TMEM16A Channel Function Does Not Influence Goblet Cell Numbers or Mucin Secretion In The Human Airway Epithelium – NACFC – Nashville – October 2019

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TMEM16A Channel Function Does Not Influence Goblet Cell Numbers or Mucin Secretion In The Human Airway Epithelium – NACFC – Nashville – October 2019

Henry Danahay1,2, Alexis Flen, Camille Ehre3, Martin Gosling1,2

1University of Sussex, Brighton, UK; 2Enterprise Therapeutics, UK; 3University of North Carolina, Chapel Hill, USA.

A role for the calcium activated chloride channel, TMEM16A, in the regulation of airway goblet cell formation and function has been recently reported (Benedetto et al., 2019; Kondo et al., 2017; Lin et al., 2015; Qin et al., 2016). Much of the published data to support a role for TMEM16A in these processes has been based on either gene silencing or the use of non-specific TMEM16A blockers (e.g. niflumic acid). It is therefore unclear whether these proposed functions are dependent on the ion channel activity of TMEM16A, through an alternative aspect of the proteins function or even a TMEM16A-independent activity of a non-selective pharmacological tool.

Using a potent TMEM16A channel blocker (Ani9) and recently identified TMEM16A potentiator compounds (Enterprise Therapeutics; proprietary), we have evaluated whether channel function can regulate goblet cell number and/or mucin secretion in primary cultures of human bronchial epithelial (HBE) cells.

Primary HBE (3 donor codes) were cultured for 2 weeks at air-liquid interface (ALI) on permeable supports and formed a well-differentiated mucociliary epithelium. On ALI day 15, cells were treated with either: 1) vehicle, 2) IL-13 (10 ng/mL) or 3) the TMEM16A potentiator, ETX001 (1 µM) with each group in either the absence or presence of the TMEM16A blocker, Ani9 (10 µM). ETX001 is a TMEM16A potentiator with EC50 values of 114 and 170 nM for the potentiation of chloride secretion in patch clamp (FRT-TMEM16A) and ion transport (CF-HBE) respectively. Ani9 fully blocks TMEM16A function in both patch clamp and HBE ion transport studies. Cells were cultured under these conditions for 96h before fixation and staining with antibodies directed against MUC5AC (goblet cells) and acetylated α-tubulin (ciliated cells). Goblet and ciliated cell numbers were quantified using an automated image acquisition (Zeiss Axiovert) and analysis system (Image J).

IL-13 induced a significant increase in the density of goblet cells based on the increased staining for MUC5AC, that was unaffected by co-administration of Ani9. Neither ETX001 or Ani9 (alone or in combination) had any effect on goblet cell numbers. Finally, the co-administration of ETX001 with IL-13 also failed to modify goblet cell numbers.

In a separate series of experiments, CF-HBE (±IL-13 pre-treatment; 2 donor codes) were treated with TMEM16A potentiator compounds for 24 hours and mucin secretion was quantified. TMEM16A potentiator compounds were without effect on the secretion of either MUC5B or MUC5B

Together, these data do not support a role for the ion channel function of TMEM16A in either the regulation of goblet cell numbers or the secretion of mucins in primary HBE.

 

Benedetto et al. (2019) FASEB J. 33(3):4502-4512

Kondo M et al. (2017) Clin Exp Allergy. 47(6):795-804.

Lin J et al. (2015) Exp Cell Res. 334(2):260-9.

Qin et al. (2016) Int Immunopharmacol. 40:106-114

EACT Increases Intracellular Calcium Levels by a Tmem16a-Independent Mechanism – NACFC – Nashville – October 2019

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EACT Increases Intracellular Calcium Levels by a Tmem16a-Independent Mechanism – NACFC – Nashville – October 2019

Sarah Lilley1, Henry Danahay1,2, Holly Charlton1, Kathryn Adley1, Roy Fox1, Martin Gosling1,2

1University of Sussex, Brighton, UK; 2Enterprise Therapeutics, UK

The N-aroylaminothiazole EACT was first described by Namkung et al. (2011) as an activator of the calcium-activated chloride channel, TMEM16A. Subsequently, EACT has been used as a tool compound by investigators to describe a wide variety of putative physiological functions of TMEM16A. The aim of the present study was to compare the pharmacology of EACT with alternative potentiators of TMEM16A that have been recently discovered by high-throughput screening.

