pharmacokinetics, metabolism, and excretion of anacetrapib, a novel inhibitor of the cholesteryl...
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Pharmacokinetics, Metabolism, and Excretion of Anacetrapib, aNovel Inhibitor of the Cholesteryl Ester Transfer Protein, in Rats
and Rhesus Monkeys
Eugene Y. Tan,1 Georgy Hartmann, Qing Chen, Antonio Pereira, Scott Bradley,2 George Doss,Andy Shiqiang Zhang, Jonathan Z. Ho, Matthew P. Braun, Dennis C. Dean,3 Wei Tang, and
Sanjeev Kumar
Departments of Drug Metabolism and Pharmacokinetics (E.Y.T., G.H., Q.C., A.P., S.B., D.C.D., W.T., S.K.), MedicinalChemistry (G.D.), and Labeled Compound Synthesis (A.S.Z., J.Z.H., M.P.B.), Merck Research Laboratories, Rahway,
New Jersey
Received June 1, 2009; accepted December 16, 2009
ABSTRACT:
The pharmacokinetics and metabolism of anacetrapib (MK-0859),a novel cholesteryl ester transfer protein inhibitor, were examinedin rats and rhesus monkeys. Anacetrapib exhibited a low clearancein both species and a moderate oral bioavailability of �38% in ratsand �13% in monkeys. The area under the plasma concentration-time curve in both species increased in a less than dose-propor-tional manner over an oral dose range of 1 to 500 mg/kg. After oraladministration of [14C]anacetrapib at 10 mg/kg, �80 and 90% ofthe radioactive dose was recovered over 48 h postdose from ratsand monkeys, respectively. The majority of the administered ra-dioactive dose was excreted unchanged in feces in both species.Biliary excretion of radioactivity accounted for �15% and urinaryexcretion for less than 2% of the dose. Thirteen metabolites, re-
sulting from oxidative and secondary glucuronic acid conjugation,were identified in rat and monkey bile. The main metabolic path-ways consisted of O-demethylation (M1) and hydroxylation on thebiphenyl moiety (M2) and hydroxylation on the isopropyl side chain(M3); these hydroxylations were followed by O-glucuronidation ofthese metabolites. A glutathione adduct (M9), an olefin metabolite(M10), and a propionic acid metabolite (M11) also were identified.In addition to parent anacetrapib, M1, M2, and M3 metaboliteswere detected in rat but not in monkey plasma. Overall, it appearsthat anacetrapib exhibits a low-to-moderate degree of absorptionafter oral dosing and majority of the absorbed dose is eliminatedvia oxidation to a series of hydroxylated metabolites that undergoconjugation with glucuronic acid before excretion into bile.
A significant amount of effort is being put into developing thera-peutic agents that are capable of lowering low-density lipoprotein(LDL) levels because increased circulating levels of LDL have beendemonstrated to increase risk for cardiovascular disease and associ-ated clinical sequelae (Kannel et al., 1979; Castelli et al., 1983;Stamler et al., 1988). Hydroxymethylglutaryl coenzyme A reductaseinhibitors (statins) remain the cornerstone of LDL-lowering therapyand have been demonstrated to reduce cardiovascular risk in humans(Shepherd et al., 1995). However, there remains a high incidence ofresidual cardiovascular events even after aggressive treatment with
LDL-lowering drugs (Bays and Stein, 2003). It has recently beenshown that, in addition to LDL, high-density lipoprotein (HDL)cholesterol is an independent factor that may modulate the risk ofcardiovascular disease. Epidemiological evidence suggests thatplasma HDL cholesterol levels are inversely correlated with athero-sclerosis and cardiovascular risk (Asztalos et al., 2004; Cobain et al.,2007). However, there are no clinical outcome data to support thishypothesis at the present time. One class of dyslipidemia agents thataffect HDL cholesterol levels are the inhibitors of cholesteryl estertransfer protein (CETP) (Hesler et al., 1987; Linsel-Nitschke and Tall,2005). CETP is a plasma glycoprotein that regulates the exchange ofcholesteryl esters and triglycerides between HDL and LDL cholester-ols (Dullaart et al., 1991; Tall, 1993), and, therefore, it has recentlybecome an attractive molecular target for the treatment of dyslipide-mia. Preliminary evidence from dalcetrapib (JTT-705, R1658; F.Hoffman-La Roche, Basel, Switzerland) and torcetrapib (Pfizer Inc.,New York, NY) indicated that CETP inhibition resulted in rapidaccumulation of cholesterol in HDL particles and, hence, caused an
1 Current affiliation: Department of Clinical Pharmacokinetics, Novartis, EastHanover, New Jersey.
2 Current affiliation: Lilly Research Laboratories, Indianapolis, Indiana.3 Current affiliation: Drug Metabolism and Pharmacokinetics, Vertex Pharma-
ceuticals, Cambridge, Massachusetts.Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.doi:10.1124/dmd.109.028696.
ABBREVIATIONS: LDL, low-density lipoprotein; HDL, high-density lipoprotein; CETP, cholesteryl ester transfer protein; LC, liquid chroma-tography; MS/MS, tandem mass spectrometry; HPLC, high-performance liquid chromatography; MRL, Merck Research Laboratories; AUC,area under the plasma concentration versus time curve; MS, mass spectrometry; CID, collision-induced dissociation; amu, atomic massunits; MK-0859, [4S,5R]-5-[3,5-bis(trifluoromethyl)phenyl]-3-{[4�-fluoro-2�-methoxy-5�-(propan-2-yl)-4-(trifluoromethyl)[1,1�-biphenyl]-2-yl]methyl}-4-methyl-1,3-oxazolidin-2-one.
0090-9556/10/3803-459–473$20.00DRUG METABOLISM AND DISPOSITION Vol. 38, No. 3Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 28696/3567558DMD 38:459–473, 2010 Printed in U.S.A.
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elevation of plasma HDL cholesterol concentration in humans (deGrooth et al., 2002; Clark et al., 2004; Dullaart et al., 2007). It hasbeen widely believed until recently that, through inhibition of CETPand elevation of HDL cholesterol, the formation of atheroscleroticplaques can be significantly reduced, thus improving the long-termclinical outcomes in cardiovascular disease. However, unfavorablecardiovascular outcome data from long-term treatment with torce-trapib have recently raised doubts as to whether CETP inhibitors willultimately provide clinical benefit (Tall, 2007), although it appearsthat cardiovascular outcomes from treatment with torcetrapib mayhave been confounded by its adverse blood pressure-raising effects inhumans (Howes and Kostner, 2007; Forrest et al., 2008).
Anacetrapib (MK-0859, [4S,5R]-5-[3,5-bis(trifluoromethyl)phenyl]-3-{[4�-fluoro-2�-methoxy-5�-(propan-2-yl)-4-(trifluoromethyl)[1,1�-biphenyl]-2-yl]methyl}-4-methyl-1,3-oxazolidin-2-one; Merck, White-house Station, NJ) (Fig. 1) is a selective inhibitor of CETP and isundergoing development as an oral drug for the treatment of primaryhypercholesterolemia and mixed hyperlipidemia. It is a potent CETPinhibitor with IC50 values of 16 and 29 nM for the CETP-mediatedtransfer of cholesteryl esters and triglycerides, respectively (J. A. Huntand P. J. Sinclair, unpublished data). In phase I and II clinical trials,anacetrapib raised HDL cholesterol levels by �130% and reduced LDLcholesterol levels by �40% with no associated major adverse events(Krishna et al., 2007, 2008; Bloomfield et al., 2009). In addition, itshowed no treatment-related effect on blood pressure in a 24-h ambula-tory blood pressure clinical trial (Krishna et al., 2007), suggesting that theblood pressure-raising effects with torcetrapib are probably molecule-and not target-specific and the clinical benefit of CETP inhibitors inatherosclerotic disease needs to be further assessed. In this regard, thesafety, tolerability, and favorable pharmacokinetic and pharmacodynamicproperties of anacetrapib have recently been assessed in a phase I clinicalstudy (Krishna et al., 2008).
In this study, we investigated the pharmacokinetics, metabolism,and excretion of anacetrapib in Sprague-Dawley rats and rhesusmonkeys, the two species that were used for the preclinical toxico-logical evaluation of this agent. Identification of excretory and circu-lating metabolites of novel, small molecule pharmaceutical agents inpreclinical toxicology species is an integral part of their developmentprogram to ascertain that toxicity of all human metabolites of theseagents is adequately characterized in preclinical studies (http://www.
fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm065014.htm). In cases where disproportionate amounts of humanmetabolites are observed relative to the preclinical toxicology species,the regulatory agencies recommend appropriate testing of these me-tabolites in separate preclinical toxicology studies. The present studieswere conducted after oral administration of [14C]anacetrapib to ratsand monkeys and metabolite profiling of radioactivity in plasma andexcreta was performed. Metabolites of anacetrapib were identified bymeans of LC-MS/MS. Where possible, metabolites were purified bypreparative LC, and the structures were confirmed by 1H NMR and/orvia comparison of HPLC retention time and mass spectral fragmen-tation with synthetic standards.
