Pharmacological allosteric agonists (calcimimetics) of the extracellular calcium-sensing receptor (CaSR) have substantial gastro-intestinal side effects and induce the expression of inflammatory markers in colon cancer cells. Here, we compared the effects of both CaSR-specific (R enantiomers) and -unspecific (S enantiomers) enantiomers of a calcimimetic (NPS 568) and a calcilytic (allosteric CaSR antagonists; NPS 2143) to prove that these effects are indeed mediated via the CaSR, rather than via off-target effects, e.g., on β-adrenoceptors or calcium channels, of these drugs. The unspecific S enantiomer of NPS 2143 and NPS S-2143 was prepared using synthetic chemistry and characterized using crystallography. NPS S-2143 was then tested in HEK-293 cells stably transfected with the human CaSR (HEK-CaSR), where it did not inhibit CaSR-mediated intracellular Ca²⁺ signals, as expected. HT29 colon cancer cells transfected with the CaSR were treated with both enantiomers of NPS 568 and NPS 2143 alone or in combination, and the expression of CaSR and the pro-inflammatory cytokine interleukin 8 (IL-8) was measured by RT-qPCR and ELISA. Only the CaSR-selective enantiomers of the calcimimetic NPS 568 and NPS 2143 were able to modulate CaSR and IL-8 expression. We proved that pro-inflammatory effects in colon cancer cells are indeed mediated through CaSR activation. The non-CaSR selective enantiomer NPS S-2143 will be a valuable tool for investigations in CaSR-mediated processes.

Idiopathic pulmonary fibrosis (IPF) is a disease with a poor prognosis and no curative therapies. Fibroblast activation by transforming growth factor β1 (TGFβ1) and disrupted metabolic pathways, including the arginine–polyamine pathway, play crucial roles in IPF development. Polyamines are agonists of the calcium/cation-sensing receptor (CaSR), activation of which is detrimental for asthma and pulmonary hypertension, but its role in IPF is unknown. To address this question, we evaluated polyamine abundance using metabolomic analysis of IPF patient saliva. Furthermore, we examined CaSR functional expression in human lung fibroblasts (HLFs), assessed the anti-fibrotic effects of a CaSR antagonist, NPS2143, in TGFβ1-activated normal and IPF HLFs by RNA sequencing and immunofluorescence imaging, respectively; and NPS2143 effects on polyamine synthesis in HLFs by immunoassays. Our results demonstrate that polyamine metabolites are increased in IPF patient saliva. Polyamines activate fibroblast CaSR in vitro, elevating intracellular calcium concentration. CaSR inhibition reduced TGFβ1-induced polyamine and pro-fibrotic factor expression in normal and IPF HLFs. TGFβ1 directly stimulated polyamine release by HLFs, an effect that was blocked by NPS2143. This suggests that TGFβ1 promotes CaSR activation through increased polyamine expression, driving a pro-fibrotic response. By halting some polyamine-induced pro-fibrotic changes, CaSR antagonists exhibit disease-modifying potential in IPF onset and development.

Introduction: Chronic obstructive pulmonary disease (COPD) is predicted to become the third leading cause of mortality worldwide. Exposure to urban particulate matter (UPM) underpins COPD development and exacerbation. We have shown, using in vivo models of inflammatory lung diseases, that airway inflammatory and remodelling changes are at least partly mediated through the CaSR, and that calcilytics abrogate them. Dendritic cells (DC) are involved in the development of COPD and their activation is accelerated by exposure to UPM, but whether the CaSR plays a role in this process is unknown. Aims: To determine whether UPM directly activates the CaSR and if calcilytics attenuate UPM-induced DC maturation and activation. Methods: Intracellular calcium responses to UPM were investigated in HEK293 cells stably transfected with the human CaSR (HEK-CaSR) or the empty vector (HEK-0), in the presence or absence of the calcilytic NPS2143. Flow cytometry and cytometric bead array were employed to evaluate maturation (%CD83) and cytokine release by human monocyte-derived DC following 24h exposure to UPM ± calcilytics. Results: UPM induced an acute, transient intracellular calcium response in the HEK-CaSR but not the HEK-0 cells, an effect inhibited by NPS2143 (p<0.001). NPS2143 significantly attenuated UPM-induced maturation (p<0.005) and release of the immunoregulatory cytokines IL-10 and IL-23p40 (p<0.001), but not IL-6 by DC. Conclusions: This study demonstrates that UPM induces maturation and activation of DC, which is inhibited experimentally by a calcilytic. These data suggest that calcilytics may alter the natural history and progression of COPD.

