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

Abstract

1. 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.

2. 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 IC₅₀ values that varied 10–100-fold. The differences observed for IC₅₀ values for the same compounds were in the following rank order: IC_{50, MDCKII-MDR1} >IC_{50, K562MDR}>IC_{50, hCMEC/D3}. Higher IC₅₀ values were obtained in cells with higher ABCB1 expression.

3. 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 IC₅₀ values on different ABCB1 expression levels.

Keywords::

• Transporter
• ABCB1
• DDI
• high-throughput
• MDCKII-MDR1
• K562MDR
• hCMEC/D3

Acknowledgments

Expert help of Felicia Kovács in the experimental procedures and Katalin Jakab MD, as well as Timea Rosta MSc in preparation of the manuscript is acknowledged.

Declaration of interest

The work was supported by Hungarian National Office for Research and Technology (Grant XTTPSRT1) (Xenobiotic Transporter Technology Platform Therapeutic and Toxicological Applications), (Grant GOP 1.3.2.) (Development of an in vivo technological platform for pharmaceutical applications by enhancing R&D capacities); and the European Community Grant EUSTROKE (HEALTH-F2-2008–202213). Calcein assay is the proprietary technology of Solvo Biotechnology.