Inhibition of inflammatory pain and cough by a novel charged sodium channel blocker

Article abstract of DOI:10.1111/bph.15531

Background and Purpose: Many pain-triggering nociceptor neurons express TRPV1 or TRPA1, cation-selective channels with large pores that enable permeation of QX-314, a cationic analogue of lidocaine. Co-application of QX-314 with TRPV1 or TRPA1 activators can silence nociceptors. In this study, we describe BW-031, a novel more potent cationic sodium channel inhibitor, and test whether its application alone can inhibit pain associated with tissue inflammation and whether this strategy can also inhibit cough. Experimental Approach: We tested the ability of BW-031 to inhibit pain in three models of tissue inflammation:- inflammation in rat paws produced by complete Freund's adjuvant or by surgical incision and a mouse ultraviolet (UV) burn model. We tested the ability of BW-031 to inhibit cough induced by inhalation of dilute citric acid in guinea pigs. Key Results: BW-031 inhibited Na_v1.7 and Na_v1.1 channels with approximately sixfold greater potency than QX-314 when introduced inside cells. BW-031 inhibited inflammatory pain in all three models tested, producing more effective and longer-lasting inhibition of pain than QX-314 in the mouse UV burn model. BW-031 was effective in reducing cough counts by 78%–90% when applied intratracheally under isoflurane anaesthesia or by aerosol inhalation in guinea pigs with airway inflammation produced by ovalbumin sensitization. Conclusion and Implications: BW-031 is a novel cationic sodium channel inhibitor that can be applied locally as a single agent to inhibit inflammatory pain. BW-031 can also effectively inhibit cough in a guinea pig model of citric acid-induced cough, suggesting a new clinical approach to treating cough.

Full text of DOI:10.1111/bph.15531

2021-BJP-0179-RP Revised May 11, 2021
Inhibition of inflammatory pain and cough by a novel charged sodium channel blocker
Running title: Inhibition of pain and cough by BW-031
Ivan Tochitsky1*, Sooyeon Jo2*, Nick Andrews1, Masakazu Kotoda1, Benjamin Doyle1,
Jaehoon Shim1, Sebastien Talbot1,3, David Roberson1, Jinbo Lee4, Louise Haste5, Stephen M.
Jordan5, Bruce D. Levy6#, Bruce P. Bean2#, Clifford J. Woolf1,2#
1 F.M. Kirby Neurobiology Research Center, Boston Children's Hospital, Boston, MA 02115
2 Department of Neurobiology, Harvard Medical School, Boston MA 02115
3 Départément de Pharmacologie et Physiologie, Université de Montréal, Canada
4 Sage Partner International, Andover MA 01810
5 Covance Inc., Woolley Rd, Alconbury, Huntingdon PE28 4HS, United Kingdom
6 Pulmonary and Critical Care Medicine, Department of Internal Medicine, Brigham and
Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA
* These authors contributed equally: Ivan Tochitsky, Sooyeon Jo.
#Co-senior authors
Correspondence to B.D.L., B.P.B. and C.J.W.
Word count: 3,276
Acknowledgements
We are grateful to Alyssa Grantham, Mary Kate Dornon, Yu Wang, Daniel Taub, Huan Wang
and Lee Barrett for technical assistance, to the Boston Children’s Hospital PK-lab for
assistance with LC/MS experiments, and to Ronald Blackman, James Ellis, and Richard
Batycky for helpful discussions and suggestions. This work was supported by the National
Institutes of Health National Institute of Neurological Diseases and Stroke [R35NS105076
(C.J.W.), R01NS036855 (B.P.B.), R01NS110860 (B.P.B.), R01HL122531 (B.D.L.)], the
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/bph.15531
This article is protected by copyright. All rights reserved.

Department of Defense [W81XWH-15-1-0480 (C.J.W. & B.B.)], Boston Biomedical
Innovation Center, the Blavatnik Biomedical Accelerator Fund, and the Boston Children’s
Hospital’s Technology Development Fund.
Author Contributions
I.T., S.J., N.A., M.K., B.D.L., B.P.B. and C.J.W. designed experiments; I.T., S.J., N.A., M.K.,
B.D., J.S., S.T., D.R., J.L., L.H., S.M.J. carried out experiments; I.T., S.J., N.A., M.K., B.D.,
J.S., L.H. and S.M.J. analyzed data; I.T., S.J., N.A., M.K., B.D., J.S., B.D.L., B.P.B. and C.J.W.
provided advice on the interpretation of data; I.T., B.D.L, B.P.B and C.J.W. wrote the
manuscript with input from all co-authors; B.D.L., B.P.B., and C.J.W. supervised the study.
All authors approved the final manuscript.
Conflict of Interest
B.D.L., B.P.B. and C.J.W. are cofounders of and equity holders in Nocion Therapeutics which
is developing charged sodium channel blockers as treatments for various disease indications,
including cough, and which has licensed BW-031 from Harvard Medical School. I.T., S.J.,
N.A., S.T. and D.R. also have founder shares in Nocion.
Declaration of Transparency and Scientific Rigour
This Declaration acknowledges that this paper adheres to the principles for transparent
reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design
and Analysis, and Animal Experimentation, and as recommended by funding agencies,
publishers and other organisations engaged with supporting research.
Data Availability
The data that support the findings of this study are available from the corresponding author
upon reasonable request.
This article is protected by copyright. All rights reserved.

Abstract
Background and Purpose: Many pain-triggering nociceptor neurons express TRPV1 or
TRPA1, cation-selective channels with large pores that enable permeation of QX-314, a
cationic analogue of lidocaine. Co-application of QX-314 with TRPV1 or TRPA1 activators
can silence nociceptors. In this study, we describe BW-031, a novel more potent cationic
sodium channel inhibitor, test whether its application alone can inhibit pain associated with
tissue inflammation, and whether this strategy can also inhibit cough.
Experimental Approach: We tested the ability of BW-031 to inhibit pain in three models of
tissue inflammation: inflammation in rat paws produced by Complete Freund’s Adjuvant or by
surgical incision and a mouse UV burn model. We tested the ability of BW-031 to inhibit cough
induced by inhalation of dilute citric acid in guinea pigs.
Key Results: BW-031 inhibited Nav1.7 and Nav1.1 channels with ~6-fold greater potency than
QX-314 when introduced inside cells. BW-031 inhibited inflammatory pain in all three models
tested, producing more effective and longer-lasting inhibition of pain than QX-314 in the
mouse UV burn model. BW-031 was effective in reducing cough counts by 78-90% when
applied intratracheally under isoflurane anesthesia or by aerosol inhalation in guinea pigs with
airway inflammation produced by ovalbumin sensitization.
Conclusion and Implications: BW-031 is a novel cationic sodium channel inhibitor that can be
applied locally as a single agent to inhibit inflammatory pain. BW-031 can also effectively
inhibit cough in a guinea pig model of citric acid induced cough, suggesting a new clinical
approach to treating cough. (246 words)
Abbreviations
TRPV1: Transient receptor potential cation channel subfamily V member 1
TRPA1: Transient Receptor Potential Cation Channel Subfamily A Member 1
This article is protected by copyright. All rights reserved.

Bullet points:
What is already known
 Entry of the cationic lidocaine derivative QX-314 through activated TRP channels can
silence nociceptive neurons.
 QX-314 co-applied with TRP channel activators can produce long-lasting inhibition of
pain and itch.
What this study adds
 A more potent cationic compound, BW-031, can inhibit inflammatory pain without co-
applied TRP activators.
 Inhaled BW-031 can also inhibit cough in guinea pigs.
Clinical significance
 Charged sodium channel blockers can be applied alone for long-lasting inhibition of
inflammatory pain.
 Charged sodium channel blockers offer a novel mechanism for inhibiting cough
associated with airway inflammation.
This article is protected by copyright. All rights reserved.

