Skip to main content

Advertisement

SpringerLink
Log in

A Randomly Coiled, High-Molecular-Weight Polypeptide Exhibits Increased Paracellular Diffusion in Vitro and in Situ Relative to the Highly Ordered α-Helix Conformer

  • Research Papers
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

No Heading

Purpose.

The current investigation was conducted to examine the effect of secondary structure of model polypeptides on their hindered paracellular diffusion.

Methods.

Poly-d-glutamic acid (PDGlu) was selected as one of the model polypeptides because of its ability to form two secondary structures; a negatively charged random coil and an α-helix with partial negative charge at pH 7.4 and 4.7, respectively. Poly-d-lysine (PDL) was selected as a positively charged random coil conformation at pH 7.4. Transport experiments were conducted across both a Caco-2 cell monolayer and the intestinal membrane of Sprague-Dawley rats. Additionally, using NMR, an estimation for the diffusion coefficient and the equivalent hydrodynamic radius for each model polypeptide was obtained.

Results.

PDGlu in the randomly coiled conformation exhibited greater paracellular transport when compared to either the same polypeptide having an α-helix secondary structure or the positively charged, randomly coiled PDL.

Conclusions.

Randomly coiled PDGlu was able to permeate through the negatively charged tight junctions of both biological membranes to a greater extent than PDGlu having an α-helix structure and suggests that molecular flexibility associated with the random coil conformation may play a more important role than overall charge and hydrodynamic radius on its hindered paracellular diffusion.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. 1. J. M. Kilby, S. Hopkins, T. M. Venetta, B. DiMassimo, G. A. Cloud, J. Y. Lee, L. Alldredge, E. Hunter, D. Lambert, D. Bolognesi, T. Matthews, M. R. Johnson, M. A. Nowak, G. M. Shaw, and M. S. Saag. Potent suppression of HIV-1 replication in humans by T20, a peptide inhibitor of gp41-mediated virus entry. Nat. Med. 4:1302–1307 (1998).

    Google Scholar 

  2. 2. S. Kobayashi, S. Kondo, and K. Juni. Permeability of peptides and proteins in human cultured alveolar A549 cell monolayer. Pharm. Res. 12:1115–1119 (1995).

    Google Scholar 

  3. 3. V. B. Lang, P. Langguth, C. Ottiger, H. Wunderli-Allenspach, D. Rognan, B. Rothen-Rutishauser, J.-C. Perriard, S. Lang, J. Biber, and H. P. Merkle. Structure-permeation relations of Met-enkephalin peptide analogues on absorption and secretion mechanisms in Caco-2 monolayers. J. Pharm. Sci. 86:846–853 (1997).

    Google Scholar 

  4. 4. Y.-L. He, S. Murby, L. Gifford, A. Collett, G. Warhurst, K. T. Douglas, M. Rowland, and J. Ayrton. Oral absorption of d-oligopeptides in rats via the paracellular route. Pharm. Res. 13:1673–1678 (1996).

    Google Scholar 

  5. 5. G. M. Pauletti, F. W. Okumu, and R. T. Borchardt. Effect of size and charge on the passive diffusion of peptides across Caco-2 cell monolayers via the paracellular pathway. Pharm. Res. 14:164–168 (1997).

    Google Scholar 

  6. 6. A. Leone-Bay, M. Sato, D. Paton, A. H. Hunt, D. Sarubbi, M. Carozza, J. Chou, J. McDonough, and R. A. Baughman. Oral delivery of biologically active parathyroid hormone. Pharm. Res. 18:964–970 (2001).

    Google Scholar 

  7. 7. P. D. Ward, T. K. Tippin, and D. R. Thakker. Enhancing paracellular permeability by modulating epithelial tight junctions. Pharm. Sci. Technol. Today 3:346–358 (2000).

    Google Scholar 

  8. 8. A. Fasano, C. Fiorentini, G. Donelli, S. Uzzau, J. B. Kaper, K. Margaretten, X. Ding, S. Guandalini, L. Comstock, and S. E. Goldblum. Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro. J. Clin. Invest. 96:710–720 (1995).

    Google Scholar 

  9. 9. J. L. Madara and J. R. Pappenheimer. Structural basis for physiological regulation of paracellular pathways in intestinal epithelia. J. Membr. Biol. 100:149–164 (1987).

    Google Scholar 

  10. 10. E. Sinaga, S. D. S. Jois, M. Avery, I. T. Makagiansar, U. S. F. Tambunan, K. L. Audus, and T. J. Siahaan. Increasing paracellular porosity by E-cadherin peptides: discovery of bulge and groove regions in the EC1-domain of E-cadherin. Pharm. Res. 19:1170–1179 (2002).

