Calcein assay: a high-throughput method to assess P-gp inhibition

Abstract

Transporter mediated drug-drug interactions (tDDI) mediated by ABCB1 have been shown to be clinically relevant. Hence, the assessment of the ABCB1 tDDI potential early in the drug development process has gained interest. We have evaluated the Calcein assay as a means of assessing the ABCB1 tDDI that is amenable to high throughout and compared it with the monolayer efflux assay. We found the Calcein assay, when performed in K562MDR cells using the protocol originally published more sensitive than digoxin transport inhibition in MDCKII-MDR1 cells. Application of the Calcein assay to cell lines containing different amounts of ABCB1, yielded IC(50) values that varied 10-100-fold. The differences observed for IC(50) values for the same compounds were in the following rank order: IC(50, MDCKII-MDR1) >IC(50, K562MDR)>IC(50, hCMEC/D3). Higher IC(50) values were obtained in cells with higher ABCB1 expression. The Calcein assay is a high-throughput alternative to digoxin transport inhibition as it appears to have a comparable selectivity but higher sensitivity than previously published digoxin transport inhibition in MDCKII-MDR1 cells. In addition, it can be performed in a barrier-specific manner highlighting the dependence of ABCB1 IC(50) values on different ABCB1 expression levels.

Screening Compounds with a Novel High-Throughput ABCB1-Mediated Efflux Assay Identifies Drugs with Known Therapeutic Targets at Risk for Multidrug Resistance Interference

“…Several efflux-based high-throughput assays for screening inhibitors of ABC transporters have been reported in recent years [21], [22], [47], [48], [49], [50]. These assays often use fluorescent substrates as a means of detecting inhibition.…”

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.…”

Itraconazole and clarithromycin inhibit P‐glycoprotein activity in primary human sinonasal epithelial cells

“…The plates were then imaged using a fluorescent microscope using excitation and emission filters of 467–498nm and 513–556nm, respectively for 500ms. A relative percent increase in calcein fluorescence over baseline media control was considered to be proportional to the degree of P‐gp inhibition as previously described . A drop in fluorescence following a previous dose‐dependent increase was considered to be an indicator of concentration related induction of cytotoxicity.…”