Background: Idiopathic pulmonary fibrosis (IPF) is a disease with very poor prognosis and no curative therapies. The G protein-coupled, calcium/cation-sensing receptor (CaSR) is activated by environmental pollutants and by arginine-derived polyamines, which are thought to play a role in IPF. Whether the CaSR is involved in the pathogenesis of pulmonary fibrosis is unknown. Objective: To investigate the CaSR as a novel drug target for the treatment of pulmonary fibrosis (PF). Methods and results: CaSR protein expression is found in the airway epithelium in the neuroepithelial bodies of the healthy and IPF human lung. Expression of arginine pathway-linked polyamines is increased in PF patient saliva samples compared to non-PF patients. Arginine pathway metabolites, ornithine and spermine, activate the CaSR in primary normal human lung fibroblasts (NHLF), effects prevented by CaSR antagonism using the calcilytic NPS2143. In NHLF calcilytic also reversed the pro-fibrotic effects of exogenous TGFβ1 administration on Rho kinase and αSMA expression, proliferation, collagen production and IL-8 secretion. Targeted CaSR ablation from fibroblasts and smooth muscle cells protects mice from spontaneously occurring, age-related lung fibrosis. Conclusions: Sustained CaSR activation in the lung drives pro-fibrotic processes, which can be reversed by calcilytic. Pharmacological and genetic CaSR blockade reduce both TGFβ1-induced and naturally occurring pro-fibrotic changes. This work provides the scientific rationale for developing inhaled calcilytics as novel therapeutics for IPF. KEY MESSAGES: Key question: How does the calcium/cation-sensing receptor (CaSR) promote pulmonary fibrosis? Bottom line: The CaSR is expressed in human IPF and experimental models of PF where receptor inhibition prevents pro-fibrotic changes and pulmonary remodeling. Why read on: CaSR blockers, calcilytics, represent a novel treatment for IPF.

Introduction: Previously we have shown that blockers of the calcium-sensing receptor (CaSR, calcilytics), reduce BHR, inflammation and remodelling in Th2/IgE- and alarmin-driven asthma models. Exposure to urban particulate matter (UPM) exacerbates asthma symptoms. It is unclear whether UPM exerts its effects via the CaSR. Aim: To determine which airway cells express the CaSR and whether UPM exerts its effects by acting directly at the CaSR. Methods: Immunohistochemistry (expression of CaSR immunoreactivity); intracellular calcium sensing (CaSR activity); flow cytometry (cellular maturation and cytokine production); qPCR (TSLP mRNA expression). Results: CaSR immunoreactivity was detected on human monocytes, myeloid dendritic cells, immature and mature monocyte-derived dendritic cells (MDDC), neutrophils, alveolar macrophages and bronchial epithelial cells (HBEC). UPM mobilised intracellular calcium in HEK-293 cells stably transfected with the human CaSR but not empty vector, an effect inhibited by calcilytics (p<0.001). Calcilytics attenuated UPM-induced MDDC maturation (p<0.0001), IL-10 release (p<0.05) and UPM-upregulated TSLP mRNA expression by HBEC (p<0.05). Conclusions: The CaSR is present in inflammatory and epithelial cells involved in asthma pathogenesis. UPM acts on the CaSR on MDDC and airway epithelial cells to induce inflammatory cytokines including alarmins, an effect likely relevant to asthma exacerbation and inhibitable by calcilytics.