Preclinical evaluation of the epithelial sodium channel inhibitor BI 1265162 for treatment of cystic fibrosis

Abstract

Background: Epithelial sodium channel (ENaC) is an important regulator of airway surface liquid volume; ENaC is hyperactivated in cystic fibrosis (CF). ENaC inhibition is a potential therapeutic target for CF. Here, we report in vitro and in vivo results for BI 1265162, an inhaled ENaC inhibitor currently in Phase II clinical development, administered via the Respimat® Soft Mist™ inhaler.

Methods: In vitro inhibition of sodium ion (Na⁺) transport by BI 1265162 was tested in mouse renal collecting duct cells (M1) and human bronchial epithelial cells (NCI-H441); inhibition of water transport was measured using M1 cells. In vivo inhibition of liquid absorption from rat airway epithelium and acceleration of mucociliary clearance (MCC) in sheep lungs were assessed. Fully differentiated normal and CF human epithelium was used to measure the effect of BI 1265162 with or without ivacaftor and lumacaftor on water transport and MCC.

Results: BI 1265162 dose-dependently inhibited Na⁺ transport and decreased water resorption in cell line models. BI 1265162 reduced liquid absorption in rat lungs and increased MCC in sheep. No effects on renal function were seen in the animal models. BI 1265162 alone and in combination with CF transmembrane conductance regulator (CFTR) modulators decreased water transport and increased MCC in both normal and CF airway human epithelial models and also increased the effects of CFTR modulators in CF epithelium to reach the effect size seen in healthy epithelium with ivacaftor/lumacaftor alone.

Conclusion: These results demonstrate the potential of BI 1265162 as a mutation agnostic, ENaC-inhibitor-based therapy for CF.

FIGURE 1

Concentration–response curves and IC₅₀ values for BI 1265162 in NCI-H441 cells. Inhibition of Na⁺ transport by BI 1265162 was tested in M1 and NCI-H441 cells. Cells were cultivated on polyester Transwell^® filters to electrically tight monolayers. Filters containing cell layers were inserted into a Ussing chamber and short-circuit current (I_SC) measured at increasing BI 1265162 final concentrations. I_SC was compared against maximum inhibition with amiloride and IC₅₀ calculated. IC₅₀: inhibition concentration that reduced the effect by 50%; M1: mouse renal collecting duct cell line.

FIGURE 2

Inhibition of water resorption by BI 1265162 in M1 cell line. Cells were cultivated on polyester Transwell^® filters to electrically tight monolayers. Apical liquid volume replaced with either saline control or saline containing 3 µM BI 1265162. Tritiated water was then added and mixed with the apical volume of the Transwell^®, and water resorption measured by scintigraphy using a radiometric dilution assay. ^#: p<0.0001, unpaired t-test.

FIGURE 3

Inhibition of lung fluid absorption by BI 1265162 in rats. Ringer lactate solution pH 5 alone or with increasing concentrations of BI 1265162 was instilled into the lungs of male Wistar rats. Three hours after instillation, animals were sacrificed and the weight of the lungs was determined. Inhibition of liquid transport was calculated from lung weights in the treated group versus lung weights of a negative control group, and an untreated control group was used as reference. Data are presented as mean±sem. ^#: p<0.0001, one-way ANOVA, Dunnett's multiple comparison test.

FIGURE 4

Effect of BI 1265162 on a) retention of tracer in lung of sheep and b) K⁺ concentration in sheep; n=2 per dose. Adult ewes were administered varying doses of BI 1265162 or vehicle using an AirLife™ nebuliser. Mucociliary clearance was measured by administration of aerosolised technetium-labelled sulfur colloid followed by γ scintigraphy. Serial images were obtained over a 2-h period, and counts were obtained from the right lung. Blood was collected and plasma potassium was determined to assess renal effects of BI 1265162. Data are presented as mean (range). ^#p=0.0027 and 0.0003 at 2 h after radiolabel administration, vehicle versus 1 μg·kg⁻¹ and 10 μg·kg⁻¹, respectively; one-way ANOVA.

FIGURE 5

Effect of BI 1265162 on a) water transport and b) MCC in normal and CF MucilAir^TM cultures. The effect of BI 1265162 with or without CFTR modulators and isoproterenol (an upregulator of CFTR expression) on water transport and MCC was evaluated in a pseudostratified, fully differentiated 3D model of human epithelium (n=5 cultures per treatment) employing bronchial cells from a CF and a non-CF donor. Water transport was measured on Days 2 and 5 of the study by weighing the membrane inserts containing the cells. MCC was measured on Day 7 by video-tracking the movements of polystyrene microbeads added to the apical surface of MucilAir™. Error bars show standard error of mean. MCC: mucociliary clearance; CF: cystic fibrosis; CFTR: cystic fibrosis transmembrane conductance regulator; ISO: isoproterenol; IVA/LUM: ivacaftor/lumacaftor. ^#: MucilAir™ culture medium + penicillin/streptomycin + 0.1% dimethyl sulfoxide. *: p<0.05; **: p<0.01; ***: p<0.001; ^¶: p<0.0001 vehicle versus BI 1265162.