Repurposing Alzheimer's and ovarian cancer drugs as sonosensitizers for glioblastoma via a positive-unlabeled learning and 3D bioprinting-based new approach methodology (NAM).

Glioblastoma (GBM) remains a lethal primary brain tumor, in part because therapeutic efficacy is limited by the blood-brain barrier (BBB) and the complex tumor microenvironment (TME). Sonodynamic therapy (SDT), i.e., use of ultrasound to activate chemical sensitizers and generate cytotoxic stress, offers a non-invasive strategy for treating deep-seated intracranial disease, but progress is constrained by the scarcity of validated sonosensitizers and the inefficiency of conventional in vitro screening methods. Here, we introduce a New Approach Methodology (NAM) that couples a neural network-based positive-unlabeled (PU) learning framework with a high-throughput, magnetic field-guided 3D bioprinting platform to accelerate identification and experimental validation of SDT-sensitizing agents. Using curated drug and small-molecule data and RDKit-derived molecular descriptors, the PU classifier identifies candidate ultrasound-responsive compounds without requiring reliable negative labels. We then validate the AI-based predictions in physiologically relevant U-87 MG glioblastoma spheroids that reproduce key TME features, including spatial heterogeneity and a hypoxic core. The NAM identifies two FDA-approved drugs, carboplatin (advanced ovarian cancer) and memantine hydrochloride (Alzheimer's disease), as effective ultrasound-responsive agents. In 3D spheroids, combining low-intensity pulsed ultrasound with either drug significantly reduces viability compared with drug-only controls, and both combinations outperform temozolomide (TMZ), the current standard chemotherapeutic. Time-resolved responses reveal distinct kinetics: memantine produces strong early cytotoxicity (24 h) enhanced by ultrasound, whereas carboplatin shows delayed but pronounced cytotoxicity (72 h), also improved by ultrasound. Together, these results establish an integrated computational-experimental NAM that enables rapid repurposing of approved drugs as SDT sensitizers and provides a scalable framework for advancing GBM therapeutic discovery while reducing reliance on animal studies.