Research ArticleArticle

In Vitro and In Vivo Metabolism and Pharmacokinetics of BMS-562086, a Potent and Orally Bioavailable Corticotropin-Releasing Factor-1 Receptor Antagonist

Lian Zhou, Randy C. Dockens, Peggy Liu-Kreyche, Scott J. Grossman and Ramaswamy A. Iyer
Drug Metabolism and Disposition June 2012, 40 (6) 1093-1103; DOI: https://doi.org/10.1124/dmd.111.043596

Abstract

The absorption, distribution, metabolism, and excretion (ADME) and the pharmacokinetic characteristics of BMS-562086 [pexacerfont; 8-(6-methoxy-2-methyl-3-pyridinyl)-2,7-dimethyl-N-[(1R)-1-methylpropyl]pyrazolo(1,5-a)-1,3,5-triazin-4-amine (DPC-A69448)] were investigated in vitro and in animals to support its clinical development. BMS-562086 was orally bioavailable in rats, dogs, and chimpanzees, with an absolute oral bioavailability of 40.1, 58.8, and 58.5%, respectively. BMS-562086 was extensively metabolized in hepatocytes from all species and completely metabolized in rats. The primary biotransformation pathways found for BMS-562086 in both liver microsomal and hepatocyte preparations and in rats were similar. These included O-demethylation, hydroxylation at the N-alkyl side chain and N-dealkylation. Multiple cytochromes P450 including CYP3A4/5 were involved in the metabolic clearance of BMS-562086. Both renal and biliary excretion played a significant role in elimination of the metabolites of BMS-562086. The involvement of other metabolic enzymes in addition to CYP3A4/5 in elimination of BMS-562086 suggests a reduced potential for drug-drug interaction through modulation of CYP3A4/5. Chimpanzees proved to be a good animal model in predicting BMS-562086 human clearance. Virtual clinical trials performed with a population-based ADME simulator suggested that a minimal dose of 100 mg daily would provide sufficient drug exposure to achieve plasma concentrations above the projected human efficacious plasma concentration of BMS-562086 (>500 nM). In summary, BMS-562086 exhibited favorable ADME and pharmacokinetic properties for further development.

Introduction

Corticotropin-releasing factor subtype 1 (CRF1) receptor mediates stress-induced activation of the hypothalamic-pituitary-adrenal axis (Owens and Nemeroff, 1991; Smagin and Dunn, 2000). In nonclinical studies, CRF1, a pituitary adrenocorticotrophic hormone secretagogue and a neurotransmitter, played a pivotal role in the regulation of endocrine, autonomic, and behavioral responses to stress (Owens and Nemeroff, 1991; Smagin and Dunn, 2000). Intracerebral ventricular administration of CRF1 to rats generated many of the behavioral and physiological effects induced by stress. Mice that are null for the CRF1 gene exhibited a reduced endocrine response to psychological stressors (Owens and Nemeroff, 1991; Smagin and Dunn, 2000). In addition, nonclinical studies suggested that CRF1 antagonists are active in the treatment of irritable bowel syndrome (IBS), a stress-related disorder (Sweetser et al., 2009). Some clinical studies have implicated CRF in the pathophysiology of both depression and anxiety. An elevated level of CRF1 in cerebrospinal fluid is observed in patients with anorexia nervosa, obsessive compulsive disorder, and post-traumatic stress disorder (Coric et al., 2010). In patients with IBS, subjects who displayed increased sensitivity to exogenous CRF1 were more likely to suffer abdominal pain and hypermotility compared with healthy cohorts (Fukudo et al., 1998).

BMS-562086 [pexacerfont; DPC-A69448; 8-(6-methoxy-2-methyl-3-pyridinyl)-2,7-dimethyl-N-[(1R)-1-methylpropyl]pyrazolo(1,5-a)-1,3,5-triazin-4-amine] is a selective CRF1 receptor antagonist. It demonstrates a potent and specific inhibitory effect (IC50 = 6.1 ± 0.6 nM) toward human CRF1 receptor and has greater than 1000-fold lower affinity (IC50 > 1000 nM) for the CRF-binding protein and biogenic amine receptors (Gilligan et al., 2009). It is active in rats (1–10 mg/kg, orally) in the defensive withdrawal and elevated plus maze models of anxiety (Gilligan et al., 2009).

An examination of the absorption, distribution, metabolism, and excretion (ADME) and the pharmacokinetic characteristics of a drug candidate is essential for its successful development. Preclinical ADME studies can provide early information on a variety of properties, including permeability, plasma protein binding, metabolic soft spots, potential formation of reactive metabolites, metabolic pathways in different species, possible metabolites formed in humans, and potential for drug-drug interactions. Furthermore, knowing the biotransformation reactions that a drug molecule may be subjected to helps in the design and conduct of safety studies for new chemical entities (Humphreys and Unger, 2006).

The ADME properties of a drug molecule influence its pharmacokinetics in animal species and humans. The pharmacokinetic data can be extrapolated from animals to predict quantitatively the likely behavior in humans, because many of the basic processes controlling pharmacokinetics are similar across species. Frequently, a reasonable dose-correlated plasma pharmacokinetic profile can further be simulated in humans. Hence, an understanding of the ADME and pharmacokinetic properties of a compound is critical in improving the chances of selecting not only the most promising drug candidate but also the most appropriate targeted doses to be tested in humans.

In this study, the ADME and pharmacokinetic properties of BMS-562086 were investigated in vitro and in animals to support its clinical development as an orally effective agent intended for the treatment of depression, anxiety disorders, and IBS. The metabolism of BMS-562086 was compared in mouse, rat, dog, monkey, and human liver microsomes and hepatocytes. The metabolic profiles and routes of excretion of BMS-562086-related components were identified in bile duct-cannulated (BDC) rats. The permeability of BMS-562086 was examined in Caco-2 cells, and its pharmacokinetic properties were determined in rats, dogs, and chimpanzees. Rats and dogs were studied because they are the two species used for long-term toxicology evaluations. Chimpanzees are viewed as a good pharmacokinetic model because their size and many of the physiological relationships that affect drug clearance, such as organ blood flow, are similar to those in humans (Wong et al., 2004). Furthermore, SimCYP population-based ADME simulator (SimCYP Ltd., Sheffield, UK) was used to make extrapolation to human pharmacokinetics by incorporating both the in vitro ADME and animal pharmacokinetic data, and the population-specific variability. Virtual clinical trials were performed to simulate the plasma pharmacokinetic profiles in humans.

Materials and Methods

Chemicals and Biologicals.

