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).