Introduction
Most clinically-used local anaesthetics are tertiary amine molecules that exist in an equilibrium
between cationic (protonated) and neutral (unprotonated) forms. The neutral molecule can gain
access to the key binding site in the channel pore (Ragsdale et al., 1996) by diffusing into the
lipid membrane surrounding the channel and then entering the channel protein through
“fenestrations” in the wall of the pore region (Payendah et al., 2011; Martin and Corry, 2014;
Gamal El-Din et al., 2018; Nguyen et al., 2019), corresponding to the “hydrophobic pathway”
proposed in a classic analysis of local anesthetic action (Hille, 1977). In a second “hydrophilic
pathway” proposed in this analysis, the neutral form of the drug can diffuse across the
membrane into the cytoplasm, where a fraction is re-protonated. From the cytoplasm, both
cationic and neutral drug molecules can enter the channel to reach the binding site in the pore,
but only when the channel is open. Consistent with this hypothesis, QX-314, a permanently
charged quaternary analogue of lidocaine, cannot inhibit neuronal sodium channels when
applied externally, but is effective when applied intracellularly (Frazier et al., 1970; Strichartz
, 1973).
Although QX-314 cannot readily diffuse across the cell membrane, it can enter some
neurons by permeating through certain cation-selective ion channels that have unusually large
pores, notably TRPV1 and TRPA1 channels (Binshtok et al., 2007; Puopolo et al., 2013;
Brenneis et al., 2014). Because these channels are selectively expressed in populations of
nociceptive (pain-sensing) primary sensory neurons, application of QX-314 together with
activators of TRPV1 or TRPA1 channels can produce selective inhibition of nociceptors with
minimal inhibition of motor neurons or non-nociceptive sensory neurons, in contrast to the
non-selective nerve block produced by lidocaine (Gerner et al., 2008; Binshtok et al., 2009;
Kim et al., 2010; Roberson et al., 2011; Brenneis et al., 2013; Zhou et al., 2014).
Pain associated with tissue inflammation is mediated in part by activation of TRPV1 and
TRPA1 channels in nociceptors (reviewed by Bautista et al., 2013; Julius, 2013), raising the
possibility that endogenous activation of these channels might be sufficient to catalyze entry
of QX-314 or other cationic sodium channel blockers in inflamed tissue, without requiring co-
application with exogenous TRP activators. An especially intriguing possible application of
the strategy is in cough, which is mediated by airway sensory neurons, including a population
of nociceptors expressing TRPV1 and TRPA1 channels (Bonvini et al., 2015; Canning et al.,
2014; Mazzone and Undem, 2016). Inhaled lidocaine is used to inhibit reflexive laryngospasm
and cough during bronchoscopy and is highly effective for acute suppression of cough in
This article is protected by copyright. All rights reserved.

patients with upper respiratory tract infections (Peleg and Binyamin, 2002), COPD (Chong et
al., 2005; Udezue, 2001), and asthma (Slaton et al., 2013; Udezue, 2001). However, lidocaine
has a short duration of action (Chong et al., 2005) and produces potential cardiac and CNS side
effects (Shirk et al., 2006) as a consequence of its high lipophilicity and ready diffusion into
the bloodstream. Also, because lidocaine blocks activity in motor neurons as well as sensory
neurons, it inhibits swallowing and the gag reflex (Noitasaeng et al., 2016), limiting its clinical
utility.
Here, we have designed and synthesized a novel cationic compound, BW-031, with
improved potency for inhibiting sodium channels compared to QX-314. We find that BW-031
when applied alone to inflamed tissue produces long-lasting inhibition of inflammatory pain in
several rat and mouse models and that inhaled BW-031 can effectively inhibit cough in a guinea
pig model of allergic airway inflammation. (571 words)
This article is protected by copyright. All rights reserved.

Methods
Chemicals. Except for BW-031, all chemicals were purchased from Sigma Aldrich or Tocris
Bioscience.
BW-031
(1-(1-(2,
6-dimethylphenylamino)-1-oxobutan-2-yl)-1-
ethylpiperidinium) was synthesized by Acesys Pharmatech according to the pathway described
in Supporting Information.
Stable cell line electrophysiology. Human embryonic kidney (HEK 293) cells stably
expressing the human Nav1.7 channel (Liu et al., 2012) were grown in Minimum Essential
Medium (MEM, ATCC) containing 10% fetal bovine serum (FBS, Sigma),
penicillin/streptomycin (Sigma), and 800 µg/ml G418 (Sigma) under 5% CO2 at 37°C. Human
embryonic kidney (HEK 293) cells stably expressing the human Nav1.1 channel (Kahlig et al.,
2010; gift of Dr. Alfred L. George, Jr.) were grown in Dulbecco’s Modified Eagle Medium
(DMEM, Thermo Fisher Scientific) containing 10% FBS (Sigma), penicillin/streptomycin
(Sigma), and 3 µg/ml Puromycin (Sigma) under 5% CO2 at 37°C. Chinese Hamster Ovary
(CHO-K1) cells stably expressing the human Nav1.8 channel and beta3 subunit (B’SYS GmbH,
Catalog CHO-NaV1.8/β3) were grown in Ham’s F-12 medium (Corning) containing 10% FBS,
penicillin/streptomycin (Sigma), and 3.5 µg/ml Puromycin (Sigma) and 350 µg/ml
Hygromycin (Sigma) under 5% CO2 at 37°C. For electrophysiological recordings, cells were
re-plated on coverslips for 1 to 6 h before recording. Whole-cell recordings were obtained using
patch pipettes with resistances of 2-2.5 MΩ when filled with the internal solution consisting of
(in mM): 61 CsF, 61 CsCl, 9 NaCl, 1.8 MgCl2, 9 EGTA, 14 creatine phosphate (tris salt), 4
MgATP, and 0.3 GTP (tris salt), 9 HEPES, pH adjusted to 7.2 with CsOH. The shank of the
electrode was wrapped with Parafilm in order to reduce capacitance and allow optimal series
resistance compensation without oscillation. Seals were obtained and the whole-cell
configuration established with cells in Tyrode’s solution consisting of (in mM): 155 NaCl, 3.5
KCl, 1.5 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, pH adjusted to 7.4 with NaOH. After
establishing whole-cell recording, cells were lifted off the bottom of the recording chamber
and placed in front of an array of quartz flow pipes (250 µm internal diameter, 350 µm external
diameter). Recordings were made using a base external solution of Tyrode’s solution with
added 10 mM TEA-Cl to inhibit small endogenous potassium currents. Solution changes were
made (in < 1 second) by moving the cell between adjacent pipes. Currents were recorded at
room temperature (21-23°C) with an Multiclamp 700B amplifier (Molecular Devices) and
filtered at 5 kHz with a low-pass Bessel filter The amplifier was tuned for partial compensation
of series resistance (typically 70-80% of a total series resistance of 4-10 MΩ), and tuning was
periodically re-adjusted during the experiment. Currents were digitized using a Digidata 1322A
This article is protected by copyright. All rights reserved.

data acquisition interface controlled by pCLAMP 9.2 software ([RRID:SCR_011323],
Molecular Devices).
Human iPSC-derived nociceptor neuron electrophysiology. Neurons were differentiated
from human induced pluripotent stem cells (IPSCs) following a previous protocol that yields
cells expressing peripherin and multiple markers of nociceptors, including expression of genes
such as SCN10A and P2XR3 (Chambers et al., 2012). Cells were cultured in 35 mm dishes
(Falcon), coated with 0.1 mg/ml poly-d-lysine (Sigma) and 10 µg/ml laminin, and grown in
DMEM/F12(1:1) media (Life Technologies) containing 10% HI FBS (Life Technologies) and
35 µg/ml Ascorbic acid (Sigma), 10 ng/ml BDNF, 10 ng/ml GDNF, 10 ng/ml NGF, 10 ng/ml
NT-3 (Life technologies) for 8 weeks. Whole-cell recordings were made using patch pipettes
with resistances of 1.5-2.5 MΩ when filled with the internal solution consisting of (in mM):
140 CsF, 10 NaCl, 1.1 EGTA, 10 HEPES, 20 D-glucose, pH adjusted to 7.2 with CsOH. The
external solution consisted of (in mM): 130 NaCl, 20 TEA-Cl, 5 KCl, 0.1 CdCl2, 2 CaCl2, 1
MgCl2, 10 D-glucose, 10 HEPES, pH adjusted to 7.4 with NaOH, which was perfused during
the recording using the ValveBank perfusion system (Automate Scientific). The size of neurons
was measured with an inverted microscope (Nikon Eclipse Ti), and neurons with a diameter of
less than 25 µm were used for the experiments. Currents were recorded using a Multiclamp
700B amplifier (Molecular Devices). Data were collected and digitized at 50 kHz using a
Digidata 1440 16-bit A/D converter controlled by pCLAMP 10.5 software (Molecular
Devices).
Electrophysiology data analysis. Data were analyzed using programs written in IGOR Pro
6.0 ([RRID:SCR_000325], Wavemetrics, Lake Oswego, OR), using DataAccess (Bruxton
Software) to read pCLAMP data files into Igor Pro. No correction was made in the reported
voltage protocols for the liquid junction potential between the internal solution and the
Tyrode’s solution in which the pipette was zeroed. Currents were corrected for linear
capacitive and leak currents, which were determined using 5 mV hyperpolarizations delivered
from the resting potential and then appropriately scaled and subtracted. Statistical analyses
were performed using IGOR Pro.
Mouse dorsal root ganglion (DRG) neuron culture and electrophysiology. DRG neurons
were cultured as previously described (Costigan et al., 1998). Animal use for culturing neurons
complied with the ARRIVE guidelines (Percie du Sert et al., 2020) and with the
recommendations made by the British Journal of Pharmacology (Lilley et al., 2020), using the
minimum number of animals to obtain the data. All procedures were approved by the
Institutional Animal Care and Use Committee (IACUC), Boston Children’s Hospital.
This article is protected by copyright. All rights reserved.