Consistent with the original reported pharmacology, EACT increased anion secretory responses in models of epithelial ion transport that could be attenuated with the TMEM16A blocker, Ani9.  Similarly, novel Enterprise Therapeutics TMEM16A potentiators from 3 structurally distinct chemical series also increased the Ani9-sensitive anion secretion in these ion transport models.

To understand the mechanism of activation of these anion secretory currents, the effects of EACT and the novel TMEM16A potentiators on levels of intracellular calcium ([Ca2+]i) were evaluated. The acute addition of EACT to primary CF-HBE induced a concentration-dependent increase in [Ca2+]i. Pre-treatment of cells with Ani9 had no effect on the EACT-induced rise in [Ca2+]i. In contrast, the novel TMEM16A potentiators had no effect on [Ca2+]i.

The observation that EACT could increase [Ca2+]i questioned the reported pharmacological mechanism of TMEM16A activation by this molecule ie. via a direct interaction with the channel. To address whether EACT could directly activate TMEM16A in the absence of an elevation of [Ca2+]i, patch-clamp studies were performed under conditions of buffered [Ca2+]i. Under these conditions, with [Ca2+]i tightly clamped, EACT showed no evidence of any activity on TMEM16A. In contrast, the novel TMEM16A potentiators all showed a potent increase in channel function.

Together, these data do not support the description of EACT as a direct TMEM16A modulator but are consistent with its activation of TMEM16A being indirect, the result of an as yet undefined mechanism leading to an elevation of [Ca2+]i. Furthermore the recent proposal that TMEM16A positively regulates [Ca2+]i (Cabrita et al., 2017) is not consistent with the lack of effect of either the TMEM16A blocker Ani9 or the novel potent and selective TMEM16A potentiators on [Ca2+]i. Our data suggest that literature reports of TMEM16A function that have relied on the use of EACT as a pharmacological tool should be interpreted with caution.

Namkung et al (2011) FASEB J 25(11):4048-4062

 

The In Vitro & In Vivo Pharmacology of Novel Tmem16a Potentiator Compounds – NACFC – Nashville – October 2019

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The In Vitro & In Vivo Pharmacology of Novel Tmem16a Potentiator Compounds – NACFC – Nashville – October 2019

Henry Danahay1,2, Sarah Lilley1, Holly Charlton1, Roy Fox1, Brian Button3, Juan Sabater4, Martin Gosling1,2

1University of Sussex, Brighton, UK; 2Enterprise Therapeutics, UK; 3University of North Carolina, Chapel Hill, USA; Mount Sinai Medical Center, Miami, USA

TMEM16A was recently identified as a calcium-activated chloride conductance and a key orchestrator of anion secretion in the human airway epithelium (Caputo et al 2008; Schroeder et al 2008; Yang et al 2008). It is now clinically established that promoting anion secretion in the airway leads to enhanced mucus clearance and reduced exacerbation frequency in CF patients and as such TMEM16A represents an important target for the next generation of mucokinetics. Importantly, positive regulators of TMEM16A function will be expected to be of benefit in all CF patients, irrespective of their CFTR mutational status.

Using 4 parallel screening approaches, we identified several chemically diverse, low molecular weight compounds that potentiated TMEM16A function. These hit compounds were validated for TMEM16A function using a patch-clamp assay under conditions where [Ca2+]i was tightly buffered at an EC20 for TMEM16A channel activity. This enabled hits that activated TMEM16A by non-specifically elevating [Ca2+]i to be rapidly filtered out from the hit list.

The efficacy of bona fide TMEM16A potentiators translated through to function in ion transport studies in CF-HBE. Pre-treatment of CF-HBE with TMEM16A potentiators for between 5 min to 96h resulted in an enhancement of Ca2+-mediated anion-secretory responses that were sensitive to the TMEM16A blocker, Ani9. Measurements of [Ca2+]i confirmed that TMEM16A potentiators had no effect on calcium mobilization, consistent with a direct effect on the channel. TMEM16A potentiators also enhanced fluid secretion in CF-HBE, measured as an increase in the height of airway surface liquid (ASL). Inhaled dosing of TMEM16A potentiators induced a dose-dependent increase mucus clearance in vivo, using a sheep model of tracheal mucus velocity.

Together, these data support the concept that potentiators of the alternative airway chloride conductance, TMEM16A, can restore both anion and fluid secretion in primary CF cells and also enhance mucociliary clearance in vivo. Enterprise Therapeutics are advancing TMEM16A potentiators into clinical development.

