Chemicals.

Bosentan, pitavastatin, pravastatin, rosuvastatin, telmisartan, and valsartan were purchased from Sequoia Research Products (Pangbourne, UK). Telmisartan acyl-β-d-glucuronide, 2-despiperidyl-2-amino repaglinide (M1), 2-despiperidyl-2(5-carboxypentylamine) repaglinide (M2), 3′-hydroxy repaglinide (M4), and repaglinide acyl-β-d-glucuronide were obtained from Toronto Research Chemicals Inc. (North York, ON, Canada). Insulin (4 mg/ml) was purchased from Invitrogen (Paisley, UK). Repaglinide, mibefradil, verapamil, and indomethacin were purchased from Sigma-Aldrich (Poole, UK). All other chemicals and reagents were obtained from Sigma-Aldrich and were of the highest grade available.

Isolation of the Rat Hepatocytes.

Rat hepatocytes were isolated from 250 to 300 g Sprague-Dawley rat livers (Charles River, Margate, Kent, UK), following the two-step collagenase perfusion method described previously (Berry and Friend, 1969). Hepatocytes were suspended in William's medium E supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 0.01% insulin at pH 7.4. Cells were counted under a microscope using a hemocytometer, and viability was assessed by using the trypan blue exclusion method. Only preparations with a viability >80% were used. Cell monolayers were checked before each experiment to ensure coverage of >80% of the well surface. The cell suspension was diluted to 400,000 cells/ml in supplemented William's medium E. Hepatocytes were plated in 24-well collagen I-coated plates (BD Biosciences, Oxford, UK) at a density of 240,000 viable cells per well. Plates were incubated for 2 h at 37°C in an atmosphere containing 5% CO₂ to allow adhesion to the collagen.

Measurement of Uptake in Rat Hepatocytes.

Uptake was measured over a range of 10 concentrations between 0.1 and 300 μM (0.1, 0.3, 1, 3, 10, 20, 30, 60, 100, and 300 μM). The maximum concentration used for telmisartan was 100 μM, because of its limited solubility in aqueous buffer. This wide range was chosen because the affinity of the seven drugs of interest toward uptake transporters in rat hepatocytes was mostly unknown (Ishigami et al., 1995; Nezasa et al., 2003; Poirier et al., 2009). The medium was removed after plating. Monolayers were rinsed twice with prewarmed serum-free Dulbecco's phosphate-buffered saline (DPBS). Substrate was dissolved in dimethyl sulfoxide (DMSO) and diluted in DPBS (maximum 1% DMSO). Incubation was started by the addition of 400 μl of substrate on top of the monolayers. After incubation for 30, 60, 90, or 120 s at 37°C, the substrate was collected for analysis. Cells were washed three times with ice-cold DPBS. Two hundred microliters of water was added to each monolayer. Samples were stored at −20°C until analysis. In each experiment, incubations were carried out in duplicate at 37°C. For each drug, experiments were performed in rat hepatocytes prepared from three separate isolations. Incubations were also carried out at 4°C to assess the passive diffusion of the substrates through the cellular membrane. At 4°C, a single well was used per time point. In preliminary studies, the use of a cocktail of OATP inhibitors (10 μM cyclosporine and 20 μM rifampicin) was investigated to assess passive permeation at 37°C in the absence of active uptake. However, substrate-dependent inhibition was observed (data not shown); therefore, the use of control incubations at 4°C was selected as an initial measurement of passive permeation. Incubations were also extended to 45–90 min to reach saturation of the active uptake. In that case, four additional time points were added between 2 and 45–90 min for at least five concentrations. To inhibit phase I metabolism of repaglinide and bosentan, a nonspecific cytochrome P450 pan-inhibitor 1-aminobenzotriazole (ABT) was added to the incubations at a concentration of 1 mM (Mico et al., 1988; Yabe et al., 2011). Previous studies in human embryonic kidney-293 cells expressing OATP1B1 have shown that ABT has no effect on the activity of uptake transporters (Plise et al., 2010). In the current study, uptake of 1 μM rosuvastatin was measured over 2 min as a control of the uptake activity of each hepatocyte preparation. The mean uptake of the 1 μM rosuvastatin control across all experiments performed in this study (n = 22) was 54.8 ± 19.5 μl/min/10⁶ cells.

Depletion Assay.

