Abstracts

Rationale: Pure cannabidiol (CBD) is efficacious in pre-clinical in vivo models of seizure (J Pharmacol Exp Ther. 2010; 332(2): 569-77).  Furthermore, recent clinical data generated by Devinsky et al. (Lancet Neurol. 2016; 15(3):270-8) support the hypothesis that pure CBD is anticonvulsant in refractory epilepsy patients. CBD’s mechanism of action is unknown and a systematic examination of its effect on human CNS-expressed voltage gated sodium channels (Nav) has yet to be described. The present study was instigated to determine the effect of physiologically relevant concentrations of a pharmaceutical formulation of pure CBD on the function of human recombinant Nav channels. Methods: HEK293 cells stably expressing human Nav 1.1, 1.2, 1.3, 1.6, or 1.7 channels were cultured at 37 oC in 5% CO2 in minimum essential medium supplemented with FBS (10% v/v), penicillin streptomycin, pyruvate, non-essential amino acids (all 1% v/v) and 200 μg/ml G418.  Media for Nav 1.2 and 1.3 cells also contained 8 μg/ml blasticidin.  Using automated patch clamp electrophysiology (IonWorks), pure CBD was examined using physiologically relevant repeat pulse trains to identify use-dependent block.  For the examination of Nav 1.1, 1.2, 1.3, and 1.6 channels, the following parameters were applied: membrane holding potential (Vh) = -85 mV, Vtest = 0 mV,  with 25 sequential test pulses of 5 ms duration at a frequency of 25 Hz. For examination of Nav 1.7, the following parameters were applied: Vh = -70 mV, Vtest = 0 mV, with 20 sequential test pulses of 10 msec duration at a frequency of 25 Hz. Pure CBD was tested at a final assay concentration range of 0.6 nM to 33 μM in quadruplicate. Tetracaine, a non-selective and use-dependent Nav channel inhibitor was examined in parallel at each Nav subtype at a final assay concentration of between 17 nM and 100 μM.  For each channel subtype, concentration response curves were constructed for inhibition of the first and last pulse of each train of 25 pulses.  Data were analyzed using IonWorks Quattro software (2.0.3.4) and GraphPad Prism (v5) and fitted to a 4 parameter logistic equation for the determination of IC50. Results: The positive control Nav channel inhibitor tetracaine inhibited all Nav subtype currents elicited by sequential depolarizing pulses in a concentration and use-dependent fashion.  In contrast, no such inhibition was observed in the presence of 0.6 nM to 33 μM CBD at Nav 1.1, 1.2, 1.3, 1.6 and 1.7. These data are summarized in Table 1. Conclusions: The anti-epileptiform activity of pure CBD at ≥10 nM has been previously demonstrated using in vitro experimental models. The data presented here confirm that pure CBD does not inhibit the function of human Nav channels at concentrations up to 33 μM. These data taken together suggest that the mechanism by which pure CBD is able to confer such anti-convulsant activity is unlikely to be mediated by inhibition of CNS-expressed voltage-gated sodium channels. Funding: This study was funded by GW Research Ltd.