Discussion
These results show that the piperidinium-containing cationic compound BW-031 is a highly effective use-dependent sodium channel inhibitor when introduced intracellularly, acting with about 6-fold greater potency than QX-314. Like QX-314 (Binshtok et al., 2007; Puopolo et al., 2013), BW-031 had no effect on sodium currents in DRG neurons if applied in the absence of large-pore channel stimulation and in contrast to lidocaine, had no effect on nerve function when applied perisciatically in the absence of inflammation. Consistent with its greater intrinsic potency compared to QX-314, BW-031 produced more effective and longer-lasting inhibition of hypersensitivity in a mouse UV-burn model.
Both QX-314 and BW-031 applied alone to inflamed tissue were effective in inhibiting inflammatory pain, consistent with extensive evidence that tissue inflammation is associated with activation of TRPV1 and TRPA1 channels by endogenous activators (Bautista et al., 2013, Julius, 2013). Besides the models of incisional pain and UV burn we studied, the activation of TRPV1 and TRPA1 channels has been found in many other examples of inflammation, including models of colitis and inflammatory bowel disease (Zielińska et al. 2015, Utsumi et al., 2018; Csekő et al., 2019; Jain et al., 2020), dermatitis (Liu et al., 2013), cystitis (DeBerry et al., 2014) and pancreatitis (Schwartz et al., 2013). Thus, there may be many possible applications of the strategy to inhibit that inflammatory pain initiated by large pore expressing sensory neurons.
Cough may be a particularly attractive application of nerve silencing by entry of charged sodium channels blockers through large pore channels. TRPV1 and TRPA1 channels are expressed in many neurons mediating cough (Belvisi et al., 2011; Birrell et al., 2009; Bonvini et al., 2015; Brozmanova et al., 2012; Forsberg et al., 1988; Grace and Belvisi, 2011; Jia et al., 2002; Laude et al., 1993; Undem et al., 2002) and are likely to be activated in many conditions triggering cough, including airway inflammation (Bessac and Jordt, 2008; Choi et al., 2018; Talbot et al., 2015; Talbot et al., 2020) and respiratory viral infection (Zaccone et al., 2016). Expression of TRPV1 channels is increased in airways of patients with chronic persistent cough (Groneberg et al., 2004) and after respiratory viral infection (Omar et al., 2017). In addition, increasing evidence suggests an important role for channels containing P2X3 proteins for mediating cough. P2X3 channels are expressed on sensory neurons innervating the lungs (Kollarik et al., 2019; Kwong et al., 2008; Mazzone and Undem, 2016) and P2X3 inhibitors reduce cough in guinea pig models (Bonvini et al., 2015; Garceau and Chauret, 2019; Pelleg et al., 2019) and appear promising in recent human cough trials (Morice et al., 2019; Smith et al., 2017; Smith et al., 2020a,b; Dicpinigaitis et al., 2020). Interestingly, recent work has demonstrated that P2X3 receptors form large-pore channels capable of passing large cations (Harkat et al., 2017), similar to TRPV1 and TRPA1 pores. Thus, it is plausible that activated P2X3-containing channels, as well as TRPV1 and TRPA1 channels, might provide a pathway for entry of BW-031 into cough-mediating neurons.
The strategy of using activated large-pore channels to introduce charged sodium channel blockers inside sensory neurons should inhibit the activity of the neurons to all subsequent stimuli and may therefore have greater efficacy than targeting single receptors like TRPV1, TRPA1, and P2X3. Once cationic sodium channel inhibitors are loaded into a cell (concentrated by the negative intracellular potential), they will not readily diffuse out through the cell membrane and can produce effects lasting for many hours, as is the case for the analgesic effect of QX-314 (Binshtok et al., 2009a; Gerner et al., 2008; Roberson et al., 2011) and as we find here for BW-031 inhibition of pain (Figures 4,5).
Overall, the ability of BW-031 to inhibit cough adds to previous evidence that targeting peripheral afferent activity by sodium channel inhibition can be an effective strategy for inhibiting cough (Muroi et al., 2011; Sun et al., 2017; Kollarik et al., 2018; Brozmanova et al., 2019; Patil et al., 2019; Undem and Sun, 2020). Using cationic sodium channel inhibitors should have the advantage of limiting the inhibition of nerve activity only to those neurons that express large-pore channels, like TRPV1, TRPA1, and P2X3, and only under those conditions, such as inflammation or noxious irritation, where these channels are activated.
The citric acid model of guinea pig cough, although widely used, clearly has limitations for predicting drug efficacy in human disease, because while TRPV1 and TRPA1 inhibitors are reasonably effective in this model (Leung et al., 2007; Mukhopadhyay et al., 2014) this has not been replicated, so far, in patients (Khalid et al., 2014; Belvisi et al., 2017; European Medicines Agency, 2013). It would be useful in future studies to explore the efficacy of BW-031 in other modes of cough induction, such as hypo-osmotic solutions or direct mechanical stimulation, which may activate different populations of nerve fibers than citric acid (Chou et al., 2018a; Morice et al., 2007).