As part of a preliminary analysis, depletion of 0.1 μM repaglinide and telmisartan was measured in rat hepatocytes plated as described above; the medium was removed after plating. Monolayers were rinsed twice with prewarmed serum-free DPBS. Substrate was dissolved in DMSO and diluted in DPBS (maximum 1% DMSO). Incubation was started by the addition of 400 μl of substrate on top of the monolayers. Reactions were stopped after 0.5, 1, 2, 5, 10, 30, 60, and 90 min for telmisartan and 0.5, 1, 2, 5, 10, 30, and 45 min for repaglinide, by the addition of 400 μl of ice-cold methanol containing 1 μM of the appropriate internal standard. Samples were stored at −20°C until analysis.

Sample Preparation and LC-MS/MS Analysis.

Cell lysates and substrate samples from the uptake experiments were thawed and quenched with an equal volume of methanol containing 1 μM of internal standard. Samples were placed for at least 1 h at −20°C before being centrifuged for 10 min at 6720g. Likewise, samples generated in the depletion assay were centrifuged in a similar manner. In both cases, 20 μl of supernatant was analyzed by LC-MS/MS as described below. All samples were analyzed on either a Waters 2795 HPLC system coupled with a Micromass Quattro Ultima mass spectrometer (Waters, Milford, MA) or a Waters 2695 HPLC system coupled with a Micromass Quattro Micro mass spectrometer. The analyte and associated internal standard were separated on a Luna C18 column (3 μm, 50 × 4.6 mm) (Phenomenex, Torrance, CA). The flow through the HPLC system was 1 ml/min and split to 0.25 ml/min before entering the mass spectrometer. All analytes were ionized by positive electrospray. LC-MS/MS analysis of each analyte is described in detail in Table 1. Samples were quantified against a standard curve, and only standards within 30% of the nominal concentration were included in the standard curve. In addition to telmisartan, the appearance of telmisartan glucuronide was monitored for modeling purposes. In the case of repaglinide, M1, M2, M4, and repaglinide glucuronide were also monitored in the cell.

Determination of Unbound Fraction in the Incubation Media.

The y intercept (t₀) of the linear regression of the media concentration over time plot was calculated at each incubation concentration. The media were assumed to be free of proteins because all hepatocytes were attached to the collagen support and the cell monolayers were rinsed thoroughly before each incubation as stated above. Therefore, fu_med was expressed as the slope of the linear regression of the unbound concentration extrapolated at t₀ versus the initial incubation concentration plot. A representative example of the fu_med estimation is illustrated for pitavastatin in Supplemental Fig. S1.

Initial Determination of Uptake Kinetic Parameters using a Conventional Two-Step Approach.

Uptake rate was calculated over 2 min at 37 and 4°C and expressed as the slope of the linear regression of the cell concentrations versus time plot. Passive diffusion (P_diff) was calculated from the uptake rates measured at 4°C as shown in eq. 1. P_diff was then inserted in eq. 2 as a constant, and the active uptake kinetic parameters were estimated by using Grafit version 6 (Erithacus Software Ltd, Horley, Surrey, UK). where v is the uptake rate; V_max is the maximum uptake rate; K_m is the affinity constant; P_diff is the passive diffusion clearance, and S is the substrate concentration. All kinetic parameters were corrected for nonspecific binding of the substrate to the incubation environment (fu_med). CL_active,u the unbound active uptake clearance, was expressed as the ratio of V_max over the unbound K_m (K_m,u). The total unbound uptake clearance (CL_uptake,u) included both the active component (CL_active,u) and clearance via passive diffusion (P_diff,u).

Determination of Uptake Kinetic Parameters Using a Mechanistic Modeling Approach.

A two-compartment model, based on the work by Poirier et al. (2008), was implemented in Matlab version 7.10 (2010) (The MathWorks, Inc., Natick, MA). Unlike the conventional two-step approach, this model allows simultaneous fitting of all concentration-time points during the experiment and relies only on measurements made at 37°C. It allows the assessment of multiple processes, namely active uptake of drugs into the hepatocytes, bidirectional passive diffusion, and intracellular binding. The uptake experiments were extended up to 45–90 min to reach steady state between concentrations in the media and intracellular compartment; analysis of uptake profiles after short and extended incubation times was performed. The scheme of steps taken to implement this model is presented in Fig. 1. The mechanistic model for the assessment of active uptake in hepatocytes is illustrated in Fig. 2A. Differential eqs. 3 and 4 show the change in cell and media concentrations over time, respectively. All parameters are expressed per well and therefore are normalized for the number of cells per well. where S_cell is the total cell concentration and S_med,u is the unbound media concentration expressed as nanomolar.

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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.

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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.

fu_cell as a parameter reflects both nonspecific intracellular binding and active processes for transport. Hence, there is an advantage of the modeling in comparison with the traditional binding assays that rely on the use of dead cells (i.e., no active transport present), providing an estimate of the nonspecific cellular binding in separation to the active process.

