Floxuridine prodrug development: Transporter affinity and enzymatic activation.

Abstract

The researches were conducted to determine the advantages of prodrug approach over a parent drug. 5'-dipeptide floxuridine prodrugs were synthesized and evaluated for their enzymatic stability in human plasma and cell homogenates and non-enzymatic stability in pH 7.4 buffer. Suitability as transporter substrates was assessed by their ability to inhibit [<super>3</super>H]Gly-Sar uptake into 3 different cells, Caco-2 cells, AsPC-1 cells, and Capan-2 cells. The transporter affinity and stability of 5'-dipeptide floxuridine prodrugs were also compared with 5'-mono ester floxuridine prodrugs. The 5'-dipeptide floxuridine prodrugs showed, higher affinity by inhibiting Gly-Sar uptake for transporters than the monoesters. All prodrugs were hydrolyzed to parent drug in all cell homogenates and human plasma. Two prodrugs, 5'-O-Valyl-Phenylalanyl-floxuridine and 5'-O-Phenylalanyl-Tyrosyl-floxuridine, were the most stable dipeptide prodrugs among them in three different cell lines, half life 62.90-83.18 min and 42.83-95.23 min, respectively. Mono amino acid/dipeptide ester prodrugs of floxuridine showed better tumor growth inhibition in two pancreatic cancer cells while the parent drug barely exhibited any tumor growth inhibition. All prodrugs showed good permeability in Capan-2 cells while the membrane permeability of floxuridine was not observed. This might be a faster metabolic pathway for FUdR and 5-FU due to enzyme upregulation. Therefore, the importance of prodrug stability cannot be emphasized enough. In prodrugs, time lag for growth inhibition of tumor cells was observed due to the various chemical/enzymatic stabilities of prodrugs. The result suggests that there are specific enzymes to activate prodrugs based on the prodrug stability and the enzyme locations in cells, in which all enzymes are well arranged in the cell compartments unlike cell homogenates. Thus, the stability of prodrugs in cell homogenates may lead to underestimation. Dipeptide prodrugs of floxuridine with improved transporter affinity and tumor growth inhibition were successfully synthesized. To design prodrugs for further investigations, not only the transporter affinity but also the stability and activation of prodrugs including enzyme substrate specificities have to be carefully considered and monitored.