Materials and Methods
Drugs, Standards, and Reagents. Anacetrapib (MK-0859) (Fig. 1) andsynthetic standards of metabolites M1, M2, M3, and M10 were synthesized byMedicinal Chemistry and Process Research at Merck Research Laboratories(MRL) (Rahway, NJ). Free base [isopropyl-1-14C]anacetrapib and [methyl-O-13CD3]anacetrapib were synthesized by the Labeled Compound Synthesisgroup (MRL). The radiochemical purity was �98% and the specific activitywas �89 �Ci/mg. Liver microsomes from monkeys were prepared at MRL.All other reagents were obtained from commercial sources with an appropriatequality.
Pharmacokinetic Studies. All experiments were performed according toprocedures approved by the Merck Research Laboratories Institutional AnimalCare and Use Committee.
Rats. Adult male Sprague-Dawley rats, weighing �280 to 330 g, werefasted overnight before the study. The animals were allowed access to food 4 hafter dosing, but water was provided ad libitum. For intravenous administra-tion, four rats received a dose of anacetrapib (dosing volume 2.5 ml/kg) viabolus injection through a catheter previously implanted into the femoral vein,followed by a 300-�l saline flush. The intravenous dose of anacetrapib wasformulated in polyethylene glycol 300-water (7:3, v/v) at a concentration of 0.2mg/ml. For oral administration, four rats received a dose via oral gavage(dosing volume 2 ml/kg). These oral doses of 5, 50, 100, and 500 mg/kg wereformulated in Imwitor 742-Tween 80 (1:1, w/w) at concentrations of 2.5, 25,50, and 250 mg/ml, respectively. Blood samples (�0.3 ml) were collected intoEDTA-containing tubes at the following time points: predose and 0.083(intravenous only), 0.25, 0.5, 1, 2, 4, 6, 8, 24, and 48 h postdose. Bloodsamples were stored on ice, and plasma was prepared by centrifugation.Plasma was transferred to a 96-well plate and stored at �70°C until analysis.
Monkeys. For pharmacokinetic studies in monkeys, three adult male rhesusmacaques (Macaca mulatta), weighing �6 to 8 kg, were fasted overnight butallowed free access to water and transferred to restraint chairs the followingmorning. Indwelling catheters were placed by percutaneous venipunctureinto the saphenous veins for collection of blood. Animals were fed andallowed access to water 4 h postdose. For intravenous administration of 0.1mg/kg anacetrapib, three monkeys received a dose at 0.5 ml/kg via bolusinjection through an intravenous catheter, followed by a 2.5-ml saline flush.This intravenous dose of anacetrapib was formulated in polyethylene glycol300-water (7:3, v/v) at a concentration of 0.2 mg/ml. For oral administra-tion, three monkeys received a dose at 2 ml/kg via nasogastric intubationand gavage, followed by a 3.5-ml saline flush. These oral doses of 1, 30,and 500 mg/kg were formulated in Imwitor 742-Tween 80 (1:1, w/w) atconcentrations of 0.5, 15, and 250 mg/ml, respectively. Blood samples (1ml) were collected into EDTA-containing Vacutainer tubes at the followingtime points: predose and 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 24, and 48 hpostdose. Blood samples were stored on ice, and plasma was prepared bycentrifugation within 30 min. Plasma was transferred to a 96-well plate andstored at �70°C until quantitative analysis.
Quantitative Analysis of Anacetrapib in Plasma. Concentrations of anace-trapib in rat and monkey plasma were determined by LC-MS/MS after proteinprecipitation. Calibration curves were generated using triplicate sets of stan-dards. These standard samples were prepared by spiking 50 �l of controlplasma with 10 �l of stock solutions containing 10, 25, 50, 250, 500, 1000, and2500 ng of anacetrapib/ml of acetonitrile-water (20:80, v/v). This yieldedcalibration standards effectively containing 2, 5, 10, 50, 100, 200, and 500 ng
N
F
F
F
O
O
O
F
F
F
FF
F
F
*
FIG. 1. Structure of [14C]anacetrapib (MK-0859). � denotes the position of the 14Clabel.
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of analyte/ml of plasma. The monkey plasma samples obtained postdose weretreated as follows. From most sample vials, 50 �l of plasma was removed foranalysis. Samples were diluted with blank plasma when they were expected tocontain levels of analyte exceeding the upper limit of quantification. To all50-�l aliquots of standard and postdose plasma samples, 10 �l of 100 ng/ml[D3,13C]anacetrapib in acetonitrile-water (20:80, v/v) were added as an internalstandard. This step was followed by protein precipitation with 200 �l ofacetonitrile. Sample mixtures were vortex-mixed for 10 min, and 600 �l ofacetonitrile was added to each. After centrifugation for 15 min at 1600g and10°C, the resulting supernatants were harvested by a Tomtec Quadra 96 model320 system and dried at 25°C under a nitrogen stream in a Zymark TurboVap96 evaporator. The residues were reconstituted in 200 �l of mobile phase,vortex-mixed, centrifuged for 15 min at 1600g and 10°C, and analyzed byLC-MS/MS.
The LC-MS/MS system comprised a Leap Technologies HTS-PALautosampler, two Shimadzu LC-10ADvp pumps (Shimadzu, Columbia,MD), an SCL-10Avp system controller, a type W six-port Valco switchingvalve, and a Sciex API 4000 mass spectrometer. Chromatographic separa-tion of the analytes was achieved on an Aquasil C18 column (2.1 � 50 mm,5-�m particle size; Thermo Fisher Scientific, Waltham, MA) using iso-cratic elution with 80% acetonitrile and 20% water containing 10 mMammonium formate and 0.1% formic acid. Total run time for a singleinjection was 3.0 min. Mass spectrometric detection of the analytes wasaccomplished using the TurboIonSpray interface operated in the positiveionization mode. Analyte response was measured by multiple reaction moni-toring of selective mass transitions for each compound. The transitions of theprotonated precursor ions to the selected product ions were m/z 6383m/z 283 foranacetrapib and m/z 6423 m/z 287 for [D3,13C]anacetrapib.
Data were acquired and processed using Sciex Analyst 1.3.1 software.Calibration curves were generated by plotting the peak area ratios of analyte tointernal standard as a function of the nominal concentrations of the standardsamples. A line of best fit was generated from the curve points by linearregression with a weighting factor of 1/x2, and concentrations of anacetrapibwere interpolated from this calibration curve. The lower limit of quantitationfor this assay was 2 ng/ml (0.00314 �M) for anacetrapib.
Calculation of Pharmacokinetic Parameters. Pharmacokinetic parame-ters were calculated by established noncompartmental methods. The area underthe plasma concentration versus time curve (AUC) was determined usingWatson LIMS software (version 6.2.0.02), with linear trapezoidal interpolationin the ascending slope and linear-log linear trapezoidal interpolation in thedescending slope. The portion of the AUC from the last measurable concen-tration to infinity was estimated from the equation, Ct/kel, where Ct representsthe last measurable concentration and kel is the elimination rate constant. Thelatter was determined from the concentration versus time curve by linearregression at the terminal phase of the semilogarithmic plot.
In Vitro Reversible Plasma Protein Binding. [14C]Anacetrapib in ethanol(2.5 �l) was added to rat or monkey plasma (497.5 �l) to achieve finalconcentrations of 1, 5, and 20 �M. Equilibrium dialysis of plasma samples wasperformed for 6 h against isotonic phosphate buffer (pH 7.4, 1.8 g/l KH2PO4,7.6 g/l Na2HPO4, and 3.9 g/l NaCl) at 37°C in 1-ml acrylic dialysis cells(Scienceware; Bel-Art Products, Pequannock, NJ). The plasma and buffer wereseparated by a membrane (Sigma-Aldrich, St. Louis, MO) that had a molecularmass cutoff of 12.4 kDa. Aliquots (100 �l) of postdialysis plasma and phos-phate buffer were measured for radioactivity by a liquid scintillation counter[Beckman LS 6500 (Beckman Coulter, Inc., Fullerton, CA) or Packard1900TR (PerkinElmer Life and Analytical Sciences, Waltham, MA)]. Estima-tion of the unbound fraction of anacetrapib was based on the ratio of radio-activity in the buffer over that in plasma samples. The limit of quantificationusing liquid scintillation counting (�2-fold above background radioactivity) inthis assay was �5 nM.
