Anti-fibrotic effects of lisinopril (ACE inhibitor) and fasudil (ROCK inhibitor) in combination for canine corneal fibrosis in vitro.

BACKGROUND: Corneal fibrosis is a leading cause of blindness in mammalian species and may result in compromised performance in sports and daily functions. This study evaluated the safety and anti-fibrotic effects of the FDA-approved drugs, angiotensin-converting enzyme inhibitor (ACE-I) lisinopril and rho-kinase inhibitor (ROCK-I) fasudil, alone and in combination, on the canine cornea using an established in vitro model. METHODS: To test the safety and efficacy of lisinopril and fasudil, primary canine corneal fibroblasts (CCFs) generated from donor corneas of healthy dogs (n = 20) were used. A series of dose-dependent and time-dependent assays with lisinopril (1-50 μM) and fasudil (1-10 nM) were performed. qRT-PCR, immunofluorescence (IF) staining, cell viability assay, cell proliferation assay, LIVE/DEAD viability/cytotoxicity assay, TUNEL assay, and total cell count were performed. RESULTS: A 25-μM lisinopril and 3-nM fasudil dose were safe, nontoxic, and optimal for therapeutic evaluations in vitro. Treatments of lisinopril or fasudil, alone or in-combination, to CCFs grown in the presence of TGF-β1 (5 ng/mL) showed inhibition of myofibroblast formation based on phase-contrast microscopy. The qRT-PCR and IF studies showed a significant decrease in expression of profibrotic markers, including α-smooth muscle actin (α-SMA; p < .0001), fibronectin (FN; p = .0002), tenascin C (TNC; p < .0001), Collagen I (Col-I; p < .0001), Collagen IIIA1 (Co-IIIA1; p < .0001), and Collagen IV (Co-lV; p < .0001). CONCLUSION: An ophthalmic formulation consisting of lisinopril and fasudil may offer a safe and effective method to treat canine corneal fibrosis. Additional studies evaluating safety and efficacy of this formulation in vivo are warranted.