An intracellular volume (V_cell) in each well of 3.9 μl/10⁶ cells was used for rat hepatocytes (Reinoso et al., 2001), and the volume of media (V_med) was 400 μl and expressed in liters. V_max is expressed in nanomoles/minutes, K_m,u is expressed in nanomoles, and P_diff,u is expressed in liters/minute. CL_active,u and CL_uptake,u were calculated as described above. Considering the saturable nature of the active transport with increasing substrate concentrations, the maximal contribution of the active process to the total uptake was estimated and expressed as the ratio of CL_active,u over CL_uptake,u.

The nominal concentrations corrected for fu_med were used as initial media concentrations. Initial cell concentrations were obtained by extrapolating the first four time points for each of the concentrations to time 0. The rationale was that not all of the drug could be washed from the cell membranes or experimental plates with DPBS during the washing steps. However, because of the differences in volumes between the cell and media compartments, the largest amount of drug found in the cells at time 0 represented <2% of the total amount of compound present in the incubation.

The solving of the rate equations was performed in Matlab version 7.10 (2010) using the ODE45 solver. Values for K_m, V_max, and P_diff obtained from the initial analysis based on the conventional two-step approach were used as a priori information in the model because of availability of the data. However, the mechanistic model could still converge to a low objective function even when uninformed priors were used, due to the richness of the data in the experimental set. A value of 0.1 was used as a priori information for fu_cell for all drugs. In a first instance, the model was allowed to optimize the four parameters with open boundaries; the greatest uncertainty was associated with the determination of fu_cell. Therefore, secondary optimization was performed by using results from the initial optimization with set boundaries, as follows: K_m,u ± 20%, V_max ± 50%, P_diff,u ± 100%, and fu_cell between 0 and 1. These boundaries were used as estimates of K_m,u and V_max were associated with low standard errors after the first round of optimization. Boundaries were greater for V_max than K_m,u to allow P_diff,u to be optimized, because these two parameters are closely linked. The use of cell and media data, individually and in combination, was investigated. However, because of the small amount of drug being taken up into the cells relative to the total amount of drug present in the incubation, depletion of parent drug from the media caused by active uptake was lower than could be accurately quantified. The use of the media concentrations as an input into the model did not improve confidence in the kinetic parameters, resulting in increased standard errors associated with the parameter estimates (data not shown). Therefore, kinetic estimates in rat hepatocytes were based on the measurements of the cell concentrations alone. Nevertheless, a minimum of 64 data points were available to estimate K_m,u, V_max, P_diff,u, and fu_cell from the rat hepatocyte experiments carried out over 2 min. This number was increased to up to 160 when experiments were extended to 45–90 min.

Unlike most studies published so far (Soars et al., 2007; Watanabe et al., 2009; Yabe et al., 2011), the model presented here takes into account the efflux of the drug from the cell into the media via passive diffusion. Comparable with previous reports, efflux caused by active transport was not included in the model, because the rat hepatocytes were plated over a short period of time, during which repolarization of the cells was limited (Hewitt et al., 2007). Internalization of efflux transporters was assumed as reported previously (Bow et al., 2008), where efflux transporters P-glycoprotein and multidrug resistance protein 2 were absent from the canalicular membrane. In addition, these transporters were not colocalized on the basolateral membrane alongside Oatp1b1. In the present study, repaglinide and telmisartan metabolites were not found in the media samples, and mass balance of the system was verified. These arguments led us to the conclusion that efflux transporters were unlikely to have a significant effect on the experimental system used here.

Determination of Uptake Kinetic Parameters and Metabolic Clearance of Telmisartan using a Mechanistic Modeling Approach.

In humans, telmisartan is transformed to a single glucuronide metabolite (Stangier et al., 2000). Because of a lack of nonspecific UDP-glucuronosyltransferase inhibitor, changes of both parent drug and glucuronide metabolite concentrations in the cell and media were monitored during the uptake experiment. To estimate the uptake kinetic parameters and the metabolic clearance of this drug simultaneously, a cell subcompartment was added in the two-compartment model, as illustrated in Fig. 2B. Changes in parent drug cell and media concentrations are shown by differential eqs. 5 and 6, respectively, whereas changes in metabolite cell concentrations over time are defined by eq. 7. where CL_met,u is the unbound metabolic clearance expressed in liters/minute. Modeling was performed in a stepwise manner as described previously (Fig. 1). CL_met obtained in the depletion assay (2.2 μl/min/10⁶ cells) was used as a priori information in this model. Only measurements made in the cell compartment (parent and metabolite) were used in the analysis; 128 data points were available to estimate K_m,u, V_max, P_diff,u, fu_cell, and CL_met,u. Initial media and cell concentrations were measured as described in the previous section. Initial metabolite cell concentrations were obtained by extrapolating concentrations obtained for the first four time points to time 0. Initial metabolite concentrations were not null, but the amount found in the cell at time 0 represented <0.01% of the total amount of drug present in the incubation. Linearity of telmisartan glucuronide formation over the length of the incubations (90 min) was confirmed. Telmisartan glucuronide was also monitored in the media, but concentrations were found to be below the limit of quantification.

