Minor cannabinoids are divided into neutral, acidic and varinic phytocannabinoids. Minor cannabinoids include cannabinol (CBN), cannabichromene (CBC), cannabigerol (CBG), cannabidioloic acid (CBDA), cannabigerolic acid (CBGA), tetrahydrocannabinolic acid (THCA), cannabinolic acid (CBNA), cannabidivarin (CBDV), tetrahydrocannabivarin (THCV), cannabigerovarin (CBGV), cannabichromevarin (CBCV), and others (Hanus, et al., 2016; Gülck and Möller, 2020). Cannabinoids appear naturally in the cannabis plant in their acidic forms and are thought to confer antioxidant and defense mechanisms (insecticidal, antimicrobial, etc.,) to the plant. Acidic cannabinoids undergo decarboxylation during heating and are converted to the corresponding neutral cannabinoids (Figure 1
). For example, THCA is converted to Δ9-THC when cannabis is smoked or vaporized. Some decarboxylation also occurs with passage of time at room temperature and during exposure to light. Cannabis products intended to contain the acidic forms of cannabinoids nearly universally also contain low levels of cannabinoids in their neutral forms. The varinic cannabinoids are considered rare but are now emerging as new targets of selective breeding. Varin compounds such as CBDV and THCV contain two fewer carbon atoms than their non-varin counterparts (CBD and Δ9-THC) endowing these cannabinoids with unique pharmacological properties (see below).
As described for the endocannabinoids, the overall pharmacological action of the minor cannabinoids often results from binding at both cannabinoid and “off target” receptors. This combination of receptor-mediated actions makes them well suited as multi-target therapeutic agents. While a number of minor cannabinoids including CBN and THCV bind to the CB1 receptor, they have significantly less binding activity when compared with Δ9-THC (Rhee, et al., 1997; Zagzoog, et al., 2020). To date, none of the minor cannabinoids have been clinically demonstrated to act as psychotropic drugs. The reported potencies of the minor cannabinoids at the CB1 and CB2 receptors and TRP channels are summarized in Table 1 and discussed in the sections below.
Neutral Cannabinoids
Cannabinol
CBN was originally isolated from Indian hemp in 1896 making it the first phytocannabinoid identified in cannabis (Wood, et al., 1886). The structural determination and total synthesis of CBN was carried out by Adams and colleagues in the 1940s (Adams, et al., 1940b). CBN is not synthesized in the cannabis plant, but is derived during the degradation of Δ9-THC. Even under ideal storage conditions, exposure to UV light and/or heat over time results in the conversion of Δ9-THC to CBN. Using 3(H)-labelled synthetic cannabinoid (e.g., HU-243, CP-55,490) displacement assays it was determined that CBN has low binding affinities for the CB1 and CB2 receptors when compared with Δ9-THC (Rhee, et al., 1997; Mahadevan, et al., 2000; Rosenthaler, et al., 2014). In addition, CBN is less potent than Δ9-THC in CB1 receptor-mediated inhibition of adenylyl cyclase, but displays equal potency in CB2 receptor-mediated inhibition (Rhee, et al., 1997). CBN is an agonist at TRPV1, TRPV2, TRPV3 and TRPV4 channels stimulating cell Ca2+ influx with the activation of Ca2+-dependent pathways (De Petrocellis, et al., 2011). It is also a potent and efficacious agonist of the TRPA1 channel. In addition, CBN acts as a potent antagonist of icilin activation of the TRPM8 channel (De Petrocellis, et al., 2011).
CBN has been identified as a potential analgesic and anti-inflammatory agent. CBN isolate has been reported to relieve chronic muscle pain disorders such as temporomandibular disorders and fibromyalgia in a rat model of myofascial pain (Wong and Cairns, 2019). For example, CBN (1 mg/ml) reduces mechanical sensitivity induced by intramuscular injection of nerve growth factor in the masseter muscle (Wong and Cairns, 2019). While CBN is not as widely recognized as CBD and Δ9-THC for its anti-inflammatory properties, it may have therapeutic benefits in treating allergic airway diseases. CBN attenuates the production of interleukins 2, 4, 5, 13 and decreases allergen mucus production in OVA-sensitized and challenged A/J mice (Jan, et al., 2003). CBN and Δ9-THC (but not CBD) can also be used to treat glaucoma since they prevent inflammation that causes elevated intraocular pressure (ElSohly, et al., 1981). In addition, preliminary data indicate that CBN decreases cell damage and acts as an antioxidant in a cell culture model of Huntington’s disease (Aiken, et al., 2004).
CBN also shows promise as an antibacterial agent and an appetite stimulant. As with other cannabinoids (e.g., CBC and CBG), CBN has been found to be highly efficacious against multiple antibiotic-resistant bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), making it a potentially viable treatment for staph infections (Appendino, et al., 2008). CBN also stimulates hyperphagia and increases food consumption and feeding time in rats (Farrimond et al., 2012a; Farrimond, et al., 2012b). Although CBN is not as potent an appetite stimulant as Δ9-THC, CBN administration is not associated with the psychotropic effects of Δ9-THC. Thus, CBN represents a non-intoxicating alternative to Δ9-THC as an appetite stimulant.
