Benzonatate inhibition of voltage-gated sodium currents

Neuropharmacology 101 (2016) 179e187

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Benzonatate inhibition of voltage-gated sodium currents M. Steven Evans a, *, G. Benton Maglinger a, Anita M. Fletcher a, Stephen R. Johnson b a b

Department of Neurology, University of Louisville, Louisville, KY, USA Department of Chemistry, University of Illinois at Springfield, Springfield, IL, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 June 2015 Received in revised form 17 August 2015 Accepted 15 September 2015 Available online 16 September 2015

Benzonatate was FDA-approved in 1958 as an antitussive. Its mechanism of action is thought to be anesthesia of vagal sensory nerve fibers that mediate cough. Vagal sensory neurons highly express the Nav1.7 subtype of voltage-gated sodium channels, and inhibition of this channel inhibits the cough reflex. Local anesthetics inhibit voltage-gated sodium channels, but there are no reports of whether benzonatate affects these channels. Our hypothesis is that benzonatate inhibits Nav1.7 voltage-gated sodium channels. We used whole cell voltage clamp recording to test the effects of benzonatate on voltage-gated sodium (Naþ) currents in two murine cell lines, catecholamine A differentiated (CAD) cells, which express primarily Nav1.7, and N1E-115, which express primarily Nav1.3. We found that, like local anesthetics, benzonatate strongly and reversibly inhibits voltage-gated Naþ channels. Benzonatate causes both tonic and phasic inhibition. It has greater effects on channel inactivation than on activation, and its potency is much greater at depolarized potentials, indicating inactivated-state-specific effects. Naþ currents in CAD cells and N1E-115 cells are similarly affected, indicating that benzonatate is not Naþ channel subtype-specific. Benzonatate is a mixture of polyethoxy esters of 4-(butylamino) benzoic acid having varying degrees of hydrophobicity. We found that Naþ currents are inhibited most potently by a benzonatate fraction containing the 9-ethoxy component. Detectable effects of benzonatate occur at concentrations as low as 0.3 mM, which has been reported in humans. We conclude that benzonatate has local anesthetic-like effects on voltage-gated sodium channels, including Nav1.7, which is a possible mechanism for cough suppression by the drug. © 2015 Elsevier Ltd. All rights reserved.

Chemical compounds studied in this article: Benzonatate (PubChem CID:7699) 4-(butylamino)benzoic acid (PubChem CID:95946) Para-aminobenzoic acid (PubChem CID:978) Tetracaine (PubChem CID:5411) Keywords: Nav1.7 voltage-gated sodium channel Cough Benzonatate Local anesthetic Patch clamp High performance liquid chromatography mass spectrometry

1. Introduction Benzonatate (Tessalon®) is a non-narcotic cough suppressant approved for human use by the United States Food and Drug Administration (FDA) in 1958 for symptomatic relief of cough in patients over 10 years of age. Benzonatate is classified as an estertype local anesthetic chemically related to tetracaine, procaine and cocaine. The mechanism of cough suppression by benzonatate is reported to be through inhibition of pulmonary stretch receptors (Pfizer Laboratories, 2014; Michelson and Schiller, 1957; Tomokazu,

Abbreviations: CAD, catecholamine A differentiated; Naþ, sodium ion; BABA, 4(butylamino)benzoic acid; TTX, tetrodotoxin; PABA, para aminobenzoic acid. * Corresponding author. Department of Neurology, University of Louisville, HSC-A Room 113, Louisville, KY 40292, USA. E-mail addresses: [email protected] (M.S. Evans), [email protected] (G.B. Maglinger), anita.m.fl[email protected] (A.M. Fletcher), [email protected] (S.R. Johnson). http://dx.doi.org/10.1016/j.neuropharm.2015.09.020 0028-3908/© 2015 Elsevier Ltd. All rights reserved.

1967; Wilson et al., 1958), whose fibers travel in the vagus nerves to the brain. Benzonatate has a numbing effect when applied directly to the oral and pharyngeal mucosa. Direct mucosal application has been used as a method for rapid oral anesthesia for awake intubation (Mongan and Culling, 1992), but for cough relief it is given orally, with drug reaching its site of action through the bloodstream after gastrointestinal absorption. Although benzonatate is reported to inhibit pulmonary stretch receptors, the mechanism of that inhibition is unknown. The primary mechanism of action of local anesthetics is inhibition of voltage-gated sodium channels. Benzonatate was patented (Matter, 1955) and approved for human use before the importance and ubiquity of these channels was fully understood, and before the first description of local anesthetic effects on voltage-gated sodium channels (TAYLOR, 1959). Whether benzonatate, any of its components, or its primary metabolite 4(butylamino)benzoic acid (BABA), affect voltage-gated sodium channels

