Mutations in the cystic fibrosis membrane conductance regulator (CFTR) gene lead to the development of cystic fibrosis, the most common fatal genetic disease in Caucasians (Ratjen and Döring, 2003). CFTR mutations result in defective ion transport in the epithelia of various organs, including the gut, pancreas, and lung. In the airway, the impairment of CFTR-mediated anion secretion (Quinton, 1989) together with the hyperabsorption of sodium via the epithelial sodium channel (ENaC) (Knowles et al., 1981; Davies et al., 2005) limits mucosal hydration, thereby reducing the volume of airway surface liquid available to support both effective ciliary beat and the constitution of an appropriately hydrated mucus gel (Boucher, 2007). A direct consequence of these changes in mucosal hydration is an impairment of airway mucus clearance by both mucociliary and cough mechanisms predisposing patients to chronic infection and inflammation resulting in a progressive loss of lung function (Boucher, 2007; Livraghi and Randell, 2007).

Several therapeutic opportunities are being tested clinically with the goal of restoring mucosal hydration in the CF airway. Inhaled hypertonic saline has been demonstrated to enhance mucociliary clearance (MCC) and to both improve lung function and reduce exacerbation frequency in CF patients (Donaldson et al., 2006; Elkins et al., 2006). The potassium-sparing diuretic amiloride, a direct blocker of ENaC, has also been demonstrated to enhance MCC in CF patients (Köhler et al., 1986; App et al., 1990), although the lack robust clinical benefit observed in several large studies has been ascribed to the short duration of action of this drug in the airway combined with its low potency (Knowles et al., 1990; Graham et al., 1993; Bowler et al., 1995). As such, long-acting amiloride derivatives such as 552-02, have been specifically designed for inhaled dosing and are presently in clinical trials for the treatment of CF lung disease (Hirsh et al., 2008). Negative regulation of ENaC function therefore represents a tangible approach for the treatment of respiratory diseases associated with impaired mucus clearance.

Recent in vitro and in vivo studies have revealed a role for protease regulation of ENaC function in transporting epithelia (Planès and Caughey, 2007). The original observation that the Kunitz-type serine protease inhibitor aprotinin attenuated ENaC activity in Xenopus kidney epithelia (Vallet et al., 1997) was extended into primary cultures of human bronchial epithelial cells by Bridges and colleagues (2001). More recently, Kunitz-type serine protease inhibitors were also demonstrated to potently attenuate ENaC function in vivo in the guinea pig airway after topical administration into the lung (Coote et al., 2008). The nature of the “channel-activating protease” (CAP) responsible for the activation of ENaC in the airway epithelium is presently unknown. A variety of coexpression studies, homology searches, and gene silencing studies support the serine protease prostasin (PRSS8) as the airway CAP (Vallet et al., 1997; Donaldson et al., 2002; Tong et al., 2004; Myerburg et al., 2008). However, additional CAPs, including matriptase (List et al., 2007), furin (Hughey et al., 2004), tissue kallikrein (Picard et al., 2008), and HNE (Caldwell et al., 2005), may also have activity in the airway relevant to ENaC regulation. The identification of low-molecular weight (LMW) inhibitors of the airway epithelial CAP would therefore represent potentially novel therapies to attenuate ENaC activity and enhance mucociliary clearance for the treatment of CF lung disease. The aim of the present study was to test the hypothesis that LMW inhibitors of the airway CAP in vitro would translate into an attenuation of ENaC activity in the airways in vivo, both mechanistically and also in terms of the predicted increase in the rate of mucociliary clearance. To this end, the present study has identified the trypsin-like serine protease inhibitor camostat (NVP-QAU145; Mospan; Foipan; FOY305, HBC276) as a potent, LMW inhibitor of several candidate CAPs in biochemical assays. Camostat is in clinical use as a trypsin-like serine protease inhibitor for the treatment of pancreatitis and postoperative reflux esophagitis and was initially demonstrated to inhibit prostasin. This activity translated into an attenuation of ENaC-mediated short-circuit current (I_SC)inprimary cultures of airway epithelial cells. In vivo, direct instillation of camostat into the airway inhibits the amiloride-sensitive tracheal potential difference in guinea pig and enhances mucociliary clearance in the conscious sheep. This profile of pharmacological activity is consistent with a protease-dependent regulation of ENaC in the airway that represents an approach to the treatment of CF lung disease.

