Evaluation of P-Glycoprotein Inhibitory Potential Using a Rhodamine 123 Accumulation Assay

Abstract

In vitro evaluation of P-glycoprotein (P-gp) inhibitory potential is now a regulatory issue during drug development, in order to predict clinical inhibition of P-gp and subsequent drug–drug interactions. Assays for this purpose, commonly based on P-gp-expressing cell lines and digoxin as a reference P-gp substrate probe, unfortunately exhibit high variability, raising thus the question of developing alternative or complementary tests for measuring inhibition of P-gp activity. In this context, the present study was designed to investigate the use of the fluorescent dye rhodamine 123 as a reference P-gp substrate probe for characterizing P-gp inhibitory potential of 16 structurally-unrelated drugs known to interact with P-gp. 14/16 of these P-gp inhibitors were found to increase rhodamine 123 accumulation in P-gp-overexpressing MCF7R cells, thus allowing the determination of their P-gp inhibitory potential, i.e., their half maximal inhibitor concentration (IC50) value towards P-gp-mediated transport of the dye. These IC50 values were in the range of variability of previously reported IC50 for P-gp and can be used for the prediction of clinical P-gp inhibition according to Food and Drug Administration (FDA) criteria, with notable sensitivity (80%). Therefore, the data demonstrated the feasibility of the use of rhodamine 123 for evaluating the P-gp inhibitory potential of drugs.

Alteration of human hepatic drug transporter activity and expression by cigarette smoke condensate

“…Additional culture for two weeks in the same medium supplemented with 2% (vol/vol) DMSO was performed in order to get a full hepatocytic differentiation of the cells (Gripon et al 2002). P-gp overexpressing mammary MCF7R cells (Jouan et al 2016) and MRP2-expressing human hepatoma HuH-7 cells (Olsavsky et al 2007), were cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies), supplemented with 10 % (vol/vol) fetal calf serum, 10 IU/mL penicillin and 10 µg/mL streptomycin. BCRP-transfected cells HEK 293 (HEK-BCRP cells) (Tournier et al 2010), kindly donated by Dr X. Decleves (Faculty of Pharmacy, University Paris-Descartes, Paris, France), were cultured in DMEM supplemented with 10% (vol/vol) fetal calf serum, 100 IU/mL amoxicillin, 100 µg/mL erythromycin and 2 mg/mL G418.…”

Effects of Anticholinergic Drugs Used for the Therapy of Overactive Bladder on P-Glycoprotein Activity

“…17) Rhodamine 123 is a lipophilic cation that is selective for P-glycoprotein-dependent transport 18) and has been widely used as an in vitro and in vivo marker of P-glycoprotein function. [19][20][21][22] Jouan et al reported that rhodamine 123 could be used for the prediction of clinical P-glycoprotein inhibition according to the Food and Drug Administration (FDA) criteria, with notable sensitivity (80%). 23) For example, the National Cancer Institute of the United States uses rhodamine 123 to screen for drugs that serve as substrates for P-glycoprotein.…”

“…[19][20][21][22] Jouan et al reported that rhodamine 123 could be used for the prediction of clinical P-glycoprotein inhibition according to the Food and Drug Administration (FDA) criteria, with notable sensitivity (80%). 23) For example, the National Cancer Institute of the United States uses rhodamine 123 to screen for drugs that serve as substrates for P-glycoprotein. 24) Van der Sandt et al reported rhodamine 123 is a substrate for both P-glycoprotein and the organic cation carrier systems in the kidney cell line.…”

Neonicotinoid pesticides poorly interact with human drug transporters

“…P‐glycoprotein/ ABCB1 ‐overexpressing mammary MCF7R cells and multidrug resistance‐associated proteins (MRP/ ABCC )‐expressing human hepatoma HuH‐7 cells were cultured in Dulbecco's modified Eagle medium (DMEM; Life Technologies), supplemented with 10% (vol/vol) fetal calf serum, 20 IU/mL penicillin, and 20 μg/mL streptomycin. Breast cancer resistance protein (BCRP/ ABCG2 )‐transfected HEK293 cells (HEK‐BCRP cells), kindly donated by Pr X. Decleves (Faculty of Pharmacy, University Paris‐Descartes, Paris, France), were cultured in DMEM supplemented with 10% (vol/vol) fetal calf serum, 100 IU/mL amoxicillin, 100 μg/mL erythromycin, and 2,000 μg/mL G418.…”