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has not yet been reported. There are nine subtypes of mammalian voltage-gated sodium channels, denoted Nav1.1 through 1.9. The tetrodotoxin (TTX)sensitive sodium channel Nav1.7 is the main sodium channel present in vagal sensory neurons, with a large contribution from the TTX-insensitive subtypes Nav1.8 and Nav1.9 (Kwong et al., 2008). Muroi et al. (Muroi et al., 2013) have shown that short hairpin RNA knockdown of Nav1.7 in the nodose ganglion of the vagus inhibits the cough reflex, suggesting that benzonatate inhibition of Nav1.7 in pulmonary sensory nerve fibers could relieve cough. Our study was designed to determine whether benzonatate has local anesthetic effects on voltage-gated sodium channels, specifically on Nav1.7. We tested this using a murine CNS cell line that highly expresses Nav1.7 (CAD cells) and compared the results to a murine neuroblastoma cell line that expresses primarily Nav1.3 (N1E-115 cells). 2. Material and methods 2.1. Cell culture Catecholamine A differentiated (CAD) cells were a gift of Raj Khanna, University of Arizona. CAD cells were grown at 37  C in 21% O2, 5% CO2, in Ham's DMEM/F12 medium (Gibco®; Life Technologies, Grand Island, NY) supplemented with with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 0.2% Glutamax (all from Life Technologies, Grand Island, NY). Cells were grown in 35 mm plastic culture dishes and passaged at a 1:4 dilution every 3e5 days after reaching 90% confluency. CAD cells can grow in a “differentiated” form when cultured without serum (Wang and Olsen, 2000). Our studies used the “undifferentiated” form. N1E-115 cells were obtained from American Type Culture Collection (Manassas, VA). N1E-115 cells were grown at 37  C in 21% O2, 5% CO2, in DMEM-high glucose with glutamine medium (Life Technologies) supplemented with 10% FBS and 1% penicillin/ streptomycin. Cells were grown in 35 mm plastic culture dishes and passaged at a 1:2 dilution every 2e3 days after reaching 90% confluency. For both lines, cells used for electrophysiological study were grown in 35 mm dishes containing 12 mm round glass coverslips. 2.2. Materials Benzonatate was obtained from Toronto Research Chemicals (Toronto, Canada), Santa Cruz Biotechnology (Dallas, Tx) and Zydus Pharmaceuticals (Pennington, NJ). Tetrodotoxin was obtained from Tocris Bioscience (Bristol, United Kingdom). 4-(butylamino)benzoic acid (BABA), para aminobenzoic acid (PABA), tetracaine and all electrolytes were obtained from Sigma (St. Louis, MO). Benzonatate concentrations of 10 mM or less were dissolved directly in the particular extracellular solution used for recording. For higher concentrations a stock solution was dissolved in 70% ethanol and added to extracellular solutions. Final ethanol concentrations were always less than 1%. Control and wash solutions always contained the same amount of ethanol as benzonatate-containing solutions and the concentrations of ethanol used did not affect Naþ currents. 2.3. HPLC-MS To examine benzonatate composition, 1.0 ng of diluted benzonatate was injected into a Waters 2795 Alliance HT High Performance Liquid Chromatograph (HPLC; Waters Corporation, Milford, MA) equipped with a combination Phenomenex Aeris™ 3.6 mm PEPTIDE XB-C18 100 Å guard cartridge coupled with Phenomenex Aeris™ 3.6 mm PEPTIDE XB-C18 100 Å column 150  2.1 mm 3.6 mm

(Phenomenex Inc., Torrance, CA) at a flow rate of 0.150 mL/min; a 2.00 min hold at 90:10 A:B (Mobil Phase A ¼ 0.01% Formic Acid; Mobil Phase B ¼ acetonitrile:methanol 1:1) followed by a binary mobile phase gradient to 10:90 A:B in 30.00 min. The effluent stream from the chromatographic separation was coupled to a Waters Quattro Ultima Triple Stage Quadrupole (TSQ) mass spectrometer (MS) with atmospheric pressure ionization and electrospray (ESI) probe, linear hexapole assembly collision cell, post acceleration conversion dynode and photomultiplier detection system (MassLynx 4.0 & QuanLynx). Full scan collection in a mass range from 150 to 1000 m/z through a quadrupole filter (Q1) was obtained with a capillary temperature of 275  C maintained at 3.5 kV. All mass spectra show the major molecular [MþH]þ ion. 2.4. Separation of benzonatate fractions To fractionate benzonatate, 1.0 mg of diluted benzonatate was injected into a BioRad BioLogic DuoFlow High Performance Liquid Chromatograph (Bio-Rad Laboratories Inc., Hercules, CA) equipped with an Agilent 5.0 mM Zorbax SB-C18 250  7.6 mm column (Agilent Technologies, Santa Clara, CA) at a flow rate of 3.0 mL/min; a 2.00 min hold at 90:10 A:B (Mobil Phase A ¼ 0.01% Formic Acid; Mobil Phase B ¼ acetonitrile:methanol 1:1) followed by a binary mobile phase gradient to 10:90 A:B in 30.00 min. The effluent stream from the chromatographic separation was coupled to a Büchi C-630 UV Monitor (254 nm) (Flawil, Switzerland) and a BioRad BioLogic Fraction Collector. The fraction collection was adjusted to collect the first one third of eluting n-ethoxy compounds, the middle one third and the latter one third; each one third represented a different overall hydrophobicity and n-ethoxy chain length group. 2.5. Whole cell recording Whole cell voltage-clamp recordings were done at room temperature using an Axopatch 200b amplifier (Molecular Devices, Sunnyvale CA). Electrodes were pulled from thin-walled borosilicate glass capillaries (1.5 OD, 1.0 ID; King Precision Glass, Inc., Claremont, CA) with a P-97 electrode puller (Sutter Instrument Co., Novato, CA) with electrode resistances of 1e2 MU when filled with internal solution. The internal solution for recording Naþ currents contained (in mM): 110 CsCl, 5 MgSO4, 10 EGTA (in CsOH), 4 ATP Na2-ATP, 25 HEPES (pH 7.2). The external solution contained (in mM): 100 NaCl, 10 tetraethylammonium chloride (TEA-Cl), 1 CaCl2, 1 CdCl2, 1 MgCl2, 10 D-glucose, 1 3,4 diaminopyridine, 0.1 NiCl2, 10 HEPES (pH 7.3). To allow complete dialysis of the pipette solution with the intracellular contents and stabilize Naþ currents, which tended to increase gradually after establishing whole cell mode, recording began after 5e10 min. Whole-cell capacitance and series resistance were compensated with the amplifier. Series resistance in whole cell mode was 2e3 MU and compensated by 50e80%. Linear leak currents were digitally subtracted using a P/4 protocol. Currents were filtered at 5 kHz using the low pass Bessel filter of the amplifier and sampled at 10e20 kHz using a Digidata 1322A interface and PClamp 10 software (Molecular Devices). Cells used for study had stable seal resistances of at least 1 GU, input resistances of at least 500 MU, and Naþ currents of at least 500 pA. The holding potential in all experiments was 80 mV unless stated otherwise. Currents were elicited at a rate of not greater than every 20 s, which eliminated use-dependent (“phasic”) effects of benzonatate on Naþ currents. In some experiments we specifically studied phasic inhibition by benzonatate, and currents were elicited at a rate of 20 Hz from a holding potential of 100 mV. For current clamp experiments the internal solution was K aspartate 130, KCl 20, MgCl 1, D-glucose 10, EGTA 1 (in KOH), and