BMS-562086, [14C]BMS-562086 (30 μCi/mg, radiochemical purity >99%, chemical purity >99%), [13C]D3-BMS-562086, and the reference standards for its three metabolites, DPH-123554 (O-demethylated metabolite), DPH-124921 (N-dealkylated metabolite), and BMS-572273 (N-dealkylated and hydrolyzed metabolite) were synthesized by Bristol-Myers Squibb Research and Development (Princeton, NJ). Caco-2 cells were obtained from the American Type Culture Collection (Manassas, VA). Dulbecco's modified Eagle's medium, HEPES, nonessential amino acids, l-glutamine, penicillin-G, and streptomycin were purchased from Invitrogen (Carlsbad, CA). Heat-inactivated fetal bovine serum was obtained from Invitrogen. Rat tail collagen-type I was purchased from BD Biosciences (San Jose, CA). Twenty-four well Transwell plates (surface area, 0.33 cm2) with a polycarbonate membrane (3.0-μm pore size) were purchased from Corning Life Sciences (Lowell, MA). Hanks' balanced salt solution was purchased from Sigma-Aldrich (St. Louis, MO). Acetic acid was purchased from Mallinckrodt Baker, Inc. (Phillipsburg, NJ). Calcium chloride, 7-ethoxycoumarin (7-EC), glucose, 7-hydroxycoumarin (7-HC), Krebs-Henseleit buffer modified salt, sodium bicarbonate, sodium hydroxide, trepan blue solution (0.4%), NADPH, and β-glucuronidase (type HA-4, from Helix aspersa) were purchased from Sigma-Aldrich. Ecolite liquid scintillation cocktail was purchased from MP Biomedicals (Solon, OH). Deionized water was prepared with a MilliQplus ultrapure water system (Millipore Corporation, Billerica, MA). All other chemicals used were reagent grade or better. Frozen rat, dog, and human sera were obtained from Bioreclamation, Inc. (Hicksville, NY). Pooled liver microsomes were purchased from BD Gentest (Woburn, MA). Hepatocytes from male beagle dogs (fresh), male cynomolgus monkeys (cryopreserved), and humans (cryopreserved from three donors) were obtained from In Vitro Technologies (Baltimore, MA). Freshly isolated mouse and rat hepatocytes were provided by Technical Support Unit, Bristol-Myers Squibb Research and Development.

Permeability across Caco-2 Cell Monolayers.

Caco-2 cells at passage 50 to 60 were seeded on polycarbonate membranes of 24-well Transwell plates at a density of 60,000 cells/cm2. The cells were cultured for 21 to 25 days in culture medium consisting of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 0.5 mM HEPES, 1% nonessential amino acids, 1% l-glutamine, 100 U/ml penicillin-G, and 100 μg/ml streptomycin. Before the permeability studies, apical (AP) and basolateral (BL) media were replaced with transport buffer (Hanks' balanced salt solution supplemented with 2% N, N-dimethylacetamide, pH 7.4). AP to BL permeability study was initiated by replacing the AP medium with the transport buffer containing 25 μM BMS-562086. All permeability experiments were performed at 37°C.

Transepithelial electrical resistance (TEER) values were measured to assess cell monolayer integrity. TEER values were obtained both at the beginning and at the end of each experiment. Only wells with TEER values between 400 and 500 Ω/cm2 throughout the experiment were used in the studies. Mannitol (25 or 100 μM) transport experiments were performed in the same manner as other transport experiments. Mannitol served as a probe of the Caco-2 cell monolayer integrity (Zhou et al., 2002).

Protein Binding in Serum.

In vitro protein binding of BMS-562086 in rat, dog, and human serum was determined by equilibrium dialysis. BMS-562086 in acetonitrile stock solution was added into serum to final concentrations of 12.8, 25.6, and 38.5 μM. The three concentrations represent the projected parent concentrations to be studied in preclinical and clinical investigations. Serum samples were dialyzed against 134 mM phosphate buffer (pH 7.4) in a dialysis membrane (Amika Corp., Holliston, MA) with a 10,000 Da molecular mass cutoff. The dialysis cells (Spectrum, Laguna Hills, CA) were rotated at 20 rpm in a water bath maintained at 37°C for 3 h. At the end of incubation, aliquots (100 μl) of buffer were taken and mixed with equal volume of serum of the species under investigation. Aliquots (20 μl) of serum samples were taken and mixed with a matrix (180 μl) contained blank serum of the species under investigation: phosphate buffer (0.134 M, pH 7.4) (1:1 v/v). The final percentage of unbound drug was expressed as the concentration in buffer divided by the concentration in serum. Serum stability of BMS-562086 was also measured with these serum samples over the 3-h incubation period.

Hepatocyte Incubations.

The cell viabilities were checked with the trypan blue exclusion method and a hemocytometer. The viability before the incubation for mouse, rat, dog, monkey, and human hepatocytes was 71, 88, 91, 68, and 78%, respectively. The cells were diluted with freshly prepared Krebs-Henseleit buffer to give a final concentration of 1.0 × 106 cells/ml. The hepatocyte incubations with BMS-562086 (25 μM) were performed in 4-ml volumes in open 20-ml glass scintillation vials in duplicate. The incubations were conducted on an orbital shaker (80 rpm) in a humidified NAPCO CO2 6000 incubator (VWR, West Chester, PA) maintained under an oxygen/carbon dioxide (95:5) atmosphere at 37°C for 3 h. The positive control incubations with 7-EC and 7-HC were performed at 100 μM. A negative control, in the absence of the hepatocytes, was also performed. A vial of hepatocyte suspension, with no compound added, was included to check for viability at 1 and 3 h. The incubations were stopped after 3 h by adding 1 volume of ice-cold acetonitrile. The reaction mixtures were sonicated for 5 min in a Branson 5210 sonicator (Branson Ultrasonics Corporation, Danbury, CT) and then centrifuged at 5000 rpm for 10 min. The supernatant fractions were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Microsomal Incubations.

For the purpose of metabolite identification, a higher BMS-562086 concentration (100 μM) was used in the mouse, rat, dog, monkey, and human liver microsomal incubations. The concentrations of microsomal proteins, NADPH, and phosphate buffer were 1 mg/ml, 1, and 100 mM, respectively. Incubations were initiated by addition of NADPH to the assay mixture. The incubations were performed in 5-ml glass test tubes at 37°C for 1 h in a shaking water bath. They were performed in duplicate at a final volume of 2 ml. Three control incubations were performed: incubation in the absence of the microsomal protein, incubation in the absence of the cofactor NADPH, and incubation with 7-EC (100 μM) as a positive control. Incubations were stopped by cooling on ice followed by addition of 2 ml of acetonitrile. The incubation mixtures were then centrifuged at 5000 rpm for 10 min. The supernatants were removed and evaporated to near dryness under a stream of nitrogen. The residues were reconstituted in 0.5 ml of acetonitrile and water (1:1) and kept at −20°C until further analysis.

For inhibition studies, human liver microsomes were incubated with BMS-562086 (1 and 25 μM) for 0, 10, 20, and 30 min in the presence or absence of ketoconazole (5 μM). The concentrations of microsomal proteins, NADPH, and phosphate buffer were the same as those used for microsomal incubations described above. For incubations in the presence of ketoconazole, human liver microsomes were preincubated for 10 min with the inhibitor before the addition of BMS-562086 and NADPH. The incubations were performed in microcentrifuge tubes at 37°C for 30 min in a shaking water bath at a final volume of 200 μl. Incubations were stopped by cooling on ice following by addition of 200 μl acetonitrile. The incubation mixture was then centrifuged at 14,000 rpm for 2 min in an Eppendorf model 5417C centrifuge Eppendorf AG (Hamburg, Germany). The supernatants were removed and evaporated to near dryness under a stream of nitrogen. The residues were reconstituted in 50 μl of water and kept at −20°C until analysis. Incubation in the absence of the microsomal protein and incubation in the absence of the cofactor NADPH were used as negative controls.

