Chemicals and Reagents.

SB939 (the chemical structure is shown in Fig. 1; the dihydrochloride salt was used in all the in vitro and in vivo studies) and SB558 [(2E)-N-hydroxy-3-(1-methyl-2-phenethyl-1H-benzimidazol-5-yl)-acrylamide hydrochloride], the internal standard, were synthesized at S*BIO Pte Ltd. (Singapore). All other reagents were of research or analytical grade. Pooled human liver microsomes (HLM) (20 mg/ml in 250 mM sucrose), pooled dog liver microsomes (DLM) from male beagle dogs (20 mg/ml in 250 mM sucrose), pooled mouse liver microsomes (MLM) from male CD-1 mice (20 mg/ml in 250 mM sucrose), NADPH regeneration system solution A (25 mM NADP⁺, 66 mM glucose 6-phosphate, 66 mM MgCl₂ in H₂O), and NADPH regeneration system solution B (40 U/ml glucose-6-phosphate dehydrogenase in 5 mM sodium citrate) were from BD Gentest (Woburn, MA). Pooled rat liver microsomes (RLM) from Wistar rats (20 mg/ml in 250 mM phosphate buffer/20% glycerol) were prepared in house.

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Fig. 1.

Chemical structure of SB939 free base [(2E)-3-[2-butyl-1-[2-(diethylamino)ethyl]-1H-benzimidazol-5-yl]-N-hydroxyarylamide].

Determination of LogD of SB939.

LogD (logarithm of distribution coefficient) was determined by partitioning a solution of SB939 between a buffer and 1-octanol mixture at approximately 25°C using the shake-flask method. The pH of the buffers used was 2.0 (HCl with 50 mM KCl), 4.5, 6.5, 7.4, and 9.1 (50 mM, sodium phosphate buffers), and the final pH measured was 2.06, 3.35, 5.43, 6.77, 7.28, 7.40, and 8.94, respectively. The concentration of SB939 in both the layers was quantified by HPLC-UV. The logarithm of the ratio of concentration of SB939 in the octanol phase and the buffer was estimated (logD).

In Vitro Plasma Protein Binding.

The in vitro plasma protein binding (PPB) of SB939 was estimated in mouse, rat, dog, and human plasma by the equilibrium dialysis method. Equilibrium dialysis was performed in a Micro-equilibrium Dialyzer (Harvard Apparatus Inc., Holliston, MA), with the two chambers (0.25 ml volume) separated by an ultrathin membrane (mol. wt. cutoff, 5 kDa; B-size; Harvard Apparatus). One of the chambers was filled with 0.25 ml of plasma containing SB939 at a concentration of 1000 ng/ml, and the other was filled with 0.25 ml of phosphate-buffered saline. Dialysis was performed in a water bath set at 37°C for 4 h. After dialysis, 0.05 ml of the dialyzed phosphate-buffered saline samples (containing unbound SB939) was extracted with 1.5 ml of methyltertiarybutylether (MTBE) for 30 min by shaking. The samples were centrifuged at 15,700g for 10 min in a refrigerated microcentrifuge (4°C), after which 1.4 ml of the supernatant was transferred to fresh Eppendorf tubes and dried at 43°C for 35 min. The residues were reconstituted in 0.1 ml of mobile phase (methanol/MilliQ-water, 60:40) and analyzed by LC-MS/MS as described under Sample Analysis. A standard solution of SB939 (500 ng/ml) prepared in mobile phase was also analyzed. The percentage unbound was calculated as follows:

The percentage bound was estimated using the following equation: %bound = 100 − %unbound.

Blood-to-Plasma Ratio.

