Advertisement

Pharmaceutical Research

, Volume 21, Issue 2, pp 278–284 | Cite as

The Transepithelial Transport of a G-CSF-Transferrin Conjugate in Caco-2 Cells and Its Myelopoietic Effect in BDF1 Mice

  • Adam Widera
  • Yun Bai
  • Wei-Chiang Shen
Article

Abstract

Purpose. The purpose of this study was to investigate the transferrin-receptor (TfR)-mediated transepithelial transport of G-CSF-transferrin (Tf) conjugate in cultured enterocyte-like Caco-2 monolayers and the myelopoietic effect of subcutaneously and orally administered G-CSF-Tf in BDF1 mice.

Methods. Caco-2 monolayers exhibiting a minimum transepithelial electrical resistance of 500 Ω PYcm2 and BDF1 mice were used as in vitro and in vivo models, respectively. TfR-mediated transcytosis was measured by using 125I-G-CSF-Tf and analyzing the downstream compartment by gamma counter. The efficacy of subcutaneously and orally administered G-CSF-Tf was determined by performing daily absolute neutrophil counts.

Results. Transport experiments in Caco-2 cells revealed that the monolayers that received 125I-G-CSF-Tf exhibited significantly higher apical-to-basolateral transport rates compared to the monolayers that received 125I-G-CSF. Inclusion of 100-fold excess unlabeled Tf reduced the extent of 125I-G-CSF-Tf transport by 80%. Chromatographic and bioactivity assays revealed that the protein recovered from the basolateral compartment was the intact conjugate, and it retained full ability to stimulate the proliferation of the granulocyte-colony stimulating factor (G-CSF) dependent cell line, NFS-60, upon reduction. Subcutaneous administration of G-CSF-Tf in BDF1 mice demonstrated that the conjugate is able to elicit a statistically significant enhancement in therapeutic effect relative to filgrastim, which includes a longer duration of action with higher absolute neutrophil counts. Oral administration of G-CSF-Tf in BDF1 mice demonstrated that G-CSF-Tf is able to elicit a significant, and apparently dose-dependent, increase in absolute neutrophil counts whereas filgrastim had no effect.

Conclusions. Our data indicate that G-CSF-Tf is transported across Caco-2 monolayers by TfR-specific processes at a rate that is significantly higher than the nonspecific flux of G-CSF. G-CSF-Tf is also able to elicit a prolonged myelopoietic effect relative to filgrastim when administered subcutaneously or orally in BDF1 mice. The development of an orally bioavailable G-CSF has the potential to provide great benefit to patients under sustained G-CSF dosing regimes.

Caco-2 G-CSF-Tf oral protein-drug delivery transepithelial transport. 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

