The preclinical absorption, distribution, metabolism, and excretion (ADME) properties of Pracinostat [(2E)-3-[2-butyl-1-[2-(diethylamino) ethyl]-1H-benzimidazol-5-yl]-N-hydroxyarylamide hydrochloride; SB939], an orally active histone deacetylase inhibitor, were characterized and its human pharmacokinetics (PK) was predicted using Simcyp and allometric scaling. SB939 showed high aqueous solubility with high Caco-2 permeability. Metabolic stability was relatively higher in dog and human liver microsomes than in mouse and rat. The major metabolites formed in human liver microsomes were also observed in preclinical species. Human cytochrome P450 (P450) phenotyping showed that SB939 was primarily metabolized by CYP3A4 and CYP1A2. SB939 did not significantly inhibit human CYP3A4, 1A2, 2D6, and 2C9 (>25 μM) but inhibited 2C19 (IC50 = 5.8 μM). No significant induction of human CYP3A4 and 1A2 was observed in hepatocytes. Plasma protein binding in mouse, rat, dog, and human ranged between ∼84 and 94%. The blood-to-plasma ratio was ∼1.0 in human blood. SB939 showed high systemic clearance (relative to liver blood flow) of 9.2, 4.5, and 1.5 l · h−1 · kg−1 and high volume of distribution at steady state (>0.6 l/kg) of 3.5, 1.7, and 4.2 l/kg in mouse, rat, and dog, respectively. The oral bioavailability was 34, 65, and ∼3% in mice, dogs, and rats, respectively. The predicted oral PK profile and parameters of SB939, using Simcyp and allometric scaling, were in good agreement with observed data in humans. Simcyp predictions showed lack of CYP3A4 and 2C19 drug-drug interaction potential for SB939. In summary, the preclinical ADME of SB939 supported its preclinical and clinical development as an oral drug candidate.
The clinical progress that has been made by hydroxamic acid derivatives such as histone deacetylase (HDAC) inhibitors is of particular interest because they are usually considered as poor drugs and are down-prioritized in lead identification campaigns because of their poor physicochemical and ADME properties. Zolinza, the first approved HDAC inhibitor, does not have optimal physicochemical and ADME properties and demonstrated poor pharmacokinetics (PK) in preclinical species, favoring intravenous administration in the initial clinical setting and then switching to an oral route at later stages in clinical development (Kelly et al., 2005). Panabinostat and Belinostat, both hydroxamic acid HDAC inhibitors, were also dosed intravenously in the initial Phase 1 studies because of their poor oral bioavailability in preclinical species (Giles et al., 2006; Steele et al., 2011). We focused on identification of an orally active, pan-HDAC inhibitor with good pharmaceutical and ADME properties that led to the discovery of Pracinostat [(2E)-3-[2-butyl-1-[2-(diethylamino)ethyl]-1H-benzimidazol-5-yl]-N-hydroxyarylamide hydrochloride; SB939]. SB939 was identified as a potent inhibitor of the HDAC enzymes with promising efficacy in preclinical models of cancer (Novotny-Diermayr et al., 2010). SB939 is currently in the Phase 2 stage of clinical development for the treatment of patients with solid tumors (Yong et al., 2011).
The pharmacokinetics (which is a function of the processes of absorption, distribution, metabolism, and elimination) of a compound is the primary determinant of its efficacy (along with potency) and safety in vivo. In the lead optimization stage of preclinical drug discovery, only compounds that have optimum ADME properties, i.e., have the potential to become a drug, are progressed into development as drug candidates. Knowledge of the physicochemical properties of a compound, integrated with its in vitro properties such as plasma protein binding, metabolic stability, cytochrome P450 (P450) inhibition/induction and isoform typing, and Caco-2 permeability and its PK in preclinical species, assist in prediction of its PK properties in humans (Smith et al., 1996; Thompson, 2000; Pelkonen and Raunio, 2005). Generation of early ADME data for the lead candidates in a drug discovery program help in prioritizing them, and in eliminating candidates with potential liabilities, for further preclinical development, because this is a resource-intensive process. The prediction of human pharmacokinetics, such as systemic clearance, volume of distribution, and concentration-time profiles, has been done by in vitro-in vivo extrapolation (Obach et al., 1997), allometry (Boxenbaum and DiLea, 1995), whole-body physiological pharmacokinetic modeling (Zhao et al., 2010), and Simcyp (Jamei et al., 2009) with reasonably good success. Simcyp was used to predict clearances of 15 clinically used drugs in humans, using in vitro metabolism data and incorporating interindividual variability known to affect metabolism, and the predicted mean oral clearances were within a 2-fold range for 93% of the drugs (Rostami-Hodjegan and Tucker, 2007). All the methods mentioned use physicochemical, in vitro ADME, and preclinical PK data, either in part or all integrated together, in performing the predictions.
