Development of inhaled CaSR NAMs for the treatment of pulmonary disorders

Asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are worldwide health issues. These are progressive and life-threatening lung diseases associated with chronic coughing, wheezing, difficulty breathing, and predisposing sufferers to severe illness, hospitalisation, and ultimately death. Existing therapies for these conditions offer symptomatic relief, thus reducing disease severity; however, none of the standard-of-care treatments is ubiquitously effective because they do not target the underlying pathology; as such, no treatment alters disease course. The G protein-coupled Calcium-Sensing receptor (CaSR) was recently implicated in orchestrating inflammatory, hyperresponsive and fibrotic responses in pulmonary disorders (PD). The CaSR is expressed on most healthy lung cells and is overexpressed in cells harvested from patients with PDs (Yarova et al. 2015). We have recently demonstrated that CaSR NAMs - negative allosteric modulators (NAM) at the CaSR - have enormous potential as a completely novel treatment of PDs, due to their disease-modifying ability to abolish airway smooth muscle hyperresponsiveness (AHR), as well as reduce airway inflammation and remodelling. Existing CaSR NAMs were initially developed for systemic use. In collaboration with Cardiff University School of Pharmacy, we have employed computer-assisted drug design to identify, synthesise and test novel CaSR NAMs to be delivered topically for PD treatment, herein referred to as New Chemical Entities (NCEs). The goal of this project was thus to identify the most suitable NCE CaSR NAM for further pre-clinical development and clinical in vivo testing using safety and efficacy in vitro biological assays and conduct a chemical synthesis review to identify which of the NCEs exhibits an optimal lung delivery profile. The activation of the CaSR in response to extracellular calcium ions (Ca2+ o) leads to an increase in intracellular calcium ion (Ca2+i) mobilisation. This response is blunted by pre- and co-exposure with CaSR NAMs. We hypothesised that selected NCE CaSR NAM exhibits a safe and potent effect for inhibiting activation of the CaSR. NCE activity was determined by single-cell intracellular Ca2+ imaging carried out on HEK-CaSR cells, which are genetically modified to stably express the human CaSR. Short and long-term live-cell imaging and cytotoxicity lactate dehydrogenase (LDH) assays were performed on primary Human Bronchial Epithelial and HEK-CaSR cells. These experiments could determine the most active NCE NAMs, E1 and C4. It was also determined via long-term exposure experiments that these compounds do not directly cause cytotoxicity to Primary Human Bronchial Epithelial cells in vitro at concentrations of