Previous research has indicated that various cannabinoids may have anti-inflammatory and antinociceptive properties. While many studies have focused on the treatment of pain through the modulation of nociceptors, specialized peripheral sensory neurons, in the skin that respond to extreme pressure and temperature as well as toxic and noxious molecules,^3,23,24,33 relatively little is known about the mechanisms of cannabinoids on Ca²⁺ mobilization and signaling in the context of inflammation and pain. There is considerable evidence that SOCE and CRAC channels may be involved in nociceptive, inflammatory, and neuropathic pain mechanisms.^34–36 In this study, we comprehensively evaluated the efficacy of various cannabinoids against SOCE in pursuit of potential anti-inflammatory and analgesic candidates. It is established that Ca²⁺ signaling mechanisms are responsible for eliciting immune cell activation and subsequent cytokine production, with the major Ca²⁺ influx pathway inducing this response to be SOCE. Hence, SOCE was the key upstream event and favorable readout in our assays to screen for inhibitors that target the pro-inflammatory cascade in immune cells. Our results clearly demonstrate that several cannabinoids are effective in reducing Ca²⁺ mobilization through the inhibition of SOCE. Moreover, we demonstrate that certain minor cannabinoids displayed a higher potency against SOCE than the widely studied major cannabinoids (CBD and THC). We further demonstrate that the carboxylic acid derivatives of the pure cannabinoids elicit a greater potency for inhibiting SOCE than their decarboxylated counterparts indicating a possible SAR. This observation is in accordance with previous studies indicating these carboxylic acid-containing cannabinoid species possess increased efficacy in inhibiting the production of anti-inflammatory mediators.^37,38

Our results identify CBGA as the most potent cannabinoid in blocking SOCE with an IC₅₀ of 530 n m and I_CRAC with an IC₅₀ of 1.3 μm. The small difference in IC₅₀ between SOCE and I_CRAC measurements may be attributed to the differences in sensitivity of the two techniques or a nonlinear relationship between CRAC channel inhibition and free cystosolic Ca²⁺ levels. It is also possible that CBGA may be affecting other regulatory components of SOCE such as, for example, CRACR2A,³⁹ resulting in stronger suppression of Ca²⁺ entry than the direct suppression of CRAC currents alone. It is possible that CBGA could additionally affect other ion channels that regulate the membrane potential (eg, K⁺ or Cl^– channels) and affect the driving force for Ca²⁺ entry. Another example of a regulatory mechanism of SOCE and a possible target of CBGA may be the enzymatic activity of the transient receptor potential melastatin 7 (TRPM7).^40,41 TRPM7 is an ion channel capable of permeating divalent cations and contains an α-kinase domain.⁴⁰ Previous studies demonstrated a link between TRPM7 kinase activity with SOCE. Faouzi et al. demonstrated a significant reduction in SOCE in TRPM7-deficient DT40 (chicken B lymphocytes) cells.⁴² This reduction was rescued by overexpression with hTRPM7-WT construct but not by the kinase dead mutant (hTRPM7-K1648R) or the kinase deleted mutant (TRPM7-ΔKinase), suggesting the significance of TRPM7 kinase activity in modulating SOCE.⁴² Additional studies further support the involvement of the TRPM7 kinase domain with SOCE. Matsushita et al. have shown that in HEK293 cells, the overexpression of TRPM7-WT but not TRPM7-kinase resulted in increased Tg-induced SOCE.⁴³ Others have shown TRPM7 kinase-dead murine T cells exhibit significantly decreased Ca²⁺ signals upon stimulation with anti-CD3/CD28 antibodies and a reduced cyclopiazonic acid (CPA)-induced SOCE.^44,45 We also show that CBGA does not block I_CRAC when applied intracellularly and acts only when applied extracellularly. While this would suggest an external or plasma membrane-delimited site of action on CRAC channels, it does not completely rule out the involvement of regulatory proteins. Whether the inhibition of SOCE is due to direct CRAC channel inhibition and/or is caused by indirect suppression via TRPM7 or auxiliary channels and regulatory proteins awaits exploration in future studies.

Our data also demonstrate that CBGA possesses high affinity to BSA, rendering it biologically inactive long-term in in vitro experiments to elicit its inhibitory effects on SOCE and subsequent T cell cytokine production. These results are in line with previous studies demonstrating the sequestering action of serum mainly due to three long-chain fatty acid binding sites located on BSA, resulting in an inverse relationship of cannabinoid activity with serum concentrations in cell viability assays.^29,30,46,47 This phenomenon of cannabinoid sequestration by BSA may account for loss of efficacy associated with long-term experiments in which standard tissue culture medium is supplemented with FBS. However, for in vivo studies, plasma proteins and serum albumins play a vital role in solubility and transportation of drugs and assessing CBGA bioavailability in vivo can be accomplished by performing spectroscopic, molecular docking, pharmacokinetic, and pharmacodynamic studies, all common practices in drug development that provide valuable information for the design of efficacy studies.^48–51

Several studies have successfully used cannabinoids or their derivatives, including CBG and CBGA, in animal models, suggesting that reasonable bioavailability of these compounds is achievable in vivo.^31,32 Acidic cannabinoids CBDA, THCA, CBGA, CBGVA, cannabichromenolic acid (CBCA), and cannabidivarinolic acid (CBDVA) have shown a rapid absorption into the plasma within 15–45 min. CBGA for example reaches a high maximal concentration of 176.14 ± 21.1 µm in murine plasma with a single intraperitoneal (i.p.) injection of just 10 mg/kg, which is above the concentration needed to achieve the inhibition of SOCE.^31,52 Anderson et al. also showed that these acidic cannabinoids are absorbed into brain tissue and the type of vehicle can change the brain-plasma ratios, as they exhibit poor brain penetration in an oil vehicle as opposed to a Tween-based vehicle. This suggests that our lead compound, CBGA, can be used to target peripheral inflammation as well as inflammation in the central nervous system. Further studies would be required to measure the bound and unbound cannabinoids and assess how different routes of administration can affect the pharmacokinetic and tissue distribution of these compounds. A better understanding of the pharmacology of these compounds would facilitate the design of more effective in vivo studies and clinical testing of cannabinoids against inflammation.^31,32