Caputo et al (2008) Science 322(5901):590-594

Schroeder et al (2008) Cell 134(6):1019-1029

Yang et al (2008) Nature 455(7217):1210-1215

 

ECFS Clinical Meeting Liverpool TMEM16A potentiator

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ECFS Clinical Meeting Liverpool TMEM16A potentiator

Objectives: TMEM16A (ANO1) is a Ca2+-activated, alternative chloride channel expressed in the human airway epithelium. A potentiator of TMEM16A is predicted to enhance the secretion of chloride and thereby increase mucus hydration and clearance in the CF lung. Our aim is to discover and test the efficacy of novel TMEM16A potentiator compounds as a new therapeutic option for the treatment of CF lung disease.

Methods: Four, parallel high-throughput screening approaches were used to identify novel TMEM16A potentiator compounds. Compounds were then optimised and tested in primary CF bronchial epithelial cells for effects on both ion transport and airway surface liquid secretion.

Results: Several classes of TMEM16A potentiators were identified from the high-throughput screens. Only compound series that potentiated TMEM16A activity in a patch-clamp electrophysiology assay and which had no effect on intra-cellular Ca2+, were progressed for lead optimization. Pre-treatment of CF-HBE with TMEM16A potentiators for between 5 min to 96h resulted in an enhancement of Ca2+-mediated anion-secretory responses that were sensitive to the TMEM16A blocker, Ani9. Measurements of [Ca2+]i confirmed that TMEM16A potentiators had no effect on calcium mobilization, consistent with a direct effect on the channel. A Series 1 TMEM16A potentiator, ETX001, increased the secretion of airway surface liquid (ASL) in CF-HBE. The ETX001-driven increase in ASL height was further enhanced in cells that had been pre-treated with IL-13 to boost TMEM16A expression. A close structural analogue of ETX001, ETX002, that is inactive on TMEM16A, did not increase ASL height.

Conclusions: Together, these data support the concept that potentiators of the alternative airway chloride conductance, TMEM16A, can restore anion conductance and fluid secretion in both primary CF cells. Enterprise Therapeutics are advancing TMEM16A potentiators into clinical development.

A systematic comparison of the profiles of inhaled ENaC blocker candidates on mucociliary clearance: are we under-dosing in clinical studies?

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A systematic comparison of the profiles of inhaled ENaC blocker candidates on mucociliary clearance: are we under-dosing in clinical studies?

Henry Danahay1, Clive McCarthy1 and Martin Gosling1

1Enterprise Therapeutics, UK

Objectives: Recent clinical studies with ENaC modulator compounds have challenged the validity of the target for treating CF lung disease. Our aim was to establish retrospective human dose predictions for several of these compounds to understand whether this might be a factor in the failure of the clinical studies to show benefit.

Methods: ENaC modulators used in recent CF clinical studies were assessed in a sheep model of mucociliary clearance (MCC) and were compared with ETD001, a novel, non-amiloride based inhaled ENaC blocker (Enterprise Therapeutics), entering clinical development in 2019. Test compounds were administered directly into the sheep airways by aerosolization and MCC was measured by gamma-scintigraphy between 4-6h after the completion of dosing. Using the dose required to achieve a maximal increase in MCC in the sheep, an estimated ‘minimal effective clinical dose’ was calculated for each ENaC modulator.

Results: Each of the compounds studied induced a dose-dependent increase in MCC, typically reaching a maximum of between 20-25% clearance of the 99mTc-sulfur colloid tracer. Combining the predicted clinical dose (based on the sheep MCC model) with the actual dose/nebuliser used in the respective clinical study (www.clintrials.gov), suggests that all the ENaC modulators tested in the clinic to date have potentially been under-dosed by as much as 90%. This analysis further assumes that the healthy sheep airways in the MCC model predict for a clinical dose in a CF lung on a 1:1 basis. If a CF lung requires a higher dose than a sheep airway, the potential for under-dosing increases.

Conclusions: The pre-clinical safety profile of ETD001 currently enables doses of between 30-40x above the minimum predicted clinically efficacious dose to be tested in clinical studies. This profile provides a good opportunity to test the ENaC therapeutic hypothesis and ultimately provide a new therapy for CF patients.

Something old and something new: ENaC & TMEM16A as targets to hydrate mucus in the CF airway

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Something old and something new: ENaC & TMEM16A as targets to hydrate mucus in the CF airway

Henry Danahay, Enterprise Therapeutics, Brighton, UK

In the absence of functional CFTR in the apical membrane, the epithelial sodium channel (ENaC) and TMEM16A, a calcium activated chloride channel, are key regulators of airway mucosal hydration. As drug targets, both ENaC blockers and TMEM16A potentiators would provide an opportunity to treat CF patients who are unsuitable for CFTR repair therapies.