Adult male C57Bl/6J mice (8-12 weeks old, Jackson Laboratories stock #000664
[RRID:IMSR_JAX:000664]) were euthanized by a slowly rising concentration of CO2 and
exsanguinated. DRGs were dissected from into Hank's balanced salt solution (HBSS) (Life
Technologies). DRGs were dissociated in 1 μg/ml collagenase A plus 2.4 U/ml dispase II
(enzymes, Roche Applied Sciences) in HEPES-buffered saline (HBSS, Sigma) for 90 min at
37 °C and then triturated down to single-cell level using glass Pasteur pipettes of decreasing
size. DRGs were then centrifuged over a 10% BSA gradient and plated on laminin-coated cell
culture dishes (Sigma). DRGs were cultured overnight in B27-supplemented neurobasal-A
medium plus penicillin/streptomycin (Life Technologies). On the day following plating, DRG
culture dishes were treated with either HBSS, 100 µM BW-031, 1 µM capsaicin or 100 µM
BW-031+1 µM capsaicin in HBSS for 30 min, followed by a 5-minute perfusion of external
solution to remove extracellular compounds.
Whole-cell current-clamp and voltage-clamp recordings were performed <24 hours after
DRG culture using an Axopatch 200A amplifier (Molecular Devices) at 25°C. Data were
sampled at 20 kHz and digitized with a Digidata 1440A A/D interface and recorded using
pCLAMP 10 software (Molecular Devices). Data were low-pass filtered at 2 kHz. Patch
pipettes were pulled from borosilicate glass capillaries on a Sutter Instruments P-97 puller and
had resistances of 1.5–3 MΩ. Series resistance was 3–10 MΩ and compensated by at least 80%
and leak currents were subtracted. Cells were classified as TRPV1+ or TRPV1- by the presence
or absence of a response to perfused 1 μM capsaicin measured in voltage clamp mode at a
holding potential of −80 mV. Cells were then held at -100mV and depolarized to -10mV with
a 100 ms step to activate Nav channels. The external solution for DRG electrophysiological
recordings consisted of (in mM): 30 NaCl, 90 Choline-Cl, 20 TEA-Cl, 3 KCl, 1 CaCl2, 1
MgCl2, 0.1 CdCl2, 10 HEPES, 10 Dextrose; pH 7.4, 320 mOsm. The internal pipette solution
consisted of (in mM): 140 CsF, 10 NaCl, 1 EGTA, 10 HEPES, 20 Dextrose; pH 7.3. No
correction was made in the reported voltage protocols for the liquid junction potential between
this solution and the external solution.
Animals for pain studies.
Animal use for pain studies comply with the ARRIVE guidelines (Percie du Sert et al., 2020)
and with the recommendations made by the British Journal of Pharmacology (Lilley et al.,
2020). Every effort was made to minimize animal suffering and the number of animals used.
Experiments were designed to use the minimum number of animals to provide sufficient
statistical power based on our previous experience with the behavioral assays, where variability
within a group of wild type mice is approximately 10 and 20% of the mean.
This article is protected by copyright. All rights reserved.

Male CD rats (7-8 weeks old) were purchased from Charles River and male C57BL/6J mice
(8-12 weeks old) were purchased from Jackson Laboratories (stock #000664
[RRID:IMSR_JAX:000664]) and housed for 1 week prior to experiments. Rats were housed 3
animals per cage and mice were housed 5 animals per cage in separate rooms with constant
temperature (23°C) and humidity (45-55%) with food and water available ad libitum. At the
end of the experiments, animals were euthanized by inhalation of a slowly rising concentration
of CO2 (30-70% over 5 minutes). All procedures were approved by the Institutional Animal
Care and Use Committee (IACUC), Boston Children’s Hospital.
Mice and rats were used for studies of pain because it enabled us to compare our results
with a large body of work studying pain in rodent models and because there is no suitable in
vitro, cellular, or computational model that can recapitulate the complexity of the neural
substrates that result in the output of pain-related behavior and which could be used to assess
in vivo biological function relevant to humans.
Male rats and mice were used for pain studies following previous practice in the lab,
reflecting the widespread belief in the field that female subjects would introduce higher
variability due to cycling gonadal hormones. It is now recognized that this assumption is likely
incorrect and has introduced a deleterious bias in the scientific literature (Mogil, 2020) and that
future studies should use equal numbers of male and female animals.
Plantar incision surgery. Rats were placed in a chamber with 5% isoflurane and monitored
until they were visibly unconscious. Once unconscious, rats were removed from the chamber
and anesthesia was maintained using 2% isoflurane delivered via nose cone. A toe pinch was
used to confirm that animals were fully anesthetized. The animals were then secured with
surgical tape at their toes and upper leg for paw stability during surgery. The plantar surface of
one hind paw was sterilized with 3 alternating wipes of betadine and ethanol. A 1.5 cm
longitudinal incision was made using a scalpel along the center of the plantar surface, beginning
1 cm from the heel and extending towards the foot pad and toes. Incision was made to the
minimal depth necessary to cut through skin and fascia to expose the underlying plantaris
muscle, approximately 1-2 mm. Once exposed the plantaris muscle was elevated for 10 seconds
with surgical forceps and gently lifted for 10 seconds. After irritation of the plantaris muscle,
the wound was closed with three sutures. After surgery animals were returned to their cage and
monitored until they fully recovered from anesthesia. Treatments were administered
subcutaneously 24 hours after injury.
Intraplantar injection of Complete Freund’s Adjuvant. Complete Freund’s Adjuvant
(CFA) was purchased from Sigma Aldrich (Cat. No. F5881). Rats were restrained and
This article is protected by copyright. All rights reserved.

subcutaneously injected in the plantar region of the left paw with 50 µL of CFA (1 mg/ml).
Animals receiving test compounds (2% QX-314 or 2% BW-031) were injected with these
compounds dissolved in 50 µL of CFA.
Plantar ultraviolet (UV) burn. Mice were anesthetized with 3% isoflurane. UV irradiation
was performed on the plantar surface of the left hind paw under maintenance anesthesia with
2% isoflurane at an intensity of 0.5 J/cm2 for 1 minute at a wavelength of 305–315 nm using a
fluorescent UV-B light source (XR UV LEDs 308nm, RayVio, Hayward, California, USA).
Peri-sciatic injection. Mice were anesthetized with 2.5% isoflurane. Upon achieving sufficient
depth of anesthesia, mice were placed in the prone position, the fur on their left hindleg was
shaved and the area was cleaned with betadine and 70% isopropanol. A 1-centimeter incision
was made in the skin on the upper thigh. The sciatic nerve was identified in the intermuscular
interval between the biceps femoris and gluteal muscle without the dissection of the superficial
fascia layers. Then, a mixture of physiological saline with 0.5% lidocaine, 0.5% QX-314 or
0.5% BW-031 (70 μL) was injected into the perineural space below the fascia using an insulin
syringe with a 30-gauge needle. The surgical wounds were closed with stainless steel wound
clips (EZ Clips, Stoelting Co.).
Behavioral measurements of sensory and motor function
Von Frey assay of mechanical sensitivity. An electronic Von Frey device (Bioseb, Model:
BIO-EVF4) was used to assess mechanical sensitivity in rats before and after paw incision
injury. Animals were habituated for 1 hour, one day prior to baseline testing. Animals were
given 30 minutes to settle before testing. An average mechanical threshold was calculated using
5 measurements taken 5 minutes apart for each animal. For baseline measurements two testing
sessions were performed on separate days prior to injury and averaged together. 50 µL of BW-
031 or saline were administered into the plantar region of the hind paw adjacent to the incision
24 hours post injury. Animals were then tested 1, 3, 5, and 24 hours post treatment. Additional
timepoints were added at 7 and 9 hours for higher concentrations of treatments.
A manual Von Frey assay was used to assess mechanical sensitivity in mice before and
after UV burn, as previously described (Lee et al., 2019). After mice were habituated to the
testing cage (7.5 × 7.5 × 15 cm) with a metal grid floor for 45 min for 2 days, baseline values
were measured using nine von Frey filaments with different bending forces (0.04, 0.07, 0.16,
0.4, 0.6, 1, 1.4, 2, and 4 g). The response patterns were collected and converted into
corresponding 50% withdrawal thresholds using the Up-Down Reader software and associated
protocol (Gonzalez-Cano et al., 2018). Based on the baseline measurement, mice were assigned
to three groups so that the baseline mechanical sensitivity among the groups was similar. Each
This article is protected by copyright. All rights reserved.

