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Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania (C.J.K., R.K.R., K.X.Y., H.S.) and Rahway, New Jersey (M.A.W., D.D., A.N.J., S.H.V., R.B.F.); Department of Clinical Pharmacology, Merck Research Laboratories, Rahway, New Jersey (J.S., J.W.); and Clinical Pharmacology Associates, Miami, Florida (K.L.)
(Received March 22, 2006; accepted May 31, 2006)
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Abstract |
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and 
that has been studied as a potential treatment for patients with type 2 diabetes. The metabolism and excretion of [14C]MK-0767 were evaluated in six human volunteers after a 5-mg (200 µCi) oral dose. Excretion of 14C radioactivity was found to be nearly equal into the urine (
50%) and feces (40%). Elimination of [14C]MK-0767 was primarily by metabolism, with minimal excretion of parent compound into the urine (<0.5% of dose) and feces (14% of the dose). [14C]MK-0767 was the major circulating compound-related entity (>96% of radioactivity) through 48 h postdose. It was also found that 91% of the total radioactivity area under the curve was due to intact MK-0767. Several minor metabolites were detected in plasma (<1% of radioactivity, each), formed by cleavage of the TZD ring and subsequent S-methylation and oxidation. All the metabolites excreted into urine were formed by TZD cleavage, whereas the major metabolite in feces was the O-demethylated derivative of MK-0767.
). In the same structural class as rosiglitazone and pioglitazone, MK-0767 is distinctly different from these compounds in that it binds to both and subtypes of the peroxisome proliferator-activated receptor (PPAR) with similar affinity (Murakami et al., 1998; Doebber et al., 2004), whereas rosiglitazone and pioglitazone were reported to bind primarily to the PPAR- receptor (Lehmann et al., 1995; Forman et al., 1997). Binding to PPAR- and - results in increased expression of genes encoding proteins involved in glucose and lipid metabolism (Mudaliar and Henry, 2001).
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,b; Liu et al., 2004; Reddy et al., 2004), whereas the in vivo metabolism in nonclinical species is the subject of a separate manuscript in preparation (S. Vincent, C. Kochansky, M. Creighton, G. Doss, B. Karanam, M. Wallace, C. Raab, H. Jenkins, R. Franklin, S. Chiu, H. Satoh, K. Awano, and M. Komuro, unpublished). The main biotransformation pathway of MK-0767 in human liver microsomes involves CYP3A4-mediated oxidative cleavage of the TZD ring, followed by S-methylation (catalyzed primarily by microsomal methyltransferase) to the methyl mercapto metabolite (M25) and subsequent S-oxidation (catalyzed by both CYP3A and flavin-containing monooxygenase) to methyl sulfoxide and sulfone amides (M16 and M20, respectively). In hepatocytes, M16, M20, and M25 are subject to esterase-mediated hydrolysis to the carboxylic acid analogs (Liu et al., 2004). |
Materials and Methods |
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40 µCi/mg) was synthesized by the Labeled Compound Synthesis Group (Merck Research Laboratories, Rahway, NJ) with 99.3% radiochemical purity (HPLC). [14C]MK-0767 was formulated as a 0.25 mg/ml solution in 10% sulfobutyl ether cyclodextrin (Captisol) and 5% glycerol in 5 mM Tris buffer, pH 8.3, and stored at –20°C until administered. Dose Administration. A single oral dose of 5 mg (200 µCi) of [14C]MK-0767 was administered to six healthy, young male volunteers, 18 to 45 years old, by the Clinical Pharmacology Associates in Miami, FL. Dosing syringes were filled with 20 ml of dosing solution (5 mg of MK-0767) and weighed, and then the dose was administered to the subject by squirting the solution directly into the mouth. This was followed by 240 ml of water. The syringes were then reweighed to determine the amount of dose administered to each subject. Before dosing, subjects had fasted for 10 h, refrained from drinking water for 1 h, and avoided exercise for 24 h. In addition, unusual or strenuous exercise was avoided for the duration of the study. Volunteers were monitored for adverse events and vital signs, and given a physical examination, 12-lead electrocardiogram, and laboratory safety tests (complete blood count, blood chemistry, and urinalysis) after administration.
