Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA.
J Pharm Biomed Anal. 2010 Apr 6;51(5):1069-77. doi: 10.1016/j.jpba.2009.12.006. Epub 2009 Dec 24.
This study was to evaluate the mechanistic effect of protein to help better interpret the permeability results for compounds with low mass balance in Caco-2 permeability assay. The absorptive or bi-directional permeability of lipophilic compounds with mass balance were measured across Caco-2 cell monolayers as well as the empty transport devices with or without protein (4% bovine serum albumin, BSA) added to the receiver side. The results from empty transport device study indicated that the filter membrane is a permeability barrier for the low mass balance compounds and protein increases permeability by improving the compound diffusivity through the filter membrane. Caco-2 permeability measured with protein provided better absorption projection. Assuming the amount of compound associated with cells as transported did not correlate with absorption. For efflux substrate identification using Caco-2 bi-directional permeability assay, protein at the receiver side had no significant effect on the conclusion regarding the tested compounds as efflux substrate but increased the permeability measurement from both transport directions. In conclusions, Caco-2 permeability results measured using protein-containing buffer at the receiver side for low mass balance compound seems to provide better correlation with in vivo absorption. The fact that protein at receiver side has minimal effect on efflux substrate identification provides scientific basis for further specific transporter characterization (such as P-gp or BCRP) using specific inhibitors, in which same concentration of inhibitor is used in both sides of the Caco-2 cell system and protein for optimal permeability assessment has to be avoided.
本研究旨在评估蛋白质的作用机制，以便更好地解释在 Caco-2 渗透率测定中质量平衡低的化合物的渗透率结果。通过向接收侧添加蛋白质（4%牛血清白蛋白，BSA），测量具有质量平衡的亲脂性化合物的吸收或双向渗透率，以及在有空运输装置和无空运输装置的情况下。空运输装置研究的结果表明，滤膜是低质量平衡化合物的渗透屏障，蛋白质通过提高化合物通过滤膜的扩散性来增加渗透性。用蛋白质测量的 Caco-2 渗透率提供了更好的吸收预测。假设与细胞结合的化合物的量与吸收无关。对于使用 Caco-2 双向渗透率测定法鉴定外排底物，接收侧的蛋白质对作为外排底物的测试化合物的结论没有显著影响，但增加了来自两个运输方向的渗透率测量。总之，对于质量平衡低的化合物，在接收侧使用含蛋白质的缓冲液测量 Caco-2 渗透率似乎与体内吸收具有更好的相关性。接收侧的蛋白质对鉴定外排底物几乎没有影响，为使用特定抑制剂进一步进行特定转运体特征描述（如 P-糖蛋白或 BCRP）提供了科学依据，在这种情况下，抑制剂的相同浓度用于 Caco-2 细胞系统的两侧，并且必须避免蛋白质以获得最佳的渗透性评估。
