Wu S J, Robinson J R
School of Pharmacy, University of Wisconsin-Madison, 53706, USA.
Pharm Res. 1999 Aug;16(8):1266-72. doi: 10.1023/a:1014809916407.
To evaluate the transcellular mechanism of novel enhancers absorption enhancement of human growth hormone (hGH), by examining the involvement of a P-glycoprotein-like efflux system, changes in membrane fluidity, and membrane damage.
Caco-2 cell monolayers were grown on Snapwell filter supports and placed in a side-by-side diffusion apparatus. Transport in both the apical to basolateral (AP to BL) and basolateral to apical (BL to AP) direction was measured at different temperatures and in the presence of potential inhibitors. Fluorescence anisotropy measurement was used to measure membrane fluidity. The fluorescence anisotropy of DPH- and TMA-DPH-labeled cell suspensions was measured at room temperature. LDH (a measure of cytosolic lactate dehydrogenase) leakage assay was used to evaluate cytotoxicity.
The bi-directional transepithelial fluxes of hGH in the presence of these novel enhancers across Caco-2 cells showed marked asymmetry. Average permeability coefficient values obtained in the apical to basolateral (AP to BL) direction were lower than those of the reverse (BL to AP) direction. On the other hand, the fluxes for hGH alone were symmetric. When P-gp-like efflux inhibitors were included in the transport medium, the permeability coefficient value of BL to AP direction was significantly decreased while the transport was increased in the reverse direction in the presence of novel enhancers. In addition, lowering the temperature to 25 degrees C completely eliminated the asymmetry of hGH transport in the presence of novel enhancers. It was also shown by fluorescence anisotropy that these novel enhancers alone only slightly increased membrane fluidity. On the other hand, upon addition of hGH to the novel enhancers, the cell membrane showed a dramatic change as compared to treatment with novel enhancers alone. The results from the LDH assay showed that the novel enhancers and/or hGH did not cause cell damage, at least up to 1 hour, and the damage seen at the 2 hour point is also much lower than other known enhancers.
This study shows that human growth hormone alone cannot be transported across Caco-2 cells, except in small quantities, by passive diffusion, but in the presence of novel enhancers, human growth hormone permeation is substantial. In addition, the asymmetry of transport of the complexed hGH appears to be due to a P-gp-like efflux system. Assuming that the present substrate specificity of the P-gp-like efflux system shows the same preference for hydrophobic molecules as p-gp, the present work also indirectly shows that human growth hormone has become more lipophilic in the presence of these novel enhancers. Furthermore, membrane fluidity data also supports the premise that these novel enhancers interact and stabilize hGH, to make them more hydrophobic and easier to be transported through cell membranes.
通过研究P - 糖蛋白样外排系统的参与情况、膜流动性的变化以及膜损伤，评估新型增强剂促进人生长激素（hGH）吸收的跨细胞机制。
将Caco - 2细胞单层培养在Snapwell滤膜支持物上，并置于侧流扩散装置中。在不同温度下以及存在潜在抑制剂的情况下，测量顶侧到基底侧（AP到BL）和基底侧到顶侧（BL到AP）方向的转运。使用荧光各向异性测量来测定膜流动性。在室温下测量DPH和TMA - DPH标记的细胞悬液的荧光各向异性。使用LDH（胞质乳酸脱氢酶的一种测量指标）泄漏测定法来评估细胞毒性。
在这些新型增强剂存在下，hGH跨Caco - 2细胞的双向跨上皮通量显示出明显的不对称性。在顶侧到基底侧（AP到BL）方向获得的平均渗透系数值低于反向（BL到AP）方向的值。另一方面，单独hGH的通量是对称的。当在转运介质中加入P - gp样外排抑制剂时，在新型增强剂存在下，BL到AP方向的渗透系数值显著降低，而反向转运增加。此外，将温度降至25摄氏度完全消除了新型增强剂存在下hGH转运的不对称性。荧光各向异性也表明，单独这些新型增强剂仅略微增加膜流动性。另一方面，在新型增强剂中加入hGH后，与仅用新型增强剂处理相比，细胞膜显示出显著变化。LDH测定结果表明，新型增强剂和/或hGH至少在1小时内不会引起细胞损伤，并且在2小时时观察到的损伤也远低于其他已知增强剂。
本研究表明，单独的人生长激素除了少量通过被动扩散外，不能穿过Caco - 2细胞，但在新型增强剂存在下，人生长激素的渗透量很大。此外，复合hGH转运的不对称性似乎是由于一种P - gp样外排系统。假设目前P - gp样外排系统的底物特异性对疏水分子的偏好与P - gp相同，本研究还间接表明在这些新型增强剂存在下人生长激素变得更具亲脂性。此外，膜流动性数据也支持这些新型增强剂与hGH相互作用并使其稳定，从而使其更具疏水性且更易于穿过细胞膜这一前提。