CBN-rich products are advertised for promoting sleep or relaxation without the impairment caused by Δ9-THC. Since CBN is a degradation product of Δ9-THC it is found in greater quantities in aged cannabis preparations. For this reason it is marketed as “the sleepy cannabinoid in old weed.” However, laboratory results obtained from sleep studies with CBN have been equivocal. In mice, CBN was reported to increase barbiturate-induced sleep time in one study (Yoshida, et al., 1995), while having no effects on sleep in another (Chesner, et al., 1974). When administered along with Δ9-THC in rats, CBN produces greater sedation compared with either cannabinoid alone (Fernandes, et al., 1974; Takahashi and Karniol, 1975). In one clinical study involving a small number of participants, the combination of Δ9-THC and CBN caused greater drowsiness than with Δ9-THC used alone (Karniol, et al., 1975). However, in a recent review of the CBN literature, Corroon (2021) found little evidence supporting a sleep promoting effect of CBN. Therefore, controlled studies are warranted to substantiate sleep-related claims of CBN containing products.
Cannabichromene
CBC is one of the most abundant minor cannabinoids found in cannabis. The structure of CBC was first determined using NMR spectroscopy by Gaoni and Mechoulam (Gaoni and Mechoulam, 1966). Although cannabinoid receptor studies using CBC are limited, the cannabinoid was initially identified as partial CB2 receptor agonist (Rosenthaler, et al., 2014). This was supported by experiments using a “hyperpolarization assay” with pituitary AtT20 cells in which CBC was found to be selective in stimulating the CB2 receptor over the CB1 receptor (Udoh, et al., 2019). In this same study, CBC was more potent and efficacious than Δ9-THC in causing cell hyperpolarization via the CB2 receptor. In contrast to these results, CBC was shown in a recent paper to display similar affinities for the CB1 and CB2 receptors and to cause both CB1 and CB2 receptor-mediated decreases in cellular cAMP levels (Zagzoog, et al., 2020). As is the case for CBN, CBC is a potent activator of the TRPA1 channel (De Petrocellis, et al., 2011). In addition, it activates TRPV3 and TRPV4 channels when applied at micromolar and submicromolar concentrations (De Petrocellis, et al., 2011). Other proposed sites of action of CBN are discussed below.
Anti-inflammatory effects of CBC were first reported in the 1980s using a rat model of edema. High doses of CBC were more efficacious than the nonsteroidal anti-inflammatory drug (NSAID) phenylbutazone in carrageenan-induced paw edema (Turner and ElSohly, 1981). CBC has been shown to reduce pain and inflammation associated with osteoarthritis in rats without the negative side effects of NSAIDs (Maione, et al., 2011). In addition, CBC attenuates lipopolysaccharide (LPS)-induced increases in nitric oxide levels in an in vitro model of colitis (Romano, et al., 2013) and reduces inflammation-induced GI motility (Izzo, et al., 2012). CBC also displays a modest anti-nociceptive effect in the mouse tail-withdrawal assay (Maione, et al., 2011; Zagzoog, et al., 2020). CBC regulates a number of cellular pathways involved in anti-nociception that include the stimulation of adenosine A1 receptors, CB1 receptors and TRPA1 channels (De Petrocellis, et al., 2011; Maione, et al., 2011). In addition, CBC has been proposed to inhibit AEA (anandamide) reuptake; thus potentiating the physiological effects of AEA (De Petrocellis, et al., 2011). Like other synergistic actions of cannabinoids, CBC has a greater anti-inflammatory response when combined with Δ9-THC than when either cannabinoid is used alone (DeLong, et al., 2010).
The anti-inflammatory actions of CBC may be important in its ability to function as a neuroprotective drug. CBC increases the viability of neural stem progenitor cells (NSPCs) in vitro through an ERK dependent mechanism (Shinjyo and Di Marzo, 2013). In addition, CBC inhibits astroglial differentiation of the NSPCs (Shinjyo and Di Marzo, 2013). NSPCs are modulated by surrounding microglial cells, brain immune cells, and astrocytes, which produce both pro- and anti-inflammatory factors. The potential anti-inflammatory and neuroprotective effects of CBC may occur through its suppression of reactive astrocytes (Covelo, et al., 2021). CBC inhibition of NSPCs differentiation into astrocytes may therefore offer a protective effect against neuro-inflammation, Alzheimer’s disease, and hepatic encephalopathy (Covelo, et al., 2021).
