The transepithelial transport of a G-CSF-transferrin conjugate in Caco-2 cells and its myelopoietic effect in BDF1 mice.
OBJECTIVE
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 ohms-cm2 and BDF1 mice were used as in vitro and in vivo models, respectively. TfR-mediated transcytosis wa 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.
Widera
Adam
A
Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90033, USA.
Bai
Yun
Y
Shen
Wei-Chiang
WC
D000284:Administration, Oral; D000818:Animals; D001692:Biological Transport; D018938:Caco-2 Cells; D002455:Cell Division / Q000187:drug effects; D004847:Epithelial Cells / Q000378:metabolism*; D000069585:Filgrastim; D016179:Granulocyte Colony-Stimulating Factor / Q000493:pharmacokinetics* / Q000494:pharmacology; D006801:Humans; D007279:Injections, Subcutaneous; D007958:Leukocyte Count; D051379:Mice; D008954:Models, Biological; D038042:Myelopoiesis / Q000187:drug effects*; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects; D011994:Recombinant Proteins; D013997:Time Factors; D014168:Transferrin / Q000493:pharmacokinetics* / Q000494:pharmacology
D011994:Recombinant Proteins; D014168:Transferrin; D016179:Granulocyte Colony-Stimulating Factor; D000069585:Filgrastim