group consisted of 10 mice, based on previous experiments showing sufficient power to detect
significance with 95% confidence. Twenty-four hours after UV irradiation, mice received a 10-
μL bolus intraplantar injection of either 2% BW-031, 2% QX-314, or vehicle (normal saline)
to the irradiated paw. The von Frey test was performed at 1, 3, 5, 7, and 24 hours after the drug
injection.
Radiant heat assay of thermal sensitivity. Thermal hypersensitivity was measured using the
plantar radiant heat test (Hargreaves et al., 1988) (Ugo Basile, Model code: 37370) in CFA
injected rats. Rats were habituated to testing enclosures for 1 hour one day prior to baseline
testing. Rats were given 30 minutes to settle before testing. An average paw withdrawal latency
was calculated using 3 measurements taken 5 minutes apart. Animals were tested 1, 4, and 24
hours after injury from CFA injection.
Toe spread assay of motor function. Mouse toe movement was evaluated in the ipsilateral
hind-paws as previously described (Ma et al., 2011) in order to assess the presence of motor
block after peri-sciatic injection of lidocaine or charged sodium channel blockers. Briefly, 5
minutes after peri-sciatic injection, mice were lifted by the tail, uncovering the hind paws for
clear observation. Under this condition, the digits spread, maximizing the space between them
(the toe spreading reflex). This reflex was scored as previously described: 0, no spreading; 1,
intermediate spreading with all toes; and 2, full spreading. Full toe spreading was defined as a
complete, wide, and sustained (at least 2 seconds) spreading of the toes. Full toe spreading was
observed in the contralateral paws for all mice tested.
Pinprick assay of sensory function. Mouse responses to pinprick were measured as
previously described (Ma et al., 2011), with modifications. Mice were placed in wire mesh
cages and habituated for 3 sessions prior to peri-sciatic injection. After peri-sciatic injection
and measurement of motor function, mice were immediately placed in wire mesh cages and an
Austerlitz insect pin (size 000) (FST, USA) was gently applied to the plantar surface of the
paw without moving the paw or penetrating the skin. The pinprick was applied three times to
the sole of the ipsilateral hind paw and three times to the sole of the contralateral hind paw. A
response was considered positive (1) when the animal briskly removed its paw. If none of the
applications elicited a positive response, the overall grade was 0.
Blinding. All behavioral measurements of sensory and motor function were performed by
investigators blinded to the drug treatment; the test order was randomized with multiple groups
being represented in each cage.
Guinea Pig Cough Experiments
This article is protected by copyright. All rights reserved.

Animals for guinea pig cough experiments. Animal use for cough studies comply with the
ARRIVE guidelines (Percie du Sert et al., 2020) and with the recommendations made by the
British Journal of Pharmacology (Lilley et al., 2020). Every effort was made to minimize
animal suffering and the number of animals used and studies were conducted in alignment with
applicable animal welfare regulations in an AAALAC-accredited facility. The cough studies
used 5-10 week-old Dunkin Hartley guinea pigs. The initial study using intratracheal
application of BW-031 under isoflurane anesthesia used female animals (range of weights of
374-505 g on the day of dosing and cough challenge), with 9 animals per each of the 4
experimental groups. The choice of female guinea pigs was based on a previous belief in the
laboratory that female guinea pigs are more susceptible to cough induction by citric acid,
although research appearing since we did this study has now shown this belief to be false
(Sterusky et al. Physiol Res 69:S171-179, 2020). The minimum number of animals was used
in order to provide sufficient statistical power based on previous experiments on citric-acid
induced cough in the laboratory. The subsequent study using ovalbumin sensitization to induce
lung inflammation used 6 male (416-580 g) and 6 female (456-557 g) animals for each
experimental group. There was no obvious difference between the initial cough numbers in
male and female animals or in the inhibition of coughing by BW-031. The number of animals
per group was increased in the ovalbumin sensitization experiments because of the expectation
that variable levels of sensitization might increase variability in the effectiveness with which
BW-031 could enter nerve terminals. Because preliminary studies showed that some guinea
pigs failed to cough in response to the citric acid challenge, each study began with 20% more
animals than were planned for the protocols and animals were first pre-screened by inhalation
of citric acid (400 mM for 7 minutes, with coughs counted during the 7 minute application and
for 10 minutes afterward) and the lowest responders were omitted from the remaining study
protocol. For the intratracheal protocol, animals with 0-1 coughs were omitted; for the
ovalbumin sensitization protocol, the 6 animals of each sex with lowest cough counts (0-3
coughs) were omitted. Pre-screening was performed a minimum of 7 days before the start of
the study protocol to allow animals to recover from any sensitization produced by the citric
acid exposure during the pre-screening. After pre-screening, the remaining animals were
allocated into each group so that each group had approximately equal group mean cough counts
measured in the pre-screening protocol.
Intratracheal drug administration and citric acid challenge. Animals were dosed via the
intratracheal route at a dose volume of 0.5 mL/kg of BW-031 dissolved in saline based on
individual bodyweights. Animals were anaesthetized (3-5% isoflurane/oxygen mix) and
This article is protected by copyright. All rights reserved.

secured to the intubation device by a cord around their upper incisor teeth. A rodent fiber optic
laryngoscope was inserted into the animal’s mouth to illuminate the posterior pharynx and
epiglottis. The tongue was released and the needle of the dosing device (Penn Century
intratracheal aerosol Microsprayer) guided through the vocal cords into the lumen of the
trachea. Following dosing, the animals were removed from the secured position and carefully
monitored until full recovery. Approximately 1 hour after intratracheal treatment, animals were
placed into whole body plethysmographs connected to a Buxco Finepointe System and exposed
to nebulized 400 mM citric acid for 7 minutes. Cough counts were recorded during the 7 minute
exposure period and for 10 minutes after nebulization period by the Buxco Finepointe system.
Cough counts were recorded in an automated fashion by the Finepoint software, to remove any
operator bias, with no involvement of the technician other than introducing the animal to the
recording chamber. Recovery from the isoflurane anesthesia was complete within an hour
based on the overt behavior of the animals, consistent with rapid recovery seen using
measurements of physiological parameters (Schmitz et al., 2016).
Ovalbumin sensitization/challenge. On Day 0 all animals were sensitized with intraperitoneal
and subcutaneous injections of chicken egg albumin (ovalbumin). Animals were administered
1 mL of a 50 mg/mL Ovalbumin (Ova) in 0.9% w/v saline solution via the intraperitoneal route
and 0.5 mL of the same solution into 2 separate subcutaneous sites (1 mL in total divided
between the left and right flank). All animals were administered a single intraperitoneal dose
of pyrilamine (15 mg/kg) at a dose volume of 1 mL/kg approximately 30 min prior to
ovalbumin challenge on Day 14 to inhibit histamine-induced bronchospasm (Featherstone et
al., 1988; Hara et al., 2005). On Day 14 animals were challenged with aerosolised ovalbumin
in 0.9% w/v saline (3 mg/mL) or 0.9% w/v saline for 15 min. Animals were placed in groups
in an acrylic box. 8 mL of ovalbumin in saline was placed in each of two jet nebulisers
(Sidestream®). Compressed air at approximately 6 L/min was passed through each nebuliser
and the output of the nebulisers passed into the box containing the animals.
Drug administration. On Day 15 approximately 24 hours after the inflammatory challenge
with ovalbumin, animals were placed in a whole-body plethysmograph (Buxco Finepointe) and
dosed with vehicle or BW-031 by inhalation using an Aeroneb nebulizer (Aerogen) over 60
minutes. Upon the completion of dosing animals were returned to their home cage for
approximately one hour before the cough challenge.
The inhaled dose of BW-031 was calculated according to the algorithm (Alexander et al.,
2008): Inhaled dose (mg/kg) = [C (mg/L) x RMV (L/min) x D (min)]/BW (kg), where C is
the concentration of drug in air inhaled, RMV is respiratory minute volume, D is the of
This article is protected by copyright. All rights reserved.