Sample Collection. Blood samples (12 ml) were collected from each subject into heparinized tubes predose and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 12, 15, 18, 24, 30, 36, 48, 60, 72, 96 h, and every 24 h up to and including 504 h postdose. Plasma was prepared within 30 min of blood draw by spinning in a centrifuge at 3000 rpm at 4°C for 15 min. Plasma was then separated into two tubes for quantitative (1 ml of plasma) and qualitative analysis (remaining plasma) and stored at –70°C. All voided urine was collected from each subject predose, and at 0 to 6, 6 to 12, 12 to 18, and 18 to 24 h, and then daily through 21 days postdose. All feces and tissue wipes were collected during predose and the 21-day period postdose. Exhaled CO2 was collected predose and 1 and 4 h postdose by subjects exhaling through a one-way valve into large scintillation vials containing 4 ml of a 1:1 mixture of 1 M hyamine hydroxide solution in methanol and ethanol containing 2 drops of 1% thymophthalein (blue indicator). The blue solution turns clear upon collection (CO2 saturation of hyamine) in approximately 40 to 60 s. After collection, Scintisafe Gel cocktail (12 ml; Fisher Scientific, Pittsburgh, PA) was added to each sample, and then the samples were stored at 4°C.
Sample Preparation. Plasma. For the quantification of MK-0767, 50 µl of plasma, 150 µl of 100 mM ammonium acetate (pH 4.0), 50 µl of methanol/water (1:1, v/v), and 50 µl of internal standard (50 ng/ml), an analog of MK-0767, were added to a polypropylene tube and vortexed. The sample mixtures were loaded onto an ISOLUTE C18 96-well solid-phase extraction cartridge plate that had been preconditioned with 0.5 ml of methanol and 0.5 ml of 100 mM ammonium acetate (pH 4.0). The plate was then washed with 0.5 ml of 5% methanol in water, followed by elution of the analytes with 0.2 ml of methanol. The eluents were evaporated to dryness under nitrogen at 40°C for 20 min. The samples were reconstituted in 0.1 ml of methanol/water (50:50, v/v), and 20-µl aliquots were analyzed by LC-MS/MS.
For metabolite profiling, equal volumes of plasma (1 ml) were combined from each subject for each selected time point (1, 2, 4, 8, 24, and 48 h). The combined plasma samples were extracted using 4 volumes of a 1:3 mixture of methanol and acetonitrile. The plasma mixtures were vortexed, sonicated, and spun in a centrifuge for 20 min at 1850g and 4°C. The supernatants were transferred to clean tubes and evaporated to dryness under nitrogen. The dry extracts were reconstituted in 0.5 ml of a 2:3:3 mixture of water/methanol/acetonitrile by vortexing, sonicating, and vortexing.
Urine. For qualitative analysis, urine was initially pooled according to weight from 0 to 168 h. Pooled urine from each subject was then combined according to radioactivity. This single pool was mixed with an equal volume of methanol, spun in a centrifuge at 1850g and 4°C for 20 min, and the supernatant was transferred to a clean tube and evaporated under nitrogen. The dried residue was reconstituted in 0.35 ml of a 2:3:3 mixture of water/methanol/acetonitrile by vortexing, sonicating, and vortexing.
Feces. For quantitative analysis of total radioactivity, each fecal sample was homogenized in approximately 2 volumes of a 1:1 mixture of ethanol/45 mM Tris solution (TRIZMA Base; Sigma-Aldrich, St. Louis, MO) using a Polytron 3100 equipped with a 20-mm probe (Kinematic). Approximately 200 to 400 mg of each fecal homogenate sample were transferred into combustion cones in triplicate and weighed. Samples were air-dried in a hood overnight.
For metabolite profiling, fecal homogenates from 0 to 120 h were pooled according to the weight excreted at each time point. Pooled homogenates from each subject were then combined according to radioactivity. Approximately 5 g of the pooled homogenate was mixed with 4 volumes of a 1:3 mixture of methanol and acetonitrile, and spun in a centrifuge at 1850g and 4°C for 20 min. The supernatant was transferred to a clean tube and evaporated under nitrogen. The dried residue was reconstituted in 0.7 ml of a 2:3:3 mixture of water/methanol/acetonitrile by vortexing and sonicating.
Quantitative Analysis. Determination of MK-0767 Plasma Concentrations by LC-MS/MS. The HPLC system consisted of a PerkinElmer (Norwalk, CT) Series 200 autosampler and a quaternary pump. Chromatographic separation was performed on a Phenomenex (Torrance, CA) Luna C18 column (50 x 2.1 mm, 5 µm) eluted isocratically with a mixture of 0.1% formic acid in water and methanol (35:65, v/v). The flow rate was 0.2 ml/min. The analyte and internal standard were detected in positive ionization mode by monitoring the transitions m/z 439.0 
m/z 264.0 for MK-0767 and m/z 415.0 m/z 264.0 for the internal standard in multiple-reaction monitoring mode. The linear dynamic range was from 4 ng/ml to 2000 ng/ml, with an intraday accuracy and precision (%CV) ranging from 99.8 to 110% and 2.3 to 4.7%, respectively. Concentrations of MK-0767 were determined from the linear least-squares fitted line of the peak area ratios of MK-0767 to the internal standard versus the concentrations of MK-0767, with reciprocal weighting (1/x) on the concentration.
