Building in vivo potency and duration of action and into a novel inhaled ENaC blocker, ETD001, presented at the European Cystic Fibrosis Society Basic Science Meeting, Loutraki, Greece, March 2018

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.

Identifying pathways regulating goblet cell metaplasia: phenotypic screening with bronchospheres, presented at the European Cystic Fibrosis Society Basic Science Meeting, Loutraki, Greece, March 2018

It is widely accepted that the composition of mucus in the CF airway, and in particular 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 drug targets that could be regulated to achieve this, we have utilised a 3D culture model of the human airway epithelium, ‘bronchospheres’. Bronchospheres are derived from primary human airway basal cells, and can be cultured to form a well-differentiated mucociliary epithelium without the need for an air-liquid interface. As such, bronchospheres are cultured in a 384 well assay format that makes them amenable to medium throughput compound screening. Treating bronchospheres with inflammatory mediators such as IL-13 induces a mucus hypersecretory phenotype with increased numbers of goblet cells and reduced numbers of ciliated cells. Our hypothesis was that the co-administration of test compounds together with IL-13 would identify compounds capable of preventing goblet cell formation with the opportunity to seed future drug discovery programs.

Bronchospheres were cultured as previously described (Danahay et al., Cell Rep. (2015) 10(2):239). On day 2 after seeding primary human airway basal cells, treatment with IL-13 ± test compounds was initiated. A library of approximately 2,000 pharmacologically active LMW compounds was used, each with a well-annotated mechanism of action. On day 8, media and treatments were topped-up and on day 14 bronchospheres were lysed and RNA isolated. QPCR was then used to assess the expression of cell-specific markers: MUC5AC (goblet cells) and FOXJ1 (ciliated cells). Compounds that induced a ≥2-fold reduction in expression of MUC5AC were classified as hits. This hit list was then refined by checking the expression of FOXJ1. Compounds that had likewise attenuated FOXJ1 expression by ≥2-fold were deprioritised as these likely represented a non-specific effect on epithelial differentiation. Compounds that either maintained or enhanced FOXJ1 expression in addition to repressing MUC5AC gene expression were prioritised for validation using traditional air-liquid interface cultures.

In total, 92 hit compounds from the bronchosphere screen were tested for effects on goblet and ciliated cell numbers in ALI HBE cultures using quantitative immunohistochemistry. Of these, 38 (41%) significantly attenuated the MUC5AC+ stained area in IL-13 treated HBE and either maintained or increased the FOXJ1+ stained area. Validated hits were then aligned based on their previously reported pharmacological activity to enable common pathways to be identified and to refine our hypotheses through further exemplification of pathway regulators.

Based on this screen, we have identified pathways that regulates the induction and maintenance of a goblet cell metaplasia in vitro and are progressing a lead optimisation program for eventual therapy in respiratory diseases associated with mucus obstruction.