Therapeutic Screening of Neurotherapeutics Efficacy and Pharmacokinetics for the Preterm Neonatal Brain

Abstract

Preterm birth occurs during a critical period of brain development, rendering extremely preterm (EP) infants born before 28 weeks’ gestation highly susceptible to hypoxic-ischemic, inflammatory, and oxidative injuries. Despite current neuroprotective interventions, effective therapies remain limited, highlighting the need to identify therapies with complementary mechanisms capable of protecting multiple brain regions and vulnerable cell populations. This thesis used an EP-equivalent ferret model for evaluating candidate therapies, investigating their mechanisms of action, and supporting clinical translation through pharmacokinetic (PK) characterization. Initial studies in the ferret organotypic whole hemisphere slice model demonstrated that the combination of azithromycin and erythropoietin produced synergistic neuroprotection across multiple brain regions, accompanied by reversal of inflammatory transcriptomic signatures and changes in microglial morphology. Subsequent head-to-head screening of eight therapeutic candidates using an optimized white matter injury model identified azithromycin, acetoacetate, caffeine, and exendin-4 as the most promising monotherapies. Bulk RNA sequencing revealed distinct mechanisms of action, with azithromycin and exendin-4 activating interferon-related immune and inflammatory responses, and caffeine suppressing cell cycle-associated pathways. To facilitate translation of promising therapy into in vivo animal studies, a sensitive Liquid Chromatography-Tandem Mass Spectrometry method was developed for simultaneous quantification of caffeine and its metabolite paraxanthine in healthy EP ferrets. PK analysis demonstrated rapid systemic absorption, clinically relevant plasma exposure, and expected metabolism following intraperitoneal administration. Collectively, this thesis establishes a translational framework that integrates ex vivo therapeutic screening, mechanistic transcriptomic analysis, and PK characterization to identify neuroprotective therapies for preterm brain injury. These findings provide a foundation for future in vivo efficacy studies and the development of clinically translatable therapies to improve neurodevelopmental outcomes in extremely preterm born infants.