Metabolism and Pharmacokinetic Studies in Animals.

All the studies were performed according to the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996).

Pharmacokinetic Studies in Rats.

One group of male Sprague-Dawley rats (n = 3, 0.34–0.35 kg b.wt.) instrumented with single jugular vein cannulas were designated for oral administration, and a second group of male Sprague-Dawley rats (n = 3, 0.34–0.35 kg b.wt.) instrumented with dual jugular vein cannulas were designated for intravenous administration. All rats were fasted for approximately 18 h before use and for 4 h after dosing. Water was provided ad libitum. BMS-562086 was administered orally by gavage to three rats at a single dose of 5 mg/kg in 0.5% aqueous methylcellulose. A single intravenous bolus dose of BMS-562086 was administered to three rats at 1 mg/kg in 20% ethanol in saline via the jugular vein cannula. Blood samples (0.2 ml/time point per animal) were collected via the jugular vein cannula for analysis of BMS-562086 at 0, 0.08 (intravenous dose only), 0.17 (intravenous dose only), 0.25, 0.5, 0.75, 1, 2, 4, 8, 12, 24, 48, 72, and 96 h postdose. Plasma was prepared from the blood samples by centrifuging for 10 min at 1000g and 5°C.

Metabolism Study in BDC Rats.

BMS-562086 (30 mg/kg) in 0.5% methylcellulose solution was orally administered to BDC male Sprague-Dawley rats (n = 3, 0.22–0.32 g b.wt.) by gavage. The animals were individually housed in metabolism cages and fasted overnight before dosing. Urine and bile were collected from the BDC rats at 0 to 12 h after dosing. Pooled urine and bile samples were prepared by combining equal percentage by weight of urine and bile from all three BDC rats. A 1-ml portion of the pooled urine and bile samples were then centrifuged at 14,000 rpm for 2 min in an Eppendorf model 5417C centrifuge (Eppendorf AG). Aliquots of the supernatants were analyzed by LC-MS/MS for metabolite identification. Glucuronide hydrolysis was conducted for bile to compare with the profile before the hydrolysis. β-Glucuronide hydrolysis was performed by incubating 100 μl of pooled bile sample with 70 μl of β-glucuronide enzyme solution (∼2618 units) in 240 μl of 0.2 M sodium acetate buffer (pH 4.8). The incubation mixture was incubated at 37°C for 16 h in a shaking incubator. The incubation mixture was evaporated to dryness in a Savant SpeedVac SPD2010 Concentrator (Thermo Fisher Scientific, Waltham, MA) at room temperature. The residue was reconstituted in 200 μl of acetonitrile/water (3:7, v/v) for LC-MS/MS analysis.

Pharmacokinetic Studies in Dogs.

Male beagle dogs (n = 3, 10.1–12.7 kg b.wt.) were used to conduct the one-way crossover study in intravenous and oral dosing. The dogs were fasted for approximately 15 h before use and for 4 h after dosing. Water was provided ad libitum. For intravenous dosing, BMS-562086 was administered as an intravenous bolus injection through a temporary percutaneous catheter into a saphenous vein at a dose of 1 mg/kg in N,N-dimethylacetamide/propylene glycol/water (1:7:2, v/v/v). A 1-week washout period was allowed between the intravenous and oral dosing. For oral dosing, a single dose of BMS-562086 was administered at 5 mg/kg in 0.5% aqueous methylcellulose by oral gavage. Blood samples (2 ml/time point per animal) were collected for analysis of BMS-562086 at 0, 0.08 (intravenous dose only), 0.17 (intravenous dose only), 0.25, 0.5, 0.75, 1, 2, 4, 8, 12, 24, 48, 72, and 96 h postdose. Plasma was prepared from the blood samples by centrifuging for 10 min at 1000g and 5°C.

Pharmacokinetic Studies in Chimpanzees.

After an overnight fast, BMS-562086 was administered orally to male chimpanzees (n = 2, 58 and 72 kg b.wt.) at a dose of 1 mg/kg in suspension of 0.5% aqueous methylcellulose and Tang orange juice (Kraft Foods Inc., Northfield, IL) containing 1% of Tween 80 (1:1, v/v). A single intravenous dose (over 10 min infusion) of BMS-562086 was administered to one male chimpanzee (60 kg b.wt.) at 0.5 mg/kg in N,N-dimethylacetamide/propylene glycol/water (1:7:2, v/v/v). Blood samples (0.2 ml/time point per animal) were collected for analysis of BMS-562086 at 0, 0.1 (intravenous dose only), 0.25 (intravenous dose only), 0.5 (intravenous dose only), 1 (intravenous dose only), 2, 4, 6, 9, 12, 24, 36, 48, 72, and 96 h postdose. Plasma was prepared from the blood samples by centrifuging for 10 min at 1000g and 5°C.

Blood to Plasma Ratio in Rats.

[14C]BMS-562086 in 0.5% (w/v) aqueous methylcellulose was administered at a target dose level of 5 mg/kg (100 μCi/kg) as a single oral gavage dose to three fasted male Long-Evans rats. Blood samples were collected at 1 h after dosing and were pooled together. A portion of the blood was centrifuged for 10 min at 1000g and 5°C for generation of plasma. Aliquots of blood (approximately 0.05 g) were placed in cones and pads, dried, and combusted. The combustions were performed using a model 387 Sample Oxidizer (PerkinElmer Life and Analytical Sciences, Waltham, MA). The resulting 14CO2 was trapped in Carbo-Sorb E (PerkinElmer Life and Analytical Sciences). After the 14CO2 trapping, Permafluor E+ scintillation cocktail (PerkinElmer Life and Analytical Sciences) was added, and the mixture was analyzed with a Tri-Carb 3100TR liquid scintillation analyzer (PerkinElmer Life and Analytical Sciences) to determine radioactivity. Aliquots of plasma (0.05 g) were mixed with Emulsifier-Safe scintillation fluid (PerkinElmer Life and Analytical Sciences) and analyzed by a Tri-Carb 3100TR liquid scintillation analyzer to determine radioactivity.

LC-MS/MS for Biotransformation Analysis.

High-performance liquid chromatography (HPLC) analysis was performed on a Shimadzu Class VP system equipped with two pumps (model LC-10AD), an autoinjector (model SIL-10AD), and a photodiode array detector (model SPD-M10A) (Shimadzu, Kyoto, Japan). Chromatographic separation was performed with a YMC PRO (Waters, Milford, MA) C18 column (2.0 × 150 mm, 3.0 μm) maintained at 35°C in an Eppendorf CH-30 column heater with an Eppendorf TC-45 temperature controller. The solvent system consisted of solvent A (10 mM ammonium acetate buffer, pH 4.1) and solvent B (acetonitrile/10 mM ammonium acetate buffer, pH 4.1, 90:10, v/v). A linear gradient system was used where solvent B was increased from 0 (0) to 100% (60 min) before re-equilibrium. The flow rate was 0.2 ml/min. For the mass spectrometric analysis, the eluent from the HPLC was directed to an LCQ DECA XP ion trap mass spectrometer (Thermo Fisher Scientific) interfaced with an electrospray ionization probe in the positive ion mode. The capillary temperature was kept at 275°C. The nitrogen gas flow rate, spray current, and voltages were adjusted to give the maximal sensitivity.

