Bruce P. Bean

Nav1.7 voltage-gated sodium channels are strongly expressed in human primary pain-sensing neurons (nociceptors), and selective Nav1.7 inhibitors have been developed as possible therapeutic agents for treating pain, so far with disappointing clinical results. In contrast, a selective Nav1.8 channel inhibitor (suzetrigine) has had successful clinical trials. Because nociceptors express both Nav1.7 and Nav1.8 channels, it is of interest to compare effects of Nav1.7 and Nav1.8 inhibitors on the excitability of human nociceptors. To compare with previous results with suzetrigine, we characterized the effects of a selective Nav1.7 inhibitor, AM-2099, on action potential generation and repetitive firing of dissociated human dorsal root ganglion (DRG) neurons, studied at 37°C. Inhibition of Nav1.7 channels by 600 nM AM-2099 generally produced a substantial depolarizing shift of action potential threshold, an increase in rheobase, a decrease in action potential upstroke velocity, decrease in action potential peak, and prolongation of the refractory period. Compared to inhibition of Nav1.8 channels, inhibition of Nav1.7 channels had larger effects on threshold and maximal upstroke velocity, while action potential peak was reduced similarly by both. Nav1.8 inhibition produced much more dramatic reduction of repetitive firing than Nav1.7 inhibition. The results show that although the excitability of human DRG neurons is affected by inhibition of Nav1.7 channels, most notably by an increase in threshold and increase in refractory period, repetitive firing of the neurons in response to strong stimuli is little affected.

Nav1.7 voltage-gated sodium channels are strongly expressed in human primary pain-sensing neurons (nociceptors) and selective Nav1.7 inhibitors have been developed as possible therapeutic agents for treating pain, so far with disappointing clinical results. In contrast, a selective Nav1.8 channel inhibitor (suzetrigine) has had successful clinical trials. Because nociceptors express both Nav1.7 and Nav1.8 channels, it is of interest to compare effects of Nav1.7 and Nav1.8 inhibitors on the excitability of human nociceptors. To compare with previous results with suzetrigine, we characterized the effects of a selective Nav1.7 inhibitor, AM-2099, on action potential generation and repetitive firing of dissociated human dorsal root ganglion neurons, studied at 37°C. Inhibition of Nav1.7 channels by 600 nM AM-2099 generally produced a substantial depolarizing shift of action potential threshold, an increase in rheobase, a decrease in action potential upstroke velocity, decrease in action potential peak, and prolongation of refractory period. Compared to inhibition of Nav1.8 channels, inhibition of Nav1.7 channels had larger effects on threshold and maximal upstroke velocity, while action potential peak was reduced similarly by both. Nav1.8 inhibition produced much more dramatic reduction of repetitive firing than Nav1.7 inhibition. The results show that although the excitability of human DRG neurons is affected by inhibition of Nav1.7 channels, most notably by an increase in threshold and increase in refractory period, repetitive firing of the neurons in response to strong stimuli is little affected. [234 words]

Primary sensory neurons that sense noxious stimuli (nociceptors) are unusual in possessing two major components of voltage‐dependent sodium channel current with different voltage dependence and kinetics. We used pharmacology to characterize the roles of defined sodium channel types in generating action potentials and controlling repetitive firing in putative peptidergic nociceptors, using mice in which CGRP‐lineage neurons are fluorescently labelled. We found that the major components of sodium current were carried by Nav1.8 channels, identified by sensitivity to VX‐548 (suzetrigine), and by Nav1.7 channels, identified by sensitivity to the toxin GsAF‐1, with an additional smaller component of current carried by tetrodotoxin‐sensitive channels resistant to GsAF‐1. Action potential clamp experiments were used to quantify the size and time course of the Nav1.8 component of sodium current during firing of single and repetitive action potentials. In most neurons, Nav1.8 current carried the majority of overall sodium entry during action potentials and nearly all of the sodium current supporting action potentials during repetitive firing. Although total tetrodotoxin‐sensitive current (mostly but not entirely carried by Nav1.7 channels) activated with smaller depolarizations and much more rapidly than Nav1.8 current, Nav1.8 current was equally important in setting the action potential threshold and was dominant in the action potential upstroke because of its greater size. By comparison with similar experiments in human dorsal root ganglion neurons, the results suggest Nav1.8 channels play a more dominant role in firing of mouse peptidergic nociceptors than in human C‐type nociceptors, suggesting limitations of pharmacology of mouse nociceptors for predicting effects on human nociceptor excitability. image Key points: The new Nav1.8 inhibitor VX‐548 (suzetrigine) was used to define the role of Nav1.8 currents in generating action potentials in mouse CGRP‐lineage dorsal root ganglion neurons. Nav1.8 current typically carried the majority of sodium current evoked by voltage steps, with remaining current mainly carried by Nav1.7 channels (identified by sensitivity to GsAF‐1) together with a small component from tetrodotoxin‐sensitive channels not inhibited by GsAF‐1. Nav1.8 current contributes to determining the action potential threshold, provides the majority of sodium current during the rising phase of the action potential and flows during the shoulder of the action potential typical of C‐fibre nociceptors. Inhibition of Nav1.8 sodium current by VX‐548 produces a counter‐intuitive decrease in refractory period, probably resulting from reduction of potassium channel activation caused by a smaller peak and narrower action potential when Nav1.8 current is inhibited.