Two aliquots of fresh human whole blood of 400 μl each were incubated with SB939 (5 μM) in a water bath maintained at 37°C, with gentle shaking at 55 rpm, for 60 min. After incubation, 50 μl of whole blood from the first aliquot was transferred to fresh Eppendorf tubes. The second aliquot of the whole blood was centrifuged at 200g for 20 min at 4°C, and 50 μl of plasma was transferred to fresh Eppendorf tubes. The whole blood and plasma samples were extracted with 1.25 ml of MTBE by vortexing for 30 min, followed by centrifugation at 16,100g for 10 min at 4°C. The supernatants were dried under a gentle stream of nitrogen, and the concentrated samples were reconstituted in 50 μl of methanol/MilliQ water (7:3) and analyzed by LC-MS/MS. An HPLC system (Agilent Technologies, Santa Clara, CA) coupled with the 3200 Q TRAP LC-MS/MS system was used to analyze the samples. SB939 was separated on a Luna C18 column (50 × 2 mm, 5 μm; Phenomenex, Torrance, CA) using a gradient mode at a flow rate of 1.0 ml/min. The mobile phase consisted of 0.1% formic acid in MilliQ water (A) and 0.1% formic acid in methanol (B). The MS instrument was operated in a positive mode. The multiple reaction monitoring transition of SB939 was 359.1→100.1 (Q1/Q3) with a declustering potential of 30 V, entrance potential 10 V, and collision energy of 30 V. The curtain gas, ion-spray voltage, temperature, nebulizer gas (GS1), and auxiliary gas (GS2) were set at 15 psi, 5500 V, 600°C, 60 psi, and 65 psi, respectively, and the interface heater was on. The samples were processed in triplicate. The analyte peak areas were used to calculate the blood-to-plasma (B/P) ratio.

Caco-2 Bidirectional Permeability Assay.

Human colon carcinoma (Caco-2) cells (passage number 60) were seeded onto Transwell assay plates, and the assay was performed with 21- to 28-day confluent monolayer cells. The incubation medium used was Hanks' balanced salt solution, pH 7.4, and incubations were carried out in an atmosphere of 5% CO₂ with a relative humidity of 95% at 37°C. The compound solutions were made by diluting 10 mM SB939 in DMSO with Hanks' balanced salt solution to give a final concentration of 5 μM (final DMSO concentration <1%). The compound was placed in the apical side to assess permeability in the A→B (apical to basolateral) direction and in the basolateral side to assess permeability in the B→A (basolateral to apical) direction. Both the apical and basolateral sides were maintained at pH 7.4. After incubation for 2 h, the apical and basolateral sides were sampled for analysis. The experiment was performed in duplicate. The concentration of SB939 was determined by LC/MS using a four-point calibration curve. The permeability coefficient (P_app) was calculated from the following equation: P_app = [dQ/dt/C₀ × A], where dQ/dt is the rate of permeation of the compound across the cells, C₀ is the concentration in donor compartment at time 0, and A is the area of the cell monolayer. The efflux ratio (ER) was estimated using the following equation: ER = P_{app, B→A}/P_{app, A→B}. Atenolol (P_app < 0.5 × 10⁻⁶ cm/s), propranolol (15 < P_app < 25 × 10⁻⁶ cm/s), and digoxin (efflux ratio >3) were used as quality controls of the monolayer batch. The integrity of the monolayer was determined by measuring the pre-experiment transepithelial electrical resistance (between 450 and 650 Ω·cm²) and using Lucifer yellow (P_app ≤ 0.5 × 10⁻⁶ cm/s).

In Vitro Metabolic Stability in Liver Microsomes.

Reactions were carried out in 96-well plates (Greiner Bio-One, Longwood, FL). The reaction mix (72 μl), containing 50 mM potassium phosphate buffer, pH 7.4, the appropriate liver microsomes (0.87 mg/ml), and the NADPH regenerating system (1.3 mM NADP⁺, 3.3 mM glucose 6-phosphate, 3.3 mM MgCl₂, 0.4 U/ml glucose-6-phosphate dehydrogenase, 50 μM sodium citrate), was preincubated at 37°C for 10 min. Reactions (in triplicate) were initiated by the addition of compound solution (50 μM, 8 μl) to the incubation mixture (compound final concentration, 5 μM; microsomes, 0.78 mg/ml). At time points 5, 15, 30, 45, and 60 min, 50-μl aliquots of the reaction mixture were quenched with 100 μl of an ice-cold stop solution containing 20% DMSO/acetonitrile. The samples were centrifuged at 4°C for 15 min at 400g. The supernatant was transferred to a 96-well plate for analysis using a generic LC/MS method. Verapamil was used as the positive control to assess the activity of the microsomal fractions from different species. The reactions were monitored based on the disappearance of SB939 over time using LC/MS as described later. Data were analyzed using GraphPad Prism (version 4.0; GraphPad Software Inc., San Diego, CA). Log-linear plots of percentage compound remaining versus time were plotted, and the slope of the curve was calculated by linear regression of the log-linear curve. The half-life (t_1/2) of metabolic stability was estimated using the first-order equation t_1/2 = 0.693/K_el, where K_el (elimination rate constant) is the slope of the linear portion of the log-linear curve. The microsomal intrinsic clearance (CL'_int) was estimated using the equation CL'_int = (0.693/t_1/2) × (volume of incubation/mg microsomal protein).

