Title Page Prevention of bleomycin induced pulmonary fibrosis by a novel anti- fibrotic peptide with relaxin like activity

Running Title: A novel, Relaxin-like anti-fibrotic peptide. Nonstandard abbreviations used in the paper: RLX – Relaxin, hRLX – Human Relaxin, SEM-standard error of the mean, IPF-idiopathic pulmonary fibrosis. This article has not been copyedited and formatted. The final version may differ from this version. Abstract Pulmonary fibrosis is a progressive and lethal lung disease characterized by accumulation of extracellular matrix and loss of pulmonary function. No cure exists for this pathologic condition, and current treatments often fail to slow its progression or relieve its symptoms. Relaxin was previously shown to induce a matrix degrading phenotype in human lung fibroblasts in vitro and to inhibit pulmonary fibrosis in vivo. A novel peptide which targets the relaxin RXFP1/LGR7 receptor was recently identified using our computational platform designed to predict novel G protein-coupled receptor peptide agonists. In this study, we examined the anti-fibrotic properties of this novel peptide, designated CGEN25009, in human cell based assays and in a murine model of bleomycin-induced pulmonary fibrosis. Similarly to relaxin, CGEN25009 was found to have an inhibitory effect on TGF-β1-induced collagen deposition in human dermal fibroblasts, while enhancing MMP-2 expression. The peptide's biological activity was also similar to relaxin in generating cellular stimulation of cAMP, cGMP and NO in the THP-1 human cell line. In vivo, 2-week administration of CGEN25009 in a preventive or therapeutic mode, i.e. concurrently with or 7 days after bleomycin treatment, respectively, caused a significant reduction of lung inflammation and injury, and ameliorated adverse airway remodeling and peri-bronchial fibrosis. The results of this study indicate that CGEN25009 displays anti-fibrotic and anti-inflammatory properties and may offer a new therapeutic option for the treatment of pulmonary fibrosis. This article has not been copyedited and formatted. The final version may differ from this version.