In this report, we describe the preclinical ADME properties of SB939. In addition, the PK parameters and profiles of SB939 were predicted in humans using 1) the Simcyp ADME simulator (Jamei et al., 2009) based on its physicochemical properties and in vitro experimental data and 2) allometric scaling (Boxenbaum, 1982) using preclinical PK data. The predicted concentration-time profiles and PK parameters obtained from both the approaches were compared with data from patients at the first time in man (FTIM) oral dose of 10 mg (Yong et al., 2011) to assess the validation of predictions.
Materials and Methods
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.
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.
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 (Papp) was calculated from the following equation: Papp = [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 = Papp, B→A/Papp, A→B. Atenolol (Papp < 0.5 × 10−6 cm/s), propranolol (15 < Papp < 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 (Papp ≤ 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 (t1/2) of metabolic stability was estimated using the first-order equation t1/2 = 0.693/Kel, where Kel (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/t1/2) × (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.
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 (t1/2) of metabolic stability was estimated using the first-order equation t1/2 = 0.693/Kel, where Kel (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/t1/2) × (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.
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).
Plasma samples from mouse, rat, and dog.
To 50 or 100 μl of plasma, 10 μl of SB558 (500 ng/ml in 50% methanol in water) internal standard was added and extracted with 1.25 or 3 ml of MTBE for 30 min on a vortexer. After extraction, the samples were centrifuged at 1200g for 10 min in a refrigerated centrifuge (Eppendorf 5415R) at 4°C. The supernatant was transferred to fresh tubes and evaporated to dryness at 35°C in a turbovap for 30 min. The dried samples were reconstituted with 0.1 ml of mobile phase (60% methanol/40% water). Sample analysis was carried out by LC-MS/MS using an Alliance HT2795 HPLC system (Waters, Milford, MA) connected to a Quattro Micro Mass Spectrometer (Waters). The mobile phase consisted of methanol and 0.1% formic acid in water (60:40). The samples were resolved on a Luna C18 column (2 × 50 mm, 5 μm; Phenomenex) maintained at 40°C, at a flow rate of 0.3 ml/min with a total run time of 5 min. The MS parameters were as follows for SB939-multiple reaction monitoring: m/z 359→m/z 100 (electrospray ionization positive); cone voltage, 35 V; collision energy, 16 eV. The corresponding parameters for SB558 (internal standard) were m/z 322→m/z 213 (electrospray ionization positive), 40 V, and 24 eV. The assay was linear between 0.5 and 1000 ng/ml (dog plasma: lower limit of quantitation, 0.5 ng/ml) and between 1.0 and 1000 ng/ml (mouse and rat plasma: lower limit of quantitation, 1.0 ng/ml), accurate and precise.
Three hundred microliters of urine [0 h (blank control), 0–4 and 4–24 h after dose] were extracted with 600 μl of methanol in 1.5-ml Eppendorf tubes. The tubes were vortexed for 30 min and centrifuged at 16,100g for 10 min at 4°C in a microcentrifuge. The supernatant was transferred to fresh Eppendorf tubes and placed in a SpeedVac for 30 min to concentrate the samples. The samples were analyzed by LC-MS/MS.
Noncompartmental PK parameters such as the volume of distribution at steady state (Vss), systemic clearance (CL), elimination half-life (t1/2), area under the plasma concentration-time curve up to the last nonzero concentration (AUC0-t), area under the plasma concentration-time curve from time 0 to infinity (AUC0-∞), the time of maximum concentration in plasma (tmax), the maximum concentration in plasma (Cmax), 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 (fg) = 1; and 3) metabolism was the main clearance mechanism. The fraction unbound in microsomes (fu,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 analysis was performed by linear regression of log-transformed values of the PK parameters (CL/F, V/F, CL, and Vss) 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.
Prediction of CL/F and V/F in humans.
CL/F and V/F showed poor correlation with body weight, and therefore predictions in humans were not attempted based on allometric relationships for CL/F and V/F. An alternative approach was chosen for the predictions. A range of CL/F and V/F was generated as follows: the mean, upper, and lower 95% confidence interval estimates of CL and Vss were divided with F values of 0.10, 0.3, 0.5, 0.75, and 1.0 (because F was unknown in humans). The predicted range of CL/F and V/F were compared with the ranges observed in humans at the 10-mg dose (Yong et al., 2011).