Materials and Methods

In Vitro Protease Inhibition

The constitutively active form of human prostasin, matriptase, and furin were produced as described previously (Boudreault et al., 1998; Friedrich et al., 2002; Shipway et al., 2004). Human trypsin (HTI, Essex Junction, VT) and human neutrophil elastase (HNE; Innovative Research of America, Inc., Sarasota, FL) were purchased from commercial sources. Enzymes were diluted into the appropriate activity buffer (prostasin, matriptase, and trypsin: 15 mM HEPES, pH 7.4, 25 mM NaCl, and 0.5% CHAPS; furin: 15 mM HEPES, pH 7.6, 25 mM NaCl, 1 mM CaCl₂, 1 mM β-mercaptoethanol, and 0.5% CHAPS; HNE: 15 mM HEPES, pH 7.6, 150 mM NaCl, and 0.75% CHAPS), added to compound solutions, and incubated at room temperature for 20 min. Fluorogenic substrates (prostasin, Ac-KHYR-amc; matriptase, Ac-RKFK-amc; furin, Ac-SNRFKR-amc; trypsin, Boc-VPR-amc; and HNE, Ac-AAPV-amc) were mixed with activity buffer and added to the enzyme/compound solution after the 20-min incubation. Fluorescence emission was quantified on a Gemini plate reader (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK) at 37°C. Excitation wavelength was 380 nm, emission wavelength was 450 nm, and cut-off was 435 nm. K_i values were determined through analysis of progression curves using BioKin PlateKi (BioKin Inc., Madison, WI). Aprotinin, soybean trypsin inhibitor (SBTI), and α-1-antitrypsin were from Sigma-Aldrich (St. Louis, MO). α1-PDX was from Calbiochem (Nottingham, UK). Camostat mesylate was purchased from Sequoia Research Products (Reading, UK). 4-Guanidino-benzoic acid 4-carboxymethyl-phenyl ester (4-GBCE) and p-guanidino-benzoic acid (PGBA) (Fig. 1) were synthesized by Novartis Global Discovery Chemistry (Horsham, UK). Placental bikunin was produced as described previously (Coote et al., 2008).

Cell Culture

Human Bronchial Epithelial Cells. Non-CF-derived human bronchial epithelial cells (HBEs) (Lonza, Workingham, UK) were cultured at an air-liquid interface as described previously (Atherton et al., 2003) on Snapwell inserts (Costar, Cambridge, UK). In some studies, from the first day of establishment of an air-liquid interface, HBEs were fed with a DMEM (50%) in Ham's F-12 media containing 2% Ultroser G (Pall BioSepra, Cergy St. Christophe, France) with gentamicin (50 μg/ml) and amphotericin B (50 ng/ml), rather than the BEGM-supplement-based media (Atherton et al., 2003). CF-derived HBEs were isolated as described previously (Devor et al., 2000) cultured under Ultroser G-containing media conditions. Cells were used between 14 to 21 days after establishing the air-liquid interface.

NCI-H441 Cells. NCI-H441 cells (American Type Culture Collection, Manassas, VA) were cultured in plastic flasks in growth medium (RPMI 1640 medium) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin mixture (1000 U/ml penicillin and 1000 μg/ml streptomycin), 1% ITS-A solution (Invitrogen, Paisley, UK), and 2 mM l-glutamine. Media were refreshed every 48 to 72 h until cells were 85% confluent. Cells were detached using trypsin-EDTA and seeded onto Snapwell inserts at a density of 1 × 10⁶ cells/insert with growth media on both apical and basolateral surfaces until confluent. Apical media were then aspirated, and the cells were cultured in differentiation media [growth media with FBS reduced to 4%, plus 10 nM l-thyroxine (Sigma Chemical, Poole, Dorset, UK), and 200 nM dexamethasone (Sigma Chemical)] at an air-liquid interface a further 7 to 14 days before use. At all stages of culture, the cells were maintained at 37°C in 5% CO₂ in an air incubator.

Guinea Pig Tracheal Epithelial Cells. Epithelial cells were isolated from the tracheas of male Dunkin-Hartley guinea pigs using a 6-h protease digest (0.15% protease type XIV; Sigma Chemical) in DMEM/Ham's F-12 containing DNase 1 (10 μg/ml; Sigma Chemical), amphotericin B (1 μg/ml), gentamicin (50 μg/ml), and 1% penicillin-streptomycin mixture. Cells were then seeded onto collagen-coated (human placental collagen type IV; Sigma Chemical) Snapwell inserts (5 × 10⁵ cell/ml) in BEGM-supplement-based media (as described for HBE culture above) containing 5% FBS and 1% penicillin-streptomycin mixture. At 24 h after seeding, cells were fed with serum-free BEGM-supplement-based media until confluent. At this time, apical media were aspirated, and the cells were fed for a further 2 days in Ultroser G-based media as described for HBE culture (see above). At all stages of culture, the cells were maintained at 37°C in 5% CO₂ in an air incubator. All cell culture products were from Invitrogen (Paisley, UK).