Δ9-THC possesses anti-tumor properties and is used for treating several different forms of cancer (Fraguas-Sánchez and Torres-Suárez, 2018). However, the psychotropic qualities of Δ9-THC limit its use as a chemotherapy agent. CBC may be beneficial in cancer treatment due to its ability to increase blood levels of AEA (see above). AEA has been shown to inhibit breast cancer cell proliferation and induce death of colon cancer cells (De Petrocellis, et al., 1998; Patsos, et al., 2005). CBC was also shown to inhibit cell migration and disrupt the cell cytoskeleton in an in vitro model of urothelial cancer (Anis, et al., 2021). In one study that examined the anti-tumor effects of several minor cannabinoids, only CBG was more potent than CBC at inhibiting the growth of several cancer cell lines (Ligresti, et al., 2006).
Cannabigerol
CBG is produced via decarboxylation of CBGA, the precursor molecule of the Δ9-THC and CBD branches of the cannabis synthesis pathway (Figure 1). Results obtained using cAMP assays revealed that CBG displays weak partial agonist activity at the CB1 and CB2 receptors (see Table 1) (Husni, et al., 2014; Navarro, et al., 2020; Zagzoog, et al., 2020). In CHO cells expressing both CB1 and CB2 receptors, CBG binds to the receptors with Kis in the low micromolar range (Navarro, et al., 2018). Of special note, in this same study CBG was found to compete with 3(H)-CP-55,490 for binding to the CB1 receptor, but not 3(H)-WIN-55,212-2 (Navarro, et al., 2018). This suggests the CBG and CP-55,490 (but not WIN-55,212-2) bind to the same orthosteric site on the receptor. CBG activates TRPV1, TRPV2, TRPV3, TRPV4 and TRPA1 channels at low micromolar concentrations (De Petrocellis, et al., 2011). CBG has also been shown to act through other off-target sites including the 5-HT1a receptor and the α2-adrenergic receptor. For example, CBG competitively antagonizes the ability of the 5-HT1a agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) to stimulate (35S)GTPγS binding in rat brain membranes (Cascio, et al., 2010). Furthermore, CBG inhibits electrically-induced contractions of the vas deferens and stimulates (35S)GTPγS binding in rat brain membranes through agonist activity at the α2-adrenergic receptor (Cascio, et al., 2010). CBG, along with acidic cannabinoids THCA and CBDA, also binds to and activates PPARγ (D'Aniello, et al., 2019) (see below).
As with other minor cannabinoids, CBG may reduce the severity of inflammatory diseases and peripheral pain. The anti-inflammatory properties of CBG are postulated to result from binding to the CB2 receptor, TRP channels, PPARγ and other targets (De Petrocellis, et al., 2011; Cascio, et al., 2010; Ruhaak, et al., 2011). There is anecdotal human and preclinical evidence for CBG having a benefit in cases of inflammatory bowel diseases including Crohn’s and ulcerative colitis. In a mouse model of colitis, CBG was found to reduce bowel inflammation, nitric oxide production [from increased nitric oxide synthase (iNOS) expression during inflammation] and oxidative stress in intestinal cells (Borrelli, et al., 2013; Pagano, et al., 2021). Similar to CBC, CBG (3 mg/kg and 10 mg/kg i.p.) produces a weak anti-nociceptive effect in mice (Zagzoog, et al., 2020). In one study this effect of CBG was inhibited by the α2-adrenergic receptor antagonist yohimbine, suggesting a role of α2-adrenergic regulation in CBG analgesia (Cascio, et al., 2010).
Inflammation and oxidative stress are both contributors to neurodegeneration, which is linked to Alzheimer’s and Huntington’s disease as well as Multiple Sclerosis (MS). CBG may protect against both neuroinflammation and oxidative stress, helping to prevent neuronal cell loss (Gugliandolo, et al., 2018). Carrillo-Salinas et al. (2014) examined the effect of the CBG analog VCE-003 on human T-cells and its efficacy in a mouse model of autoimmune MS. When tested in vitro, VCE-003 inhibited antigen-induced T-cell proliferation, cell cycle progression and the expression of surface activation markers. VCE-003 also prevented the expression of the pro-inflammatory enzyme iNOS in microglia. In animals, VCE-003 attenuated MS through activating CB2 and PPARγ receptors (Carrillo-Salinas, et al., 2014). CBG was also investigated in a mouse model of Huntington’s disease induced using 3-nitropropionate (3-NP) (Valdeolivas, et al., 2015). Treatment with CBG (10 mg/kg) reduced levels of the pro-inflammatory cytokines IL-6 and tumor necrosis factor α in the 3-NP treated mice. CBG also partially improved motor deficits and preserved striatum neurons in the R2/6 transgenic model of Huntington’s disease (Valdeolivas, et al., 2015).