Assays of BMS-562086 in Plasma of Rats, Dogs, and Chimpanzees.

Plasma from pharmacokinetic studies in rats, dogs, and chimpanzees was analyzed for unchanged BMS-562086 by a validated LC-MS/MS method. The internal standard used was [13C]D3-BMS-562086. A 70-μl aliquot of plasma/internal standard mixture was extracted by adding 3 volumes of acetonitrile. Then the sample was vortexed for 2 min, followed by centrifugation at 14,000 rpm for 2 min in an Eppendorf model 5417C centrifuge (Eppendorf AG). The supernatant was directly analyzed by LC-MS/MS. The HPLC system consisted of two Class VP Shimadzu pumps (model LC-10AD) and a PE 200 LC autosampler (PerkinElmer Life and Analytical Sciences). A YMC ODS AQ (Waters) C18 column (2.0 × 50 mm, 5.0 μm) was used with an isocratic solvent system consisting of 20% solvent A, 0.1% formic acid in water, and solvent B, 0.1% formic acid in acetonitrile. The flow rate was 0.3 ml/min. The entire column elute was directly introduced into a Quattro mass spectrometer (Waters) equipped with an electrospray ionization probe. Analyses were performed in the positive ion mode. Ultrahigh-purity nitrogen was used as the nebulizing gas and desolvation gas. Other parameters were adjusted as needed to achieve maximal sensitivity. Multiple reaction monitoring mode was used for detection of BMS-562086 and its internal standard [13C]D3-BMS-562086. The transition m/z 341.02 → m/z 285.04 was used for BMS-562086 detection, and m/z 344.96 → m/z 289.04 was used for [13C]D3-BMS-562086 detection. Both BMS-562086 and its internal standard [13C]D3-BMS-562086 were eluted at retention time 1.09 min. The standard curve range for the plasma assay was 1.00 to 1000 ng/ml. Spiked analytical quality control (QC) samples were analyzed along with the study samples for assessment of the accuracy and precision of each analytical run. The QC samples at low, medium, and high concentration range of the standard curve were included in the analytical run in a minimum of two replicates. Deviations from the predicted concentrations for at least two thirds of the QC samples were within ±15% of their nominal values at all concentration levels, with at least one QC sample at each level meeting the acceptance criteria. The lower limits of quantification of the assay were 1.00 ng/ml.

Pharmacokinetic Analysis.

Plasma concentration versus time data for BMS-562086 in rats, dogs, and chimpanzees were analyzed by a noncompartmental method using Kinetica 4.2 within the eToolbox (version 2.2; Thermo Fisher Scientific). The peak concentration (Cmax) and the time to peak concentration (Tmax) were obtained from experimental observations. The terminal log-linear phase of the concentration-time curve was identified by least-squares linear regression of at least three data points that yield maximal G criteria. The t1/2 of the terminal log-linear phase was calculated as ln2/k, where k is the absolute value of the slope of the terminal log-linear phase, known as the elimination rate constant. The area under the concentration-time curve, AUC(INF), was determined by using the mixed log-linear trapezoidal method. The plasma clearance (CLp) was calculated as the quotient of dose and AUC(INF) after intravenous administration. Terminal volume of distribution (Vz) was obtained from dividing the CLp by the elimination rate constant after intravenous administration. The absolute oral bioavailability (F) was estimated as the ratio of dose-normalized AUC values after oral and intravenous doses.

Permeability Analysis.

The apparent permeability coefficients (Papp, cm/s) through Caco-2 cell monolayers were calculated according to the following equation: Embedded Image where dQ/dt (nmol/s) is the appearance rate of the compound in the receiver compartment (the BL compartment for AP to BL transport); A is the surface area of the monolayer (1 cm2); and C0 is the initial concentration of the compound administered in the donor compartment (the AP compartment for AP to BL transport). The appearance rates (dQ/dt) were calculated by plotting the amounts of compound transported to the receiver side versus time and determining the slopes of these plots in the linear range. The correlation coefficients (r2) obtained from the least-squares linear regression analysis were in the range of 0.96 to 1.00.

Statistical Analysis.

Student's t test was used for statistical analysis with a P value of less than 0.05 being considered statistically significant.

Prediction of Human Clearance and Volume of Distribution.

The allometric scaling method (Mahmood and Balian, 1996) was used to predict CLp and volume of distribution values in humans. In the simple allometric scaling method, body weights of the animals were used to allometrically scale total CLp values from rats, dogs, and chimpanzees to humans on a log-log scale by using the equation below, where BW is species body weight (0.34, 11.2, 60, and 70 kg for rats, dogs, chimpanzees, and humans, respectively) and a and b are the coefficient and exponent of the allometric equation, respectively: CL = a × BWb.

Likewise, allometric scaling was used to estimate human Vz by using both the body weight of the animals and the corresponding animal terminal volume of distribution as follows: Vz = a × BWb, where BW is species body weight (0.34, 11.2, 60, and 70 kg for rats, dogs, chimpanzees, and humans, respectively) and a and b are coefficient and exponent of the allometric equation, respectively.

Prediction of Human Plasma Profiles.

The SimCYP population-based ADME simulator (version 10; SimCYP Ltd.) was used to perform human plasma profile simulation. The built-in advanced dissolution, absorption, and metabolism model and the minimal physiology-based pharmacokinetic model were used to construct the final prediction model. A healthy female volunteer population was used for the demographics data as provided in the software. Human plasma profiles were simulated only in women to be consistent with the clinical trial populations defined by preclinical safety evaluation (Sweetser et al., 2009; Coric et al., 2010). Each dose (oral, once daily for 3 weeks) was simulated with 10 trials and 10 subjects in each trial, which led to a total of 100 simulations.

Results

Permeability of BMS-562086 across Caco-2 Cell Monolayers.

A permeability coefficient (Papp) of 1.7 × 10−5 cm/s was obtained for transport of BMS-562086 in the AP to BL direction across Caco-2 cell monolayers. Mannitol, a probe of the Caco-2 cell monolayer integrity, had a Papp value of 2.1 × 10−7 cm/s for AP to BL in the studies, which was comparable to the reported value in the literature (Zhou et al., 2002).

Serum Protein Binding.

The extent of protein binding determined in fresh rat, dog, and human sera was 93.6, 95.2, and 96.1%, respectively, suggesting high protein binding for BMS-562086. BMS-562086 did not show concentration-dependent protein binding over the concentration range tested (12.8–38.5 μM). Degradants of BMS-562086 were not detected in the serum samples over the 3-h incubation period, indicating BMS-562086 was stable under the conditions used for protein binding.

