Preclinical evaluation of inhaled calcilytics as a potential anti-asthma therapy

P. L. Yarova¹, P. Huang¹, V. S. Telezhkin², M. Schepelmann¹, E. J. Kidd¹, W. R. Ford¹, K. J. Broadley¹, J. P. Ward³, C. J. Corrigan³, P. J. Kemp¹, D. Riccardi¹. ¹Cardiff University, Cardiff, United Kingdom, ²Newcastle University, Newcastle upon Tyne, United Kingdom, ³Kings College London, London, United Kingdom.

Introduction: Asthma is a heterogeneous disease that is characterised by airway hyperresponsiveness, obstruction and inflammation. Conventional bronchodilators and corticosteroids do not help all the patients and can lead to unwanted side effects, therefore new therapies are needed. We have recently shown that inhaled calcilytics, inhibitors of the extracellular calcium-sensing receptor (CaSR), can suppress airway hyperresponsiveness and inflammation in asthma animal models (1). Four calcilytics developed for the treatment of osteoporosis and abandoned due to lack of efficacy for this indication can be repurposed for inflammatory lung diseases. Here we carried out drug safety, efficacy and specificity studies, to select the best available calcilytic, delivered topically, as a novel anti-asthma therapeutic.

Methods: Custom-synthetized calcilytics, the quinazolin-2-one AXT914 (Novartis), and the amino-alcohols JTT-305 (JT/Merck), NPSP-795 (GSK/Shire) and Ronacaleret (GSK), were administered at 3μM as 1h daily inhalations to male Balb/c mice for five days. For in vivo observations, blood pressure was measured using tail-cuff system (CODA), bronchoalveloar lavage fluid, blood serum, and lungs were collected to determine inflammation, Ca²⁺ levels, and lung structure. For ex vivo experiments, mouse tracheas were mounted in a wire myograph (DMT), and rising concentrations of calcilytics were applied to the bath. Human embryonic kidney 293 (HEK) cells expressing α1C subunit of large conductance calcium channel (Lca) were used to investigate the effects of the calcilytics on Lca by the patch-clamp voltage-clamp technique.

Results: Repeated exposure to inhaled calcilytics had no effect on blood pressure, serum calcium levels, or lung structure; however, AXT914 and JTT-305 evoked a small increase in leucocyte infiltration into the lungs compared to vehicle (N=6, p<0.05). Application of rising concentrations of calcilytics to the tracheas (pre-contracted with acetylcholine; Imax=80%) was ineffective up to 1μM, whilst 30μM of JTT-305, NPSP-795, and Ronacaleret evoked a decrease of the tone when compared to the vehicle control (19%, 24% and 52%, respectively; N=3-8, p<0.05). Nifedipine-sensitive Lca current was not affected by exposure to either 10μM of NPSP-795 or AXT914 (N=3-6, p>0.05).

Conclusions: Pharmacologically relevant doses of calcilytics delivered to the lung, do not affect serum calcium homeostasis, blood pressure, or airway tone. Supramaximal doses of calcilytics exhibit mild relaxant properties that are not attributed to off-target effect on Lca. Small inflammation evoked by repeat exposures to AXT914 and JTT305 suggests that an alternative method to drug nebulisation should be considered for lung delivery of these two calcilytics.

References:

1. Yarova PL et al. (2015). Science TM 7: 284ra60.