Prediction of CL and Vss in humans.
The mean CL and Vss and the range were predicted for a 70-kg human using the mean, upper, and lower 95% confidence interval estimates for the corresponding allometric exponents and coefficients.
Prediction of t1/2 of SB939 in humans.
No correlations were observed between the elimination half-life and body weight among the preclinical species (data not shown). Therefore, the elimination t1/2 was predicted in humans using the mean, upper, and lower 95% confidence interval estimates of Vss and CL using the following equation:
The in vitro ADME properties of SB939 are summarized in Table 2. The mean logD7.4 of SB939 was 2.07. SB939 showed high in vitro PPB in mouse, rat, and human plasma (range, ∼92–94%), with slightly lower values in dog. The measured B/P ratio was ∼1 in human blood. SB939 displayed high permeability and low efflux in the Caco-2 bidirectional permeability assay. In microsomal metabolic stability studies, SB939 was relatively stable in human and dog microsomes, when compared with that in mice and rat. In the P450 isoform phenotyping studies using recombinant human P450s, SB939 was found to be mainly metabolized by CYP3A4 and CYP1A2. In the inhibition assay using human P450s, SB939 did not inhibit CYP1A2, 2C9, 2D6, and 3A4 but inhibited 2C19 (IC50 = 5.8 μM).
Metabolite profiling studies in vitro, after incubation of SB939 with liver microsomal fractions, indicated the formation of metabolites in humans, mouse, dog, and rat (Fig. 2). Major metabolite peaks observed, in addition to the parent (M), were M1 and M6 (HLM); M1, M6, and M8 (MLM and DLM); and M1, M4, and M8 in RLM (Fig. 2). In addition, minor metabolite peaks were also observed in all the species. The EMS spectra for the major and minor metabolite peaks (observed in Fig. 2) for HLM, MLM, DLM, and RLM are shown in Supplemental Figs. 1 to 4, respectively. Table 3 summarizes the metabolites of SB939 (MH+ with m/z 359) identified in the microsomal incubations. In HLM, MLM, DLM, and RLM, the major metabolites formed were the N-deethylation (M1) and bis-N-deethylation (M6) products, with M6 detected in trace amounts in RLM. An oxidation product of SB939 (M4) was found to be a major metabolite in RLM and was not detected in HLM, MLM, and DLM. In addition to this, a major metabolite also observed in MLM, DLM, and RLM was the amide formation due to reduction of the hydroxamic acid group in SB939 (M8), which was detected in trace amounts in HLM. Oxidative metabolites M2, M3, M5, and M7 were also detected with varying intensities in different species (Table 3). In general, the metabolites formed in human were seen in mice, rat, or dog. On the basis of their MS and MS/MS spectra (EPI spectrum for metabolites formed in RLM and MLM is shown in Fig. 3), a metabolic pathway as outlined in Fig. 4 was proposed. Two fragment ions at m/z 260 and m/z 100 were of diagnostic value in identifying the changes to the structure in the molecule. The ion at m/z 100 remains unchanged if the triethylamino side chain attached to benzimidazole is intact in the molecule, thus aiding in characterizing M1, M5, and M6 metabolites. The oxidation in the core benzimidazole part alters the fragment ion m/z 260 providing evidence of metabolic change in this part of the molecule which assisted in identifying metabolites M2, M3, M5, and M7.
The potential of SB939 to induce human CYP3A4 and 1A2 was assessed in vitro using freshly cultured human hepatocytes, and the data are summarized in Table 4. The mean fold induction of CYP3A4 by SB939 at 0.1, 1.0, and 10.0 μM was <40% of the induction caused by the positive controls rifampicin and dexamethasone. The fold induction of CYP1A2 by SB939, at all concentrations, was <40% of the induction caused by the positive control omeprazole. As per the Pharmaceutical Research and Manufacturers of America perspective study (Bjornsson et al., 2003) SB939 does not have the potential to induce CYP3A4 and 1A2 in vivo.
The PK profiles of SB939 in mice, rats, and dogs, after a single intravenous dose and oral dosing, are shown in Fig. 5. The noncompartmental analysis PK parameters of SB939 in mice, rats, dogs, and humans are summarized in Table 5.