Inhaled ENaC blockers are predicted to attenuate fluid absorption out of the airway mucosa and improve mucus clearance. Clinical studies using the ENaC blocker amiloride, were first reported >30 years ago although since then novel inhibitors have failed to deliver efficacy in Phase 2. In contrast, positive modulators of TMEM16A as an alternative anion conductance to compensate for the loss of CFTR function in the airway epithelium are only now becoming available to begin to ask whether this mechanism could provide adequate anion and fluid secretion to hydrate airway mucus.

Why have novel, inhaled ENaC blockers failed to deliver any clinical benefit in Phase 2 studies? Is ENaC simply just not a good target for restoring mucus hydration or have we not developed the right drug candidates yet? Human genetics would support the negative regulation of ENaC as being a core mechanism by which to enhance mucus clearance, in which case the drug candidates are presumably the issue. To this end, we have evaluated the in vivo potency and efficacy of several ENaC blockers that have entered clinical development. A common finding is that doses used for the clinical studies are low when compared to the predicted ‘minimally efficacious dose’ derived from the pre-clinical dose-response relationships. This may be because of safety concerns commonly associated with ENaC blockers, specifically the potential for inducing hyperkalaemia as a side effect of ENaC block in the kidney. Moving forward, perhaps we should consider advancing only candidates with a broader safety window, offering the potential to dose significantly higher than a predicted minimally efficacious dose? Furthermore, clinical studies to confirm target engagement and duration of action would help to understand whether ENaC does represent a meaningful target to inhibit and thereby restore mucus hydration.

TMEM16A-based drug discovery is at a much earlier stage. High-throughput screening has identified novel potentiators of the channel. These compounds do not directly open the channel but rather ‘sensitize’ it to elevations in [Ca2+]i under the control of normal physiology. We can demonstrate that TMEM16A potentiators will enhance airway epithelial anion and fluid secretion and improve mucus clearance with the opportunity to treat patients currently unsuitable for CFTR repair drugs.

The pharmacology of novel TMEM16A potentiator compounds

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The pharmacology of novel TMEM16A potentiator compounds

Henry Danahay1,2, Sarah Lilley1, Holly Charlton1, Roy Fox1, Brian Button3, Martin Gosling1,2
1University of Sussex, Brighton, UK; 2Enterprise Therapeutics, UK; 3University of North Carolina, Chapel Hill, USA

TMEM16A was recently identified as a calcium-activated chloride conductance and a key orchestrator of anion secretion in the human airway epithelium (Caputo et al 2008; Schroeder et al 2008; Yang et al 2008). It is now clinically established that promoting anion secretion in the airway leads to enhanced mucus clearance and reduced exacerbation frequency in CF patients and as such TMEM16A represents an important target for the next generation of mucokinetics. Importantly, positive regulators of TMEM16A function will be expected to be of benefit in all CF patients, irrespective of their CFTR mutational status.

Using 4 parallel screening approaches, we identified several chemically diverse, low molecular weight compounds that potentiated TMEM16A function. These hit compounds were validated for TMEM16A function using a patch-clamp assay under conditions where [Ca2+]i was tightly buffered at an EC20 for TMEM16A channel activity. This enabled hits that activated TMEM16A by non-specifically elevating [Ca2+]i to be rapidly filtered out from the hit list.

The efficacy of bona fide TMEM16A potentiators translated through to function in ion transport studies in CF-HBE. Pre-treatment of CF-HBE with TMEM16A potentiators for between 5 min to 96h resulted in an enhancement of Ca2+-mediated anion-secretory responses that were sensitive to the TMEM16A blocker, Ani9. Measurements of [Ca2+]i confirmed that TMEM16A potentiators had no effect on calcium mobilization, consistent with a direct effect on the channel.

A Series 1 TMEM16A potentiator, ETX001, increased the secretion of airway surface liquid (ASL) in CF-HBE. The ETX001-driven increase in ASL height was further enhanced in cells that had been pre-treated with IL-13 to boost TMEM16A expression. A close structural analogue of ETX001, ETX002, that is inactive on TMEM16A, did not increase ASL height.
Together, these data support the concept that potentiators of the alternative airway chloride conductance, TMEM16A, can restore anion conductance and fluid secretion in both primary CF cells. Enterprise Therapeutics are advancing TMEM16A potentiators into clinical development.

