Proline prodrug of melphalan targeted to prolidase, a highly specific dipeptidase overexpressed in melanoma
Anticancer agents exhibit poor selectivity towards cancer cells thus leading to significant systemic toxicity. Enzyme-prodrug targeting strategy could potentially reduce systemic toxicity and increase the therapeutic index of chemotherapeutic agents. In this project, bioinformatic tools such as Perl®, Visual Basic® , Cluster® and TreeView ® were used to analyze public gene expression databases to identify potential enzyme targets. The analyses indicated prolidase to be a desirable enzyme target based on its high expression in melanoma cell lines and specificity for dipeptide substrates containing proline at the carboxy terminus. RT-PCR expression of prolidase was determined in tumor cell lines and the feasibility of targeting prolidase with prodrugs of anticancer agents was tested by (a) synthesizing prodrugs of melphalan that comprised linkage of the carboxy terminus of melphalan to the N-terminus of L- and D-stereoisomers of proline, and (b) determining their bioconversion and anti-proliferative activities, in vitro, in selected cancer cell lines. The results of hydrolysis studies of the L- and D-proline prodrugs of melphalan, designated as prophalan-L and prophalan-D respectively, indicated a significantly higher rate of activation of prophalan-L compared to prophalan-D in the cancer cell homogenates. The hydrolytic activity against prophalan-L (r2 = 0.86) as well as the cytotoxicity of prophalan-L (r 2 = 0.88) correlated highly with the expression levels of prolidase in the cancer cell lines, while prophalan-D was ineffective at comparable concentrations. The uptake of melphalan and the prodrugs did not exhibit a significant difference in the evaluated cell lines demonstrating activation of prodrug to melphalan to be the required step for cytotoxic effect. The two prodrugs and melphalan were then evaluated in vivo in the C57BL6 murine melanoma model for their therapeutic efficacy and toxicity. The tumor growth profiles demonstrated significant difference between prophalan-D and melphalan/prophalan-L treated mice but insignificant difference between melphalan and prophalan- L treated mice. However, prophalan-L was significantly less toxic than melphalan, while no significant difference in toxicity was seen for the prodrugs and control (buffered saline). Thus, prophalan- L, a stable prodrug of melphalan, exhibits a higher therapeutic index than melphalan by significantly reducing systemic toxicity while maintaining the therapeutic efficacy.