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Fig. 1. Benzonatate is a mixture. A: HPLC chromatogram of benzonatate from our suppliers confirms that benzonatate is a mixture with varying numbers of ethoxy units. The peak annotations indicate number of ethoxy units. The inset shows the nonaethoxy compound of benzonatate. B: The mass spectra confirm the [MþH]þ ion of three different variants. The reported molecular weight (M) for benzonatate is 603.7 Da with nine ethoxy units and is only one of several n-ethoxy compounds in the mixture. The overall hydrophobicity of the molecule increases with an increase in ethoxy unit chain length indicated by the increase in retention time. Our electrophysiological experiments used the mixture.

HEPES 10. The external solution was NaCl 145, KCl 5 mM, CaCl2 2, MgCl2 1, D-glucose 10, HEPES 10. Cells were manually held at approximately 70 mV and action potentials elicited with depolarizing pulses. Cells were perfused with external solution at 0.5 ml per minute using a syringe pump, through a chamber having a volume of 0.5 ml. Test solutions were applied through five-barrel square glass tubes and a perfusion system with computer-controlled valves (Warner VC-6MCS Perfusion Valve Control System; Warner Instruments, Hamden, CT). Test solutions in current clamp experiments were given using pressure application through patch pipettes with a PicoSpritzer III system (Parker Hannefin, Pine Brook, NJ). 2.6. Data analysis Analysis and curve fitting was performed with Clampfit 10 (Molecular Devices), SigmaPlot 11 (Systat Software, Inc., San Jose,

CA), and Excel (Microsoft Corporation, Redmond, WA). Data are shown as mean ± S.E.M. Statistical significance was determined using ANOVA and post-hoc Student's two tailed t tests. The statistical significance level was P < 0.05. 3. Results 3.1. Benzonatate is a mixture Benzonatate (CAS 104-31-4) in FDA product labeling is stated to have the formula 2, 5, 8, 11, 14, 17, 20, 23, 26-nonaoxaoctacosan-28yl p-(butylamino) benzoate with a molecular weight of 603.7 g/mol (Pfizer Laboratories, 2014). This compound has a side chain of nine repeated ethoxy units. However, benzonatate as defined in the original US Patent for the drug (Matter, 1955) is a mixture of nethoxy compounds where n may vary from 3 to 17. We performed HPLC-MS analysis of pharmaceutical-grade benzonatate (Zydus Pharmaceuticals) and benzonatate from two chemical suppliers

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Fig. 2. Benzonatate, but not its metabolite, blocks voltage-gated sodium currents. A: Benzonatate (100 mM) reversibly blocked Naþ currents. In CAD cells Naþ currents are primarily due to Nav1.7 type channels. Currents were elicited by 100 ms depolarizing steps from 80 to 10 mV every 20 s. B: Results from 10 CAD cells show that benzonatate (100 mM) produced 84% block. (Bars in this and following figures are S.E.M.) C: 4(butylamino)benzoic acid (BABA), the primary metabolite of benzonatate, does not inhibit Naþ currents at a concentration of 100 mM.

