Therapeutic administration of inhaled INS1009, a treprostinil prodrug formulation, inhibits bleomycin-induced pulmonary fibrosis in rats

Abstract Idiopathic pulmonary fibrosisis a progressive and lethal disease and while there are now two approved drugs(Esbriet ® and Ofev ® ) additional effective treatments are still needed. Recently, prostacyclin analogs such as iloprostand treprostinil(TRE) have been shown to exert some protection against bleomycin-induced pulmonary fibrosisin mice when administered in a prophylactic regimen. In this study, we evaluated the effect of the inhaled treprostinilprodrug hexadecyl- treprostinil(C16TR) formulated in a lipid nanoparticle (INS1009) administered therapeutically in a fibrotic rat model. Male Fischer 344 rats challenged with intra-tracheal saline instillation were then treated with daily inhaled phosphate buffered saline (PBS) while rats challenged with bleomycinsulfate (3.5–4.0 mg/kg) instillation were treated with either daily inhaled PBS, daily inhaled INS1009 (10, 30, or 100 μg/kg), or twice-daily orally with the anti-fibrotic compound pirfenidone(100 mg/kg). Dosing started on day 10 post- bleomycinchallenge and continued until day 27 after bleomycin. Lungs were harvested 24 h after the last dose of treatment for evaluation of lung hydroxyprolinecontent and pulmonary histology. Lung hydroxyprolinecontent increased from 421 μg/lung lobe in saline challenged and PBS treated animals to 673 μg/lung lobe in bleomycinchallenged and PBS treated rats. Treatment of bleomycinchallenged rats with 10, 30, or 100 μg/kg INS1009 dose-dependently reduced lung hydroxyprolinecontent to 563, 501, and 451 μg/lung lobe, respectively, and pirfenidonedecreased hydroxyprolinecontent to 522 μg/lung lobe. Histologically, both INS1009 (100 μg/kg) and pirfenidone(100 mg/kg) reduced the severity of subepithelial fibrosis. Single dose pharmacokinetic (PK) studies of inhaled INS1009 in bleomycinchallenged rats showed dose-dependent increases in lung C16TR concentration and plasma TRE on day 10 post- bleomycinchallenge. Multiple dose PK studies of inhaled INS1009 showed dose-dependent increases only in lung C16TR concentration on day 27 post- bleomycinchallenge. We also investigated the effects of TRE on the cytokine transforming growth factor-β 1 (TGF-β 1 )-stimulated collagen gene and protein expressions in cultured human lung fibroblasts, assessed by real-time PCR and Sirius Redstaining, respectively. In human fibroblasts, TRE (0.001–10 μM) inhibited TGF-β 1 (20 ng/mL)-induced expression of collagen mRNA and protein in a concentration-dependent manner. These results demonstrated that inhaled INS1009, administered in a therapeutic dosing paradigm, dose-dependently (10–100 μg/kg) inhibited bleomycin-induced pulmonary fibrosisin rats. This effect may involve direct actions of TRE in suppressing collagen expression in lung fibroblasts.