Optimisation of a novel series of ENaC inhibitors, leading to the selection of the long-acting inhaled clinical candidate ETD001, a potential new treatment for cystic fibrosis.

Cystic Fibrosis (CF) results from the loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR), an ion channel of key importance in the airway epithelia. CFTR helps control optimal hydration of the airways, a crucial requirement for healthy lungs. CFTR modulators have recently been approved as an effective treatment option for many genetic variants of CF.

ETD001: A novel inhaled ENaC blocker with an extended duration of action in vivo.

Background: Inhibiting ENaC in the airways of people with cystic fibrosis (pwCF) is hypothesized to enhance mucociliary clearance (MCC) and provide clinical benefit. Historically, inhaled ENaC blockers have failed to show benefit in pwCF challenging this hypothesis. It is however unknown whether the clinical doses were sufficient to provide the required long duration of action in the lungs and questions whether a novel candidate could offer advantages where others have failed?

Methods: Dose-responses with the failed ENaC blockers (VX-371, BI 1265162, AZD5634, QBW276) together with ETD001 (a novel long acting inhaled ENaC blocker) were established in a sheep model of MCC and were used to predict clinically relevant doses that would provide a long-lasting enhancement of MCC in pwCF.

Niclosamide does not modulate airway epithelial function through blocking of the calcium activated chloride channel, TMEM16A.

Niclosamide and benzbromarone have been described as inhibitors of the calcium activated chloride channel, TMEM16A, and on this basis have been considered and tested as clinical candidates for the treatment of airway diseases. However, both compounds have previously demonstrated activity on a range of additional biological targets and it is unclear from the literature to what extent any activity on TMEM16A may contribute to efficacy in these models of airway disease. The aim of the present study was therefore to examine the pharmacology and selectivity of these clinical candidates together with a structurally unrelated TMEM16A blocker, Ani9, in a range of functional assays to better appreciate the putative role of TMEM16A in the regulation of both epithelial ion transport and the development of an airway epithelial mucus secretory phenoptype.

Discovery of Icenticaftor (QBW251), a Cystic Fibrosis Transmembrane Conductance Regulator Potentiator with Clinical Efficacy in Cystic Fibrosis and Chronic Obstructive Pulmonary Disease.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel are established as the primary causative factor in the devastating lung disease cystic fibrosis (CF). More recently, cigarette smoke exposure has been shown to be associated with dysfunctional airway epithelial ion transport, suggesting a role for CFTR in the pathogenesis of chronic obstructive pulmonary disease (COPD). Here, the identification and characterization of a high throughput screening hit as a potentiator of mutant human F508del and wild-type CFTR channels is reported.

Safety and efficacy of the cystic fibrosis transmembrane conductance regulator potentiator icenticaftor (QBW251).

Background: This is the first-in-human study of icenticaftor, an oral potentiator of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) channel. Restoration of CFTR activity has shown significant clinical benefits, but more studies are needed to address all CFTR mutations.

Methods: Safety, pharmacodynamics/pharmacokinetics of icenticaftor were evaluated in a randomized, double-blind, placebo-controlled study in healthy volunteers.

Efficacy and Safety of the CFTR Potentiator Icenticaftor (QBW251) in COPD: Results from a Phase 2 Randomized Trial.

Rationale: Excess mucus plays a key role in COPD pathogenesis. Cigarette smoke-induced cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction may contribute to disease pathogenesis by depleting airway surface liquid and reducing mucociliary transport; these defects can be corrected in vitro by potentiating CFTR.

Objective: To assess the efficacy of the CFTR potentiator icenticaftor in improving airflow obstruction in COPD patients with symptoms of chronic bronchitis.

Potentiating TMEM16A does not stimulate airway mucus secretion or bronchial and pulmonary arterial smooth muscle contraction.

The calcium-activated chloride channel (CaCC) TMEM16A enables chloride secretion across several transporting epithelia, including in the airways. Additional roles for TMEM16A have been proposed, which include regulating mucus production and secretion and stimulating smooth muscle contraction. The aim of the present study was to test whether the pharmacological regulation of TMEM16A channel function, could affect any of these proposed biological roles in the airways.

TMEM16A: An Alternative Approach to Restoring Airway Anion Secretion in Cystic Fibrosis?

