Determining P-glycoprotein-drug interactions: Evaluation of reconstituted P-glycoprotein in a liposomal system and LLC-MDR1 polarized cell monolayers

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• 作者：Donald L. Melchior^a ; ^{Donald.Melchior@glsynthesis.com} ; Frances J. Sharom^b ; Raymond Evers^c ; George E. Wright^a ; Joseph W.K. Chu^b ; Stephen E. Wright^a ; Xiaoyan Chu^c ; Jocelyn Yabut^c
• 关键词：ABC ; ATP-binding cassette ; AMP-PNP ; adenosine 5&prime ; -(尾 ; 纬-imido)triphosphate ; AUC ; area under the curve ; BSA ; bovine serum albumin ; CHAPS ; 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate ; Fl-t-pgp ; Fluorosome-trans-pgp ; MIANS ; 2-(4-maleimid
• 刊名：Journal of Pharmacological and Toxicological Methods
• 出版年：2012
• 期刊代码：28_10568719
• 类别：pha
• 出版时间：March-April, 2012
• 卷：65
• 期：2
• 页码：64-74
• 文件大小：1260 K

摘要

Introduction

P-Glycoprotein (ABCB1, MDR1) is a multidrug efflux pump that is a member of the ATP-binding cassette (ABC) superfamily. Many drugs in common clinical use are either substrates or inhibitors of this transporter. Quantitative details of P-glycoprotein inhibition by pharmaceutical agents are essential for assessment of their pharmacokinetic behavior and prevention of negative patient reactions. Cell-based systems have been widely used for determination of drug interactions with P-glycoprotein, but they suffer from several disadvantages, and results are often widely variable between laboratories. We aimed to demonstrate that a novel liposomal system employing contemporary biochemical methodologies could measure the ability of clinically used drugs to inhibit the P-glycoprotein pump. To accomplish this we compared results with those of cell-based approaches.

Methods

Purified transport-competent hamster Abcb1a P-glycoprotein was reconstituted into a unilamellar liposomal system, Fluorosome-trans-pgp, whose aqueous interior contains fluorescent drug sensors. This provides a well-defined system for measuring P-glycoprotein transport inhibition by test drugs in real time using rapid fluorescence-based technology.

Results

Inhibition of ATP-driven transport by Fluorosome-trans-pgp employed a panel of 46 representative drugs. Resulting IC₅₀ values correlated well (r² = 0.80) with K_d values for drug binding to purified P-glycoprotein. They also showed a similar trend to transport inhibition data obtained using LLC-MDR1 cell monolayers. Fluorosome-trans-pgp IC₅₀ values were in agreement with published results of digoxin drug-drug interaction studies in humans.

Discussion

This novel approach using a liposomal system and fluorescence-based technology is shown to be suitable to study whether marketed drugs and drug candidates are P-glycoprotein inhibitors. The assay is rapid, allowing a 7-point IC₅₀ determination in < 6 min, and requires minimal quantities of test drug. The method is amenable to robotics and offers a cost advantage relative to conventional cell-based assays. The well-defined nature of this assay also obviates many of the inherent complications and ambiguities of cell-based systems.