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Elucidation of the Anti-Fibrotic Activity of a Repurposed Drug for Idiopathic Pulmonary Fibrosis
Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease with significant mortality and morbidity, with few effective and well tolerated pharmacological treatments. Currently, IPF is the most common idiopathic interstitial pneumonia with a median survival time of 2-4 years following diagnosis. Two anti-fibrotic therapies, pirfenidone and nintedanib, are FDA-approved for IPF treatment. However, the therapeutics are cost prohibitive in numerous countries and induce significant adverse side effects that are difficult for patients to tolerate. Therefore, the demand for a novel, effective, well tolerated anti-fibrotic therapeutic remains. To this end, spironolactone, an established mineralocorticoid receptor (MR) antagonist, has been identified as a potential novel therapy for IPF. Spironolactone is an off-patent drug for fibrotic activity reduction, clinically safe, and a cost-effective option for widespread, scalable implementation.
The overall goal of this research project was to investigate the major anti-fibrotic signalling pathways and subsequent pharmacological effects of spironolactone. Thus, we aimed to access its suitability as an IPF pharmacological treatment. The investigation of the pharmacological activity and mechanisms of action of spironolactone as an anti-fibrotic is necessary in order for us to proceed to phase 1 clinical studies.
This thesis investigated the anti-fibrotic activity of spironolactone on IPF-derived fibroblasts and alveolar epithelial cells in a ‘Scar-in-a-jar’ fibrogenesis assay. A specific focus undertaken was on evaluating pro-fibrotic markers as possible therapeutic targets and investigation of the classical mechanism of spironolactone. Foremost, the efficacy of spironolactone in reducing collagen deposition from pulmonary fibroblasts was successfully demonstrated on Wi38 cells. IPF-derived fibroblasts displayed a significant reduction of collagen deposition after treatment with spironolactone alone and after combined therapies with spironolactone and pirfenidone. Spironolactone alone successfully demonstrated its potential in reversing collagen tissue formation. Significant reductions in COL1A1 and ACTA2 gene expression were observed following combination therapy exposure, in comparison to untreated controls, displaying potential for spironolactone in reducing myofibroblast activation. This study indicated that spironolactone is producing its anti-fibrotic activity via a MR-independent signalling pathway.
Having established a suitable in vitro dose of spironolactone for alveolar epithelial cells, spironolactone, and combination therapy with pirfenidone reduced A549 cell α-SMA production. The study indicates that spironolactone has the potential to reduce epithelial- mesenchymal transition (EMT), as observed in IPF pathogenesis. A significant outcome of EMT in alveolar epithelial cells is the collective migration of cells which aids the aberrant wound healing response in IPF. Employing a scratch migration assay we observed that spironolactone alone and combination therapy reduced cell migration. Additionally, we observed that the gene expression of ACTA2 and FGFR1, pro-fibrotic genes were reduced in our rapid fibrogenesis model after treatment with spironolactone. The study indicated that the anti- fibrotic activity of spironolactone on alveolar epithelial cells is through an MR-independent pathway.
In conclusion, this thesis demonstrates the anti-fibrotic activity of spironolactone in IPF- derived fibroblasts and alveolar epithelial cells, thus suggesting further exploratory work in an in vitro fibrogenesis model. Taken together, the findings in this research study indicate the potential of spironolactone to proceed to preclinical studies, with the long-term goal of repurposing spironolactone as an IPF pharmacological treatment.
Irish Lung Fibrosis Association
First SupervisorDr. Cian O’Leary
Second SupervisorProf. Killian Hurley
CommentsSubmitted for the Award of Masters of Science: Research to the Royal College of Surgeons in Ireland, 2022
Published CitationReardon J,. Elucidation of the Anti-Fibrotic Activity of a Repurposed Drug for Idiopathic Pulmonary Fibrosis [MSc Thesis] Dublin: Royal College of Surgeons in Ireland; 2022
- Master of Science (MSc): Research
Date of award2022-06-01
- Master of Science (MSc): Research
- Respiratory Medicine