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

Pulm Pharmacol Ther. 2018 Apr:49:95-103. doi: 10.1016/j.pupt.2018.01.012. Epub 2018 Feb 2.

Abstract

Idiopathic pulmonary fibrosis is 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 iloprost and treprostinil (TRE) have been shown to exert some protection against bleomycin-induced pulmonary fibrosis in mice when administered in a prophylactic regimen. In this study, we evaluated the effect of the inhaled treprostinil prodrug 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 bleomycin sulfate (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-bleomycin challenge and continued until day 27 after bleomycin. Lungs were harvested 24 h after the last dose of treatment for evaluation of lung hydroxyproline content and pulmonary histology. Lung hydroxyproline content increased from 421 μg/lung lobe in saline challenged and PBS treated animals to 673 μg/lung lobe in bleomycin challenged and PBS treated rats. Treatment of bleomycin challenged rats with 10, 30, or 100 μg/kg INS1009 dose-dependently reduced lung hydroxyproline content to 563, 501, and 451 μg/lung lobe, respectively, and pirfenidone decreased hydroxyproline content 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 bleomycin challenged rats showed dose-dependent increases in lung C16TR concentration and plasma TRE on day 10 post-bleomycin challenge. Multiple dose PK studies of inhaled INS1009 showed dose-dependent increases only in lung C16TR concentration on day 27 post-bleomycin challenge. We also investigated the effects of TRE on the cytokine transforming growth factor-β1 (TGF-β1)-stimulated collagen gene and protein expressions in cultured human lung fibroblasts, assessed by real-time PCR and Sirius Red staining, respectively. In human fibroblasts, TRE (0.001-10 μM) inhibited TGF-β1 (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 fibrosis in rats. This effect may involve direct actions of TRE in suppressing collagen expression in lung fibroblasts.

Keywords: Fibroblasts; Hexadecyl-treprostinil; INS1009; Inhalation treatment; Pulmonary fibrosis; Rats.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Administration, Inhalation
  • Animals
  • Antihypertensive Agents / administration & dosage*
  • Antihypertensive Agents / pharmacokinetics
  • Antihypertensive Agents / pharmacology
  • Bleomycin / administration & dosage
  • Bleomycin / toxicity
  • Cells, Cultured
  • Disease Models, Animal
  • Dose-Response Relationship, Drug
  • Epoprostenol / administration & dosage
  • Epoprostenol / analogs & derivatives*
  • Epoprostenol / pharmacokinetics
  • Epoprostenol / pharmacology
  • Fibroblasts / drug effects
  • Fibroblasts / metabolism
  • Humans
  • Hydroxyproline / metabolism
  • Idiopathic Pulmonary Fibrosis / drug therapy*
  • Idiopathic Pulmonary Fibrosis / physiopathology
  • Lipids / chemistry
  • Male
  • Nanoparticles*
  • Prodrugs
  • Pyridones / pharmacology
  • Rats
  • Rats, Inbred F344
  • Real-Time Polymerase Chain Reaction

Substances

  • Antihypertensive Agents
  • Lipids
  • Prodrugs
  • Pyridones
  • hexadecyl-treprostinil
  • Bleomycin
  • pirfenidone
  • Epoprostenol
  • Hydroxyproline