    Google Scholar 

  11. 11. Y. Matsukawa, V. H. L. Lee, E. D. Crandall, and K.-J. Kim. Size-dependent dextran transport across rat alveolar epithelial cell monolayers. J. Pharm. Sci. 86:305–309 (1997).

    Google Scholar 

  12. 12. Y. Horibe, K. Hosoya, K.-J. Kim, T. Ogiso, and V. H. L. Lee. Polar solute transport across the pigmented rabbit conjunctiva: size dependence and the influence of 8-bromo cyclic adenosine monophosphate. Pharm. Res. 14:1246–1251 (1997).

    Google Scholar 

  13. 13. A. N. O. Dodoo, S. Bansal, D. J. Barlow, F. C. Bennet, R. C. Hider, A. B. Lansley, M. J. Lawrence, and C. Marriott. Systematic investigations of the influence of molecular structure on the transport of peptides across cultured alveolar cell monolayers. Pharm. Res. 17:7–14 (2000).

    Google Scholar 

  14. 14. D. Hollander, D. Ricketts, and C. A. R. Boyd. Importance of ‘probe’ molecular geometry in determining intestinal permeability. Can. J. Gastroentrol. 2:35A–38A (1988).

    Google Scholar 

  15. 15. W. Rubas, M. Cromwell, T. Gadek, D. Narindray, and R. Mrsny. Structural elements which govern the resistance of intestinal tissues to compound transport. Mat. Res. Soc. Sym. Proc. 331:179–185 (1994).

    Google Scholar 

  16. 16. A. Adson, T. J. Raub, P. S. Burton, C. L. Barsuhn, A. R. Hilgers, K. L. Audus, and N. F. H. Ho. Quantitative approaches to delineate paracellular diffusion in cultured epithelial cell monolayers. J. Pharm. Sci. 83:1529–1536 (1994).

    Google Scholar 

  17. 17. F. W. Okumu, G. M. Pauletti, D. G. Vander Velde, T. J. Siahaan, and R. T. Borchardt. The effect of charge and conformation on the permeability of a hexapeptide across monolayers of a cultured human intestinal epithelial cell (Caco-2 cells). Pharm. Res. 12:S302 (1995).

    Google Scholar 

  18. 18. G. T. Knipp, D. G. Vander Velde, T. J. Siahaan, and R. T. Borchardt. The effect of solution conformation and charge on the paracellular permeability of model pentapeptides across Caco-2 cell monolayers. Pharm. Res. 12:S303 (1995).

    Google Scholar 

  19. 19. G. T. Knipp, D. G. Vander Velde, T. J. Siahaan, and R. T. Borchardt. The effect of β-turn structure on the passive diffusion of peptides across Caco-2 cell monolayers. Pharm. Res. 14:1332–1340 (1997).

    Google Scholar 

  20. 20. F. W. Okumu, G. M. Pauletti, D. G. Vander Velde, T. J. Siahaan, and R. T. Borchardt. Effect of restricted conformational flexibility on the permeation of model hexapeptides across Caco-2 cell monolayers. Pharm. Res. 14:169–175 (1997).

    Google Scholar 

  21. 21. S. Gangwar, S. D. S. Jois, T. J. Siahaan, D. G. Vander Velde, V. J. Stella, and R. T. Borchardt. The effect of conformation on membrane permeability of an acyloxyalkoxy-linked cyclic prodrug of a model hexapeptide. Pharm. Res. 13:1657–1662 (1996).

    Google Scholar 

  22. 22. N. Greenfield and G. D. Fasman. Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry 8:4108–4116 (1969).

    CAS  PubMed  Google Scholar 

  23. 23. W. C. Johnson Jr. and I. Tinoco Jr. Circular dichroism of polypeptide solutions in the vacuum ultraviolet. J. Am. Chem. Soc. 94:4389–4390 (1972).

    Google Scholar 

  24. 24. X. Boulenc, E. Marti, H. Joyeux, C. Roques, Y. Berger, and G. Fabre. Importance of the paracellular pathway for the transport of a new bisphosphonate using the human Caco-2 monolayers model. Biochem. Pharmacol. 46:1591–1600 (1993).

    Google Scholar 

  25. 25. R. L. Kacich, R. H. Renston, and A. L. Jones. Effects of cytochalasin D and colchicine on the uptake, translocation, and biliary secretion of horseradish peroxidase and [14C] sodium taurocholate in the rat. Gastroenterology 85:385–394 (1983).

