In Vitro Metabolism of Mk-0767 [( )-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2- Methoxy-n-[[(4-trifluoromethyl) Phenyl]methyl]benzamide], a Peroxisome Proliferator-activated Receptor / Agonist. I. Role of Cytochrome P450, Methyltransferases, Flavin Monooxygenases, and Esterases

The metabolism of MK-0767, ( )-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl) phenyl]methyl]benzamide, a thiazolidinedione (TZD)-containing peroxisome proliferatoractivated receptor / agonist, was studied in liver microsomes and hepatocytes from humans and rat, dog, and rhesus monkey, to characterize the enzyme(s) involved in its metabolism. The major site of metabolism is the TZD ring, which underwent opening catalyzed by CYP3A4 to give the mercapto derivative, M22. Other metabolites formed in NADPH-fortified liver microsomes included the TZD-5-OH derivative (M24), also catalyzed by CYP3A4, and the O-desmethyl derivative (M28), whose formation was catalyzed by CYP2C9 and CYP2C19. Metabolite profiles from hepatocyte incubations were different from those generated with NADPH-fortified microsomal incubations. In addition to M22, M24, and M28, hepatocytes generated several S-methylated metabolites, including the methyl mercapto (M25), the methyl sulfoxide amide (M16), and the methyl sulfone amide (M20) metabolites. Addition of the methyl donor, S-adenosyl methionine, in addition to NADPH, to microsomal incubations enhanced the turnover and resulted in metabolite profiles similar to those in hepatocyte incubations. Collectively, these results indicated that methyltransferases played a major role in the metabolism of MK-0767. Using enzyme-specific inhibitors, it was concluded that microsomal thiol methyltransferases play a more important role than the cytosolic thiopurine methyltransferase. Baculovirus-expressed human flavin-containing monooxygenase 3, as well as CYP3A4, oxidized M25 to M16, whereas further oxidation of M16 to M20 was catalyzed mainly by CYP3A4. Esterases were involved in the formation of the methyl sulfone carboxylic acids, minor metabolites detected in hepatocytes. MK-0767, ( )-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxyN-[[4-trifluoromethyl) phenyl]methyl]benzamide, is a novel thiazolidinedione (TZD) derivative (Fig. 1) that was being developed as an oral antidiabetic agent and was recently discontinued due to preclinical toxicity. MK-0767 has been shown to exert potent hypoglycemic and hypolipidemic actions in obese Zucker fatty rats by virtue of its potent binding to the peroxisome proliferator-activated receptors (PPARs) and (Murakami et al., 1998). PPARs are members of the nuclear receptor family and have recently been proposed to play a key role in lipid and carbohydrate homeostasis. Three PPAR isoforms have been identified: PPAR , PPAR , and PPAR . PPAR is expressed in several tissues that have a high lipid catabolism activity, such as the liver, and has been shown to be activated by compounds such as fibrates, resulting in oxidation and stimulation of the uptake of fatty acids and synthesis of lipoproteins. PPAR is expressed abundantly in adipose tissues and has been shown to be activated by TZD derivatives, resulting in the stimulation of lipolysis of circulating triglycerides and subsequent uptake of fatty acids into the adipose cell. PPAR is expressed ubiquitously in many tissues, but the highest expression is in the gut, kidney, and heart. However, it currently lacks connection to any important clinical manifestation (Kersten et al., 2000). The objective of this study was to delineate the in vitro metabolic pathways and identify the enzymes involved in the metabolism of MK-0767. A combination of different approaches was used, namely, enzyme-specific chemical inhibitors and monoclonal antibodies, as well as immunoinhibition and metabolism by recombinant enzymes. Materials and Methods Chemicals and Reagents. MK-0767 and synthetic standards of its major metabolites (Fig. 1), the mercapto derivative, M22, the methyl mercapto, M25, the methyl sulfoxide amide M16, the methyl sulfone amide, M20, the Odesmethyl metabolite, M28, and the methyl sulfoxide carboxylic acids, M5 and M9, were synthesized at Kyorin Pharmaceutical Co., Ltd. (Tokyo, Japan). [TZD-5-C]MK-0767 (Fig. 1) was synthesized by the Labeled Compound Synthesis Group at Merck Research Laboratories (Rahway, NJ). [H]SAM Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.104.000034. ABBREVIATIONS: MK-0767, ( )-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl) phenyl]methyl]benzamide; PPAR, peroxisome proliferator-activated receptor; TZD, thiazolidinedione; BNPP, bis-p-nitrophenylphosphate; P450, cytochrome P-450; TMT, thiol methyltransferase; TPMT, thiopurine methyltransferase; FMO, flavin monooxygenase; SAM, S-adenosyl methionine; SAH, S-adenosyl homocysteine; DCMB, 2,3-dichloro-methylbenzylamine. 