Treatment of Epithelial Cells with CAP Inhibitors and Measurement of Short-Circuit Current

Before the addition of protease inhibitors, any apical fluid was aspirated from the surface of the cells while maintained in the culture plate. Vehicle (100 μl; 50% DMEM in BEGM with no supplements) or test compounds were then added to the apical surface. Cells were maintained at 37°C in 5% CO₂ in an air incubator for 90 min (unless otherwise stated). At the completion of the compound incubation step, Snapwell inserts were mounted in vertical diffusion chambers (Costar UK) and were bathed with continuously gassed Ringer's solution (5% CO₂ in O₂; pH 7.4) maintained at 37°C containing 120 mM NaCl, 25 mM NaHCO₃, 3.3 mM KH₂PO₄, 0.8 mM K₂HPO₄, 1.2 mM CaCl₂, 1.2 mM MgCl₂, and 10 mM glucose. The solution osmolarity was always between 280 and 300 mOsmol/kg H₂O for all physiological salt solutions used. CAP inhibitor compound concentrations were maintained in the apical chamber throughout the study unless otherwise stated. Cells were voltage-clamped to 0 mV (model EVC4000; WPI, Stevenage, UK) and the I_SC was measured. R_T was measured by applying a 1- or 2-mV pulse at 30-s intervals and calculating R_T by Ohm's law. Data were recorded using a PowerLab workstation (ADInstruments Ltd., Chalgrove, Oxfordshire, UK). The baseline I_SC together with the responses to the addition of amiloride (10 μM apical side), forskolin (0.6 μM apical and basolateral sides), or trypsin (excess to apical side) was then recorded. Data are expressed as either absolute mean I_SC or ΔI_SC values ± S.E.M. (in microamperes per square centimeter). Aprotinin (200 μg/ml; 31 μM) was used as a positive control in most studies, and concentrations of test compounds required to inhibit 50% of the aprotinin-sensitive I_SC were calculated using GraphPad Prism (GraphPad Software Inc., San Diego, CA). A one-way analysis of variance (ANOVA) with a post hoc Dunnett's test, with significance assumed when p < 0.05, was used for making comparisons between test compounds and vehicle controls.

Guinea Pig Tracheal Potential Difference Model

All studies were performed in accordance with the guidelines of the United Kingdom Home Office on the operation of the Animals (Scientific Procedures) Act 1986 and were approved by the local Ethical Review Process. The guinea pig tracheal potential difference (TPD) was measured as described recently for the testing of negative regulators of ENaC function in the airway (Coote et al., 2008). In brief, male Dunkin-Hartley guinea pigs (300–650 g) were placed under short-term inhalation anesthesia (halothane/N₂O) and were administered 200 μl of either vehicle (5% glucose solution; Baxter Healthcare, Newbury, Berkshire, UK) or test compound by intratracheal instillation (via the mouth) using a modified oral gavage needle (Vet-Tech Solutions, Congleton, Cheshire, UK). Animals were then allowed to recover and were fully ambulatory before the TPD procedure. At various times after the completion of intratracheal dosing with test compounds, animals were placed under surgical anesthesia using a midazolam, ketamine, and xylazine regimen. The trachea was then visualized near the sternum using minimal blunt dissection, and a lateral incision was made in the trachea near the sternum. The bevelled end of an exploring agar (3.5% in Hanks' balanced salt solution) electrode was inserted into the lumen of the trachea, and a reference electrode was placed adjacent to the trachea in electrical contact with the flesh of the throat. Potential difference measurements were taken via an IsoMil (WPI) and were recorded using a PowerLab workstation (ADInstruments Ltd.). After the measurement of TPD, animals were sacrificed by anesthetic overdose and exsanguination. Data are expressed as mean absolute potential difference values ± S.E.M. (millivolts) and were compared using a one-way ANOVA with a post hoc Dunnett's test, with significance assumed when p < 0.05. Dose-response data were fitted using GraphPad Prism (GraphPad Software Inc.), and the dose required to induce 50% of the compound effect (ED₅₀) was calculated.