Nav1.7 voltage-gated sodium channels are strongly expressed in human primary painsensing neurons (nociceptors) and selective Nav1.7 inhibitors have been developed as possible therapeutic agents for treating pain, so far with disappointing clinical results. In contrast, a selective Nav1.8 channel inhibitor (suzetrigine) has had successful clinical trials. Because nociceptors express both Nav1.7 and Nav1.8 channels, it is of interest to compare effects of Nav1.7 and Nav1.8 inhibitors on the excitability of human nociceptors. To compare with previous results with suzetrigine, we characterized the effects of a selective Nav.7 inhibitor, AM-2099, on action potential generation and repetitive firing of dissociated human dorsal root ganglion neurons, studied at 37°C. Inhibition of Nav1.7 channels by 600 nM AM-2099 generally produced a substantial depolarizing shift of action potential threshold, an increase in rheobase, a decrease in action potential upstroke velocity, decrease in action potential peak, and prolongation of refractory period. Compared to inhibition of Nav1.8 channels, inhibition of Nav1.7 channels had larger effects on threshold and maximal upstroke velocity, while action potential peak was reduced similarly by both. Nav1.8 inhibition produced much more dramatic reduction of repetitive firing than Nav1.7 inhibition. The results show that although the excitability of human DRG neurons is affected by inhibition of Nav1.7 channels, most notably by an increase in threshold and increase in refractory period, repetitive firing of the neurons in response to strong stimuli is little affected. Significance statement: Nav1.7 sodium channels are highly expressed in primary pain-sensing neurons and humans with null mutations in Nav1.7 channels have loss of pain sensation. However, unlike the Nav1.8 inhibitor suzetrigine, Nav1.7 inhibitors have so far not reached clinical use. We compared effects of Nav1.7 on electrical excitability of human dorsal root ganglion neurons with those of suzetrigine and found that while Nav1.7 inhibition affects spike threshold more than suzetrigine, there is little effect on repetitive firing with strong stimuli.

Site 1 sodium channel blockers (S1SCBs), such as tetrodotoxin (TTX) and neosaxitoxin, are ultrapotent local anesthetics with low tissue toxicity. Their duration of action is relatively brief, but increasing the dose can lead to systemic toxicity. Here, we report that selected flavonoids—puerarin (PUE), naringenin, and kaempferol—prolong nerve block from S1SCBs 4- to 25-fold. Using both tympanic membrane (TM) permeation and sciatic nerve fluorescence distribution models, we demonstrate the flavonoids increase drug penetration across biological barriers (the TM and the barriers in and around nerve), suggesting that they act as chemical permeation enhancers (CPEs). Importantly, flavonoids exhibited minimal tissue toxicity compared to conventional CPEs. Coencapsulation of TTX and PUE into a liposomal delivery system further prolonged local anesthesia to over 25 d from a single injection. These findings establish flavonoid compounds as a safe class of CPEs and provide a platform of long-acting, nonopioid pain therapies. Flavonoids may be attractive alternatives to conventional CPEs in biomedical applications.

Nav1.8 voltage-gated sodium channels are strongly expressed in human primary pain-sensing neurons (nociceptors) and a selective Nav1.8 inhibitor VX-548 (suzetrigine) has shown efficacy for treating acute pain in clinical trials. Nociceptors also express other sodium channels, notably Nav1.7, raising the question of how effectively excitability of the neurons is reduced by inhibition of Nav1.8 channels alone. We used VX-548 to explore this question, recording from dissociated human dorsal root ganglion neurons at 37 °C. Applying VX-548 at 10 nM (about 25 times the IC ₅₀ determined using cloned human Nav1.8 channels at 37 °C) had only small effects on action potential threshold and upstroke velocity but substantially reduced the peak and shoulder. Counterintuitively, VX-548 shortened the refractory period—likely reflecting reduced potassium channel activation by the smaller, narrower action potential—sometimes resulting in faster firing. Generally, repetitive firing during depolarizations was diminished but not eliminated by VX-548. Voltage clamp analysis suggested two reasons that repetitive firing often remains in 10 to 100 nM VX-548. First, many neurons had such large Nav1.8 currents that even 99% inhibition leaves nA-level Nav1.8 current that could help drive repetitive firing. Second, Nav1.7 current dominated during initial spikes and could also contribute to repetitive firing. The ability of human neurons to fire repetitively even with >99% inhibition of Nav1.8 channels may help explain the incomplete analgesia produced by even the largest concentrations of VX-548 in clinical studies.

