Panou D A, Pedersen S F, Kristensen M, Nielsen H M
Center for Biopharmaceuticals and Biobarriers in Drug Delivery (BioDelivery), Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Section for Cell Biology and Physiology, Department of Biology, Faculty of Science, University of Copenhagen, Copenhagen, Denmark.
Front Drug Deliv. 2023 Aug 24;3:1221628. doi: 10.3389/fddev.2023.1221628. eCollection 2023.
Absorption of therapeutic peptides like glucagon-like peptide or insulin for diabetes therapy upon oral administration is highly restricted by the tight junction (TJ) proteins interconnecting the cells comprising the intestinal epithelium. An approach to improve transepithelial permeation of such biopharmaceuticals via the paracellular pathway is to use functional excipients, which transiently modulate the TJs. Here, we investigated the membrane-interacting peptide, penetramax, and the divalent cation chelator, ethylene glycol tetraacetic acid (EGTA) at different concentrations, to reveal and compare their cellular modes of action when increasing the transepithelial permeation of drug macromolecules. The epithelial integrity was studied in real time along with dextran permeation across differentiated epithelial Caco-2 cell monolayers. TJ protein expression and cytoskeleton organization were investigated during and after exposure to penetramax or EGTA. Based on orthogonal methods, we show that penetramax acts by a mechanism that immediately and transiently widens the paracellular space, resulting in size selective permeant passage and with subsequent reconstitution of the epithelium. At the same time, the expression and organization of different TJ proteins are modulated reversibly. In contrast, the effect of EGTA on modulating the paracellular space is slower and TJ protein unspecific, and without clear permeant size selectivity. Overall, these data provide in-depth insights for understanding intestinal barrier dynamics of importance when evaluating new or existing excipients for oral delivery of biopharmaceuticals, such as peptide therapeutics.
口服给药时，用于糖尿病治疗的胰高血糖素样肽或胰岛素等治疗性肽的吸收受到紧密连接（TJ）蛋白的高度限制，这些蛋白连接构成肠上皮的细胞。一种通过细胞旁途径改善此类生物药物经上皮渗透的方法是使用功能性辅料，其可短暂调节紧密连接。在此，我们研究了不同浓度的膜相互作用肽penetramax和二价阳离子螯合剂乙二醇四乙酸（EGTA），以揭示并比较它们在增加药物大分子经上皮渗透时的细胞作用模式。我们实时研究了上皮完整性以及葡聚糖在分化的上皮Caco-2细胞单层中的渗透情况。在暴露于penetramax或EGTA期间及之后，研究了紧密连接蛋白表达和细胞骨架组织。基于正交方法，我们表明penetramax通过一种机制起作用，该机制可立即且短暂地扩大细胞旁空间，导致大小选择性的渗透物通过，并随后重建上皮。同时，不同紧密连接蛋白的表达和组织被可逆地调节。相比之下，EGTA对调节细胞旁空间的作用较慢且对紧密连接蛋白无特异性，并且没有明确的渗透物大小选择性。总体而言，这些数据为理解肠屏障动力学提供了深入见解，这在评估用于口服递送生物药物（如肽类治疗药物）的新辅料或现有辅料时具有重要意义。
