Xia C Q, Shen W C
Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles 90089-9121, USA.
Pharm Res. 2001 Feb;18(2):191-5. doi: 10.1023/a:1011032502097.
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.
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.
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.
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.
研究GTP酶抑制剂 tyrphostin 8（T-8）对培养的肠上皮样Caco-2细胞中转铁蛋白受体（TfR）介导的胰岛素-转铁蛋白（In-Tf）偶联物转胞吞作用以及对链脲佐菌素（STZ）诱导的糖尿病大鼠胃肠道（GI）对In-Tf吸收的影响。
分别将Caco-2细胞和糖尿病大鼠用作体外和体内模型。使用125I-In-Tf测量TfR介导的转胞吞作用。通过降血糖作用证明糖尿病大鼠体内胰岛素的吸收。使用一键式血糖监测系统测定大鼠血糖水平。
T-8通过增强滤膜生长的Caco-2细胞单层中TfR介导的转胞吞作用，增加了In-Tf偶联物从顶端到基底外侧的转运，并且这种增强作用比先前报道的布雷菲德菌素A（BFA）诱导的作用更高、更快。转运研究期间跨上皮电阻（TEER）的测量表明，T-8对细胞紧密连接的破坏作用小于BFA。通过口服给药后在STZ诱导的糖尿病大鼠中的降血糖作用评估In-Tf的胃肠道吸收。T-8或BFA均可改善口服In-Tf对STZ诱导的糖尿病大鼠的降血糖作用。然而，T-8的作用比BFA更强，尤其是在给药后7小时。单独的非偶联胰岛素或胰岛素-人血清白蛋白（In-HSA）偶联物或与T-8联合使用在口服给药后均未显示任何降血糖作用，表明T-8增强In-Tf的降血糖活性是由于TfR介导的转胞吞作用的选择性增强。
我们的数据表明，T-8可作为转铁蛋白偶联物用于增加胰岛素的胃肠道吸收。T-8作为TfR介导的转胞吞作用的增强剂，优于先前报道的BFA。T-8对In-Tf的转运增加更高，对上皮细胞的毒性更低。我们的研究结果为促进胰岛素以及其他肽或蛋白质药物的胃肠道吸收提供了一种替代方法。