references

  1. 1.
    T. Kuwabara, Y. Kato, S. Kobayashi, H. Suzuki, and Y. Sugiyama. Nonlinear pharmacokinetics of a recombinant human granulocyte colony-stimulating factor derivative (nartograstim): species differences among rats, monkeys and humans. J. Pharmacol. Exp. Ther. 271:1535-1543 (1994).Google Scholar
  2. 2.
    B. I. Lord, L. B. Woolford, and G. Molineux. Kinetics of neutrophil production in normal and neutropenic animals during the response to filgrastim (r-metHu G-CSF) or filgrastim SD/01 (PEG-r-metHu G-CSF). Clin. Cancer Res. 7:2085-2090 (2001).Google Scholar
  3. 3.
    W. Halpern, T. A. Riccobene, H. Agostini, K. Baker, D. Stolow, M. L. Gu, J. Hirsch, A. Mahoney, J. Carrell, E. Boyd, and K. J. Grzegorzewski. Albugranin, a recombinant human granulocyte colony stimulating factor (G-CSF) genetically fused to recombinant human albumin induces prolonged myelopoietic effects in mice and monkeys. Pharm. Res. 19:1720-1729 (2002).Google Scholar
  4. 4.
    Y. W. Chien and A. K. Banga. Potential developments insystemic delivery of insulin. Drug Dev. Ind. Pharm. 15:1601-1634 (1989).Google Scholar
  5. 5.
    W. A. Ritschel, G. B. Ritschell, and G. Sathyan. Insulin drug delivery systems: rectal gels. Res. Commun. Chem. Pathol. Pharmacol. 62:103-112 (1988).Google Scholar
  6. 6.
    A. Adjei and P. Gupta. Pulmonary delivery of therapeutic peptides and proteins. J. Controlled Rel. 29: (1994).Google Scholar
  7. 7.
    R. V. Morgan and M. A. Huntzicker. Delivery of systemic regular insulin via ocular route in dogs. J. Ocul. Pharmacol. Ther. 12:515-526 (1996).Google Scholar
  8. 8.
    Z. Shao, Y. Li, R. Krishnamoorthy, T. Chermak, and A. K. Mitra. Different effects of anionic, cationic, nonionic and physiologic surfactants on the dissociation, α-chymotryptic degradation, and enteral absorption of insulin hexamers. Pharm. Res. 10:243-251 (1993).Google Scholar
  9. 9.
    A. Yamamoto, T. Taniguchi, K. Rikyuu, T. Tsuji, M. Fujita, S. Murakami, and S. Muranishiet. Effects of various protease inhibitors on the intestinal absorption and degradation of insulin in rats Pharm. Res. 11:1496-1500 (1994).Google Scholar
  10. 10.
    V. H. L. Lee. Protease inhibitors and penetration enhancers as approaches to modify peptide absorption. J. Controlled Rel. 13:213-223 (1990).Google Scholar
  11. 11.
    V. H. L. Lee, A. Yamamoto, and U. B. Kompella. Mucosal penetration enhancers for facilitation of peptide and protein drug absorption. CRC Crit. Rev. Therap. Drug Delivery Sys. 8:91-192 (1991).Google Scholar
  12. 12.
    I. Morishita, M. Mortishita, K. Takayama, Y. Machida, and T. Nagai. Enternal insulin delivery by microsheres in 3 different formulations using Eudragit L100 ans S100. Int. J. Pharm. 93:29-37 (1993).Google Scholar
  13. 13.
    H. A. Huebers and C. A. Finch. The physiology of transferrin and transferrin receptors. Physiol. Rev. 67:520-582 (1987).Google Scholar
  14. 14.
    C. Q. Xia, J. Wang, and W. C. Shen. Hypoglycemic effect of insulin-transferrin conjugate in streptozotocin-induced diabetic rats. J. Pharmacol. Exp. Ther. 295:594-600 (2000).Google Scholar
  15. 15.
    D. Banerjee, P. R. Flanagan, J. Cluett, and L. S. Valberg. Transferrin receptors in the human gastrointestinal tract. Relationship to body iron stores. Gastroenterology 91:861-869 (1986).Google Scholar
  16. 16.
    K. L. Azari and R. E. Feeney. Resistance of metal complexes of conalbumin and transferrin to proteolysis and thermal denaturation. J. Biol. Chem. 232:293-302 (1958).Google Scholar
  17. 17.
    H. A. Huebers, E. Huebers, E. Csiba, W. Rummel, and C. A. Finch. The significance of transferrin for intestinal iron absorption. Blood 61:283-290 (1983).Google Scholar
  18. 18.
    C. N. Roy and C. A. Enns. Iron homeostasis: new tales from the crypt. Blood 96:4020-4027 (2000).Google Scholar
  19. 19.
    L. M. Souza, T. C. Boone, J. Gabrilove, P. H. Lai, K. M. Zsebo, D. C. Murdock, V. R. Chazin, J. Bruszewski, H. Lu, and K. K. Chen. Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells. Science 232:61-65 (1986).Google Scholar
  20. 20.
    A. Widera, K. J. Kim, E. D. Crandall, and W-C. Shen. Transcytosis of GCSF-transferrin across rat alveolar epithelial cell monolayers. Pharm. Res. 20:1231-1238 (2003).Google Scholar
  21. 21.
    N. Shirafuji, S. Asano, S. Matsuda, K. Watari, F. Takaku, and S. Nagata. A new bioassay for human granulocyte colony-stimulating factor (hG-CSF) using murine myeloblastic NFS-60 cells as targets and estimation of its levels in sera from normal healthy persons and patients with infectious and hematological disorders. Exp. Hematol. 17:116-119 (1989).Google Scholar
  22. 22.
    T. Mosmann. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55-63 (1983).Google Scholar
  23. 23.
    F. Heckner. Practical Microscopic Hematology. Urban & Schwarzenberg, Baltimore, 1982.Google Scholar
  24. 24.
    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).Google Scholar
  25. 25.
    K. L. Audus, R. L. Bartel, I. J. Hidalgo, and R. T. Borchart. The use of cultured epithelial and endothelial cells for drug transport and metabolism studies. Pharm. Res. 7:435-451 (1990).Google Scholar
  26. 26.
    A. Quaroni and J. Hockman. Development of intestinal cell culture models for drug transport and metabolism studies. Adv. Drug Delivery Rev. 22:3-52 (1996).Google Scholar
  27. 27.
    L. L. Gan and D. R. Thakker. Application of the Caco-2 model in the design and development of orally active drugs: elucidation of biochemical and physiological barriers posed by the intestinal epithium. Adv. Drug Delivery Rev. 23:77-98 (1997).Google Scholar
  28. 28.
    D. Shah and W. C. Shen. Transcellular delivery of an insulin-transferrin conjugate in enterocyte-like Caco-2 cells. J. Pharm. Sci. 85:1306-1311 (1996).Google Scholar
  29. 29.
    J. Wan, M. E. Taub, D. Shah, and W. C. Shen. Brefeldin A enhances receptor-mediated transcytosis of transferrin in filter-grown Madin-Darby canine kidney cells. J. Biol. Chem. 267:13446-13450 (1992).Google Scholar
  30. 30.
    C. Q. Xia and W. C. Shen. Tyrphostin-8 enhances transferrin receptor-mediated transcytosis in Caco-2-cells and inreases hypoglycemic effect of orally administered insulin-transferrin conjugate in diabetic rats. Pharm. Res. 18:191-195 (2001).Google Scholar
  31. 31.
    L. Li and J. Kaplan. Alteration in the organ distribution of iron by truncated transferrin: implications for iron chelation therapy. J. Lab. Clin. Med. 130:271-277 (1997).Google Scholar
  32. 32.
    H. Tanaka and T. Kaneko. Pharmacokinetics of recombinant human granulocyte colony-stimulating factor in mice. Blood 79:536-539 (1992).Google Scholar
  33. 33.
    M. A. Winkler, J. O. Price, P. D. Foglesong, and W. H. West. Biodistribution and plasma survival in mice of anti-melanoma monoclonal antibody cross-linked to OKT3. Cancer Immunol. Immunother. 31:278-284 (1990).Google Scholar

Copyright information

© Plenum Publishing Corporation 2004

Authors and Affiliations

  1. 1.Department of Pharmaceutical Sciences, School of PharmacyUniversity of Southern CaliforniaLos Angeles

Personalised recommendations