| Human recombinant insulin was conjugated at a 1:1 ratio to iron loaded human transferrin (Tf) via a disulfide linkage. Free insulin could be released from insulin-transferrin conjugate (In-Tf) upon the reduction of the disulfide bond in liver. In-Tf displayed a slow, dose-dependent, and prolonged hypoglycemic effect after oral administration in fasted streptozotocin (STZ)-induced diabetic rats. Evidence indicated that the gastrointestinal (GI) absorption of In-Tf was mediated by Tf receptors (TfR). Furthermore, intact In-Tf and free insulin was detected in the serum of rats at 4 h after oral administration of the conjugate, indicating that In-Tf can overcome the absorption barriers in the GI tract. However, the slow rate of TfR-mediated transcytosis in the intestinal epithelium due to its TfR polarized distribution impedes the development of this approach. Therefore, a known GTPases inhibitor, tyrphostin 8 (T-8), was tested for enhancing the GI absorption of In-Tf by promoting TfR-mediated transport. The enhancing effect of T-8 on TfR-mediated transcytosis was compared with that of brefeldin A (BFA), and the mechanism of the enhancement was investigated using Caco-2 and MDCK cells. Our data indicated that T-8 increased the GI absorption of In-Tf and was better than BFA in enhancing TfR-mediated transcytosis. T-8 produces a higher increase on the transport of In-Tf and a lower toxicity in Caco-2 cells. T-8-induced enhancement was reversible, dose-dependent and cell type-specific. T-8 increased TfR-mediated transcytosis in Caco-2 but not in MDCK cells, whereas BFA was effective in both cell lines. Since the sorting compartments for apical membrane proteins are different in MDCK and Caco-2 cells, i.e., trans-Golgi network (TGN) in MDCK cells and both TGN and basolateral endosomes in Caco-2 cells, our results indicate that T-8 interferes the intracellular processing of TfR predominantly at basolateral endosomes. These results demonstrate that transepithelial transport via TfR-mediated transcytosis is a feasible approach for developing the oral delivery of insulin, as well as other peptide drugs. Furthermore, TfR-mediated transport could be further improved by using inhibitors of GTPase for altering intracellular processing of endocytosed Tf. |