In Vitro Blood/Plasma Partition Ratio. [14C]Anacetrapib in ethanol (2.5�l) was added to rat or monkey blood and plasma (497.5 �l each) to achievefinal concentrations of 1, 5, and 20 �M. The resulting blood and plasmasamples (0.5 ml each) were incubated at 37°C for 30 min. Blood samples thenwere subjected to centrifugation at 3000 rpm (Beckman GS6R) for 10 min toobtain plasma. Aliquots (0.1 ml) of plasma samples were measured for radio-activity by a scintillation counter (Beckman LS 6500). The blood/plasma
partition ratio was estimated by comparison of the radioactivity in plasmaprepared from the treated blood samples with that in control plasma.
Excretion of [14C]Anacetrapib in Bile Duct-Cannulated Animals. Rats.Four bile duct-cannulated male Sprague-Dawley rats (�0.4 kg) were dosedorally with 10 mg/kg [14C]anacetrapib via oral bolus gavage. The dose, with afinal drug concentration of 5 mg/ml, was formulated in Imwitor 742-Tween 80(1:1, w/w) and contained 85 �Ci/ml radioactivity.
Cumulative bile and urine samples were collected into tared bottles duringthe time intervals from 0 to 4, 4 to 8, 8 to 24, and 24 to 48 h after dosing. Feceswere collected at 24-h intervals for 2 days. Terminal blood samples (�3 ml)were collected into EDTA-containing tubes at 48 h after oral dosing. Plasmawas prepared by centrifugation of the blood sample and was stored at �20°Cuntil analysis.
Monkeys. Three bile duct-cannulated male rhesus monkeys (9.7–10.7 kg)were orally dosed at 10 mg/kg [14C]anacetrapib via nasogastric intubation andoral gavage, followed by a 3.5-ml saline flush. The oral dose was formulatedin Imwitor 742-Tween 80 (1:1, w/w) at a drug concentration of 10 mg/ml and16 �Ci/ml radioactivity. Bile was collected into tared bottles during the timeintervals from 0 to 4, 4 to 8, 8 to 24, 24 to 48, 48 to 72, and 72 to 96 h afterdrug administration. Urine was collected in a similar manner from 0 to 8, 8 to24, 24 to 48, 48 to 72, and 72 to 96 h after dosing. Feces were collected at 24-hintervals for 4 days. Predose bile, urine, and plasma samples also werecollected. Blood (2 ml) was collected into heparinized Vacutainer tubes at0.25, 0.5, 1, 2, 4, 6, 8, 24, 48, 72, and 96 h after dosing, and plasma wasprepared by centrifugation. All samples were stored at �20°C until analysis.
Sample Processing and Radiometric Analysis. Aliquots of bile or urinefrom a single animal were pooled proportionally based on excreta volumesover 0 to 48 h. Bile or urine samples for LC-MS analysis were prepared bymixing equal volumes of the corresponding pooled single animal excretasamples. Aliquots (1 ml) of pooled feces homogenate from each animal over0 to 48 h were extracted twice by adding 5 ml of acetonitrile, followed bycentrifugation and separation of supernatants, which were then evaporated todryness under N2. The residues were reconstituted in 0.3 ml of 75% aqueousacetonitrile (v/v) for LC-MS/MS analysis. The recovery of radioactivity wasapproximately 80%. Plasma was pooled from each animal over 0 to 48 h.Aliquots (1 ml) of the pooled samples were mixed with 5 ml of acetonitrile byvortexing. The mixture was centrifuged, and the resulting supernatants wereseparated and evaporated to dryness under N2. The residues were reconstituted in0.3 ml of 75% aqueous acetonitrile (v/v) for LC-MS/MS analysis. The extractionefficiency of radioactivity from plasma samples was approximately 70%.
Aliquots of bile (50 �l) or urine (100 �l) were mixed with 6 ml ofScintiSafe Gel scintillation cocktail (Thermo Fisher Scientific), and the radio-activity was measured by a liquid scintillation counter (Beckman LS 6500).Total radioactivity in feces was determined by combustion of fecal homoge-nates (feces-water ratio of 1:2, w/v) with a Packard oxidizer (model 307;PerkinElmer Life and Analytical Sciences) and scintillation counting.
Excretion Mass Balance of [14C]Anacetrapib in Monkeys. Excretionmass balance of [14C]anacetrapib in monkeys after a single oral dose wasdetermined at Charles River Laboratories Preclinical Services (Wilmington,MA). This study was conducted in compliance with the U.S. Food and DrugAdministration Good Laboratory Practice Regulations as set forth in Title 21of the U.S. Code of Federal Regulations, Part 58, and was approved by theMerck Research Laboratories Institutional Animal Care and Use Committee.In addition, this study was inspected by the Charles River Laboratories QualityAssurance Unit to assure conformity with the Good Laboratory PracticeRegulations promulgated by the U.S. Food and Drug Administration.[14C]Anacetrapib in Imwitor 742:Tween 80 (1:1, w/w) was administered orallyby gavage to each animal at a target dose of 5 mg/kg (75 �Ci/kg) in a dosevolume of 2 ml/kg. Body weights of animals that were used in dose calcula-tions were determined on the day of dosing. The dosing tubes were flushedwith 6 ml of water immediately after dosing, removed from the animal, andthen retained for extraction of residual radioactivity. The amount of doseformulation administered to each animal was determined from predose andpostdose syringe weights. Animals were fasted for approximately 10 to 11 hbefore dosing. Food was returned to the animals after collection of the 4-hpostdose blood sample. Blood samples (6 ml/time point) were collected beforedosing and at 2, 4, 6, 8, 24, and 48 h after dosing. Blood samples werecollected from an indwelling venous catheter and vascular access port or by
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venipuncture of a femoral vein into tubes containing dipotassium EDTA andwere mixed by inversion of tubes. Urine and feces were collected separatelyfrom 0 to 8 and 8 to 24 h postdose and then over 24-h intervals through 672 hpostdose. Urine was collected into containers, which were surrounded by dryice, and feces were collected at room temperature. Cage debris (mostlycomposed of residual food and hair) was collected daily after each postdoseexcreta collection. After collection of the cage debris, the floor, screen, and panof each cage were rinsed, and the rinse was collected into the cage debriscontainer. After the final collections at 672 h after dosing, the cages werewashed thoroughly with 50% (v/v) reagent alcohol, and the wash was collectedinto a separate container for each cage. The cages were wiped with gauze pads,and the gauze pads were placed into a separate container for each cage. Allsample weights were recorded. Blood samples were kept chilled on wet icebefore centrifugation to obtain plasma samples. All other samples were storedat �20 � 5°C, unless processed or analyzed immediately after collection.
Sample analysis. Triplicate weighed aliquots (approximately 0.2–0.4 geach) of feces, cage debris, and cage wash homogenate were placed into conesand pads, dried for at least 4 h at ambient temperature, combusted, and thenanalyzed by liquid scintillation counting. These samples were combusted by anOxidizer model 307 (PerkinElmer Life and Analytical Sciences). The resulting[14C]CO2 was trapped in 9 ml of Carbo-Sorb E (PerkinElmer Life andAnalytical Sciences) and mixed with 9 ml of Permafluor E� (PerkinElmerLife and Analytical Sciences) scintillation fluid. The combustion efficiencywas determined for each combustion session. Plasma and urine samples weremixed with 15 ml of Emulsifier-Safe (PerkinElmer Life and Analytical Sci-ences) scintillation fluid. All samples were analyzed for total radioactivity byliquid scintillation counting for 2 min in a Beckman model LS 6000 or LS6500 liquid scintillation counter). Scintillation counter data were automaticallycorrected for counting efficiency using an external standardization techniqueand an instrument-stored quench curve generated from a series of sealedquench standards. A value of zero was assigned to samples for which radio-activity (disintegrations per minute) was less than or equal to twice thebackground disintegrations per minute; for other samples, background radio-activity was subtracted before concentrations of radioactivity and percentagesof the radioactive dose recovered were calculated. Amounts of radioactivity incombusted experimental samples were adjusted for the efficiency of thecombustion system, which was determined from oxidized and nonoxidizedstandards counted along with the experimental samples. The anacetrapib-equivalent concentration of radioactivity in a sample was calculated by divid-ing the concentration of radioactivity in the sample by the specific activity andwere expressed as nanogram equivalents per gram. The total radioactivityrecovered in a matrix was expressed as a percentage of the actual administeredradioactive dose. Statistical analyses were limited to the calculation of themean and S.D., as appropriate.