Metabolism in Hepatocytes.

The biotransformation profiles in mouse, rat, dog, monkey, and human hepatocytes after incubation with BMS-562086 are shown in Fig. 1. Metabolites of BMS-562086 were numbered on the basis of their chronologies of identification and did not follow HPLC elution order. HPLC analysis of the positive control samples, 7-EC and 7-HC, showed that the rates of formation of the 7-HC from 7-EC and formation of 7-HC glucuronide and 7-HC sulfate from 7-HC were comparable to historical data, suggesting competent oxidative and conjugative enzyme activities in hepatocytes (data not shown).

Fig. 1.
Fig. 1.

Biotransformation profiles in mouse, rat, dog, monkey, and human hepatocytes after incubation of 25 μM BMS-562086 for 3 h.

Approximately 75, 68, 61, 83, and 40% of parent disappeared from the incubation of BMS-562086 (25 μM) with mouse, rat, dog, monkey, and human hepatocytes for 3 h, respectively. The rate of disappearance of BMS-562086 followed the order of monkeys > mice ≈ rats ≈ dogs > humans. There were a total of 15 metabolites identified. Five oxidative metabolites, DPH-123554, DPH-124921, BMS-572273, M3, and M6 were observed in hepatocytes across all five species. Metabolite M9, an oxidative metabolite that was present in human hepatocytes, was also formed in rat and dog hepatocytes, but not in mouse and monkey hepatocytes. Furthermore, glucuronides (M1, M4, M12, and M13) and sulfate conjugate (M10) were detected in animal species, but not in human hepatocytes.

Metabolism in Microsomes.

The oxidative metabolites identified in human microsomal incubations, namely, DPH-123554, DPH-124921, BMS-572273, M3, M6, and M9, were also present in the human hepatocyte incubation mixtures and were detected at least in mouse microsomal preparation (Table 1). No conjugative metabolites were observed in the microsomal incubations because the microsomes were not fortified with cofactors necessary for conjugation reactions.

View this table:
TABLE 1

LC-MS/MS characterization of BMS-562086 and its metabolites identified in mouse, rat, dog, monkey, and human microsomes and hepatocytes, and in BDC rats

A relative formation of BMS-562086 (25 μM) incubated in human liver microsomes for 30 min in the presence or absence of ketoconazole (5 μM) is shown in Fig. 2. DPH-123554 and M6 were the major metabolites identified (measured by UV at 280 nm) in human liver microsomes. The formation of metabolites DPH-123554, M6, and M9 in human liver microsomes was highly sensitive to ketoconazole inhibition (∼80% inhibition) (Fig. 2). The formation of metabolites DPH-124921, BMS-572273, and M3 was not sensitive to ketoconazole inhibition (Fig. 2).

Fig. 2.
Fig. 2.

A relative formation of metabolites of BMS-562086 (25 μM) in the presence (filled symbol) or absence (open symbol) of ketoconazole (5 μM) in human liver microsomes after a 30-min incubation.

Metabolism in BDC Rats.

The biotransformation profiles of 0- to 12-h pooled urine, 0- to 12-h pooled bile, and 0- to 12-h pooled bile hydrolyzed with glucuronidase are shown in Fig. 3. Unchanged BMS-562086 was not detected in either rat urine or bile. A number of oxidative and conjugated metabolites were detected in urine and bile. Five oxidative metabolites (DPH-123554, DPH-124921, BMS-572273, M2, and M3) and two glucuronide metabolites (M1 and M4) were detected in rat urine. Five oxidative metabolites (DPH-123554, BMS-572273, M2, M3, and M6) and three glucuronide metabolites (M1, M4, and M13) were detected in rat bile. Glucuronide metabolites (M4 and M13) were the major metabolites in bile.

Fig. 3.
Fig. 3.

The biotransformation profiles of 0- to 12-h pooled urine (A), 0- to 12-h pooled bile (B), and 0- to 12-h pooled bile hydrolyzed with glucuronidase (C) after single oral administration of 30 mg/kg BMS-562086 to rats (n = 3).

The chromatographic pattern of rat bile changed upon incubation with β-glucuronidase. The peaks for M4 and M13 reduced considerably accompanied by the concomitant appearance of the corresponding aglycone peaks M11 and M9, respectively (Fig. 4, B and C). Likewise, the peak for metabolite M1 reduced markedly with the appearance of an aglycone peak M14. The disappearance of metabolites M1, M4, and M13 after hydrolysis by β-glucuronidase confirmed that all three metabolites are glucuronide conjugates. All the metabolites, except M14, that were detected in rat urine and bile were also observed in rat hepatocyte incubation.

Fig. 4.
Fig. 4.

Plasma concentration versus time profiles for BMS-562086 in rats, dogs, and chimpanzees after a single intravenous and oral administration. Plasma BMS-562086 was measured by HPLC-MS/MS. The inserts show the plasma concentration profiles at 1 to 4 h.

Pharmacokinetics of BMS-562086 in Rats, Dogs, and Chimpanzees.

The plasma concentration versus time profiles for BMS-582086 after a single intravenous and oral administration to rats, dogs, and chimpanzees are shown in Fig. 4. The pharmacokinetic parameters of BMS-562086 after the intravenous and oral administration to rats, dogs, and chimpanzees are summarized in Table 2. After the intravenous bolus dose, the plasma BMS-562086 concentrations exhibited a multiexponential decline in rats, dogs, and chimpanzees. The CLp of BMS-562086 was higher in rats (17.9 ml · min−1 · kg−1) and dogs (11.6 ml · min−1 · kg−1) than in chimpanzees (2.0 ml · min−1 · kg−1). Assuming the value of CLp of BMS-562086 approximates the value of CLb in these three species, BMS-562086 has an estimated hepatic extraction ratio of 0.32, 0.38, and 0.08 in rats, dogs, and chimpanzees, respectively (calculated by dividing CLp by respective hepatic blood flow, 55.2, 30.9, and 25.5 ml · min−1 · kg−1 for rats, dogs, and chimpanzees) (Davies and Morris, 1993; Wong et al., 2004). The assumption that CLb is equal to CLp is reasonable at least in rats, where the blood to plasma concentration ratio of BMS-562086-equivalent radioactivity was 0.95 at 1 h postdose. The average Vz of BMS-582086 in the rats, dogs, and chimpanzee was 14.9, 28.2, and 4.2 l/kg, respectively. The Vz was approximately 22-, 47-, and 7-fold greater than total body water for rats (0.67 l/kg), dogs (0.60 l/kg), and chimpanzees (0.60 l/kg), respectively, indicating extensive extravascular distribution. The terminal plasma elimination t1/2 values ranged from 13 to 43 h in rats, dogs, and chimpanzees. After oral administration, peak plasma concentrations (Tmax) were achieved in less than 2 h for all three species, indicating a rapid oral absorption. BMS-562086 was orally bioavailable in rats, dogs, and chimpanzees, with an absolute oral bioavailability of 40.1, 58.8, and 58.5%, respectively.

View this table:
TABLE 2

Pharmacokinetic parameters (mean ± S.D.) of BMS-562086 in rats, dogs, and chimpanzees

LC-MS/MS Characterization of the Metabolites.

