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Pharmaceutical Research

, Volume 22, Issue 9, pp 1510–1518 | Cite as

Floxuridine Amino Acid Ester Prodrugs: Enhancing Caco-2 Permeability and Resistance to Glycosidic Bond Metabolism

  • Christopher P. Landowski
  • Xueqin Song
  • Philip L. Lorenzi
  • John M. Hilfinger
  • Gordon L. Amidon
Research Paper

Abstract

Purpose

The aim of this study was to synthesize amino acid ester prodrugs of 5-fluoro-2′-deoxyuridine (floxuridine) to enhance intestinal absorption and resistance to glycosidic bond metabolism.

Methods

Amino acid ester prodrugs were synthesized and examined for their hydrolytic stability in human plasma, in Caco-2 cell homogenates, and in the presence of thymidine phosphorylase. Glycyl-l-sarcosine uptake inhibition and direct uptake studies with HeLa/PEPT1 cells [HeLa cells overexpressing oligopeptide transporter (PEPT1)] were conducted to determine PEPT1-mediated transport and compared with permeability of the prodrugs across Caco-2 monolayers.

Results

Isoleucyl prodrugs exhibited the highest chemical and enzymatic stability. The prodrugs enhanced the stability of the glycosidic bond of floxuridine. Thymidine phosphorylase rapidly cleaved floxuridine to 5-fluorouracil, whereas with the prodrugs no detectable glycosidic bond cleavage was observed. The 5′-l-isoleucyl and 5′-l-valyl monoester prodrugs exhibited 8- and 19-fold PEPT1-mediated uptake enhancement in HeLa/PEPT1 cells, respectively. Uptake enhancement in HeLa/PEPT1 cells correlated highly with Caco-2 permeability for all prodrugs tested. Caco-2 permeability of 5′-l-isoleucyl and 5′-l-valyl prodrugs was 8- to 11-fold greater compared with floxuridine.

Conclusions

Amino acid ester prodrugs such as isoleucyl floxuridine that exhibit enhanced Caco-2 transport and slower rate of enzymatic activation to parent, and that are highly resistant to metabolism by thymidine phosphorylase may improve oral delivery and therapeutic index of floxuridine.

Key Words

Caco-2 permeability floxuridine prodrugs metabolism PEPT1 thymidine phosphorylase 

Abbreviations

HeLa/PEPT1

HeLa cells overexpressing PEPT1

PEPT1

oligopeptide transporter

Notes

Acknowledgment

This work was supported by grant NIGMD-1R01GM 37188.