In addition to demonstrating the SOCE inhibitory effects of pure CBGA, we investigated whether these effects on SOCE by CBGA could be altered with the introduction of a second cannabinoid. The majority of Cannabis research has been performed on the major cannabinoids, but evidence supports the notion of minor cannabinoids, terpenes, lignans, and flavonoids may contribute to a synergistic interaction commonly reffered to as the “entourage effect.”^53,54 Previous studies have demonstrated that Cannabis-derived extracts present a better efficacy than pure individual cannabinoids, with extracts rich in CBD and/or THC containing small concentrations of minor cannabinoids, such as CBG and THCA.⁵⁵ Moreover, Blasco-Benito et al. investigated this entourage effect by comparing pure cannabinoids and botanical extracts with corresponding concentrations of terpenes with standard chemotherapeutic drugs used in different breast cancer therapies.^52,55 Interestingly, the authors found that in all cases the botanical extract outperformed the pure cannabinoids in cell cultures but not in vivo and both the botanical extract and pure cannabinoids had no impact on standard chemotherapeutic therapies.⁵⁵ Our study investigated the potential entourage effect between our most potent cannabinoid, CBGA, in combination with one additional cannabinoid. While our results showed some supra- or subadditive effects on SOCE, these were rather moderate and generally did not exceed 10%. A possible explanation for this could be that experiments by Blasco-Benito et al. compared a botanical extract and pure cannabinoids with the lone similarity being the concentration of THC, suggesting that an unknown combination of phytochemicals of considerable complexity may be at play.⁵⁵ Furthermore, our study of the entourage effect combines our lead cannabinoid, CBGA, with one additional cannabinoid and does not take into account the possibility of other phytochemicals such as terpenes, lignans, and flavonoids contributing to the biological activity. This could explain the lack of large “entourage effects” between different cannabinoids in our isobolographic assays. We favor the view that entourage effects may not necessarily manifest themselves as targeting a single mechanism such as SOCE, but more likely be caused by the complementary effects of various phytochemicals on multiple mechanisms or targets within a given cell or even across multiple cell types contributing to chronic inflammation.

Indeed, our results also highlight the similarities and differences between the panel of pure cannabinoids screened against SOCE within several different immunological cell lines. We thoroughly screened each cannabinoid at different doses to determine IC₅₀ values in the various immune cell types, all of which exhibited the presence of SOCE. The results show varying degrees of IC₅₀ values for different cannabinoids within a single cell line as well as across cell lines, suggesting significant diversity in molecular targets that underlie or modulate SOCE in these cell types. Previous research has demonstrated that SOCE regulation involves not only complexities in the composition of primary SOCE molecules itself (STIM1 and STIM2 acting as ER Ca²⁺ sensors and Orai1/2/3 as homo- or even heteromeric CRAC channel units), but also additional cell-specific environments and multiple modulatory mechanisms. These mechanisms include post translational modifications of different targets critical for SOCE such as phosphorylation, ubiquitination, and glycosylation of Orai and STIM isoforms, in addition to other interacting proteins possibly involved in recruitment and translocation of Orai1 and STIM1 to ER/PM junctions.^56–64 As to which molecular target or combination of targets these cannabinoids may be modulating has yet to be determined.

From these data, it is also interesting to note that the pure cannabinoids can be grouped by their potency against specific cell types. The heat map table (Figure 11A) demonstrates that cannabinoids containing carboxylic acid functional groups possess the highest potency for SOCE inhibition in T cells. The radar chart (Figure 11C) shows that certain cannabinoids have similar IC₅₀ values within specific cell types. It is tempting to speculate that the mechanism of action by cannabinoids in these specific cell types could be similar. Future experiments should take into consideration not only the potency of each cannabinoid but the specific cell type that the cannabinoid exerts its activity against. Thus, certain cannabinoids appear to have very specific activities on SOCE in different cell types: (1) CBGA appears to be relatively potent in most cell types, but highly potent in T cells and human mast cells. (2) CBNA and CBCA are highly specific and potent in T cells and relatively ineffective in the other cell types tested. (3) THCA, THC, and CBDV are highly potent and selective in human mast cells, but not very effective in all other cell types. This could be used to develop targeted therapeutic uses for diseases in which these cell types play important roles.

In summary, we have shown the SOCE inhibitory activity of cannabinoids through a combination of methods including HTS assays, T cell activation assays, cytokine production, and electrophysiological mesurements. We have also discovered a previously unrecognized mechanism of action due to inhibition of CRAC channels and the resulting suppression of SOCE. This SOCE inhibitory activity could be instrumental in developing alternative therapies for T cell-mediated inflammation. Importantly, we show that carboxylated cannabinoids elicit a more potent inhibition of SOCE compared to the decarboxylated forms, which in general lack psychoactivity and may therefore be more desirable as therapeutics. Additionally, we show the differences in potency between cannabinoids within the same cell line as well as across cell lines and that the entourage effect could be influenced by not only the specific cannabinoids but also the specific cell types being targeted. Taken together, these data illustrate the complexity and promising potential of cannabinoid and Cannabis-based therapies against SOCE-mediated inflammation.