Qualitative Analysis with LC-MS/MS, Radioflow Detector, and NMR.The LC-MS/MS system consisted of a Shimadzu SIL-10ADvp autosam-pler, a Shimadzu SCL-10Avp system controller, two Shimadzu LC-10ADvp pumps, a Packard flow scintillation analyzer (500TR series), anda LCQ XP Plus ion trap mass spectrometer (Thermo Fisher Scientific). Themass spectrometer was operated with an electrospray interface in eitherpositive or negative ion detection mode, and data were acquired usingXcalibur software (version 1.2; Thermo Fisher Scientific). The MS spectrawere recorded in a data- and list-dependent mode. A Zorbax RX-C8 HPLCcolumn (4.6 � 250 mm; 4 �m; Waters, Milford, MA) was used forchromatographic separation. The mobile phases A and B were 5% aqueousacetonitrile (v/v) and 95% aqueous acetonitrile (v/v), respectively, bothcontaining 0.1% formic acid and 1 mM ammonium acetate. The gradientprogram began at 5% B for first 3 min and was increased linearly to 35%B in 2 min, to 42% B in 43 min, to 75% B in 17 min, and finally to 100%B in 5 min, followed by a hold at 100% B for 5 min. The flow rate was 1.5ml/min, and the eluate was diverted in a 5:1 ratio to the Packard flowscintillation analyzer and the LCQ mass spectrometer, respectively. Scin-tillation cocktail (Packard Ultima Flo-M) was pumped at a rate of 3.8ml/min to the radiometric detector. NMR spectra of anacetrapib and itsmetabolites were recorded on a Varian Inova 600 MHz NMR spectrometer.
Results
All animals were observed frequently during the course of thestudies and appeared to be healthy, with no overt adverse signs.
Pharmacokinetics of Anacetrapib. Rats. The pharmacokineticparameters of anacetrapib in rats after intravenous and oral adminis-tration are presented in Tables 1 and 2, and the mean plasma concen-tration versus time curves are shown in Fig. 2. After an intravenous
Time (h)
0 10 20 30 40 50
Pla
sma
Con
cent
ratio
n (µ
M)
0.001
0.01
0.1
1
10
100
IV 0.5mpkPO 5mpkPO 50mpkPO 100mpkPO 500mpk
FIG. 2. Mean plasma concentration-time curves of anacetrapib in rats after intra-venous and oral doses. Anacetrapib was administered intravenously in polyethyleneglycol 300-water (7:3, v/v) or orally in Imwitor 742-Tween 80 (1:1, w/w) toovernight-fasted male Sprague-Dawley rats. Plasma concentrations of anacetrapibwere determined by LC-MS/MS assay, and means � S.D. for n � 3 to 4 animalsare shown. mpk, milligrams per kilogram.
TABLE 1
Pharmacokinetic parameters for anacetrapib in rats and monkeys afterintravenous administration
The intravenous doses were formulated in polyethylene glycol 300-water (70:30, v/v).Data are means � S.D.
Species Dose AUC(0–)a CL Vdss t1/2
mg/kg �M � h ml/min/kg l/kg h
Rat 0.5 5.9 � 1.5 2.3 � 0.6 1.1 � 0.6 12 � 1Monkey 0.1 24 � 6.7 0.1 � 0.3 0.3 � 0.1 34 � 5
CL, plasma clearance; Vdss, volume of distribution at steady state; t1/2, terminal phasehalf-life.
a Percentage of AUC values extrapolated for rats and monkeys were 3 and 37%, respectively.
TABLE 2
Pharmacokinetic parameters for anacetrapib in rats and monkeys afteroral administration
Data are means � S.D.
Species Dose AUC(0–)a Cmax Tmax Foral
b
mg/kg �M � h � M h %
Rat 5 23 � 2 5 � 1 3 � 1 3850 82 � 29 11 � 2 3 � 1 N.D.
100 183 � 38 26 � 4 4 � 2 N.D.500 362 � 134 21 � 3 5 � 3 N.D.
Monkey 1 26 � 3 2 � 1 5 � 1 1130 365 � 104 42 � 22 4 � 0 N.D.
500 1500 � 581 205 � 151 5 � 1 N.D.
Cmax, observed peak plasma concentration after oral dosing; Tmax, time to reach Cmax; F,bioavailability; N.D., not determined.
a Percentage of AUC extrapolated ranged from 0.4 to 22%.b Bioavailability for rats and monkeys was determined by a relative comparison of the mean
AUC values associated with the intravenous and the lowest oral doses.
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dose of 0.5 mg/kg, the mean values for systemic plasma clearance,steady-state volume of distribution, and terminal half-life were 2.3ml/min/kg, 1.1 l/kg, and 12 h, respectively. After oral dosing at 5mg/kg, the bioavailability of anacetrapib was 38%. Exposures (AUC)increased in a less than dose-proportional manner from 23 �M � h at5 mg/kg to 362 �M � h at 500 mg/kg. In this dose range, the peakplasma level (Cmax) ranged from 5 to 26 �M and the time to reachpeak plasma level (Tmax) ranged from 3 to 4.5 h.
Monkeys. A summary of the pharmacokinetic parameters of anace-trapib in monkeys after an intravenous dose of 0.1 mg/kg and oraldoses of 1, 30, and 500 mg/kg is presented in Tables 1 and 2, and themean plasma concentration versus time curves are shown in Fig. 3.The mean values for systemic plasma clearance, steady-state vol-ume of distribution, and half-life were 0.11 ml/min/kg, 0.31 l/kg,and 34 h, respectively. After oral dosing at 1 mg/kg, the bioavail-ability of anacetrapib was 11%. Similar to rats, the mean plasmaAUC values increased in a less than dose-proportional mannerfrom 26 �M � h at 1 mg/kg to 1500 �M � h at 500 mg/kg. The Tmax
values ranged from 4 to 5 h, and the Cmax values ranged from 2 to205 �M at these doses.
In Vitro Reversible Plasma Protein Binding and Blood/PlasmaPartition Ratio. The unbound fraction of anacetrapib in rat andmonkey plasma was less than 0.5% over a concentration range of 1 to20 �M. The average blood/plasma partition ratios of anacetrapib overthe same concentration range were 0.6 in the rat and 0.7 in themonkey.
Excretion of [14C]Anacetrapib in Bile Duct-Cannulated Ani-mals. Rats. The excretion of [14C]anacetrapib-associated radioactivitywas examined in bile duct-cannulated male Sprague-Dawley rats, andthe data are summarized in Table 3. After oral dosing of [14C]anace-trapib at 10 mg/kg, �96% of the administered radioactive dose wasrecovered over a period of 48 h, of which 15% was recovered in bile, 1%in urine, and 80% in feces. The intact parent compound excreted in bileaccounted for �1% of the dose, but it was not detected in urine. Basedon a high recovery of the intact parent compound (63% of dose) in feces,which probably represents unabsorbed drug, the extent of oral absorptionof [14C]anacetrapib appears to be low to moderate at best.
Monkeys. After oral dose of [14C]anacetrapib to bile duct-cannulatedmale monkeys at 10 mg/kg, nearly 100% of the dose was recovered overa period of 96 h. Similar to rats, 13% of the dose was recovered in bile,1% in urine, and a much larger proportion, 90%, in feces. A large amountof the intact parent compound (74% of radioactivity) was recovered infeces, suggesting that, similar to rats, the extent of [14C]anacetrapib oralabsorption was low in monkeys as well.
Excretion Mass Balance of [14C]Anacetrapib in Monkeys. In theexcretion mass balance study in monkeys, the excretion of [14C]anace-trapib-associated radioactivity was monitored for a total 28 days. Totalrecoveries of radioactivity for all matrices are summarized in Table 4.Fecal excretion was the major route of elimination of drug-related radio-activity. Mean recoveries of the radioactive dose through 672 h afterdosing were 75% in feces and 1.2% in urine. The majority of the fecalradioactivity was excreted within 48 h after dosing, but elimination ofradioactivity in feces continued through 672 h after dosing. Cage residuescontained an average total of 3.6% of the dosed radioactivity. The meantotal recovery of the radioactive dose from all matrices collected through672 h after dosing was 79.6%.
In Vivo Metabolite Profiles. Rats. Representative HPLC chro-matograms for metabolites of [14C]anacetrapib in rat plasma, bile, andfeces are shown in Fig. 4. The contributions of various biliary andfecal metabolites to the overall elimination of anacetrapib dose areshown in Table 5. In rat urine, only 1 to 2% of the administeredradioactive dose was recovered, and the radioactivity levels were toolow for meaningful analysis.