The concept that increasing airway hydration leads to improvements in mucus clearance and lung function in cystic fibrosis has been clinically validated with osmotic agents such as hypertonic saline and more convincingly with cystic fibrosis transmembrane conductance regulator (CFTR) repair therapies. Although rapidly becoming the standard of care in cystic fibrosis (CF), current CFTR modulators do not treat all patients nor do they restore the rate of decline in lung function to normal levels. As such, novel approaches are still required to ensure all with CF have effective therapies.

TMEM16A Potentiation: A Novel Therapeutic Approach for the Treatment of Cystic Fibrosis.

Enhancing non-CFTR (cystic fibrosis transmembrane conductance regulator)-mediated anion secretion is an attractive therapeutic approach for the treatment of cystic fibrosis (CF) and other mucoobstructive diseases. To determine the effects of TMEM16A potentiation on epithelial fluid secretion and mucociliary clearance. The effects of a novel low-molecular-weight TMEM16A potentiator (ETX001) were evaluated in human cell and animal models of airway epithelial function and mucus transport.

Emerging Concepts and Therapies for Mucoobstructive Lung Disease.

A spectrum of intrapulmonary airway diseases, for example, cigarette smoke-induced bronchitis, cystic fibrosis, primary ciliary dyskinesia, and non-cystic fibrosis bronchiectasis, can be categorized as "mucoobstructive" airway diseases. A common theme for these diseases appears to be the failure to properly regulate mucus concentration, producing mucus hyperconcentration that slows mucus transport and, importantly, generates plaque/plug adhesion to airway surfaces. These mucus plaques/plugs generate long diffusion distances for oxygen, producing hypoxic niches within adherent airway mucus and subjacent epithelia.

Investigation of the Role of Protein Kinase D in Human Rhinovirus Replication.

Picornavirus replication is known to cause extensive remodeling of Golgi and endoplasmic reticulum membranes, and a number of the host proteins involved in the viral replication complex have been identified, including oxysterol binding protein (OSBP) and phosphatidylinositol 4-kinase III beta (PI4KB). Since both OSBP and PI4KB are substrates for protein kinase D (PKD) and PKD is known to be involved in the control of Golgi membrane vesicular and lipid transport, we hypothesized that PKD played a role in viral replication. We present multiple lines of evidence in support of this hypothesis.

NVP-QBE170: an inhaled blocker of the epithelial sodium channel with a reduced potential to induce hyperkalaemia.

Background And Purpose: Inhaled amiloride, a blocker of the epithelial sodium channel (ENaC), enhances mucociliary clearance (MCC) in cystic fibrosis (CF) patients. However, the dose of amiloride is limited by the mechanism-based side effect of hyperkalaemia resulting from renal ENaC blockade. Inhaled ENaC blockers with a reduced potential to induce hyperkalaemia provide a therapeutic strategy to improve mucosal hydration and MCC in the lungs of CF patients.

Notch2 is required for inflammatory cytokine-driven goblet cell metaplasia in the lung.

The balance and distribution of epithelial cell types is required to maintain tissue homeostasis. A hallmark of airway diseases is epithelial remodeling, leading to increased goblet cell numbers and an overproduction of mucus. In the conducting airway, basal cells act as progenitors for both secretory and ciliated cells.

Phosphoinositide 3-kinase alpha-dependent regulation of branching morphogenesis in murine embryonic lung: evidence for a role in determining morphogenic properties of FGF7.

Branching morphogenesis is a critical step in the development of many epithelial organs. The phosphoinositide-3-kinase (PI3K) pathway has been identified as a central component of this process but the precise role has not been fully established. Herein we sought to determine the role of PI3K in murine lung branching using a series of pharmacological inhibitors directed at this pathway.

Respiratory syncytial virus can infect basal cells and alter human airway epithelial differentiation.

Respiratory syncytial virus (RSV) is a major cause of morbidity and mortality worldwide, causing severe respiratory illness in infants and immune compromised patients. The ciliated cells of the human airway epithelium have been considered to be the exclusive target of RSV, although recent data have suggested that basal cells, the progenitors for the conducting airway epithelium, may also become infected in vivo. Using either mechanical or chemical injury models, we have demonstrated a robust RSV infection of p63+ basal cells in air-liquid interface (ALI) cultures of human bronchial epithelial cells.

Reduced sodium transport with nasal administration of the prostasin inhibitor camostat in subjects with cystic fibrosis.

