Comparative evaluation of the biological properties of reducible and acid-sensitive folate prodrugs of a highly potent doxorubicin derivative.

Two new water-soluble folate receptor-targeted drug conjugates that contain the highly active doxorubicin derivative N-(5,5-diacetoxybut-1-yl)doxorubicin were designed and evaluated for their biological activity against folate receptor positive tumours. The prodrugs were designed to contain an acid-sensitive hydrazone bond KO019 or in addition a disulphide bond KO013 in order to elucidate the importance of the pre-determined breaking point for their in vitro and in vivo properties. Fluorescence microscopy studies confirmed higher uptake of the prodrugs in folate receptor positive KB cells than in the folate receptor negative A549 lung cancer cells. In subsequent in vivo studies in the folate receptor positive KB xenograft model, KO019 was as active as the free drug but significantly less toxic when dosed at twice the dose of the free drug whereas KO013 showed no anticancer efficacy. As an explanation, we could show by HPLC that the prodrug KO013 that additionally contains a disulphide bond undergoes rapid disulphide exchange in murine plasma in the order of 40% after 5h at 37°C in contrast to KO019 which was essentially stable after a 5h incubation.