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Pharmacokinetics. Intact MK-0767 and total radioactivity plasma concentrations were plotted versus time. Area under the curve to the time of last detectable MK-0767 concentration (AUC0-t; t ranged from 144 to 240 h) was calculated for both MK-0767 and total radioactivity using the linear-log trapezoidal method. Cmax and Tmax were determined by inspection, whereas the apparent half-life (t1/2) for intact MK-0767 was estimated using linear regression of the terminal portion of the log-transformed concentration-time curve.
Qualitative Analysis. Metabolite profiles were generated for plasma, urine, and feces using LC-MS/MS and radiometric detection. Samples were injected onto a Luna phenyl-hexyl (4.6 x 150 mm, 4-µm particle size) column (Phenomenex) using a PerkinElmer Series 200 autosampler. Analytes were eluted from the column with a mobile phase consisting of solvent A (10 mM ammonium acetate and 0.1% acetic acid in water) and solvent B (10 mM ammonium acetate and 0.1% acetic acid in 7:93 methanol/acetonitrile) pumped by two solvent dedicated micro pumps (PerkinElmer Series 200) at a flow rate of 1 ml/min. The column was eluted with an isocratic hold of 5% B for 3 min, followed by a 45-min linear gradient to 50% B, a 5-min linear gradient to 60% B, a 2-min linear gradient to 95% B, and a 7-min isocratic hold at 95% B. The column was equilibrated for 10 min at 5% B before the next run. Analytes were detected using a Sciex API3000 mass spectrometer equipped with a turbo ion-spray source. Radioactivity in the column eluent was measured either on-line using a PerkinElmer 625A Flow Scintillation Analyzer, or by collecting 30-s fractions with a Gilson (Middleton, WI) FC204 fraction collector, mixing with 6 ml of Scintisafe Gel cocktail (Fisher Scientific), and counting the fractions off-line in a 1900TR Liquid Scintillation Analyzer (PerkinElmer). The eluent was split so that one-fifth of the flow was directed to the mass spectrometer with the remaining going to the scintillation analyzer or fraction collector. Metabolite identification was achieved by multiple reaction monitoring of known mass to charge ratio transitions of MK-0767 and metabolites after collision-activated disassociation and by comparison with known standards or previously identified metabolites (Liu et al., 2004).
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Results |
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90%, with 50% and 40% excreted into urine and feces, respectively, over the 21-day collection period (Table 1). Exhaled air was collected 1 and 4 h postdose and examined for [14C]CO2. Radioactivity levels were not above background.
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An intravenous dose was not given in this study. A previous study with unlabeled compound in the sulfobutyl ether cyclodextrin formulation used in this study resulted in a bioavailability of 96% (data not shown), which deemed an i.v. dose of [14C]MK-0767 not necessary.
Plasma Concentrations. Mean plasma concentrations of total radioactivity and MK-0767 versus time are shown in Fig. 2. MK-0767 was the main circulating radioactive entity upon comparison of MK-0767 levels with total radioactivity. Parent compound accounted for 91% of the total plasma radioactivity area under the curve (AUC) over 216 h (Table 2).
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[14C]MK-0767 and Metabolites in Plasma, Urine, and Feces. Plasma. A representative HPLC radiochromatogram of pooled plasma from six human subjects 24 h after oral administration of [14C]MK0767 is shown in Fig. 3. Essentially identical profiles were obtained at all plasma time points profiled (1, 2, 4, 8, 24, and 48 h). [14C]MK-0767 was the major radioactive component at all time points (>96%). Several metabolites, also identified in nonclinical species, were detected by LC-MS/MS. Metabolites M13, M16, M20, and M25 were detected at all time points at levels <1% of circulating radioactivity for each metabolite (Table 3).
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Urine. Metabolites accounted for >99% of the radioactivity found in pooled 0- to 168-h urine (50% of total dose; Fig. 4). The methyl sulfoxide amide (M16) was the major metabolite, accounting for 52% of urinary radioactivity, followed by the methyl sulfone amide (M20, 19%), the isomeric methyl sulfoxide carboxylic acids M5 and M9 (4 and 8%, respectively), M10 (4%), M13 (2%), M11 (1.5%), and M24 (1%). Unchanged parent compound represented <0.5% of the radioactivity in urine.
