Minor Cannabinoids: Biosynthesis, Molecular Pharmacology and Potential Therapeutic Uses

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 K_is 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-HT_1a receptor and the α₂-adrenergic receptor. For example, CBG competitively antagonizes the ability of the 5-HT_1a 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).

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).

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-HT_1A 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-HT_1A 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-HT_1A 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.