Protein Binding.

S 55746 (Fig. 1) was provided by Servier Laboratories. The unbound fraction in plasma (f_u,p) of [14C]-S 55746 was determined in animals and humans by dialysis using the Rapid Equilibrium Dialysis Device System (Thermo Fisher Scientific, Waltham, MA) at concentrations of 500, 5000, and 50,000 ng/ml for all species. Aliquots of spiked plasma were placed into the sample chamber, and samples of nonspiked dialysis buffer were added to the buffer chamber. The plates were covered and incubated at 37°C at 500 rpm on an orbital shaker (for 1, 3, 4, and 5 hours). All incubations were performed in triplicate and analyzed by liquid scintillation counting.

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Fig. 1.

Chemical structure of S 55746.

Blood-to-Plasma Partition.

The blood-to-plasma concentration ratio (R_bl:p) was determined in triplicate per concentration for all species. Prior to spiking blood, a small aliquot of blood was taken for the determination of the hematocrit. Spiked blood samples were incubated on a roller mixer for 1 hour at 37°C and then centrifuged at room temperature. Finally, plasma was transferred into appropriate tubes to be analyzed by liquid scintillation counting to allow the determination of drug concentration. The R_bl:p was calculated using the following equation:(1)where C_bl and C_p are, respectively, the drug concentration in blood and plasma.

Microsome and Hepatocyte Clearance Determination.

Microsomal incubations were carried out for different times (0, 7, 17, 30, and 60 minutes) at 37°C with human liver microsomes (HLMs) with S 55746 at a final concentration of 0.1 and 40 μM. Reactions were initiated by the addition of NADPH after a 10-minute preincubation time. During incubation, aliquots were sampled at each incubation time and the enzymatic reaction was stopped by protein precipitation using methanol.

For hepatocytes, S 55746 was incubated for different time points (0, 5, 10, 20, 30, 60, and 100 minutes) in William’s EC Medium (Thermo Fisher Scientific). Reactions were initiated by the addition of S 55746. During incubation, aliquots were sampled at each incubation time, and the enzymatic reaction was stopped by protein precipitation using acetonitrile.

Samples were then centrifuged and the supernatants transferred to analysis plates, followed by analysis using liquid chromatography coupled with mass spectrometry in tandem (LC-MS/MS).

The K_m and V_m parameters of S 55746 in HLMs and in hepatocytes were determined via substrate disappearance kinetics. Since the enzyme activity decreases with time, V_m can be described by:(2)where V_m0 is the initial V_m and k_d is the inactivation constant for the degradation of enzyme activity.

The model used to describe the disappearance of the substrate (S) was therefore:(3)The data were analyzed with the nonlinear analysis software WinNonlin version 5.1 (Pharsight Corporation). The K_m and V_m parameters were estimated with the inclusion of microsomal binding data.

The determination of the intrinsic clearance was calculated using the following formulas according to microsome or hepatocyte experiments:(4)(5)where C_u is the unbound concentration, CL_int,vivo is the intrinsic clearance for the whole organ, CL_{int,vitro,mic} is the intrinsic clearance determined with the microsomal system, and MPPGL represents the microsomal protein per gram of liver of the relevant species, and:(6)where CL_{int,vitro,hep} is the intrinsic clearance measured by the hepatocyte system and HPGL represents the number of hepatocytes per gram of liver.

Identification of Enzymes Involved in Human Hepatic Metabolism.

Cytochrome contribution was determined via clearance experiments with S 55746 and recombinant human cytochromes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. S 55746 was incubated with each individual recombinant human cytochrome for 0, 7, 17, 30, or 60 minutes.

The relative contribution of each cytochrome enzyme to the oxidative part of the microsomal clearance of S 55746 was estimated by comparing the CL_int of each individual recombinant human cytochrome (CL_int, rhCYP) to the sum of individual recombinant human cytochrome CL_int (∑CL_int, rhCYP):(7)A relative activity factor (RAF) was estimated from the ratio of the CL_int values for cytochrome-specific probe substrates using recombinant human cytochrome and using HLMs using the following equation:(8)The concentration of bactosomes to be used in the incubation was calculated from the concentration of HLMs used as the standard for intrinsic clearance measurement, using the following equation:(9)where [rhCYP] is the bactosomal protein concentration and [HLM] is the microsomal protein concentration.