After intravenous administration, SB939 showed a biexponential disposition (Fig. 5a) with high systemic clearance [CL exceeding its liver blood flow (LBF) (Davies and Morris, 1993)] and high steady-state volume of distribution [Vss > 0.7 l/kg (Davies and Morris, 1993)], with a t1/2 of 2.3 h. After a single oral dose, SB939 showed rapid absorption and multiexponential decline, with a t1/2 of 2.4 h and an absolute oral bioavailability (F) of 34%.
SB939 displayed multiexponential disposition with first-order kinetics after a single intravenous dose (Fig. 5b). The CL was high [CL exceeding its LBF (Davies and Morris, 1993)], with high Vss [Vss > 0.7 l/kg (Davies and Morris, 1993)] and a mean t1/2 of 0.9 h. After a single oral dose, SB939 showed rapid absorption, multiexponential decline with a mean t1/2 of 2 h, and poor F (Table 5).
After intravenous administration, SB939 displayed multiexponential disposition (Fig. 5c) with high CL (∼81% LBF) and Vss [Vss > 0.6 l/kg (Davies and Morris, 1993)] and a t1/2 of 3.9 h. After a single oral dose, SB939 showed rapid absorption, multiexponential decline with a mean t1/2 of 4.1 h, and F = 65% (Table 5).
After oral dosing of SB939 in rats, the major metabolite observed in urine was a glucuronidated product of SB939 (M9) at the retention time of 13.35 min (Fig. 6a). The EPI scan confirmed the presence of SB939 glucuronide with characteristic fragment ions at m/z 359.1 and m/z 100.1 corresponding to the protonated molecular ion of SB939 and its characteristic fragment, respectively (Fig. 6b).
The PK of SB939 was predicted, at the FTIM dose of 10 mg, in healthy humans (n = 100) using the Simcyp simulator with the input parameters shown in Table 1. The results are shown in Fig. 7 and Table 6. SB939 was predicted to display rapid absorption followed by biexponential disposition with first-order kinetics. The observed mean profile from the starting 10-mg dose (Yong et al., 2011) was in reasonably good agreement with the predicted data (Fig. 7). The mean predicted AUC and Cmax was 1.4-fold higher and 1.6-fold less, respectively, than the observed mean values (Table 6). The observed mean AUC was within the range of the predicted 95% confidence interval. The predicted mean CL/F (37 l/h) was comparable to the observed mean value of 45 l/h. The mean terminal t1/2 was ∼4-fold longer than the observed mean value (Yong et al., 2011). The predicted mean CL and Vss were 0.14 l · h−1 · kg−1 and 4.4 l/kg, respectively.
On the basis of in vitro P450 phenotyping, inhibition, and induction results, simulations were performed to assess the potential effects of inhibition and induction of CYP3A4 on the PK of SB939 and the effect of SB939 on PK of omeprazole (substrate for 2C19) in humans. The results are summarized in Fig. 8 and Supplemental Fig. 5. The predicted PK profile of SB939 at the recommended dose of 60 mg was in agreement with the observed mean data from cancer patients (Yong et al., 2011), except that the terminal phase was slightly steeper than the mean and 95% confidence interval curves (Supplemental Fig. 5a). The fold difference between the mean predicted and observed values for AUC, tmax, Cmax, and CL/F was <2-fold (Table 6). When coadministered with ketoconazole, the predicted median AUC ratio of SB939 was 1.16 (Fig. 8a), and the predicted mean PK profile of SB939 when administered alone was similar to that when coadministered with ketoconazole (Supplemental Fig. 5b), suggesting lack of DDI potential for SB939 with CYP3A inhibitors. The predicted median AUC ratio of SB939 when coadministered with rifampicin was 0.83 (Fig. 8b), and the predicted mean PK profile of SB939 was not significantly changed (Supplemental Fig. 5c), suggesting lack of potential DDI with CYP3A inducers. SB939 did not appear to affect the PK of omeprazole significantly (Fig. 8c; Supplemental Fig. 5d), in terms of the AUC ratio (median, 1.36) and PK profiles.
The PK parameters of SB939 were also predicted in humans using allometric scaling. The allometric relationships for CL and Vss and predicted values in humans for a 70-kg individual are shown in Fig. 9 and Table 7. The relationship between CL and body weight (Fig. 9a) and between Vss and body weight showed good linearity (Fig. 9b). The mean allometric exponent and coefficient for CL were 0.72 and 2871, respectively, and the corresponding values for Vss were 1.1 and 2799, respectively (Table 7). The predicted mean and 95% confidence interval range in humans for CL and Vss were 62 l/h (31–123 l/h) and 313 l (99–277 l), respectively (Table 7).
