key: cord-0305233-tjld4s5b
authors: Tochitsky, Ivan; Jo, Sooyeon; Andrews, Nick; Kotoda, Masakazu; Doyle, Benjamin; Shim, Jaehoon; Talbot, Sebastien; Roberson, David; Lee, Jinbo; Haste, Louise; Jordan, Stephen M.; Levy, Bruce D.; Bean, Bruce P.; Woolf, Clifford J.
title: Inhibiting cough by silencing large pore-expressing airway sensory neurons with a charged sodium channel blocker
date: 2020-12-07
journal: bioRxiv
DOI: 10.1101/2020.12.07.414763
sha: b4f88d632b069ba39066e82142b280b549d14028
doc_id: 305233
cord_uid: tjld4s5b

Although multiple diseases of the respiratory system cause cough, there are few effective treatments for this common condition. We previously developed a strategy to treat pain and itch via the long-lasting inhibition of nociceptor sensory neurons with QX-314, a cationic sodium channel blocker that selectively enters only into activated nociceptors by permeating through the endogenous TRPV1 and TRPA1 large pore ion channels they express. In this study we design and characterize BW-031, a novel cationic compound with ∼6-fold greater potency than QX-314 for inhibiting sodium channels when introduced inside cells and with minimal extracellular activity. We show that inhalation of aerosolized BW-031 effectively inhibits citric acid-induced cough in an allergic inflammation guinea pig cough model. These data support the use of charged sodium channel blockers for the selective inhibition of airway sensory neurons with activated large pore channels as a novel targeted therapy for treating cough.

neurons that express large pore channels, only those whose terminals are accessible from the 232 surface of the airway. 233 Our approach of exploiting activated large-pore channels to introduce charged sodium 234 channel blockers inside activated sensory neurons will inhibit the activity of the neurons to 235 subsequent different stimuli and may therefore have greater efficacy than targeting single 236 receptors. Once cationic sodium channel inhibitors are loaded into a cell (concentrated by the 237 negative intracellular potential), they will not readily diffuse out through the cell membrane and 238 may produce effects lasting for many hours, as is the case for the analgesic effect of QX-314 239 (Binshtok et al., 2009a; Gerner et al., 2008; Roberson et al., 2011) and as we find here for BW-240 031 (Fig. 2b,c) . 241 From the current experiments, we do not know which exact population of sensory neurons 242 BW-031 silences to inhibit the citric acid-induced cough or which large-pore entry pathways are 243 most important. Citric acid-induced coughing in the guinea pig is mediated by C-fibers expressing TRPV1 and TRPA1 channels (Canning, 2006; Canning et al., 2004) , both of which 245 are activated by citric acid, and also by a population of A-delta fibers (Canning et al., 2004) , 246 likely through activation of ASIC channels (Kollarik et al., 2007) . In principle, the relative role 247 of different large-pore channels could be tested by examining whether specific inhibitors of 248 TRPA1, TRPA1, or P2X3 channels prevent the effect of inhaled BW-031, but as such inhibitors 249 will themselves reduce coughing, interpreting such experiments would be challenging. It is also 250 uncertain to what degree different large pore channels are activated in human cough conditions. 251 Nevertheless, a sizeable fraction of human patients with chronic cough show amplification of the 252 cough evoked by TRPV1 or TRPA1 agonists (Long et al., 2019b) , and TRPV1 is up-regulated in 253 the airway nerves of some patients with chronic cough (Groneberg et al., 2004) , suggesting that 254 these channels are likely active in certain human cough conditions. The efficacy of P2X3 255 inhibitors against cough in recent clinical trials also indicates activation of this channel in 256 patients (Smith et al., 2020a; Smith et al., 2020b) .

