Pharmaceutical Research

, Volume 14, Issue 2, pp 169–175 | Cite as

Effect of Restricted Conformational Flexibility on the Permeation of Model Hexapeptides Across Caco-2 Cell Monolayers

  • Franklin W. Okumu
  • Giovanni M. Pauletti
  • David G. Vander Velde
  • Teruna J. Siahaan
  • Ronald T. Borchardt


Purpose. To determine how restricted conformational flexibility of hexapeptides influences their cellular permeation characteristics.

Methods. Linear (Ac-Trp-Ala-Gly-Gly-X-Ala-NH2; X = Asp, Asn, Lys) and cyclic (cyclo[Trp-Ala-Gly-Gly-X-Ala]; X = Asp, Asn, Lys) hexapeptides were synthesized, and their transport characteristics were assessed using the Caco-2 cell culture model. The lipophilicities of the hexapeptides were determined using an immobilized artificial membrane. Diffusion coefficients used to calculate molecular radii were determined by NMR. Two-dimensional NMR spectroscopy, circular dichroism, and molecular dynamic simulations were used to elucidate the most favorable solution structure of the cyclic Asp-containing peptide.

Results. The cyclic hexapeptides used in this study were 2−3 times more able to permeate (e.g., Papp = 9.3 ± 0.3 × 10−8 cm/sec, X = Asp) the Caco-2 cell monolayer than were their linear analogs (e.g., Papp = 3.2 ± 0.3 × 10−8 cm/sec, X = Asp). In contrast to the linear hexapeptides, the flux of the cyclic hexapeptides was independent of charge. The cyclic hexapeptides were shown to be more lipophilic than the linear hexapeptides as determined by their retention times on an immobilized phospholipid column. Determination of molecular radii by two different techniques suggests little or no difference in size between the linear and cyclic hexapeptides. Spectroscopic data indicate that the Asp-containing linear hexapeptide exists in a dynamic equilibrium between random coil and β-turn structures while the cyclic Asp-containing hexapeptide exists in a well-defined compact amphophilic structure containing two β-turns.

Conclusions. Cyclization of the linear hexapeptides increased their lipophilicities. The increased permeation characteristics of the cyclic hexapeptides as compared to their linear analogs appears to be due to an increase in their flux via the transcellular route because of these increased lipophilicities. Structural analyses of the cyclic Asp-containing hexapeptide suggest that its well-defined solution structure and, specifically the existence of two β-turns, explain its greater lipophilicity.