Time (h)
0 10 20 30 40 50
Pla
sma
Con
cent
ratio
n (µ
M)
0.01
0.1
1
10
100
1000
IV 0.1mpkPO 1mpkPO 30mpkPO 500mpk
FIG. 3. Mean plasma concentration-time curves of anacetrapib in monkeys afterintravenous and oral doses. Anacetrapib was administered intravenously in poly-ethylene glycol 300-water (7:3, v/v) or orally in Imwitor 742-Tween 80 (1:1, w/w)to overnight-fasted male rhesus monkeys. Plasma concentrations of anacetrapibwere determined by LC-MS/MS assay, and means � S.D. for n � 3 animals areshown. mpk, milligrams per kilogram.
TABLE 3
Excretion of [14C] radioactivity in bile duct-cannulated rats (n � 4) and monkeys (n � 3) after oral administration of 10 mg/kg [14C]anacetrapib
Data are means � S.D.
Time (h)
Radioactive Dose Excreted
Rat Monkey
Bile Urine Feces Bile Urine Feces
%
0–24 10 � 2.6 0.6 � 0.1 36 � 16 8.9 � 4.8 0.8 � 0.4 64 � 1324–48 4.3 � 2.3 0.1 � 0 44 � 16 2.5 � 0.5 0.1 � 0.0 25 � 1548–96 N.D. N.D. N.D. 1.1 � 0.1 0.1 � 0.1 1.8 � 1.4Total 15 � 2.8 0.7 � 0.2 80 � 13 13 � 4.5 1.0 � 0.5 90 � 12
TABLE 4
Recovery of radioactivity through 672 h after 5 mg/kg and 75 �Ci/kg oral doseof [14C]anacetrapib to male rhesus monkeys (n � 4)
Matrix Time Recovery of Radioactivity
h % dose
Urine 0–672 1.2 � 0.2Feces 0–672 74.7 � 7.5Cage debris 0–672 3.4 � 1.7Cage wash 672 0.1 � 0.1Cage wipe 672 0.06 � 0.06Total 0–672 79.6 � 5.8
463METABOLISM AND EXCRETION OF ANACETRAPIB IN RATS AND MONKEYS
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Rat plasma. The profile from rat plasma samples pooled over48 h after dosing showed four radioactive peaks that were identi-fied as anacetrapib and the oxidative metabolites M1, M2, and M3.The parent drug was the major radioactive component, accountingfor �38% of the total radioactivity. The metabolites M1, M2, andM3 accounted for �17, �14, and �31% of the radioactivity,respectively.
Rat feces. The metabolite profile from pooled rat feces was similarto that from rat plasma in that parent anacetrapib, M1, M2, and M3
were detected. In feces, parent drug accounted for 63% of the totalradioactive dose. M1, M2, and M3 accounted for 4.8, 2.7, and 5.6%of the dose, respectively.
Rat bile. Approximately 81% of the radioactivity excreted in bilecould be identified. As shown in the representative HPLC radio-chromatogram (Fig. 4), anacetrapib was extensively metabolized inthe rat, giving rise to at least nine metabolites in rat bile. The intactparent compound excreted over 48 h accounted for 4.7% of theradioactivity excreted in bile which, in turn, corresponds to 0.7%of the total administered radioactive dose. All identified metabo-lites represented �11% of the dose. As was shown by LC-MS/MSanalysis (see also Identification of Metabolites), biliary metabolitesresulted from O-demethylation on the biphenyl moiety (M1) andhydroxylation on the isopropyl side chain (M3) and subsequentglucuronidation on the aromatic and aliphatic hydroxyl groups(M4, M5, M6, M7, and M8). In addition, a glutathione adduct (M9)and an olefinic metabolite (M10) were detected. The three mostabundant metabolites in rat bile were M4, M5, and M7 and theseaccounted for 1.7, 3.5, and 3.3% of the radioactive dose, respec-tively. The quantities of all other metabolites were less than 1% ofthe dose in each case.
Monkeys. Representative HPLC chromatograms for metabolitesof [14C]anacetrapib in monkey plasma, bile, and feces are shown inFig. 5. The mean percentages of biliary and fecal metabolites relativeto the administered dose are shown in Table 5. Similar to rats, only 1 to2% of the administered radioactive dose was recovered in monkey urine,and radioactivity concentrations in urine were too low for meaningfulanalysis.
Monkey plasma. Based on radiometric data, intact parent drug wasthe only circulating entity in monkey plasma. The presence of tracelevels of metabolites, similar to those seen in rat plasma (M1, M2, and
FIG. 4. HPLC radiochromatographic profiles of anacetrapib and metabolites in 48-h pooled samples of plasma, bile, and feces of rats after a 10 mg/kg oral dose of[14C]anacetrapib.
TABLE 5
Relative abundance of biliary and fecal metabolites of anacetrapib in rat andmonkey after oral administration of 10 mg/kg [14C]anacetrapib
Analytes
Percentage of Radioactivity Dosea
Rat Monkey
Bile Feces Bile Feces
Anacetrapib 0.7 63 N.D. 74M1 0.2 4.8 N.D. 2.8M2 N.D. 2.7 N.D. 2.7M3 0.7 5.6 0.1 5.0M4 1.7 N.D. 1.1 N.D.M5 3.5 N.D. 0.9 N.D.M6 0.5 N.D. 0.5 N.D.M7 3.3 N.D. 0.5 N.D.M8 0.5 N.D. 0.7 N.D.M9 0.5 N.D. N.D. N.D.M10 0.2 N.D. N.D. N.D.M11 N.D. N.D. 0.4 N.D.M12 N.D. N.D. 2.8 N.D.M13 N.D. N.D. 0.7 N.D.
N.D., not detected.a Calculation was based on dose recovery during the first 48-h period. Approximately 1 to 2%
of the dose was detected in urine, and the radioactivity in urine was too low for meaningfulanalysis.
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M3), cannot be completely ruled out; limitations in sample volumeand the sensitivity of the radiometric assay did not allow us to obtainmore conclusive metabolite data.
Monkey feces. Parent anacetrapib was the major radioactive com-ponent in monkey feces, accounting for 74% of the administered dose.Radiochromatographic analysis of the feces (Fig. 5) also showedminor amounts of M1, M2, and M3, which accounted for 2.8, 2.7, and5.0% of the radioactive dose, respectively.
Monkey bile. In monkey bile, approximately 80% of the totalexcreted radioactivity could be identified. Unchanged parent drug wasnot detected, suggesting that absorbed anacetrapib probably under-went complete metabolic turnover in monkey liver before excretion.The vast majority of the radioactivity excreted in monkey bile couldbe attributed to metabolites resulting from oxidative metabolism (O-demethylation and aromatic and aliphatic hydroxylation) and subse-quent conjugation with glucuronic acid. One metabolite (M11) re-sulted from oxidation of the isopropyl side chain to a propionic acidderivative. Of the nine metabolites identified, the most abundant werethe glucuronic acid conjugates M4 and M12, which accounted for 1.1and 2.8% of the administered radioactive dose, respectively. The otherglucuronic acid conjugates (M5, M6, M7, M8, and M13) and thepropionic acid metabolite M11 each accounted for less than 1% of thedose.
Mass Spectral Fragmentation of Anacetrapib. Full-scan MSof anacetrapib showed a molecular ion at m/z 638 [M � H]�. Asshown in Fig. 6A, the CID product ion spectrum of m/z 638produced characteristic fragment ions at m/z 325, 314, and 283.The m/z values of fragment ions indicated that fragmentation of theparent molecule occurs through cleavage of the bond between thenitrogen of the oxazolidinone and the methylene carbon linkage tothe biphenyl moiety, generating fragment ions at m/z 325 and m/z314. High-resolution MS analysis of the most abundant fragment at
m/z 283 (data not shown) indicated an elemental formula of[C15H11F4O]�, suggesting that it was derived from the 4�-fluoro-5�-isopropyl-2�-methoxy-4-(trifluoromethyl)biphenyl moiety ofthe molecule.
Identification of Metabolites. To determine structures of metab-olites, LC-MS/MS with radiometric detection was used. Where pos-sible, metabolites were isolated and purified by means of semiprepara-tive HPLC for structure elucidation by 1H NMR. The CIDfragmentation and 1H-NMR spectra for anacetrapib are presented inFig. 6. Likewise, fragmentation spectra for M1, M2, and M3 (the threemetabolites that circulate in rat and human plasma) are presented inFig. 7 for illustrative purposes. The mass spectral fragmentation and1H-NMR data for all of the remaining metabolites are summarized inTables 6 and 7, respectively. In addition, chemical structures of anumber of metabolites (M1, M2, M3, M4, M6, M8, M10, and M12)were confirmed by comparing their chromatographic retention timesand mass spectral fragmentation with those of the correspondingsynthetic standards. A proposed scheme of metabolic pathways ofanacetrapib in rats and monkeys is presented in Fig. 8.
