Date Published: August 16, 2013
Publisher: Public Library of Science
Author(s): Annie Albin Lumen, Libin Li, Jiben Li, Zeba Ahmed, Zhou Meng, Albert Owen, Harma Ellens, Ismael J. Hidalgo, Joe Bentz, Andreas Hofmann.
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.
It is well established that transporters play an important role in absorption, distribution, metabolism and elimination of drugs. Inhibition of drug transporters can affect drug safety and efficacy. The International Transporter Consortium published a white paper reviewing the clinically important drug transporters and summarizing which in vitro methods are suitable for assessing drug-drug interaction (DDI) risks . P-glycoprotein (P-gp) is listed as one of the ABC transporters of emerging clinical importance. The risk for a DDI resulting from P-gp inhibition is assessed by determining the in vitro inhibitor concentration required to reduce probe-substrate transport by 50%, i.e. the IC50. Digoxin is typically used in in vitro inhibition studies as a clinically relevant P-gp probe substrate since it has a narrow therapeutic window and digoxin clinical drug-drug interactions have been ascribed to P-gp inhibition. Inhibition of digoxin transport is often determined using confluent polarized cell lines expressing high levels of P-gp such as Caco-2 , MDCK-MDR1-NKI (from the Netherlands Cancer Institute) , MDCK-MDR1-NIH (from NIH)  and LLC-PK1 (from the Netherlands Cancer Institute) .
Digoxin is a widely prescribed cardiovascular drug with a narrow therapeutic index and therefore not very tolerant of drug-drug interactions. Since digoxin is a P-gp substrate, digoxin drug-drug interactions are often ascribed to P-gp inhibition. It is common practice in the pharmaceutical industry to assess the risk for a clinically relevant digoxin drug-drug interaction by determining the in vitro P-gp inhibitory potency using P-gp expressing cell lines . We have demonstrated kinetically that digoxin transport across MDCK-MDR1-NKI cells involves a basolateral and an apical uptake transporter as well as P-gp, although we were not able to determine the identity of that transporter, other than that it was inhibited by GF120918 . Recently, it has been reported that digoxin is a substrate for uptake transporters in other cell lines as well . The involvement of other transporters besides P-gp in digoxin transport across P-gp expressing confluent monolayers in MDCK-MDR1-NKI cells raises two questions: (1) does the experimentally measured IC50 in these cells represent inhibition of P-gp, inhibition of the uptake transporters, or a combination of the two and (2) do other P-gp expressing cell lines also express a digoxin uptake transporter.
The structural mass action kinetic analysis has been used to demonstrate that the IC50 for inhibition of digoxin transport across MDCK-MDR1-NKI, MDCK-MDR1-NIH, Caco-2 and CPT-B2 cells is a convolution of inhibition of P-gp and of a kinetically identified basolateral digoxin uptake mechanism. Three possible sources are discussed for this kinetically identified uptake mechanism: (1) inhibition of an uptake transporter, (2) inhibitor binding to the basolateral outer membrane directly reducing digoxin passive permeability and (3) aggregation between digoxin and inhibitor in basolateral chamber resulting in reduced free digoxin concentrations. The weight of evidence presented here supports the existence of a basolateral digoxin uptake transporter in all cell lines and that GF120918 and CsA can bind to this uptake transporter, inhibiting digoxin uptake by the cells. This model is shown in Fig. 9. A contribution from hetero-dimerization model cannot be completely excluded for ketoconazole and verapamil by these data. Further studies are required. Without this mass action kinetic analysis we developed using the MDCK-MDR1-NKI cells yielding elementary rate constants, the deconvolution of the inhibition curves shown here, the kinetic evidence of the existence of widely expressed basolateral transporters and the inhibition of these basolateral transports by some, if not all, P-gp substrates could not have been accomplished. We believe this kinetic analysis, with its powerful diagnostic tests for the kinetic requirement of other transporters, will continue to contribute to the current discussion on the relative importance of transporters and passive permeability in transport biology . Our work supports the importance of transporters and of passive permeability by providing a direct analytical kinetic method to measure their relative contributions simultaneously.