Supernatants were analyzed using a TurboFlow LC-MS/MS Platinum Mass Spectrometer (Thermo Fisher Scientific) to assay S 55746 concentrations. Orthogonal assays using microsomes and CYP inhibitors were conducted to confirm the results.

Assessment of Mechanism-Based Inhibition in HLMs.

The possible existence of mechanism-based inhibition (Obach et al., 2007) of CYP3A4 was also investigated. HLMs were prewarmed with S 55746 (0, 0.25, 0.5, 1, 2, 3, 10, and 30 μM) for 5 minutes at 37°C. The preincubation was initiated by the addition of NADPH or buffer, and at different preincubation times (0, 2, 4, 6, 8, 12, and 14 minutes) an aliquot of the preincubate was collected and added to a vial containing midazolam (Sekiguchi et al., 2011). After an incubation of 7 minutes, reactions were terminated by the addition of methanol. Samples were placed on ice before centrifugation. Supernatants were analyzed using LC-MS/MS for the measurement of hydroxymidazolam concentration. Calculations and regression analysis were performed using Excel 2003 (Microsoft Corporation, Redmond, WA) and Xlfit4 (IDBS UK, Guildford, UK).

A value for k_inact (maximum inactivation rate constant) and K_app (concentration that produces the half-maximal rate of inactivation) was determined by nonlinear regression analysis. The data were fitted to the following equation:(10)where k_obs is the slope observed when representing the natural logarithm of the percentage of remaining activity against the preincubation time and [I] is the inhibitor concentration.

Induction.

Isolated human hepatocytes cultured on type I collagen plates were used to assess the induction potential of S 55746. For 3 days, these human hepatocytes were treated with S 55746 at 0.1, 1, 2.5, 5, or 10 μM omeprazole (a known inducer of human CYP1A), phenobarbital (a known inducer of human CYP2B6), or rifampicin (a known inducer of human CYP3A). After 3 days, the hepatocytes were incubated with the cytochrome P450 substrates phenacetin (CYP1A2), bupropion (CYP2B6), and midazolam (CYP3A4) for the assessment of enzyme activity.

After the incubation, hepatocytes were harvested to isolate RNA, analyzed by quantitative reverse-transcription polymerase chain reaction. The obtained activities were compared with those of control cultures not exposed to the test compounds or to the reference cytochrome inducers. The results were expressed as fold induction compared with the control conditions or as a percentage of the reference inducer cytochrome activity or mRNA expression increase. When a concentration-dependent increase of cytochrome mRNA was observed and a maximum fold change was reached during the experiments, the data (fold induction) were processed for the determination of induction parameters (maximal induction, maximal fold induction, and the concentration at which there is a 50% maximal induction effect) by nonlinear regression fitting of the data.

Apparent Permeability Determination.

The Caco-2 cell line (Hilgers et al., 1990) was cultured in Dulbecco’s modified Eagle’s medium containing 10% (v/v) of fetal calf serum, 1% l-glutamine, nonessential amino acids, antibiotics, and amphotericin. Cells were seeded at 90,000 cells/cm² onto a microporous membrane of 24-multiwell insert plates so that apical (A) and basolateral (B) media would be separated by the cell monolayer. Cells were maintained at 37°C in an atmosphere of 95% air and 5% CO₂ and used between 21 and 30 days postseeding. S 55746 was incubated at 10 µM in the apical chamber. Samples (duplicate) were collected at 30 and 90 minutes in the basolateral chamber and analyzed by LC-MS/MS. Reference compounds for apparent permeability P_{app A-B} (cimetidine, propranolol) were used to determine the calibration curve of the Caco-2 model.

Table 1 shows the in vitro and in silico parameters for all species.

Distribution.

In the full PBPK distribution model, we assumed that the drug distributes instantaneously and homogeneously within each tissue and that its uptake in each tissue is limited by the blood flow (perfusion limited). The rate of change of the drug in a noneliminating tissue is described by the following differential equation (Nestorov, 2003; Jones et al., 2009):(11)where Q_T is the tissue blood flow and V_T is the volume of the tissue. The subscript T denotes the tissue, the subscript a represents the artery, bl denotes the blood, and v denotes the vein. C_a,bl and C_v,bl,T refer to the concentration in the arterial blood entering the tissue and the concentration in the venous blood leaving the tissue, respectively, and Kp_T:bl is the partition coefficient between the tissue and the blood.

