(747d) Targeted Enzyme Prodrug Therapy for Prostate Cancer – a Comparative Study of L-Methioninase, Purine Nucleoside Phosphorylase, and Cytosine Deaminase
Prostate cancer is the most common non-skin malignancy and the second leading cause of cancer-related death in American men, yet remains essentially incurable. Since the introduction of prostate specific antigen (PSA) screening, the lethality of prostate cancer stems not from a lack of early detection but more commonly from the failure of loco-regional therapies creating a need for improved systemic therapies. Currently, most single-agent anticancer drugs face challenges due to increased multidrug resistance, pharmacokinetic limitations, and restricted clinical dosage or frequency of administration due to cytotoxicity in non-cancerous tissues.
Enzyme prodrug therapy shows promise for the treatment of solid tumors such as prostate cancer, but current approaches lack effective/safe delivery strategies. To address this, we have developed three enzyme-containing fusion proteins targeted via annexin V (AV) to phosphatidylserine exposed on the tumor vasculature and tumor cells, using the enzymes L-methioninase (MT), purine nucleoside phosphorylase (PNP), or cytosine deaminase (CD). In enzyme prodrug therapy, the fusion protein is allowed to bind to the tumor before a nontoxic drug precursor, a prodrug, is introduced. Upon interaction of the prodrug with the bound enzyme, an anticancer compound is formed, but only in the direct vicinity of the tumor, thereby mitigating the risk of side effects while creating high intratumoral drug concentrations. Prostate cancer is especially suited to this targeting strategy as prostate carcinomas have been shown to have approximately twice the vascular density of healthy prostate tissue. The applicability of these enzyme prodrug systems to treating prostate cancer has remained unexplored. Additionally, target availability may increase with the addition of low-dose docetaxel treatment to the enzyme prodrug treatment. Therefore, in this study we examined the binding strength and the cytotoxic efficacy of these enzyme prodrug systems on human prostate cancer cell line PC-3 and on human endothelial cells grown to mimic the tumor vasculature. We also examined the effect on PC-cells of low-dose docetaxel on the cytotoxic efficacy for the three enzyme prodrug systems.
Enzymatically active fusion proteins were recombinantly produced in Escherichia coli and purified. The binding to cells of all three fusion proteins was relatively strong, with dissociation constants of 1.5 nM and lower for both the endothelial and PC-3 cells. In the in vitro cytotoxicity studies, the system using the MT-AV fusion protein performed the best, giving greater than 95% killing of endothelial cells grown to mimic the tumor vasculature and near complete killing of the PC-3 cells. In the presence of only the selenomethionine prodrug, there was a slight or no effect on these cells. Low-dose docetaxel treatment was found to increase the cytotoxic effect of the annexin V-targeted therapeutics for the PNP-AV and CD-AV systems. In conclusion, the enzyme prodrug system using MT-AV appears to be the most promising as an effective treatment of prostate cancer.