Sheep Mucociliary Clearance Model

All procedures were approved by the Mount Sinai Animal Research Committee to ensure the humane care and treatment of experimental animals. Adult ewes (25–45 kg) were restrained in an upright position in specialized body harness in modified carts. The head of the animal was immobilized, and after local anesthesia of the nasal passage was induced with 2% lidocaine, the animals were nasally intubated with a standard endotracheal tube (7.5 mm in diameter; Mallinckrodt, Hazelwood, MO). A flexible bronchoscope was used to guide the tube and verify its position in the trachea. After intubation, the animals were allowed rest for approximately 20 min before administration of any agent. All aerosols were generated using a Raindrop Nebulizer (Nellcor Puritan-Bennett, Carlsbad, CA), which produces a droplet with a MMAD of approximately 1.1 μm. To maximize central deposition in the airways, a dosimeter was used to deliver the radiolabeled material. The nebulizer was connected to the dosimeter system consisting of a solenoid valve and a source of compressed air (20 psi). The output of the nebulizer was connected to a T-piece, with one end attached to a respirator (Harvard Apparatus Inc., Holliston, MA). The system was activated for 1 s at the onset of the inspiratory cycle of the respirator, which was set at an inspiratory/expiratory ratio of 1:1 and a rate of 20 breaths/min. A tidal volume of 300 ml was used to deliver the test compounds or vehicle (water) to dryness. Aerosolized technetium-labeled sulfur colloid (^99mTc-SC) was used to measure the effects of the various doses of test compounds or control on mucociliary clearance. Approximately 20 mCi of ^99mTc-SC in a total volume of 2 ml of sterile saline was placed in the nebulizer. A tidal volume of 500 ml was used to deliver the ^99mTc-SC for 3 min. A gamma camera (Dyna Cam; Picker Corp., Nothford, CT) integrated with a computer was used to record and analyze the clearance of ^99mTc-SC over 2 h. During the time period between administration of the agent and nebulization of the radioaerosol, the animals were connected to a Bennett humidifier (Nellcor Puritan-Bennett) to avoid desiccation of the airways. Periodic gavages with tap water via a nasogastric tube were performed to avoid dehydration. After ^99mTc-SC nebulization, the animals were immediately extubated and positioned in their natural upright position underneath the gamma camera so that the field of image was perpendicular to the animals' spinal cord. After acquisition of a baseline image, serial images were obtained over a 2-h period at 5-min intervals for the first hour and then every 15 min for the next hour. All images were obtained and stored in the computer for analysis. An “area of interest” was traced over the image corresponding to the right lung of the animals, and counts were recorded. The left lung was excluded from analysis because its corresponding image is superimposed over the stomach and counts could be affected by swallowed radiolabeled mucus. The counts were corrected for decay and the percentage of radioactivity remaining relative to the baseline image was calculated at serial time points. Data are expressed as the percentage of inhaled radiolabel ^99mTc-SC cleared from a the right lung over time. Differences in clearance of ^99mTc-SC were compared at both 60 and 120 min after radioaerosol administration using a one-way ANOVA. A significant difference between groups was followed up with a Student-Newman-Keuls test to determine pairwise differences, with significance assumed when p < 0.05.

Results

In Vitro Inhibition of Candidate CAPs. The effects of both macromolecular and low-molecular weight serine protease inhibitors on proteases described previously as candidate CAPs were studied. The Kunitz-type protease inhibitors, aprotinin and placental bikunin, both inhibited human prostasin and matriptase (Table 1) in contrast to SBTI and α1-anti-trypsin. Furin activity was unaffected by any of the macromolecular inhibitors studied. Camostat (Fig. 1) also inhibited the activity of prostasin and matriptase, although with significantly greater potency on matriptase. 4-GBCE (Fig. 1), the major metabolite of camostat, also inhibited both prostasin and matriptase, although with a reduced potency on both proteases compared with parent. There was no effect of camostat or 4-GBCE on the activity of furin or HNE. All of the inhibitors studied inhibited the activity of trypsin.

Protease Inhibitor Regulation of ENaC Activity in Human Airway Epithelial Cells. Consistent with previous reports that have studied the effects of macromolecular protease inhibitors on non-CF human airway epithelial ion transport (Bridges et al., 2001; Donaldson et al., 2002), the amiloride-sensitive I_SC was sensitive to inhibition by the Kunitz-type inhibitors aprotinin (31 μM) and placental bikunin (1 μM) but was insensitive to SBTI (10 μM) and α1-antitrypsin (1 μM) after a 90-min preincubation (Fig. 2). Using HBEs isolated from four non-CF donors, aprotinin (31 μM; 90 min) induced a mean 65.5 ± 2.4% attenuation of the amiloride-sensitive I_SC (n = 12 independent studies using 50 cell inserts), with an IC₅₀ value of 809 nM (mean of two independent studies) (Fig. 2, a–c). Placental bikunin (1 μM; 90 min) inhibited the amiloride-sensitive I_SC by 64.4 ± 1.8% (n = 4), with an IC₅₀ value of 10 nM (Fig. 2d). The prohormone convertase inhibitor α1-PDX (0.1 to 10 μM) was without effect on the amiloride-sensitive I_SC, inducing a maximal mean 17.4 ± 7.0% decrease in current at the highest concentration studied (10 μM; p = 0.22). At 10 μM, α1-PDX significantly attenuated the transepithelial resistance (220 ± 18 Ω · cm² compared with 495 ± 85 Ω · cm² in the vehicle control group; p = 0.02; n = 4) and was not therefore examined at higher concentrations. Over the course of these studies, there was no effect of aprotinin (31 μM) on the transepithelial resistance (-0.2 ± 7.1% change from vehicle-treated controls). The post-amiloride, forskolin-stimulated I_SC response was likewise unaffected by macromolecular protease inhibitor pretreatment (Fig. 2b; mean data not shown).

