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

• Published in: Doklady. Biochemistry and biophysics 2025-01
• Main Authors: Tranova, Yu S, Slepnev, A A, Chernykh, I V, Shchulkin, A V, Mylnikov, P Yu, Popova, N M, Povetko, M I, Yakusheva, E N
• Format: Article
• Language: English
• ISSN: 1608-3091; 1608-3091
• DOI: 10.1134/S1607672924701266

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 o