In Vitro Metabolite Profiling Using Liver Microsomes.

The microsomal incubations were carried out in a total volume of 0.5 ml. The incubation mixture consisted of 100 mM potassium phosphate buffer, pH 7.4, microsomal protein (DLM and HLM at 1.0 mg/ml; MLM and RLM at 2.0 mg/ml), and SB939 (50.0 μM). The reaction was initiated by the addition of NADPH to a final concentration of 2.0 mM. After addition of NADPH, the tubes were immediately transferred and incubated in a water bath at 37°C with shaking. The incubations were carried out for 1 h. A blank microsomal sample containing only buffer, microsomes, and NADPH was used as a control. Reactions were terminated by mixing with 2.0 ml of chilled acetonitrile. All samples were centrifuged at 15,700g for 5 min at 4°C. The supernatants were transferred to fresh tubes and dried under a gentle stream of nitrogen. The dried samples were reconstituted in mobile phase and analyzed by LC-MS/MS as described below. EMS and EPI scan modes were used in identification of metabolites.

LC-MS/MS Analysis.

A HPLC system (1100 series; Agilent Technologies) containing an Agilent 1100 G1312A pump, a G1329A autosampler, and a G1316A column oven, coupled to a 3200 Q TRAP Mass Spectrometer (Applied Biosystems, Foster City, CA), was used for analysis. The MS/MS analysis was performed in positive mode on the 3200 Q TRAP LC-MS/MS system (Applied Biosystems) with a TurboIonSpray probe. The source temperature was set at 600°C, with declustering potential at 30 eV, entrance potential at 10 eV, collision energy at 30 V, curtain gas at 10 psi, ion spray energy at 5000 V, nebulizer gas (GS1) at 60 psi, and turbo gas (GS2) at 65 psi. The samples were resolved on a Zorbax-Eclipse XDB-C18 column (250 × 4.6 mm internal diameter; 5-μm particle size; Agilent Technologies), maintained at 25°C. The mobile phase consisted of A [0.1% formic acid in water, pH 3.4 (solvent A)] and B [acetonitrile (solvent B)] and was used in a gradient mode (100% A at 0 min, followed by 5% B at 3.1 min, 15% B at 20 min, and 100% B at 27 min; the mobile phase was brought to 100% A by 27.01 min and maintained for 30 min) at a flow rate of 1.0 ml/min.

In Vitro P450 Isoform Phenotyping Using Recombinant Human P450s.

Bactosomes (final P450 concentration of 100 pmol/ml CYP1A2, 25 pmol/ml CYP2C9, 100 pmol/ml CYP2C19, 50 pmol/ml CYP2D6, and 25 pmol/ml CYP3A4), 0.1 M phosphate buffer, pH7.4, and SB939 (final concentration, 5 μM; final DMSO concentration, 0.25%) were preincubated at 37°C before the addition of NADPH (final concentration, 1 mM) to initiate the reaction. Incubations were also performed using control Bactosomes (no P450 enzymes present) to reveal any nonenzymatic degradation. The final incubation volume was 25 μl. SB939 was incubated for 0, 5, 15, 30, and 45 min with each isoform. Ethoxycoumarin, dextromethorphan, diazepam, testosterone, and diclofenac were used as positive controls to assess the activity of CYP1A2, -2D6, -2C19, -3A4, and -2C9, respectively. The reactions were stopped by the addition of 50 μl of methanol containing internal standard at the appropriate time points. The incubation plates were centrifuged at 600g for 20 min at 4°C to precipitate the proteins. The supernatants were analyzed by LC-MS/MS. Log-linear plots of the peak area ratio (corrected for any loss in the incubations with the control Bactosomes) versus time were plotted, and the slope of the curve was calculated by linear regression of the log-linear curve. The half-life (t_1/2) of metabolic stability was estimated using the first-order equation t_1/2 = 0.693/K_el, where K_el (elimination rate constant) is the slope of the linear portion of the log-linear curve. The microsomal intrinsic clearance (CL'_int) was estimated using the equation CL'_int = (0.693/t_1/2) × (volume of incubation/pmol P450).

In Vitro P450 Inhibition Studies in HLM.