    Google Scholar 

  26. 26. I. Legen and A. Kristl. pH and energy dependent transport of ketoprofen across rat jejunum in vitro. Eur. J. Pharm. Biopharm. 56:87–94 (2003).

    Google Scholar 

  27. 27. M. Tomita, Y. Hotta, R. Ohkubo, and S. Awazu. Polarized transport was observed not in hydrophilic compounds but in dextran in Caco-2 cell monolayers. Biol. Pharm. Bull. 22:330–331 (1999).

    Google Scholar 

  28. 28. I. J. Hidalgo, A. Kato, and R. T. Borchardt. Binding of epidermal growth factor by human colon carcinoma cell (Caco-2) monolayers. Biochem. Biophys. Res. Commun. 160:317–324 (1989).

    Google Scholar 

  29. 29. H. J. Baker. The Laboratory Rat. Academic Press, New York, 1979.

    Google Scholar 

  30. 30. I. Komiya, J. Y. Park, A. Kamani, N. F. H. Ho, and W. I. Higuchi. Quantitative mechanistic studies in simultaneous fluid flow and intestinal absorption using steroids as model solutes. Int. J. Pharm. 4:249–262 (1980).

    Google Scholar 

  31. 31. S. Park, M. E. Johnson, and L. W.-M. Fung. NMR analysis of secondary structure and dynamics of a recombinant peptide from the N-terminal region of human erythroid α-spectrin. FEBS Lett. 485:81–86 (2000).

    Google Scholar 

  32. 32. H. N. Nellans. (B) Mechanisms of peptide and protein absorption. (1) paracellular intestinal transport: modulation of absorption. Adv. Drug Deliv. Rev. 7:339–364 (1991).

    Google Scholar 

  33. 33. X. Zhou, Y. X. Li, N. Li, and J. S. Li. Glutamine enhances the gut-trophic effect of growth hormone in rat after massive small bowel resection. J. Surg. Res. 99:47–52 (2001).

    Google Scholar 

  34. 34. Y. Dou, S. Gregersen, J. Zhao, F. Zhuang, and H. Gregersen. Morphometric and biomechanical intestinal remodeling induced by fasting in rats. Dig. Dis. Sci. 47:1158–1168 (2002).

    Google Scholar 

  35. 35. A. M. Landel. Stability studies on fluorescein isothiocyanate-bovine serum albumin conjugate. Anal. Biochem. 73:280–289 (1976).

    Google Scholar 

  36. 36. L. Hovgaard, E. J. Mack, and S. W. Kim. Insulin stabilization and GI absorption. J. Control. Release 19:99–108 (1992).

    Google Scholar 

  37. 37. U. Schröder, K.-E. Arfors, and O. Tangen. Stability of fluorescein labeled dextrans in vivo and in vitro. Microvasc. Res. 11:33–39 (1976).

    Google Scholar 

  38. 38. N. Salamat-Miller, M. Chittchang, A. K. Mitra, and T. P. Johnston. Shape imposed by secondary structure of a polypeptide affects its free diffusion through liquid-filled pores. Int. J. Pharm. 244:1–8 (2002).

    Google Scholar 

  39. 39. M. P. Bohrer, W. M. Deen, C. R. Robertson, J. L. Troy, and B. M. Brenner. Influence of molecular configuration on the passage of macromolecules across the glomerular capillary wall. J. Gen. Physiol. 74:583–593 (1979).

    Google Scholar 

  40. 40. M. P. Bohrer, G. D. Patterson, and P. J. Carroll. Hindered diffusion of dextran and ficoll in microporous membranes. Macromolecules 17:1170–1173 (1984).

    Google Scholar 

  41. 41. M. El-Sayed, M. F. Kiani, M. D. Naimark, A. H. Hikal, and H. Ghandehari. Extravasation of poly(amidoamine) (PAMAM) dendrimers across microvascular network endothelium. Pharm. Res. 18:23–28 (2001).

    Google Scholar 

  42. 42. M. E. Lane, C. M. O’Driscoll, and O. I. Corrigan. The relationship between rat intestinal permeability and hydrophilic probe size. Pharm. Res. 13:1554–1558 (1996).

    Google Scholar 

  43. 43. Y. Tanaka, Y. Taki, T. Sakane, T. Nadai, H. Sezaki, and S. Yamashita. Characterization of drug transport through tight-junctional pathway in Caco-2 monolayer: comparison with isolated rat jejunum and colon. Pharm. Res. 12:523–528 (1995).

