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Glioblastomas have been historically difficult to treat with poor long-term survival. With novel strategies focused on targeting hypoxia-inducible factor (HIF) regulatory pathways, recent evidence has shown that Acriflavine (ACF) can effectively target glioma invasiveness and recurrence. However, local delivery of ACF and its combinatory effects with Temozolomide (TMZ) and radiation therapy (XRT) have not yet been optimized. In this study we test a novel polymeric matrix that can gradually release ACF at the tumor bed site in combination with systemic TMZ and XRT. In vitro cytotoxicity assays of ACF in combination with TMZ and XRT were performed on rodent and human cell lines with CCK-8 and flow cytometry. In vitro drug release was measured and intracranial safety was assessed in tumor-free animals. Finally, efficacy was assessed in an intracranial gliosarcoma model and combination therapy with TMZ and XRT evaluated. Combination therapy of ACF, TMZ, and XRT was able to reduce cell viability and induce apoptosis in glioma cells. In vitro and in vivo release of ACF was measured in benchtop and animal models. Efficacy was established in an in vivo gliosarcoma model in which intracranial ACF (p 0.01) significantly improved median survival and the combination therapy of ACF, TMZ and XRT (p 0.01) significantly improved median survival and led to long-term survival (LTS). We provide evidence that ACF, combined with TMZ and XRT, led to LTS in an intracranial model of rat gliosarcoma. These findings, in combination with the use of a novel polymeric matrix that allows more gradual drug delivery, constitute a first step in the translation of this novel strategy to human use.

Glioblastoma multiforme (GBM) has few clinically approved therapeutic regimens. One of these therapeutic options includes placing biodegradable wafers releasing BCNU (Gliadel®) into the tumor bed at the time of surgical removal of the tumor. Due to the significant benefit this polymer technology has had clinically, we have prepared wafers releasing Temozolomide (TMZ), an anticancer drug used systemically for treating GBM. TMZ delivered via polymer wafer could be used as a complementary treatment with or as an alternative to Gliadel®. TMZ is an alkylating agent which is water soluble. To remain comparable with the preclinical studies that led to Gliadel® the same size of wafers were formulated with TMZ. Wafers were loaded with 50% w/w TMZ in poly(lactic acid-glycolic acid) (PLGA) and showed reliable release of high dose TMZ for a period of 4 weeks. To achieve this 30-day release of the highly water soluble drug, we developed an encapsulation method, where the drug powder was first coated with the polymer to form core-shell particles in which the coating shell served as a rate controlling membrane for the drug particles. Wafers were also made with a co-loading of TMZ and BCNU. All wafers were tested in vivo by treating an intracranial 9 L gliosarcoma model in F344 rats. Rats that were either untreated or treated with blank wafer died within 11 days while the median survival for rats treated with systemic TMZ was 18 days. The group that received the BCNU alone wafer had a median survival of 15 days, the group that received the TMZ wafer alone had a median survival of 19 days, and the group treated with the BCNU-TMZ wafer had a median survival of 28 days with 25% of the animals living long term (p .0038 vs. Control; p .001 vs. Blank Polymer). These findings demonstrate the potential of this newly designed wafer for treating GBM. Moreover, this concept, can pave the way for other drug combinations that may improve the clinical application of numerous agents to treat solid tumors.