Caputo et al (2008) Science 322(5901):590-594
Schroeder et al (2008) Cell 134(6):1019-1029
Yang et al (2008) Nature 455(7217):1210-1215

EACT increases intracellular calcium levels by a TMEM16A-independent mechanism

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EACT increases intracellular calcium levels by a TMEM16A-independent mechanism

Sarah Lilley1, Henry Danahay1,2, Holly Charlton1, Kathryn Adley1, Roy Fox1, Martin Gosling1,2
1University of Sussex, Brighton, UK; 2Enterprise Therapeutics, UK

The N-aroylaminothiazole EACT was first described by Namkung et al. (2011) as an activator of the calcium-activated chloride channel, TMEM16A. Subsequently, EACT has been used as a tool compound by investigators to describe a wide variety of putative physiological functions of TMEM16A. The aim of the present study was to compare the pharmacology of EACT with alternative potentiators of TMEM16A that have been recently discovered by high-throughput screening.

Consistent with the original reported pharmacology, EACT increased anion secretory responses in models of epithelial ion transport that could be attenuated with the TMEM16A blocker, Ani9. Similarly, novel TMEM16A potentiators from 3 structurally distinct chemical series also increased the Ani9-sensitive anion secretion in these ion transport models.

To understand the mechanism of activation of these anion secretory currents, the effects of EACT and the novel TMEM16A potentiators on levels of intracellular calcium ([Ca2+]i) were evaluated. The acute addition of EACT to primary CF-HBE induced a concentration-dependent increase in [Ca2+]i. Pre-treatment of cells with Ani9 had no effect on the EACT-induced rise in [Ca2+]i. In contrast, the novel TMEM16A potentiators had no effect on [Ca2+]i.

The observation that EACT could increase [Ca2+]i questioned the reported pharmacological mechanism of TMEM16A activation by this molecule ie. via a direct interaction with the channel. To address whether EACT could directly activate TMEM16A in the absence of an elevation of [Ca2+]i, patch-clamp studies were performed under conditions of buffered [Ca2+]i. Under these conditions, with [Ca2+]i tightly clamped, EACT showed no evidence of any activity on TMEM16A. In contrast, the novel TMEM16A potentiators all showed a potent increase in channel function.

Together, these data do not support the description of EACT as a direct TMEM16A modulator but are consistent with its activation of TMEM16A being indirect, the result of an as yet undefined mechanism leading to an elevation of [Ca2+]i. Furthermore the recent proposal that TMEM16A positively regulates [Ca2+]i (Cabrita et al., 2017) is not consistent with the lack of effect of either the TMEM16A blocker Ani9 or the novel potent and selective TMEM16A potentiators on [Ca2+]i. Our data suggest that literature reports of TMEM16A function that have relied on the use of EACT as a pharmacological tool should be interpreted with caution.

Namkung et al (2011) FASEB J 25(11):4048-4062

Gordon Research Conference ‘Modeling and Targeting Mucociliary Function in Health and Disease: Genes to Cells to Interaction Networks and Tissues’ February 2019

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Identifying pathways & compounds regulating goblet cell metaplasia in vitro

Henry Danahay1, Roy Fox2 and Martin Gosling1
1Enterprise Therapeutics, UK; 2University of Sussex, UK

It is widely accepted that the composition of mucus in the CF airway, and the hydration status, significantly affects its clearance and thereby the potential for form plugs, restrict airflow and create a nidus for chronic microbial colonisation. A variety of largely ion channel-based strategies are being employed to promote mucosal hydration e.g. CFTR repair, ENaC blockers and TMEM16A potentiators. An alternative, but complimentary approach would be to reduce the excessive production and secretion of the mucin proteins that contribute to the solids component of the mucus gel. One approach to reduce excessive mucus production in the diseased lung is to reduce the number of mucus producing goblet cells in the airways.

To identify pathways that could regulate goblet cell formation in primary human bronchial epithelial (HBE) cells, we used ‘bronchospheres’ (Danahay et al., Cell Rep. [2015] 10(2):239) to run a screen of approximately 2,000 compounds. IL-13 treatment of the organoids increased the expression of goblet cell markers, including MUC5AC, and compounds that prevented this increase were identified as hits. This list was then refined, removing compounds that also decreased the expression of the ciliated cell marker FOXJ1, as these likely represented a non-specific, negative effect on epithelial differentiation. The remaining compounds were then validated for effects in air-liquid interface cultures of primary HBE, of which ˜40% significantly reduced an IL-13 induced increase in MUC5AC+ goblet cell numbers using quantitative immunohistochemistry.