The predicted and the observed ranges of CL/F and V/F in humans are shown in Table 8. The predicted range for CL/F and V/F was ∼13- and 33-fold, respectively. The observed CL/F and V/F range in humans at 10 mg was in the lower range of the predicted values (Table 8). The predicted range of elimination t1/2 in humans was very close to the observed range (Table 8).
SB939 is a highly soluble (>100 mg/ml in water for the HCl salt) and a moderately lipophilic weak base. The high permeability and low efflux ratio of SB939 in the Caco-2 assay, in addition to its high solubility, suggested that it may show high intestinal absorption in humans. The low efflux ratio also indicated that SB939 may not be a substrate for P-glycoprotein transporters. The Caco-2 bidirectional assay is routinely used in preclinical development to assess the permeability of drug candidates (Dressman et al., 2008). The Caco-2 model has been reported to predict the fraction absorbed by the intestine (fa) for highly permeable compounds with passive transport (Fagerholm, 2007). Drugs that are lipophilic bases tend to show high PPB (Smith et al., 1996). The high PPB of SB939 (84–94%) may be due to its moderately high logD and weak basic nature. SB939 showed species differences in metabolic stability in liver microsomal incubations. The in vivo clearance was predicted (relative to the corresponding LBF) to be high in rats, moderate in mice, and low in dogs and humans. This also suggested that SB939 may have high first-pass effect and lower bioavailability across the liver (Fh) in rats and mice and moderate to high Fh in dogs and humans. In vitro metabolite identification studies have been known to predict the metabolites formed in vivo with reasonable accuracy (Pelkonen and Raunio, 2005). Although the metabolite profiles of SB939 were similar across the species tested, the profiles (based on their abundance) were most similar in mice, dogs, and humans, indicating that the mouse and dog would be the appropriate species for toxicology testing of SB939.
The plasma CL of SB939 in mice and rats was higher than their corresponding LBF (Davies and Morris, 1993), suggesting high metabolic clearances, consistent with the in vitro metabolic stability data in RLM and MLM, and the existence of extrahepatic clearance. It is possible that SB939, a hydroxamic acid, may also be cleared extrahepatically. Vorinostat and Givinostat, both hydroxamic acids, were cleared by metabolic and renal pathways in preclinical species (Sandhu et al., 2007; Furlan et al., 2011). Furthermore, in line with other hydroxamic acid functionalized HDAC inhibitors, which are known to undergo glucoronidation at the hydroxamic acid group, the formation of the glucuronide product of SB939 as a major Phase 2 metabolite was observed in rat urine. Investigation of glucoronide formation in human and preclinical species and the UDP glucuronosyltransferases involved in the glucoronidation are planned and results will be published elsewhere. The high Vss of SB939 could be due to its lipophilic basic nature, leading to binding to the negatively charged phospholipids of the cell membrane. The Vss of basic drugs has been shown to be positively correlated with lipophilicity in humans (Smith et al., 1996). The high Vss of SB939 in mice was consistent with the results from its tissue distribution pattern in the tumor-bearing mice, and its distribution into tumors correlated well with its efficacy, in addition to excellent PK/pharmacodynamic relationships for efficacy and biomarker, in murine xenograft models (Novotny-Diermayr et al., 2010). SB939 showed rapid absorption in mice, rats, and dogs that could be due to its high solubility and permeability. The bioavailability was moderate in mice despite the CL exceeding the LBF, suggesting that SB939 may be cleared by extrahepatic mechanisms or that the hepatic metabolizing enzymes were saturated by the high portal concentrations resulting from the high oral dose. The plasma clearance can exceed liver blood flow when the B/P ratio was >1 (Yang et al., 2007). The B/P ratio of SB939 in mouse was ∼1 (in-house data), suggesting that the B/P ratio was not contributing to the observed high CL in mice. The poor F in rats may be explained by the high intrinsic clearance observed in vitro leading to high first-pass extraction.