It has been considered that the drug might be eliminated only from liver where the rate of change of the drug can be expressed by:(12)where the subscript h denotes the liver, CL_int,vivo denotes the intrinsic in vivo clearance, and C_u,h denotes the C_u in the liver.

The equations of Poulin and Theil (2002a,b), modified by Berezhkovskiy (2004), and the equations of Rodgers et al. (2005a,b) and Rodgers and Rowland (2006, 2007) were compared with predicted Kp_T:p values from physicochemical parameters and the resulting volume of distribution in plasma:

(13)

Clearance.

The well-stirred model (Rowland et al., 1973; Pang and Rowland, 1977) was used to determine hepatic clearance CL_h,bl from the intrinsic clearance CL_int, the hepatic blood flow Q_h, and the unbound fraction in blood f_u,bl:(14)where f_u,bl was calculated by dividing the f_u,p by the blood-to-plasma ratio.

Oral Absorption.

The oral absorption in humans was predicted using the ADAM (advanced dissolution, absorption, and metabolism) model (Jamei et al., 2009b; Patel et al., 2014) implemented in Simcyp. The ADAM model is a multicompartmental gastrointestinal transit model. The gastrointestinal tract is treated as a one-stomach, seven-small-intestine, and one-colon compartment, and the drug can exist in several states simultaneously: unreleased, undissolved, dissolved, or degraded. In this model, data from the P_app experiment are used to predict the effective permeability across the enterocyte (Sun et al., 2002), and the integration of the dissolution profile of the formulation can be used to determine the fraction of the pharmaceutically active compound released from the formulation over time.

Strategy for Interspecies Qualification.

Before performing PBPK simulations in humans, a qualification exercise was performed on different preclinical species. For this qualification with rat and dog PBPK models, the mouse physiologic model parameters were replaced with the corresponding values for rat and dog, and the drug-specific parameters measured in mouse-specific in vitro systems (e.g., CL_int, f_u,p) were replaced with the corresponding values measured in rat and dog in vitro systems. Simulations were carried out based on the dose and administration schedule in the in vivo studies, and the rat and dog PBPK model predictions were evaluated for their goodness of prediction based on the model evaluation criteria described below (see Prediction Performances and Parameter Analyses). Intravenous PK data from two preclinical species, rat and dog, were available to qualify the PBPK approach and to validate interspecies extrapolation.

Mouse Studies.

SCID mice (female) were treated with S 55746 by intravenous bolus of 1, 3, 10, or 25 mg/kg. Blood samples (from the tail or saphenous vein) between 0.083 and 30 hours after administering a dose were collected in two to five mice per time point.

Rat Studies.

PK studies in Wistar rat (male) were conducted after an intravenous infusion administered at the following doses: 0.5, 1.5, or 5 mg/kg. Blood samples (from the tail vein) were collected from 0.17 to 24 hours after administration. Three animals were used for each dose level.

Dog Studies.

In PK studies in Beagle dog (male), S 55746 was given after intravenous administration of a single bolus. Doses of 0.2 and 1.5 mg/kg were administered to dogs. S 55746 was quantified from 0.17 to 24 hours after administration. Data from three dogs were available for each dose.

Noncompartmental Analysis.

For in vivo studies, the S 55746 plasma concentration-time profiles were analyzed by noncompartmental analysis, and the area under the curve (AUC) was calculated using the log-linear trapezoidal rule.

Assessment of Prediction Accuracy.

The accuracy of the PBPK prediction was evaluated on AUC and plasma concentrations. The goodness of prediction in each preclinical species was evaluated based on the average fold error (AFE) and on the root mean square error (RMSE) (Sheiner and Beal, 1981). The fold error, AFE, and RMSE were calculated as follows:(15)(16)(17)Where PRED is the prediction, OBS is the observation, and n is the number of samples.

To account for the interindividual variability existing in clinical data, prediction performances were assessed using the 95th confidence interval of the simulated AUC and C_max (Abduljalil et al., 2014).