The LC-MS/MS characterization of BMS-562086 and its metabolites identified in mouse, rat, dog, monkey, and human microsomes and hepatocytes, and in BDC rats, are shown in Table 1. The mass spectral fragmentation patterns for BMS-562086 and its metabolites are illustrated on the structures listed at the top of Table 1. The identification for BMS-562086, DPH-123554, DPH-124921, and BMS-572273 was based on its tandem mass spectrometry (MS/MS) fragmentation and comparison of its chromatographic retention times with the reference standards. The structures of the other metabolites were proposed by their LC-MS/MS fragmentation patterns.

BMS-562086 showed a protonated molecular ion at m/z 341. The base peak in the product ion spectrum was observed at m/z 285, consistent with a neutral loss of a butylene molecule (C4H8, 56 Da). A loss of a molecule of C4H9NHCN (98 Da) from the protonated molecular ion gave rise to a product ion at m/z 243.

DPH-123554, formed by O-demethylation of BMS-562086, had a protonated molecular ion at m/z 327. Its product ion spectrum yielded one abundant ion at m/z 271 and one minor ion at m/z 229, attributable to a neutral loss of a butylene molecule (C4H8) and a C4H8NHCN molecule from the protonated molecular ion, respectively.

DPH-124921, formed by N-dealkylation of BMS-562086, showed a protonated molecular ion at m/z 285. The product ion spectrum obtained from m/z 285 exhibited two abundant ions at m/z 270, a loss of •CH3; and m/z 243, a loss of NH2CN.

BMS-572273, formed by N-dealkylation followed by hydrolysis of BMS-562086, had a protonated molecular ion at m/z 286, which is 1 Da higher than that of DPH-124921. The product ion spectrum of m/z 286 showed a loss of HOCN, resulting in a fragment ion at m/z 243.

Metabolite M1 was identified as a glucuronide conjugate of the oxidized and N-dealkylated product of BMS-562086, namely, a glucuronide conjugate of the oxidized product of DPH-124921. The protonated molecular ion of metabolite M1 was observed at m/z 477. Fragmentation of the molecular ion at m/z 477 yielded a major ion at m/z 301, attributable to a neutral loss of 176 Da from the protonated molecular ion, indicating that M1 was a glucuronide conjugate. The ion at m/z 301 is 16 Da higher than the protonated molecular ion of DPH-124921 (m/z 285), suggesting that the ion at m/z 301 structurally represented an oxidized product of DPH-124921. An additional loss of a H2O molecule from the ion at m/z 301 generated a minor ion at m/z 283 in the product ion spectrum.

Metabolite M2 was identified as a product of bis-oxidation followed by dehydrogenation of BMS-562086. The oxidation positions were determined to have occurred on the N-alkyl side chain of the molecule. The protonated molecular ion of metabolite M2 was observed at m/z 371, which is 30 Da higher than that of BMS-562086. Upon MS2 analysis, M2 showed a product ion at m/z 243, the same fragment ion as that of BMS-562086, DPH-124921, and BMS-572273, suggesting that they all share the same core aromatic ring structure. Therefore, the oxidation positions were assigned on the N-alkyl side chain of the molecule.

Metabolite M3 was an oxidation and O-demethylation product of BMS-562086 (the oxidized product of DPH-123554) with the oxidation site occurring on the N-alkyl side chain of the molecule. The protonated molecular ion of metabolite M3 was observed at m/z 343, which is 16 Da higher than that of DPH-123554 (O-demethylation of BMS-562086). Two prominent product ions were observed in the product ion spectrum upon fragmentation of the molecular ion at m/z 343. The presence of a product ion at m/z 229, which was also a product ion of DPH-123554, suggested that the oxidation has occurred on the N-alkyl side chain of the molecule. The base peak in the product ion spectrum, m/z 272, was consistent with the replacement of the N-alkyl side chain of the molecular ion with an −OH group.

Metabolite M4 was determined to be a glucuronide conjugate of the bis-oxidation product of BMS-562086 (a glucuronide conjugate of metabolite M11). The protonated molecular ion of M4 was observed at m/z 549. The base peak in the product ion spectrum, m/z 373, was generated by a neutral loss of 176 Da, indicating that M4 is a glucuronide conjugate. The MS3 spectrum of M4 (m/z 549 → m/z 373 →) contained product ions at m/z 355, 302, and 259, which are consistent with a bis-oxidation product of BMS-562086 (metabolite M11, to be characterized below), supporting the assignment of M4 as a glucuronide conjugate of the bis-oxidation product of BMS-562086.

Metabolites M5, M6, and M7 were positional isomers, each identified as the product of bis-oxidation coupled with dehydrogenation of DPH-123554 (the O-demethylated BMS-562086). The oxidation position was tentatively assigned to occur on one of the three methyl groups on the pyrazolotriazine ring moiety. M5, M6, and M7 were chromatographically adjacent to one another, and all had a protonated molecular ion at m/z 357, which is 30 Da higher than that of DPH-123554 (or 16 Da higher than that of the parent). The spectrum of molecular ion at m/z 357 for all three metabolites showed fragment ions at m/z 329, 286, and 243. The ion at m/z 329 was the characteristic ion, produced by a loss of CO (28 Da), suggesting the presence of a −COOH group in the structure (formed by bis-oxidation coupled with dehydrogenation). The product ion at m/z 243 was likely produced by a loss of C5H9N2• (97 Da) and OH• (17 Da) from the protonated molecular ion. The product ion at m/z 286 was proposed to arise from a loss of C5H8NH, a molecule derived from the N-alkyl side chain.

Metabolites M8 and M9 were positional isomers of monohydroxylation products of BMS-562086. The hydroxylation positions were determined to occur on the N-alkyl side chain. Both metabolites showed a protonated molecular ion at m/z 357, 16 Da higher than that of the parent. The major product ions at m/z 286 and 243 are the same fragment ions as those of M2, suggesting that M2, M8, and M9 have the same core aromatic ring structure. Because metabolites M8 and M9 fragmented in a similar pattern, the exact oxidation position could not be further established.

Metabolite M10 was identified as a sulfate conjugate of the hydroxylated BMS-562086. M10 had a protonated molecular ion at m/z 437. Fragmentation of m/z 437 yielded a predominant ion at m/z 357, attributable to a neutral loss of 80 Da from the molecular ion, which indicated that metabolite M10 is a sulfate conjugate. MS3 analysis of m/z 357 (m/z 437 → m/z 357 →), produced fragments at m/z 301 and 283, consistent with a neutral loss of a butylene molecule (C4H8, 56 Da) and a further loss of H2O from the protonated molecule of hydroxylated BMS-562086, respectively. The fact that a butylene molecule (C4H8) was observed (m/z 357 → m/z 301), as seen in the fragmentation of protonated molecular ion of BMS-562086 (m/z 341 → m/z 285, a loss of 56 Da), suggested that the hydroxylation position was not on the N-alkyl side chain.