References

  1. 1.
    Kawaguchi, T., Saito, M., Suzuki, Y., Nambu, N., Nagai, T. 1985Specificity of esterases and structure of prodrug esters. II. Hydrolytic regeneration behavior of 5-fluoro-2′-deoxyuridine (FUdR) from 3′,5′-diesters of FUdR with rat tissue homogenates and plasma in relation to their antitumor activityChem. Pharm. Bull. (Tokyo)3316521659Google Scholar
  2. 2.
    Nishizawa, Y., Casida, J. E. 19653′,5′-Diesters of 5-fluoro-2′-deoxyuridine: synthesis and biological activityBiochem. Pharmacol.1416051619CrossRefPubMedGoogle Scholar
  3. 3.
    Tobias, S. C., Borch, R. F. 2001Synthesis and biological studies of novel nucleoside phosphoramidate prodrugsJ. Med. Chem.4444754480CrossRefPubMedGoogle Scholar
  4. 4.
    Wei, Y., Yan, Y., Pei, D., Gong, B. 1998A photoactivated prodrugBioorg. Med. Chem. Lett.824192422CrossRefPubMedGoogle Scholar
  5. 5.
    Vig, B. S., Lorenzi, P. J., Mittal, S., Landowski, C. P., Shin, H. C., Mosberg, H. I., Hilfinger, J. M., Amidon, G. L. 2003Amino acid ester prodrugs of floxuridine: synthesis and effects of structure, stereochemistry, and site of esterification on the rate of hydrolysisPharm. Res.2013811388CrossRefPubMedGoogle Scholar
  6. 6.
    Landowski, C. P., Vig, B. S., Song, X., Amidon, G. L. 2005Targeted delivery to PEPT1-overexpressing cells: acidic, basic, and secondary floxuridine amino acid ester prodrugsMol. Cancer Ther.4659667PubMedGoogle Scholar
  7. 7.
    Laar, J. A., Rustum, Y. M., Ackland, S. P., Groeningen, C. J., Peters, J. 1998Comparison of 5-fluoro-2′-deoxyuridine with 5-fluorouracil and their role in the treatment of colorectal cancerEur. J. Cancer34296306CrossRefPubMedGoogle Scholar
  8. 8.
    Birnie, G. D., Kroeger, H., Heidelberger, C. 1963Studies of fluorinated pyrimidines. XVIII. The degradation of 5-fluoro-2′-deoxyuridine and related compounds by nucleoside phosphorylaseBiochemistry13566572CrossRefGoogle Scholar
  9. 9.
    Moranand, R. G., Heidelberger, C. 1979Determinants of 5-fluorouracil sensitivity in human tumorsBull. Cancer667983PubMedGoogle Scholar
  10. 10.
    Mukherjee, K. L., Boohar, J., Wentland, D., Ansfield, F. J., Heidelberger, C. 1963Studies of fluorinated pyrimidines. XVI. Metabolism of 5-fluorouracil-2-C14 and 5-fluoro-2′-deoxyuridine-2-C14 in cancer patientsCancer Res.234966Google Scholar
  11. 11.
    Chaudhuri, N. K., Mukherjee, K. L., Heidelberger, C. 1959Studies on fluorinated pyrimidines. VII. The degradative pathwayBiochem. Pharmacol.1328341CrossRefGoogle Scholar
  12. 12.
    Harbers, E., Chaudhuri, N. K., Heidelberger, C. 1959Studies on fluorinated pyrimidines. VIII. Further biochemical and metabolic investigationsJ. Biol. Chem.23412551262PubMedGoogle Scholar
  13. 13.
    Kanzawa, F., Hoshi, A., Kuretani, K., Saneyoshi, M., Kawaguchi, T. 1981Antitumor activity of 3′,5′-diesters of 5-fluoro-2′-deoxyuridine against murine leukemia L1210 cellsCancer Chemother. Pharmacol.61923CrossRefPubMedGoogle Scholar
  14. 14.
    Fletcher, C., Bean, B. 1985Evaluation of oral acyclovir therapyDrug Intel. Clin. Pharm.19518524Google Scholar
  15. 15.
    Beauchamp, L. M., Orr, G. F., Miranda, P., Burnette, T., Kernitsy, T. A. 1992Amino acid ester prodrugs of acyclovirAntivir. Chem. Chemother.3157164Google Scholar
  16. 16.
    Han, H., Vrueh, R. L., Rhie, J. K., Covitz, K. M., Smith, P. L., Lee, C. P., Oh, D. M., Sadee, W., Amidon, G. L. 19985′-Amino acid esters of antiviral nucleosides, acyclovir, and AZT are absorbed by the intestinal PEPT1 peptide transporterPharm. Res.1511541159CrossRefPubMedGoogle Scholar
  17. 17.
    Sugawara, M., Huang, W., Fei, Y. J., Leibach, F. H., Ganapathy, V., Ganapathy, M. E. 2000Transport of valganciclovir, a ganciclovir prodrug, via peptide transporters PEPT1 and PEPT2J. Pharm. Sci.89781789CrossRefPubMedGoogle Scholar
  18. 18.
    Shin, H. C., Landowski, C. P., Amidon, G. L. 2003Transporters in the GI tractWaterbeemd, L. H.Lennernäs, H.Artursson, P. eds. Drug Bioavailability/Estimation of Solubility, Permeability and Absorption (Series: Methods and Principles in Medicinal Chemistry) Vol. 18WileyWeinheim, Germany245287Google Scholar
  19. 19.
    Hsu, C. P., Hilfinger, J. M., Walter, E., Merkle, H. P., Roessler, B. J., Amidon, G. L. 1998Overexpression of human intestinal oligopeptide transporter in mammalian cells via adenoviral transductionPharm. Res.1513761381CrossRefPubMedGoogle Scholar
  20. 20.
