Biopharmaceutics and Pharmaceutical Technology, Saarland University, 66123 Saarbrucken, Germany.
Mol Pharm. 2010 Jun 7;7(3):642-51. doi: 10.1021/mp900191s.
Previous work conducted in our laboratories established the notion that TPGS 1000 (d-alpha-tocopheryl polyethylene glycol 1000 succinate), a nonionic surfactant, modulates P-glycoprotein (P-gp) efflux transport via P-gp ATPase inhibition. The current in vitro research using Caco-2 cells was conducted to further explore the P-gp ATPase inhibition mechanism. Using a monoclonal CD243 P-gp antibody shift assay (UIC2), we probed P-gp conformational changes induced via TPGS 1000. In the presence of TPGS 1000, UIC2 binding was slightly decreased. TPGS 1000 does not appear to be a P-gp substrate, nor does it function as a competitive inhibitor in P-gp substrate efflux transport. The reduction in UIC2 binding with TPGS 1000 was markedly weaker than with orthovanadate, data ruling out trapping P-gp in a transition state by direct interaction with one or both of the P-gp ATP nucleotide binding domains. An intracellular ATP depletion mechanism could be ruled out in the UIC2 assay, and by monitoring intracellular ATP levels in the presence of TPGS 1000. Indicating slow distribution of TPGS 1000 into the membrane, and in agreement with an intramembranal or intracellular side of action, Caco-2 cell monolayer experiments preincubated with TPGS 1000 produce stronger substrate inhibitory activity than those conducted by direct substrate and surfactant coapplication.
先前在我们实验室进行的工作确立了这样一种观点，即 TPGS1000（d-α-生育酚聚乙二醇 1000 琥珀酸酯），一种非离子表面活性剂，通过抑制 P-糖蛋白（P-gp）ATP 酶来调节 P-gp 外排转运。本研究使用 Caco-2 细胞进行了进一步的体外研究，以探索 P-gp ATP 酶抑制机制。通过使用单克隆 CD243 P-gp 抗体移位测定法（UIC2），我们研究了 TPGS1000 诱导的 P-gp 构象变化。在 TPGS1000 的存在下，UIC2 结合略有减少。TPGS1000 似乎不是 P-gp 的底物，也不是 P-gp 底物外排转运的竞争性抑制剂。与钒酸盐相比，TPGS1000 减少 UIC2 结合的作用明显较弱，这排除了 P-gp 通过与一个或两个 P-gp ATP 核苷酸结合域的直接相互作用而被捕获在过渡态的可能性。在 UIC2 测定中可以排除细胞内 ATP 耗竭机制，并且可以通过监测 TPGS1000 存在时细胞内 ATP 水平来排除这种机制。这表明 TPGS1000 缓慢分布到膜中，并且与跨膜内或细胞内作用一致，用 TPGS1000 预孵育的 Caco-2 细胞单层实验产生比直接应用底物和表面活性剂更强的底物抑制活性。
