Simultaneous assessment of uptake and metabolism in rat hepatocytes: a comprehensive mechanistic model

Abstract

Kinetic parameters describing hepatic uptake in hepatocytes are frequently estimated without appropriate incorporation of bidirectional passive diffusion, intracellular binding, and metabolism. A mechanistic two-compartment model was developed to describe all of the processes occurring during the in vitro uptake experiments performed in freshly isolated rat hepatocytes plated for 2 h. Uptake of rosuvastatin, pravastatin, pitavastatin, valsartan, bosentan, telmisartan, and repaglinide was investigated over a 0.1 to 300 μM concentration range at 37°C for 2 or 45-90 min; nonspecific binding was taken into account. All concentration-time points were analyzed simultaneously by using a mechanistic two-compartment model describing uptake kinetics [unbound affinity constant (K(m,u)), maximum uptake rate (V(max)), unbound active uptake clearance (CL(active,u))], passive diffusion [unbound passive diffusion clearance (P(diff,u))], and intracellular binding [intracellular unbound fraction (fu(cell))]. When required (telmisartan and repaglinide), the model was extended to account for the metabolism [unbound metabolic clearance (CL(met,u))]. The CL(active,u) ranged 8-fold, reflecting a 11-fold range in uptake K(m,u), with telmisartan and valsartan showing the highest affinity for uptake transporters (K(m,u) <10 μM). Both P(diff,u) and fu(cell) span over two orders of magnitude and reflected the lipophilicity of the drugs in the dataset. An extended incubation time allowed steady state to be reached between media and intracellular compartment concentrations and reduced the error in certain parameter estimates observed with shorter incubation times. Active transport accounted for >70% of total uptake for all drugs investigated and was 4- and 112-fold greater than CL(met,u) for telmisartan and repaglinide, respectively. Modeling of uptake kinetics in conjunction with metabolism improved the precision of the uptake parameter estimates for repaglinide and telmisartan. Recommendations are made for uptake experimental design and modeling strategies.

Fig. 1.

Schematic representation of the input data and steps required for the simultaneous assessment of uptake and metabolism in hepatocytes by using a mechanistic two-compartment model. C₀ represents concentration at time 0.

Fig. 2.

A, two-compartment model (eqs. 3 and 4) describing the change in cell and media concentration of the parent drug over time because of active uptake (K_m,u and V_max), passive diffusion (P_diff,u), and intracellular binding (fu_cell) in a plated rat hepatocyte assay. S_cell and S_med,u represent the total cell and unbound media concentrations, respectively. B, extended mechanistic two-compartment model (eqs. 8–10) describing the interplay of active uptake (K_m,u and V_max), passive diffusion (P_diff,u), intracellular binding (fu_cell), and metabolism (CL_met1,u and CL_met2,u) in plated rat hepatocyte assay. S_Met1,cell and S_Met2,cell represent the total concentration of the metabolites in the cell. In the case of telmisartan, a single metabolite (met1) was taken into account for the modeling of its uptake and metabolism.

Fig. 3.

Formation of M1 (○), M2 (●), M4 (□), and repaglinide glucuronide (RPGG) (■) in plated rat hepatocytes over a range of concentrations (A) and at 100 μM in the presence (empty bar) or absence (filled bar) of 1 mM ABT (B). Metabolite concentrations were monitored in the cells over 2 min. M1, M2, and M4 formation rates are represented as mean ± S.D. of three experiments in the absence of ABT. All other data are results from a single experiment carried out in duplicate.

Fig. 4.

Representative kinetic profile of rosuvastatin uptake in plated rat hepatocytes at 10 concentrations (0.1–300 μM) over 2-min incubation (A) and 45-min incubation (B). Lines represent the predicted uptake profile based on a mechanistic two-compartmental model describing the changes in drug concentrations in both the cells and the incubation media over time (eqs. 3 and 4). Data points are mean of duplicate measurements.

Fig. 5.

Relationship between LogD_7.4 and P_diff,u (A) and fu_cell (B) in rat hepatocytes for seven OATP substrates. Estimates were obtained by using a two-compartment model describing the change of drug concentrations in both the cell and the incubation media over 2-min incubations for nonmetabolized drugs. Estimates for repaglinide and telmisartan were generated by using an extended two-compartment model incorporating metabolism. Data are represented as mean ± S.D. of three experiments with the exception of repaglinide (n = 1). 1, valsartan; 2, pravastatin; 3, rosuvastatin; 4, bosentan; 5, pitavastatin; 6, repaglinide; 7, telmisartan.

Fig. 6.

Representative uptake kinetic profiles after 2-min incubation for rosuvastatin and pitavastatin over a range of substrate concentrations based on the conventional two-step approach (eqs. 1 and 2) (A and C) or the mechanistic modeling approach (eq. 3) (B and D). In A and C, total and passive uptake rates were obtained from measurements at 37 and 4°C, respectively. Active uptake was expressed as the difference between total uptake and passive diffusion. In B and D, only measurements at 37°C were used to estimate uptake kinetics; active transport and passive diffusion were delineated by using the mechanistic model (eq. 3). ● and solid lines represent measured and predicted total uptake estimated from the kinetic parameters obtained with each approach, respectively. Dashed and dotted lines represent cellular uptake caused by active transport and passive diffusion, respectively. ○ represent measurements obtained at 4°C. Data points are mean of duplicate measurements.

Fig. 7.

Representative cellular concentrations of repaglinide (A), repaglinide glucuronide (B), and M2 (C) obtained when measured in plated rat hepatocytes at 10 concentrations (0.1–300 μM) over 45-min incubations without 1-aminobenzotriazole. Lines represent the predicted uptake profile based on the extended mechanistic two-compartmental model (Fig. 2B) describing the changes in drug and metabolite concentrations in both the cells and the incubation media over time. Data points are mean of duplicate measurements.