Enhanced cancer cell growth inhibition by dipeptide prodrugs of floxuridine: Increased transporter affinity and metabolic stability

Dipeptide monoester prodrugs of floxuridine were synthesized, and their chemical stability in buffers, resistance to glycosidic bond metabolism, affinity for PEPT1, enzymatic activation and permeability in cancer cells were determined and compared to those of mono amino acid monoester floxuridine prodrugs. Prodrugs containing glycyl moieties were the least stable in pH 7.4 buffer (t(1/2) < 100 min). The activation of all floxuridine prodrugs was 2- to 30-fold faster in cell homogenates than their hydrolysis in buffer, suggesting enzymatic action. The enzymatic activation of dipeptide monoester prodrugs containing aromatic promoieties in cell homogenates was 5- to 20-fold slower than that of other dipeptide and most mono amino acid monoester prodrugs (t(1/2) similar to 40 to 100 min). All prodrugs exhibited enhanced resistance to glycosidic bond metabolism by thymidine phosphorylase compared to parent floxuridine. In general, the 5'-O-dipeptide monoester floxuridine prodrugs exhibited higher affinity for PEPT1 than the corresponding 5'-O-mono amino acid ester prodrugs. The permeability of dipeptide monoester prodrugs across Caco-2 and Capan-2 monolayers was 2- to 4-fold higher than the corresponding mono amino acid ester prodrug. Cell proliferation assays in AsPC-1 and Capan-2 pancreatic ductal cell lines indicated that the dipeptide monoester prodrugs were equally as potent as mono amino acid prodrugs. The transport and enzymatic profiles of 5'-L-phenylalanyl-L-tyrosyl-floxuridine, 5'-L-phenylalanyl-L-glycyl-floxuridine, and 5'-L-isoleucyl-L-glycyl-floxuridine suggest their potential for increased oral uptake, delayed enzymatic bioconversion and enhanced resistance to metabolism to 5-fluorouracil, as well as enhanced uptake and cytotoxic activity in cancer cells, attributes that would facilitate prolonged systemic circulation for enhanced therapeutic action.