Abstract Rationale The transcription factor Forkhead box O3 (FoxO3), often linked to longevity, is also implicated in the progression of age-related diseases including idiopathic pulmonary fibrosis (IPF). In IPF, FoxO3 is significantly downregulated and excluded from the cell nucleus in human tissue and experimental models. Moreover, FoxO3 knockdown in healthy lung models induces IPF-like phenotypes, while FoxO3 knockout in mouse bleomycin models exacerbates lung damage. Conversely, indirect FoxO3 activation reduces the fibrotic burden. These studies implicate FoxO3 as a critical integrator of pro-fibrotic signalling, suggesting pharmacological reconstitution as a novel treatment strategy. Thus, Refoxy Pharmaceuticals is developing novel, first-in-class selective small molecule activators of FoxO3 for the treatment of IPF and other age-related diseases. Lead compound FXY-1 shows disease-modifying potential in cellular in-vitro, ex-vivo, and animal models. Methods 1) Impact on fibroblast-to-myofibroblast transition (FMT): IPF-derived human lung fibroblasts stimulated with TGF-β were incubated with FXY-1 for 3 days. α-SMA production was quantified by immunostaining. 2) Impact on epithelial-to-mesenchymal transition (EMT): human bronchiolar epithelial cells stimulated with TGF-β were incubated with FXY-1 for 3 days. Supernatants were analysed for metalloproteases and pro-collagens by ELISA. 3) Ex-vivo assessment using precision cut lung slices (PCLS) prepared from biopsy tissue (62-year-old male donor, ex-smoker, 30 pack history). Treatment effect of FXY-1 over a dose range was measured via secreted fibrosis-related proteins in supernatants; tissues were assessed via pseudo-bulk RNA sequencing. 4) Mouse lung bleomycin models: FXY-1 was dosed orally from day 1, day 7, and day 14 (post intratracheal bleomycin installation) over a 21-day period. Lung tissue was subject to histopathological assessment by Ashcroft score and stained for collagen and α-SMA production. Results 1) FXY-1 reduced α-SMA production in fibroblasts at IC5010µM comparable to Nintedanib in the same experiment. 2) Collagen production was inhibited at sub-micromolar IC50 and MMP9 expression induced at low micromolar concentrations, whereas Nintedanib treatment induced collagen expression and reduced MMP9 levels. 3) In the PCLS model, collagen, fibronectin, and MMP2 levels were significantly impacted at 30 µM with no effect on cell viability. 4) In the mouse bleomycin models, FXY-1 treatment as low as 10mg/kg/day resulted in improvements in Ashcroft score with all endpoints significantly impacted at doses ≤100mg/kg/day. Conclusions Taken together these data present a robust case for the advancement of a selective FoxO3 activator to clinical assessment as novel and differentiated treatment for IPF. This abstract is funded by: Refoxy Pharmaceutical GmbH
Bhamra et al. (Fri,) studied this question.