Simcyp-simulated oral PK profiles of SB939, at the FTIM dose of 10 mg in healthy human populations, compared reasonably well, within the limits of 95% confidence range, with the observed mean PK profile in cancer patients at 10 mg (Yong et al., 2011) except for the terminal t1/2, which was overpredicted. The predicted mean CL/F was comparable to the observed value. Interindividual variability in exposures has been documented for anticancer drugs in cancer patients, which was attributed to P450 allelic polymorphisms (for drugs cleared by P450s), differential expression of P450s in cancer tissues (e.g., CYP3A), physiological changes caused by tumor invasion, and coadministration of multiple drugs (Fujita, 2006). In contrast to the single-point predictions made by allometric scaling (Tang and Mayersohn, 2006), for clearance and volume of distribution, Simcyp has the advantage of providing estimates with a variability range (with extreme cases) expected based on covariates (e.g., demographics, disease) that are known to influence pharmacokinetic parameters. Thus, an estimate provided with a range is more realistic than single-point estimates and provides an indication of the variability that can be expected in real populations (Jamei et al., 2009). SB939 was predicted to display high Vss (>0.6 l/kg) and low systemic clearance (∼12% LBF) in humans.
SB939 was found to be metabolized by CYP3A4 and 1A2 in vitro and did not show the potential to induce CYP3A and 1A2. Because CYP3A is the most abundant P450 in human liver and intestine (Rostami-Hodjegan and Tucker, 2007), the potential of SB939 PK being affected by the potent CYP3A inhibitor (ketoconazole) and inducer (rifampicin) was simulated in Simcyp. The magnitude of the AUC ratio was used to assess the significance of in vivo DDI (Bjornsson et al., 2003). The predicted AUC ratio of SB939 indicated that SB939 did not show the potential to be affected by ketoconazole and rifampicin coadministration. Although CYP2C19 was inhibited by SB939 in vitro, the predicted AUC ratio of omeprazole (a substrate for 2C19) was not significant, suggesting a lack of DDI for SB939 in vivo. Further clinical pharmacology studies need to be conducted to verify the predictions of DDI for SB939.
Prediction of CL/F and V/F in humans by interspecies scaling was not attempted because of poor correlations observed for CL/F and V/F between preclinical species. This may be explained by the poor F of SB939 in rats compared with the higher F in mice and dogs. Tang and Mayersohn (2006), in a comparison of predictability of CL and CL/F in humans based on allometric scaling, found that the average prediction error was higher for CL/F, and one of the possible reasons was attributed to variation in F across species. Systemic clearance and Vss showed good allometric relationships between mouse, rat, and dog. The mean values for the allometric exponent for CL and Vss were close to the expected allometric exponents for CL and Vss (Boxenbaum and DiLea, 1995). The observed range of CL/F and V/F of SB939 in humans (Yong et al., 2011) was closer to the lower range of predicted values using CL, Vss, and varying F. Intravenous PK studies have not been performed in humans to confirm the predictions of CL and Vss made using both the approaches.
In summary, the physicochemical characteristics and preclinical ADME data of SB939 supported its development as an oral drug candidate. Based on the in vitro metabolite profiles and preclinical PK properties, the mouse and dog were recommended for toxicology and safety pharmacology studies with SB939. The predicted PK profiles and parameters of SB939, using Simcyp and allometric scaling, in healthy human populations were in reasonably good agreement with observed data from cancer patients. SB939 is currently undergoing multiple Phase 2 trials in solid tumors (http://clinicaltrials.gov).
Participated in research design: Ethirajulu.
Conducted experiments: Wang, Sangthongpitag, Yong Hu, Wu, Xin, Goh, and Sun New.
Contributed new reagents or analytic tools: Wang.
Performed data analysis: Yeo, Khalid Pasha, Venkatesh, and Jayaraman.
Wrote or contributed to the writing of the manuscript: Jayaraman and Ethirajulu.
We thank Tony Ng for assistance in performing the in vivo experiments.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
- histone deacetylase
- area under the plasma concentration-time curve from time zero to the last measured nonzero concentration
- area under the plasma concentration-time curve from time zero to infinity
- B/P ratio
- blood-to-plasma ratio
- peak concentration in plasma
- systemic clearance
- oral clearance
- enhanced mass spectrum
- enhanced product ion
- oral bioavailability
- fraction escaping hepatic metabolism
- fraction of unbound drug in plasma
- fraction of unbound drug in microsomal incubations
- liver blood flow
- plasma protein binding
- volume of distribution at steady state
- apparent volume of distribution
- absorption, distribution, metabolism, and excretion
- (2E)-3-[2-butyl-1-[2-(diethylamino)ethyl]-1H-benzimidazol-5-yl]-N-hydroxyarylamide hydrochloride
- first time in man
- human liver microsomes
- dog liver microsomes
- rat liver microsomes
- mouse liver microsomes
- liquid chromatography-tandem mass spectrometry
- permeability coefficient
- dimethyl sulfoxide
- drug-drug interaction.
- Received July 4, 2011.
- Accepted August 26, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics