiolic acid (Δ9-THCa), tetrahydrocannabivarin (THCV), cannabigerol (CBG), cannabichromene (CBC), and cannabidivarin (CBDV)2–5.

CB1R and CB2R, their endogenous agonists 2-arachidonoylglycerol (2-AG) and anandamide (AEA), and the associated anabolic and catabolic enzymes constitute the endocannabinoid system1,5. CB1R activation inhibits nociception and locomotor activity, activates reward pathways, and regulates mood, memory and cognition, and central hormone homeostasis1. CB2R activation inhibits the inflammatory response in lymphocytes and microglia1. Interest in the development of compounds that target CB1R and CB2R is at an all-time high because of their multiple physiological roles and consequent associations with many different disease states. To date, this interest has focused mainly on synthetic compounds that target CB1R such as antagonists/inverse agonists (e.g. rimonabant), negative allosteric modulators (e.g. Org27569), and positive allosteric modulators (e.g. GAT211 and ZCZ011); as well as inhibitors of cannabinoid catabolic enzymes that increase 2-AG or AEA levels17,18. Comparatively, little research has been done to isolate and characterize the wide variety of naturally-occurring cannabinoids of Cannabis (i.e. phytocannabinoids)2,19. The work that has been done indicates that many of these ligands are active at CB1R, CB2R, and other receptors2,19. Within the last three years, an influx of high-quality
studies has examined the pharmacology of subsets of these phytocannabinoids for their specific activity, receptor affinity, ligand bias, and neuroprotective properties in rodent models19–22. Previously, our group has characterized the pharmacology of endogenous, synthetic, and plant-derived cannabinoids in vitro and in vivo10,11,15,23,24. In the present study, we utilized high- and medium-throughput assay systems to screen phytocannabinoids against one another and a reference compound, CP55,940, to directly assess their pharmacodynamic activity.

The primary aim of the present study was to explore the CB1R-dependent, CB2R-dependent, and in vivo pharmacology of 8 phytocannabinoids: Δ9-THC, Δ9-THCa, THCV, CBD, cannabidiolic acid (CBDa), CBDV, CBG, and CBC (Fig. 1). All compounds were assayed for the displacement of [3H]CP55,940, inhibition of forskolin-stimulated cAMP accumulation, and βarrestin2 recruitment in Chinese hamster ovary (CHO) cells stably expressing either human CB1R or CB2R. Compounds were assessed for signaling bias between inhibition of cAMP and βarrestin2 and selectivity between the two cannabinoid receptors. Compounds were further screened in the in vivo tetrad assays for cataleptic, hypothermic, anti-nociceptive, locomotive, and anxiety-modifying activities. We observed that all compounds tested displayed some degree of activity at CB1R or CB2R, with several being weak partial agonists. As Canada and other jurisdictions increasingly permit the use of Cannabis for medicinal and non-medicinal purposes, this research provides critical insight about the therapeutic potential and utility of phytocannabinoids.