scholarly journals INFLUENCE OF POLYSACCHARIDE COMPLEXES OF PLANTS OF CENTRAL RUSSIA ON THE P-GLYCOPROTEIN ACTIVITY IN VITRO

2020 ◽  
pp. 73-81
Author(s):  
Ivan Vladimirovich Chernykh ◽  
Aleksey Vladimirovich Shchul'kin ◽  
Yekaterina Yevgen'yevna Kirichenko ◽  
Sergey Konstantinovich Pravkin ◽  
Yelena Nikolayevna Yakusheva
Keyword(s):  
High Performance ◽  
Protein Activity ◽  
Apparent Permeability ◽  
Melilotus Officinalis ◽  
Polysaccharide Complex ◽  
Transport Medium ◽  
Transporter Protein ◽  
Central Russia ◽  
P Glycoprotein

The purpose of this work was to study the effect of polysaccharide complexes isolated from tansy flowers (Tanacetum vulgare L., fam. Asteraceae) and melilotus herb (Melilotus officinalis L., fam. Fabaceae) on P-glycoprotein (Pgp, ABCB1 protein) activity in vitro. On Caco-2 cell line the effect of polysaccharide complex isolated from tansy flowers and melilotus herb on Pgp activity was studied. In vitro Pgp activity was assessed by the transport of fexofenadine in the transwell system. High performance liquid chromatography with UV detection at wavelength 220 nm was used to determine fexofenadine concentration in the transport medium. It was revealed that when polysaccharide isolated from tansy flowers was added to the transport medium in concentrations 10 and 100 μM the ratio of the apparent permeability coefficients of fexofenadine b-a/a-b decreased by 1.81 and 2.65 times, respectively, compared with the series of isolated transport of fexofenadine, which indicated decreased Pgp functional activity under the polysaccharide action. The polysaccharide complex of the melilotus herb did not change the b-a/a-b ratio in any of the applied concentrations, thus it did not affect the activity of this transporter. It is advisable to continue the study of tansy flower polysaccharide complex as an inhibitor of Pgp transporter protein in order to assess the possibility of its clinical use for the treatment of pharmacoresistant forms of cancer by overcoming the phenomenon of multidrug resistance of cells.

Evaluation of female sex hormones influence on the protein-transporter p-glycoprotein functioning in vitro

2020 ◽  
Vol 66 (6) ◽  
pp. 444-449
Author(s):  
A.V. Shchulkin ◽  
I.V. Chernykh ◽  
N.M. Popova ◽  
A.A. Slepnev ◽  
E.N. Yakusheva
Keyword(s):  
Sex Hormones ◽  
Constitutive Androstane Receptor ◽  
Direct Inhibition ◽  
Protein Activity ◽  
Transporter Protein ◽  
Female Sex ◽  
Female Sex Hormones ◽  
P Glycoprotein ◽  
Increased Activity

The effects of female sex hormones estradiol and progesterone on P-glycoprotein (Pgp) functioning have been investigated using Caco-2 cells. Pgp activity was analyzed in a transwell system by the transport of its substrate, fexofenadine. The amount of the transporter protein was analyzed by enzyme immunoassay. Incubation of Caco-2 cells with 10 μM estradiol and incubation for 3 days increased activity and synthesis of Pgp. Moreover, this effect was suppressed by the inhibitor of the constitutive androstane receptor (CAR) CINPA 1. Incubation of these cells with 100 μM progesterone for 3 days increased Pgp synthesis, but its activity remained unchanged due to non-genomic (direct) inhibition of Pgp molecule by gestagen. The pregnan-X receptor inhibitor (PXR), ketoconazole suppressed the inducing effect of progesterone on Pgp synthesis. The combination of 10 μM estradiol and 100 μM progesterone increased Pgp synthesis, but did not increase the transporter protein activity, due to direct inhibition of the Pgp molecule by progestogen. Thus, it was found that estradiol increased activity and synthesis of Pgp by stimulating CAR, and progesterone stimulated transporter protein synthesis by activating PXR.

scholarly journals Development and validation of the method for the quantitative determination of methotrexate in a transport medium by HPLC-MS/MS

2021 ◽  
pp. 45-51
Author(s):  
P. Yu. Mylnikov ◽  
Yu. Tranova ◽  
A. V. Shchulkin ◽  
E. N. Yakusheva
Keyword(s):  
Quantitative Determination ◽  
Gradient Elution ◽  
Sample Volume ◽  
Transport Medium ◽  
Mass Selective Detector ◽  
Transporter Protein ◽  
Wide Range ◽  
Separation Temperature

