Abstract 1895: Ciclopirox prodrug for the prevention and therapy of non-muscle invasive bladder cancer

Objective: Ciclopirox prodrug for the prevention of Non-Muscle Invasive Bladder Cancer (NMIBC) represents a potential breakthrough in the management of high-grade, NMIBC. If successful, this represents the first systemic approach to manage NMIBC. Ciclopirox olamine, the active pharmaceutical ingredient in several topical antifungal drug products, has demonstrated anticancer activity in blood and solid tumors. We developed a phosphoryloxymethyl prodrug of ciclopirox (CPX) for efficient intravenous delivery, thereby avoiding dose-limiting gastrointestinal toxicities and first-pass effect associated with oral administration. Methods: In vitro anticancer activity of CPX was performed in both NMIBC (T24) and MIBC (253-JBV) cell lines. Cell proliferation was determined by hexoseaminidase assay. Stemness was determined by the Spheroid assay. Flow cytometric analyses was performed for cell cycle analysis. RT-PCR array analysis was performed to identify signaling pathways. Real Time PCR, western blot and immunofluorescent studies were performed for determining gene expression. Results: Pharmacokinetic studies conducted in mice, rats and dogs demonstrated that the drug is rapidly and completely metabolized and eliminated via the urine. In vitro, CPX inhibited growth of T24 and 253-JBV cells at 4 and 2 μM, respectively coupled with S-phase cell cycle arrest. FITC conjugated Annexin V-coupled flow cytometry studies showed that CPX induces apoptotic cell death. CPX also suppressed bladdosphere formation, suggesting that stem cells are affected. Notch signaling pathway plays a significant role in stem cell behavior, and previous studies have suggested that the pathway may be a therapeutic target for bladder cancer. Notch receptor activation, which occurs following ligand binding, involves specific intracellular cleavage by the γ-secretase complex. First, RT-PCR array analyses suggested that CPX inhibits the Notch signaling pathway. Furthermore, CPX significantly inhibited the expression of the γ-secretase complex proteins Presenilin1, Nicastrin, APH-1 and PEN-2. In addition, there was a reduction in the expression of downstream targets of the pathway including Hes1 and Cyclin-D1. These data suggest that CPX significantly inhibits Notch intracellular signaling pathway proteins. Ectopic expression of the activated/cleaved Notch protein reversed CPX mediated inhibition of cell proliferation. Conclusion: CPX is a novel chemotherapeutic and preventive agent for bladder cance