Preclinical and clinical studies show CBD is anticonvulsant; however, its mechanism of action is unclear and likely involves numerous epilepsy drug targets (Devinsky et al. 2016; Pisanti et al. 2017). Many clinically effective anticonvulsants inhibit Na_V channels, and purified CBD non-selectively inhibits these channels (Ghovanloo et al. 2018). However, as cannabis contains 500 phytochemicals including phytocannabinoids and terpenes, many cannabis-based products being used to treat epilepsy contain constituents beyond CBD (Nuutinen 2018). Our study shows that a CBD-dominant nutraceutical product that contains a mixture of phytocannabinoids and terpenes potently modulated various functional properties of the sodium channel subtypes Na_V1.1–Na_V1.8.

The IC₅₀ values of the hemp-derived NP across Na_V1.1-Na_V1.7 ranged from 1.6 to 4.2 μg NP/mL, which equates to CBD IC₅₀ values of 93–245 nM. Our data show that purified CBD inhibited a subset of Na_V channels with IC₅₀ values in the micromolar range. Thus, NP is an order of magnitude more potent than purified CBD at these Na_V channels, implying that either another component of the NP potently inhibits the channels or that the various components within the NP work cooperatively to inhibit channel function. Whether the other phytocannabinoid or terpene constituents of NP alone directly affect Na_V channel subtypes is unknown, although there is evidence that Δ⁹-THC inhibits sodium channels at micromolar concentrations (Turkanis et al. 1991) and linalool inhibits Na_V channels at high micromolar concentrations (Leal-Cardoso et al. 2010). Mounting evidence supports synergistic interactions between the many molecular constituents of cannabis-derived products, consistent with the notion of an “entourage effect” (Russo 2019; Russo 2011). Future studies could explore whether an “entourage effect” or synergism between components within the NP contributes to its inhibition of Na_V channel function.

NP (3 μg NP/mL) inhibited peak current amplitude across this superfamily of human sodium channels, with the exception Na_V1.8, where no change was seen, and Na_V1.3 where peak current amplitude was enhanced. However, hemp seed oil vehicle (3 μg hemp seed oil/mL) also enhanced peak current amplitude of Na_V1.3 suggesting the effect was not specific to the NP. While postnatal expression of Na_V1.3 is very low in rodent brain tissue, human tissue distribution studies have shown that Na_V1.3 is retained in the adult brain (Cheah et al. 2013; Gazina et al. 2010). In adult brain, Na_V1.3 expression is present in both excitatory neurons and inhibitory interneurons (Whitaker et al. 2001). Further, de novo SCN3A that are both GOF and LOF have been implicated in childhood epilepsies (Chen et al. 2015; Holland et al. 2008; Lamar et al. 2017; Vanoye et al. 2014). NP and/or hemp seed oil could provide a potential therapeutic benefit in patients with LOF Na_V1.3 mutations. NP had no effect on peak current amplitude of Na_V1.8, which is in contrast to previous work that shows both tonic and use-dependent inhibition of Na_V1.8 channels in murine primary sensory neurons by 2 μM CBD (Zhang and Bean 2021). Since the highest concentration of CBD examined in the NP was only 175 nM, it may explain why no effect was observed. Future studies could also determine whether interactions between NP and sodium channels exhibit state-dependence.

NP had variable effects on the biophysical properties of activation, steady-state fast inactivation and recovery from fast inactivation for the channel isoforms. NP did not affect the activation properties of Na_V1.1, Na_V1.2, Na_V1.4 or Na_V1.7; however, it did cause hyperpolarizing shifts in the midpoint of activation for Na_V1.3 and Na_V1.8, indicative of enhanced channel availability. A depolarizing shift in the conductance curve for Na_V1.5 was observed with NP, thus impeding channel function. The overall inhibitory effects of NP on Na_V1.5, the cardiac sodium channel, provides some cause for concern. However, purified CBD is well tolerated in humans with no clinically significant ECG changes even at peak plasma concentrations >2 μM, consistent with purified CBD only modestly inhibiting Na_V1.5 (IC₅₀ of 3.8 μM) (Ghovanloo et al. 2018). While there is scant data available evaluating plasma exposure levels in patients using nutraceutical hemp extracts, a recent clinical trial reported that a CBD-dominant cannabis herbal extract attained a plasma concentration of ~200 nM in childhood epilepsy patients when dosed up to 10–12 mg/kg CBD per day (Huntsman et al. 2019). No serious adverse events were reported in this study; however, it would be prudent for future safety studies administering CBD-dominant cannabis based-products to incorporate ECG measures. However, it is unlikely that this product would affect sodium channels when administered as a nutraceutical product where a common dosage is 15 mg CBD twice daily, amounting to a ~0.5 mg/kg per day CBD dose (Capano et al. 2020). NP treatment resulted in hyperpolarizing shifts in the midpoint of inactivation for all channel subtypes, except Na_V1.2. Hyperpolarizing shifts of channel inactivation mean that the channels move into an inactivated state more readily, indicative of the channels being functionally inhibited.

Recovery from steady-state fast inactivation, for Na_V1.1, Na_V1.2, Na_V1.4, Na_V1.5, Na_V1.6 and Na_V1.7, was significantly slower in the presence of NP and is also consistent with functional inhibition of these channels. Notably, these effects appear to be independent of the NP as the hemp seed oil vehicle produced the same pattern of results on channel inactivation, suggesting other components of the hemp seed oil, which is devoid of phytocannabinoids and terpenes, exert these effects.

Our results suggest that NP might be further explored to treat epilepsies which are sensitive to inhibition of voltage-gated sodium channels, including LGS where GOF mutations have been identified in SCN2A (Na_V1.2) and SCN8A (Na_V1.6) (Epi4K. 2013). However, it is difficult to reconcile our observation that NP inhibits Na_V1.1 channel function with research suggesting that CBD-dominant hemp extracts have anticonvulsant effects in DS patients who in 80% of cases have a LOF SCN1A mutation (Na_V1.1) (Depienne et al. 2009). Use of sodium channel inhibitors is contraindicated in DS because inhibiting Na_V1.1 would exacerbate LOF SCN1A mutations by reducing the neuronal inhibition exerted by GABAergic interneurons (Ogiwara et al. 2007; Stein et al. 2019). Interestingly, in DS patient-derived pluripotent stem cells, 50 nM CBD attenuated the excitability of excitatory neurons and potentiated the excitability of inhibitory neurons, opposing effects resulting in reestablishment of normal network activity (Sun and Dolmetsch 2018). Furthermore, therapies that potentiate Na_V1.1 channel function abolish seizures and reduce mortality in a mouse model of DS (Richards et al. 2018). It remains possible that the promiscuous target activity of the cannabinoids and terpenes in NP affects other epilepsy-relevant drug targets such as GABA_A receptors (Anderson et al. 2019a; Bakas et al. 2017), G-protein coupled receptor 55 (GPR55) (Kaplan et al. 2017) and TRPV1 channels (Gray et al. 2020) which overshadow any deleterious effects of Na_V1.1 channel inhibition. Alternatively, the brain concentrations of the NP might be insufficient to inhibit Nav1.1 in vivo.