Metabolite M1. In negative ion mode, M1 gave a molecular ion atm/z 622, corresponding to a loss of 16 mass units relative to parentanacetrapib in positive ion mode, suggesting that this was the O-demethylated derivative. The CID spectrum of m/z 622 showed twomajor fragments at m/z 578 and 312 (Fig. 7A), which were presum-ably formed via the loss of elements of CO2 from the oxazolidinonering and by cleavage between the oxazolidinone ring and the meth-ylene linkage to the biphenyl, respectively.
Metabolites M2 and M3. Both M2 and M3 gave molecular ions atm/z 638 in negative ion mode, indicating a net addition of 2 Da toanacetrapib; this presumably arose from concurrent O-demethylationand monohydroxylation. Comparison of the CID product ion spectraof the two metabolites showed that M3 generated a fragment ion at
FIG. 5. HPLC radiochromatographic profiles of anacetrapib and metabolites in plasma, bile and feces of monkeys after a 10 mg/kg oral dose of [14C]anacetrapib.
465METABOLISM AND EXCRETION OF ANACETRAPIB IN RATS AND MONKEYS
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m/z 620 due to loss of water, which was not the case for M2 (Fig. 7, B andC). From this finding and from additional fragment ions at m/z 325 (M2) andm/z 312 (M3), it was concluded that for M2, hydroxylation had occurred onthe biphenyl moiety, whereas M3 probably incorporated a hydroxyl group onthe isopropyl side chain. The proposed structures were further confirmedthrough comparing the chromatographic retention times and mass spectralfragmentation of synthetic standards of the putative metabolites with those ofM2 and M3.
Metabolite M4. CID mass spectral analysis of M4 (molecular ion atm/z 798 in negative mode) indicated loss of glucuronic acid, giving
rise to a fragment ion at m/z 622 corresponding to the O-desmethyl-ated analog M1 (Table 6). The NMR spectra confirmed M4 to be theglucuronic acid conjugate of the O-desmethyl analog, as the methoxysignal (near 3.7 ppm in the parent spectrum) was noticeably absent(Table 7). Subsequent glucuronidation on the phenolic hydroxyl wasalso confirmed by the presence of the glucuronic acid signals. Thechemical shift of the anomeric proton (near 5.0 ppm) is consistentwith a phenolic O-glucuronide structure (Table 7).
Metabolite M5. M5 was the most abundant metabolite in rat bileand was also present in large quantities in monkey bile. M5 (molec-
FIG. 6. CID product ion spectrum (A) and 1H NMR spectrum (B) of anacetrapib.
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ular ion at m/z 814 in negative ion mode) exhibited an addition of �16amu relative to M4. In the CID spectrum, a fragment ion at m/z 638indicated loss of the glucuronic acid moiety (Table 6). The MS3 CID
fragment spectrum of m/z 638 (not shown) did not demonstrate loss ofa water molecule and was in fact identical to the fragmentationspectrum of M2. These data suggest that the additional hydroxylation
A)
200 250 300 350 400 450 500 550 600
m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
312.0
578.2
292.0518.1324.1 579.2268.1 558.1498.1
538.1
213.0 456.8365.7 407.4
312.0
M-H- m/z 622
N
F
F
F
HO
O
O
F
F
F
FF
F
F
312
578
324
B)
200 250 300 350 400 450 500 550 600
m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
325.1
594.1
282.0
305.1326.1281.0 595.2
M-H- m/z 638
312.0
N
F
F
F
HO
O
O
F
F
F
FF
F
F
OH
594
-2H, 325
312
200 250 300 350 400 450 500 550 600
m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
312.0
620.1
594.1
292.0576.1
536.1
M-H- m/z 638 -H2O
N
F
F
F
HO
O
O
F
F
F
FF
F
F
OH
594
312
312 - HF
594
312
C)
FIG. 7. CID product ion spectrum of M1 (A), M2 (B), and M3 (C).
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in M5 had occurred on the aromatic moiety and that M2 probablyrepresented the aglycone portion of the M5 structure; however, theexact site of the glucuronic acid conjugation at either of the twohydroxy moieties of the catechol was not identified.
Metabolite M6. Mass spectral analysis of M6 (molecular ion at m/z828 in negative mode) indicated a change in molecular weight of 192amu (�16 � 176 amu) relative to the parent. In the CID spectrum, the
loss of glucuronic acid gave a fragment ion at m/z 652 (Table 6). Thepresence of the methoxy group was confirmed by the peak near 3.7 ppmin the NMR spectrum (Table 7). Hydroxylation at one of the isopropylmethyls was readily apparent from the loss of one methyl signal (near 1.1ppm in the parent spectrum) and appearance of a new set of signalsconsistent with a CH2OH moiety (near 3.6 and 4.0 ppm). Glucuronida-tion was confirmed by the presence of the glucuronic acid signals. The
TABLE 6
Summary of key mass spectral data for anacetrapib and its metabolites in the rat and monkey
Parent or Metabolite Chemical Structure and Proposed Fragmentation Scheme Molecular Ion M-H�� Molecular Ion M � H�� Characteristic CID Fragment Ions
m/z m/z m/z
Parent N
F
F
F
O
O
O
F
F
F
FF
F
F
314
325
283
638 325, 314, 283
M1N
F
F
F
OH
O
O
F
F
F
FF
F
F
312
578
324
622 578, 312
M2N
F
F
F
OH
O
O
F
F
F
FF
F
F
OH
594
-2H, 325
312
638 594, 325
M3N
F
F
F
OH
O
O
F
F
F
FF
F
F
OH
594
312
638 620, 594, 312
M4 N
F
F
F
O
O
O
F
F
F
FF
F
F
622
Glu
798 622
Continued
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TABLE 6—Continued
Parent or Metabolite Chemical Structure and Proposed Fragmentation Scheme Molecular Ion M-H�� Molecular Ion M � H�� Characteristic CID Fragment Ions
M5N
F
F
F
OH
O
O
F
F
F
FF
F
F
OH
638
Glu
814 638, 594, 325
M6N
F
F
F
O
OO
F
F
FF
F
F
F
O Glu
828 652
M7 N
F
F
F
HO
OO
F
F
FF
F
F
FOH
OH
Glu
830 654
M8 N
F
F
F
O
O
O
F
F
F
FF
F
F
OH
OH
654
-H2O
636
Glu
830 654, 636
M9a
OH
F
FF
N
O
O
F
OH
F
F
F
FFF
S Cys
Gly
Glu
816
638-H2O
798
945 816, 798, 638, 308
M10 N
F
F
F
O
OO
F
F
FF
F
F
FGlu
796 620
Continued
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location of the glucuronic acid moiety was determined to be on the newlyintroduced hydroxyl group, as the chemical shift of the anomeric proton(near 4.2 ppm) is consistent with an aliphatic O-glucuronide structure.
Metabolite M7. M7 (molecular ion at m/z 830 in negative mode)demonstrated the addition of �32 amu relative to M4. In the CIDspectrum, the loss of a glucuronic acid moiety gave a fragment ion atm/z 654 (Table 6). As with M6, hydroxylation at one of the isopropylcarbons was evident by the appearance of –CH2OH proton signalsin the 1H-NMR spectrum (Table 7). In addition, hydroxylation ofthe fluorinated aromatic ring was apparent from the loss of one ofthe corresponding proton signals. Based on a comparison of themeasured 1H-19F coupling constant (7.6 Hz) to those in the parentspectrum (4JHF � 8.8 Hz versus 3JHF � 12.5 Hz), the peak near 6.8ppm was assigned as the proton meta to the fluorine, indicating thathydroxylation had occurred at the carbon in between the fluorineand the existing hydroxyl (Table 7). The slight upfield shift of thisproton signal from the parent spectrum (near 7.1 ppm) is consistent withthe addition of a hydroxyl group at its para position. Glucuronidation wasconfirmed by the presence of the glucuronic acid signals, but the locationof the glucuronide could not be precisely determined. The aliphatichydroxyl can be excluded because the chemical shift of the anomericproton (near 4.8 ppm) is too far downfield to be an aliphatic glucuronide(see 4.2 ppm for M6); thus, the glucuronic acid moiety is probablyattached to one of the aromatic hydroxyls.