As noted previously, CBG is effective in suppressing cancer cell growth (Ligresti, et al., 2006). In a murine colon cancer model, CBG was found to promote cancer cell death and inhibit the growth of tumors (Borrelli, et al., 2014). This inhibition was mimicked by TRPM8 channel antagonists (Borrelli, et al., 2014). Additionally, in vitro experiments using leukemia cell lines suggest this anti-cancer activity is enhanced when CBG is combined with other cannabinoids such as CBD (Scott, et al., 2013). Clinical studies are currently underway to determine if these results are translatable to treatment in humans. Individuals living with cancer and AIDS commonly experience anorexia and cachexia. CBG represents a non-psychoactive alternative to Δ9-THC for treating anorexia since it stimulates appetite and increases food consumption (Brierley, et al., 2017). Interestingly, CBG as part of a whole plant cannabis extract is more potent in stimulating appetite than CBG as an isolate (Brierley, et al., 2017). Thus, these results provide additional evidence that synergism of minor cannabinoids with other components of the cannabis plant may enhance their clinical efficacy (Russo, 2011).
Cannabinoid Acids
Cannabidioloic Acid
CBDA was first isolated in 1955 and its structure elucidated in 1965 (Mechoulam and Gaoni, 1965). CBDA has a low affinity for both the CB1 and CB2 receptors based on 3[H]-CP-55,490 displacement assays (Zagzoog, et al., 2020). However, it shows moderate efficacy in inhibiting adenylyl cyclase through these receptors (Navarro, et al., 2020; Zagzoog, et al., 2020). In addition, CBDA is one of several minor cannabinoids (along with THCA and THCV) that functions as allosteric regulators at 5-HT1a receptors (Bolognini, et al., 2013). CBDA enhances 8-OH-DPAT-stimulated (35S)GTPγS binding to 5-HT1a receptors expressed in rat brain and CHO cell membranes possibly by binding to an allosteric site on the receptor (Bolognini, et al., 2013). CBDA was reported to be 1,000 times more potent than CBD in stimulating (35S)GTPγS binding at the 5-HT1a receptor. Based on in silico docking experiments, it was predicted that CBDA, along with the cannabinoids CBGA and CBG, bind to PPARs (D'Aniello, et al., 2019). In vitro reporter assays carried out with CHO cells confirmed that all three minor cannabinoids activate PPARα and PPARγ (D'Aniello, et al., 2019).
CBDA produces dose-dependent anti-hyperalgesia and anti-inflammatory effects in a rodent model of carrageenan-induced hind paw inflammation (Rock, et al., 2018). The anti-hyperalgesia effect of CBDA is blocked by AMG9810, an antagonist of the TRPV1 channel. In addition, when combined with Δ9-THC, low doses of CBDA are more effective in preventing hyperalgesia and reducing inflammation. CBDA may also produce anti-inflammatory effects via cyclooxygenase 2 (COX-2) enzyme inhibition; the same mechanism of action as the NSAID Celecoxib (Takeda, et al., 2008; Ruhaak, et al., 2011). CBDA inhibits the COX-2 enzyme in vitro with an EC50 of 2 µM and has 9-fold greater selectivity in inhibiting the COX-2 enzyme over the COX-1 enzyme. This selective inhibition is dependent on the presence of the carboxylic acid moiety in the CBDA molecule (Takeda, et al., 2008).
CBDA has anti-nausea effects at low doses in mice that are mediated via agonist activity at CNS 5-HT1A receptors (Pertwee, et al., 2018). CBDA was found to be 1000-fold more potent than CBD in reducing nausea-induced conditioned gaping disgust responses (Rock et al., 2020). The drug HU-580, a stable analogue of CBDA that is not metabolized to CBD, also reduces LiCl-induced conditioned gaping (Pertwee, et al., 2018). In addition to suppressing acute nausea, CBDA decreases anticipatory nausea and vomiting which occurs upon re-exposure to a contextual stimulus previously associated with acute nausea (e.g., a chemotherapy session) (Limebeer, et al., 2014). CBDA combined with ondansetron, a commonly used antiemetic drug, enhances ondansetron’s effect when applied at low doses (Rock and Parker, 2013).
Anderson et al. (2019) examined the anti-seizure activity of CBDA using Scn1aRX/+ mouse model of Dravet Syndrome. The Scn1aRX/+ mice develop generalized tonic-clonic seizures in response to elevated body temperature and thus recapitulate the seizures observed in children with Dravet Syndrome. When administered using i.p. injection (10 and 30 mg/kg), CBDA raised the temperature threshold required for seizures in the mice (Anderson, et al., 2019). CBDA also displayed dose-dependent protection in rats against electroshock-induced seizures (Goerl, et al., 2021). While clinical trials have not been reported, CBDA may be more effective than CBD in reducing seizures in humans. According to a patent application by GW Pharmaceuticals, the makers of Epidiolex® (a sublingual spray containing 100 mg of CBD/100 ml of solution), CBDA displays greater bioavailability and potency in treating epilepsy (GW PHARMA LTD, 2015).
