Journal
PHARMACEUTICAL RESEARCH
Volume 20, Issue 8, Pages 1231-1238Publisher
KLUWER ACADEMIC/PLENUM PUBL
DOI: 10.1023/A:1025005232421
Keywords
transferrin; alveolar epithelial cells; protein drug delivery; pulmonary drug delivery; BFA
Funding
- NHLBI NIH HHS [HL38621, HL38658, HL64365, HL38578] Funding Source: Medline
- NIGMS NIH HHS [GM 63647] Funding Source: Medline
- BHP HRSA HHS [AHA GIA9950442N] Funding Source: Medline
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Purpose. The purpose of this study was to use primary cultured rat alveolar epithelial cell monolayers to examine the potential of using transferrin receptor (TfR)-mediated transcytosis for noninvasive systemic protein drug delivery via the pulmonary route. Methods. Freshly isolated rat type II pneumocytes were plated onto tissue culture-treated polycarbonate 12-mm Transwells. AEC monolayers (greater than or equal to 2500 Omegacm(2)) were treated with keratinocyte growth factor (10 ng/mL) for maintenance of type II cell-like characteristics. Filgrastim (GCSF)-Tf conjugates were prepared using the linkers SPDP and DPDPB. TfR-specific binding and uptake were determined using I-125-Tf and Fe-59-Tf treatment, respectively. Apical-to-basolateral (A-to-B) transferrin receptor (TfR)-mediated transcytosis was determined by dosing the apical compartment with 1.5 mug/mL of I-125-Tf or I-125-GCSF-Tf. Nonspecific TfR-independent transport of I-125-Tf and I-125-GCSF-Tf was determined in parallel by including 150 mug/mL of nonradiolabeled Tf. Basolateral samples (500 muL) were taken at 2, 4, and 6 h post-dosing, subjected to 15% trichloroacetic acid precipitation, and assayed in a Packard gamma counter. TfR-specific transport was determined as the difference between total and nonspecifc transport. The effects of brefeldin-A (BFA) on TfR distribution and (A-to-B) transport of I-125-Tf, I-125-GCSF and I-125-GCSF-Tf was studied by including the agent in the apical fluid at 1 mug/mL. Results. BFA treatment resulted in a small significant reduction in TfR at the basolateral surface of type II cell-like monolayers, while it had no effect on TfR distribution in type I cell-like monolayers. In contrast, BFA treatment significantly altered the endocytosis of TfR, reducing the basolateral uptake of Fe-59-Tf while greatly increasing the apical uptake of Fe-59-Tf. BFA treatment, however, did not affect the TfR-specific uptake of Fe-59-Tf in type I cell-like monolayers. TfR-specific apical-to-basolateral transcytosis of I-125-Tf and I-126-GCSF-Tf conjugates was significantly enhanced in the presence of BFA in type II cell-like monolayers, whereas it had no effect on apical-to-basolateral transport of I-125-GCSF. BFA-enhanced transport of I-125-GCSF-Tf was approximately 3-fold higher than that of I-125-GCSF in the presence or absence of BFA. Moreover, I-125-GCSF transport in the presence of BFA was not significantly different from non-specific I-125-GCSF-Tf transport. Chromatographic analyses and bio-assays revealed that GCSF-Tf was not degraded during transport via TfR-specific processes, and that GCSF retained biologic activity when liberated from the conjugate via dithiothreitol reduction. Conclusion. This study suggests the possibility of using TfR-mediated transcytosis for systemic delivery of therapeutic proteins via the alveolar epithelium.
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