PRCA-08 A NOVEL BBB-PERMEABLE ANALOG FOR BREAST CANCER BRAIN METASTASES

Abstract Breast cancer brain metastasis (BCBM) accounts for 10-30% of the breast cancer metastatic burden and is associated with dismal prognoses, having a median survival time of 7.9 months. The poor outcomes stem from limited understanding of BCBM and the lack of blood-brain barrier (BBB) and blood-tumor barrier (BTB) penetrant drugs. This highlights the imperative to develop new therapies for BCBM. Truncated GLI1 (tGLI1), a tumor-specific GLI1 variant and terminal effector of the SHH pathway, has been identified as a key driver of breast cancer stem cells (CSCs) and a promoter of BCBM. The FDA-approved antifungal ketoconazole (KCZ) was discovered to selectively target tGLI1-positive breast cancer cells in vitro and inhibit tGLI1-driven brain metastases in vivo. However, the use of KCZ raises safety concerns due to its potent inhibition of the CYP3A4 enzyme, thereby leading to liver toxicity, adrenal insufficiency, and drug interactions. To address these issues, we modified the structure of KCZ and developed KCZ-analog, a novel tGLI1 inhibitor candidate. KCZ-analog was found to selectively target tGLI1-positive breast CSCs, while it lost the ability to inhibit CYP3A4 activity in vitro. Characterization of KCZ-analog revealed that it binds directly and specifically to tGLI1 but does not inhibit DNA binding. Through an in vivo maximum tolerated dose (MTD) study, we found that KCZ-analog showed no liver toxicity (normal serum ALT) or adrenal insufficiency (normal serum ACTH) levels. Toxicological analyses confirmed no toxic effects on other vital organs, and neither KCZ-analog nor KCZ affected blood cell count. Moreover, a perfusion study showed that KCZ-analog has enhanced bioavailability and can cross the BBB and BTB. Furthermore, a mouse metastasis model demonstrated that systemic administration of KCZ-analog suppressed the progression of tGLI1-positive BCBM. Our study supports further characterization of the pharmacological properties of KCZ-analog and its potential in the prevention of BCBM in vivo.