Kotze Soné, Goss Kai-Uwe, Ebert Andrea
Department of Computational Biology and Chemistry, Helmholtz Centre for Environmental Research (UFZ), Permoserstraße 15, Leipzig 04318, Germany.
Institute of Chemistry, University of Halle-Wittenberg, Kurt-Mothes-Straße 2, Halle 06120, Germany.
Int J Pharm X. 2024 Jul 8;8:100269. doi: 10.1016/j.ijpx.2024.100269. eCollection 2024 Dec.
MDCK/Caco-2 assays serve as essential in vitro tools for evaluating membrane permeability and active transport, especially mediated by P-glycoprotein (P-gp). Despite their utility, challenges remain in quantifying active transport and using the efflux ratio (ER) to determine intrinsic values for active efflux. Such an intrinsic value for P-gp facilitated efflux necessitates knowing whether this transporter transports the neutral or ionic species of a compound. Utilising MDCK-MDR1 assays, we investigate a method for determining transporter substrate fraction preference by studying ER pH-dependence for basic, acidic and non-dissociating compounds. These results are compared with model fits based on various assumptions of transporter species preference. As an unexpected consequence of these assays, we also give evidence for an additional influx transporter at the basolateral membrane, and further extend our model to incorporate this transport. The combined influences of paracellular transport, the previously unaccounted for basolateral influx transporter, as well as potential pH effects on the transporter impedes the extraction of intrinsic values for active transport from the ER. Furthermore, we determined that using inhibitor affects the measurement of paracellular transport. While clear indications of transporter species preference remain elusive, this study enhances understanding of the MDCK system.
MDCK/Caco-2 试验是评估膜通透性和主动转运（尤其是由 P-糖蛋白（P-gp）介导的主动转运）的重要体外工具。尽管它们很有用，但在量化主动转运以及使用外排比率（ER）来确定主动外排的内在值方面仍然存在挑战。P-gp 促进外排的这种内在值需要知道该转运蛋白转运的是化合物的中性还是离子形式。利用 MDCK-MDR1 试验，我们通过研究碱性、酸性和非解离性化合物的 ER 对 pH 的依赖性，研究了一种确定转运蛋白底物分数偏好的方法。将这些结果与基于转运蛋白物种偏好的各种假设的模型拟合进行比较。作为这些试验的一个意外结果，我们还提供了证据证明在基底外侧膜存在一种额外的内流转运蛋白，并进一步扩展我们的模型以纳入这种转运。细胞旁转运、先前未考虑的基底外侧内流转运蛋白以及转运蛋白上潜在的 pH 效应的综合影响阻碍了从 ER 中提取主动转运的内在值。此外，我们确定使用抑制剂会影响细胞旁转运的测量。虽然转运蛋白物种偏好的明确迹象仍然难以捉摸，但这项研究增强了对 MDCK 系统的理解。
