O2172: In utero treprostinil administration reverses pulmonary arteriolar hypermuscularizaton in the ovine model of congenital diaphragmatic hernia

Background: Congenital diaphragmatic hernia (CDH) results in severe morbidity and mortality due to pulmonary hypertension (PH) and pulmonary hypoplasia. Treprostinil is a prostacyclin-analogue which is clinically approved for the treatment of PH, and we have previously shown that prenatal maternal administration of treprostinil results in significant improvement in the pulmonary vasculature hypermuscularization phenotype in the rat and rabbit models of CDH. Here we show that vascular remodeling can be achieved with direct fetal treprostinil administration which resulted in significantly reduced hypermuscularization of pulmonary arteries in the ovine model of CDH.

Methods: At 72-80 days gestation (dGA), excision of the left hemidiaphragm was performed in ovine fetuses. Each fetus was randomized into treatment groups: 1) subcutaneous fetal treprostinil treatment or 2) no drug administration. At 105-115 dGA, subcutaneous pumps were placed in the right or left flank of the experimental cohort with pumps programmed to deliver 20ng/kg/min continuously. At 135-140 dGA all lambs were delivered via Cesarean section and lung tissue was harvested. Lungs were flushed with cold saline administered via the main pulmonary artery and were then instilled with formalin through vascular and tracheal cannulation, processed, paraffin-embedded, sectioned and stained with haematoxylin-eosin (H&E) and α-smooth muscle actin (α-SmA). Vascular morphometry consisted of manual measurement of the proportionate medial wall thickness (%MWT) of pulmonary arterioles < 100µm internal diameter, a surrogate marker of PH and the primary outcome of this study. Groups were compared using one-way ANOVA and Dunnett's multiple comparisons test. Statistical significance was defined as p < 0.05.

Results: Prenatal exposure to treprostinil reduced overall %MWT in CDH lambs from 50.92 to 46.13 µM (p = 0.0104, Dunnett's test) (Figure 2). Drug exposure did not affect lung hypoplasia or alveolar development, and no adverse maternal or fetal events were noted related to treprostinil administration.

Conclusion: Pulmonary hypertension is the major cause of mortality and a leading cause of morbidity in CDH patients. Medial wall thickening of the pulmonary vascular bed is a hallmark of disease severity and the primary endpoint of this study. These results demonstrate safety and efficacy of direct fetal pharmacotherapy for the treatment of congenital pulmonary hypertension in the ovine model of CDH. Drug administration was initiated during the pseudoglandular phase of lung development, which coincides with the time point of diagnosis in over two thirds of cases, making intervention feasible at this time point. This preclinical translational study in a large animal model provides further support for clinical applications of treprostinil as a treatment approach for CDH-induced pulmonary hypertension. Furthermore, this study reports a novel method of direct fetal pharmacotherapy which may have implications for treatment of a number of congenital disorders.