Method for Testing Drugs Belonging to Substrates and Inhibitors of the Transporter Protein BCRP on CACO-2 Cells.

IF 0.8 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY
Yu S Tranova, A A Slepnev, I V Chernykh, A V Shchulkin, P Yu Mylnikov, N M Popova, M I Povetko, E N Yakusheva
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引用次数: 0

Abstract

Introduction: 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.

Aim: The aim of the study was to develop a methodology for testing drugs for belonging to BCRP substrates and inhibitors in vitro.

Materials and methods: 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.

Results: 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.

Conclusions: 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.

CACO-2细胞转运蛋白BCRP底物和抑制剂药物的检测方法。
乳腺癌耐药蛋白(Breast cancer resistance protein, BCRP)是一种外排膜转运蛋白,控制着大量药物的药代动力学。它的活性在服用一些内源性和外源性物质时可能发生变化,从而使其成为药物相互作用的一个环节。目的:本研究的目的是开发一种在体外测试属于BCRP底物和抑制剂的药物的方法。材料和方法:在过表达BCRP的Caco-2细胞上进行培养,在由顶室和基底侧室组成的transwell系统中进行培养。细胞在顶室底部播种,顶室为半透膜。首先,在1、5、10和50 μM的浓度范围内,BCRP底物甲氨蝶呤、米托蒽醌和槲皮素在基底室向顶室方向(Papp b-a)和相反方向(Papp a-b)的转运情况进行了评估。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。为了证实BCRP参与了它们的转运,我们在转运介质中加入了转运抑制剂利血平,浓度为50 μM。采用高效液相色谱-质谱联用(HPLC-MS/MS)分析培养皿中底物的浓度。结果:甲氨蝶呤(1 μM)、米托蒽醌(1 μM)和槲皮素(1 ~ 10 μM)分别加入transwell系统的顶腔和底外侧腔后,受体腔内均未检测到其含量。在甲氨蝶呤浓度为5 μM时,Papp b-a/Papp a-b比值为3.38±0.08,表明转运蛋白参与了其转运。当甲氨蝶呤浓度分别为10 μM和50 μM时,Papp b-a/Papp a-b比值降至2以下。在米托蒽醌浓度为5 μM时,Papp b-a/Papp a-b比值为2.72±0.16。当浓度增加到10 μM时,Papp b-a/Papp a-b比值增加到6.18±0.08。当物质浓度为50 μM时,该指数有所下降,但仍保持在2以上。槲皮素浓度为50µM时,Papp b-a/Papp比值小于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 = 0.0002),表明消除了物质转运中的不对称性。在米托蒽醌浓度为10µM时,利血平使其Papp b-a/Papp a-b比值降低3.36倍(p < 0.0001)。这些结果表明BCRP参与控制这两种物质通过细胞单层的转移。结论:建立了一种以甲氨蝶呤(5 μM)和米托蒽醌(10 μM)为标记底物,利血平(50 μM)为抑制剂,检测药物与BCRP底物和抑制剂相关性的方法,并在Caco-2细胞上进行了实验。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Doklady Biochemistry and Biophysics
Doklady Biochemistry and Biophysics 生物-生化与分子生物学
CiteScore
1.60
自引率
12.50%
发文量
68
审稿时长
6-12 weeks
期刊介绍: Doklady Biochemistry and Biophysics is a journal consisting of English translations of articles published in Russian in biochemistry and biophysics sections of the Russian-language journal Doklady Akademii Nauk. The journal''s goal is to publish the most significant new research in biochemistry and biophysics carried out in Russia today or in collaboration with Russian authors. The journal accepts only articles in the Russian language that are submitted or recommended by acting Russian or foreign members of the Russian Academy of Sciences. The journal does not accept direct submissions in English.
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