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Discussion |
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MK-0767 was eliminated mainly by metabolism, followed by excretion of the metabolites into urine and feces, with 14% of the dose recovered as parent compound in the feces. Approximately 50% of the dose was recovered in urine as metabolites, most of which were formed by oxidative TZD cleavage (M16, which was the major urinary metabolite, M20, M5, and M9) or complete loss of the TZD (M10). The only urinary metabolite with an intact TZD ring was M24, which accounted for 1% of the urinary radioactivity (0.5% of the dose). Parent compound represented <0.5% of the urinary radioactivity. The radioactivity in feces (40% of the dose) was composed primarily of MK-0767 and its O-desmethyl derivative, M28, as the major radioactive components, 14% and 4.5% of the dose, respectively. Also excreted into human feces, but at lower levels, were the hydroxylated products M30 and M31. These metabolites were detected in feces but not bile from intravenously dosed rats, dogs, and monkeys, and as discussed elsewhere, most likely they were excreted as labile conjugates into bile (unpublished results).
The oral bioavailability of MK-0767 from the cyclodextrin formulation used in this study was determined in a separate study to be 96% (unpublished data). Thus, most likely, the MK-0767 excreted into feces represents primarily absorbed material that was excreted either unchanged or as a labile conjugate. As reported elsewhere (Reddy et al., 2004; unpublished results), labile conjugates of MK-0767 and its phase I metabolites were detected in rat, dog, and monkey bile. Specifically, rat bile was shown to contain a dihydrohydroxy-S-glutathionyl conjugate on the trifluoromethylbenzyl-ring of MK-0767, which upon incubation with rat intestinal contents was converted to MK-0767 (Reddy et al., 2004).
MK-0767 has been shown to be eliminated largely by TZD-ring cleavage in all species (S. Vincent, C. Kochansky, M. Creighton, G. Doss, B. Karanam, M. Wallace, C. Raab, H. Jenkins, R. Franklin, S. Chiu, H. Satoh, K. Awano, and M. Komuro, manuscript in preparation). This is somewhat different from the elimination of other TZD-containing compounds like rosiglitazone (Bolton et al., 1996; Cox et al., 2000), pioglitazone (Krieter et al., 1994; Maeshiba et al., 1997), and troglitazone (Kawai et al., 1997). Interestingly, there have been several recent articles building on the work of Kassahun et al. (2001) with TZD-ring scission of troglitazone. Pioglitazone was recently shown to undergo TZD-ring opening to reactive products in rat, dog, and human liver microsomes and rat but not human hepatocytes (Shen et al., 2003; Baughman et al., 2005). In addition, Reddy et al. (2005) identified a mechanism for TZD-ring opening using an MK-0767 analog, MRL-A, which involves P450-mediated S-oxidation followed by spontaneous scission to a sulfenic acid intermediate. This intermediate then undergoes reduction by glutathione, NADPH, and/or disproportionation to the free thiol (Reddy et al., 2005). Thus, it would be expected that MK-0767 would follow an analogous mechanism to form the free thiol (M22) before undergoing the subsequent S-methylation and oxidation steps (Fig. 1). This facile methylation of the mercapto intermediate (M22) most likely attenuates any potential risk for idiosyncratic or other toxic reactions resulting from TZD-ring cleavage of MK-0767.
To summarize, in humans, [14C]MK-0767 was the major circulating entity after a 5 mg/200 µCi oral dose. Elimination occurred by metabolism, followed by nearly equal excretion of radioactivity into urine and feces. Metabolites excreted into urine were formed primarily by TZD cleavage, whereas the major radioactive components in feces were MK-0767 and its O-demethylated derivative.
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Acknowledgments |
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Footnotes |
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ABBREVIATIONS: MK-0767, (±)-5-[2,4-dioxothiazol-5-yl)methyl]-2-methoxy-N-[4-trifluoromethyl] benzamide; HPLC, high-performance liquid chromatography; LC-MS/MS, liquid chromatography-tandem mass spectrometry; AUC, area under the curve; PPAR, peroxisome proliferator-activated receptor; TZD, thiazolidinedione.
Address correspondence to: Christopher J. Kochansky, Merck Research Laboratories, P.O. Box 4, Sumneytown Pike, West Point, PA 19486. E-mail: christopher_kochansky{at}merck.com
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References |
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/ agonist, displays robust antihyperglycemic and hypolipidemic activities. Biochem Biophys Res Commun 318: 323–328.[CrossRef][Medline] and . Proc Natl Acad Sci USA 94: 4312–4317. (PPAR ). J Biol Chem 270: 12953–12956. (PPAR-) and PPAR-. Diabetes 47: 1841–1847.[Abstract]This article has been cited by other articles:
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  R. K. Rippley, K. X. Yan, N. D. Matthews, H. E. Greenberg, G. A. Herman, and J. A. Wagner Human Pharmacokinetics and Interconversion of Enantiomers of MK-0767, a Dual PPAR{alpha}/{gamma} Agonist J. Clin. Pharmacol., March 1, 2007; 47(3): 323 - 333. [Abstract] [Full Text] [PDF]
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