Cannabis sativa contains many related compounds known as phytocannabinoids. The main psychoactive and nonpsychoactive compounds are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), respectively. Much of the evidence for clinical efficacy of CBD-mediated antiepileptic effects has been from case reports or smaller surveys. The mechanisms for CBD's anticonvulsant effects are unclear and likely involve noncannabinoid receptor pathways. CBD is reported to modulate several ion channels, including sodium channels (Nav). Evaluating the therapeutic mechanisms and safety of CBD demands a richer understanding of its interactions with central nervous system targets. Here, we used voltage-clamp electrophysiology of HEK-293 cells and iPSC neurons to characterize the effects of CBD on Nav channels. Our results show that CBD inhibits hNav1.1–1.7 currents, with an IC50 of 1.9–3.8 μm, suggesting that this inhibition could occur at therapeutically relevant concentrations. A steep Hill slope of ∼3 suggested multiple interactions of CBD with Nav channels. CBD exhibited resting-state blockade, became more potent at depolarized potentials, and also slowed recovery from inactivation, supporting the idea that CBD binding preferentially stabilizes inactivated Nav channel states. We also found that CBD inhibits other voltage-dependent currents from diverse channels, including bacterial homomeric Nav channel (NaChBac) and voltage-gated potassium channel subunit Kv2.1. Lastly, the CBD block of Nav was temperature-dependent, with potency increasing at lower temperatures. We conclude that CBD's mode of action likely involves 1) compound partitioning in lipid membranes, which alters membrane fluidity affecting gating, and 2) undetermined direct interactions with sodium and potassium channels, whose combined effects are loss of channel excitability.

Molecular determinants of ion channel tetramerization are well characterized, but those involved in heteromeric channel assembly are less clearly understood. The heteromeric composition of native channels is often precisely controlled. Cyclic nucleotide-gated (CNG) channels from rod photoreceptors exhibit a 3:1 stoichiometry of CNGA1 and CNGB1 subunits that tunes the channels for their specialized role in phototransduction. Here we show, using electrophysiology, fluorescence, biochemistry, and X-ray crystallography, that the mechanism for this controlled assembly is the formation of a parallel 3-helix coiled-coil domain of the carboxy-terminal leucine zipper region of CNGA1 subunits, constraining the channel to contain three CNGA1 subunits, followed by preferential incorporation of a single CNGB1 subunit. Deletion of the carboxy-terminal leucine zipper domain relaxed the constraint and permitted multiple CNGB1 subunits in the channel. The X-ray crystal structures of the parallel 3-helix coiled-coil domains of CNGA1 and CNGA3 subunits were similar, suggesting that a similar mechanism controls the stoichiometry of cone CNG channels.

Selective block of Na1.7 promises to produce non-narcotic analgesic activity without motor or cognitive impairment. Several Na1.7-selective blockers have been reported, but efficacy in animal pain models required high multiples of the IC for channel block. Here, we report a target engagement assay using transgenic mice that has enabled the development of a second generation of selective Nav1.7 inhibitors that show robust analgesic activity in inflammatory and neuropathic pain models at low multiples of the IC. Like earlier arylsulfonamides, these newer acylsulfonamides target a binding site on the surface of voltage sensor domain 4 to achieve high selectivity among sodium channel isoforms and steeply state-dependent block. The improved efficacy correlates with very slow dissociation from the target channel. Chronic dosing increases compound potency about 10-fold, possibly due to reversal of sensitization arising during chronic injury, and provides efficacy that persists long after the compound has cleared from plasma.

ABSTRACT:We report on a novel series of aryl sulfonamides that act as nanomolar potent, isoform-selective inhibitors of the human sodium channel hNa V 1.7. The optimization of these inhibitors is described. We aimed to improve potency against hNa V 1.7 while minimizing off-target safety concerns and generated compound 3. This agent displayed significant analgesic effects in rodent models of acute and inflammatory pain and demonstrated that binding to the voltage sensor domain 4 site of Na V 1.7 leads to an analgesic effect in vivo. Our findings corroborate the importance of hNa V 1.7 as a drug target for the treatment of pain. KEYWORDS: Sodium channel, Na V 1.7, Na V 1.5, pain, aryl sulfonamide, formalin model, cold allodynia T he sodium channel Na V 1.7 belongs to a family of transmembrane voltage gated sodium channels, which consists of nine isoforms in mammals (Na V 1.1 to Na V 1.9).1−4 Na V 1.7 plays a crucial role in pain sensation, and there is strong genetic evidence linking Na V 1.7 and its encoding SCN9A gene to painful disorders in humans. Gain-of-function mutations in the SCN9A gene result in painful conditions such as inherited erythromelalgia, paroxysmal extreme pain disorder, and idiopathic small fiber neuropathies. In contrast, loss-of-function mutations in the SCN9A gene were found to be the genetic cause of a rare disorder called congenital insensitivity to pain, characterized by a complete loss of the ability to sense painful stimuli. It is noteworthy that no significant side effects have been reported in people lacking Na V 1.7, such as cognitive, motor, or non-nociceptive sensory impairments other than anosmia, giving further support to the concept of Na V 1.7 antagonists as analgesics.1−4 The predominant expression of the Na V 1.7 isoform in the PNS may offer a pathway to limit CNS-related adverse effects by developing compounds that do not cross the blood−brain barrier. Combined, these observations and findings have made Na V 1.7 a promising target for drug development for the treatment of pain. Indeed, there has been tremendous interest in the development of small molecule Na V 1.7 inhibitors as analgesics, particularly isoform-selective inhibitors, and coverage of the progress has been the subject of several excellent reviews. 1−7 In recent years, a series of aryl sulfonamides as Na V inhibitors have been reported that appear to be highly selective for Na V 1.7 over the cardiac ion channel Na V 1.5. [4][5][6]8 Since block of the Na V 1.5 channel may lead to arrhythmia and thus limit the therapeutic potential of nonselective Na V 1.7 inhibitors, isoform-selective inhibitors have attracted considerable interest due to their potential to avoid these adverse events.3,5 An example is aryl sulfonamide PF-04856264 ( Figure 1), which selectively blocks Na V 1.7 over Na V 1.5 and Na V 1.3.