Metabolite M8. In the HPLC radiochromatogram (Fig. 5), M8 elutedin two separate radioactive peaks, M8A and M8B, with a 2-min differ-
ence in retention times. The Q1 full scan and CID fragment ion spectraof the two peaks were found to be identical. M8A and M8B demonstratedthe addition of �32 amu relative to M4, and a molecular ion at m/z 830was seen in negative ion mode. The CID spectra for both analytes showedfragment ions at m/z 654 (loss of glucuronic acid) and m/z 636 (loss ofglucuronic acid and water) (Table 6). The NMR spectra of both metab-olites indicated that hydroxylation had occurred at one of the isopropylmethyls and at the benzylic carbon for the same reasons outlined in thedescription of metabolites M6 and M12, respectively (Table 7). Thesimilarity observed for all the physical data suggests that M8A and M8Bwere diastereomers, differing only in the stereochemistry of the benzyliccarbon.
Metabolite M9. M9 was found only in rat bile. The mass spectrumof M9 showed a molecular ion at m/z 945 in positive mode, reflectinga net addition of 307 amu to parent anacetrapib, which can berationalized as demethylation (�14 Da), hydroxylation (�16 Da), andaddition of glutathione (�305 Da). The CID spectrum displayedfragment ions with characteristic masses of 816 and 638 (Table 6),indicating the loss of pyroglutamate (129 amu) and glutathione (307amu); the loss of the entire glutathionyl moiety under CID indicatesthat it is probably attached to an aliphatic carbon. The most likelystructure of M9 shown in Table 6 is consistent with the abovefragmentation spectrum and can be rationalized by the addition ofglutathione to a putative electrophilic p-quinone methide intermediate.
Metabolite M10. M10 was also found in trace amounts only in ratbile. The molecular ion (m/z 796 in negative ion mode) in the full scan
TABLE 6—Continued
Parent or Metabolite Chemical Structure and Proposed Fragmentation Scheme Molecular Ion M-H�� Molecular Ion M � H�� Characteristic CID Fragment Ions
M11N
F
F
F
O
O
O
F
F
F
FF
F
F
OH
O
269
666 646, 269
M12 N
F
F
F
O
OO
F
F
FF
F
F
F
OH
Glu
814 638, 620
M13 N
F
F
F
HO
OO
F
F
FF
F
F
F3 O + Glu
846 670
Glu, glucuronic acid.a The regiochemistry of hydroxylation and glutathione addition shown is tentative and is based only on mass spectral fragmentation and on the collective knowledge of the overall metabolic routes
of anacetrapib.
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showed a mass difference of �2 amu relative to the mass of M4, theglucuronic acid conjugate of the O-demethyl metabolite M1, indicat-ing either dehydrogenation on an aliphatic moiety (isopropyl sidechain), or loss of water from an aliphatic hydroxyl group throughin-source CID. The NMR spectra of M10 contained no signals indic-ative of hydroxylation. Instead, one of the isopropyl methyl signalshad shifted from 1.2 to 2.1 ppm, and the other signal was missingentirely. A new signal corresponding to two protons appeared near 5.2ppm. Such signals are consistent with the conversion of the isopropylgroup to a propene moiety (Table 7).
Metabolite M11. The metabolites M11, M12, and M13 were foundonly in monkey bile. The MS full scan in negative ion mode detecteda molecular ion at m/z 666, corresponding to the net addition 30 amuto the mass of the parent drug. The fragment ions in the CID spectrum,with characteristic loss of 44 amu (corresponding to elements of CO2),suggested the presence of a carboxylic acid group on the isopropylside chain of the biphenyl moiety (Table 6).
Metabolite M12. M12 was found to be the most abundant metab-olite in monkey bile, accounting for approximately 3% of the admin-istered dose. M12 (molecular ion at m/z 814) exhibited the additionof �16 amu relative to M4. In the CID spectrum, fragment ions at m/z638 and 620 indicated sequential losses of the glucuronic acid andwater. Furthermore, the loss of the isopropyl methine proton signal(seen in the parent 1H NMR spectra near 3.2 ppm) indicated theformation of the tertiary alcohol from hydroxylation at the benzyliccarbon (Table 7). Chemical shift of the anomeric proton of theglucuronic acid moiety indicates conjugation on the biphenyl ring.
Metabolite M13. The molecular ion signal at m/z 846 in negative ionmode indicated a mass change of �48 � 176 relative to the O-demethylatedmetabolite M1. The fragment ions in the CID spectrum suggested that threehydroxylations and a conjugation with glucuronic acid had occurred on thebiphenyl moiety of the molecule (Table 6).
Discussion
The objective of this study was to examine the pharmacokineticsand disposition of anacetrapib in rats and monkeys, the two speciesthat are being used for the preclinical toxicological evaluation of thisdrug candidate.
The pharmacokinetics of anacetrapib were characterized by lowsystemic plasma clearance (2.3 and 0.1 ml/min/kg in the rat andmonkey, respectively) and a long half-life, ranging from 12 to 34 h.The oral bioavailability of anacetrapib was low to moderate (13% inmonkeys and 38% in rats) and the increase in systemic exposure wasless than dose-proportional in both species; this was probably relatedto low aqueous solubility of the drug that leads to dissolution limitedabsorption after oral administration.
In excretion studies using bile duct-cannulated rats and monkeys, itwas shown that almost complete recovery of drug-related radioactiv-ity was achieved over a time period of 48 h in rats or 96 h in monkeysafter an oral dose, with the majority of the radioactive dose excretedin feces. An additional study monitoring the excretion of radioactivityover 28 days in rhesus monkeys led to the same conclusion, althoughin this latter study the total radioactivity recovery was slightly lower(�80%) than that obtained in bile duct-cannulated monkeys(�100%). The reason for the somewhat lower apparent excretionrecovery in intact monkeys relative to that in bile duct-cannulatedanimals is not clear.
The in vivo metabolic profile of anacetrapib seemed to be consis-tent across species. In both the rat and the monkey, oxidative metab-olism was predominant and involved O-demethylation and multiplehydroxylations on the biphenyl moiety; in contrast, the oxazolidinoneand the 3,5-bis(trifluoromethyl)phenyl moieties of the molecule weremetabolically stable. The major metabolites in rat bile were productsof O-demethylation (M1) and aromatic hydroxylation (M2), whichsubsequently underwent glucuronidation on the catechol hydroxylgroups (M5 and M7) before excretion into bile. In monkey bile, themost abundant metabolite (M12) was a product of O-demethylationfollowed by glucuronidation on the resulting phenolic hydroxyl groupand concomitant hydroxylation on the isopropyl side chain. Further-more, the metabolites M1, M2, and M3 were found to circulate in ratplasma at significant levels relative to those of the parent. Theirpresence in monkey plasma seems likely but could not be conclu-sively proven. The fact that these three metabolites also appeared inthe feces of bile duct-cannulated rats and monkeys suggests that theywere either excreted into the gut lumen by mechanisms other thanbiliary excretion (e.g., intestinal excretion) or formed by the metabolicactivity of intestinal bacteria.
Additional (minor) metabolic pathways present included dehydro-genation of the isopropyl side chain to yield an olefin metabolite(detected as glucuronic acid conjugate M10 in rats) or its oxidation toa propionic acid derivative (M11, in monkeys). Furthermore, traceamounts of a glutathione adduct (M9), which is probably formed viacapture of a quinone methide intermediate at the biphenyl moiety byglutathione, were detected in the rat. The structures of the majority ofmetabolites identified in rats and monkeys indicate that the O-demeth-ylation of anacetrapib to form M1 is the core first step in the bio-transformation of this drug in both species, and several of the othermetabolites represent secondary and tertiary products of further oxi-dation of M1 and their conjugation with glucuronic acid.
TABLE 7
Proton NMR chemical shifts of anacetrapib and metabolites
Chemical shifts measured at 600 MHz in CD3CN-D2O (10:1). Rotamers (atropisomers)are indicated by asterisks whenever resolved. Nonequivalent CH2 protons are indicatedby primes.
