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.

Figure 1. Model of a confluent cell monolayer, with the apical membrane on top and the basolateral membrane below, where it binds to the polycarbonate insert.

Passive permeability occurs in both directions. P-gp expressed on the apical membrane transports substrate from the inner apical membrane monolayer into the apical chamber. The concentration of substrate in the apical and basolateral chambers, C_A and C_B, are measured, while the concentration of substrate in the inner plasma membrane, C_PC, and the cytosol, C_C, are predicted as part of the mass action modeling and data fitting process.

Figure 2. Transport data of various P-gp substrates and their fitted curves across the MDCK-MDR1-NKI cell monolayers, data acquired at GSK.

Fig. 2A, 0.3 µM verapamil (VRP); Fig. 2B, 0.3 µM ketoconazole (KCZ); and Fig. 2C, 0.02 µM cyclosporine-A (CsA). The B∶B>A and A∶B>A denotes the concentration of drug in the basolateral chamber and the apical chamber respectively, when the donor chamber was the basolateral chamber, i.e., transport runs B>A. Similar nomenclature is followed for the A>B transport curves. All B>A data are shown by squares and solid lines. All A>B data are shown by triangles and dashed lines. Data shown are the average of triplicates, with standard deviation error bars. The fitted parameters are shown in Table 2.

Figure 3. Inhibition of 0.03 µM digoxin B>A transport at 2 hr across the MDCK-MDR1-NKI cell monolayer with increasing concentrations of GF120918, data acquired at GSK.

Data shown are the average of triplicates, with standard deviation error bars. The dashed line shows the predicted inhibition when GF120918 only binds to P-gp, i.e. not to the basolateral digoxin uptake transporter. The solid line shows the fit when GF120918 binds to P-gp and the basolateral digoxin uptake transporter. K_QB is the effective association constant of GF120918 to the basolateral digoxin uptake transporter defined by Eq. (3), Table 3. K_QB combines the uptake transporter surface density, the binding constant of the inhibitor to the uptake transporter and, if the inhibitor accesses the binding site from the membrane, the inhibitor partition coefficient.

Figure 4. Simulated inhibition of 0.1 µM digoxin B>A transport across the MDCK-MDR1-NKI cells by an inhibitor under three scenarios of inhibitor binding to digoxin in the donor and receiver chambers.

Fig. 4A shows the case where K_CQ = 0, i.e. digoxin transport was inhibited solely by P-gp inhibition. The curve is sigmoidal. Fig. 4B shows the case where K_CQ = 3e3 M⁻¹, i.e. digoxin transport was inhibited by P-gp inhibition and by loss of free digoxin in the donor chamber due to heterodimerization to the inhibitor. The curve is not sigmoidal. Fig. 4C shows the case where K_CQ = 1e6 M⁻¹, i.e. digoxin transport was inhibited by P-gp inhibition and by loss of free digoxin in the donor chamber due to strong heterodimerization to the inhibitor. The curve is sigmoidal with a large inhibitor concentration plateau, i.e. PC plateau, of no transported digoxin, since there is no free digoxin.

Figure 5. Inhibition of 10 µM digoxin B>A transport across cell monolayers, as measured on the apical side after 1 hr with increasing concentrations of GF120918 using the MDCK-MDR1-NIH cell monolayers (Fig. 5A), Caco-2 monolayers (Fig. 5B), and CPT-B2 monolayers (Fig. 5C), data acquired at Absorption Systems.

Open symbols represent experimental data sets. The dotted lines shows the fitted inhibition assuming that the inhibitor binds only to P-gp, i.e. K_QB = 0. The solid lines show the fit using the values of K_QB shown in Table 3.

Figure 6. Inhibition of 10 µM digoxin B>A transport across the MDCK-MDR1-NIH cell monolayers, as measured on the apical side after 1 hr with increasing concentrations of cyclosporine A (Fig. 6A), ketoconazole (Fig. 6B) and verapamil (Fig. 6C), data acquired at Absorption Systems.

Open symbols represent experimental data sets. The dotted lines shows the fitted inhibition assuming that the inhibitor binds only to P-gp, i.e. K_QB = 0. The solid lines show the fit using the values of K_QB shown in Table 3.

Figure 7. Transport data of vinblastine and their fitted curves across the MDCK-MDR1-NKI cell monolayers, data acquired at GSK.

The same nomenclature is used as in Fig. 2. Fig. 7A shows the 6 hour experiment for 0.3 µM vinblastine. The dotted line shows the best fit with just P-gp inhibition. With BT, a good fit could be obtained, but not a unique fit, since the transport curves were essentially straight. Fig. 7B shows a stitched 18 hour experiment for 10 µM vinblastine, which was required to get unique fits for the kinetic parameters. The fitted parameters are shown in Table 2.

Figure 8. Simulation of +GF120918 passive permeabilities where the kinetic necessity of a basolateral digoxin uptake transporter could be validated.

Simulated transport data was created across the MDCK-MDR1-NKI cells for a virtual hybrid molecule with the P-gp rate constants of verapamil and the basolateral transporter clearance of loperamide (k_A = 0, k_B = 100 s⁻¹). The passive permeability was varied between 200–1000 nm/s. The substrate concentration range used for fitting was 3 nM to 30 µM. The simulated data were analyzed using only P-gp, i.e. only k_r and k₂ were used to fit the data. The coefficient of variation, CV, for the best fit at each passive permeability value was obtained. When the fits deteriorated to a coefficient of variation, CV, above 0.03, or 3%, the kinetic need for the basolateral digoxin uptake transporter becomes plausible .

Figure 9. Model of transporters that are kinetically significant for digoxin transport and the inhibition of digoxin transport for all cell lines studied here.

Starting from the basolateral chamber, the basolateral digoxin uptake transporter, denoted BT, transports digoxin into the cytosol more rapidly than bilayer permeability. This clearance is measured in the absence of inhibitor and denoted k_B(s⁻¹). Inhibitor binding to BT according to a binding constant denoted K_QB (M⁻¹), defined in Eq. 4 and shown in Table 3, can inhibit this digoxin transport. Efflux of digoxin from the cell into the apical compartment is mediated by P-gp, according to the digoxin kinetic parameters, Table 2. This efflux can be inhibited by competitive binding of the inhibitor to P-gp, according to its binding constant K_Q, which is equal to K_C in Table 2, since it is the inhibitor in this case.