For each P450, the reaction mixture consisted of potassium phosphate buffer (100 mM, pH 7.4), HLM (0.25 mg/ml for CYP1A and CYP3A4, 0.5 mg/ml for CYP2C19 and CYP2D6, 1 mg/ml for CYP2C9), an appropriate probe substrate (i.e., 0.5 μM ethoxyresorufin for CYP1A, 120 μM tolbutamide for CYP2C9, 25 μM mephenytoin for CYP2C19, 5 μM dextromethorphan for CYP2D6, and 2.5 μM midazolam for CYP3A4), varying concentrations of the reference inhibitor (i.e., α-naphthoflavone for CYP1A, sulfaphenazole for CYP2C9, tranylcypromine for CYP2C19, quinidine for CYP2D6, and ketoconazole for CYP3A4) or SB939, and 1 mM NADPH in a total volume of 200 μl. The reactions were started by the addition of NADPH. The plates were incubated at 37°C for 5 min (CYP1A and CYP3A4), 60 min (CYP2C9 and CYP2C19), or 30 min (CYP2D6). For the CYP1A incubation, the reactions were terminated by the addition of methanol, and the formation of the metabolite resorufin was monitored by fluorescence (excitation wavelength, 535 nm; emission wavelength, 595 nm). For the CYP2C9, CYP2C19, CYP2D6, and CYP3A4 incubations, the reactions were terminated by the addition of methanol containing an internal standard. The samples were then centrifuged, and the supernatants were combined, for the simultaneous analysis of 4-hydroxytolbutamide (CYP2C9), 4-hydroxymephenytoin (CYP2C19), dextrophan (CYP2D6), 1-hydroxymidazolam (CYP3A4), and the internal standard by LC-MS/MS. The enzyme activity of each P450 was estimated based on the product (metabolite) appearance over time. The metabolites were quantified using calibration curves of corresponding authentic standard of the metabolite. Concentration response curves were plotted, and the IC₅₀ values were estimated using a sigmoid (nonlinear regression) model in GraphPad Prism.

In Vitro Human P450 Induction in Hepatocytes.

The freshly isolated hepatocytes were added to a 24-well plate so that the final seeding density was 0.15 × 10⁶ cells/cm² in Williams E medium. The cells were incubated at 37°C, 95% humidity, 5% CO₂ for 48 h before the addition of the test compound and control inducers. The control inducers were dexamethasone (50 μM) and rifampicin (10 μM) for CYP3A4 and omeprazole (50 μM) for CYP1A. Solutions of the control inducers (10 or 50 μM) and SB939 (0.1, 1.0, and 10 μM) were prepared in culture medium with a final DMSO concentration of 0.1%. Negative controls were included that contained 0.1% DMSO in culture medium. At the end of the 48-h culture period, the medium was removed; replaced with the prewarmed medium containing the control inducers (10 or 50 μM), SB939 (0.1, 1, and 10 μM), or negative controls (0.1% DMSO); and incubated for an additional 72 h. Medium was changed with fresh test compound or negative control every 24 h. The experiment was done in triplicate. At the end of the 72-h incubation, the medium was replaced with the appropriate probe substrates midazolam (20 μM) and ethoxyresorufin (20 μM) for CYP3A4 and CYP1A, respectively, and incubated for 30 and 60 min for midazolam and ethoxyresorufin, respectively. At the end of the incubation, the supernatant was removed from the plate and mixed with an equal volume of methanol (containing internal standard for CYP3A4 only). Standard curves of the metabolites 1-hydroxymidazolam (0.001–1 μM) and resorufin (0.001–2.5 μM) were prepared in culture medium. An aliquot of the standards was added to a plate that contained an equal volume of methanol (containing internal standard). The samples and standards were then centrifuged at ∼600g for 20 min at 4°C. The supernatant was analyzed using LC-MS/MS for CYP3A4 and fluorescence for CYP1A (excitation wavelength, 535 nm; emission wavelength, 595 nm). To determine whether the levels of metabolite formation were statistically significantly higher in the test compound samples compared with the appropriate negative controls, a one-way ANOVA with Dunnett's post test was performed. For the positive control compounds, an unpaired, one-tailed t test was used to determine whether the levels of metabolite formation were statistically significantly higher in the positive controls compared with the appropriate negative controls. In both cases, a p value of less than 0.05 was determined to be significant. To determine the significance of induction potential, the induction of CYP3A4 and 1A2 by SB939 at each concentration was expressed as a percentage of the fold induction of the corresponding positive controls.

Pharmacokinetic Studies.

