title: Tyrphostin-8 enhances transferrin receptor-mediated transcytosis in Caco-2- cells and inreases hypoglycemic effect of orally administered insulin-transferrin conjugate in diabetic rats.
abstract: OBJECTIVE
  To investigate the effect of tyrphostin 8 (T-8), a GTPase inhibitor, on transferrin receptor (TfR)-mediated transcytosis of insulin-transferrin (In-Tf) conjugate in cultured enterocyte-like Caco-2 cells and on gastrointestinal (GI) absorption of In-Tf in streptozotocin (STZ)-induced diabetic rats.
  
  
  METHODS
  Caco-2 cells and diabetic rats were used as in vitro and in vivo models, respectively. TfR-mediated transcytosis was measured using 125I-In-Tf. The absorption of insulin in diabetic rats was demonstrated by the hypoglycemic effect. Rat blood glucose level was determined using a ONE TOUCH blood glucose monitoring system.
  
  
  RESULTS
  T-8 increased apical-to-basolateral transport of In-Tf conjugate by enhancing TfR-mediated transcytosis in filter-grown Caco-2 cell monolayer, and this enhancement was higher and faster than the previously reported brefeldin A (BFA)-induced effect. The measurement of transepithelial electrical resistance (TEER) during the transport study showed that T-8 was less destructive on the cell tight junction than BFA. The GI absorption of In-Tf was evaluated by its hypoglycemic effect after oral administration in STZ-induced diabetic rats. The glucose-lowering effect of orally administered In-Tf in STZ-induced diabetic rats was improved by either T-8 or BFA. However, the effect of T-8 was more potent than that of BFA, especially at 7 h after administration. Either non-conjugated insulin or insulin-human serum albumin (In-HSA) conjugate by itself or in combination with T-8 did not show any hypoglycemic effect after oral administration, indicating that T-8-enhanced hypoglycemic activity of In-Tf was due to a selective enhancement of TfR-mediated transcytosis.
  
  
  CONCLUSIONS
  Our data indicated that T-8 could be used to increase the GI absorption of insulin as a transferrin conjugate. T-8, as an enhancer of TfR-mediated transcytosis, is better than the previously reported BFA. T-8 produces a higher increase on the transport of In-Tf and a lower toxicity on epithelial cells. Our findings provide an alternative approach to promote the GI absorption of insulin, as well as other peptide or protein drugs.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles 90089-9121, USA.
authors.forename: C Q
authors.initials: CQ
authors.lastname: Xia
authors.forename: W C
authors.initials: WC
authors.lastname: Shen
mesh_terms: D000818:Animals; D018938:Caco-2 Cells; D002465:Cell Movement / Q000187:drug effects; D003921:Diabetes Mellitus, Experimental / Q000139:chemically induced / Q000378:metabolism*; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D007003:Hypoglycemia / Q000209:etiology; D007004:Hypoglycemic Agents / Q000009:adverse effects / Q000494:pharmacology*; D007328:Insulin / Q000009:adverse effects / Q000737:chemistry / Q000493:pharmacokinetics*; D007408:Intestinal Absorption; D051381:Rats; D017207:Rats, Sprague-Dawley; D011990:Receptors, Transferrin / Q000187:drug effects / Q000502:physiology*; D014168:Transferrin / Q000009:adverse effects / Q000737:chemistry / Q000493:pharmacokinetics*; D020032:Tyrphostins / Q000009:adverse effects / Q000494:pharmacology*
chemical_list: D004791:Enzyme Inhibitors; D007004:Hypoglycemic Agents; D007328:Insulin; D011990:Receptors, Transferrin; D014168:Transferrin; D020032:Tyrphostins; C075061:tyrphostin 8