Digoxin transport was investigated using a bidirectional transcellular transport assay across a monolayer of Caco-2 cells. Cells were equilibrated in assay buffer (Hanks' Balanced Salt Solution with HEPES 15 mM adjusted to pH 7.4 with NaOH) for 30 minutes before [3H]digoxin (1 μM) was added to the donor compartment (apical side of the monolayer in apical-to-basal transport experiments, or the basal side in basal-to-apical experiments). BI 425809 (1, 3, 10, 30, or 100 μM) or zosuquidar (1 μM) was added to both the donor compartment and the receiver compartment on the opposite side of the monolayer. The assay was initiated after 30 minutes of preincubation with digoxin. Samples were collected from the donor compartment at −30, 0, and 90 minutes and from the receiver compartment at 0, 30, 60, and 90 minutes. Sample radioactivity was measured using a liquid scintillation counter.
The permeability coefficient (Papp) value was calculated using the transport rate and the initial concentration of radioactivity in the donor compartment using the following equation, where Papp is the permeability coefficient (cm/s), Ct0 is the initial radioactivity concentration in the donor compartment at time t0 (dpm/mL), A is the area of the filter (cm2), VR is the volume of buffer in the receiver compartment (mL), and ΔCR/Δt is the change in radioactivity concentration over time in the receiver compartment (dpm/[mL·s]):
The transport rate ∆CR/∆t was calculated based on the linear part of the compound concentration in the receiver compartment over time curve.
The efflux ratio (ER) for digoxin was calculated as the ratio of the permeability coefficients for basal-to-apical (BtoA) and apical-to-basal (AtoB) transport, using the following equation:
The concentration of inhibitor resulting in 50% inhibition of P-gp was calculated based on iterative nonlinear regression analysis of the dose-response relationship, which was performed using XLfit (version 5.3.1.3; IDBS, Guildford, United Kingdom). IC50 values were calculated, assuming standard (hyperbolic) Michaelis-Menten kinetics, using the following equation (where ER is the observed ER, h is the slope factor, I is the concentration of inhibitor [μM], ERmax is the ER at I = 0, and ERmin is the ER at I = infinity):
The permeability coefficient (Papp) value was calculated using the transport rate and the initial concentration of radioactivity in the donor compartment using the following equation, where Papp is the permeability coefficient (cm/s), Ct0 is the initial radioactivity concentration in the donor compartment at time t0 (dpm/mL), A is the area of the filter (cm2), VR is the volume of buffer in the receiver compartment (mL), and ΔCR/Δt is the change in radioactivity concentration over time in the receiver compartment (dpm/[mL·s]):
The transport rate ∆CR/∆t was calculated based on the linear part of the compound concentration in the receiver compartment over time curve.
The efflux ratio (ER) for digoxin was calculated as the ratio of the permeability coefficients for basal-to-apical (BtoA) and apical-to-basal (AtoB) transport, using the following equation:
The concentration of inhibitor resulting in 50% inhibition of P-gp was calculated based on iterative nonlinear regression analysis of the dose-response relationship, which was performed using XLfit (version 5.3.1.3; IDBS, Guildford, United Kingdom). IC50 values were calculated, assuming standard (hyperbolic) Michaelis-Menten kinetics, using the following equation (where ER is the observed ER, h is the slope factor, I is the concentration of inhibitor [μM], ERmax is the ER at I = 0, and ERmin is the ER at I = infinity):