Cannabigerolic Acid
CBGA is the precursor cannabinoid to THCA, CBDA, and CBCA (see Figure 1). Since CBGA is decarboxylated over time to CBG, it is rarely found in significant amounts in mature cannabis flowers. Thus, harvesting hemp very early yields higher levels of CBGA compared to later in the plant’s life. In addition, some cultivars have increased yields of CBGA through selective breeding to inhibit its transformation into other cannabinoids during the plant’s maturation (Garfinkel, et al., 2021). Similar to CBDA, CBGA displays low affinity for both the CB1 and CB2 receptors (Navarro, et al., 2020). Nonetheless, CBGA is equally as efficacious as Δ9-THC in decreasing intracellular cAMP levels though the CB1 receptor (Table 1) (Navarro, et al., 2020). However, unlike Δ9-THC, it is not effective in recruiting βarr2 to the CB1 receptor (Navarro, et al., 2020). CBGA has important off target effects including activating PPARs (D'Aniello, et al., 2019). In addition, fractions of Cannabis sativa containing high amounts of CBG/CBGA inhibit the aldose reductase enzyme (Smeriglio, et al., 2018).
While less in known about the therapeutic uses of CBGA compared with the other minor cannabinoids, it may play a role in controlling diabetes mellitus and preventing the cardiovascular complications that can accompany Type 2 diabetes (D'Aniello, et al., 2019). Through activation of PPARs, CBGA can improve lipid metabolism and reduce the accumulation of adipose tissue; thus reducing insulin resistance in the Type 2 patient (Gao, et al., 2015). Type 2 diabetes is considered a “coronary artery disease equivalent” and mortality in Type 2 diabetes primarily results from cardiovascular events including acute coronary syndrome (ACS). By inhibiting the enzyme aldose reductase, CBGA improves cardiac glucose metabolism and reduces the risk of ACS (Smeriglio, et al., 2018). Synthetic inhibitors of aldose reductase have severe side effects including elevations in blood liver enzymes from hepatotoxicity, as well as nausea and vomiting. Therefore, plant-derived CBGA offers a promising alternative to these inhibitors.
CBGA may also be beneficial in treating some types of cancer. A cannabis fraction containing high amounts of CBGA was reported to have cytotoxic activity against colon cancer cells (Nallathambi, et al., 2018). Interestingly, synergistic toxic effects were observed when CBGA was given with a cannabis fraction high in THCA. These two fractions also prevented the growth and proliferation of adenomatous colon polyps that are colon cancer precursors. When tested at micromolar concentrations, CBGA was also shown to have cytotoxic actions in human leukemia cancer cell lines (Scott, et al., 2013). In further support of cannabinoid synergism, the IC50 for CBGA leukemia cell toxicity was reduced when co-applied with CBD (Scott, et al., 2013; Scott, et al., 2017).
Tetrahydrocannabinolic Acid
THCA is a non-psychotropic cannabinoid that is converted to Δ9-THC through decarboxylation by exposure to heat (Figure 1
). Since THCA is a precursor to Δ9-THC, and because no sample of THCA is completely free of Δ9-THC, possession of this cannabinoid could be prosecuted under the U.S. government Federal Analogue Act. THCA displays roughly 60- and 125-fold lower affinity for the CB1 and CB2 receptors compared with Δ9-THC (McPartland, et al., 2017). While high concentrations of THCA inhibit forskolin-stimulated increases in cAMP through the CB1 receptor, it produces no inhibition through the CB2 receptor (McPartland, et al., 2017). Nagal et al. (2017) compared the effects of several cannabinoids, including CBDA, CBGA and THCA on PPAR activity. When compared with CBDA and CBGA, THCA has the highest binding affinity for PPARγ (Nadal, et al., 2017). In addition, THCA is more potent than other minor cannabinoids in inducing PPARγ-mediated transcriptional activity.
THCA was recently shown to possess potent anti-inflammatory activity in mice fed a high fat diet (HFD) (Palomares, et al., 2020; Carmona-Hidalgo, et al., 2021). THCA treatment reduced the expression of inflammatory molecules including tumor necrosis factor alpha (TNF-α4) and cytokine interleukin 10 (IL-10) in the HFD mice. This effect was mediated via PPARγ stimulation (Palomares, et al., 2020). THCA also improved glucose tolerance and attenuated liver fibrosis in the HFD mice (Carmona-Hidalgo, et al., 2021). Using an in vitro COX-1/COX-2 assay it was determined that Δ9-THCA inhibits both COX-1 and COX-2 enzymes with a concentration causing 50% inhibition (IC50) in the high micromolar range (Ruhaak, et al., 2011). Nallathambi et al. (2017) reported that cannabis fractions containing high amounts of THCA produce anti-inflammatory effects (e.g., reduction in IL-8) in several colon epithelial cell lines and in colon tissue biopsies. Anti-inflammatory effects of THCA were inhibited by treatment with the GPR55 antagonist CID16020046, but not by the CB1 and CB2 receptor antagonist rimonabant and SR144528 (Nallathambi, et al., 2017). In addition to its anti-inflammatory properties, THCA also has anti-nausea and antiemetic properties in mice at doses much lower than Δ9-THC (Rock, et al., 2013a). Thus, THCA may present a non-psychotropic alternative to Δ9-THC for treating nausea and vomiting.
