Utilization of membrane vesicle preparations to study drug–ABC transporter interactions

Glavinas, Hristos; Méhn, Dóra; Jani, Márton; Oosterhuis, B.; Herédi-Szabó, Krisztina; Krajcsi, Péter

doi:10.1517/17425255.4.6.721

Cited by 84 publications

(40 citation statements)

References 62 publications

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“…An important limitation for some of these cell lines is that functional expression of drug transporters, particularly OAT1 (SLC22A6) and OAT3 (SLC22A8), or other renal characteristics can be lost (46), although some of the recent advanced molecular biology tools may be useful to overcome such issues (47). Cell membrane vesicles can be isolated from tissue samples, cell lines and recombinantly expressed transporter systems (48,49), allowing multiple or individual transporters to be studied. The inside-out vesicular transport assay is particularly useful for studying transport mediated by efflux transporters, which can be challenging when in standard cellbased assays.…”

Section: Kidney Slicesmentioning

confidence: 99%

“…The use of membrane vesicles is limited to drugs with low lipophilicity/passive permeability. Drugs with high lipophilicity/permeability will show high levels of nonspecific binding to lipid bilayer of the vesicle and may not be trapped within the vesicles following uptake, making it difficult to measure transporter-related uptake rates (48).…”

Section: Kidney Slicesmentioning

confidence: 99%

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Key to Opening Kidney for In Vitro–In Vivo Extrapolation Entrance in Health and Disease: Part I: In Vitro Systems and Physiological Data

Scotcher

Jones²,

Posada

et al. 2016

AAPS J

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ABSTRACT.The programme for the 2015 AAPS Annual Meeting and Exhibition (Orlando, FL; 25-29 October 2015) included a sunrise session presenting an overview of the state-of-the-art tools for in vitro-in vivo extrapolation (IVIVE) and mechanistic prediction of renal drug disposition. These concepts are based on approaches developed for prediction of hepatic clearance, with consideration of scaling factors physiologically relevant to kidney and the unique and complex structural organisation of this organ. Physiologically relevant kidney models require a number of parameters for mechanistic description of processes, supported by quantitative information on renal physiology (system parameters) and in vitro/in silico drug-related data. This review expands upon the themes raised during the session and highlights the importance of high quality in vitro drug data generated in appropriate experimental setup and robust system-related information for successful IVIVE of renal drug disposition. The different in vitro systems available for studying renal drug metabolism and transport are summarised and recent developments involving state-of-the-art technologies highlighted. Current gaps and uncertainties associated with system parameters related to human kidney for the development of physiologically based pharmacokinetic (PBPK) model and quantitative prediction of renal drug disposition, excretion, and/or metabolism are identified.

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Section: Kidney Slicesmentioning

confidence: 99%

Section: Kidney Slicesmentioning

confidence: 99%

Key to Opening Kidney for In Vitro–In Vivo Extrapolation Entrance in Health and Disease: Part I: In Vitro Systems and Physiological Data

Scotcher

Jones²,

Posada

et al. 2016

AAPS J

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“…The most commonly used cell line for expressing transporter protein and preparing vesicles is insect Sf9 cells (Glavinas et al, 2008). However, the membrane composition in the insect Sf9 cell plasma membrane is different from than that for mammalian plasma membranes and has been shown to significantly affect transporter functionality (Pál et al, 2007).…”

mentioning

confidence: 99%

High-Activity P-Glycoprotein, Multidrug Resistance Protein 2, and Breast Cancer Resistance Protein Membrane Vesicles Prepared from Transiently Transfected Human Embryonic Kidney 293-Epstein-Barr Virus Nuclear Antigen Cells

Karlsson¹,

Heddle²,

Rozkov³

et al. 2010

Drug Metab Dispos

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ABSTRACT:Membrane-bound transporter proteins play an important role in the efflux of drugs from cells and can significantly influence the pharmacokinetics of drug molecules. This study describes the production of large amounts of high-activity transporter membrane vesicles from human embryonic kidney 293-Epstein-Barr virus nuclear antigen cells transiently transfected using a Gateway-adapted pCEP4 plasmid. Transfections were scaled up to 10-liter cell cultures, and vesicle preparations were optimized using ultracentrifugation with a sucrose cushion, which enabled us to produce hundreds of milligrams of membrane vesicles expressing human efflux transporter proteins Pglycoprotein (P-gp)/multidrug resistance 1 (ABCB1), multidrug resistance protein 2 (MRP2) (ABCC2), and breast cancer resistance protein (BCRP) (ABCG2). Assays were developed and optimized for analyzing the ATP-dependent functionality of the transporters using probe substrates and specific inhibitors. Excellent signal/noise ratios of ATP-stimulated uptake for P-gp, MRP2, and BCRP vesicles were obtained, indicating high expression of functioning transporters. The uptake kinetics of the transporters was investigated by determining K m and V max using the model substrates N-methylquinidine (P-gp), estradiol-17␤-glucuronide (MRP2), and estrone-3-sulfate (BCRP). The ATP-dependent transport was inhibited by the model inhibitors verapamil (P-gp), benzbromarone (MRP2), and sulfasalazine (BCRP). The vesicles are thus well suited to screen for possible substrates and inhibitors in high throughput systems or are used for detailed mechanistic investigations of transporter kinetics of specific substances.