Several of these validated hits were identified as not only preventing the IL-13 induced increase in MUC5AC+ goblet cells but also showed the potential to increase ciliated cell numbers. These compounds were prioritized and further tested in primary HBE to understand whether: 1) their pharmacology was specific to an IL-13 driven goblet cell phenotype, and 2) whether they could reverse an established goblet metaplasia.

Examples were demonstrated to attenuate the increase in MUC5AC+ goblet cells that was driven by the TLR5 agonist, flagellin as well as the type III interferon, IL-28. Furthermore, IL-28 also induced a profound increase in the number of MUC5B+ cells that could also be attenuated by some of the compounds.
A small number of the most promising compounds were then introduced to HBE that had an established goblet cell phenotype. Under conditions where HBE had been treated with IL-13 for 14 days to elevate goblet cell numbers, the introduction of test compounds between days 15-28, in the continued presence of IL-13, was able to reverse the increase in goblet cells and to also recover ciliated cell numbers.

Based on the output of this screen, we have identified compounds that can both prevent and reverse a goblet cell phenotype in primary HBE. Next steps will be to understand whether these effects can translate into in vivo models of a goblet cell metaplasia.

32nd Annual North American Cystic Fibrosis Conference (NACFC) 18-20 October-Denver – Submission 210

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Submission: 210 – PHARMACOKINETIC AND PHARMACODYNAMIC PROFILE OF A NOVEL INHALED ENAC BLOCKER, ETD001

 

Henry Danahay1, Clive McCarthy1, William Abraham2, Holly Charlton3, Sarah Lilley3, Roy Fox3 and Martin Gosling1

1Enterprise Therapeutics, UK; 2Mount Sinai Medical Center, USA; 3University of Sussex, UK

 

Inhaled drugs are typically cleared very rapidly from the airway mucosa. Inhaled amiloride, accelerated mucociliary clearance in clinical studies, but with a half-life in the airway mucosa of <30 min, it was a short acting effect which did not translate into clinical efficacy. To achieve a long duration of action in the clinic with novel inhaled ENaC blockers, it will be necessary to maintain compound levels in the airway lumen at a concentration that will maintain target engagement for a sufficient period of time.

The aim of the present project, was to discover novel inhaled ENaC blocker compounds. A key element of the optimisation phase was to understand lung-specific pharmacokinetics, and how these related to in vivo efficacy. To this end, two key models were used. The first employed intra-tracheal delivery of test compound into the lungs of rats with sampling of compound levels in the airway, lung tissue and plasma. Compounds were then tested for duration of action and efficacy in the sheep model of mucociliary clearance (MCC).

Over the course of this project, we discovered numerous novel and potent ENaC blockers with a range of pharmacodynamic, pharmacokinetic and physicochemical properties. Of these, ETD001 was selected as a development candidate based upon its overall profile. Notably, ETD001 was not the most potent compound, with an in vitro potency (in nM) of: 40, 31 and 30 on human, sheep and rat ENaC respectively. ETD001 did however show a long residence in the airway lumen of the rat, which was accompanied by a potent and long lasting acceleration of MCC in the sheep following inhaled delivery. Other, more potent examples of novel ENaC blockers, showed significantly greater on-target potency (<5 nM) but failed to show efficacy in the sheep, which could be correlated with a diminished retention of compound in the airway lumen.

Based on the observations that ETD001 showed a long duration of action in vivo and that this was due to sustained compound levels in the airway lumen, we tested the hypothesis that repeat inhaled dosing would further increase the potency of the compound. After a single inhaled dose of ETD001, maximum efficacy in the sheep was observed at 13 µg/kg. In contrast, following twice daily dosing of ETD001 for 3.5 days, the maximal efficacious dose was reduced to 3 µg/kg, a 4-fold increase in potency.

In parallel and consistent with the sheep efficacy data, a 7 day repeat dose study in the rat revealed that lung levels of ETD001 were increased between days 1-7 in a dose-dependent manner. Furthermore, there was no change in blood potassium levels induced by ETD001 at any of these dose levels studied.

In summary, ETD001 is a novel inhaled ENaC blocker with a long duration of action in the airway lumen. Safety and efficacy data support a human tolerated dose that is 30-40 fold over the predicted minimum efficacious human dose. This is significant in view of the potential under-dosing of the Vertex ENaC blocker, VX-371, in a recent negative Phase 2 study.