As discussed previously for CBC and CBG, THCA may also exhibit neuroprotective properties that could be beneficial in the treatment of neurodegenerative diseases. THCA improved neuronal viability through a PPARγ-dependent pathway in an in vitro model of Huntington’s disease (Nadal, et al., 2017). THCA also caused an improvement in hind limb dystonia and locomotor activity in mice treated with 3-NPA. These neuroprotective actions of THCA were significantly reduced when mice were co-administered the PPARγ antagonist T0070903. In contrast, THCA had no effects on the survival of dopaminergic neurons in a 1-methyl-4-phenyl pyridinium (MPP+) cell culture model of Parkinson’s disease (Moldzio, et al., 2012). Since THCA undergoes decarboxylation to Δ9-THC, it is possible that the reported neuroprotective effects of THCA in Huntington’s disease may have resulted from contamination by Δ9-THC (Sagredo, et al., 2011). In addition, THCA displays poor brain penetration properties when tested using two vehicles (vegetable oil and Tween 80) (Anderson, et al., 2019); a limitation that could reduce its clinical efficacy.
Anecdotal reports have long suggested that THCA acts as an anticonvulsant. Over 40 years ago Karler and Turkanis reported that THCA (200 mg/kg) reduces seizures in the mouse maximal electroshock test (Karler and Turkanis, 1979). In a more recent mouse study the anticonvulsant effects of THCA were found to vary depending on the seizure model utilized and whether Δ9-THC was given along with the THCA (Benson, et al., 2020). When used alone, THCA (2, 30, and 100 mg/kg) was ineffective in the 6-Hz threshold (6-HzT) model of psychomotor seizures, but had anticonvulsant activity when given with Δ9-THC. Conversely, THCA used alone or with Δ9-THC did not reduce hyperthermia-induced seizures in the Scn1aRX/+ mice model (compared with the protective effects of CBDA described above). More encouraging results were reported with THCA in a clinical study. The frequency and duration of seizures were reduced in four case reports of children using low doses of THCA (0.1–1 mg/kg per day) in conjunction with conventional antiepileptic drugs and full spectrum cannabis (Sulak, et al., 2017). In contrast, Epidiolex® (CBD), which is approved by the U.S. Food and Drug Administration (FDA) for treating epilepsy, is dosed from 5 to 25 mg/kg per day. Thus, THCA may be ten to hundred times more potent in reducing seizures. However, increased doses of THCA did not improve efficacy in this clinical study (Sulak, et al., 2017). Furthermore, formulations of THCA containing high levels of the terpenoid α-linalool were more efficacious than formulations containing low levels of the terpenoid. Thus, other components of the THCA formulation may have accounted for the beneficial effects. Finally, symptoms and seizure activity worsened in one patient after increasing the THCA dose (Sulak, et al., 2017).
Varinic Cannabinoids
Cannabidivarin
CBDV is found in landrace cannabis strains that have relatively high amounts of CBD and low amounts of Δ9-THC. Prior to its isolation in 1969, it was assumed that all naturally occurring cannabinoids contained a pentyl side chain, rather than the propyl chain found in CBDV and THCV. CBDV displays low binding affinity for the CB1 and CB2 receptors (Rosenthaler, et al., 2014; Husni, et al., 2014). Consistent with this, high concentrations of CBDV are needed for CB1 receptor stimulation of (35S)GTPγS binding, inhibition of cAMP synthesis and recruitment of βarr2 (Husni, et al., 2014; Navarro, et al., 2020; Zagzoog, et al., 2020). Overall, CBDV is a more potent and efficacious agonist at CB2 receptors (Navarro, et al., 2020; Zagzoog, et al., 2020). CBDV displays a similar pharmacological profile for TRP channels as CBN, CBG and THCV; activating TRPV1, TRPV2, TRPV3, TRPV4 and TRPA1 channels while inhibiting the TRPM8 channel (De Petrocellis, et al., 2008; De Petrocellis, et al., 2011). Other important off target sites for CBDV include the de-orphanized receptors GPR55 and GPR6. Binding of CBDV to the GPR55 receptor stimulates ERK1/2 phosphorylation and inhibits LPS-mediated signaling effects occurring through the GPR55 receptor (Anavi-Goffer, et al., 2012). These effects of CBDV are comparable to those of Δ9-THC. GPR6 is a constitutively active receptor that couples to Gs to stimulate adenylyl cyclase and recruits βarr2. CBDV acts as an inverse agonist at the GPR6 receptor causing significant inhibition of βarr2 recruitment at concentrations of 1 and 10 µM (Laun, et al., 2018).
