Abstract 4436: Biological evaluation of folate prodrugs with a highly toxic derivative of doxorubicin

Abstract

Abstract Folic acid is itself not biologically active but it is essential for the proliferation of cells. Cellular uptake of folate in the body is primarily mediated by the folate receptor (FR), a membrane-bound protein for which three isomers have been identified. The isomer FR-α is highly over-expressed in many human tumors. Since FR-α has a high affinity for binding and transporting physiologic levels of folic acid into cells, coupling a drug through a cleavable linker to folic acid can yield a prodrug that can be delivered inside tumor cells. Many studies have focused on reducible or acid-sensitive based linkers that are cleaved in the endosomes and/or cytoplasm. For the development of new folate receptor-targeted prodrugs, the highly active doxorubicin derivative N-(5,5-diacetoxybut-1-yl)doxorubicin was chosen to avoid potential saturation of the folate receptor. This acyclic doxorubicin diacetate undergoes an ester-mediated hydrolysis producing 2-pyrrolinodoxorubicin that is 500-1000 times more active than doxorubicin. Two prodrugs were designed to contain an acid-sensitive hydrazone bond (1) or in addition a disulfide bond (2) in order to elucidate the importance of the pre-determined breaking point for their in vitro and in vivo activity. In a 24h MTT assay, 1 was 5-fold more active than 2 in folate-positive KB cells in the nanomolar range whereas no activity was observed in the folate-negative A549 lung cancer cells, an observation that correlated well with the uptake of 1 and 2 in fluorescence microscopy studies. In subsequent in vivo studies in the folate-positive KB model, 1 was as active as the free drug but significantly less toxic when dosed at twice the dose of the free drug whereas 2 showed no anticancer efficacy. As an explanation, we could show by HPLC that prodrug 2 that additionally contains a disulfide bond undergoes rapid disulfide exchange in murine and human plasma in the order of 40% after 5 h at 37°C in contrast to 1 which was essentially stable after a 5 h incubation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4436. doi:10.1158/1538-7445.AM2011-4436