    Google Scholar 

  44. 44. U. Bock, C. Kolac, G. Borchard, K. Koch, R. Fuchs, P. Streichhan, and C.-M. Lehr. Transport of proteolytic enzymes across Caco-2 cell monolayers. Pharm. Res. 15:1393–1400 (1998).

    Google Scholar 

  45. 45. A. R. Hilgers, R. A. Conradi, and P. S. Burton. Caco-2 cell monolayers as a model for drug transport across the intestinal mucosa. Pharm. Res. 7:902–910 (1990).

    Google Scholar 

  46. 46. A. Wada. Helix-coil transformation and titration curve of poly-l-glutamic acid. Mol. Phys. 3:409–416 (1960).

    Google Scholar 

  47. 47. D.-C. Kim, P. S. Burton, and R. T. Borchardt. A correlation between the permeability characteristics of a series of peptides using an in vitro cell culture model (Caco-2) and those using an in situ perfused rat ileum model of the intestinal mucosa. Pharm. Res. 10:1710–1714 (1993).

    Google Scholar 

  48. 48. S. Yee. In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man-fact or myth. Pharm. Res. 14:763–766 (1997).

    Google Scholar 

  49. 49. P. Artursson and J. Karlsson. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells. Biochem. Biophys. Res. Commun. 175:880–885 (1991).

    Google Scholar 

  50. 50. W. Rubas, M. E. M. Cromwell, Z. Shahrokh, J. Villagran, T.-N. Nguyen, M. Wellton, T.-H. Nguyen, and R. J. Mrsny. Flux measurements across Caco-2 monolayers may predict transport in human large intestinal tissue. J. Pharm. Sci. 85:165–169 (1996).

    Google Scholar 

  51. 51. P. Artursson. Cell cultures as models for drug absorption across the intestinal mucosa. Crit. Rev. Ther. Drug Carrier Syst. 8:305–330 (1991).

    Google Scholar 

  52. 52. A. H. Dantzig and L. Bergin. Uptake of the cephalosporin, cephalexine by a dipeptide transport carrier in the human intestinal cell line, Caco-2. Biochim. Biophys. Acta 1027:211–217 (1990).

    Google Scholar 

  53. 53. H. Lennernäs, K. Palm, U. Fagerholm, and P. Artursson. Comparison between active and passive drug transport in human intestinal epithelial (Caco-2) cells in vitro and human jejunum in vivo. Int. J. Pharm. 127:103–107 (1996).

    Google Scholar 

  54. 54. H. Lennernäs, S. Nylander, and A.-L. Ungell. Jejunal permeability: a comparison between the Ussing chamber technique and the single-pass perfusion in humans. Pharm. Res. 14:667–671 (1997).

    Google Scholar 

  55. 55. P. Artursson, A.-L. Ungell, and J.-E. Löfroth. Selective paracellular permeability in two models of intestinal absorption: cultured monolayers of human intestinal epithelial cells and rat intestinal segments. Pharm. Res. 10:1123–1129 (1993).

    Google Scholar 

  56. 56. J. L. Madara, D. Barenberg, and S. Carlson. Effects of cytochalasin D on occluding junctions of intestinal absorptive cells: further evidence that the cytoskeleton may influence paracellular permeability and junctional charge selectivity. J. Cell Biol. 102:2125–2136 (1986).

    Google Scholar 

  57. 57. G. M. Pauletti, S. Gangwar, G. T. Knipp, M. M. Nerurkar, F. W. Okumu, K. Tamura, T. J. Siahaan, and R. T. Borchardt. Structural requirements for intestinal absorption of peptide drugs. J. Control. Rel. 41:3–17 (1996).

    Google Scholar 

Download references

Author information

Author notes

  1. Nazila Salamat-Miller

    Present address: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas, 66047, USA

Authors and Affiliations

  1. Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri, 64110, USA

    Nazila Salamat-Miller, Montakarn Chittchang, Ashim K. Mitra & Thomas P. Johnston

Authors
  1. Nazila Salamat-Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Montakarn Chittchang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ashim K. Mitra
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thomas P. Johnston
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Thomas P. Johnston.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Salamat-Miller, N., Chittchang, M., Mitra, A. et al. A Randomly Coiled, High-Molecular-Weight Polypeptide Exhibits Increased Paracellular Diffusion in Vitro and in Situ Relative to the Highly Ordered α-Helix Conformer. Pharm Res 22, 245–254 (2005). https://doi.org/10.1007/s11095-004-1192-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-004-1192-4

Key words:

Search

Navigation