In common with the Kunitz-type inhibitors, camostat also attenuated the amiloride-sensitive I_SC in primary normal human bronchial epithelial cells by 76.2 ± 5.8%, with an IC₅₀ value of 58 ± 29 nM (n = 3 independent studies using 15 cell inserts) (Fig. 3, a and b). Transepithelial resistance was not significantly affected by the highest concentration of camostat studied (30 μM; 10.9 ± 4.7% increase over vehicle control). The forskolin-stimulated increase in I_SC was likewise unaffected by camostat (30 μM) (19.7 ± 5.5% increase over vehicle control; p > 0.05). 4-GBCE, the primary metabolite of camostat, also inhibited the amiloride-sensitive I_SC in normal HBEs, with an IC₅₀ value of 400 nM (Fig. 3, c and d). Next, the activity of camostat was confirmed in HBEs from four ^{ΔF508/ΔF508}CFTR donors, generating a mean IC₅₀ value of 23 ± 3 nM (n = 4 independent studies using 21 cell inserts) (Fig. 4). In CF HBE, forskolin-induced a small (<2 μA/cm²) and variable increase in I_SC that was also unaffected by pretreatment with either aprotinin or camostat.

We next addressed whether ENaC activity could be regulated by a CAP-mediated mechanism in the small airway epithelium, using NCI-H441 cells as a model of the bronchiolar epithelium (Shaul et al., 1994). Aprotinin induced a mean 78.3 ± 2.7% decrease in amiloride-sensitive I_SC (n = 3 independent studies using 15 cell inserts), with an IC₅₀ value of 959 nM, whereas there was no significant effect of SBTI (10 μM) or α1-antitrypsin (1 μM) (Fig. 5, a and b). Camostat attenuated the amiloride-sensitive I_SC in NCI-H441 cells by 58.1 ± 4.7%, with an IC₅₀ value of 98 nM (Fig. 5c).

To increase confidence that camostat and related LMW CAP inhibitors were functioning through a CAP inhibitory mechanism, studies were performed to assess the sidedness and reversibility of the compound activity. When added to the basolateral surface of normal HBEs, neither aprotinin nor camostat significantly influenced the magnitude of the amiloride-sensitive I_SC, supporting the selectivity of camostat for an apically located target (Fig. 6a). Consistent with an inhibitory activity on an apically located CAP(s), the addition of an excess of trypsin to the apical surface of the HBEs, treated previously for 90 min with camostat, induced a significant rise in the amiloride-sensitive I_SC (50.1 ± 1.5 μA/cm²) that was not observed in the vehicle-treated cells (1.2 ± 0.6 μA/cm²) (Fig. 6, b–e).

Temporal Analysis of CAP Inhibitor Activity on HBE Cells. Extended incubations of HBE with aprotinin or camostat were performed to assess whether the magnitude of the inhibition of the amiloride-sensitive I_SC could be increased beyond the 60 to 70% observed with a 90-min treatment. Normal (non-CF) HBEs were exposed to supramaximal concentrations of either apical aprotinin (31 μM) or camostat (3 μM) for 90 min or 6, 24, and 48 h (media and test compounds were refreshed at 6 and 24 h). Aprotinin induced a time-dependent attenuation of the I_SC, inhibiting 74.3 ± 1.5% of the amiloride-sensitive I_SC at 90 min (p < 0.01) that had further increased to an 87.9 ± 0.7% inhibition by 24 h (p < 0.01). There was no further inhibition of amiloride-sensitive I_SC by 48 h (89.9 ± 1.3%) (Fig. 7a). Likewise, extended incubation with camostat induced a time-dependent attenuation of the amiloride-sensitive I_SC, with a 59.3 ± 1.6% (p < 0.01), 72.0 ± 1.5% (p < 0.01), and 80.7 ± 1.2% (p < 0.01) inhibition observed at 90 min, 6 h, and 24 h, respectively (Fig. 7b).

