Hydroxypyridone anti-fungals selectively induce myofibroblast apoptosis in an in vitro model of hypertrophic scars.

Hypertrophic scars are a common complication of burn injuries, yet there are no medications to prevent their formation. During scar formation, resident fibroblasts are transformed to myofibroblasts which become resistant to apoptosis. Previously, we have shown that hydroxypyridone anti-fungals can inhibit transformation of fibroblasts, isolated from hypertrophic scars, to myofibroblasts. This study aimed to investigate if these drugs can also target myofibroblast persistence. Primary human dermal fibroblasts, derived from burn scar tissue, were exposed to transforming growth factor beta-1 (TGF-ß1) for 72 h to induce myofibroblast transformation. The cells were then incubated with three hydroxypyridone anti-fungals (ciclopirox, ciclopirox ethanolamine and piroctone olamine; 0.03-300 µM) for a further 72 h. The In-Cell ELISA method was utilised to quantify myofibroblast transformation by measuring alpha-smooth muscle actin (α-SMA) expression and DRAQ5 staining, to measure cell viability. TUNEL staining was utilised to assess if the drugs could induce apoptosis. When given to established myofibroblasts, the three hydroxypyridones did not reverse myofibroblast transformation, but instead elicited a concentration-dependent decrease in cell viability. TUNEL staining confirmed that the hydroxypyridone anti-fungals induced apoptosis in established myofibroblasts. This is the first study to show that hydroxypyridone anti-fungals are capable of inducing apoptosis in established myofibroblasts. Together with our previous results, we suggest that hydroxypyridone anti-fungals can prevent scar formation by preventing the formation of new myofibroblasts and by reducing the number of existing myofibroblasts.

Phenotypic screening identifies hydroxypyridone anti-fungals as novel medicines for the prevention of hypertrophic scars.

Although hypertrophic scarring affects ∼91% of burn patients annually, there is no drug to prevent this common complication. Hypertrophic scars are a result of dysregulated wound healing, characterised by persistent myofibroblast transformation and the excessive accumulation of extracellular matrix (ECM). Due to the multi-mechanistic nature of the scarring process, target-based approaches for identifying novel drugs have failed. Primary human dermal fibroblasts, derived from burn scar tissue, were exposed to transforming growth factor-beta 1 (TGF-ß1) to induce myofibroblast transformation. A phenotypic screening assay, measuring alpha-smooth muscle actin (α-SMA) expression, was developed to screen 1,954 approved drugs. Drugs that elicited >80% inhibition of α-SMA expression, and >80% cell viability were progressed as candidate drugs. Anti-myofibroblast activity of the candidates was confirmed before investigating their effects on extracellular matrix (ECM) production and keratinocyte epithelial-mesenchymal transition (EMT). TGF-ß1 induced myofibroblast transformation in primary human dermal fibroblasts (Emax = >3 ng/mL). The assay was optimised and validated (Z' = 0.59), before screening 1,954 approved drugs. 90 drugs were identified as hits and hydroxypyridone anti-fungals selected for further testing. Concentration-response curves for these drugs confirmed their concentration-dependent anti-myofibroblast activity (IC50 = 1.4 - 16.7 µM). Hydroxypyridone anti-fungals were also found to successfully reduce ECM production and keratinocyte EMT. This is the first study to screen approved drugs in primary human dermal fibroblasts. Hydroxypyridone anti-fungals were found to prevent myofibroblast transformation, ECM production and keratinocyte EMT suggesting they could be repurposed to prevent hypertrophic scarring.