Efflux-Aware Human BBB Assembloid Assay for Brain-Penetrant Therapeutic Discovery in Medulloblastoma

About This Grant

Project Summary/Abstract Medulloblastoma (MB) is the most common malignant brain tumor in children and often disseminates early within the central nervous system. Among the four molecular subgroups, Groups 3 and 4 carry the greatest metastatic burden and poorest outcomes. Although digoxin is an approved cardiac glycoside with a well-defined pharmacokinetic (PK) and safety profile and shows activity in Group 3 and Group 4 patient-derived models, translation is limited by its narrow therapeutic index and P-glycoprotein(P-gp)–mediated efflux at the blood brain barrier. We treat radiotherapy as a programmable trigger to localize drug action. Our radiotherapy-activated pro- digoxin chemotypes mask the sugar moiety with N-alkyl-4-picolinium or phenyl-azide carbonate groups to reduce transporter recognition while preserving the lactone required for target engagement; ionizing radiation cleaves the trigger in the treated field to release native digoxin, aiming to raise intratumoral exposure while keeping systemic levels within pediatric therapeutic ranges. The R61 phase will synthesize and analytically qualify two predefined pro-digoxin chemotypes and establish a stability-indicating liquid chromatography–tandem mass spectrometry method to quantify intact pro-drug, released digoxin, and scission products. Using induced pluripotent stem cell–derived brain and vessel organoid assembloids that recapitulate a functional human blood brain barrier, we will quantify radiotherapy-triggered conversion, directional permeability, and P-gp efflux to derive a dose-to-release function and a dosing-to-beam timing window. A focused pilot PK study will confirm brain exposure and lock pharmacodynamic (PD) sampling for Na⁺/K⁺-ATPase target engagement and radiation-linked biomarkers. The R33 phase will implement a preregistered two-by-two design in paired primary and recurrent Group 3 and Group 4 intracranial patient-derived orthotopic xenograft models, using volumetric magnetic resonance imaging to track burden and progression and therapeutic drug monitoring to maintain clinically relevant digoxin-equivalent troughs. Embedded regional bioanalysis will verify in-tissue conversion in irradiated brain, and a focused safety module will monitor cardiac and neurologic tolerance. Critically, R33 will deliver an integrated PK → PD → Efficacy model that links exposure and target engagement to tumor control, identifies the optimal drug-to-radiation schedule and minimal effective duration, and back-translates active mouse exposures to pediatric therapeutic drug-monitoring ranges. By coupling a human neurovascular platform, field-restricted activation chemistry, and quantitative mass spectrometry with rigorous in vivo testing, this program seeks to expand the therapeutic index for children with Group 3 and Group 4 MB and establish a generalizable path from mechanism to schedule to outcome for transporter-limited agents in pediatric neuro-oncology.