Inositol polyphosphate derivative inhibits Na+ transport and improves fluid dynamics in cystic fibrosis airway epithelia

Abstract

Amiloride-sensitive, epithelial Na(+) channel (ENaC)-mediated, active absorption of Na(+) is elevated in the airway epithelium of cystic fibrosis (CF) patients, resulting in excess fluid removal from the airway lumen. This excess fluid/volume absorption corresponds to CF transmembrane regulator-linked defects in ENaC regulation, resulting in the reduced mucociliary clearance found in CF airways. Herein we show that INO-4995, a synthetic analog of the intracellular signaling molecule, D-myo-inositol 3,4,5,6-tetrakisphosphate, inhibits Na(+) and fluid absorption across CF airway epithelia, thus alleviating this critical pathology. This conclusion was based on electrophysiological studies, fluid absorption, and (22)Na(+) flux measurements in CF airway epithelia, contrasted with normal epithelia, and on electrophysiological studies in Madin-Darby canine kidney cells and 3T3 cells overexpressing ENaC. The effects of INO-4995 were long-lasting, dose-dependent, and more pronounced in epithelia from CF patients vs. controls. These findings support preclinical development of INO-4995 for CF treatment and demonstrate for the first time the therapeutic potential of inositol polyphosphate derivatives.

Fig. 1

INO-4995 and INO-4913. The structure of the pro-drug, INO-4995, is depicted on the right side of the figure (3). INO-4995 ester groups are hydrolyzed by intracellular esterases releasing the active compound, INO-4913 (2). INO-4995 and INO-4913 are analogs of the endogenous signaling molecule, Ins(3,4,5,6)P₄ (1).

Fig. 2

Dose dependence of the long-term effect of INO-4995 on spontaneous short-circuit current (I_sc) and resistance in cystic fibrosis (CF) and non-CF airway epithelia. Monolayers of differentiated CF nasal epithelial cultures were exposed to INO-4995 applied to solution bathing the mucosal membrane for 2 h. The monolayers were washed and then returned to air-liquid interface (ALI) for 22 h before being mounted in Ussing chambers for testing. In these experiments, INO-4995 inhibited the basal I_sc in CF human nasal airway epithelia with an EC₅₀ of 10–20 μM. Vehicle [PBS containing 1% 1:1, DMSO+DMSO containing 5% (wt/vol) Pluronic F-127] present in both treatment and control for the 2-h exposure period. A: representative experiment contrasting basal I_sc in monolayers incubated for 2 h with two different concentrations of INO-4995 vs. vehicle control and measured 22 h after exposure: 6 traces are shown, 3 from monolayers pretreated with 40 μM INO-4995 (dotted line), 1 from monolayer pretreated with 10 μM INO-4995 (dashed line), and 2 vehicle-treated controls (solid lines). B: comparison of the dose dependence of INO-4995 inhibition of basal I_sc in human nasal epithelia (CF vs. non-CF). Control CF (n = 14); control non-CF (n = 16). Differences between the means of the CF vs. non-CF samples were evaluated for statistical significance via Student’s unpaired t-test. *P = 0.035; **P = 0.0012; ***P < 0.001. C: comparison of various doses of INO-4995 on transepithelial resistance in CF human nasal epithelia. Data are means ± SD; n = 3–17 monolayers.