The citric acid model of guinea pig cough, although widely used, clearly has limitations for 258 predicting drug efficacy in human disease, because while TRPV1 and TRPA1 inhibitors are quite 259 effective in this model (Leung et al., 2007; Mukhopadhyay et al., 2014) this has not been The charged local anesthetic strategy does not necessarily require generating compounds 269 with selectivity only for blocking certain sodium channels, because the selectivity for silencing 270 specific sensory neurons is based instead on targeting only those neurons with activated large-271 pore channels. We used Nav1.7 inhibition for our initial in vitro tests of BW-031 partly because 272 of evidence that Nav1.7 channels are important in cough-mediating neurons (Kollarik et al., 273 2018; Muroi et al., 2011; Patil et al., 2019; Sun et al., 2017; Undem and Sun, 2020) ; however, we 274 do not know what types of sodium channels are most important for the effects of BW-031 on cough inhibition. Recent work has shown an important distinction between the sodium channels 276 responsible for initiating action potentials in nerve terminals and those responsible for axonal 277 conduction (Kollarik et al., 2018; Muroi et al., 2011) . Conduction in airway C-fiber axons is 278 mediated by tetrodotoxin-sensitive sodium channels, most likely mainly Nav1.7 channels 279 (Kollarik et al., 2018; Muroi et al., 2011) ; however, action potential initiation at peripheral 280 terminals is mediated in different neuronal types by either mainly tetrodotoxin-resistant Nav1.8 281 channels (jugular C-fibers) or by Nav1.7 channels (nodose C-fibers and A-delta fibers (Kollarik Plantar incision surgery. Rats were placed in a chamber with 5% isoflurane and monitored 479 until they were visibly unconscious. Once unconscious, rats were removed from the chamber and 480 anesthesia was maintained using 2% isoflurane delivered via nose cone. A toe pinch was used to 481 confirm that animals were fully anesthetized. The animals were then secured with surgical tape 482 at their toes and upper leg for paw stability during surgery. The plantar surface of one hind paw 483 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 485 and extending towards the foot pad and toes. Incision was made to the minimal depth necessary 486 to cut through skin and fascia to expose the underlying plantaris muscle, approximately 1-2 mm. 487 Once exposed the plantaris muscle was elevated for 10 seconds with surgical forceps and gently 488 lifted for 10 seconds. After irritation of the plantaris muscle, the wound was closed with three 489 sutures. After surgery animals were returned to their cage and monitored until they fully 490 recovered from anesthesia. Treatments were administered subcutaneously 24 hours after injury. BIO-EVF4) was used to assess mechanical sensitivity in rats before and after paw incision 512 injury. Animals were habituated for 1 hour, one day prior to baseline testing. Animals were given 513 30 minutes to settle before testing. An average mechanical threshold was calculated using 5 514 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-516 031 or saline were administered into the plantar region of the hind paw adjacent to the incision 517 24 hours post injury. Animals were then tested 1, 3, 5, and 24 hours post treatment. Additional 518 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 520 UV burn, as previously described (Lee et al., 2019) . After mice were habituated to the testing 521 cage (7.5 × 7.5 × 15 cm) with a metal grid floor for 45 min for 2 days, baseline values were injected rats. Rats were habituated to testing enclosures for 1 hour one day prior to baseline 535 testing. Rats were given 30 minutes to settle before testing. An average paw withdrawal latency 536 was calculated using 3 measurements taken 5 minutes apart. Animals were tested 1, 4, and 24 537 hours after injury from CFA injection. preliminary studies showed that some guinea pigs failed to cough in response to the citric acid 571 challenge, each study began with 20% more animals than were planned for the protocols and 572 animals were first pre-screened by inhalation of citric acid (400 mM for 7 minutes, with coughs 573 counted during the 7 minute application and for 10 minutes afterward) and the lowest responders 574 were omitted from the remaining study protocol. For the intratracheal protocol, animals with 0-1 575 coughs were omitted; for the ovalabumin sensitization protocol, the 6 animals of each sex with lowest cough counts (0-3 coughs) were omitted. Pre-screening was performed a minimum of 7 577 days before the start of the study protocol to allow animals to recover from any sensitization 578 produced by the citric acid exposure during the pre-screening. After pre-screening, the remaining 579 animals were allocated into each group so that each group had approximately equal group mean 580 cough counts measured in the pre-screening protocol. Step 1：Preparation To a mixture of 1 (10.0g, 59.88mmol) was added SOCl2 (60mL, c=1.0). The mixture was heated to reflux. After completion, the reaction mixture was concentrated under reduce pressure to give intermediate 2 (9.2g,yield=82.8%) as a yellow oil.

