Transport Inhibition of Digoxin Using Several Common P-gp Expressing Cell Lines Is Not Necessarily Reporting Only on Inhibitor Binding to P-gp

Abstract

We have reported that the P-gp substrate digoxin required basolateral and apical uptake transport in excess of that allowed by digoxin passive permeability (as measured in the presence of GF120918) to achieve the observed efflux kinetics across MDCK-MDR1-NKI (The Netherlands Cancer Institute) confluent cell monolayers. That is, GF120918 inhibitable uptake transport was kinetically required. Therefore, IC50 measurements using digoxin as a probe substrate in this cell line could be due to inhibition of P-gp, of digoxin uptake transport, or both. This kinetic analysis is now extended to include three additional cell lines: MDCK-MDR1-NIH (National Institute of Health), Caco-2 and CPT-B2 (Caco-2 cells with BCRP knockdown). These cells similarly exhibit GF120918 inhibitable uptake transport of digoxin. We demonstrate that inhibition of digoxin transport across these cell lines by GF120918, cyclosporine, ketoconazole and verapamil is greater than can be explained by inhibition of P-gp alone. We examined three hypotheses for this non-P-gp inhibition. The inhibitors can: (1) bind to a basolateral digoxin uptake transporter, thereby inhibiting digoxin's cellular uptake; (2) partition into the basolateral membrane and directly reduce membrane permeability; (3) aggregate with digoxin in the donor chamber, thereby reducing the free concentration of digoxin, with concomitant reduction in digoxin uptake. Data and simulations show that hypothesis 1 was found to be uniformly acceptable. Hypothesis 2 was found to be uniformly unlikely. Hypothesis 3 was unlikely for GF120918 and cyclosporine, but further studies are needed to completely adjudicate whether hetero-dimerization contributes to the non-P-gp inhibition for ketoconazole and verapamil. We also find that P-gp substrates with relatively low passive permeability such as digoxin, loperamide and vinblastine kinetically require basolateral uptake transport over that allowed by +GF120918 passive permeability, while highly permeable P-gp substrates such as amprenavir, quinidine, ketoconazole and verapamil do not, regardless of whether they actually use the basolateral transporter.

Applicability of free drug hypothesis to drugs with good membrane permeability that are not efflux transporter substrates: A microdialysis study in rats

“…Good permeability of quinidine has also been reported by other investigators. In a study with MDCK‐MDR1‐NKI cell lines, the permeability was about 60 × 10 −6 cm/sec for quinidine 40 . In another study, the permeability was about 14 × 10 −6 cm/sec for quinidine, when Caco‐2 cells were used 41 …”

ITC Commentary on the Prediction of Digoxin Clinical Drug–Drug Interactions from In Vitro Transporter Assays

“…By contrast, transcellular flux in the basolateral‐to‐apical direction is appreciable because it is a transport‐mediated process across both basolateral and apical membranes. Basolateral uptake of digoxin has been shown to involve sodium‐dependent (Na‐dep) processes in HEK293 cells 5 and Caco‐2 cells, 8 and active uptake has been demonstrated on a kinetic level in MDCKII cells 9 and hepatocytes 10 . ( b ) Transport‐mediated clinical disposition of digoxin.…”

Quantitative and Mechanistic Assessment of Model Lipophilic Drugs in Micellar Solutions in the Transport Kinetics Across MDR1-MDCK Cell Monolayers

“…However, in a different analysis focused on relative transport activity for Digoxin in the 2 MDR1-MDCK cell lines, it was inferred that surface Pgp density was greater in the NKI line than NIH. 38 The reasons for these discrepancies are not known but may arise from differences in cell culture conditions or some other unknown factor. Nevertheless, as stated earlier, based on our observations the MDR1-MDCK cell, whatever the origin, may represent a better Pgp model for drug screening than Caco-2 cells, especially for use of surfactants as solubilization aids.…”