exposure in minutes, BW is bodyweight in kg, with RMV (in L/min) calculated as 0.608 x
BW (kg)0.852
.
Cough/respiratory function measurement. One hour following the end of vehicle or drug
administration on Day 15, the animals were placed into a whole-body plethysmograph
connected to a Buxco Finepointe system. Animals were then exposed to nebulized 400 mM
citric acid for 7 minutes. Cough counts were recorded throughout the 7 minute exposure period
and for 10 minutes following the end of nebulization period. Animals were euthanized within
approximately 60 min following the end of the cough challenge recording period by an
overdose of pentobarbitone administered by the intraperitoneal route.
Tissue sampling. Upon euthanasia, 2 mL of blood were sampled from the descending vena
cava from each animal. The blood was allowed to stand at room temperature for a minimum of
60 minutes but less than 120 minutes to allow the clotting process to take place. Samples were
then centrifuged at 2000 g for 10 minutes at 25°C and the resulting serum was frozen at -80C
for subsequent analysis of BW-031 concentrations. Also following termination an incision was
made in the neck and the muscle layers were separated by blunt dissection and the trachea
isolated. A small incision was made in the trachea and a tracheal cannula inserted. The cannula
was secured in place with a piece of thread. The lungs were then removed and the left lung lobe
tied off and removed. The right lung was lavaged with 3 mL of phosphate buffered saline (PBS)
at room temperature. The PBS was left in the airway for 10 seconds whilst the organ was gently
massaged before being removed, this was repeated twice further. In total, three lots of 3 mL of
PBS was used to lavage the right lung.
BAL immune cell quantification. A total and differential cell count of the BAL was
performed using the XT-2000iV (Sysmex UK Ltd). The sample was vortexed for
approximately 5 seconds and analyzed. A total and differential cell count (including
eosinophils, neutrophils, lymphocytes and mononuclear cells (includes monocytes and
macrophages)) was reported as number of cells per right lung per animal.
Liquid Chromatography/Mass Spectrometry (LC/MS). Serum samples were kept at -80°C
until being assayed, at which time they were thawed at room temperature. Each serum sample
was added to 100 µL of 80:20 (acetonitrile:water) solution and the mixture placed in a 1.5 mL
Eppendorf Safe-Lock tubes prefilled with zirconium oxide beads (Next Advance Inc., Troy,
NY). After vortexing for 30 seconds and sonicating for 10 minutes, the homogenate was
centrifuged at 10,000 rpm for 10 minutes. The supernatant was then separated into a new
Eppendorf tube to be stored at -80°C until the time for analysis. To 20 µL of liquid sample, 10
This article is protected by copyright. All rights reserved.

µL of internal standard (bupivacaine 10 ng/mL in acetonitrile:water (50:50)) were added, plus
170 µL of methanol chilled at 4°C. After vortexing for 30 seconds, the mixture was centrifuged
at 10,000 rpm for 10 minutes. The supernatant solution was then transferred to a clean
Eppendorf tube and evaporated to dryness under vacuum at 50 oC for 40 minutes. The residue
was reconstituted with 100 µL of the starting mobile phase, i.e. aqueous 0.1% formic
acid:methanol (90:10), and vortexed for 30 seconds. This solution was transferred to amber
screw neck vials and setup in the refrigerated autosampler tray of the chromatograph for
injection. This whole procedure was also applied to spiked calibrators and quality control QC
samples used in the quantification and validation methods. At least three injections were carried
out from each vial. Bupivacaine, used as internal standard, methanol LC/MS grade, and formic
acid LC/MS grade were purchased from Sigma-Aldrich St. Louis, MO, USA). Pure MilliQ
water at 18 MΩ-cm was obtained by reserve osmosis with a Direct-Q3 UV water purifier
(Millipore SAS, France).
The quantification of BW-031 in serum fluid samples was carried out with an Acquity H
Class UPLC chromatographer with a XEVO TQ MS triple quadrupole mass spectrometer
detector (Water Corp., Milford, MA, USA). The assay used an Acquity UPLC BEH C18 1.7
µm 2.1x100 mm column with a VanGuard 2.1X 5 mm guard column, both kept at constant
35oC. The mobile phase was ran on a gradient of A: aqueous 0.1% formic acid and B: methanol
starting at time zero with a A:B proportion of 90:10, at 3 min 10:90, and at 4.2 min 90:10 until
the end of the run at 6 minutes. The flow rate was set at 0.3 mL/min, with an injection volume
of 3 µL, and a post-run organic wash of 5 seconds. Multiple reaction monitoring (MRM) was
used for the mass spectrometry acquisition in positive electrospray ionization (ESI) mode. The
mass transitions monitored were m/z 263.22→86.02 and m/z 289.09→140.2, for BW-031 and
bupivacaine respectively. The cone voltages were 36V and 30V, and the collision energies
24eV and 12eV, also respectively. The whole LC/MS system was controlled by the MassLynx
v.4.2 software ([RRID:SCR_014271], Water Corp., Milford, MA, USA), including the
TargetLynx Application Manager for data processing and analytes quantification. Good
linearity and reproducibility were achieved in the range of 1–100 ng/mL, and the precision and
accuracy of the method were 2.74% and 98.6%, respectively. The lower limit of quantification
was 1.8 ng/mL.
Cardiotoxicity. Frozen human IPSC-derived cardiomyocytes (Cor.4U cardiomyocytes,
[RRID:CVCL_Y550]) were purchased from Axiogenesis AG (currently NCardia AG). Cor.4U
cells were thawed and plated at a density of 10000 cells/well into 384-well plates that were
This article is protected by copyright. All rights reserved.

pre-coated with 10 µg/mL bovine fibronectin in sterile phosphate buffered saline, pH 7.4.
Cor.4U Culture Medium (Axiogenesis) was used to maintain the cells in culture for 7 days and
was changed daily. Cells exhibited synchronous beating on day 3 after plating. On day 7 after
plating, the medium was changed to BMCC medium (Axiogenesis). The EarlyTox
Cardiotoxicity Kit (Molecular Devices) was used to measure calcium flux as a proxy for
cardiomyocyte beating activity112. Cor.4U cells were incubated with the EarlyTox calcium
sensitive dye in BMCC media for 1 hour, and then the plates were transferred to the
FDSS700EX plate reader (Hamamatsu Photonics). The baseline calcium flux was measured
for 5 minutes and then charged local anesthetics dissolved in BMCC media or media alone
were added to the wells using a robot. 10 minutes after compound treatment, the calcium flux
was measured again. All measurements were performed at 37°C under 95% CO2/5% O2.
Calcium flux parameters were measured using the Hamamatsu Analysis Software.
Data and statistical analysis
The data and statistical analysis comply with the recommendations of the British Journal of
Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018).
Statistical analysis was carried out only for data where sample size was at least 5 for each
group. All experimental data points including outliers were included in the scatter plots and
data analysis. The analysis was carried out using GraphPad Prism 8 software
([RRID:SCR_002798], GraphPad Software Inc., San Diego, CA, USA). All data are expressed
as the mean ± standard deviation (SD). Statistical comparisons were made using a paired two-
tailed Student's t test for two groups or ANOVA for multiple groups, using the Kolmogorov–
Smirnov test to evaluate normality and Bartlett and Brown–Forsythe tests for homogeneity of
variance. Post hoc tests were conducted only if the F in ANOVA achieved P< 0.05, and there
was no significant variance inhomogeneity. In most cases in which there was variance
inhomogeneity (data in Figures 3A, 6B, 7B), we achieved homogeneity of variance by
transforming raw data using logarithmic transformation prior to using ANOVA. The resulting
Bartlett’s tests were non-significant indicating success of the transformation method. In a case
(Figure 8A) in which several transforms could not be found to normalize the variance (as shown
by significant Bartlett’s tests after each transform method), we employed non-parametric
Kruskall Wallis ranks analysis. For experiments with repeated measurements (Figures 4 and
5), the Greenhouse-Geisser test was used to test for deviations from sphericity in the repeated-
measurement experiments and in cases where a significant deviation occurred (Greenhouse-
Geisser epsilon<0.99), the Greenhouse-Geisser correction was applied. Details of the statistical
This article is protected by copyright. All rights reserved.

tests used for the data in each figure are given in Supporting Information. There is only one
threshold for statistical significance (P < 0.05) in all analysis.
Nomenclature of Targets and Ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in
http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS
Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the
Concise Guide to PHARMACOLOGY 2019/20 (Alexander et al., 2019).
This article is protected by copyright. All rights reserved.

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
particularly high potency for sodium channel inhibition (Bräu et al., 1998; Scholz et al., 1998).
Based on this, 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. The currents remaining after
partial inhibition by 30 µM BW-031 applied intracellularly had unchanged voltage-dependence
of activation and a voltage-dependence of availability (assayed using 50-ms conditioning
pulses) that was slightly shifted (~11 mV) in the hyperpolarizing direction (Supplementary
Figure 1); a possible interpretation is that channels with BW-031 trapped inside are completely
unable to pass current, so that the activation curve represents channels with no drug, but that
the fraction of channels containing BW-031 can change during the 50-ms conditioning pulses,
as if strong hyperpolarizations can to some extent “squeeze” BW-031 out of BW-031-
containing channels. 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). Intracellular
BW-031 inhibited Nav1.1 channels with a similar potency to Nav1.7 channels (Figures 2a,b).
Intracellular BW-031 also inhibited heterologously-expressed Nav1.8 channels (Figures 2c,d)
but with greatly reduced potency compared to Nav1.7 and Nav1.1 channels, with intracellular
300 µM BW-031 producing 30±18% inhibition (mean±SD, n=5), similar to the effect of 3 µM
BW-031 on Nav1.7 channels (37±9% 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;
This article is protected by copyright. All rights reserved.