(Santa Cruz Biotechnology and Toronto Research Chemicals). We found that each was a mixture of compounds (Fig. 1). Benzonatate from each supplier had identical effects on Naþ currents. Data from each is included in these results and analyzed together. For our experiments, the reported benzonatate concentrations are calculated by assuming that the average molecular weight of the mixture was 603.7, the molecular weight of the 9-ethoxy compound. 3.2. Benzonatate blocks Naþ currents and action potentials We examined benzonatate effects on Naþ currents using whole cell recording in CAD cells. CAD cells are a cell line derived from a murine tyrosine-hydroxylase-positive neuronal tumor. They express tetrodotoxin-sensitive Naþ channels composed primarily of Nav1.7 with much smaller contributions from Nav1.1 and Nav1.3 (Wang et al., 2011; King et al., 2012). We found that Naþ currents in CAD cells were completely and reversibly blocked by tetrodotoxin (1 mM, N ¼ 5 CAD cells, data not shown), consistent with previous reports that CAD cells contain only tetrodotoxin-sensitive Naþ channels (Wang et al., 2011). Naþ currents were quickly and reversibly inhibited by benzonatate (100 mM; N ¼ 6 CAD cells; Fig. 2A and B). Consistent with its inhibition of Naþ currents, benzonatate also inhibited action potential firing. CAD cells studied in current clamp mode fired single action potentials when stimulated with a depolarizing pulse. Benzonatate (100 mM, N ¼ 6 cells) blocked action potential firing (Supplemental Figure 1). Lower benzonatate concentrations (1 mM) reduced action potential upstroke velocity and action potential peak but did not block spike firing (data not shown, with these effects seen in N ¼ 3 of 4 CAD cells tested at 1 mM). 3.3. Block is concentration- and state-dependent Local anesthetics are thought to preferentially bind to the

inactivated state of the Naþ channel. The channels are more inactivated at depolarized potentials, thereby making the degree of Naþ current antagonism by local anesthetics dependent on both concentration and voltage. Naþ current antagonism by benzonatate was both concentration-dependent and voltage-dependent (Fig. 3). Naþ currents were elicited using 50 ms depolarizations to 0 mV every 20 s from a holding potential of either 100 mV (N ¼ 5 CAD cells) or 70 mV (N ¼ 5 CAD cells; Fig. 3A). At 100 mV, Naþ current inactivation is minimal, but at 70 mV there is partial inactivation. Small inhibitory effects were seen with concentrations as low as 0.3 mM, especially when cells were held at 70 mV. 1000 mM benzonatate produced nearly 100% inhibition of Naþ currents (data not shown). Concentration-response curves fit to these data indicate a 50% inhibitory concentration (IC50) of 5.9 mM at a holding potential of 100 mV and 1.4 mM at a holding potential of 70 mV. We compared the voltage-dependence of inhibition by benzonatate (10 mM) to that of tetracaine (1 mM; Fig. 3C and D), which had similar potency at these concentrations. Benzonatate (10 mM) and tetracaine (1 mM) were applied to CAD cells (N ¼ 5 CAD cells for both drugs) and the effects of changes in voltage compared to nodrug controls (N ¼ 5) given the same voltage paradigm. Benzonatate and tetracaine at these concentrations both produced 40% inhibition at a holding potential of 100 mV. When the holding voltage was stepped to 50 mV the amount of inhibition compared to no-drug controls increased to 74% for benzonatate and 89% for tetracaine. Both tetracaine and benzonatate had markedly greater inhibitory effects when cells were held at 50 mV (many channels inactivated) compared to 100 mV (few channels inactivated), consistent with inactivation-state-dependent blockade. Since effects of benzonatate are relatively rapidly reversible, similar effects were seen in an experimental paradigm in which benzonatate (10 mM, N ¼ 5) was washed off between voltage changes (Supplemental Fig. 2).

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Fig. 3. Benzonatate Naþ current block is concentration- and state-dependent. A: Naþ currents were elicited from a steady holding potential of either 100 mV (open circles) or 70 mV (closed circles). Benzonatate was applied with stepwise increases in concentration (0.3, 1, 3, 10, 30 and 100 mM) every 2 min and then washed out. Percent inhibition was calculated compared to pre-drug control values. There was a clear increase in inhibition with increasing benzonatate concentrations, indicating concentration-dependence. The amount of inhibition was greater when the cells were held at 70 mV than when held at 100 mV, indicating voltage-dependence. B: Concentration-response curves were calculated from these data, showing that the IC50 for Naþ current block by benzonatate was 5.9 mM at 100 mV but considerably less, 1.4 mM, at 70 mV. C: For this experiment, benzonatate (10 mM, filled circles) or tetracaine (1 mM, filled triangles) were applied at time 0 s, and continued without interruption through the entire experiment. The results were compared to no-drug controls (open circles). The initial holding potential of 100 mV was stepped to 50 mV (indicated by the gray box) at 120 s and then back to 100 mV at 260 s. Controls have less current at depolarized potentials due to partial voltage-gated inactivation and less ionic driving force. Compared to controls, both benzonatate and tetracaine show much greater Naþ current block at 50 mV than at 100 mV, consistent with inactivation-state-dependent channel blockade. D: These traces are examples of Naþ currents at the times and voltages indicated in the graph in C.

3.4. Metabolites do not block Naþ currents In aqueous solution the ester bond of benzonatate spontaneously hydrolyzes, and in vivo hydrolysis is catalyzed by plasma and liver esterases, especially by plasma pseudocholinesterase. Its metabolites are BABA (Lin et al., 1999) and low molecular weight polyethylene glycol monomethyl ethers. BABA undergoes decarboxylation to butylaniline which oxidizes to form other metabolites (Chalardsunthornvatee and Thomas, 1961). Other ester-type local anesthetics are metabolized to para aminobenzoic acid (PABA). We tested the effects of 100 mM BABA on Naþ currents, and it had no effect (Fig. 2C). PABA (100 mM, N ¼ 10) had no effect on Naþ currents and neither BABA (100 mM) nor PABA (100 mM) pre-application or co-application affected benzonatate (100 mM) inhibition of Naþ currents (data not shown).

