PROJECT SUMMARY Necrotizing enterocolitis (NEC), an acute inflammatory necrosis of the intestinal tract, is the most common acquired gastrointestinal and surgical emergency for preterm very low-birth weight infants in the neonatal intensive care unit. Despite considerable advances in neonatal care, NEC remains a devastating disease (mortality rates range from 15% to 30%) that lacks a cure. Management is largely nonspecific and includes the administration of broad-spectrum antibiotics, initiation of bowel rest and the provision of fluid and inotropic support to maintain cardiorespiratory function. Surgical intervention is required in up to 50% of the NEC cases and typically includes the removal of necrotic intestine. Normalizing the gut microbiome with antibiotics and probiotics to remove the inflammatory and vascular toxicities caused by endotoxins in abnormal gut cultivars is a popular recent treatment strategy. In addition, studies have shown that swallowed amniotic fluid is anti- inflammatory, matures the fetal gut, and may prevent infection. Importantly, there are no treatments that mimic amniotic fluid or its function in the fetal gut. Plakous has developed and patented methods to extract and preserve the cytokines and growth factors stored within the placental disc. The result is an acellular preparation of full-term, post-delivery human placenta that we have named Protego-PDTM. Our methods yield high concentrations of chemokines with a much lower pro-inflammatory chemokine composition compared to term amniotic fluid. We hypothesize that an orally administered therapeutic similar to 16-20 week amniotic fluid during the transition between birth and normal feeding volumes will bolster and sustain gut maturation while reducing the hyperinflammatory milieu which drives intestinal mucosal injury of the premature gut that becomes NEC. Our preliminary data indicate that Protego-PDTM can increase gut cell number and differentiation as well as modulate their response to lipopolysaccharides by decreasing TNF-α secretion. In addition, Protego-PDTM has been shown to increase survival and decrease NEC incidence in a piglet model of the disease. In this FastTrack SBIR, we propose to develop assays to ensure quality testing of our product, assess dosing and safety profile examination in preparation for regulatory clearance. These objectives will be accomplished through the following aims: 1) To develop a bioactivity assay to test the potency of Protego- PDTM, 2) To evaluate Protego-PDTM efficacy and dose-response in a piglet model of NEC, 3) To evaluate the safety of Protego-PDTM in a standard toxicology testing assessment in rodents. Successful completion of this project will allow Plakous to put together an Investigational New Drug dossier necessary in the approval process of a Biologic Therapeutic for Protego-PDTM. Additionally, the data will be presented to the Office of Orphan Products to support the designation of Protego-PDTM as a therapeutic for NEC as a Rare Pediatric Disease.

PROJECT NARRATIVE Necrotizing enterocolitis (NEC) is a devastating intestinal disease that affects ~5% of preterm neonates. Despite advancements in neonatal care, mortality remains high (30–50%) and it lacks a cure. Plakous has developed a cell-free preparation of full-term, post-delivery human placenta that closely resembles second- trimester amniotic fluid that can bolster and sustain gut maturation to prevent NEC. In this SBIR, Plakous will develop assays to ensure quality testing of the product, and assess dosing and safety profile examination in preparation for regulatory clearance.