Symposium 24: Enhancing CFTR functions- What’s In The Pipeline Blocking sodium channels. Marcus Mall, MD. Talk from the 35 th European Cystic Fibrosis Congress; June 6-9, 2012; Dublin, Ireland

The imbalance of sodium absorption and chloride secretion is critical in the pathogenesis of CF lung disease, leading to airway surface fluid depletion, mucus plugging and the subsequent inflammation and infection characteristic of CF lung disease. In the Enhancing CFTR Functions-What’s In The Pipeline symposium, Marcus Mall, MD of the Division of Pediatric Pulmonology, Translational Lung Research Center, University of Heidelberg in Germany, described on-going research.

"The low volume hypothesis suggests that dehydration of the airway surface liquid (ASL) in the lung is caused by an imbalance of both chloride and sodium ions. This imbalance leads to mucostasis in the airway lumen and provides an environment for bacterial colonization that allows chronic infections. The low volume hypothesis was validated in a mouse model, which showed increased sodium transport in the airways of mice with CF-like disease, which is very similar to what we see in children in early CF lung disease," he explained.

Based on this principle, a promising new class of potent human epithelial sodium channel (ENaC) blockers, which includes P643, is currently undergoing active research. These compounds are structurally distinct from amiloride, a potassium-sparing diuretic that blocks ENaC but did not show long term effectiveness in CF ENaC-Tg mice trials. Amiloride caused a mucolytic effect in the proximal and distal airways in neonatal ENaC-Tg mice, and an anti-inflammatory effect (reduced neutrophilia and interleukin 8), but without any effect on mucus obstruction and inflammation in adult ENaC-Tg mice.

By contrast, investigations using novel ENaC blockers revealed robust therapeutic effect, including reducing mucus obstruction and inflammatory cell reduction, in adult ENaC-Tg mice. P643 inhibited ENaC with IC₅₀ of 30nM in human bronchial epithelial cells in vitro, as well as in vivo using a guinea pig model. In a sheep model, it also accelerated mucociliary clearance. P643 is about 50x more potent and more readily absorbed than amiloride; the effects translate into keeping fluid longer in the lumen of the airways, Dr. Mall said.

Similar effects in the active regulation if ENaC can be attained through manipulation of serine proteases and anti-proteases, he continued. "Normally, anti-proteases are diluted by high volume conditions, allowing membrane-bound proteases to activate the sodium channel and absorb the excess volume. Under low-volume conditions, however, the anti-proteases become concentrated and inhibit protease activity. This aggravates airway surface dehydration in CF airways."

Furthermore, chronic neutrophil inflammation increases the activity of neutrophil elastase, which further activates ENaC, thereby aggravating ENaC-medicated ASL depletion. This suggests that inhibitors, especially low molecular weight inhibitors of both channel activating protease and neutrophil elastase, may be viable approaches to hydrating the airways in patients with CF.

The channel activating protease (CAP) inhibitor camostat has been shown to inhibit ENaC function in airway epithelia, both in vitro and in vivo using a sheep model. CAP was also shown to enhance prolonged accelerated mucociliary clearance, in a dose dependent fashion, according to the early results using this approach.

"In addition to CAP inhibitors, there are also several small-molecule neutrophil inhibitors currently in development. These protease inhibitors may actually emerge as an alternative to ENaC blockers in the near future", Dr Mall said.

Dr. Peter Mogayzel and Dr. Marcus Mall, Division of the Pediatric Pulmonology, Translational Lung Research Center, at the University of Heidelberg in Heidelberg, Germany, discuss Blocking Sodium Channels.

Workshop 14: Early Stage Therapies Kick-Starting the Stop-Mutations; Michael Wilschanski MD. Talk from the 35 th ECFS; June 6-9, 2012; Dublin, Ireland.

In the Early Stage Therapies workshop, Fredrick Van Goor, PhD, Head of Biology, CF Research Program at Vertex Pharmaceuticals described as in vitro study using ivacaftor on CFTR mutations other than the Class III G551D mutation.

"Ivacaftor is a broad acting CFTR potentiator in vitro. It shows promise as a CFTR potentiating agent for multiple mutant CFTR forms with residual CFTR function beyond G551D. These data suggest we may be able to define a class of patients, based on the molecular and functional phenotype, for clinical study," he explained.

His investigation normalized mutant CFTR syntheses by using host Fisher rat thyroid (FRT) cell lines with a single integration site, resulting in cells with very similar levels of mutation expression. In these cell lines, he found a wide range of baseline chloride transport in the absence of ivacaftor.

Using the CFTR2 database, Dr. Van Goor plotted the sweat chloride levels associated with the individual mutations against those from the in vitro system and saw a good qualitative relationship between in vivo measures of CFTR function (sweat chloride) and the baseline chloride transport, suggesting it was a reasonable system to evaluate baseline chloride transport in responses to CFTR modulators.

After acute addition of ivacaftor, the great majority of the mutant CFTR forms tested responded from baseline by at least 10%, a potentially therapeutic level of change in chloride transport that has been associated with milder disease forms. While some mutant CFTR forms had little or no response to ivacaftor, those that exhibited a good response had either a processing defect or normal amounts of CFTR at the cell surface.

Dr. Van Goor synthesized P67L (class IV conductance mutation) and A455E (Class V reduced CFTR synthesis mutation) cell lines, observing an overall 6-10% of residual baseline chloride transport. These mutant forms responded to ivacaftor by at least 10% from baseline.

Cells with Class IV mutations have CFTR protein at the cell surface but a decreased (in the 30-70% range) level of channel conductance, with sweat chloride levels consistent with residual CFTR function and a lower PI rate. The R347A mutation showed, in spite of a low chloride transport of 5% at baseline, a 10% response to ivacaftor. Other Class IV mutation cell lines, such as R334W, had higher sweat chloride levels consistent with minimal CFTR function, higher rates of PI, a lower level of baseline chloride transport in vitro, and a smaller increase in response to ivacaftor.

In Class II mutations (i.e. F508del) very little or no mature CFTR reaches the cell surface, resulting in higher level of sweat chloride values, a higher incidence of PI and no or minimal measurable chloride transport. The effect of ivacaftor on chloride transport on Class II CFTR cell line mutations was low.

Mutant CFTR forms are a heterogeneous group associated with multiple different molecular defects and a range in the disease phenotype, according to Dr. Van Goor, who continued by explaining, "This is different from the very homogeneous mutant group associated with Class III mutations, in which all of the defects are related to channel gating. The mutant CFTR forms all had minimum chloride transport in the absence of ivacaftor and all had a significant response on a similar level to ivacaftor, as G551D did."