3.5. Phasic inhibition occurs at low concentrations Local anesthetics cause “tonic” inhibition of Naþ currents when currents are elicited at a slow rate, and “phasic” inhibition when currents are elicited at a rapid rate (Butterworth and Strichartz,

1990). We studied phasic inhibition by eliciting Naþ currents using 100 to 10 mV depolarizations for 10 ms at 20 Hz for 5 s. Significant phasic inhibition of Naþ currents occurred at low concentrations of benzonatate (1 mM, Fig. 4). In CAD cells, this protocol elicited some phasic inhibition even in control cells with no drug application. The magnitude of phasic inhibition was markedly increased by benzonatate (Fig. 4B, results from N ¼ 5 CAD cells shown). At this benzonatate concentration, phasic inhibition was about 50%, but tonic inhibition was much smaller, only 10%.

3.6. Inactivation is affected more than activation Naþ currents undergo voltage-gated activation followed by voltage-gated inactivation. Naþ currents may be inhibited by reducing their initial activation, or by reducing their recovery from inactivation. The currentevoltage (IeV) relationship in CAD cells was studied using 100 ms depolarizations from 80 mV to various voltages from 80 mV to þ40 mV (Fig. 5A). In every cell studied, benzonatate (10 mM, N ¼ 10) reduced Naþ currents at every potential, but the shape of the IeV curve was little affected (Fig. 5B).

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Fig. 4. Benzonatate at low concentrations causes phasic inhibition of voltage-gated sodium currents. A: Top CAD cells were tested for phasic inhibition of Naþ currents. In this example, benzonatate (1 mM) caused a small decrease in Naþ current of the first impulse (tonic inhibition), and progressively greater reductions in current with continued rapid stimulation (phasic inhibition). Bottom Phasic inhibition in this cell gradually increased with continued stimulation, and had not plateaued at the end of the 5 s train. Phasic inhibition partially reversed with washing. B: Phasic inhibition was tested with benzonatate (1 mM), which caused approximately 50% inhibition during the train of pulses. (“Ratio” indicates ratio of Naþ current to first current elicited in the train. Scale bar in A is 500 pA  1 ms.)

Fig. 5. Benzonatate affects voltage-gated inactivation of sodium currents more than activation. A: In this example, benzonatate (10 mM) reduced Naþ currents at every potential, but B: it did not affect the shape of the IeV curve. C: The current-conductance relationship was calculated from the data in A, and the points fitted using the Boltzmann equation. Benzonatate did not change the voltage-sensitivity of activation. D: Summary of effects of benzonatate (10 mM) on voltage-gated activation. On the average, benzonatate caused a slight shift of the activation curve to the right. E: Voltage-gated inactivation was studied using 500 ms conditioning pulses followed by a 10 ms test pulse (see inset in E). Benzonatate increased inactivation of Naþ currents during the conditioning pulses. A summary of effects of benzonatate (10 mM) on voltage-gated inactivation in 10 CAD cells is shown in the graph. Effects on inactivation were much more marked than on activation, with a 13 mV shift of the inactivation curve to the left.

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At a concentration of 1 mM benzonatate did not detectably affect either voltage-gated activation or inactivation. At 10 mM, benzonatate affected both phenomena, but voltage-gated inactivation (Fig. 5E) was affected much more than activation (Fig. 5D). The voltage-sensitivity of activation was studied by fitting the current-conductance curve with a Boltzman equation. There was no shift in activation in the example shown (Fig. 5C) and only a small shift in activation to more depolarized levels after benzonatate (10 mM) application in all cells studied (Fig. 5D, average of N ¼ 10 CAD cells). The voltage-sensitivity of inactivation was studied using 500 ms conditioning pulses to voltages from 120 mV to 0 mV prior to a 10 ms test pulse to þ10 mV (see inset in Fig. 5E). With benzonatate (10 mM) there was a shift in the inactivation curve to more hyperpolarized levels in every cell studied (Fig. 5C). The average shift was 13 mV (Fig. 5E, N ¼ 10 CAD cells). 3.7. Block is not selective for Nav1.7 Benzonatate at clinically-used doses is a specific remedy for cough. Muroi et al. (Muroi et al., 2013) have shown that selective inhibition of Nav1.7 in the nodose ganglion inhibits cough. We have shown that benzonatate strongly inhibits Naþ currents in CAD cells, indicating that it strongly inhibits Nav1.7. To determine whether benzonatate may be specifically selective for Nav1.7 we also studied tonic and phasic inhibition of Naþ currents in N1E-115 mouse neuroblastoma cells. These cells express primarily Nav1.3, with no detectable Nav1.7 (Jo and Bean, 2011). We found that tonic inhibition by benzonatate (100 mM, 80% inhibition, N ¼ 5 cells) and phasic inhibition by benzonatate (10 mM, 20% inhibition, N ¼ 5 cells) was quite similar in N1E-115 cells and CAD cells (Supplemental Fig. 3). These results indicate that benzonatate block of Naþ current is not selective for Nav1.7 over Nav1.3. 3.8. Benzonatate fractions have modestly different effects on Naþ currents Benzonatate is a mixture of different polyethoxy esters of 4(butylamino)benzoic acid. These compounds are expected to have very similar chemical properties, but will differ in molecular weight and corresponding properties such as hydrophobicity. Using HPLC, benzonatate was separated into three fractions based on relative hydrophobicity and molecular weight (Supplemental Fig. 4). Each fraction represents a group of n-ethoxy repeats. The low hydrophobicity fraction comprises primarily the 5, 6 and 7-ethoxy repeats. The intermediate hydrophobicity fraction comprises primarily the 8 and 9-ethoxy repeats. The high hydrophobicity fraction comprises primarily the 10, 11 and 12-ethoxy repeats. The effects on Naþ currents of the fractions with low, intermediate, and high hydrophobicity were compared. The overall concentration is expressed in micrograms/ml. 0.2 mg of each fraction was available for these studies and the number of experiments was limited. We compared effects on tonic inhibition of Naþ currents in CAD cells and N1E-115 cells with 0.60 mg/ml (similar to 1 mM benzonatate) and 12 mg/ml (similar to 20 mM benzonatate) of each fraction. We found that in both CAD and N1E-115 cells each fraction produced detectable inhibition of Naþ currents, with 12 mg/ml producing significantly more inhibition than 0.6 mg/ml in each case (Supplemental Figs. 5 A and B). Two factor ANOVA indicated a strong effect of concentration (N ¼ 3 cells per fraction, two concentrations per fraction; concentration F 48.3, P ¼ 0.000015, df ¼ 1) and of fraction type (fraction F 6.6, P ¼ 0.01, df ¼ 2). In CAD cells the effects of both concentrations on tonic inhibition were greatest with the intermediate, and less with both the low and high hydrophobicity fractions (low compared to intermediate statistically