In a separate study, non-CF HBEs were treated with either vehicle, aprotinin (31 μM), or camostat (30 μM) for 90 min at which time the apical dosing solution was aspirated, and the mucosa was rinsed three times with warmed phosphate-buffered saline (0.5 ml). The amiloride-sensitive I_SC was then measured using individual groups of HBEs in Ussing chambers at 1, 3, and 6 h after washing (Fig. 7c). Amiloride-sensitive I_SC was attenuated by both aprotinin (61.5 ± 2.4% inhibition; p = 0.006) and camostat (54.5 ± 1.3% inhibition; p < 0.01) in the cells examined immediately upon completion of the treatment with CAP inhibitor. By 1 h after washing, the amiloride-sensitive I_SC remained significantly attenuated in both groups, although the degree of inhibition had diminished to 36.3 ± 3.7% (p < 0.01) in the aprotinin group but was maintained at 69.3 ± 1.9% (p < 0.01) by camostat. By 3 h after washing, the amiloride-sensitive I_SC had returned to control levels in the aprotinin group (5.2 ± 17.7% inhibition; p > 0.05), whereas inhibition was maintained at 57.5 ± 7.4% (p < 0.01) by camostat. The inhibitory effect of camostat on the amiloride-sensitive I_SC was still apparent at 6 h after washing (70.8 ± 6.5%; p < 0.01).

Effects of Camostat on the Bioelectric Properties of the Guinea Pig Airway Epithelium in Vitro and in Vivo. Before in vivo testing, the activity of camostat on cultured guinea pig tracheal epithelial cells was examined. Freshly isolated and cultured guinea pig tracheal epithelial cells displayed a spontaneous amiloride-sensitive I_SC, with an ENaC blocker potency order of 552-02 (1.8 ± 0.3 nM) > benzamil (19 ± 1 nM) > amiloride (271 ± 37 nM) >> 5-(N-ethyl-N-isopropyl)amiloride (>30,000 nM) (mean IC₅₀ values shown in parentheses; n = 4–9 independent experiments). The amiloride-sensitive I_SC was attenuated by both aprotinin (63.8 ± 1.5% at 310 μM) and camostat (86.3 ± 1.6% at 10 μM), with IC₅₀ values of 530 and 46 nM, respectively (Fig. 8, a and b). 4-GBCE did not significantly attenuate the amiloride-sensitive I_SC (Fig. 8c).

Camostat was next tested for activity on the tracheal potential difference in guinea pigs in vivo. In the present studies, vehicle-dosed guinea pigs displayed a spontaneous TPD of approximately -10 mV, consistent with published values in this region of the trachea (Coote et al., 2008). At 2 h after intratracheal dosing, camostat attenuated the TPD from -10.3 ± 1.0 mV in vehicle-dosed animals to a maximum of -4.7 ± 0.5 mV (p < 0.001; n = 6–8 animals/group), with a calculated ED₅₀ value of 3 μg/kg i.t. (Fig. 9a). In a separate study to test the activity of camostat at 5 h after dosing, the TPD was attenuated from -9.9 ± 0.6 to -5.7 ± 0.4 mV (p < 0.01; n = 5–9 animals/group), with an ED₅₀ value of 26 μg/kg i.t. (Fig. 9b).

To confirm whether camostat was attenuating the guinea pig TPD through an ENaC-mediated pathway, camostat was coadministered with amiloride. Amiloride (6000 μg/kg i.t.) and camostat (100 μg/kg i.t.) attenuated the TPD from -14.4 ± 0.7 to -6.7 ± 0.8 mV and -7.0 ± 0.1 mV, respectively (p < 0.001). When combined into the same dosing formulation, the combination of both agents at these doses attenuated TPD to -6.5 ± 0.2 mV (p < 0.001), a value that was not different from that obtained with either agent alone (p > 0.05; n = 6–7 animals/group) (Fig. 9c). 4-GBCE (trifluoroacetate salt), at a dose of 150 μg/kg i.t., was without significant effect on the TPD measured at 2 h after dosing (Fig. 9d).

