Comparison of 3 Assay Systems Using a Common Probe Substrate, Calcein AM, for Studying P-gp Using a Selected Set of Compounds

Abstract

The multidrug resistance protein 1 (MDR1) transporter is the most abundantly investigated adenosine triphosphate (ATP)-Binding Cassette (ABC) transporter protein. Multiple assay systems were developed to study MDR1-mediated transport and possible drug-drug interactions. Yet, as different probe substrates are used in these assays, it is difficult to directly compare the results. In this study, a common probe substrate was applied in 3 assay systems developed to study MDR1: the cellular dye efflux assay, the ATPase assay, and the vesicular transport assay. This probe substrate is calcein acetoxymethyl ester (calcein AM), the acetoxymethyl ester derivative of the fluorescent dye, calcein. Using a common probe allows the investigation of the effect of passive permeability on the result obtained by testing various compounds. In this study, 22 compounds with different logP values were tested in the above-mentioned 3 assay systems. The vesicular transport assay proved most sensitive, detecting 18 of 22 interactions with the protein. The ATPase assay detected 15 interactions, whereas the cellular dye efflux assay was the least sensitive with only 10 hits. A correlation was found between the hydrophobicity of the compound and the ratio of cellular and vesicular transport IC(50) values, indicating the effect of passive permeability on the result. Based on hydrophobicity, the current study provides guidelines on applying the most correct tool for studying MDR1 interactions.

Multilayer Spheroids To Quantify Drug Uptake and Diffusion in 3D

“… 10 , 11 Thus, there is a need for new in vitro models to predict drug toxicity and unwanted drug–drug interactions and a need for models to discover new and more effective inhibitors of efflux transporters associated with multidrug resistance. Although monolayers of cells have been useful for defining some of these transport parameters and have been used to identify inhibitors of efflux pumps, such as Pgp, 11 , 18 , 21 there is growing recognition that 2D cell culture may not adequately replicate the biological complexities of the 3D in vivo environment.…”

“…10,11 Thus, there is a need for new in vitro models to predict drug toxicity and unwanted drug−drug interactions and a need for models to discover new and more effective inhibitors of efflux transporters associated with multidrug resistance. Although monolayers of cells have been useful for defining some of these transport parameters and have been used to identify inhibitors of efflux pumps, such as Pgp, 11,18,21 there is growing recognition that 2D cell culture may not adequately replicate the biological complexities of the 3D in vivo environment. 3D models have been used to investigate transport properties, molecular gradients, and cellular gradients, but often these methods require complex experimental and analytical procedures such as two-photon microscopy, 33 radiolabeled probes, 23 tissue sectioning, 24 or mathematical models.…”

“…Current methods to quantify 3D uptake and diffusion in tissues are cumbersome and include the use of microelectrode sensors or radiolabeled molecules, and are not amenable to higher throughput analyses. 21 − 25 …”

Vincristine-induced expression of P-glycoprotein in MOLM-13 and SKM-1 acute myeloid leukemia cell lines is associated with coexpression of nestin transcript

“…Thus, in P-gp-positive cells, the efflux activity of this transporter blocks calcein/AM entry and antagonizes the intracellular retention of fluorescent calcein. The decrease in calcein retention has often been used as a measure of drug efflux activity of transporters present in the plasma membrane of MDR cells (Glavinas et al 2011;Szeremy et al 2011), and in the case of P-gp, may be antagonized by P-glycoprotein inhibitors such as verapamil, cyclosporine A and ketoconazole (Eneroth et al 2001;Orlicky et al 2004). Depression of calcein retention within the intracellular space is visible in both VCRresistant cell lines (MOLM-13/VCR and SKM-1/VCR) compared with VCR-sensitive cells (MOLM-13 and SKM-1) (Fig.…”

Characterization of toxicologically significant efflux transporters and their interactions with drugs

“…For example, in the case of ABCB1 and ABCC1 CalceinAM is used [68][69][70], while in the case of ABCG2, the most frequently applied dye is Hoechst 33342 [71].…”