    Han, H. K., Oh, D. M., Amidon, G. L. 1998Cellular uptake mechanism of amino acid ester prodrugs in Caco-2/hPEPT1 cells overexpressing a human peptide transporterPharm. Res.1513821386CrossRefPubMedGoogle Scholar
  21. 21.
    Gao, J., Hugger, E. D., Beck-Westermeyer, M. S., Borchardt, R. T. 2000Estimating intestinal mucosal permeation of compounds using Caco-2 cell monolayersEnna, S. J.Williams, M.Ferkany, J. W.Kenakin, T.Porsolt, R. D.Sullivan, J. P. eds. Current Protocols in PharmacologyWileyNew York721723Google Scholar
  22. 22.
    Weller, S., Blum, M. R., Doucette, M., Burnette, T., Cederberg, D. M., Miranda, P., Smiley, M. L. 1993Pharmacokinetics of the acyclovir pro-drug valaciclovir after escalating single- and multiple-dose administration to normal volunteersClin. Pharmacol. Ther.54595605PubMedGoogle Scholar
  23. 23.
    Ganapathy, M. E., Huang, W., Wang, H., Ganapathy, V., Leibach, F. H. 1998Valacyclovir: a substrate for the intestinal and renal peptide transporters PEPT1 and PEPT2Biochem. Biophys. Res. Commun.246470475CrossRefPubMedGoogle Scholar
  24. 24.
    Faria, T. N., Timoszyk, J. K., Stouch, T. R., Vig, B. S., Landowski, C. P., Amidon, G. L., Weaver, C. D., Wall, D. A., Smith, R. L. 2004A novel high-throughput pept1 transporter assay differentiates between substrates and antagonistsMol. Pharm.16776CrossRefPubMedGoogle Scholar
  25. 25.
    Sawada, K., Terada, T., Saito, H., Hashimoto, Y., Inui, K. I. 1999Recognition of l-amino acid ester compounds by rat peptide transporters PEPT1 and PEPT2J. Pharmacol. Exp. Ther.291705709PubMedGoogle Scholar
  26. 26.
    Shin, H. C., Landowski, C. P., Sun, D., Vig, B. S., Kim, I., Mittal, S., Lane, M., Rosania, G., Drach, J. C., Amidon, G. L. 2003Functional expression and characterization of a sodium-dependent nucleoside transporter hCNT2 cloned from human duodenumBiochem. Biophys. Res. Commun.307696703CrossRefPubMedGoogle Scholar
  27. 27.
    Sun, D., Lennernas, H., Welage, L. S., Barnett, J. L., Landowski, C. P., Foster, D., Fleisher, D., Lee, K. D., Amidon, G. L. 2002Comparison of human duodenum and Caco-2 gene expression profiles for 12,000 gene sequences tags and correlation with permeability of 26 drugsPharm. Res.1914001416CrossRefPubMedGoogle Scholar
  28. 28.
    Woodman, P. W., Sarrif, A. M., Heidelberger, C. 1980Specificity of pyrimidine nucleoside phosphorylases and the phosphorolysis of 5-fluoro-2′-deoxyuridineCancer Res.40507511PubMedGoogle Scholar
  29. 29.
    Norman, R. A., Barry, S. T., Bate, M., Breed, J., Colls, J. G., Ernill, R. J., Luke, R. W., Minshull, C. A., McAlister, M. S., McCall, E. J., McMiken, H. H., Paterson, D. S., Timms, D., Tucker, J. A., Pauptit, R. A. 2004Crystal structure of human thymidine phosphorylase in complex with a small molecule inhibitorStructure (Camb.)127584CrossRefGoogle Scholar
  30. 30.
    Bennett, E. M., Li, C., Allan, P. W., Parker, W. B., Ealick, S. E. 2003Structural basis for substrate specificity of Escherichia coli purine nucleoside phosphorylaseJ. Biol. Chem.2784711047118CrossRefPubMedGoogle Scholar
  31. 31.
    Kim, I., Song, X., Vig, B. S., Mittal, S., Shin, H. C., Lorenzi, P. J., Amidon, G. L. 2004A novel nucleoside prodrug activating enzyme: substrate specificity of biphenyl hydrolase-like proteinMol. Pharm.1117127CrossRefPubMedGoogle Scholar
  32. 32.
    Fukushima, S., Kawaguchi, T., Nishida, M., Juni, K., Yamashita, Y., Takahashi, M., Nakano, M. 1987Selective anticancer effects of 3′,5′-dioctanoyl-5-fluoro-2′-deoxyuridine, a lipophilic prodrug of 5-fluoro-2′-deoxyuridine, dissolved in an oily lymphographic agent on hepatic cancer of rabbits bearing VX-2 tumorCancer Res.4719301934PubMedGoogle Scholar
  33. 33.
    Xia, Z., Wiebe, L. I., Miller, G. G., Knaus, E. E. 1999Synthesis and biological evaluation of butanoate, retinoate, and bis(2,2,2-trichloroethyl)phosphate derivatives of 5-fluoro-2′-deoxyuridine and 2′,5-difluoro-2′-deoxyuridine as potential dual action anticancer prodrugsArch. Pharm. (Weinheim)332286294CrossRefGoogle Scholar

Copyright information

© Springer Science + Business Media, Inc. 2005

Authors and Affiliations

  • Christopher P. Landowski
    • 1
  • Xueqin Song
    • 1
  • Philip L. Lorenzi
    • 1
  • John M. Hilfinger
    • 2
  • Gordon L. Amidon
    • 1
  1. 1.Department of Pharmaceutical Sciences, College of PharmacyUniversity of MichiganAnn ArborUSA
  2. 2.TSRL, Inc.Ann ArborUSA

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