Step 2：Preparation To a solution of 3 (5.0g, 41.3mmol, 1.0eq) in DCM (100ml, c=0.5) was added pyridine (4.9g, 61.95mmol, 1.5eq). To the solution was added 2 (9.2g, 49.59mmol, 1.2eq) in DCM (40mL, c=1.2). The reaction mixture was stirred at room temperature over night. Then to the solution was added water (50mL). The organic phase was washed with brine, dried over Na2SO4, filtered and concentrated under reduce pressure. The residue was washed with n-hexane to give intermediate 4 (7.8g, yield=70%, HPLC: 98.6%). To a solution of NaH (0.35g, 14.8mmol, 2.0eq) in THF (37mL, c=0.4) was added 5 (0.75g, 8.8mmol, 1.2eq). To the solution was added 4 (2.0g, 7.4mmol, 1.0eq) in THF (20mL, c=0.37). The reaction mixture was then stirred at room temperature over night. To the suspension was added water (20mL) and EA (50mL). The organic phase was washed with water (50mL×2). Then the organic phase was adjusted to pH 2, extracted with EA(40mL×2). The aqueous fractions were combined and adjusted to pH 9, then extracted with EA (40×2). The combined organic fractions was washed with brine, dried over Na2SO4, filtered and concentrated under reduce pressure. The residue was washed with n-hexane to give the intermediate 6 (0.48g, yield=24%, HPLC: 99.3%) as a solid.

Step 4：Preparation Intermediate 6 (0.48g, 1.75mmol, 1.0 eq) and MeCN (9mL, c=0.2) was added in sealed tube. To this solution, EtI (2mL, 14.0 eq) was added. After addition, the reaction mixture was stirred at 90℃ for 10h. After completion, the reaction mixture was concentrated under reduce pressure. The residue was purified by column chromatography to give intermediate 7 (470mg, yield=62.6%, HPLC: 99%) as a solid.

Step 4：Preparation To a solution of 7 (200mg, 0.465mmol, 1.0 eq) in deionized water (3ml, c=0.15) was added

Refractory Chronic Cough: Results from a Randomized, Controlled, Phase 2b Clinical Trial

B14 CLINICAL TRIALS ACROSS PULMONARY DISEASE

Nav current was evoked by 20-ms depolarizations from -100 to 0 mV at the indicated frequencies. b, Collected results for hNav1.1 inhibition by 100 µM intracellular BW-031 (red, n=5) compared with control (black, n=5) and with inhibition of hNav1.7 (n=6, replotted from Fig. 1e) c, Use-dependent inhibition of hNav1.8 channels by 300 µM intracellular BW-031 (red) compared to recording with control intracellular solution (black)

mV at the indicated frequencies. d, Collected results (mean±SEM) of hNav1.8 inhibition by 300

µM intracellular BW-031 (red, n=5) compared with control (black, n=5) and with inhibition of hNav1.7 by 100 µM BW-031 (n=6, replotted from Fig. 1e) Data are mean±SEM and statistics are calculated from two-tailed Mann-Whitney Test. ns p>0.05, *p<0.05, and **p<0.01. Supplementary Fig. 2. BW-031 does not inhibit Nav currents in TRPV1 -DRG neurons in

Bars represent mean±SEM for each condition, while the individual data points are displayed as open circles. d, Toe spread assay of motor function in mice after peri-sciatic injection of 0.5% lidocaine, 0.5% QX-314 or 0.5% BW-031. Only lidocaine produces robust block of motor function in naïve mice

Only lidocaine produces robust sensory analgesia in naïve mice. N=10 male mice per group, Fisher's exact test (5 min time point), p=4.1x10 -6 , ***p<0.001. Data are mean±SEM. f, Hargreaves assay of hindpaw thermal (white), 2% QX-314 dissolved in CFA (blue) or 2% BW-031 dissolved in CFA (red). Both QX-314 and BW-031 produce robust thermal analgesia. Two-way repeated measures ANOVA with treatment as the between groups factor and time as the within groups factor

After addition, the reaction mixture was stirred at room 1094 temperature overnight. The suspension was then filtered and the filtrate was lyophilized to give 1095 compound 8 (141mg, yield=89.8%) as a solid

NMR (300 MHz, D2O): δ 7.117 (m, 3H), 4.056 (dd, J=8.1 Hz, 1H), 3.712~3.808 (m, 1H), 3.656 1097