Characterisation and microparticle formulation of a novel CAP inhibitor, QUB-TL1

Introduction: Impaired mucociliary clearance (MCC) in cystic fibrosis leads to chronic cycles of infection and inflammation, which in turn causes impairment of respiratory processes and damage to the lung tissue (1). MCC may be corrected through inhibition of trypsin-like, channel activating proteases (CAPs) such as trypsin, furin and prostasin, which activate the epithelial sodium channel (ENaC), dysregulation of which causes dehydration of the airways surface liquid (2). A novel CAP inhibitor, QUB-TL1, has demonstrated efficacy in restoration of mucocilliary clearance (3). It is prudent to consider that inhibition of CAPs may have a synergistic effect on inflammatory pathways closely linked within the CF lung. Aims and Objectives: The objective of this study was to investigate the effects of QUB-TL1 upon inflammation and develop a potential delivery method for the peptide. Methods: CF airway epithelial cells were treated with the novel compound (QUBTL1), in the presence and absence of known immunostimulant, Tumour Necrosis Factor alpha (TNFα) for 24 hrs (4). Supernatant from each treatment was analysed for concentration of cytokines IL-6 and IL-8 by enzyme-linked immunosorbent assay (ELISA). RNA harvested from the treated cells was assayed for its effects on cytokines, in addition to a number of other receptors involved in inflammatory processes such as PAR-2, NFκB, TLR4 and A20, at the level of expression by both qPCR and RT-PCR methods. The biotinylated compound was then bound to streptavidin-coated microparticles, applied to cells and assayed by fluorigenic assay and ELISA. In addition, the cytotoxic effect of particles on cells was assessed via LDH assay. Results and Discussion: QUB-TL1 showed efficacy in the reduction of TNFα mediated IL-6 and IL-8 release (n=3, p<0.05), with an approximate two-fold reduction observed for both. Through qPCR, the compound showed a significant effect in reducing expression of PAR-2, IL-6, NFκB and A20 in particular. The streptavidin coated microparticles were found to retain the ability to inhibit trypsin and reduce cytokine release, similar to free inhibitor. The particles also exhibited a negligible amount of toxicity in vitro. Conclusion: The incorporation of the compound onto microparticles has promise as a means of delivery, which presents an opportunity to locally deliver CAP inhibitors to the lung surface via inhalable formulations. QUB-TL1 appears to have multiple effects in vitro, not only in the suppression of ENaC and rehydration of the airways but in enabling modulation of the enhanced cycle of inflammation which causes overt lung damage in CF. Modulation of this sort could in turn improve quality of life and reduce hospitalization. Thus this inhibitor demonstrates great potential in treatment of CF lung disease. 1. Geborek A, Hjelte L. (2011) J Cyst Fibros,10(3), 187-192. 2. Thibodeau PH, Butterworth MB. (2013) Cell Tissue Res, 351, 309-323. 3. Reihill JA et al (2016) Am J Respir Crit Care Med. First published online 25 Mar 2016 as DOI: 10.1164/rccm.201511-2216OC. 4. Haddad, J.J., (2002). Biochem Bioph Res Co 296:847-85