Repurposing clonixin and flunixin as anti-fibrotic candidates: Computational and preclinical evaluation in bleomycin-induced pulmonary fibrosis in rats.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease that poses a significant health concern not only in humans but also in veterinary medicine, particularly in large animals, where effective treatment options remain scarce. This study explores the anti-fibrotic potential of two widely used non-steroidal anti-inflammatory drugs (NSAIDs), Clonixin (CLX) and Flunixin (FLX), through an integrated computational and in vivo approach relevant to veterinary applications. Molecular docking followed by dynamics for 100 ns was performed using Schrödinger Glide and Desmond, respectively, and results were comparable to Pirfenidone (PFD), an approved antifibrotic agent. Further, the in vivo efficacy of CLX and FLX was assessed in a bleomycin-induced pulmonary fibrosis model in Sprague Dawley rats, serving as a translational model for fibrotic lung diseases in animals. To evaluate phase-specific therapeutic effects, treatments were administered during either the inflammatory (days 1-7) or fibrotic (days 8-14) phases. Notably, both CLX and FLX demonstrated pronounced anti-fibrotic effects. This was evident during the fibrotic phase, significantly reducing lung coefficient, bronchoalveolar immune cell infiltration, oxidative stress, airway resistance, and hydroxyproline content. Histopathological evaluations confirmed reduced collagen deposition and improved lung architecture, as reflected by lower Ashcroft scores. Molecular docking and dynamics simulations revealed that CLX and FLX interacted with active site residues of TGF-β and formed more stable and persistent complexes compared to PFD, supporting their stronger binding and potential anti-fibrotic efficacy. These findings highlight the potential of CLX and FLX as repurposed anti-fibrotic agents, opening avenues for therapeutic exploration in chronic respiratory fibrotic conditions. Nonetheless, as the present study was conducted in a rodent model, translation to veterinary practice cannot yet be substantiated. Rigorous investigations in large animal models are essential to validate efficacy, safety, and clinical applicability before their consideration for veterinary use.