Adam Widera1, Yun Bai, Wei-Chiang Shen. 1. Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90033, USA.
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 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.
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 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.
Authors: Nikhil J Kavimandan; Elena Losi; Jeffrey J Wilson; Jennifer S Brodbelt; Nicholas A Peppas Journal: Bioconjug Chem Date: 2006 Nov-Dec Impact factor: 4.774