Metabolite M11, the aglycone of metabolite M4, was identified as a bis-oxidation product of BMS-562086. M11 had a protonated molecular ion at m/z 373 (32 Da higher than that of BMS-562086) and major MS2 product ions at m/z 355, 302, and 259. The product ion at m/z 355 was consistent with a loss of H2O from the protonated molecular ion at m/z 373. The product ion at m/z 302 resulted from a loss of the N-alkyl side chain followed by addition of a hydroxyl group. A loss of a molecule of (HO)C4H9NHCN (98 Da) from the protonated molecular ion gave rise to a product ion at m/z 259.

Metabolite M12 was identified as a glucuronide conjugate of an oxidation product of BMS-562086. Metabolite M12 had a protonated molecular ion at m/z 533, which is 176 Da higher than that of the oxidation product of BMS-562086. MS3 analysis of m/z 357 (m/z 533 → m/z 357 →) produced fragments at m/z 301, consistent with a neutral loss of a butylene molecule (C4H8, 56 Da). The fact that a butylene molecule (C4H8) was observed (m/z 357 → m/z 301), as seen in the fragmentation of protonated molecular ion of BMS-562086 (m/z 341 → m/z 285, a loss of 56 Da), suggested that the oxidation position was not on the N-alkyl side chain.

Metabolite M13 was identified as the glucuronide conjugate of M9, an oxidation product of BMS-562086. The protonated molecular ion of metabolite M13 was observed at m/z 533. Fragmentation on m/z 533 gave an ion at m/z 357, attributable to a neutral loss of 176 Da from the molecular ion, indicating that M13 is a glucuronide conjugate. The MS3 analysis of m/z 357 (m/z 533 → m/z 357 →), produced fragments at m/z 243 and 286, a fragmentation pattern similar to that of M9. These data, along with the fact that the reduced peak for M13 after hydrolysis of rat bile by β-glucuronidase was accompanied by the concomitant appearance of the peak for M9 (Fig. 4, B and C), suggested that Metabolite M13 was the glucuronide conjugate of M9.

Metabolite M14, the aglycone of M1, was identified as an oxidized and N-dealkylated product of BMS-562086. Metabolite M14 was observed in rat bile only after hydrolysis by β-glucuronidase. Its fragmentation pattern has been assigned earlier, along those of M1.

Prediction of Human Pharmacokinetic Parameters.

Human plasma CL and Vz values predicted for BMS-562086 by the allometric scaling method are shown in Fig. 5. The human plasma CL and Vz values predicted for BMS-562086 were 2.97 ml · min−1 · kg−1 and 7.79 l/kg (208 ml/min and 545 liter for a 70-kg human), respectively. The coefficient (a) and exponent (b) of the allometric equation used for the estimation of CL were 14.80 and 0.62, respectively. The coefficient (a) and exponent (b) of the allometric equation used for the estimation of Vz were 16.74 and 0.82, respectively. The correlation coefficients (R2) for the estimation of CL and Vz were 0.88 and 0.87, respectively.

Fig. 5.
Fig. 5.

Prediction of plasma clearance (left) and steady-state volume of distribution (right) of BMS-562086 in humans by interspecies allometric scaling.

Prediction of Human Plasma Profiles.

The human projected plasma concentration-time profile of BMS-562086 after a once-daily oral dose of 100 mg of BMS-562086 for 3 weeks was simulated using the SymCYP population-based ADME simulator (Fig. 6). The simulated plasma concentration-time profile indicated that the steady-state plasma concentration was achieved by the end of week one, with the total mean plasma concentration values in the range of 925 to 1300 nM (trough to peak). The individual trials suggested the variability was likely moderate, with trough-peak values in the range of 725 to 1530 nM in the healthy female volunteer population.

Fig. 6.
Fig. 6.

Human projected plasma concentration-time profiles of BMS-562086 after a once-daily oral dose of 100 mg of BMS-562086 for 3 weeks were simulated using a SymCYP population-based ADME simulator. Simulations were performed with 10 virtual trials and 10 subjects in each trial, which led to a total of 100 virtual trials.

Discussion

Metabolism.

The primary biotransformation pathways found for BMS-562086 in both liver microsomal and hepatocyte preparations and in rats included: 1) O-demethylation to DPH-123554, 2) hydroxylation at the N-alkyl side chain to metabolites M8 and M9, and 3) N-dealkylation to DPH-124921. BMS-562086 was further metabolized via multiple pathways including oxidation, glucuronidation, and sulfation of the oxidative products, and a combination of the above pathways. The proposed biotransformation pathways for BMS-562086 are shown in Fig. 7. Because the primary biotransformation pathways are oxidative in nature, cytochrome P450 enzymes play a critical role in the overall metabolic clearance of BMS-562086. Furthermore, the absence of direct conjugative metabolites suggests a limited role for the conjugative enzymes in overall metabolic clearance. The formation of the two prominent primary metabolites, DPH-123554 and M9, was highly sensitive to ketoconazole inhibition in human liver microsomes, indicative of the significant involvement of CYP3A4/5 in the BMS-562086 metabolic elimination. The formation of the primary metabolite DPH-124921 was not sensitive to ketoconazole inhibition, suggesting the metabolic elimination of BMS-562086 was not entirely dependent on CYP3A4/5. Taken together, the metabolic clearance of BMS-562086 was mediated by multiple cytochromes P450 including CYP3A4/5. Even though the fractions metabolized through these pathways are not known, the involvement of multiple enzymes in the overall metabolic clearance should reduce the potential for drug-drug interaction through modulation of a single elimination pathway.

Fig. 7.
Fig. 7.

Proposed biotransformation pathways for BMS-562086. The proposed MS/MS fragmentation is marked with dashed lines. The major fragment ions are also denoted on the structures.

No unique human metabolites were detected in human liver microsomes and hepatocytes. The in vitro metabolic profiles were qualitatively similar across mice, rats, dogs, and humans. The similarity among biotransformation profiles across the five species for BMS-562086 helps in justifying the selection of the animal species to be used for toxicological evaluation. DPH-123554 and DPH-124921 were identified as the prominent metabolites of BMS-562086 in both liver microsomal and hepatocyte preparations and in vivo in rats. Both metabolites were inactive toward rat CRF binding with IC50 values >1000 nM, suggesting that they are unlikely to contribute to the pharmacology of BMS-562086. BMS-562086 was significantly metabolized in hepatocytes in all species. BMS-562086 was not detected in rat urine and bile, indicating the absorbed compound was metabolized completely before elimination in rats. BMS-562086-derived metabolites were found in both urine and bile of rats, suggesting both urinary and biliary pathways were involved in the elimination of the metabolites in rats (Fig. 3). Because of the significant contribution of metabolism to its overall elimination, an evaluation of the impact on pharmacokinetics of BMS-562086 in subjects with hepatic impairment is warranted in future clinical studies. On the other hand, pharmacokinetics of BMS-562086 in subjects with renal impairment is not expected to be altered because of the lack of contribution of renal excretion to its total clearance.

