Results
Among local anesthetics with an amino-amide scaffold like lidocaine, compounds like bupivacaine and mepivacaine in which the amino group is part of a piperidine ring have higher potency for sodium channel inhibition (Bräu et al., 1998; Scholz et al., 1998). Based on this observation, we designed and synthesized compounds containing a cationic charge in a piperidinium ring and identified one, BW-031 (Figure 1a), as a compound that inhibited Nav1.7 sodium channels with substantially higher potency than QX-314 when applied intracellularly (Figures 1b-c). As for QX-314 (Strichartz, 1973; Schwarz et al., 1977; Yeh, 1978), inhibition by BW-031 progressively accumulates with each cycle of activation and deactivation of the sodium channel, as if the intracellular blocker can enter the channel only when it is open and is effectively trapped within the channel after the channel closes. BW-031 had minimal effect on Nav1.7 currents when applied extracellularly (Figures 1d-e), suggesting that, like QX-314, it cannot effectively enter sodium channels through the narrow ion selectivity filter in the outer pore region of the channel or diffuse across the cell membrane. Intracellular BW-031 inhibited native sodium currents in nociceptors differentiated from human induced pluripotent stem cells (hiPSCs) with very similar potency as for heterologously expressed Nav1.7 channels (Figures 1f-g). BW-031 inhibited Nav1.1 channels with a similar potency to Nav1.7 channels (Figures 2a,b). However, BW-031 was considerably less effective in inhibiting heterologously-expressed Nav1.8 channels (Figures 2c,d), with intracellular 300 µM BW-031 producing only 30 ± 8% inhibition (n=5), similar to the effect of 30 µM BW-031 on Nav1.7 channels (37 ± 4% inhibition, n=6). The much weaker effect on Nav1.8 channels was unexpected, because previous work showed that the uncharged piperidine-containing anesthetics bupivacaine and mepivacaine have generally similar effects on native TTX-resistant sodium channels in DRG neurons and heterologously-expressed Nav1.8 channels as on a variety of TTX-sensitive channels (Bräu et al., 1998; Scholz et al., 1998; Scholz et al., 2000; Leffler et al, 2010).
BW-031 applied externally to mouse DRG neurons inhibited sodium currents only when it was applied together with capsaicin to activate TRPV1 channels (Figure 3a) and the combined application of BW-031 and capsaicin had no effect on sodium currents in DRG neurons lacking expression of TRPV1 channels, as tested by the response to 1 µM capsaicin (Figure 3b). Thus, like QX-314 (Binshtok et al., 2007; Brenneis et al., 2013, 2014; Stueber et al., 2016), BW-031 can permeate through activated TRPV1 channels to block sodium channels from the inside of the cell.
We next tested the possibility that BW-031 applied alone might be able to inhibit nociceptors in several rodent models of inflammatory pain. BW-031 effectively reduced hypersensitivity in a rat model of inflammation induced by paw injection of Complete Freund’s Adjuvant (CFA), in which inflammation activates TRPV1 and TRPA1 channels (Garrison and Stucky, 2014; Asgar et al., 2015; Kanai et al., 2007; Lennertz et al., 2012). In this model, latency of paw withdrawal to a thermal stimulus was decreased at 1 hour and even more at 4 hours, and BW-031 blocked this effect at both times (Figure 4a). Similarly, BW-031 also blocked mechanical hyperalgesia in a more clinically-relevant rat paw incision model of surgical pain (Brennan et al., 1996; Barabas and Stucky, 2014) in which the incision produced pronounced mechanical hyperalgesia when assayed 24 hours later. BW-031 injected near the incision greatly reduced the mechanical hyperalgesia, with strong effects at 3 hours and 5 hours after BW-031 injection that then progressively declined at later times (Figure 4b). Figure 5a shows results from a mouse model of UV-burn-induced inflammatory pain (Yin et al., 2016) where inflammatory mediators activate TRPV1 and TRPA1 channels in nociceptors (Acosta et al., 2014; Yin et al., 2016). Plantar UV-burn results in pronounced mechanical allodynia 24 hours later, at which time intra-plantar injection of 2% BW-031 produced robust mechanical analgesia lasting for at least 7 hours, with considerably longer-lasting effects than QX-314. Interestingly, in both the mouse UV burn model and the rat CFA paw-injection model, BW-031 not only reversed the tactile hypersensitivity resulting from the injury but also produced substantial long-lasting analgesia relative to the control situation, indicating a general inhibition of nociceptors at the site of administration to the inflamed tissue.
To test the selectivity of BW-031 to inhibit neurons only in conditions in which TRPV1, TRPA1 or other large-pore channels are activated, we performed perisciatic injections in naïve mice, with perisciatic injection of lidocaine as a positive control that inhibits neuronal activity without any requirement for activation of large-pore channels. We found that neither BW-031 nor QX-314 produced any block of either sensory or motor function, in contrast to the transient inhibition of both by lidocaine (Figures 5b,c), consistent with a requirement for activated TRP or other large-pore channels for neuronal inhibition by the charged blockers.
