Nav1.8, an analgesic target for nonpsychotomimetic phytocannabinoids

Abstract

Pain impacts billions of people worldwide, but treatment options are limited and have a spectrum of adverse effects. The search for safe and nonaddictive pain treatments has led to a focus on key mediators of nociceptor excitability. Voltage-gated sodium (Nav) channels in the peripheral nervous system-Nav1.7, Nav1.8, and Nav1.9-play crucial roles in pain signaling. Among these, Nav1.8 has shown promise due to its rapid recovery from inactivation and role in repetitive firing, with recent clinical studies providing proof-of-principal that block of Nav1.8 can reduce pain in humans. We report here that three nonpsychotomimetic cannabinoids-cannabidiol (CBD), cannabigerol (CBG), and cannabinol (CBN)-effectively inhibit Nav1.8, suggesting their potential as analgesic compounds. In particular, CBG shows significant promise due to its ability to effectively inhibit excitability of peripheral sensory neurons. These findings highlight the therapeutic potential of cannabinoids, particularly CBG, as agents that may attenuate pain via block of Nav1.8, warranting further in vivo studies.

Keywords: cannabidiol; cannabigerol; cannabinol; sensory neurons; voltage-gated sodium channel.

Fig. 1.

Comparative concentration–response curves for each cannabinoid against endogenous Nav1.8. (A) The IC₅₀ curves of CBD, CBG, and CBN on mouse Nav1.8 from Nav1.9^−/− animals. Each neuron underwent a step-wise protocol to determine exact midpoint (V_1/2) in the presence of 500 nM TTX. Then, each concentration of a noted cannabinioid+500 nM TTX was perfused over the course of 200 s, at 0.2 Hz. Fractional block was measured after 100 ms at the V_1/2 at the test-pulse and was fit with Hill equation (n = 5 to 10). (B) Representative sample current traces. Data shown as mean ± SEM.

Fig. 2.

Cannabinoid characterization in Nav1.8. All recordings for CBD, CBG, and CBN were obtained at 1, 5, and 15 µM, respectively. (A) Current dissection of Nav1.8 and peak current inhibition by each compound. (B) State dependence measurements. (C) Peak conductance and (D) current density measurements. (E) Voltage dependence of activation and inactivation. (F) Recovery from inactivation. (G and H) show use-dependent inactivation from −100 and −70 mV holding-potentials. Data shown as mean ± SEM. *indicate statistical significance. (I) shows the effects of each compound as calculated values on the indicated biophysical parameters to compare inhibitory effects.

Fig. 3.

Expression profile. (A) Rat pup DRG neurons were transfected with Halo-Nav1.8, cultured for 6 d in microfluidic chambers, exposed to JF549i ligand, and fixed with PFA. Confocal images of somas and axons were acquired, (B and C) and the fluorescent signal was quantified. Concentrations for CBD, CBG, and CBN are 1, 5, and 15 µM respectively. Data shown as mean ± SEM. ns indicate not statistically significant.

Fig. 4.

TTX-resistant analysis. (A) Distribution of capacitances of WT rat DRG neurons for current density and inactivation measurements. (B) Peak current density comparison. (C and D) Current densities in TTX (500 nM) and TTX + CBG (5 µM) plotted as a function of potential, in matched-paired. (E and F) Inactivation curves. (G) Midpoints of single vs. double Boltzmann curves from neuronal populations, in TTX vs. TTX + CBG. (H) ∆ hyperpolarization of each midpoint. Data shown as mean ± SEM. *indicate statistical significance.

Fig. 5.

TTX-resistant current dissection. (A) Distribution of capacitances sizes from neurons of WT rat DRG neurons. (B) Current dissection shown as mean peak amplitudes of Nav currents from −120, −100, and −50 mV in pharmacological conditions. (C) CBG (5 µM) effect at noted potentials. (D) TTX-resistant state-dependent inhibition. (E) Two-pulse protocol used to measure inhibition and representative current traces. (F) Inhibition of TTX-resistant current binned by size. Data shown as mean ± SEM. *indicate statistical significance.

Fig. 6.

Excitability and summary. (A) All measurements are matched-paired in WT rat DRG neurons, at 5 µM CBG. Sample AP traces. (B) Cell capacitance distribution (Right axis), and maximal number of APs in Veh vs. CBG (Left Axis). (C) Collected results for the number of APs during 1 s of current injections in Veh vs. CBG (5 µM). (D) WT rat DRG neuron suprathreshold oscillation. All measurements are matched-paired in WT rat DRG neurons, at 5 µM CBG (no TTX). The cells were current-clamped using a ramp protocol from −50 pA to +60 pA over the course of 500 ms. Two of the cells displayed suprathreshold oscillation events (indicated by arrows). Five µM CBG blocked both AP spikes, as well as oscillation events. The point plot shows the quantification of AP and oscillation events before and after CBG perfusion. Data shown as mean ± SEM. *indicate statistical significance. (E) Summary of the effects of each of the tested cannabinoids on each component of the depolarizing phase of nociceptive AP. CBD and CBN have not been directly evaluated on Nav1.9, thus, their inhibition is indicated as dashed lines. All three inhibit Nav1.7; however, given their small magnitude of state dependence, Nav1.7 may be a smaller part of their effect on APs. All three inhibit Nav1.8, but as CBG demonstrated the most robust use-dependent inhibition, it is indicated as the most effective. CBD is second because it demonstrates the most potent effect in vitro (20, 33).