CBD (marketed as Epidiolex®) was approved by the U.S. Food and Drug Administration (FDA) in 2018 for preventing epileptic seizures in Lennox-Gastaut syndrome and Dravet syndrome in children. CBDV, a structural homolog of CBD, possesses anti-epileptic properties when tested in animals and humans. When examined in vitro in rat brain slices, CBDV attenuates epileptiform local field potentials induced by 4-amino pyridine (Hill, et al., 2012). In vivo, CBDV (200 mg/kg per day) significantly reduces PTZ-induced seizure activity in the rats (Hill, et al., 2012). However, when used alone, CBDV has no effect on pilocarpine-induced seizures, but requires the co-administration of valproate or phenobarbital to be effective. Consistent with this, Amada et al. (2013) reported that CBDV significantly decreases PTZ-induced seizure severity. In addition, CBDV suppresses the expression of several epilepsy-related genes in animals that respond to CBDV anti-epileptic treatment (Amada, et al., 2013). A human trial to assess the efficacy, safety, and tolerability of CBDV in adults with focal seizures was recently conducted by GW Pharmaceuticals, the maker of Epidiolex® (Brodie, et al., 2021). The drug GPW42006 (800 mg b.i.d.), containing CBDV as its major component, reduced the frequency of seizures by 41%. However, similar reductions in focal seizure frequency were observed in the CBDV and placebo (38%) groups. There was also no differences between CBDV and placebo groups for any specific seizure type. Therefore, higher doses and longer durations of treatment of GPW42006 will be needed in future clinical trials to better access the benefits of CBDV.
CBDV has been investigated as a treatment for symptoms associated with autism spectrum disorder (ASD) such as repetitive behaviors, cognitive challenges and issues with communication and social functioning (Mouro, et al., 2019). Mice carrying mutations in the MeCP2 gene and MeCP2 null mice develop Rett Syndrome (RTT), a neurodevelopment disease related to ASD. CBDV treatment (2 mg/kg) was found to rescue both behavioral and phenotypic changes in the RTT mice model (Vigli, et al., 2018). These CBDV effects included improvements in motor coordination, locomotion and brain weight. When administered using a life-long treatment schedule, CBDV also prolonged survival and delayed the appearance of neurological and motor deficits in MeCP2 null mice (Zamberletti, et al., 2019a). CBDV also reversed memory deficits in these mice. Similar behavioral improvements were reported using CBDV in a valproic acid-induced model of ASD (Zamberletti, et al., 2019b).
CBDV may also have utility in the treatment of Duchenne muscular dystrophy (DMD) and in preventing nausea. In a recent study, CBDV was found to improve muscle quality and locomotion and to slow muscle degeneration in male dystrophic mdx mice (Iannotti, et al., 2019). Muscle improvement by CBDV (and CBD) was accompanied by anti-inflammatory and pro-autophagic effects. Both CBDV (200 mg/kg) and THCV (20 mg/kg) are effective in reducing LiCl-induced conditioned gaping in rats (Rock, et al., 2013b). In contrast, CB1 receptor inverse agonists such as SR141716 and AM251 are known to enhance nausea. Thus, CBDV and THCV do not function in vivo as CB1 receptor inverse agonists. In conclusion, CBDV has Δ9-THC-like, antiemetic effects in rodents consistent with a CB1 receptor agonist, but without the psychotropic activity of Δ9-THC.
Tetrahydrocannabivarin
THCV is derived from cannabigerovarin acid (CBGVA), one of the two primary minor cannabinoid precursors, the other being CBGA (Figure 1). THCA synthase converts CBGVA to THCVA, which is then decarboxylated to the neutral compound THCV when exposed to heat or light (Hanus, et al., 2016). THCV is typically found in very small amounts in cannabis flowers, though breeders are developing strains with higher concentrations. While THCV binds to both CB1 and CB2 receptors, its pharmacological effects remain controversial. In some studies, THCV has been reported to act as an antagonist/inverse agonist at the CB1 and CB2 receptors. In (35S)GTPγS binding assays measured with rodent brain preparations, THCV acts as an antagonist to WIN-55,212-2 (Dennis, et al., 2008). In addition, THCV antagonizes CP-55,940-induced stimulation of (35S)GTPγS binding in rodent brain and CHO cell membranes (Thomas, et al., 2005). In contrast, more recent studies using CHO cells have demonstrated that THCV functions as a partial agonist at the cannabinoid receptors to inhibit cAMP formation and to stimulate βarr2 recruitment (Zagzoog, et al., 2020). Computational docking experiments revealed that THCV interacts with the same residues as Δ9-THC in the orthosteric site of the CB1 receptor (Jung, et al., 2018). However, the pentyl side chain of Δ9-THC protrudes into a sub-pocket of the binding site. THCV containing a propyl side chain does not have this interaction (Jung, et al., 2018). This difference and the distinct binding energies of the two ligands might account for the higher affinity of Δ9-THC for the CB1 receptor.
As with other minor cannabinoids, THCV has off target actions at TRP channels and 5-HT1A receptors. THCV acts as an agonist at TRPV1-4 and TRPA1 channels, while acting as an antagonist at the TRPM8 channel (De Petrocellis, et al., 2011). THCV (at 100 nM) was found to enhance (H3)-8-OH-DPAT binding to the 5-HT1A receptor and to increase the potency of 8-OH-DPAT-stimulated (35S)GTPγS binding to cell membranes (Cascio, et al., 2015). Thus, both THCV and CBDA appear to function as positive allosteric regulators of the 5-HT1A receptor.