Position Parent M4 M6 M7 M8 M10 M12
ppm
a 7.682 7.687 7.660 7.682 7.679 7.688 7.682a* 7.697 7.647 7.641 7.645b 7.350 7.423 7.406 7.389 7.449 7.451 7.441b* 7.345 7.377 7.356 7.419 7.407 7.396 7.393c 7.655 7.661 7.673 7.661 7.687 7.685 7.689c* 7.657 7.639 7.638 7.686 7.641 7.636 7.638d 6.786 6.965 6.810 6.975 7.025 6.972d* 6.802 7.036 6.831 7.059 7.104 7.069e 7.093 7.098 7.161 6.875 7.395 7.182 7.383e* 7.086 7.093 7.131 7.363 7.166 7.353f 7.783 7.788 7.796 7.790 7.807 7.793 7.811f* 7.812 7.827 7.825 7.843 7.835 7.838g 7.950 7.943 7.948 7.940 7.934 7.945 7.942g* 7.960 7.942h 5.442 5.459 5.502 5.504 5.552 5.444 5.542h* 5.615 5.705 5.642 5.692 5.762 5.738 5.743i 3.781 3.790 3.821 3.792 3.773 3.821 3.784i* 3.927 3.907 3.824 3.997 3.988 3.993j 4.589 4.710 4.561 4.737 4.790 4.718 4.756j* 4.582 4.610 4.541 4.644 4.621 4.598 4.618j� 4.117 4.208 4.133 4.183 4.210 4.229 4.182j�* 3.923 4.080 4.040 4.092 4.105 4.123 4.110K 0.431 0.414 0.450 0.476 0.428 0.428 0.430k* 0.275 0.358 0.424 0.432 0.402 0.404M 1.188 1.194 1.215 1.160 1.451 2.063 1.460m* 1.417 1.486N 1.124 1.164 4.020 3.575 3.663 5.206 1.524n* 4.001 3.699 5.185 1.505n� 3.569 3.526 3.579n�* 3.582 3.588O 3.129 3.124 3.324 3.142P 3.699 3.707gluc1 5.025 4.229 4.689 5.046 5.040 5.026gluc1* 4.850 4.807 4.871 4.883 4.868
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As discussed in the companion article on the metabolism of anace-trapib in humans (Kumar et al., 2010), the only metabolites detectedin biological matrices (plasma and feces) from healthy human volun-teers given a single 150-mg oral dose of [14C]anacetrapib were M1,M2, and M3. All of these metabolites were present at much lowerlevels than those of the parent in human plasma (�14% of totalplasma, radioactivity). Furthermore, similar to animals, the majorityof the administered [14C]anacetrapib-associated radioactivity (91%)was excreted in feces in humans as the intact parent drug, whichprobably reflects unabsorbed drug. Metabolites (M1, M2, and M3)were the only other drug-related entities detected in feces, with eachaccounting for �5% of the administered dose. Given that M1, M2,and M3 were formed in both rats and monkeys and were found tocirculate in rat plasma at levels in excess of those observed in humans,we conclude that these metabolites have been adequately qualified inthe preclinical safety studies of anacetrapib (Guidance for Industry onSafety Testing of Drug Metabolites, U.S. Department of Health andHuman Services, Center for Drug Evaluation and Research, Food andDrug Administration, February 2008; http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm065014.htm).
Data on animal and human disposition and metabolism of torce-trapib (Pfizer Inc.), another CETP inhibitor, which progressed intolate clinical development before it was discontinued as a result ofunfavorable cardiovascular outcomes, have also been published re-cently (Dalvie et al., 2008; Prakash et al., 2008). A comparison of theabsorption, distribution, metabolism and excretion profile for anace-trapib and torcetrapib reveals significant differences between thedisposition and metabolism of these two CETP inhibitors. The twodrugs appear strikingly different in their routes of elimination andtheir metabolic profiles. The findings of this study indicate thatanacetrapib is primarily excreted in feces as unchanged drug in bothrats and monkeys, with minimal excretion of the radioactive dose inurine. In comparison, the elimination of torcetrapib-associated radio-activity is more balanced in both rats and monkeys, with both urinaryand fecal excretion of metabolites playing an important role in elim-ination of drug-related material (Prakash et al., 2008). This differencein routes of excretion of drug-related material between the two agentsseems to be related to differences in their metabolic pathways. Themetabolism of anacetrapib involves oxidative O-demethylation andhydroxylation at multiple sites on the biphenyl and isopropyl moieties
N
F
F
F
O
O
O
F
F
FF
F
F
F
N
F
F
F
O
O
O
F
F
FF
F
F
F
N
F
F
F
O
O
O
F
F
FF
F
F
F
N
F
F
F
HO
O
O
F
F
FF
F
F
F
N
F
F
F
O
O
O
F
F
FF
F
F
F
N
F
F
F
HO
O
O
F
F
FF
F
F
F
N
F
F
F
HO
O
O
F
F
FF
F
F
F
N
F
F
F
O
O
O
F
F
FF
F
F
F
N
F
F
F
HO
O
O
F
F
FF
F
F
F
N
F
F
F
HO
O
O
F
F
FF
F
F
F
N
F
F
F
HO
O
O
F
F
FF
F
F
F
N
F
F
F
O
O
O
F
F
FF
F
F
F
OH
OH
Anacetrapib
M12
M6
M9
M3
M4
M13
M5
M8
M2
M11
M1
O Glu
Glu
Glu
HO
O
OH
OH
OH
Glu
OH
N
F
F
F
O
O
O
F
F
FF
F
F
F
M10
N
F
F
F
HO
O
O
F
F
FF
F
F
F
M7
OH
*
SG
Glu
OH
Glu
3 O + Glu OH
Glu
FIG. 8. Proposed metabolic pathways of anacetrapib in rats and monkeys. � denotes the position of the 14C radiolabel. For sake of simplicity, only one pathway of theseveral possible for the formation of M7, M8, M10, M12, and M13 is depicted. Glu, glucuronic acid; SG, glutathione.
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as shown in Fig. 8; these oxidative metabolites are then conjugatedwith glucuronic acid before excretion into bile/feces. Furthermore, theplasma radioactivity profiles after [14C]anacetrapib administration inboth rats and monkeys are dominated by the parent drug; smalleramounts of three oxidative metabolites M1, M2, and M3 circulate inrat plasma, whereas metabolites do not appear to account for signif-icant amounts of drug-related material in monkey plasma. Torcetrapibexhibits a complex metabolic fate with as many as 28 primary andsecondary metabolites identified in preclinical species (Prakash et al.,2008). A key feature of the metabolism of torcetrapib appears to berelated to oxidative cleavage in its core structure between the bistri-fluoromethybenzyl and dihydroquinoline moieties, which results inthe formation of low molecular weight metabolites including variousfurther oxidized and/or conjugated derivatives of 2-methyl-6-trifluomethylquinoline and bistrifluoromethylbenzoic acid from the two halves of theparent compound. These low molecular weight metabolites are moresuited to excretion via the renal pathway and appear to account for theobserved differences in routes of excretion of drug-related materialbetween torcetrapib and anacetrapib. In contrast, the overall structureof anacetrapib seems to stay largely intact after metabolism, and themetabolites have a relatively high molecular mass (�600 Da), whichis more suited for excretion via the biliary/fecal pathway. Further-more, in contrast to anacetrapib, the plasma radioactivity profile after[14C]torcetrapib administration is dominated by metabolites in bothrats and monkeys. In particular, plasma exposure to low molecularweight metabolites of torcetrapib including 2-methyl-6-trifluoro-methyl quinoline and bistrifluoromethylbenzoic acid and their sec-ondary oxidized and/or conjugated derivatives is much greater relativeto that of the parent drug (Prakash et al., 2008). The relevance of theseabundant low molecular weight circulating metabolites of torcetrapibto its toxicological profile remains to be determined.
In conclusion, the pharmacokinetics and metabolism of [14C]anace-trapib were investigated in rats and monkeys. The majority of theadministered radioactivity was excreted via the fecal route and wascomposed largely of the unabsorbed parent drug, along with smallamounts of oxidative metabolites. The metabolic pathways identifiedincluded O-demethylation to M1, which was probably further hy-droxylated at the biphenyl and isopropyl moieties to form a number ofother oxidative metabolites; many of these metabolites were conju-gated with glucuronic acid before excretion into bile. Collective dataon the disposition of anacetrapib in rats and monkeys indicate thatanacetrapib probably exhibits a low-to-moderate degree of oral ab-sorption across both species and a major pathway of elimination of theabsorbed drug is through oxidative metabolism followed by excretionof metabolites (and their conjugates) via the biliary/fecal route.
Acknowledgments. We thank Rachel Ortiga, John Strauss, andSuzanne Ciccotto-Gilbert (Department of Drug Metabolism and Phar-macokinetics at Merck) for assistance with rat studies and GenevieveAndrews-Kelly, Bonnie Friscino, Cesaire Gai, Susan Iliff, Tu Trinh,and Aihua Zhang (Laboratory Animal Resources at Merck) for helpwith monkey studies.
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Address correspondence to: Dr. Sanjeev Kumar, Department of Drug Me-tabolism and Pharmacokinetics, Merck Research Laboratories, 126 E. LincolnAve., RY80-141, Rahway, NJ 07065. E-mail: [email protected]
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