Relevance. BCRP is an efflux transporter protein that plays an important role in the pharmacokinetics of a wide range of drugs. The BCRP activity in vitro experiments is assessed by the transport of transporter protein substrates (methotrexate, etc.) across the bilipid membrane of cells overexpressingBCRP, for example, Caco-2 cells. The aim is to develop and validate a method for the quantitative determination of the BCRP substrate, methotrexate, in the transport medium of Caco-2 cells by HPLC-MS/MS. Methods. The work was performed on an Ultimate 3000 HPLC chromatograph (ThermoFisher, USA) with a TSQ Fortis tandem mass-selective detector (ThermoFisher, USA). The conditions of chromatographic analysis were as follows: column UCT Selectra C18 4.6 mm * 100 mm 5um, 100A, Selectra C18 Guard Cartridges SLC-18GDC46-5UM, separation temperature 35 °С, flow rate 0.3 ml/min, injected sample volume - 2 μl, analysis time - 10 min. Used a gradient elution: the ratio of the solution of 0.1 % formic acid and acetonitrile was at 0 min 75 and 25 %; 0.4 min 60 and 40 %; 6 minutes 20 and 80 %; 8 minutes 75 and 25 %. Under these conditions, the retention time of methotrexate is 3.11 minutes. Detection conditions: methotrexate - positive ionization mode, 455.15 m / z → 308.125 m / z, collision energy 22.99 V, source fragmentation 5, CID gas pressure 2 mTorr. The extraction of methotrexate from the transport medium (Hanks solution with 25 mM Hepes and 1% dimethyl sulfoxide) after incubation with Caco-2 cells for 3 h was carried out with a mixture of methanol + water in a ratio of 1: 1. Results. The developed method was validated according to the following parameters: selectivity, linearity, accuracy, precision, limit of quantitative determination, sample transfer, sample stability. The confirmed analytical range of the method was 60 -10,000 nmol / L in the transport medium. Conclusions: a method for the quantitative determination of methotrexate in the transport medium of Caco-2 cells by HPLC-MS / MS was developed and validated.

Effects of Ginkgo biloba Extract Ingestion on the Pharmacokinetics of Talinolol in Healthy Chinese Volunteers

2009 ◽  
Vol 43 (5) ◽  
pp. 944-949 ◽  
Author(s):  
Lan Fan ◽  
Gong-You Tao ◽  
Guo Wang ◽  
Yao Chen ◽  
Wei Zhang ◽  
...  
Keyword(s):  
Oral Dose ◽  
Single Oral Dose ◽  
Ginkgo Biloba ◽  
High Performance ◽  
Plasma Concentrations ◽  
Ginkgo Biloba Extract ◽  
Maximum Plasma ◽  
Time Curve ◽  
P Glycoprotein

Background Ginkgo biloba extract (GBE), the best selling herbal medicine in the world, has been reported to inhibit P-glycoprotein in vitro. However, the effects of GBE on P-glycoprotein activity in humans have not been clarified. Objective To investigate the effects of single and repeated GBE ingestion on the oral pharmacokinetics of talinolol, a substrate drug for P-glycoprotein in humans. Methods Ten unrelated healthy male volunteers were selected to participate in a 3-stage sequential study. Plasma concentrations of talinolol from 0 to 24 hours were measured by high-performance liquid chromatography after talinolol 100 mg was administrated alone, with a single oral dose of GBE (120 mg), and after 14 days of repeated GBE ingestion (360 mg/day). Results A single oral dose of GBE did not affect the pharmacokinetics of talinolol. Repeated ingestion of GBE increased the talinolol maximum plasma concentration (Cmax) by 36% (90% CI 10 to 68; p = 0.025), the area under the concentration-time curve (AUC)0-24 by 26% (90% CI 11 to 43; p = 0.008) and AUC0-∞ by 22% (90% CI 8 to 37; p = 0.014), respectively, without significant changes in elimination half-life and the time to Cmax. Conclusions Our results suggest that long-term use of GBE significantly influenced talinolol disposition in humans, likely by affecting the activity of P-glycoprotein and/or other drug transporters.

scholarly journals A dynamic in-vitro model of the intestinal epithelium for the investigation of P-glycoprotein activity

2020 ◽  
Author(s):  
Joana Costa ◽  
Vanessa Almonti ◽  
Ludovica Cacopardo ◽  
Daniele Poli ◽  
Simona Rapposelli ◽  
...  
Keyword(s):  
Inhibitory Effect ◽  
Therapeutic Effects ◽  
Protein Activity ◽  
Glycoprotein P ◽  
Phenyl Derivative ◽  
Natural Substances ◽  
P Glycoprotein ◽  
Pre Treatment ◽  
Vitro Model