Nav1.8 sodium channels are expressed in pain-sensing neurons, and some Nav1.8 inhibitors significantly reduce pain in clinical trials. Several Nav1.8 inhibitors have an unusual state dependence whereby inhibition is relieved by depolarization. We compared the state-dependent action of several Nav1.8 channel inhibitors to test whether inhibition is relieved during action potential (AP) firing under physiological conditions to produce “reverse use dependence.” A-887826 inhibition was substantially relieved by AP waveforms applied at 20 Hz at 37°C. In contrast, there was no relief during AP trains with suzetrigine (VX-548) or LTGO-33, even though inhibition could be effectively removed by long, strong depolarizations. These differences were explained by differences in the voltage dependence and kinetics with which the compounds dissociate from depolarized channels and rebind to resting state channels. Suzetrigine required the strongest depolarizations for relief (midpoint +33 mV) and relief was slow (tau >300 ms at +20 mV), so almost no relief occurred during an AP waveform. Relief from A-887826 required weaker depolarizations (midpoint +13 mV) and was much faster, so some relief occurred during each AP waveform and accumulated during 20-Hz trains. LTGO-33 required the weakest depolarizations for relief (midpoint −11 mV) and relief was even faster than for A-887826, but reinhibition between AP waveforms was far faster than for A-887826, so that relief did not accumulate during AP trains at 20 Hz. The results show that, unlike A-887826, there is no use-dependent relief of inhibition by suzetrigine or LTGO-33 with physiological voltage waveforms at physiological temperatures, but each for different reasons.

Nav1.8 channels are an attractive therapeutic target for pain because they are prominent in primary pain-sensing neurons with little expression in most other kinds of neurons. Recently, two Nav1.8-targeted compounds, VX-150 and VX-548, have shown efficacy in clinical trials for reducing pain. We examined the characteristics of Nav1.8 inhibition by these compounds. The active metabolite form of VX-150 (VX-150m) inhibited human Nav1.8 channels with an IC₅₀ of 15 nM. VX-548 (suzetrigine) was even more potent (IC₅₀ 0.27 nM). Both VX-150m and VX-548 had the unusual property of "reverse use-dependence", whereby inhibition could be relieved by repetitive depolarizations, a property seen before with another Nav1.8 inhibitor, A-887826. The relief of VX-548 inhibition by large depolarizations occurred with a time constant of ~40 ms that was not concentration-dependent. Re-inhibition at negative voltages occurred with a rate that was nearly proportional to drug concentration, consistent with the idea that relief of inhibition reflects dissociation of drug from the channel and re-inhibition reflects re-binding. The relief of inhibition by depolarization suggests a remarkably strong and unusual state-dependence for both VX-150m and VX-548, with very weak binding to channels with fully activated voltage sensors despite very tight binding to channels with voltage sensors in the resting state. Significance Statement The Nav1.8 sodium channel is a current target for new drugs for pain. This work describes the potency, selectivity, and state-dependent characteristics of inhibition of Nav1.8 channels by VX-150 and VX-548, compounds that have recently shown efficacy for relief of pain in clinical trials but whose mechanism of interaction with channels has not been described. The results show that the compounds share an unusual property whereby inhibition is relieved by depolarization, demonstrating a state-dependence different than most sodium channel inhibitors.

Many neurons in the mammalian brain show pacemaking activity: rhythmic generation of action potentials in the absence of sensory or synaptic input. Slow pacemaking of neurons releasing modulatory transmitters is easy to rationalize. More surprisingly, many neurons in the motor system also show pacemaking activity, often rapid, including cerebellar Purkinje neurons that fire spontaneously at 20–100 Hz, as well as key neurons in the basal ganglia, including subthalamic nucleus neurons and globus pallidus neurons. Although the spontaneous rhythmic firing of pacemaking neurons is phenomenologically similar to cardiac pacemaking, the underlying ionic mechanism in most neurons is quite different than for cardiac pacemaking. Few spontaneously active neurons rely on HCN ‘pacemaker’ channels for their activity. Most commonly, a central element is ‘persistent’ sodium current, steady‐state subthreshold current carried by the same voltage‐dependent sodium channels that underlie fast action potentials. Persistent sodium current is a steeply voltage‐dependent current with a midpoint near −60 mV, which results in regenerative spontaneous depolarization once it produces a net inward current when summed with all other background currents, often at voltages as negative as −70 mV. This ‘engine’ of pacemaking is present in almost all neurons and must be held in check in non‐pacemaking neurons by sufficiently large competing outward currents from background potassium channels. The intrinsic propensity of neurons to fire spontaneously underlies key normal functions such as respiration and generates the complex background oscillatory circuits revealed in EEGs, but can also produce out‐of‐control oscillations of overall brain function in epilepsy, ataxia and tremor. image