Effects of Camostat on Mucociliary Clearance in the Sheep. The effects of inhaled camostat on the airway clearance of ^99mTc-SC were studied in conscious sheep. Camostat or vehicle was dosed either 2 or 5 h before the administration of ^99mTc-SC, and mucociliary clearance was then monitored for 120 min. A single inhaled dose of either 3 or 9 mg of camostat, dosed at 2 h before the assessment of clearance, was sufficient to significantly increase the 60 min ^99mTc-SC clearance (p < 0.001; n = 3–5 animals/group). By 120 min after ^99mTc-SC administration, the lower dose of 1.5 mg of camostat had also significantly enhanced clearance (p < 0.005) (Fig. 10a). Larger doses of camostat were required to observe activity at the 5-h assessment. Doses of either 30 or 60 mg of camostat significantly enhanced clearance 60 min into the assessment (p < 0.001; n = 3–5 animals/group). The 15-mg dose had significantly increased clearance by the end of the 120-min assessment (Fig. 10b).

Discussion

Desiccation of the airway epithelial mucosa has been widely linked to the development of lung disease in CF as a consequence of the loss of CFTR-mediated chloride secretion together with ENaC-mediated Na⁺ absorption (Boucher, 2007). Acute treatment with the inhaled ENaC blocker amiloride (Köhler et al., 1986; App et al., 1990) has been demonstrated to enhance mucociliary clearance in CF patients and healthy controls, consistent with a mechanism that increases airway mucosal hydration. The long-term clinical benefit of inhaled amiloride has, however, been inconsistent, potentially because of the low potency and short duration of action in the airways of a compound originally designed to be rapidly eliminated by the kidney (Burrows et al., 2006). Furthermore, a single study has reported a negative impact of inhaled amiloride on the beneficial effects of inhaled hypertonic saline (Donaldson et al., 2006). Consequently, new therapeutic candidates that will attenuate airway ENaC function are required to enable the clinical significance of ENaC function in the CF lung to be tested.

The observation that ENaC activity is positively regulated by CAPs has enabled the evaluation of an alternative therapeutic approach to inhibit ENaC function that is independent of direct channel blockade. In the present study, we report the identification of camostat, a LMW inhibitor of airway epithelial CAPs, that is in clinical use as a trypsin-like serine protease inhibitor (Ishi et al., 1980) for the treatment of pancreatitis and postoperative reflux esophagitis. Camostat was initially identified as a potent inhibitor of prostasin and was subsequently selected for additional profiling.

The inhibitory activity of camostat on ENaC function was confirmed in both normal HBE (four donors) and CF HBE from five donors carrying the ^{ΔF508/ΔF508}CFTR mutation. The role of CAP regulation of ENaC was also confirmed in NCI-H441 cells, a human small airway epithelial cell line of Clara cell origin (Shaul et al., 1994). Camostat and 4-GBCE, together with aprotinin and placental bikunin, potently attenuated amiloride-sensitive I_SC in HBEs, with a potency order of placental bikunin > camostat >4-GBCE > aprotinin >> SBTI =α1-antitrypsin. This potency order was not consistent with that observed with any of the putative candidate CAPs examined in biochemical assays. For example, α1-antitypsin was without effect on amiloride-sensitive I_SC in the HBE and NCI-H441 models but potently inhibited matriptase in the biochemical assay (K_i = 1 nM), a profile that would be inconsistent with a salient role for matriptase as an airway CAP. The prohormone convertase furin has been demonstrated to positively regulate ENaC activity in both engineered cell systems and also murine kidney epithelia (Hughey et al., 2004). In the present study, the HBE-active CAP inhibitors did not inhibit furin catalytic activity in the biochemical assays. Furthermore, the furin inhibitor α1-PDX failed to significantly influence ENaC function in HBE at concentrations up to 10 μM. These observations are therefore inconsistent with a salient role for furin as a rate-limiting CAP in the HBE model. Each of the HBE-active inhibitors did, however, demonstrate inhibitory activity toward prostasin, although with a divergent potency order between the biochemical and cell-based assays. The enhanced potency of the LMW inhibitors in the cell-based models could reflect the increased incubation time relative to the biochemical assay. Camostat and 4-GBCE are pseudoirreversible inhibitors that will form reversible covalent acylenzyme intermediates (Ramjee et al., 2000). An increased incubation time could therefore enhance the observed potency of this class of inhibitors. Alternatively, this divergent potency order could question the relevance of prostasin as a salient CAP in these model systems.

Consistent with activity on an apically located CAP, aprotinin and camostat only attenuated amiloride-sensitive I_SC when added to the apical surface of HBE. Furthermore, the apical addition of an excess of trypsin reversed the aprotinin and camostat-induced attenuation of the I_SC, supporting the hypothesis that camostat was working through a CAP inhibitor mechanism. Muto et al. (1994) reported that the serine protease inhibitor nafamostat and two of its metabolites (PGBA and 6-amidino-2-napthol) directly blocked ENaC. PGBA is also a metabolite of camostat and the potential for a direct ENaC blocking mechanism needed to be evaluated. If camostat/PGBA was directly blocking ENaC, trypsin would not have been predicted to recover the I_SC. PGBA (10 μM) also failed to affect amiloride-sensitive I_SC in HBEs on either acute addition in Ussing chambers, or after a 90-min incubation. The exogenous addition of HNE to CAP inhibitor treated airway epithelial cells has also been reported to restore ENaC function in an analogous fashion to trypsin (Caldwell et al., 2005). With reported high levels of HNE in the CF airways (Ordoñez et al., 2003), this “CAP redundancy” could represent an issue with the CAP inhibitor approach.