Scholz and Vogel, 2000; Leffler et al, 2010). Previous experiments with native Nav1.8
channels in rat DRG neurons showed ~30% use-dependent inhibition by internal 200 µM QX-
314 with 0.75 Hz stimulation (Leffler et al., 2005) which by comparison with effects of 100
µM QX-314 on Nav1.7 channels (Figure 1b,c) raises the possibility that Nav1.8 channels may
be less sensitive than Nav1.7 channels to inhibition by intracellular charged blockers in general,
but more work will be required to examine this in detail.
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, 2013) 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.
This article is protected by copyright. All rights reserved.

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.,
2018; 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., 2019). 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. Animals dosed with 7.53 mg/kg BW-031 had cough counts of 0.9±1.3
(n=9) in a 17-minute period, compared to 9.4±7.3 in control animals (n=9), with complete
suppression of coughing in 5 of the 9 animals dosed with 7.53 mg/kg BW-031.
This article is protected by copyright. All rights reserved.

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±5.5 in
control (n=12) to 2.2±3.1 with BW-031 (n=12), with complete suppression of cough in 7 of the
12 animals treated with BW-031.
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±160 nM (n=12) (Figure 8a), far lower than the average serum level of
~15 M (3.6 g/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,c).
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).
Discussion
These results show that the novel 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-
This article is protected by copyright. All rights reserved.

lasting inhibition of hypersensitivity in a mouse UV-burn model. The overall concordance of
potency between the in vitro studies with cloned Nav1.7 channels and in vivo pain models
suggests that future screening of novel compounds in this class can start with in vitro studies
on cell lines, reducing the need for animal studies in the initial phases of drug discovery.
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 potential clinical
applications of the strategy to inhibit inflammatory pain. An unfortunate limitation of our
studies on pain is that we used only male rats and mice, reflecting a previously widespread
belief in the field that female subjects might introduce higher variability due to cycling gonadal
hormones (a belief now known to be misguided and likely deleterious to clinical translation of
rodent research (Mogil, 2020)), so that we cannot be sure our results will generalize to females.
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., 2018; 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
This article is protected by copyright. All rights reserved.

P2X3-containing channels, as well as TRPV1 and TRPA1 channels, might provide a pathway
for entry of BW-031 into cough-mediating neurons. However further work will be needed to
test directly whether BW-031 can pass through TRPA1 channels, as previously shown for QX-
314 by both direct measurements of permeation (Brenneis et al., 2014) and functional effects
when co-applied with TRPA1 activators (Lennertz et al., 2012; Stueber et al., 2016; Yamaki et
al., 2021), or through P2X3 channels.
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., 2009; Gerner et al., 2008; Roberson et al., 2011)
and as we find here for BW-031 inhibition of inflammatory pain (Figures 4,5).
Overall, the ability of BW-031 to inhibit cough adds to previous evidence that targeting
peripheral nerve 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. In mice,
silencing nociceptors by QX-314 can reduce lung inflammation induced by both ovalbumin
and house dust mite sensitization (Talbot et al., 2015), consistent with a significant role of
nociceptors in exacerbating inflammation by release of pro-inflammatory neuropeptides (Chiu
et al., 2012; Talbot et al., 2015; Crosson et al., 2021), raising the possibility that BW-031 and
other more potent charged sodium channel blockers have the potential to be disease-modifying
in human allergic airway inflammation.
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
This article is protected by copyright. All rights reserved.

stimulation, which may activate different populations of nerve fibers than citric acid (Chou et
al., 2018; Morice et al., 2007).
This article is protected by copyright. All rights reserved.

References
Acosta, M.C., Luna, C., Quirce, S., Belmonte, C., and Gallar, J. (2014). Corneal Sensory Nerve
Activity in an Experimental Model of UV Keratitis. Investigative Ophthalmology & Visual
Science 55, 3403-3412.
Adner, M., Canning, B.J., Meurs, H., Ford, W., Ramos Ramírez, P., van den Berg, M.P.M.,
Birrell, M.A., Stoffels, E., Lundblad, L.K.A., Nilsson, G.P., et al. (2020). Back to the
future: re-establishing guinea pig in vivo asthma models. Clinical science (London,
England : 1979) 134, 1219-1242.
Alexander, D.J., Collins, C.J., Coombs, D.W., Gilkison, I.S., Hardy, C.J., Healey, G.,
Karantabias, G., Johnson, N., Karlsson, A., Kilgour, J.D., et al. (2008). Association of
Inhalation Toxicologists (AIT) working party recommendation for standard delivered dose
calculation and expression in non-clinical aerosol inhalation toxicology studies with
pharmaceuticals. Inhalation toxicology 20, 1179-1189.
Alexander, S.P.H., Mathie, A., Peters, J.A., Veale, E.L., Striessnig, J., Kelly, E., Armstrong,
J.F., Faccenda, E., Harding, S.D., Pawson, A.J., Sharman, J.L., Southan, C., Davies, J.A.,
CGTP Collaborators. (2019) THE CONCISE GUIDE TO PHARMACOLOGY 2019/20:
Ion channels. Br J Pharmacol. 176:S142-S228.
Asgar, J., Zhang, Y., Saloman, J.L., Wang, S., Chung, M.K., and Ro, J.Y. (2015). The role of
TRPA1 in muscle pain and mechanical hypersensitivity under inflammatory conditions in
rats. Neuroscience 310, 206-215.
Barabas, M.E., and Stucky, C.L. (2013). TRPV1, but not TRPA1, in primary sensory neurons
contributes to cutaneous incision-mediated hypersensitivity. Mol Pain 9: 9.
Bautista, D.M., Pellegrino, M., and Tsunozaki, M. (2013). TRPA1: A gatekeeper for
inflammation. Annu Rev Physiol 75: 181–200.
Belvisi, M.G., Birrell, M.A., Wortley, M.A., Maher, S.A., Satia, I., Badri, H., Holt, K., Round,
P., McGarvey, L., Ford, J., et al. (2017). XEN-D0501, a Novel Transient Receptor Potential
Vanilloid 1 Antagonist, Does Not Reduce Cough in Patients with Refractory Cough.
American journal of respiratory and critical care medicine 196, 1255-1263.
Belvisi, M.G., Dubuis, E., and Birrell, M.A. (2011). Transient receptor potential A1 channels:
insights into cough and airway inflammatory disease. Chest 140, 1040-1047.
Bessac, B.F., and Jordt, S.-E. (2008). Breathtaking TRP channels: TRPA1 and TRPV1 in
airway chemosensation and reflex control. Physiology (Bethesda) 23, 360-370.
Binshtok, A.M., Bean, B.P., and Woolf, C.J. (2007). Inhibition of nociceptors by TRPV1-
mediated entry of impermeant sodium channel blockers. Nature 449, 607-610.
Binshtok, Alexander M., Gerner, P., Oh, Seog B., Puopolo, M., Suzuki, S., Roberson, David
P., Herbert, T., Wang, C.-F., Kim, D., Chung, G., et al. (2009a). Coapplication of Lidocaine
and the Permanently Charged Sodium Channel Blocker QX-314 Produces a Long-lasting
Nociceptive Blockade in Rodents. Anesthesiology 111, 127-137.
Birrell, M.A., Belvisi, M.G., Grace, M., Sadofsky, L., Faruqi, S., Hele, D.J., Maher, S.A.,
Freund-Michel, V., and Morice, A.H. (2009). TRPA1 agonists evoke coughing in guinea
pig and human volunteers. American journal of respiratory and critical care medicine 180,
1042-1047.
Bonvini, S.J., Birrell, M.A., Smith, J.A., and Belvisi, M.G. (2015). Targeting TRP channels for
chronic cough: from bench to bedside. Naunyn-Schmiedeberg's archives of pharmacology
388, 401-420.
Bräu, M.E., Vogel, W., and Hempelmann, G. (1998). Fundamental properties of local
anesthetics: half-maximal blocking concentrations for tonic block of Na+ and K+ channels
in peripheral nerve. Anesthesia and analgesia 87, 885-889.
Brennan, T.J., Vandermeulen, E.P., and Gebhart, G.F. (1996). Characterization of a rat model
of incisional pain. Pain 64, 493-501.
This article is protected by copyright. All rights reserved.