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significant with 0.6 mg/ml, P ¼ 0.04 two tailed t test, N ¼ 3; high compared to intermediate statistically significant P ¼ 0.03, N ¼ 3). At a higher concentration of 12 mg/ml there was only a nonsignificant trend toward greater effects of the intermediate fraction. In N1E-115 cells, there was no apparent difference in the effects of the different fractions, but there was a strong effect of concentration (P ¼ NS, two tailed t test, N ¼ 3 cells per fraction, two concentrations per fraction). We also compared effects of each fraction on phasic inhibition at 0.60 mg/ml in CAD and N1E-115 cells (Supplemental Figs. 5C and D). One factor ANOVA showed significantly different effects of the fractions in both CAD cells (N ¼ 3 CAD cells per fraction; F 55.1, P ¼ 0.00014, df ¼ 2) and in N1E-115 cells (N ¼ 3 N1E-115 cells per fraction; F ¼ 16.7, P ¼ 0.0035, df ¼ 2). The low hydrophobicity fraction produced minimal phasic inhibition, the high hydrophobicity fraction moderate inhibition and the intermediate hydrophobicity fraction the most phasic inhibition, in both CAD and N1E115 cells. These results indicate that all benzonatate components have detectable effects on Naþ currents even in concentrations as low as 1 mM. The intermediate hydrophobicity fraction was most effective for both tonic and phasic inhibition in CAD cells and more effective on phasic inhibition in N1E-115 cells. The intermediate fraction contains the 9-ethoxy benzonatate component that Matter (Matter, 1955) reported to be most effective for cough inhibition. Our results suggest that the 9-ethoxy component may also be especially potent with respect to Naþ channel inhibition. 4. Discussion Benzonatate is a popular orally administered FDA-approved cough suppressant. In 2013 approximately 25 million physician prescriptions were written in the US for cough medicines, and 7.2 million for benzonatate. It is an unusual drug because it is not a pure compound but consists of a mixture of various polyethoxy esters of 4-(butylamino)benzoic acid. Its mechanism of action has been poorly understood. Our study has shown that benzonatate inhibits voltage-gated sodium channels, with similar effects on both Nav1.7 and Nav1.3 subtypes. A benzonatate fraction of intermediate hydrophobicity, containing the 9-ethoxy component most effective in cough, is also most potent in inhibition of Na þ channels. Benzonatate is metabolized by plasma and liver esterases to BABA (Wetherell and French, 1986). Our study showed that BABA does not inhibit voltage-gated sodium channels and does not inhibit the potency of co-applied benzonatate. Like other local anesthetics, benzonatate exhibits both tonic and phasic inhibition and its effects depend on both drug concentration and inactivation state of the channel. Its potency is somewhat less than that of tetracaine, another ester-type local anesthetic. Inhibitory effects on Na þ channels occur with concentrations of less than 1 mM. Our results are consistent with local anesthetic effects on sensory nerve fibers of the respiratory system being the mechanism of cough inhibition by benzonatate. Benzonatate is thought to work through a peripheral mechanism, inhibition of pulmonary stretch receptors. The cough reflex is complex (Canning, 2010). Cough can be initiated by a variety of sensory stimuli, chemical and mechanical, and is mediated by different fiber types. Sensory afferents responsible for cough initiation originate in the pharynx or respiratory passages, travel via the vagus nerve to the nodose and jugular ganglia of the vagus and then to the brainstem. The initial sensory stimulation then reflexly elicits the inspiratory phase of cough. During inspiration, myelinated pulmonary stretch receptors are activated. Stretch receptors signal the degree of lung inflation and reflexly elicit the expiratory, phase of cough. Muroi et al. (Muroi et al., 2013) demonstrated that the