THCV has been shown to reduce inflammation and inflammatory pain in mice. THCV attenuated signs of inflammation induced by intraplantar injection of carrageenan in mouse hind paws and reduced hyperalgesia from formalin hind paw injection (Bolognini, et al., 2010). Surprisingly, the ability of THCV to relieve formalin-induced hyperalgesia was significantly attenuated by both the CB1 receptor-selective antagonist rimonabant, and CB2 receptor-selective antagonist SR144528 (Bolognini, et al., 2010). Thus, when tested in vivo, THCV exhibits both CB1 and CB2 receptor agonist activity. These anti-inflammatory actions of THCV were further supported by in vitro experiments using peritoneal-derived macrophages (Romano, et al., 2016). THCV was found to suppress inflammatory pathways by down-regulating LPS-induced expression of iNOS, COX-2 and interleukin 1β. THCV mediated suppression of nitrite production (from nitric oxide) was prevented by pretreatment of the macrophages with SR144528.
THCV has also shown promise as an anti-epileptic agent and in the treatment of neurodegeneration in Parkinson’s disease. Hill et al. (2010) used an extracellular multi-electrode array (MEA) assay to study the effects of THCV on spontaneous epileptiform bursting in rat brain slices. Pretreatment of the brain slices with THCV (20 µM) reduced burst complex incidence and the amplitude and frequency of paroxysmal depolarizing shifts (Hill, et al., 2010). In addition, in rats treated in vivo with pentylenetetrazole (PTZ) to induce seizures, THCV (0.25 mg/kg) reduced the incidence of seizures. Approximately 33% of animals treated with THCV exhibited a complete absence of PTZ seizures (compared with 13% of control treated rats). Garcia et al. (2011) reported that THCV reduces slow motor movements in rats with 6-hydroxydopamine (6-OHDA)-induced Parkinson’s disease. In addition, 2 weeks of treatment with THCV reduced microglial activation and preserved nigrostriatal dopaminergic neurons in the 6-OHDA Parkinson’s model (García, et al., 2011). Thus, THCV may be a useful treatment for Parkinson’s disease by preventing neuronal degradation and alleviating associated symptoms.
THCV regulates blood glucose levels suggesting it might be useful in weight reduction and treating diabetes. In mice with dietary-induced obesity (DIO), THCV improved fasting plasma glucose and glucose tolerance in a dose-dependent manner (Wargent, et al., 2013). In addition, THCV increased insulin sensitivity in genetically (ob/ob) obese mice. While THCV increased energy expenditure in the DIO and ob/ob mice, it did not reduce food intake or overall body weight (Wargent, et al., 2013). This contrasts with a previous study where intraperitoneal administration of THCV caused hypophagia and weight loss in rodents (Riedel, et al., 2009). Importantly, a clinical trial evaluated the effects of THCV and CBD on 62 subjects with type 2 diabetes (Jadoon, et al., 2016). Although THCV had no effect on plasma HDL levels, it significantly decreased fasting plasma glucose levels and improved pancreatic β-cell function in the type 2 patients. In addition to these findings, THCV was reported to increase the response to aversive stimuli in humans in regions of the brain (amygdala, insula and mid orbitofrontal cortex) involved in food aversion (Tudge, et al., 2015). This suggests that THCV may aid in appetite suppression and weight loss without the side effects (depression, anxiety, etc.,) caused by the CB1 receptor antagonist rimonabant (Mitchell and Morris, 2007).
Various minor cannabinoids including THCV, CBC, CBG and CBDV have shown promise in the treatment of skin disorders and are being investigated for the treatment of atopic dermatitis, psoriasis, scleroderma, acne hair growth and pigmentation disorders, keratin diseases, skin tumors, and pruritus (Tubaro, et al., 2010; Oláh, et al., 2016; Tóth, et al., 2019). It is postulated that these cannabinoids produce anti-acne effects by regulating homeostatic sebaceous lipogenesis and by exerting anti-proliferative and anti-inflammatory actions. In vitro experiments have shown that THCV inhibits the proliferation of human SZ95 sebocytes (Oláh, et al., 2016). This anti-proliferative effect of THCV occurs through a CBD-like mechanism of action; increasing intracellular Ca2+ and stimulating ERK1/2 following TRPV4 channel activation. In addition, THCV exhibits powerful anti-inflammatory properties by reducing levels of arachidonic acid (AA), needed for lipogenesis (Oláh, et al., 2016). THCV also suppresses lipid synthesis in the sebaceous glands, providing relief to acne sufferers whose condition is triggered by excessive oil production (Oláh, et al., 2016; Tóth, et al., 2019). In conclusion, THCV and other minor cannabinoids will continue to be evaluated for the management of acne.