Brenneis, C., Kistner, K., Puopolo, M., Jo, S., Roberson, D., Sisignano, M., Segal, D., Cobos,
E.J., Wainger, B.J., Labocha, S., et al. (2014). Bupivacaine-induced cellular entry of QX-
314 and its contribution to differential nerve block. British journal of pharmacology 171,
438-451.
Brenneis, C., Kistner, K., Puopolo, M., Segal, D., Roberson, D., Sisignano, M., Labocha, S.,
Ferreiros, N., Strominger, A., Cobos, E.J., et al. (2013). Phenotyping the function of
TRPV1-expressing sensory neurons by targeted axonal silencing. The Journal of
neuroscience : the official journal of the Society for Neuroscience 33, 315-326.
Brozmanova, M., Mazurova, L., Ru, F., Tatar, M., and Kollarik, M. (2012). Comparison of
TRPA1-versus TRPV1-mediated cough in guinea pigs. European journal of pharmacology
689, 211-218.
Brozmanova, M., Svajdova, S., Pavelkova, N., Muroi, Y., Undem, B.J., and Kollarik, M.
(2019). The voltage-gated sodium channel Na(V)1.8 blocker A-803467 inhibits cough in
the guinea pig. Respiratory physiology & neurobiology 270, 103267.
Canning, B.J. (2006). Anatomy and neurophysiology of the cough reflex: ACCP evidence-
based clinical practice guidelines. Chest 129, 33s-47s.
Canning, B.J., Chang, A.B., Bolser, D.C., Smith, J.A., Mazzone, S.B., and McGarvey, L.
(2014). Anatomy and neurophysiology of cough: CHEST Guideline and Expert Panel
report. Chest 146, 1633-1648.
Canning, B.J., Mazzone, S.B., Meeker, S.N., Mori, N., Reynolds, S.M., and Undem, B.J.
(2004). Identification of the tracheal and laryngeal afferent neurones mediating cough in
anaesthetized guinea-pigs. The Journal of physiology 557, 543-558.
Chambers, S.M., Qi, Y., Mica, Y., Lee, G., Zhang, X.J., Niu, L., Bilsland, J., Cao, L., Stevens,
E., Whiting, P., et al. (2012). Combined small-molecule inhibition accelerates
developmental timing and converts human pluripotent stem cells into nociceptors. Nature
biotechnology 30, 715-720.
Chiu, I.M., Hehn, C.A. von, and Woolf, C.J. (2012). Neurogenic inflammation and the
peripheral nervous system in host defense and immunopathology. Nat Neurosci 15: 1063–
1067.
Choi, J.Y., Lee, H.Y., Hur, J., Kim, K.H., Kang, J.Y., Rhee, C.K., and Lee, S.Y. (2018). TRPV1
Blocking Alleviates Airway Inflammation and Remodeling in a Chronic Asthma Murine
Model. Allergy, asthma & immunology research 10, 216-224.
Chong, C.F., Chen, C.C., Ma, H.P., Wu, Y.C., Chen, Y.C., and Wang, T.L. (2005). Comparison
of lidocaine and bronchodilator inhalation treatments for cough suppression in patients with
chronic obstructive pulmonary disease. Emergency medicine journal : EMJ 22, 429-432.
Chou, Y.-L., Mori, N., and Canning, B.J. (2018). Opposing effects of bronchopulmonary C-
fiber subtypes on cough in guinea pigs. American Journal of Physiology-Regulatory,
Integrative and Comparative Physiology 314, R489-R498.
Costigan, M., Mannion, R.J., Kendall, G., Lewis, S.E., Campagna, J.A., Coggeshall, R.E.,
Meridith-Middleton, J., Tate, S., and Woolf, C.J. (1998). Heat shock protein 27:
developmental regulation and expression after peripheral nerve injury. The Journal of
neuroscience : the official journal of the Society for Neuroscience 18, 5891-5900.
Crosson, T., Wang, J.-C., Doyle, B., Merrison, H., Balood, M., Parrin, A., et al. (2021). FcεR1-
expressing nociceptors trigger allergic airway inflammation. J Allergy Clin Immunol. Jan
13:S0091-6749(21)00008-7. doi: 10.1016/j.jaci.2020.12.644. Online ahead of print
Csekő, K., Beckers, B., Keszthelyi, D., and Helyes, Z. (2019). Role of TRPV1 and TRPA1 Ion
Channels in Inflammatory Bowel Diseases: Potential Therapeutic Targets?
Pharmaceuticals (Basel) 12: 48.
This article is protected by copyright. All rights reserved.

Curtis, M.J., Alexander, S., Cirino, G., Docherty, J.R., George, C.H., Giembycz, M.A., et al.
(2018). Experimental design and analysis and their reporting II: updated and simplified
guidance for authors and peer reviewers. Br J Pharmacol 175: 987–993.
DeBerry, J.J., Schwartz, E.S., and Davis, B.M. (2014). TRPA1 mediates bladder hyperalgesia
in a mouse model of cystitis. Pain 155: 1280–1287.
Dicpinigaitis, P.V., McGarvey, L.P., and Canning, B.J. (2020). P2X3-Receptor Antagonists as
Potential Antitussives: Summary of Current Clinical Trials in Chronic Cough. Lung 198,
609-616.
European Medicines Agency (2013). A Phase 2a, Multi-Centre, Randomised, Double-Blind,
Parallel Group, Placebo-Controlled Study to Evaluate Efficacy, Safetyand Tolerability of
Inhaled GRC 17536, Administered for 4 Weeks, in Patientswith Refractory Chronic
Cough. EU Clinical Trials Registry 2016.
Featherstone, R.L., Hutson, P.A., Holgate, S.T., and Church, M.K. (1988). Active sensitization
of guinea-pig airways in vivo enhances in vivo and in vitro responsiveness. The European
respiratory journal 1, 839-845.
Forsberg, K., Karlsson, J.A., Theodorsson, E., Lundberg, J.M., and Persson, C.G. (1988).
Cough and bronchoconstriction mediated by capsaicin-sensitive sensory neurons in the
guinea-pig. Pulmonary pharmacology 1, 33-39.
Frazier, D.T., Narahashi, T., and Yamada, M. (1970). The site of action and active form of
local anesthetics. II. Experiments with quaternary compounds. J Pharmacol Exp Ther 171:
45–51.
Gamal El-Din, T.M., Lenaeus, M.J., Zheng, N., and Catterall, W.A. (2018). Fenestrations
control resting-state block of a voltage-gated sodium channel. Proc Natl Acad Sci U S A
115: 13111–13116.
Garrison, S.R., and Stucky, C.L. (2014). Contribution of transient receptor potential ankyrin 1
to chronic pain in aged mice with complete Freund’s adjuvant-induced arthritis. Arthritis
Rheumatol 66: 2380–2390.
Garceau, D., and Chauret, N. (2019). BLU-5937: A selective P2X3 antagonist with potent anti-
tussive effect and no taste alteration. Pulm Pharmacol Ther 56, 56-62.
Gerner, P., Binshtok, A.M., Wang, C.F., Hevelone, N.D., Bean, B.P., Woolf, C.J., and Wang,
G.K. (2008). Capsaicin combined with local anesthetics preferentially prolongs
sensory/nociceptive block in rat sciatic nerve. Anesthesiology 109, 872-878.
Gonzalez-Cano, R., Boivin, B., Bullock, D., Cornelissen, L., Andrews, N., and Costigan, M.
(2018). Up-Down Reader: An Open Source Program for Efficiently Processing 50% von
Frey Thresholds. Frontiers in pharmacology 9, 433.
Grace, M.S., and Belvisi, M.G. (2011). TRPA1 receptors in cough. Pulm Pharmacol Ther 24,
286-288.
Groneberg, D.A., Niimi, A., Dinh, Q.T., Cosio, B., Hew, M., Fischer, A., and Chung, K.F.
(2004). Increased expression of transient receptor potential vanilloid-1 in airway nerves of
chronic cough. American journal of respiratory and critical care medicine 170, 1276-1280.
Hara, J., Fujimura, M., Myou, S., Oribe, Y., Furusho, S., Kita, T., Katayama, N., Abo, M.,
Ohkura, N., Herai, Y., et al. (2005). Comparison of cough reflex sensitivity after an inhaled
antigen challenge between actively and passively sensitized guinea pigs. Cough 1, 6-6.
Harding, S.D., Sharman, J.L., Faccenda, E., Southan, C., Pawson AJ, Ireland S, Gray AJG,
Bruce L, Alexander SPH, Anderton S, Bryant C, Davenport AP, Doerig C, Fabbro D, Levi-
Schaffer F, Spedding M, Davies JA, NC-IUPHAR (2018). The IUPHAR/BPS Guide to
PHARMACOLOGY in 2018: updates and expansion to encompass the new guide to
IMMUNOPHARMACOLOGY. Nucleic Acids Res. 46, D1091-1106.
Hargreaves, K., Dubner, R., Brown, F., Flores, C., and Joris, J. (1988). A new and sensitive
method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32, 77-88.
This article is protected by copyright. All rights reserved.