All the animal studies were done as per approved protocols by the Institutional Animal Care and Use Committee at the Biological Resource Centre in Singapore.

Intravenous and Oral Pharmacokinetics in Mice.

Female BALB/c mice (age, 10–12 weeks; 18–20 g b.wt.) were used in the study. Food and water were given ad libitum. SB939 was administered intravenously, via the tail vein, at a dose of 10 mg/kg as a solution (2 mg/ml) in saline at a dose volume of 5 ml/kg. The oral dose, 50 mg/kg, was administered by gavage at a dose volume of 10 ml/kg in 0.5% methylcellulose and 0.1% Tween 80 at a concentration of 5 mg/ml. At defined time points (5 or 10 min, 30 min, and 1, 2, 4, 8, and 24 h after dose), groups of three mice per time point were sacrificed by an overdose of CO₂, and blood was collected by cardiac puncture and placed in tubes containing K₃EDTA as the anticoagulant. Plasma was obtained by centrifuging the blood samples at 800g for 10 min and storing at −60 to −80°C until analysis.

Intravenous and Oral Pharmacokinetics in Rats.

Male Wistar rats (age, 6–8 weeks; 220–335 g b.wt.) were used in the study. Three rats were used for the intravenous and oral studies each, in a parallel design. One day before the study, the jugular vein was cannulated in all the rats to be used. Rats were fasted overnight before dosing and fed 4 h after dose. Water was given ad libitum. SB939 (2 mg/kg) was administered intravenously as a bolus dose through the jugular vein, at a dose volume of 0.8 ml/kg (2.5 mg/ml) in saline. The oral dose (10 mg/kg) was administered by gavage at a dose volume of 4 ml/kg as a 2.5 mg/ml suspension in 0.5% methylcellulose and 0.1% Tween 80. From each rat, serial blood samples (∼0.2 ml per sample) were drawn at 5, 15, 30, and 45 min and at 1, 1.5, 2, 3, 4, 6, and 24 h after dose and placed in tubes containing K₃EDTA as the anticoagulant. Plasma was obtained by centrifuging the blood samples at 800g for 10 min and storing at −60 to −80°C until analysis.

Intravenous and Oral Pharmacokinetics in Dogs.

Six male beagle dogs (age, 1–2 years; 8–17 kg b.wt.) were used in the study. Three dogs were used in the intravenous and oral studies each, in a parallel design. The dogs were fasted overnight before dose and fed 4 h after dose in the oral study. Water was given ad libitum. The intravenous dose (2 mg/kg) was given as a bolus (0.5 ml/kg) in saline at a concentration of 4 mg/ml. The oral dose (10 mg/kg) was administered by gavage at a dose volume of 2.5 ml/kg as a suspension (4 mg/ml) in 0.5% methylcellulose and 0.1% Tween 80. At defined time points (before dose and 2, 5, 15, and 30 min and 1, 2, 4, 6, 8, and 24 h after dose), serial blood samples (∼1 ml) were collected from the foreleg vein via a butterfly catheter and placed into a chilled polypropylene tube with K₃EDTA as the anticoagulant. Samples were centrifuged at 4°C at a speed of 800g for 15 min, and plasma was harvested and stored at −60 to −80°C until analysis.

In Vivo Metabolism Study in Rats to Identify Glucoronidation Products.

Three male Wistar rats (age, 6–8 weeks; 220–280 g b.wt.) were housed individually in metabolic cages, designed to permit the separate collection of urine and feces, on a 12-h light cycle at 21 to 22°C and 40 to 60% humidity. Animals were supplied with water and a commercial diet ad libitum before the study initiation. SB939 was administered, orally by gavage, as a single dose of 100 mg/kg in 0.5% methylcellulose and 0.1% Tween 80 in water (10 ml/kg dose volume). Before administration, urine was collected for 24 h as a blank control. After dosing, urine was collected over a period of 24 h (in two sampling periods: 0–4 and 4–24 h).

Sample Analysis.

Pharmacokinetic Analysis.

Noncompartmental PK parameters such as the volume of distribution at steady state (V_ss), systemic clearance (CL), elimination half-life (t_1/2), area under the plasma concentration-time curve up to the last nonzero concentration (AUC_0-t), area under the plasma concentration-time curve from time 0 to infinity (AUC_0-∞), the time of maximum concentration in plasma (t_max), the maximum concentration in plasma (C_max), oral clearance (CL/F), and the apparent volume of distribution (V/F) were estimated using WinNonlin (version 4.0; Pharsight, Mountain View, CA). The terminal elimination rate constant was estimated from the terminal linear part of the log-linear plot (a minimum of three points on the linear phase). The linear trapezoidal method was used to compute the AUC. For rats and dogs, PK parameters were estimated in individual animals and averaged. For mice, the PK parameters were estimated using the mean concentration data. The absolute oral bioavailability, F (%), was calculated using the following equation:

Prediction of Human Pharmacokinetics Using Simcyp.

