Department of Analytical Environmental Chemistry, Helmholtz Centre for Environmental Research (UFZ), Permoserstraße 15, Leipzig 04318, Germany.
Department of Analytical Environmental 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.
Eur J Pharm Sci. 2023 Dec 1;191:106592. doi: 10.1016/j.ejps.2023.106592. Epub 2023 Sep 24.
Intrinsic membrane permeability is one of several factors that critically determine the intestinal absorption of a chemical. The intrinsic membrane permeability of a chemical is usually extracted from transwell experiments with Caco-2 or MDCK cells, preferably by the pK-Flux method, which is considered the method of choice when aqueous boundary layer effects need to be excluded. The pK-Flux method has two variants, the iso-pH method, where apical and basolateral pH are equal, and the gradient-pH method, where apical and basolateral pH are different. The most commonly used method is the gradient-pH method, as it is intended to reflect the pH-conditions in the gastrointestinal tract. However, concentration-shift effects caused by the applied pH-difference between apical and basolateral compartment in the gradient-pH method have not been considered in the evaluation of the experimental data in the past. Consequently, incorrect intrinsic membrane permeabilities have been determined. In this work, we present a revised method for extracting the intrinsic membrane permeability from gradient-pH data that considers concentration-shift effects in the basolateral aqueous boundary layer and filter as well as in the cytosol. Furthermore, we propose the use of the iso-pH method, where only concentration-shift effects in the cytosol need to be considered, as an alternative to the gradient-pH method. We use the five lipophilic bases amantadine, chloroquine, propranolol, venlafaxine and verapamil as examples to compare gradient-pH method and iso-pH method with regard to the extractability of the intrinsic membrane permeability. For lipophilic bases, the iso-pH method proves to be advantageous. All intrinsic membrane permeabilities determined in this work were substantially higher than the intrinsic membrane permeabilities reported in literature.
内在膜通透性是决定化学物质肠道吸收的几个关键因素之一。化学物质的内在膜通透性通常通过 Caco-2 或 MDCK 细胞的 Transwell 实验提取，最好使用 pK-Flux 法，当需要排除水相边界层效应时，该方法被认为是首选方法。pK-Flux 法有两种变体，等 pH 法，其中顶侧和基底外侧 pH 相等，和 pH 梯度法，其中顶侧和基底外侧 pH 不同。最常用的方法是 pH 梯度法，因为它旨在反映胃肠道中的 pH 条件。然而，在过去评估实验数据时，梯度-pH 法中顶侧和基底外侧隔室之间应用的 pH 差异引起的浓度转移效应尚未被考虑。因此，确定了不正确的内在膜通透性。在这项工作中，我们提出了一种从 pH 梯度数据中提取内在膜通透性的修正方法，该方法考虑了基底外侧水相边界层和过滤器以及细胞质中的浓度转移效应。此外，我们建议使用等 pH 法，其中只需考虑细胞质中的浓度转移效应，作为 pH 梯度法的替代方法。我们使用五种亲脂性碱基金刚烷胺、氯喹、普萘洛尔、文拉法辛和维拉帕米作为示例，比较 pH 梯度法和等 pH 法对内在膜通透性的提取能力。对于亲脂性碱基，等 pH 法证明是有利的。本工作中确定的所有内在膜通透性都明显高于文献报道的内在膜通透性。