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“…Because of the difficulty in getting the confluent cell monolayer system to yield a K I simply, simulations of inside-out plasma membrane vesicles (Glavinas et al, 2008) were undertaken. These vesicles have been proposed as a simpler system for fitting a K I or a Michaelis constant K m , depending on the experiment, because the binding site is directly exposed to the incubation medium.…”

Section: Physical Mechanism For the Overestimate Equationmentioning

confidence: 99%

“…The importance of membrane transporters in the metabolism and disposition of drugs is clear (Chang and Benet, 2005;Collett et al, 2005;Endres et al, 2006;Shitara et al, 2006;Bartholomé et al, 2007;Balimane et al, 2008;Glavinas et al, 2008;Kurnik et al, 2008;Nies et al, 2008). Assessing drug-drug interaction risk is an important aspect of drug development, which is often quantified by the concentration of inhibitor required to reduce probe substrate transport by 50%, reported as the IC 50 (Gao et al, 2001;Zong and Pollack, 2003;Rautio et al, 2006).…”

mentioning

confidence: 99%

If the K_I Is Defined by the Free Energy of Binding to P-Glycoprotein, Which Kinetic Parameters Define the IC₅₀ for the Madin-Darby Canine Kidney II Cell Line Overexpressing Human Multidrug Resistance 1 Confluent Cell Monolayer?

Lumen¹,

Acharya²,

Polli³

et al. 2009

Drug Metab Dispos

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ABSTRACT:From previous fits of drug transport kinetics across confluent Madin-Darby canine kidney II cell line overexpressing human multidrug resistance 1 cell monolayers, we found that a drug's binding constant to P-glycoprotein (P-gp) was significantly smaller than its IC 50 when that drug was used as an inhibitor against another P-gp substrate. We tested several IC 50 candidate functions, including the standard function, the Kalvass-Pollack function, and the efflux ratio, to determine whether any of them yielded an IC 50 ‫؍‬ K I , as would be expected for water-soluble enzymes. For the confluent cell monolayer, the IC 50 /K I ratio is greater than 1 for all candidate functions tested. From the mass action kinetic model, we have derived a simple approximate equation that shows how the IC 50 /K I ratio depends on the elementary rate constants from our mass action model. Thus, the IC 50 will differ between cell lines and tissues, for the same probe substrate and inhibitor, if there are different membrane concentrations of P-gp, or the probe substrate's elementary rate constants, partition coefficient, binding constant to P-gp, passive permeability, and ability to access the other transporters (if any) in the two cell lines. The mass action model and the approximate equation for the IC 50 /K I ratio derived here can be used to estimate the elementary rate constants needed to extrapolate in vitro drug-drug interactions for compounds to the in vivo environment.

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Utilization of membrane vesicle preparations to study drug–ABC transporter interactions

Cited by 84 publications

References 62 publications

Key to Opening Kidney for In Vitro–In Vivo Extrapolation Entrance in Health and Disease: Part I: In Vitro Systems and Physiological Data

Key to Opening Kidney for In Vitro–In Vivo Extrapolation Entrance in Health and Disease: Part I: In Vitro Systems and Physiological Data

High-Activity P-Glycoprotein, Multidrug Resistance Protein 2, and Breast Cancer Resistance Protein Membrane Vesicles Prepared from Transiently Transfected Human Embryonic Kidney 293-Epstein-Barr Virus Nuclear Antigen Cells

If the K_I Is Defined by the Free Energy of Binding to P-Glycoprotein, Which Kinetic Parameters Define the IC₅₀ for the Madin-Darby Canine Kidney II Cell Line Overexpressing Human Multidrug Resistance 1 Confluent Cell Monolayer?

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Utilization of membrane vesicle preparations to study drug–ABC transporter interactions

Cited by 84 publications

References 62 publications

Key to Opening Kidney for In Vitro–In Vivo Extrapolation Entrance in Health and Disease: Part I: In Vitro Systems and Physiological Data

Key to Opening Kidney for In Vitro–In Vivo Extrapolation Entrance in Health and Disease: Part I: In Vitro Systems and Physiological Data

High-Activity P-Glycoprotein, Multidrug Resistance Protein 2, and Breast Cancer Resistance Protein Membrane Vesicles Prepared from Transiently Transfected Human Embryonic Kidney 293-Epstein-Barr Virus Nuclear Antigen Cells

If the KI Is Defined by the Free Energy of Binding to P-Glycoprotein, Which Kinetic Parameters Define the IC50 for the Madin-Darby Canine Kidney II Cell Line Overexpressing Human Multidrug Resistance 1 Confluent Cell Monolayer?

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If the K_I Is Defined by the Free Energy of Binding to P-Glycoprotein, Which Kinetic Parameters Define the IC₅₀ for the Madin-Darby Canine Kidney II Cell Line Overexpressing Human Multidrug Resistance 1 Confluent Cell Monolayer?