Consistent with previous reports, CAP inhibition attenuated approximately 55 to 75% of the amiloride-sensitive I_SC by 90 min in our human airway epithelial cell models. An important question was whether this degree of channel inhibition could be increased with an extended CAP inhibitor exposure, because it might be reasonably assumed that a greater inhibition of ENaC function would have a more profound impact mucosal hydration. Extending the incubation time of HBEs with either aprotinin or camostat beyond 90 min resulted in a further significant attenuation of amiloride-sensitive I_SC that was maximal by 24 h. We next asked how prolonged the CAP inhibitor-mediated attenuation of ENaC function was and how camostat compared with aprotinin. Camostat maintained a maximal inhibitory activity on ENaC for at least 6 h after compound washout, in contrast to aprotinin that had lost all inhibitory activity by 3 h. The prolonged activity of camostat over aprotinin may be because of different mechanisms of protease inhibition. Kunitz-type inhibitors aprotinin and placental bikunin are competitive, reversible inhibitors, whereas camostat and 4-GBCE are pseudoirreversible inhibitors (Ramjee et al., 2000). It is possible that camostat forms a slowly reversible covalent complex with its target CAP, thereby maintaining CAP inhibition after free, unbound compound has been washed away. Together, these observations are consistent with camostat inducing a prolonged CAP inhibition, providing an opportunity to maximize attenuation of ENaC function.

Coote et al. (2008) recently demonstrated sensitivity of the guinea pig TPD to Kunitz-type macromolecular serine protease inhibitors in vivo. In the present study, aprotinin demonstrated a similar in vitro potency in cultured GPTEC to that observed in HBE. Camostat activity was also confirmed in the GPTECs, with a potency similar to that observed in the HBEs. Having confirmed cross-species reactivity with the airway CAP, camostat was tested in vivo for effects on airway ENaC function. In vivo, camostat (mesylate) potently attenuated the guinea pig TPD, with ED₅₀ values of 3 and 26 μg/kg i.t. at 2 and 5 h after compound dosing, respectively. This compares favorably with the acute potency of amiloride (ED₅₀ of 14 μg/kg i.t.) in this model (Coote et al., 2008). It is noteworthy that at a dose of 6000 μg/kg i.t. amiloride failed to significantly attenuate the TPD by 4 h after dosing in this model (K. Coote and H. Danahay, unpublished observations), consistent with camostat having a more prolonged efficacy than amiloride in vivo. When camostat was coadministered with a maximal dose of amiloride, by 2 h after dosing there was no greater effect of the combined agents together than was observed with either alone, consistent with camostat attenuating ENaC function in the guinea pig trachea. The observation that 4-GBCE failed to significantly attenuate TPD in guinea pigs, at a dose 50-fold higher than the camostat ED₅₀ value, suggests that in vivo efficacy is because of the activity of camostat rather than its primary metabolite.

We next tested whether inhaled camostat would enhance MCC in the sheep, as has been demonstrated with direct ENaC blockers (Hirsh et al., 2004, 2008). A 1.5-mg administered dose of camostat (mesylate) significantly increased MCC between 2 to 4 h after compound administration. The degree of maximal MCC enhancement (achieved with a 3-mg dose) was similar to the maximum achieved with a direct ENaC blocker (Hirsh et al., 2004, 2008) in a similar model system. A dose of 30 mg of camostat was required to maximally enhance MCC between 5 to 7 h after compound dosing. Amiloride is rapidly cleared from sheep (Mentz et al., 1986) and human airways (Noone et al., 1997), with estimated half-lives in airway surface fluid of 10 to 23 min, respectively, and with a short pharmacodynamic action. As such, inhaled camostat represents an opportunity to achieve a prolonged mucokinetic activity in the lung relative to amiloride.

In summary, we have identified camostat as a LMW inhibitor of the airway CAP(s) in both normal and CF airway epithelia. That camostat attenuated ENaC function in the airways in vivo and enhanced MCC with a prolonged duration of action provides additional validation to promote this compound toward a clinical evaluation of its mucokinetic potential for the treatment of CF lung disease.