ADME studies in preclinical species and humans with radiolabeled material are typically performed to provide quantitative understanding of the role of each metabolic pathway in the elimination of a compound. However, earlier studies in discovery are conducted with nonradiolabeled compound. Nonetheless, the results of the metabolic in vitro and in vivo investigation provide key information for the appropriate placement of the radiolabel. If the label is incorporated in a metabolically labile moiety of the molecule, there is a chance that it may be liberated through metabolism. If the labile moiety is similar to an endogenous compound, it may get incorporated into cellular components. These events could lead to poor recovery of radioactivity and could pose safety concern because there could be extended tissue exposure to radioactivity. The metabolites derived from BMS-562086 were produced by modifications mainly of the side-chain groups, whereas the core pyrazolotriazine ring structure was intact in all of the metabolites identified. Therefore, introducing the radiolabel on the pyrazolotriazine ring rather than the side chains would ensure a good recovery of radioactivity and would not lead to endogenous incorporation of the label.

Absorption.

Permeability of BMS-562086 across Caco-2 cell monolayers in the absorption direction was high and comparable to the value for compounds that exhibit good absorption in humans (Zhou et al., 2002). Permeability of BMS-562086 in the secretory direction was not done in this study. Thus, it is not known whether BMS-562086 is a substrate of efflux transporter such as P-glycoprotein, according to Caco-2 results. However, preliminary work with isolated rat duodenum, jejunum, ileum, and colon muscle layers demonstrated that the BL-to-AP permeability was not higher than the AP-to-BL permeability, suggesting that BMS-562086 may not be a substrate of efflux transporters (data not shown). The high permeability determined for BMS-562086 by the Caco-2 experiments was consistent with its good oral bioavailability observed in animals. The absolute oral bioavailability of BMS-562086 achieved 40.1, 58.8, and 58.5% in rats, dogs, and chimpanzees, respectively. Both high permeability and an extensive metabolism profile of BMS-562086 agree well with the observation made by Benet (2010) who recognized that the major route of elimination for highly permeable drugs was mainly through metabolism.

Prediction of Human Pharmacokinetics.

The extraction ratio characterized BMS-562086 as a moderate hepatic clearance compound in rats and dogs, but a low hepatic clearance compound in chimpanzees. The chimpanzees are the closest living relatives to humans and are viewed as a valuable surrogate model for human ADME studies (Wong et al., 2004). The chimpanzee was chosen as a pharmacokinetic model in this study to aid in the selection of drug candidates for clinical development.

The plasma clearance of BMS-562086 was predicted to be 2.97 ml · min−1 · kg−1 for a 70-kg human. The predicted CLp represented a hepatic extraction ratio of 0.14 if compared with a hepatic blood flow of 20.7 ml · min−1 · kg−1 in humans and assumed that the CLp had a similar value to CLb. The predicted Vz value in humans was 545 liter (7.79 l/kg) for a 70-kg human, a value approximately 13-fold greater than that of the total body water (42 liter). Thus, the human clearance and Vz values predicted by the allometric scaling method suggested low plasma clearance and significant extravascular distribution for BMS-562086 in humans. The elimination t1/2 was expected to be long in humans. The allometric equation for clearance prediction gave an exponent of 0.62, which was between the lower and upper limit values of 0.55 and 0.70 to indicate a reasonable good clearance prediction (Mahmood and Balian, 1996). This prediction was validated in the single ascending dose studies where the predicted human clearance of BMS-562086 matched the observed human clearance (data not shown). The human plasma concentration-time profiles of BMS-562086 were simulated using the SymCYP population-based ADME simulator (Fig. 6). The simulation results suggested BMS-562086 was able to achieve the mean trough concentration values above the projected human efficacious concentration of >500 nM after a 100-mg daily oral dose (Coric et al., 2010). Furthermore, the mean trough concentration values for each individual trial were all above 500 nM, suggesting that the targeted concentration would be achieved in a majority of subjects rather than just an “average one” (Cubitt et al., 2011). The simulated steady-state concentrations (925–1300 nM) were within 2-fold of the observed clinical serum concentrations of BMS-562086 at weeks 4 to 6, where the average mean concentration was 1630 nM after a 100-mg daily oral administration to patients with generalized anxiety disorder (Sweetser et al., 2009; Coric et al., 2010).

In summary, BMS-562086 exhibited favorable ADME and pharmacokinetic properties, making it a suitable candidate for further development. BMS-562086 was rapidly absorbed in rats, dogs, and chimpanzees with good oral bioavailability. Metabolism studies suggested that multiple pathways, including CYP3A4/5-dependent and -nondependent reactions, mediated the metabolic clearance of BMS-562086. BMS-562086 was metabolized completely before elimination in rats. Both renal and biliary excretion played a role in the elimination of BMS-562086 metabolites. A 100-mg daily dose was predicted to be the minimal daily dose to provide sufficient drug exposure to exhibit efficacy. The compound was therefore chosen to advance to clinical development as a potentially effective agent intended for the oral treatment of generalized anxiety disorder and IBS (Sweetser et al., 2009; Coric et al., 2010).

Authorship Contributions

Participated in research design: Zhou, Dockens, Grossman, and Iyer.

Conducted experiments: Zhou and Liu-Kreyche.

Contributed new reagents or analytic tools: Zhou and Liu-Kreyche.

Performed data analysis: Zhou and Dockens.

Wrote or contributed to the writing of the manuscript: Zhou, Dockens, and Iyer.

Footnotes

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

    http://dx.doi.org/10.1124/dmd.111.043596.

  • ABBREVIATIONS:

    CRF
    corticotropin-releasing factor
    IBS
    irritable bowel syndrome
    BMS-562086
    8-(6-methoxy-2-methyl-3-pyridinyl)-2,7-dimethyl-N-[(1R)-1-methylpropyl]pyrazolo(1,5-a)-1,3,5-triazin-4-amine
    ADME
    absorption, distribution, metabolism, and excretion
    BDC
    bile duct-cannulated
    DPH-123554
    O-demethylated metabolite of BMS-562086
    DPH-124921
    N-dealkylated metabolite of BMS-562086
    BMS-572273
    N-dealkylated and hydrolyzed metabolite of BMS-562086
    AP
    apical
    BL
    basolateral
    TEER
    transepithelial electrical resistance
    7-EC
    7-ethoxycoumarin
    7-HC
    7-hydroxycoumarin
    LC-MS/MS
    liquid chromatography-tandem mass spectrometry
    HPLC
    high-performance liquid chromatography
    QC
    quality control
    Cmax
    maximal observed concentration
    Tmax
    time of maximal observed concentration
    AUC(INF)
    area under the plasma concentration-time curve from time zero extrapolated to infinite time
    CLb
    blood clearance
    CLp
    plasma clearance
    dQ/dt
    appearance rate
    VZ
    terminal volume of distribution
    F
    oral bioavailability.

  • Received October 29, 2011.
  • Accepted March 1, 2012.

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METABOLISM AND PHARMACOKINETICS OF BMS-562086

Lian Zhou, Randy C. Dockens, Peggy Liu-Kreyche, Scott J. Grossman and Ramaswamy A. Iyer
Drug Metabolism and Disposition June 1, 2012, 40 (6) 1093-1103; DOI: https://doi.org/10.1124/dmd.111.043596
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