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cough reflex elicited by pressure requires intact Nav1.7 in nodose ganglion neurons. It is likely that all types of vagal sensory neurons, including the myelinated fibers mediating pulmonary stretch reflexes, strongly express Nav1.7 (Kwong et al., 2008). Our results are compatible with the notion that benzonatate may inhibit cough through inhibition of Nav1.7 channels in either pharyngeal or pulmonary afferent fibers or in the pulmonary stretch receptors. The first description of benzonatate was in US Patents issued to Matter (Matter, 1955) in 1955, describing a series of polyethoxy esters of 4(butylamino)benzoic acid. Chemically it resembles the ester-type local anesthetics such as tetracaine, procaine and cocaine. Our results indicate that it has the effects on Naþ channels expected of local anesthetics, and with a potency similar to that of another ester-type local anesthetic, tetracaine. Most local anesthetics are weak bases. They are tertiary amines and charged at physiological pH. Benzonatate is a secondary amine and so is neutral at physiological pH. It has a pKa of 3.47, similar to benzocaine, another neutral ester-type local anesthetic, which has a pKa of 2.78. However, benzonatate is physiologically quite dissimilar to benzocaine in that it displays significant phasic-type inhibition, but benzocaine does not (Neumcke et al., 1981; Bekkers et al., 1984; Schneider and Dubois, 1986). At the time benzonatate was developed, antitussive effects were thought to require selectivity for pulmonary stretch and tactile receptors over thermoreceptors (BUCHER, 1956). Hence, benzonatate was developed with a long polyethylene glycol monomethyl ether group, with the expectation that would enhance its affinity for myelin over the unmyelinated fibers responsible for thermal sensation (Chappel et al., 1963). The addition of the n-ethoxy groups conferred selectivity for stretch receptors with n from 6 to 18, with a minimal activity on thermoreceptors at n ¼ 9 (BUCHER, 1956). It is reasonable to speculate that the n-ethoxy chain of benzonatate also contributes to its ability to modulate sodium channels. Nonphysiological amphiphiles containing a polyethoxy chain can alter hydrophobic coupling between sodium channel and their surrounding membranes. This induces a hyperpolarizing shift in channel inactivation (Lundbaek et al., 2004), thereby reducing sodium currents. Our study has confirmed that benzonatate is a mixture of compounds with varying numbers of ethoxy units in the side chain. Benzonatate has long been thought to be a local anesthetic because of its numbing effects and chemistry, and we have shown that benzonatate, the mixture, has effects like local anesthetics on Naþ channels. Polyethylene glycols differ very little in their chemistry, but there are important differences in their physical properties. Hence, the length of the ethoxy chain in the different benzonatate components is not expected to affect their chemical properties but may affect their physical properties like hydrophobicity. Differences in hydrophobicity can potentially affect anesthetic potency, rate of onset, and type of inhibition (Butterworth and Strichartz, 1990). Although benzonatate is a mixture, the ‘title compound’ with 9 ethoxy units has been shown to have the greatest antitussive effect (Matter, 1955). To determine whether the various compounds contained in benzonatate may have differential effects on Naþ channels, we separated benzonatate into low, intermediate and high hydrophobicity fractions. In CAD cells, which express primarily Nav1.7, all fractions displayed tonic inhibition of Naþ currents at both concentrations tested. However, the intermediate fraction was the most potent for both tonic and phasic inhibition. That result is consistent with previous results showing that intermediate ethoxy chain lengths of 9 were most effective in suppressing responses of pulmonary stretch receptors (BUCHER, 1956; Bein and Bucher, 1957) and cough (Herzog, 1956), and is consistent with our initial hypothesis that effects on Nav1.7 may account for cough suppression. Nevertheless, all fractions had clear effects in

