Osmolytes and ion transport modulators: new strategies for airway surface rehydration

Abstract

Mucociliary clearance (MCC) in CF lung disease is limited by airway dehydration, leading to persistent bacterial infection and inflammation in the airways. Agents designed to rehydrate the airway mucosa lead to improved MCC. Hyperosmolar agents, such as hypertonic saline and mannitol, create a luminal osmotic gradient, drawing water into the dehydrated ASL. Ion transport modulators function to activate alternative chloride channels and/or to block sodium hyperabsorption that occurs through a dysregulated ENaC channel. Combinations of these therapies may result in a synergistic improvement in airway hydration, and thus, restore MCC. Active ongoing phase II and III trials of new pharmacotherapeutics are covered in this review.

Figure 1:

(A) XZ-confocal image of a cross section of airway epithelial cells stained with calcein, a fluorescent green dye, and ASL stained with Texas Red Dextran, a cell impermeable red dye. (B) After administration of an osmotic stimulus (HS), there is an increase in ASL height along with a decline in cell height. (C) HS aerosolized to the surface of HBE cultures results in a significant but transient increase in ASL height, lasting under an hour. N=8. Rectangle represents duration of HS administration (8μg NaCl/cm²/min for 15 min).

Figure 2:

Effects of variation in HS delivery rates on ASL volumes responses. (A) Comparison of ASL heights achieved with three rates of nebulized HS (low, 3μg NaCl/cm²/min; moderate, 8μg NaCl/cm²/min; and high, 18 μg NaCl/cm²/min) with duration varied to hold constant total delivered mass of salt (demonstrated by length of bar at top). (B) Both absolute height change and total hydration (integrated area over time) favored the mid-range dosing regimen but did not reach statistical significance (A; P=0.388). (C,D,E) Dashed lines indicates the expected rise in ASL height if all salt deposited resulted in an equimolar flux of water into the ASL. Solid black lines indicate the actual change in ASL height observed during experiments. The failure of these two lines to overlay indicates an inhibition of the transport of water from the epithelial cells into the ASL. (C) Low dose nebulization (3μg NaCl/cm²/min). Theoretical slope 4.72 μm/min versus experimental slope of 0.45 μm/min, p<0.0001. (D) Medium dose nebulization (8μg NaCl/cm²/min). Theoretical slope 14.92 μm/min versus experimental slope of 8.66 μm/min, p=0.0002. (E) High dose nebulization (18μg NaCl/cm²/min). Theoretical slope 51.49 μm/min versus experimental slope of 20.78 μm/min, p=0.0004. N=4 per experimental condition.

Figure 3:

Effect of sodium reabsorption on HS-mediated ASL height. (A) Experimental data of hypertonic saline (at 8μg NaCl/cm²/min) in the absence and presence of a potent sodium channel blocker (VX-371 at 50 μg/ml). (B) Mathematical model predicting the results of HS effect with normal ENaC conductance (solid line, consistent with experimental data) and 15% of ENaC channel normal conductance (dashed line).

Figure 4:

Cell volume changes in response to osmotic stimulus of HS at varying HS deposition rates. A 20% decrease in cell height in response to the hypertonic stimulus was observed at all rates. Cell height did not recover during the time frame of the experimentation (60 minutes). Cell volume reduction was a consistent finding regardless of rate of HS administration. (n=4 for each condition).

Figure 5:

Incubation with mercury chloride apically or basolaterally resulted in a diminished ASL response to aerosolized HS (at 8μg NaCl/cm²/min) compared with native cultures. (A) Experimental data. (B) Mathematical model of experimental data. N=8 for each condition. (C) Cell height change to aerosolized HS in the absence (control) or presence of selective block of the apical or basolateral cell membrane with mercury chloride.

Figure 6:

Sequential ASL volume responses to aerosolized 7% HS for 15 min (8μg NaCl/cm²/min). (A) Sequential ASL volume responses to aerosolized HS. A smaller ASL response to a second administration of aerosolized HS was observed when two identical HS doses were separated by 15 minutes. Break in x-axis indicate time between two doses. For each dose, HS was nebulized at 8 μg NaCl/cm²/min, administered continuously for 15 minutes. The ASL height of the second peak was about 60% of the first peak. (B) A hypotonic saline rinse was interposed between HS administration. The hypotonic solution administration produced cellular swelling associated with improved ASL volume responses to a subsequent administration of HS (~140% of first dose). (C) Interposition of an isotonic rinse between the first and second HS administration. The same second dose effect is not seen when doses of HS were separated by an isotonic saline bolus. ASL height of second peak persisted ~50% of the first peak. (D) In model simulation, the water permeability of the HBE apical membrane during the second dose was reduced to 10% of the basal water permeability. N=4 for each condition.

Figure 7:

The effects of hyperconcentrated mucus on HBE surfaces on nebulized HS (at 8μg NaCl/cm²/min) in inducing ASL volume responses. (A) In the normal (2% mucus) state (solid line), there was a 3-fold increase in ASL volume during nebulization. HBE cultures with hyperconcentrated mucus (12%) (Dashed line) exhibited a 7-fold increase in ASL above baseline ASL. The duration of ASL being increased above basal levels was also increased in hyperconcentrated mucus HBE cultures. (B) Rates of reabsorption (time to baseline ASL, in μm/min) were significantly different between the normal and the CF-like hyperconcentrated mucus cultures. (C) Two sequential doses of HS administered to hyperconcentrated cultures. A similar response in ASL height was noted for the two HS administrations. (D) Dashed line indicates the expected rise in ASL height if all salt deposited remained on the surface and resulted in an equimolar flux of water into the ASL. Solid line indicates the actual change in ASL height observed during experiments. Compare to Figures 2C–E: In the presence of an intact mucus layer (12% solids), actual and expected ASL height more closely approximated each other early in the HS delivery interval. Theoretical slope 14.48 μm/min versus experimental 7.61 μm/min, p=0.03.

Figure 8:

Increasing the durability of HS delivery. (A) Experimental data showing the effect of HS alone (8 μg NaCl/cm²/min) or in the presence of a long-term sodium channel blocker (VX-371) over 8 hours. (B) Mathmatical modeling shown the effect of inhibiting 85% of ENaC current (i.e. 15% residual activity) on ASL height over 8 hours. Note that the effect of HS alone was so transient that the increase in ASL height (seen at 30 minutes; refer to Figure 1B) was completed by the first sampling point at one hour.