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Abstract：
OBJECTIVE: According to the regulatory guidelines, one of the critical steps in using in-vitro permeability methods for permeability classification is to demonstrate the suitability of the method. Here, suitability of the permeability method by using a monolayer of cultured epithelial cells was verified with different criteria.
METHODS: Imaging with a transmission electron microscope was used for characterisation of the cells. Monolayer integrity was confirmed by transepithelial electrical resistance measurements and permeability of zero permeability marker compounds. Real-time polymerase chain reaction was employed to evaluate expression levels of 84 known transporters. Samples for bidirectional permeability determination were quantified by ultra-performance liquid chromatography.
RESULTS: The Caco-2 cells grow in an intact monolayer and morphologically resemble enterocytes. Genes of 84 known transporters were expressed at different levels; furthermore, expression was time depended. Functional expression of efflux transporter P-glycoprotein was confirmed. We established a correlation between permeability coefficients of 21 tested drug substances ranging from low, moderate and high absorption with human fraction absorbed literature data (R2 = 0.84).
CONCLUSIONS: Assay standardisation assures the consistency of experimental data. Only such fully characterised model has the ability to accurately predict drug\'s intestinal permeability at the early stage of research or for the BCS-based biowaiver application.
METHODS: Imaging with a transmission electron microscope was used for characterisation of the cells. Monolayer integrity was confirmed by transepithelial electrical resistance measurements and permeability of zero permeability marker compounds. Real-time polymerase chain reaction was employed to evaluate expression levels of 84 known transporters. Samples for bidirectional permeability determination were quantified by ultra-performance liquid chromatography.
RESULTS: The Caco-2 cells grow in an intact monolayer and morphologically resemble enterocytes. Genes of 84 known transporters were expressed at different levels; furthermore, expression was time depended. Functional expression of efflux transporter P-glycoprotein was confirmed. We established a correlation between permeability coefficients of 21 tested drug substances ranging from low, moderate and high absorption with human fraction absorbed literature data (R2 = 0.84).
CONCLUSIONS: Assay standardisation assures the consistency of experimental data. Only such fully characterised model has the ability to accurately predict drug\'s intestinal permeability at the early stage of research or for the BCS-based biowaiver application.
摘要：
目标：根据监管指南，使用体外渗透性方法进行渗透性分类的关键步骤之一是证明该方法的适用性。这里,用不同的标准验证了使用单层培养的上皮细胞的通透性方法的适用性。
方法:使用透射电子显微镜成像来表征细胞。单层完整性通过跨上皮电阻测量和零渗透性标记化合物的渗透性得到证实。采用实时聚合酶链反应评估84种已知转运蛋白的表达水平。用于双向渗透性测定的样品通过超高效液相色谱法定量。
结果：Caco-2细胞以完整的单层生长，形态类似于肠上皮细胞。84个已知转运蛋白的基因表达水平不同；表达取决于时间。证实了外排转运蛋白P-糖蛋白的功能性表达。我们建立了21种测试药物的渗透系数之间的相关性，范围从低，中等和高吸收与人体部分吸收文献数据(R2=0.84)。
结论:测定标准化确保了实验数据的一致性。只有这种充分表征的模型能够在研究的早期阶段或基于BCS的生物防腐剂应用中准确预测药物的肠道通透性。
方法:使用透射电子显微镜成像来表征细胞。单层完整性通过跨上皮电阻测量和零渗透性标记化合物的渗透性得到证实。采用实时聚合酶链反应评估84种已知转运蛋白的表达水平。用于双向渗透性测定的样品通过超高效液相色谱法定量。
结果：Caco-2细胞以完整的单层生长，形态类似于肠上皮细胞。84个已知转运蛋白的基因表达水平不同；表达取决于时间。证实了外排转运蛋白P-糖蛋白的功能性表达。我们建立了21种测试药物的渗透系数之间的相关性，范围从低，中等和高吸收与人体部分吸收文献数据(R2=0.84)。
结论:测定标准化确保了实验数据的一致性。只有这种充分表征的模型能够在研究的早期阶段或基于BCS的生物防腐剂应用中准确预测药物的肠道通透性。