Background: Prostasin, a trypsin-like serine protease, is a channel-activating protease and major regulator of epithelial sodium channel-mediated sodium absorption. Its direct inhibition by camostat represents a potential approach to inhibiting sodium transport in cystic fibrosis (CF).

Methods: To determine whether a topical formulation of camostat represents an efficacious and tolerable approach to reducing Na+ transport in the CF airway, we conducted a two-part randomized, double-blind, placebo-controlled, crossover, ascending single-dose study to evaluate the pharmacodynamics, safety, and pharmacokinetics of camostat administered through a nasal spray pump in subjects with CF.

Development of primary human nasal epithelial cell cultures for the study of cystic fibrosis pathophysiology.

Cultured primary epithelial cells are used to examine inflammation in cystic fibrosis (CF). We describe a new human model system using cultured nasal brushings. Nasal brushings were obtained from 16 F508del homozygous patients and 11 healthy controls.

Discovery of a novel chemotype of potent human ENaC blockers using a bioisostere approach. Part 2: α-Branched quaternary amines.

We report the synthesis and biological evaluation of a series of novel α-branched pyrazinoyl quaternary amines for their ability to block ion transport via the epithelial sodium channel (ENaC) in human bronchial epithelial cells (HBECs). Compound 12 g has an IC(50) of 30 nM and is highly efficacious in the Guinea-pig tracheal potential difference (TPD) model of ENaC blockade with an ED(50) of 1 μg kg(-1) at 1h. In addition the SAR results demonstrate for the first time the chiral nature of the binding site of human ENaC.

Discovery of a novel chemotype of potent human ENaC blockers using a bioisostere approach. Part 1: quaternary amines.

We report the identification of a novel series of human epithelial sodium channel (ENaC) blockers that are structurally distinct from the pyrazinoyl guanidine chemotype found in prototypical ENaC blockers such as amiloride. Following a rational design hypothesis a series of quaternary amines were prepared and evaluated for their ability to block ion transport via ENaC in human bronchial epithelial cells (HBECs). Compound 11 has an IC(50) of 200nM and is efficacious in the Guinea-pig tracheal potential difference (TPD) model of ENaC blockade with an ED(50) of 44μgkg(-1) at 1h.

ENaC-mediated effects assessed by MRI in a rat model of hypertonic saline-induced lung hydration.

Background And Purpose: The epithelial sodium channel (ENaC) regulates airway mucosal hydration and mucus clearance. The lack of such regulation in cystic fibrosis patients leads to desiccation of the airway lumen, resulting in mucostasis that establishes the environment for infections. Osmotic agents and negative ENaC regulators can be used to restore mucosal hydration.

Regulation of airway mucosal hydration.

Ion channels control the hydration status of the airway epithelium through apical anion secretion and cation absorption, which is accompanied by osmotically obligated water. The key channels in this process are the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), which is principally responsible for Cl(-) secretion by airway epithelial cells, and the epithelial Na(+) channel (ENaC), which is responsible for the absorption of Na ions. In CF, defective CFTR-mediated Cl(-) secretion and an accompanying upregulation in ENaC-mediated Na absorption results in a reduction in airway surface liquid volume, leading to poorly hydrated mucus and impaired mucociliary clearance.

In vivo assessments of mucus dynamics in the rat lung using a Gd-Cy5.5-bilabeled contrast agent for magnetic resonance and optical imaging.

Dysfunctions in mucociliary clearance are associated with the accelerated loss of lung function in several respiratory diseases. Approaches enabling the in vivo visualization of mucus dynamics in rodents at high resolution and sensitivity would be beneficial for experimental lung research. We describe the synthesis and characterization of two bilabeled amino dextran-based probes binding specifically to mucin.

Active site conformational changes of prostasin provide a new mechanism of protease regulation by divalent cations.

Prostasin or human channel-activating protease 1 has been reported to play a critical role in the regulation of extracellular sodium ion transport via its activation of the epithelial cell sodium channel. Here, the structure of the extracellular portion of the membrane associated serine protease has been solved to high resolution in complex with a nonselective d-FFR chloromethyl ketone inhibitor, in an apo form, in a form where the apo crystal has been soaked with the covalent inhibitor camostat and in complex with the protein inhibitor aprotinin. It was also crystallized in the presence of the divalent cation Ca(+2).