| The anticancer drug, floxuridine, exhibits poor oral bioavailability partially due to extensive first pass metabolism by pyrimidine nucleoside phosphorylases to a less potent metabolite, 5-fluorouracil. The objective of this work was to design amino acid ester prodrugs of floxuridine to enhance intestinal absorption and minimize ester and glycosidic bond metabolism. In designing prodrugs to improve oral bioavailability, it was important to know what transporters or metabolizing enzymes prodrugs would encounter after reaching the absorptive surface of the intestine. Microarray expression profiling revealed that transporter expression in the Caco-2 cell model is most similar to that found in the human ileum. In terms of metabolizing enzyme expression, this model best resembles the colon. Based on peptide transporter and metabolizing enzyme expression in the intestine, floxuridine prodrugs were designed to take advantage of carrier mediated transport and resist metabolism. Twenty-seven floxuridine prodrugs were synthesized with diversity in promoiety structure, stereochemistry, and esterification site to understand the structural features involved in optimal prodrug absorption via peptide transporters, as well as how different promoieties are hydrolyzed and how they may affect parent drug metabolism. Floxuridine permeability across Caco-2 cells was enhanced up to 11-fold when the 5' position was esterified with an isoleucine or valine amino acid. The 5'-Val, 5'-Phe, 5'-Leu, and 5' -Ile prodrugs exhibited permeability values between 1--5 x 10-6 cm/s, similar to that observed with valacylovir. PEPT1 prodrug transport correlated well with permeability across Caco-2 monolayers suggesting peptide carrier enhancement. The promoiety and the esterification site most significantly influenced carboxylesterase mediated activation rates. Carboxylesterase displayed the greatest catalytic efficiency for prodrugs containing a phenylalanine residue and was less active against prodrugs containing valine or isoleucine, regardless of the esterification site. Floxuridine was rapidly cleaved by thymidine phosphorylase, whereas gemcitabine and all the ester prodrugs resisted breakdown. Therefore, the rate limiting step to prodrug metabolism is ester bond hydrolysis which subsequently releases the parent drug. This suggests that stable ester prodrugs may provide a mechanism to control undesired floxuridine metabolism. The combined strategy of increasing the intestinal uptake and reducing the metabolism of floxuridine may lead to a more effective orally administered cancer drug. |