Guinea pigs are the standard pre-clinical model for studying cough (Adner et al., 2020; Bonvini et al., 2015; Lewis et al., 2007; Morice et al., 2007) as the main features of airway innervation are similar in guinea pigs and humans (West et al., 2015; Mazzone and Undem, 2016). Coughing in guinea pigs can be mediated both by a subset of bronchopulmonary C-fibers and by a distinct mechanically-sensitive and acid-sensitive subtype of myelinated airway mechanoreceptors (Canning, 2006; Canning et al., 2014; Canning et al., 2004; Chou et al., 2018b; Mazzone et al., 2009; Mazzone and Undem, 2016). The neurons mediating the C-fiber pathway have strong expression of both TRPV1 and TRPA1 channels (Bonvini et al., 2015; Canning et al., 2014; Mazzone and Undem, 2016), and coughing in both guinea pigs and humans can be evoked by both TRPV1 agonists like capsaicin (Bonvini et al., 2015; Brozmanova et al., 2012; Kanezaki et al., 2012; Laude et al., 1993) and by TRPA1 agonists (Birrell et al., 2009; Bonvini et al., 2015; Kanezaki et al., 2012; Long et al., 2019b). The importance of this population of TRPV1 and TRPA1-expressing neurons in at least some forms of cough suggested the possibility that loading charged sodium channel inhibitors into these neurons might inhibit cough.
We used two different experimental protocols to test whether BW-031 can inhibit cough in guinea pigs when applied under conditions in which TRPV1 and TRPA1 channels are likely to be activated. In the first, we delivered a small volume (0.5 mL/kg) of different doses of BW-031 intratracheally to animals under transient isoflurane anesthesia (Figure 6), relying on the ability of isoflurane to activate TRPV1 and TRPA1 channels (Matta et al., 2008; Kichko et al., 2015). One hour after the administration of BW-031, coughing was induced by inhalation of aerosolized citric acid, which induces a low rate of coughing (typically 0.5-1 cough/minute (Tanaka and Maruyama, 2005)), and coughs were measured using whole-body plethysmography. BW-031 produced a dose-dependent reduction in the number of coughs evoked by citric acid, with administration of 7.53 mg/kg BW-031 reducing cough counts during a 17-minute period from 9.4±2.4 in control to 0.9±0.5 with BW-031 (n=9, p=0.005, Tukey’s post-hoc test) (Figure 6b), with a complete suppression of coughing in 5 of the 9 animals tested.
With these encouraging results, we next tested BW-031 in a potentially more translationally-relevant guinea pig model of ovalbumin-induced allergic airway inflammation, which produces activation and upregulation of both TRPV1 and TRPA1 channels in the airways (Liu et al., 2015; McLeod et al., 2006; Watanabe et al., 2008). Guinea pigs were sensitized by intraperitoneal and subcutaneous injections of ovalbumin (Figure 7a). Fourteen days later, inhaled ovalbumin induced allergic airway inflammation, reflected by increased immune cell counts in the bronchoalveolar lavage (BAL) measured one day after the ovalbumin-challenge (Figure 7b). Nebulized BW-031 was administered to restrained awake guinea pigs via snout-only inhalation chambers one day after the allergen challenge, and cough was then induced by citric acid one hour after the inhalation of BW-031. BW-031 strongly inhibited the citric acid-induced cough in a dose-dependent manner (Figure 7c). At the highest dose tested (17.6 mg/kg), BW-031 reduced cough counts over 17 minutes from 10±1.6 in control to 2.2±0.89 with BW-031 (n=12, p=0.0009, Tukey’s post-hoc test), with complete suppression of cough in 7 of the 12 animals.
The hydrophobicity of local anesthetics like lidocaine enables ready absorption from lung tissue into the blood. In principle, the absorption of cationic compounds like BW-031 might be expected to be much less. The highest dose of inhaled BW-031 (17.6 mg/kg) resulted in a serum concentration of 419±46 nM (n=12) (Figure 8a), far lower than the average serum level of 15 µM (3.6 µg/mL) lidocaine measured following lidocaine spray anesthesia used for bronchoscopy (Labedzki et al., 1983). The serum concentration of BW-031 after aerosol inhalation was many orders of magnitude below the concentration at which any effect of BW-031 was seen on contraction of human IPSC-derived cardiomyocytes (3 mM; Figure 8b). Thus, inhaled BW-031 should have a high therapeutic index with regard to in vivo cardiotoxicity, which is a significant concern with inhaled lidocaine (Horáček and Vymazal, 2012).