CAD cells. In addition, all fractions had approximately the same effects in N1E-115 cells, which contain primarily Nav1.3, so channel-subtype-specific effects of whole benzonatate as well as its components appear to be minimal. Benzonatate is administered orally, and for it to affect cough through local anesthetic effects on sensory afferents it must be absorbed by the gut and transported in the bloodstream to the respiratory system. Benzonatate serum concentrations in humans have been reported only rarely. After overdose, one report indicated a concentration of 4.1 mM (2.5 mg/ml) in a patient after ingestion of 36 capsules of 100 mg benzonatate (Crouch et al., 1998), and another reported a blood level of 58 mM (35 mg/ml) in a fatal overdose (Cohan et al., 1986). Research subjects ingesting 200 mg by topical oral administration had a peak serum concentration of less than 3.3 mM (2 mg/ml) (Kelly et al., 1993). Analysis of human levels is complicated by the presence of numerous similar compounds in the clinically-used mixture and a relatively short half-life due to metabolism by plasma and liver esterases, primarily pseudocholinesterase. It has been suggested that measurement of its more stable metabolite BABA may be a better measure of benzonatate dosage (Lin et al., 1996). We found significant inhibition of Naþ currents by benzonatate at 1 mM or less, indicating that benzonatate effects on Naþ channels occur at serum concentrations that are attained in humans. Benzonatate effects on cough suppression were originally shown in the 1950's and its ability to selectively suppress pulmonary stretch receptors when given systemically used whole animal preparations. The mechanism of that selective effect is still unclear, but our data are consistent with Nav1.7 channel inhibition as a possible mechanism, since benzonatate inhibits Naþ channels at concentrations found in humans, it strongly affects channels firing in a phasic manner, which would affect rapidly firing stretch receptors preferentially, and the intermediate fraction of benzonatate is most effective both for Naþ channel inhibition and for cough suppression. Although our results indicate that Nav1.7 type channels are strongly affected by benzonatate, they are not selectively affected compared to the Nav1.3 type. Confirmation of the hypothesis will require specific study of pulmonary stretch receptors. Although inhibition of Naþ channel function in pulmonary stretch receptors is an attractive possible mechanism for cough inhibition by benzonatate, the cough reflex is complex, and the complete cellular mechanism may vary for different coughproducing stimuli. Inhibition of vagal sensory fibers that detect irritative stimuli is another possible mechanism. The gag reflex does not involve pulmonary stretch receptors, and this mechanism could explain the reported ability of benzonatate to suppress gag (Lineback, 1962), such as during bronchoscopy (Gregoire et al., 1958; Cattaneo, 1959; Feinsilver, 1961) or overdose, and cough during tracheostomy (Fior, 1957). Lidocaine, another local anesthetic, has antitussive effects that may involve inhibition of transient receptor potential ankyrin 1 (TRPA1) and transient receptor potential vanilloid 1 (TRPV1) channels in bronchial nerve fibers (Docherty et al., 2013; Rivera-Acevedo et al., 2013; Piao et al., 2009; Leffler et al., 2011), although neither of these effects has yet been described for benzonatate. Benzonatate has long been used for cough suppression, and due to its chemistry and numbing effects has been classified as a local anesthetic. Unlike other local anesthetics it is dosed orally rather than parenterally, and exerts its therapeutic effects far from its point of entry, not “locally”. Our study has shown that benzonatate, like other local anesthetics, has inhibitory effects on voltage-gated Naþ channels. Benzonatate effects on voltage-gated Naþ channels are not selective for specific Nav subtypes, although certain of its polyethoxy components have more potency than others. Benzonatate inhibition of Naþ channel function occurs at concentrations

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shown to be attained in humans, making local anesthetic effects on peripheral sensory nerve fibers mediating cough a plausible mechanism of action. 5. Conclusions - The antitussive benzonatate is a mixture. - Benzonatate strongly inhibits voltage-gated sodium currents. - Benzonatate is not selective for the Nav1.7 subtype important in the cough reflex. - Benzonatate inhibition is reversible, concentration-dependent and channel-state-dependent. - Benzonatate affects channel inactivation more than activation. - Benzonatate causes both tonic and phasic inhibition of sodium currents. - Sodium channel inhibition is a possible mechanism for cough suppression by benzonatate. Acknowledgments The authors wish to thank Richard A. Bryant, MD, Assistant Professor of Anesthesiology, The Ohio State University for his helpful reading of this manuscript. Funding was provided by University of Louisville startup research funds to MSE and University of Illinois at Springfield startup research funds to SRJ. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.neuropharm.2015.09.020. References Bein, H.J., Bucher, K., 1957. Anesthetic effects on pulmonary stretch receptors and other nervous substrates; pharmacology of tessalon. Helv. Physiol. Pharmacol. Acta 15, 55e62. Bekkers, J.M., Greeff, N.G., Keynes, R.D., Neumcke, B., 1984. The effect of local anaesthetics on the components of the asymmetry current in the squid giant axon. J. Physiol. 352, 653e668. BUCHER, K., 1956. New effect mechanism of the antitussive drug tessalon. Schweiz Med. Wochenschr. 86, 94e96. Butterworth, J.F., Strichartz, G.R., 1990. Molecular mechanisms of local anesthesia: a review. Anesthesiology 72, 711e734. Canning, B.J., 2010. Afferent nerves regulating the cough reflex: mechanisms and mediators of cough in disease. Otolaryngol. Clin. North Am. 43, 15e25 vii. Cattaneo, A.D., 1959. Experiences with tessalon in peroral endoscopy. Minerva Anestesiol. 25, 373e375. Chalardsunthornvatee, P., Thomas, R.E., 1961. The stability of solutions of amethocaine hydrochloride. Aust. J. Pharm. 800e802. Chappel, C.I., Von Seeman, C., 1963. In: Ellis, G.P., West, G.B. (Eds.), Antitussive Drugs, Progress in Medicinal Chemistry. Bath, Great Britain, Butterworth & Co., Limited, pp. 89e133. Cohan, J.A., Manning, T.J., Lukash, L., Long, C., Ziminski, K.R., Conradi, S.E., 1986. Two fatalities resulting from Tessalon (benzonatate). Vet. Hum. Toxicol. 28, 543e544. Crouch, B.I., Knick, K.A., Crouch, D.J., Matsumura, K.S., Rollins, D.E., 1998. Benzonatate overdose associated with seizures and arrhythmias. J. Toxicol. Clin. Toxicol. 36, 713e718. Docherty, R.J., Ginsberg, L., Jadoon, S., Orrell, R.W., Bhattacharjee, A., 2013. TRPA1 insensitivity of human sural nerve axons after exposure to lidocaine. Pain 154, 1569e1577. Feinsilver, O., 1961. The use of tessalon in the preparation of the patient for bronchoscopy and bronchography. Curr. Ther. Res. Clin. Exp. 3, 451e456. Fior, R., 1957. Treatment of cough in otorhinolaryngology by a new derivative of

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