Fig. 3

INO-4995 inhibits basal I_sc in Madin-Darby canine kidney (MDCK) cells overexpressing human epithelial Na⁺ channels (hENaC). A: representative I_sc tracings are shown for MDCK cells incubated for 22 h in INO-4995 (100 μM; dashed line) or vehicle control (solid line). Amiloride (10⁻⁴ M) was added to both monolayers after baseline currents were recorded and the decrease in I_sc was recorded. Amiloride was then washed out of the luminal solution, and currents returned to basal values that were again sensitive to inhibition by amiloride (10⁻⁴ M). The amiloride-sensitive current represents ~82% of the baseline current in control cells and 91% in INO-4995-treated cells, demonstrating that the major current in both of these conditions is mediated by ENaC. B: summary data of the amiloride-sensitive current in INO-4995 treated monolayers (open bars) and vehicle-treated monolayers (solid bars). All values are means ± SE, n = 8. INO-4995 incubation results in a significantly reduced amiloride-sensitive current in these cells compared with vehicle-treated control cells (P < 0.01). C: patch-clamp recording of an excised outside-out patch from a 3T3 cell expressing rat αβγ-ENaC subunits. Channel transitions from selected consecutive traces at −60 mV are shown before (Control, sweep 13) and during bath application of INO (20 μM, INO, sweeps 14–18). An abrupt decrease in channel activity was observed within 1.5-min drug exposure. Inset: recording from the same patch (−40 mV) showing bath amiloride (10 μM, horizontal line) reversibly inhibited channel activity; horizontal and vertical scale bars are 5 s and 0.2 pA, respectively. Slope conductance (7.6 picoSiemens in 150 mM Li⁺) and amiloride-inhibited channel activity confirmed ENaC identity. Dashed lines through traces represent closed-channel current level. D: open channel diary of recording shown in C. Open probability (P_o) ranged from 0.13 to 0.80 (sweeps 1-13, control) and showed no evidence of time-dependent reduction in P_o before drug exposure (INO, see bar). E: summary effects of INO on ENaC activity. Channel activity (NP_o, N = number of channels) was reduced ~28% in the presence of INO relative to control (Control NP_o = 2.55 ± 0.56 vs. INO NP_o = 1.84 ± 0.58, means ± SE; n = 9, P = 0.042). Solid lines connect symbols of NP_o recorded before and after 1- to 5-min bath exposure to INO (20 μM) from the same patch. Experiments were performed at ~23°C.

Fig. 4

Effect of INO-4995 on basal I_sc in apically permeabilized monolayers. A: amiloride inhibits basal I_sc and obliterates difference between control and INO-4995-treated monolayers. I_sc responses in monolayers pretreated with INO-4995 are contrasted with control monolayers incubated with vehicle control. Nystatin was used to permeabilize the apical membrane, as described in METHODS. INO-4995 (10 μM) was administered to the monolayers for 2 h, removed, and cells were mounted in Ussing chambers 4 h later. Subsequent permeabilization of the apical membrane with nystatin evokes an identical shift in I_sc in both control (solid line) and INO-4995-treated (dashed line) monolayers. This current is completely inhibitable by blocking the Na⁺-K⁺-ATPase with ouabain. B: effect of INO-4995 is abolished after permeabilization of the apical membrane with nystatin. Subsequent change following amiloride (100 μM) addition is indicative of effectiveness of the nystatin permeabilization. Ouabain-sensitive current is indistinguishable between INO-4995-treated (dashed line) and control (solid line) monolayers. C: no difference is seen with INO-4995 pretreatment (10 μM, 2 h, tested after 22 h) on I_sc across apically permeabilized monolayers with a basolateral to apical K⁺ grandient. Amilo-ride (100 μM) was present during the experiment. Nystatin (360 = μg/ml) was added at the indicated time.

Fig. 5

INO-4995 inhibits fluid absorption in CF airway epithelia. A: dose response of amiloride inhibition of absorption rate in cultured CF human nasal epithelial cells. Average absorption rates were calculated from Δ [Blue Dextran] over 18 h of amiloride exposure, as described in METHODS. Data are means ± SD (n = 6 for each condition). Control: vehicle. *P < 0.05; **P < 0.001; ***P < 0.005. B: dose response curve for INO-4995 inhibition of fluid absorption measured with the Blue Dextran assay, as described in METHODS. Data show the INO-4995 dose-dependent change in fluid absorption rates from the control values, means ± SD. Control: vehicle [1% 1:1, DMSO+DMSO containing 5% (wt/vol) Pluronic F-127] present during the 2-h exposure period. Differences between means of treated and control samples were evaluated for statistical significance using Student’s unpaired t-test: *P = 0.038; **P = 5.1 × 10⁻⁶; ***P = 1.8 × 10⁻⁸. C: comparison of the effect of a 2-h exposure with 50 μM INO-4995 vs. 50 μM amiloride on absorption rate after 42 h in cultured CFHNE. Data are means ± SD for n = 6 monolayers. Statistical significance (P = 0.0007) vs. control was determined by Student’s unpaired t-test. Vehicle [Krebs-Ringer containing 0.1% 1:1, DMSO+DMSO containing 5% (wt/vol) Pluronic F-127] present in both treatment and control for the 2-h exposure period.