Pharmaceutical Research

, Volume 15, Issue 9, pp 1382–1386 | Cite as

Cellular Uptake Mechanism of Amino Acid Ester Prodrugs in Caco-2/hPEPT1 Cells Overexpressing a Human Peptide Transporter

  • Hyo-kyung Han
  • Doo-Man Oh
  • Gordon L. Amidon


Purpose. This study characterized the cellular uptake mechanism and hydrolysis of the amino acid ester prodrugs of nucleoside antiviral drugs in the transiently transfected Caco-2 cells overexpressing a human intestinal peptide transporter, hPEPTl (Caco-2/hPEPTl cells).

Methods. Amino acid ester prodrugs of acyclovir and AZT were synthesized and their apical membrane permeability and hydrolysis were evaluated in Caco-2/hPEPTl cells. The cellular uptake mechanism of prodrugs was investigated through the competitive inhibition study in Caco-2/hPEPTl cells.

Results. L-Valyl ester of acyclovir (L-Val-ACV) was approximately ten fold more permeable across the apical membrane than acyclovir and four times more permeable than D-valyl ester of acyclovir (D-Val-ACV). Correspondingly, L-valyl ester of AZT (L- Val-AZT) exhibited three fold higher cellular uptake than AZT. Therefore, amino acid ester prodrugs significantly increased the cellular uptake of the parent drugs and exhibited the D,L-stereoselectivity. Furthermore, prodrugs were rapidly hydrolyzed to the parent drugs by the intracellular hydrolysis, following the apical membrane transport. In the inhibition studies, cephalexin and small dipeptides strongly inhibited the cellular uptake of L-Val-ACV while L-valine had no effect, indicating that the peptide transporter is primarily responsible for the apical membrane transport of L-Val-ACV. In addition, the cellular uptake of L-Val-ACV was five times higher in Caco-2/hPEPT 1 cells than the uptake in the untransfected Caco-2 cells, implying the cellular uptake of L-Val-ACV was related to the enhancement of the peptide transport activity in Caco-2/hPEPTl cells.

Conclusions. Caco-2/hPEPTl system is an efficient in vitro model for the uptake study of peptidyl derivatives. Amino acid ester prodrugs significantly improved the cellular uptake of the parent drugs via peptide transport mechanism and were rapidly converted to the active parent drugs by the intracellular hydrolysis.

amino acid ester Caco-2 cells cellular uptake peptide transporter permeability prodrugs 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    S. Weller, M. R. Blum, M. Doucette, T. Burnette, D. M. Cederberg, P. de Miranda, and M. L. Smiley. Pharmacokinetics of the acyclovir prodrug valaciclovir after escalating single and multiple dose administration to normal volunteers. Clin. Pharmacol. Ther. 54:595–605 (1993).PubMedGoogle Scholar
  2. 2.
    R. H. Lupia, N. Ferencz, J. L. Lertora, S. K. Aggarwal, W. J. George, and K. C. Agrawal. Comparative Pharmacokinetics of Two Prodrugs of Zidovudine in Rabbits: Enhanced Levels of Zidovudine in Brain Tissue. Antimicrob. Agents Chemother. 37:818–829 (1993).PubMedGoogle Scholar
  3. 3.
    L. M. Beauchamp, G. F. Orr, P. de Miranda, T. Burnette, and T. A. Krenitsky. Amino acid ester prodrugs of acyclovir. Antiviral. Chem. Chemother. 3:157–164 (1992).Google Scholar
  4. 4.
    H-k. Han and G. L. Amidon. Intestinal absorption of valacyclovir, a novel prodrug of acyclovir, in the rat jejunum. Pharm. Res. 13:S-246 (1996).Google Scholar
  5. 5.
    I. J. Hidalgo, T. J. Raub, and R. T. Borchardt. Characterization of the human colon carcinoma cell line(Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology 96:736–749 (1989).PubMedGoogle Scholar
  6. 6.
    I. Tamai, H. Takanaga, H. Maeda, Y. Sai, T. Ogihara, H. Higashida, and A. Tsuji. Participation of a proton-cotransporter, MCT1, in the intestinal transport of monocarboxylic acids. Biochem. Biophys. Res. Commun. 214:482–489 (1995).PubMedGoogle Scholar
  7. 7.
    J. Mesonero, M. Matosin, D. Cambier, M. J. Rodriguez-Yoldi, and E. Brot-Laroche. Sugar-dependent expression of the fructose transporter GLUT5 in Caco-2 cells. Biochem. J. 312:757–762 (1995).PubMedGoogle Scholar
  8. 8.
    M. Hu. Comparison of uptake characteristics of thymidine and zidovudine in a human intestinal epithelial model system. J. Pharm. Sci. 82:829–833 (1993).PubMedGoogle Scholar
  9. 9.
    S. Chong, S. A. Dando, K. M. Soucek, and R. A. Morrison. In vitro permeability through Caco-2 cells is not quantitatively predictive of in vivo absorption for peptide-like drugs absorbed via the dipeptide transporter system. Pharm. Res. 13:120–123 (1996).PubMedGoogle Scholar
  10. 10.
    W. Liu, S. Ramamoorthy, Y. J. Fei, N. E. Ganapathy, M. A. Hediger, V. Ganapathy, and F. H. Leibach. Molecular cloning of PEPT2, a new member of the H+/peptide cotransporter family from human kidney. Biochim. Biophys. Acta. 1235:461–466 (1995).PubMedGoogle Scholar
  11. 11.
    R. Liang, Y. J. Fei, P. D. Prasad, S. Ramamoorthy, H. Han, T. L. Yang-Feng, M. A. Hediger, V. Ganapathy, and F. H. Leibach. Human intestinal H+/peptide cotransporter: cloning, functional expression and chromosomal localization. J. Biol. Chem. 270:6456–6463 (1995).PubMedGoogle Scholar
  12. 12.
    K. Y. Covitz, G. L. Amidon, and W. Sadée. Human dipeptide transporter, hPEPT1, stably transfected into chinese hamster ovary cells. Pharm. Res. 13:1631–1634 (1996).PubMedGoogle Scholar
  13. 13.
    M. C-P. Hsu, J. M. Hilfinger, E. Walter, H. P. Merkle, B. J. Roessler, and G. L. Amidon. Overexpression of Human Intestinal Oligopeptide Transporter in Mammalian Cells via Adenoviral Transduction. (in press).Google Scholar
  14. 14.
    O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275 (1951).PubMedGoogle Scholar
  15. 15.
    M. Hu, P. Subramanian, H. I. Mosberg, and G. L. Amidon. Use of the peptide carrier system to improve the intestinal absorption of L-α-methyldopa: carrier kinetics, intestinal permeabilities, and in vitro hydrolysis of dipeptidyl derivatives of L-α-methyldopa. Pharm. Res. 6:66–70 (1989).PubMedGoogle Scholar
  16. 16.
    K. C. Meadows and J. B. Dressman. Mechanism of acyclovir uptake in rat jejunum. Pharm. Res. 7:299–303 (1990).PubMedGoogle Scholar

Copyright information

© Plenum Publishing Corporation 1998

Authors and Affiliations

  • Hyo-kyung Han
    • 1
  • Doo-Man Oh
    • 1
  • Gordon L. Amidon
    • 1
  1. 1.College of PharmacyThe University of MichiganAnn Arbor

Personalised recommendations