Harkat, M., Peverini, L., Cerdan, A.H., Dunning, K., Beudez, J., Martz, A., Calimet, N., Specht,
A., Cecchini, M., Chataigneau, T., et al. (2017). On the permeation of large organic cations
through the pore of ATP-gated P2X receptors. Proceedings of the National Academy of
Sciences of the United States of America 114, E3786-e3795.
Hille, B (1977). Local anesthetics: hyrophilic and hydrophobic pathways for the drug-receptor
reaction. The Journal of General Physiology 69,497-515.
Horáček, M., and Vymazal, T. (2012). Lidocaine not so innocent: Cardiotoxicity after topical
anaesthesia for bronchoscopy. Indian J Anaesth 56, 95-96.
Jain, P., Materazzi, S., De Logu, F., Rossi Degl’Innocenti, D., Fusi, C., Li Puma, S., et al.
(2020). Transient receptor potential ankyrin 1 contributes to somatic pain hypersensitivity
in experimental colitis. Sci Rep 10: 8632.
Jia, Y., McLeod, R.L., Wang, X., Parra, L.E., Egan, R.W., and Hey, J.A. (2002). Anandamide
induces cough in conscious guinea-pigs through VR1 receptors. British journal of
pharmacology 137, 831-836.
Julius, D. (2013). TRP channels and pain. Annual review of cell and developmental biology
29, 355-384.
Kanai, Y., Hara, T., Imai, A., and Sakakibara, A. (2007). Differential involvement of TRPV1
receptors at the central and peripheral nerves in CFA-induced mechanical and thermal
hyperalgesia. Journal of Pharmacy and Pharmacology 59, 733-738.
Kanezaki, M., Ebihara, S., Gui, P., Ebihara, T., and Kohzuki, M. (2012). Effect of cigarette
smoking on cough reflex induced by TRPV1 and TRPA1 stimulations. Respiratory
medicine 106, 406-412.
Khalid, S., Murdoch, R., Newlands, A., Smart, K., Kelsall, A., Holt, K., Dockry, R.,
Woodcock, A., and Smith, J.A. (2014). Transient receptor potential vanilloid 1 (TRPV1)
antagonism in patients with refractory chronic cough: a double-blind randomized
controlled trial. The Journal of allergy and clinical immunology 134, 56-62.
Kahlig, K.M., Saridey, S.K., Kaja, A., Daniels, M.A., George, A.L. Jr, Wilson, M.H. (2010)
Multiplexed transposon-mediated stable gene transfer in human cells. Proc Natl Acad Sci
USA. 107:1343-1348.
Kichko, T.I., Niedermirtl, F., Leffler, A., and Reeh, P.W. (2015). Irritant volatile anesthetics
induce neurogenic inflammation through TRPA1 and TRPV1 channels in the isolated
mouse trachea. Anesth Analg 120: 467–471.
Kim, H.Y., Kim, K., Li, H.Y., Chung, G., Park, C.-K., Kim, J.S., et al. (2010). Selectively
targeting pain in the trigeminal system. Pain 150: 29–40.
Kollarik, M., Sun, H., Herbstsomer, R.A., Ru, F., Kocmalova, M., Meeker, S.N., and Undem,
B.J. (2018). Different role of TTX-sensitive voltage-gated sodium channel (NaV1)
subtypes in action potential initiation and conduction in vagal airway nociceptors. The
Journal of Physiology 596, 1419-1432.
Kwong, K., Kollarik, M., Nassenstein, C., Ru, F., and Undem, B.J. (2008). P2X2 receptors
differentiate placodal vs. neural crest C-fiber phenotypes innervating guinea pig lungs and
esophagus. American journal of physiology Lung cellular and molecular physiology 295,
L858-865.
Labedzki, L., Ochs, H.R., Abernethy, D.R., and Greenblatt, D.J. (1983). Potentially toxic serum
lidocaine concentrations following spray anesthesia for bronchoscopy. Klin Wochenschr
61: 379–380.
Laude, E.A., Higgins, K.S., and Morice, A.H. (1993). A comparative study of the effects of
citric acid, capsaicin and resiniferatoxin on the cough challenge in guinea-pig and man.
Pulmonary pharmacology 6, 171-175.
This article is protected by copyright. All rights reserved.

Lee, S., Jo, S., Talbot, S., Zhang, H.B., Kotoda, M., Andrews, N.A., Puopolo, M., Liu, P.W.,
Jacquemont, T., Pascal, M., et al. (2019). Novel charged sodium and calcium channel
inhibitor active against neurogenic inflammation. eLife 8. e48118.
Leffler, A., Herzog, R.I., Dib-Hajj, S.D., Waxman, S.G., and Cummins, T.R. (2005).
Pharmacological properties of neuronal TTX-resistant sodium channels and the role of a
critical serine pore residue. Pflugers Arch 451: 454–463.
Leffler A, Reckzeh J, Nau C. (2010) Block of sensory neuronal Na+ channels by the secreolytic
ambroxol is associated with an interaction with local anesthetic binding sites. Eur J
Pharmacol. 630:19-28.
Lennertz, R.C., Kossyreva, E.A., Smith, A.K., and Stucky, C.L. (2012). TRPA1 mediates
mechanical sensitization in nociceptors during inflammation. PloS one 7, e43597.
Leung, S.Y., Niimi, A., Williams, A.S., Nath, P., Blanc, F.-X., Dinh, Q.T., and Chung, K.F.
(2007). Inhibition of citric acid-and capsaicin-induced cough by novel TRPV-1 antagonist,
V112220, in guinea-pig. Cough 3, 1-5.
Lewis, C.A., Ambrose, C., Banner, K., Battram, C., Butler, K., Giddings, J., Mok, J., Nasra, J.,
Winny, C., and Poll, C. (2007). Animal models of cough: literature review and presentation
of a novel cigarette smoke-enhanced cough model in the guinea-pig. Pulm Pharmacol Ther
20, 325-333.
Lilley, E., Stanford, S.C., Kendall, D.E., Alexander, S.P.H., Cirino, G., Docherty, J.R., et al.
(2020). ARRIVE 2.0 and the British Journal of Pharmacology: Updated guidance for 2020.
Br J Pharmacol 177: 3611–3616.
Liu, B., Escalera, J., Balakrishna, S., Fan, L., Caceres, A.I., Robinson, E., et al. (2013). TRPA1
controls inflammation and pruritogen responses in allergic contact dermatitis. FASEB J 27:
3549–3563.
Liu, P., Jo, S., and Bean, B.P. (2012). Modulation of neuronal sodium channels by the sea
anemone peptide BDS-I. Journal of neurophysiology 107, 3155-3167.
Liu, Z., Hu, Y., Yu, X., Xi, J., Fan, X., Tse, C.-M., Myers, A.C., Pasricha, P.J., Li, X., and Yu,
S. (2015). Allergen challenge sensitizes TRPA1 in vagal sensory neurons and afferent C-
fiber subtypes in guinea pig esophagus. Am J Physiol Gastrointest Liver Physiol 308,
G482-G488.
Long, L., Yao, H., Tian, J., Luo, W., Yu, X., Yi, F., Chen, Q., Xie, J., Zhong, N., Chung, K.F.,
et al. (2019). Heterogeneity of cough hypersensitivity mediated by TRPV1 and TRPA1 in
patients with chronic refractory cough. Respiratory research 20, 112.
Ma, C.H., Omura, T., Cobos, E.J., Latremoliere, A., Ghasemlou, N., Brenner, G.J., van Veen,
E., Barrett, L., Sawada, T., Gao, F., et al. (2011). Accelerating axonal growth promotes
motor recovery after peripheral nerve injury in mice. The Journal of clinical investigation
121, 4332-4347.
Martin, L.J., and Corry, B. (2014). Locating the route of entry and binding sites of benzocaine
and phenytoin in a bacterial voltage gated sodium channel. PLoS Comput Biol 10:
e1003688.
Matta, J.A., Cornett, P.M., Miyares, R.L., Abe, K., Sahibzada, N., and Ahern, G.P. (2008).
General anesthetics activate a nociceptive ion channel to enhance pain and inflammation.
Proc Natl Acad Sci U S A 105: 8784–8789.
Mazzone, S.B., Reynolds, S.M., Mori, N., Kollarik, M., Farmer, D.G., Myers, A.C., and
Canning, B.J. (2009). Selective expression of a sodium pump isozyme by cough receptors
and evidence for its essential role in regulating cough. The Journal of neuroscience : the
official journal of the Society for Neuroscience 29, 13662-13671.
Mazzone, S.B., and Undem, B.J. (2016). Vagal Afferent Innervation of the Airways in Health
and Disease. Physiological reviews 96, 975-1024.
This article is protected by copyright. All rights reserved.