The Simcyp population-based ADME simulator is a software tool that simulates or predicts the pharmacokinetic profiles and parameters of a compound, based on its physicochemical and preclinical in vitro and/or in vivo ADME properties, in human populations (Jamei et al., 2009). Most importantly, Simcyp also predicts the variability (including the extremes) of the PK parameters and profiles in human populations using its extensive database of physiological parameters associated with different demographics, race, and disease populations, thereby enabling the performance of virtual clinical trials in thousands of patients (Jamei et al., 2009). Simcyp version 9.3 was used for predicting the PK parameters and profiles of SB939. The list of input parameters used in the simulations is shown in Table 1. The following assumptions were made: 1) the major plasma binding protein was α1 acid glycoprotein (SB939 is a weak base); 2) fraction unbound in enterocytes (f_g) = 1; and 3) metabolism was the main clearance mechanism. The fraction unbound in microsomes (f_u,mic) was calculated using the Turner method in Simcyp. The advanced dissolution, absorption, and metabolism model was selected for simulation of PK profiles. The formulation selected was immediate release (for a 10-mg capsule). The PK simulations were run using the physiologically based distribution model (method 2, because the basic pKa of SB939 was >7.0). For elimination, the recombinant enzyme option was selected (CYP3A4 and 1A2 were the main enzymes that metabolized SB939 in vitro)_. Simulations were performed as 10 trials in a healthy volunteer population (10 subjects in each trial; total subjects in population, 100). A single oral dose of 10 mg (the first time in man dose) was administered to the population, and the simulation was carried out for a period of 24 h. The terminal half-life was estimated from the predicted concentration time profiles.

Simulation of Drug-Drug Interaction Studies Using Simcyp.

On the basis of the results from the P450 isoform typing, inhibition, and induction studies with SB939, the following simulations were performed to predict the drug-drug interaction (DDI) potential for SB939: 1) PK of SB939 at 60 mg [the recommended therapeutic dose (Yong et al., 2011)]; 2) effect of ketoconazole on the oral PK of SB939: ketoconazole (inhibitor) was dosed at 400 mg q.d. for 4 days, and SB939 was administered as a single oral dose of 60 mg on the fourth day along with ketoconazole. The PK profiles and parameters of SB939 were predicted in the presence and absence of ketoconazole; 3) effect of rifampicin (inducer) on the PK of SB939: rifampicin was dosed at 600 mg q.d. for 5 days, followed by a single 60-mg dose of SB939 on the fifth day along with rifampicin. The PK profiles and parameters of SB939 were predicted in the presence and absence of rifampicin; 4) effect of SB939 on the PK of omeprazole: SB939 was dosed at 60 mg q.d. every other day for 1 week (three doses), followed by a single oral dose of omeprazole (dose, 20 mg) along with the last dose of SB939. The PK profiles and parameters of omeprazole were predicted with and without SB939. The input parameters for SB939 were the same as described in the previous section. The input files for ketoconazole, rifampicin, and omeprazole were from the Simcyp library. The doses and regimen for the DDI studies were chosen based on the Pharmaceutical Research and Manufacturers of America guidance (Bjornsson et al., 2003). Simulations were performed as 10 trials in a healthy volunteer population (10 subjects in each trial; total subjects in population, 100). The AUC ratio, defined as the ratio of AUC of substrate in the presence of the inhibitor/inducer to the AUC of substrate alone, was used to assess the magnitude of DDI (Bjornsson et al., 2003).

Allometric Scaling.

Allometric analysis was performed by linear regression of log-transformed values of the PK parameters (CL/F, V/F, CL, and V_ss) against log-transformed body weights (Boxenbaum, 1982). Mean values for PK and body weights were used for mice, and individual values were used for rats, dogs, and humans. The allometric exponent (slope) and coefficient (intercept) and the regression coefficient (r²) were estimated for each PK parameter. The 95% confidence intervals were estimated for the exponents and the intercepts. The departures from linearity were evaluated at 95% confidence levels.