Study of Influence of Estradiol on the Activity of P-Glycoprotein in Vitro

Erokhina, Pelageya D.; Abalenikhina, Yu. V.; Shchulkin, A. V.; Chernykh, I. V.; Kotlyarova, A. A.; Правкин, С К; Slepnev, A. A.; Yakusheva, E. N.

doi:10.23888/hmj202083329-336

Cited by 7 publications

(2 citation statements)

References 10 publications

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“…Клетки линии HepG2 культивировали при 37º С и 5% содержании СО 2 в Дульбекко модифицированной среде Игла (DMEM) с высоким содержанием глюкозы (4500 мг/л) ("Sigma-Aldrich", Германия), содержащей L-глутамин (4 мМ) ("Sigma-Aldrich", Германия), 10% фетальной бычьей сыворотки ("Sigma-Aldrich", Германия), 100 ЕД/мл пенициллина и 100 мкг/мл стрептомицина ("Sigma-Aldrich", Германия) [16,[35][36]. Смена питательной среды производилась каждые 48 ч.…”

Section: материалы и методыunclassified

HepG2 cell line as a model for studying of the statins’ hepatic uptake

Erokhina

Slepnev

Mylnikov

et al. 2023

jour

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Statins (β-hydroxy-β-methyl-glutaryl-CoA reductase inhibitors) are the main class of drugs for the treatment of dyslipidemia. To improve the safety of therapy with their use, a test system is needed to assess their penetration into hepatocytes using OATP1B1/OATP1B3 transporter proteins. The aim of the study was to develop a method for assessing the penetration of statins into hepatocytes on HepG2 cells (human hepatocellular carcinoma). Materials and methods. Cells were cultured in 6- and 24-well plates. The presence of OATP1B1 in HepG2 cells was assessed using the Western blot method. Penetration of statins into cells was analyzed using atorvastatin as an example. It was added to the cell monolayer at concentrations of 1 and 10 μM and incubated for 30 minutes. The cells were then removed from the wells, lysed in various ways, and the amount of atorvastatin was determined by high performance liquid chromatography with tandem mass selective detection (HPLC-MS/MS). Results. Western blot showed the presence of OATP1B1 in HepG2 cells, the main protein that transports statins into hepatocytes. The best way to lyse the cells was a three-cycle freeze-thaw cycle at -80 °C. The analytical range of the method for the quantitative determination of atorvastatin in the lysate of HepG2 cells by HPLC-MS/MS was 0.5-200 nmol/l, which made it possible to perform transport experiments with the addition of atorvastatin at a concentration of 1 μM and incubation for up to 30 min. The use of the OATP1B1/OATP1B3 inhibitor rifampicin (100 μM) reduced the penetration of atorvastatin into HepG2 cells, which confirms the adequacy of the proposed method. Conclusions. A technique for assessing the penetration of statins into hepatocytes on HepG2 cells has been developed.

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Section: материалы и методыunclassified

HepG2 cell line as a model for studying of the statins’ hepatic uptake

Erokhina

Slepnev

Mylnikov

et al. 2023

jour

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“…Для оценки активности BCRP в качестве субстрата было предложено анализировать трансмембранный перенос кверцетина -низкотоксичного вещества группы флавоноидов природного происхождения, относящегося к препаратам витамина Р. Испытания in vitro выполняют на клетках линии Сасо-2 (аденокарцинома ободочной кишки человека) после их 21дневного инкубирования на полупроницаемой мембране трансвелл-системы. Данная культура в указанных условиях приобретает морфологическое и функциональное сходство с энтероцитами человека, гиперэкспрессирующими BCRP и гликопротеин-P [4][5][6]. Изучение транспорта кверцетина требует разработки и валидации метода количественного анализа вещества в соответствующей матрице.…”

Section: Introductionunclassified

Method for quantitative quercetin detection using HPLC-MS/MS

Tranova¹,

Mylnikov²,

Shchulkin³

et al. 2022

HMJ

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INTRODUCTION: Human breast cancer resistance protein (BCRP, ABCG2) is a transport protein of ABC superfamily of transporters that uses ATP energy of for its work. BCRP plays an important role in the pharmacokinetics of drugs, therefore all new drugs are recommended to be tested for belonging to its substrates, inducers and inhibitors. The influence of substances on the activity of this transport protein in vitro is evaluated by a change of transmembrane transfer of its substrates, one of which being quercetin. This, in turn, requires the development and validation of a method for its quantitative analysis in an appropriate matrix. AIM: To develop a method for the quantitative determination of quercetin using high performance liquid chromatography (HPLC) with tandem mass selective detection (MS/MS) with full validation. MATERIALS AND METHODS: The method was developed on Ultimate 3000 HPLC equipped with TSQ Fortis (Thermo Fisher, USA) MS/MS detector. The conditions of chromatographic analysis were as follows: column UCT Selectra C18 4.6 mm*100 mm 5μm, 100 A, Selectra C18 Guard Cartridges SLC-18 GDC46-5UM. The analysis time was 11 min at separation temperature 35°С, flow rate 0.5 ml/min, injected sample volume 5 μl. Gradient elution regime was used: on 0th minute, the ratio of 0.1% formic acid solution and acetonitrile was 70% and 30%; 0.3 min — 30% and 70%; 4 min — 1% and 99%; 9 min — 70% and 30%. Quercetin retention time was 3.91 minutes. Detection conditions: negative ionization mode, 301 m/z → 150.9 m/z with collision energy 22 V, 301 m/z →178.9 m/z with collision energy 17 V, source fragmentation 5 V, electrospray voltage 3 000 V, CID gas pressure 1 mTorr, Sheath gas 50 Arb, Aux gas 10 Arb, Sweep gas 10 Arb, ion transfer tube temperature 300°C, vaporizer temperature 350°C. The matrix was transport medium after incubation for 3 hours with Caco-2 cells overexpressing BCRP. In this medium, the transport of quercetin through the cell monolayer was further evaluated in vitro. Precipitation of protein and isolation of quercetin from the transport medium was realized by a mixture of water and acetonitrile in a ratio of 1:1. RESULTS: The developed method was validated by the following parameters: selectivity, linearity, accuracy, precision, limit of quantification, sample transfer, matrix effect, sample stability. The analytical range of the technique was 5–500 nmol/l. In this case, the correlation coefficient was more than 0.99. The limit of detection and the lower limit of quantification of quercetin (LLQQ) were 1 and 5 nmol/l, respectively. The calculation of intra- and inter-cycle accuracy and precision showed that these parameters do not exceed 20% for the concentration corresponding to the lower limit of quantitative determination, and 15% for other concentrations. The analyte demonstrated stability in triple freeze-defreeze cycle at -80°C, in storage at -80°C for 60 days, after sample preparation and being kept in the autosampler for 24 hours. There was no sample transfer and no matrix effect. CONCLUSION: A method for the quantitative determination of quercetin using HPLC-MS/MS in a transport medium was developed and fully validated.

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