peptide delivery conformation Caco-2 cells membrane permeability NMR CD 


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  1. 1.
    N. C. Wrighton, F. X. Farrell, R. Chang, A. K. Kashyap, F. P. Barbone, L. S. Mulcahy, D. L. Johnson, R. W. Barett, L. K. Jolliffe, and W. J. Dower. Science 273:397–544 (1996).Google Scholar
  2. 2.
    G. M. Pauletti, S. Gangwar, G. T. Knipp, M. M. Nerurkar, F. W. Okumu, K. Tamura, T. J. Siahaan, and R. T. Borchardt. J. Controlled Release 41:3–17 (1996).Google Scholar
  3. 3.
    V. H. L. Lee, R. D. Traver, and M. E. Taub. in: V. H. L. Lee (ed.), Peptide and Protein Drug Delivery, Marcel Dekker, New York, 1991, pp. 303–357.Google Scholar
  4. 4.
    M. J. Humphrey. in: S. S. Davis, L. Illum and E. Tomlinson (eds.), Delivery Systems for Peptide Drugs, Plenum Press, New York, 1986, pp. 139–151.Google Scholar
  5. 5.
    X. H. Zhou. J. Controlled Release 29:239–252 (1994).Google Scholar
  6. 6.
    J. P. Yevich. in: P. Krogsgaard-Larsen and H. Bundgaard (eds.), A Textbook of Drug Design and Development, Harwood Academic Publishers, Langhorne, 1991, pp. 606–630.Google Scholar
  7. 7.
    R. A. Conradi, P. S. Burton, and R. T. Borchardt. in: V. Pliska, B. Testa and H. Van de Waterbeemd (eds.), Lipophilicity in Drug Action and Toxicology, VCH, Weinheim, 1996, pp. 233–250.Google Scholar
  8. 8.
    R. A. Conradi, A. R. Hilgers, N. F. H. Ho, and P. S. Burton. Pharm. Res. 9:435–439 (1992).Google Scholar
  9. 9.
    P. S. Burton, R. A. Conradi, A. R. Hilgers, N. F. H. Ho, and L. L. Maggiora. J. Controlled Release 19:87–93 (1992).Google Scholar
  10. 10.
    J. Drewe, G. Fricker, J. Vonderscher, and C. Beglinger. Br. J. Pharmacol. 108:298–303 (1993).Google Scholar
  11. 11.
    S. Lundin and P. Artursson. Int. J. Pharm. 64:181–186 (1990).Google Scholar
  12. 12.
    D. T. Thwaites, B. H. Hirst, and N. L. Simmons. Pharm. Res. 10:674–681 (1993).Google Scholar
  13. 13.
    G. M. Pauletti, F. W. Okumu, and R. T. Borchardt. Pharm. Res. 14:169–175 (1997).Google Scholar
  14. 14.
    G. T. Knipp, N. F. H. Ho, C. L. Barsuhn, and R. T. Borchardt. Pharm. Res. 12:S-302 (1995).Google Scholar
  15. 15.
    A. Adson, T. J. Raub, P. S. Burton, C. L. Barsuhn, A. R. Hilgers, K. L. Audus, and N. F. H. Ho. J. Pharm. Sci. 83:1529–1536 (1994).Google Scholar
  16. 16.
    D. Barlow and T. Satoh. J. Controlled Release 29:283–291 (1994).Google Scholar
  17. 17.
    I. J. Hidalgo, T. J. Raub, and R. T. Borchardt. Gastroenterology 96:736–749 (1989).Google Scholar
  18. 18.
    G. M. Pauletti, S. Gangwar, F. W. Okumu, T. J. Siahaan, V. J. Stella, and R. T. Borchardt. Pharm. Res. 13:1615–1623 (1996).Google Scholar
  19. 19.
    H. Rink. Tetrahedron Lett. 28:3787–3790 (1987).Google Scholar
  20. 20.
    G. Lu, S. Mojsov, J. P. Tam, and R. B. Merrifield. J. Org. Chem. 46:3433–3436 (1981).Google Scholar
  21. 21.
    J. Diago-Meseguer and A. L. Palomo-Coll. Synthesis 1980:547–551 (1980).Google Scholar
  22. 22.
    S. Gangwar, S. D. S. Jois, T. J. Siahaan, D. G. Vander Velde, V. J. Stella, and R. T. Borchardt. Pharm. Res. 13:1655–1660 (1996).Google Scholar
  23. 23.
    G. M. Pauletti, S. Gangwar, B. Wang, and R. T. Borchardt. Pharm. Res. 14: 11–17 (1997).Google Scholar
  24. 24.
    R. W. Woody. in: V. J. Hruby (ed.), The Peptides, Academic Press, New York, 1985, pp. 15–114.Google Scholar
  25. 25.
    K. Wüthrich, NMR of Proteins and Nucleic Acids, John Wiley & Sons, New York (1986).Google Scholar
  26. 26.
    V. F. Bystrov. Progr. NMR Spectrosc. 10:41–81 (1976).Google Scholar
  27. 27.
    K. D. Fine, C. A. Santa Ana, J. L. Portes, and J. S. Fordtran. Gastroenterology 108:983–989 (1995).Google Scholar
  28. 28.
    F. W. Okumu, G. M. Pauletti, D. G. Vander Velde, T. J. Siahaan, and R. T. Borchardt. Pharm. Res. 12:S-302 (1995).Google Scholar
  29. 29.
    G. M. Pauletti, F. W. Okumu, D. G. Vander Velde, and R. T. Borchardt. Pharm. Res. 13:S-235 (1996).Google Scholar
  30. 30.
    J. Hochman and P. Artursson. J. Controlled Release 29:253–267 (1994).Google Scholar
  31. 31.
    J. R. Pappenheimer and K. Z. Reiss. J. Memb. Biol. 100:123–136 (1987).Google Scholar

Copyright information

© Plenum Publishing Corporation 1997

Authors and Affiliations

  • Franklin W. Okumu
  • Giovanni M. Pauletti
  • David G. Vander Velde
  • Teruna J. Siahaan
  • Ronald T. Borchardt

There are no affiliations available

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