Transport inhibition of digoxin using several common P-gp expressing cell lines is not necessarily reporting only on inhibitor binding to P-gp.

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.

If the KI is defined by the free energy of binding to P-glycoprotein, which kinetic parameters define the IC50 for the Madin-Darby canine kidney II cell line overexpressing human multidrug resistance 1 confluent cell monolayer?

From previous fits of drug transport kinetics across confluent Madin-Darby canine kidney II cell line overexpressing human multidrug resistance 1 cell monolayers, we found that a drug's binding constant to P-glycoprotein (P-gp) was significantly smaller than its IC(50) when that drug was used as an inhibitor against another P-gp substrate. We tested several IC(50) candidate functions, including the standard function, the Kalvass-Pollack function, and the efflux ratio, to determine whether any of them yielded an IC(50) = K(I), as would be expected for water-soluble enzymes. For the confluent cell monolayer, the IC(50)/K(I) ratio is greater than 1 for all candidate functions tested. From the mass action kinetic model, we have derived a simple approximate equation that shows how the IC(50)/K(I) ratio depends on the elementary rate constants from our mass action model. Thus, the IC(50) will differ between cell lines and tissues, for the same probe substrate and inhibitor, if there are different membrane concentrations of P-gp, or the probe substrate's elementary rate constants, partition coefficient, binding constant to P-gp, passive permeability, and ability to access the other transporters (if any) in the two cell lines. The mass action model and the approximate equation for the IC(50)/K(I) ratio derived here can be used to estimate the elementary rate constants needed to extrapolate in vitro drug-drug interactions for compounds to the in vivo environment.