Tranova Yu S, Slepnev A A, Chernykh I V, Shchulkin A V, Mylnikov P Yu, Popova N M, Povetko M I, Yakusheva E N
Ryazan State Medical University, Ryazan, Russian Federation.
Dokl Biochem Biophys. 2025 Feb;520(1):89-95. doi: 10.1134/S1607672924701266. Epub 2025 Jan 22.
Breast cancer resistance protein (BCRP) is an efflux membrane transporter that controls the pharmacokinetics of a large number of drugs. Its activity may change when taking some endo- and exogenous substances, thus making it a link in drug interactions.
The aim of the study was to develop a methodology for testing drugs for belonging to BCRP substrates and inhibitors in vitro.
The work was performed on Caco-2 cells overexpressing BCRP, the cultivation was performed in a transwell system consisting of the apical and basolateral chambers. Cells were seeded at the bottom of the apical chamber, which is a semi-permeable membrane. Primarily, the transport of BCRP substrates-methotrexate, mitoxantrone, and quercetin-was evaluated in the concentration range of 1, 5, 10, and 50 μM in the direction from the basal chamber to the apical one (Papp b-a) and in the opposite direction (Papp a-b). The Papp b-a/Papp a-b ratio more than 2 characterizes the involvement of transporter proteins in the transcellular transport of substances. To confirm the involvement of BCRP in their transport, an experiment was carried out with the addition of a transporter inhibitor, reserpine, at a concentration of 50 μM to the transport medium. The concentration of substrates in the chambers was analyzed by HPLC-MS/MS.
After the addition of methotrexate (1 μM), mitoxantrone (1 μM), and quercetin (1-10 μM) to the apical or basolateral chambers of the transwell system, their content in the recipient chamber was not detected. At methotrexate concentration of 5 μM, the Papp b-a/Papp a-b ratio was 3.38 ± 0.08, which indicates the involvement of transporters in its transfer. When methotrexate was added to the donor chamber at concentrations of 10 and 50 μM, the Papp b-a/Papp a-b ratio decreased to values below 2. At mitoxantrone concentration of 5 μM, the Papp b-a/Papp a-b ratio was 2.72 ± 0.16. An increase in the concentration to 10 μM led to an increase in the Papp b-a/Papp a-b ratio to 6.18 ± 0.08. At the substance concentration of 50 μM, the index decreased but remained above 2. At the quercetin concentration of 50 µM, the Papp b-a/Papp ratio was below 2. Reserpine reduced the Papp b-a/Papp a-b ratio of methotrexate 3.31 times (p = 0.0002), which indicates the elimination of asymmetry in the transport of the substance. At a mitoxantrone concentration of 10 µM, reserpine reduced its Papp b-a/Papp a-b ratio 3.36 times (p < 0.0001). These results indicate the involvement of BCRP in the control of the transfer of both substances through the cellular monolayer.
A method of testing drugs for affiliation to BCRP substrates and inhibitors using methotrexate (5 μM) and mitoxantrone (10 μM) as marker substrates and reserpine (50 μM) as inhibitor was developed and tested on Caco-2 cells.
乳腺癌耐药蛋白（BCRP）是一种外排膜转运蛋白，可控制大量药物的药代动力学。服用某些内源性和外源性物质时，其活性可能会发生变化，因此它成为药物相互作用中的一个环节。
本研究的目的是开发一种体外检测药物是否为BCRP底物和抑制剂的方法。
实验在过表达BCRP的Caco-2细胞上进行，培养在由顶侧室和基底外侧室组成的Transwell系统中。细胞接种在顶侧室底部，该底部为半透膜。首先，评估BCRP底物甲氨蝶呤、米托蒽醌和槲皮素在1、5、10和50 μM浓度范围内从基底室到顶侧室方向（Papp b-a）以及相反方向（Papp a-b）的转运情况。Papp b-a/Papp a-b比值大于2表明转运蛋白参与了物质的跨细胞转运。为证实BCRP参与了它们的转运，在转运培养基中加入浓度为50 μM的转运抑制剂利血平进行实验。通过HPLC-MS/MS分析各室中底物的浓度。
在Transwell系统的顶侧室或基底外侧室中加入甲氨蝶呤（1 μM）、米托蒽醌（1 μM）和槲皮素（1 - 10 μM）后，未在接受室中检测到它们的含量。当甲氨蝶呤浓度为5 μM时，Papp b-a/Papp a-b比值为3.38 ± 0.08，这表明转运蛋白参与了其转运。当甲氨蝶呤以10和50 μM的浓度加入供体室时，Papp b-a/Papp a-b比值降至2以下。当米托蒽醌浓度为5 μM时，Papp b-a/Papp a-b比值为2.72 ± 0.16。浓度增加到10 μM导致Papp b-a/Papp a-b比值增加到6.18 ± 0.08。当物质浓度为50 μM时，该指标下降但仍高于2。当槲皮素浓度为50 µM时，Papp b-a/Papp比值低于2。利血平使甲氨蝶呤的Papp b-a/Papp a-b比值降低了3.31倍（p = \alpha），这表明消除了该物质转运的不对称性。当米托蒽醌浓度为10 µM时，利血平使其Papp b-a/Papp a-b比值降低了3.36倍（p < \alpha）。这些结果表明BCRP参与了这两种物质通过细胞单层的转运控制。
开发了一种使用甲氨蝶呤（5 μM）和米托蒽醌（10 μM）作为标记底物以及利血平（50 μM）作为抑制剂来检测药物是否为BCRP底物和抑制剂的方法，并在Caco-2细胞上进行了测试。