Abstract Multidrug resistance is still an obstacle for chemotherapeutic treatments. One of the proteins involved in this phenomenon is the P-glycoprotein, P-gp, which is known to be responsible for the efflux of therapeutic substances from the cell cytoplasm. To date, the identification of a drug that can efficiently inhibit P-gp activity remains a challenge, nevertheless some studies have identified natural compounds suitable for that purpose. Amongst them, curcumin has shown an inhibitory effect on the protein in in vitro studies using Caco-2 cells.To understand if physiological flow can modulate membrane protein activity, we studied the uptake of a P-gp substrate under static and dynamic conditions. Caco-2 cells were cultured in bioreactors and in Transwells and the basolateral transport of Rhodamine-123 assessed in the two systems as a function of P-gp activity. Experiments were performed with and without pre-treatment of the cells with an extract of curcumin or an arylmethyloxy-phenyl derivative to evaluate the inhibitory effect of the natural substance with respect to a synthetic compound.The results indicated that the P-gp activity of the cells cultured in the bioreactors was intrinsically lower, and that the effect of both natural and synthetic inhibitors was up modulated by the presence of flow. Our study underlies the fact that the use of more sophisticated and physiologically relevant in vitro models can bring new insights on the therapeutic effects of natural substances such as curcumin.

Inhibitory Effects of Benzaldehyde, Vanillin, Muscone and Borneol on P-Glycoprotein in Caco-2 Cells and Everted Gut Sac

Pharmacology ◽  
2018 ◽  
Vol 101 (5-6) ◽  
pp. 269-277 ◽  
Author(s):  
Shixiang Wang ◽  
Nan Tan ◽  
Cuicui Ma ◽  
Jie Wang ◽  
Pu Jia ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Prescription Drugs ◽  
High Performance ◽  
Mrna Level ◽  
Herbal Medicines ◽  
Mrna Levels ◽  
Inhibitory Effects ◽  
Apparent Permeability ◽  
P Glycoprotein ◽  
Everted Gut Sac

Aims: In clinical practice, herbal medicines have played an important role in the modulation of drug transporters through the combination of conventional prescription drugs, which necessitates the elucidation of herb-drug interactions. The present study was designed to investigate the inhibitory effects and mechanisms of benzaldehyde, vanillin, muscone, and borneol on P-glycoprotein (P-gp). Methods: The effects of the 4 compounds on the intracellular accumulation of rhodamine-123 (Rho-123) in vinblastine-treated Caco-2 (VB-Caco-2) cells were studied by monitoring fluorescence intensity through a flow cytometry assay, and the effects of these compounds on Rho-123 transport through VB-Caco-2 monolayers and Rho-123 intestinal absorption in the rat everted gut sac were investigated by high-performance liquid chromatography. Moreover, P-gp expression in VB-Caco-2 cells was assessed using flow cytometry and Western blot analysis, and the relative ABCB1 mRNA level was determined by Real-time RT-PCR. Key Findings: The results showed that benzaldehyde, vanillin, muscone, and borneol significantly increased Rho-123 uptake in VB-Caco-2 cells, increased the absorption rate and apparent permeability coefficient of Rho-123 in rat jejunum and ileum, and decreased the efflux ratio of Rho-123 from 6.52 to less than 2 during transport across VB-Caco-2 cell monolayers. In addition, these compounds reduced the protein and ABCB1 mRNA levels of P-gp in VB-Caco-2 cells. Conclusions: These data indicate that benzaldehyde, vanillin, muscone and borneol could effectively reverse multidrug resistance via inhibiting the P-gp function and expression pathway. The data provide fodder for further investigation into the interaction between the 4 compounds and other drugs transported by P-gp.

scholarly journals A dynamic in-vitro model of the intestinal epithelium for the investigation of P-glycoprotein activity

2020 ◽  
Author(s):  
Joana Costa ◽  
Vanessa Almonti ◽  
Ludovica Cacopardo ◽  
Daniele Poli ◽  
Simona Rapposelli ◽  
...  
Keyword(s):  
Inhibitory Effect ◽  
Therapeutic Effects ◽  
Protein Activity ◽  
Synthetic Compound ◽  
Phenyl Derivative ◽  
Natural Substances ◽  
P Glycoprotein ◽  
Pre Treatment ◽  
Vitro Model

Abstract Multidrug resistance is still an obstacle for chemotherapeutic treatments. One of the proteins involved in this phenomenon is the P-glycoprotein, P-gp, which is known to be responsible for the efflux of therapeutic substances from the cell cytoplasm. To date, the identification of a drug that can efficiently inhibit P-gp activity remains a challenge, nevertheless some studies have identified natural compounds suitable for that purpose. Amongst them, curcumin has shown an inhibitory effect on the protein in in vitro studies using Caco-2 cells. Aiming at understanding the role of physiological flow on the modulation of membrane protein activity, we studied the uptake of a P-gp substrate under static and dynamic conditions. Caco-2 cells were cultured in bioreactors and in Transwells and the basolateral transport of Rhodamine-123 assessed in the two systems as a function of P-gp activity. Experiments were performed with and without pre-treatment of the cells with an extract of curcumin or an arylmethyloxy-phenyl derivative to evaluate the inhibitory effect of the natural substance with respect to a synthetic compound. The results indicated that the P-gp activity of the cells cultured in the bioreactors was intrinsically lower, and that the effect of both natural and synthetic inhibitors was up modulated by the presence of flow. Our study underlies the fact that the use of more sophisticated and physiologically relevant in vitro models can bring new insights on the therapeutic effects of natural substances such as curcumin.

