Figure 3: Impact of the efflux transporter P-gp on intracellular bioavailability (F_ic).

(a) Schematic representation of cell types used in this study: MDCK cells where canine P-gp has been knocked-out using CRISPR/Cas 9 (cP-gp-KO); MDCK cells transfected with human P-gp, expressing both canine and human P-gp. (b) Comparison between F_ic in cP-gp-KO and in P-gp-transfected MDCK cells at 0.5 μM compound concentration. Negatively charged compounds at pH 7.4 are represented as triangles, neutral and zwitterionic species are represented by circles, and positively charged compounds are represented by squares. Substrates of P-gp are highlighted in yellow. Nelfinavir is represented in parentheses as its F_ic is outside the range of this plot in P-gp-transfected cells. (c) Relationship between simvastatin acid concentration and F_ic in P-gp-transfected MDCK cells (yellow filled squares), P-gp-KO MDCK cells (blue circles), and P-gp-KO MDCK cells pre-incubated (15 min) followed by co-incubation with 10 μM cyclosporine A (gray triangles). (d) Kinetic cell model simulations that best described experimental observations of concentration-dependence F_ic for simvastatin acid (conditions: passive permeability = 10 × 10⁻⁶ cm/s; V_max,uptake = 1000 pmol/min/mg protein; K_m,uptake = 10 μM; V_max,efflux = 1000 pmol/min/mg protein; K_m,efflux = 100 μM).