Determination of Uptake Kinetic Parameters and Metabolic Clearance of Repaglinide using a Mechanistic Modeling Approach.

Repaglinide is a CYP2C8 and CYP3A4 substrate, and to date six phase I and one phase II metabolites have been identified (Bidstrup et al., 2003; Gan et al., 2010). To identify the major metabolites generated in rat hepatocytes, formation of M1, M2, M4, and repaglinide glucuronide was monitored in the cells during uptake studies. M2 and repaglinide glucuronide were identified as the two major metabolites in rat hepatocytes (Fig. 3A) and considered for subsequent modeling purposes. Two cell subcompartments were added to the mechanistic two-compartment model to simultaneously describe uptake, passive diffusion, and formation of these two metabolites. Equation 8 defines the changes in repaglinide cell concentrations, whereas changes in media concentrations are as described in the case of telmisartan (eq. 6). Repaglinide glucuronide and M2 cell concentrations over time are shown by eqs. 9 and 10, respectively. where S_met,gluc is repaglinide glucuronide cell concentrations; S_met,M2 is the M2 cell concentrations; CL_met,gluc,u is the unbound metabolic clearance caused by the formation of repaglinide glucuronide; and CL_met,M2,u is the unbound metabolic clearance caused by the formation of M2. Initial media, cell, and metabolite concentrations were calculated as explained previously. This model was tested with three different experimental scenarios to identify its limitations.

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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.

Uptake of repaglinide and formation of repaglinide glucuronide were estimated from experiments conducted in the presence of ABT (scenario 1). Then, CL_met,gluc,u and CL_met,M2,u were estimated simultaneously from an incubation carried out without ABT, where both metabolites were monitored in addition to repaglinide (scenario 2). Finally, CL_met,gluc,u and CL_met,M2,u were replaced in eqs. 9 and 10 by Michaelis-Menten kinetic parameters (K_m,M2,u and V_max,M2 and K_m,gluc,u and V_max,gluc, respectively) to describe the potential saturation of the metabolic activity (scenario 3). CL_met obtained in the depletion assay was used as a priori information in this model. In all cases, only measurements made in the cell compartments (parent and metabolites) were used in the analysis. Formation of all metabolites was linear over the length of the incubation (45 min). Depending on the scenario investigated, between 240 and 360 data points were available to estimate K_m,u, V_max, P_diff,u, fu_cell, CL_met,gluc,u, and CL_met,M2,u. Any differences in parameter estimates and associated error depending on whether uptake and metabolic processes were considered in isolation or combined were investigated.

Identifiability of the Kinetic Parameters Estimated from the Various Models.

Identifiability of the kinetic parameters was verified by using Differential Algebra for Identifiability of Systems (DAISY) software (Bellu et al., 2007). Estimates from all of the models described previously were found to be only locally identifiable, when only observations made in the cell compartment were used. However, one of the two potential sets of kinetic estimates was predicted to always contain a nonsensical negative K_m,u value. By setting boundaries to ensure only positive values of the kinetic parameters were taken into account, a unique set of estimates could be defined.

Statistical Analysis.

Standard error associated with each parameter estimate was computed in Matlab version 7.10 (2010) by using a Jacobian approach reported previously (Landaw and DiStefano, 1984). Coefficients of variation (CVs) for each estimate were subsequently calculated and expressed as percentages to assess the quality of the parameter estimates. The arithmetic mean, standard error, and CV were calculated from experiments conducted using rat hepatocytes isolated on three different occasions. Goodness of fit was assessed by visual inspection of the data and minimum objective function value. To quantify the improvement of the fitting when metabolism was added to the model, the rms error (rmse) and the geometric mean fold error (gmfe) were calculated by using eqs. 11 and 12 for results obtained with telmisartan when incubations were performed over 2 min and for repaglinide when incubations were carried out over 45 min.

Collation of LogD_7.4.

LogD_7.4 values determined experimentally were collated from the literature for each drug investigated. Values used in this study and corresponding references are presented in Supplemental Table S1.