scholarly journals Inhibitory Effect of Berberine on Broiler P-glycoprotein Expression and Function: In Situ and In Vitro Studies

2019 ◽  
Vol 20 (8) ◽  
pp. 1966 ◽  
Author(s):  
Yujuan Zhang ◽  
Li Guo ◽  
Jinhu Huang ◽  
Yong Sun ◽  
Fang He ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Inhibitory Effect ◽  
In Vitro Models ◽  
Apparent Permeability ◽  
Glycoprotein P ◽  
P Glycoprotein ◽  
And Function ◽  
Xenobiotic Receptor

Overcoming P-glycoprotein (P-gp) efflux is a strategy to improve the absorption and pharmacokinetics of its substrate drugs. Berberine inhibits P-gp and thereby increases the bioavailability of the P-gp substrate digoxin in rodents. However, the effects of berberine on P-gp in chickens are still unclear. Here, we studied the role of berberine in modulating broilers P-gp expression and function through both in situ and in vitro models. In addition, molecular docking was applied to analyze the interactions of berberine with P-gp as well as with chicken xenobiotic receptor (CXR). The results showed that the mRNA expression levels of chicken P-gp and CXR decreased in the ileum following exposure to berberine. The absorption rate constant of rhodamine 123 increased after berberine treatment, as detected using an in situ single-pass intestinal perfusion model. Efflux ratios of P-gp substrates (tilmicosin, ciprofloxacin, clindamycin, ampicillin, and enrofloxacin) decreased and the apparent permeability coefficients increased after co-incubation with berberine in MDCK-chAbcb1 cell models. Bidirectional assay results showed that berberine could be transported by chicken P-gp with a transport ratio of 4.20, and this was attenuated by verapamil (an inhibitor of P-gp), which resulted in a ratio of 1.13. Molecular docking revealed that berberine could form favorable interactions with the binding pockets of both CXR and P-gp, with docking scores of −7.8 and −9.5 kcal/mol, respectively. These results indicate that berberine is a substrate of chicken P-gp and down-regulates P-gp expression in chicken tissues, thereby increasing the absorption of P-gp substrates. Our findings suggest that berberine increases the bioavailability of other drugs and that drug-drug interactions should be considered when it is co-administered with other P-gp substrates with narrow therapeutic windows.

scholarly journals In vitro and in situ effects of atorvastatin and ezetimibe on P-glycoprotein expression and function

2016 ◽  
Vol 11 (4) ◽  
pp. 911
Author(s):  
Mehran Mesgari Abbasi ◽  
Hadi Valizadeh ◽  
Hamed Hamishehkar ◽  
Maryam Bannazadeh Amirkhiz ◽  
Parvin Zakeri-Milani
Keyword(s):  
Intestinal Permeability ◽  
High Performance ◽  
Video Clip ◽  
Permeability Model ◽  
Active Efflux ◽  
Single Pass ◽  
P Glycoprotein ◽  
And Function

<p class="Abstract">P-glycoprotein (P-gp) is a membrane transporter responsible for the active efflux from the cell. Inhibition of the activity may lead to clinically significant drug-drug interactions. This study was performed to investigate the effects of atorvastatin and ezetimibe on the function and expression of P-gp. The <em>in vitro</em> rhodamine-123 (Rho123) efflux assay and Western blot in Caco-2 cells, and the <em>in situ</em> rat single-pass intestinal permeability model followed by high performance liquid chromatography were developed. Rho123 intracellular accumulation in 100 µM of atorvastatin- and ezetimibe-treated cells was significantly higher than that in control cells (p&lt;0.05). P-gp expression was decreased by 100 µM atorvastatin and ezetimibe. Intestinal effective permeability of digoxin in the presence of atorvastatin (3 and 100 µM), ezetimibe (10 and 100 µM) was significantly increased (p&lt;0.05). Both drugs  inhibited P-gp activity in vitro and<em> in situ</em>. Atorvastatin and ezetimibe down-regulated the expression of P-gp <em>in vitro</em>. </p><p class="Abstract"><strong>Video Clip of Methodology</strong>:</p><p class="Abstract"><a href="https://youtube.com/v/BQuz1ER3_NQ">Single-pass intestinal permeability</a>: 17 min 26 sec</p><p> </p>

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