0090-9556/04/3209-1015–1022$20.00 DRUG METABOLISM AND DISPOSITION Vol. 32, No. 9 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 34/1172936 DMD 32:1015–1022, 2004 Printed in U.S.A. 1015 at A PE T Jornals on O cber 9, 2017 dm d.aspurnals.org D ow nladed from (S-adenosyl-L-methionine-methyl-H), SAM, S-adenosyl homocysteine (SAH), bis-p-nitrophenylphosphate (BNPP), paraoxon, anisic acid, and 2,3dichloro-methylbenzylamine (DCMB) were obtained from Sigma-Aldrich (St. Louis, MO). All other chemicals and reagents were obtained from commercial sources and were of analytical grade. Liver Preparations, Recombinant and Purified Enzymes, and Antibodies. Human liver microsomal preparations were prepared from six individual livers by differential centrifugation (Raucy and Lasker, 1991). Aliquots from each preparation were pooled on the basis of equivalent protein concentrations. Rat, dog, and rhesus monkey liver microsomes were prepared from fresh or frozen control tissue. Microsomes containing recombinant P450 isozymes, with coexpressed P450 reductase, were obtained from Dr. Tom Rushmore (Merck Research Laboratories, West Point, PA). Microsomes containing baculovirus-expressed human recombinant FMO3 were obtained from BD Gentest (Woburn, MA). Human and rat hepatocytes were isolated, based on a two-step perfusion procedure (Pang et al., 1997), and exhibited a viability greater than 80%, as determined by the trypan blue exclusion test. The cells were suspended in Krebs-bicarbonate buffer. Frozen human, rat, and dog liver cytosol and S9 preparations were purchased from In Vitro Technologies (Baltimore, MD). Ascites fluid containing monoclonal antibodies to P450 isoforms were obtained from Dr. T. Rushmore (Merck Research Laboratories, West Point, PA). Porcine and rabbit carboxy esterases were purchased from Sigma-Aldrich. Liver Microsomal Incubations. [C]MK-0767 (1–5 M) was incubated with human, rat, dog, and rhesus liver microsomes at a protein concentration of 1 to 2 mg/ml in 0.1 M potassium phosphate buffer pH 7.4, containing 1 mM EDTA and 5 mM MgCl2. Incubations were initiated by the addition of an NADPH-regenerating system, consisting of 10 mM glucose 6-phosphate, 10 mM NADP, and 1.4 units/ml of glucose-6-phosphate dehydrogenase. The reactions were carried out for 30 to 60 min at 37°C in a shaking water bath. [C]MK-0767 was added as an acetonitrile solution, the final solvent concentration not exceeding 2%. To generate methylated metabolites, microsomal incubations were fortified with a 100 M concentration of the methyl donor, SAM (solutions of 10 mM prepared in water), before addition of the NADPHregenerating system. The microsomal metabolism of some of the intermediate metabolites of MK-0767 was also studied. The mercapto metabolite, M22, was incubated at a concentration of 10 M in the presence of 100 M [H]SAM and in the presence or absence of an NADPH-regenerating system at 37°C for 30 min. The methylmercapto metabolite, M25, and the methyl sulfoxide amide, M16, were incubated at 10 M in the presence of an NADPHregenerating system at 37°C for 30 min. Typically, the total volume of the incubations was 200 l and reactions were quenched with 50 l of acetonitrile. Hepatocyte Incubations with [C]MK-0767. Rat, dog, and human hepatocytes (1–2 million cells/ml) were incubated with 1–5 M [C]MK-0767. Reactions were carried out for 30 to 60 min at 37°C. Typically, the incubation volumes were 500 l and the reactions were terminated with 100 l of acetonitrile. Incubations of [C]MK-0767 and M22 with Human Liver S9 Fraction. The compounds were incubated with human liver S9 fraction (2–5 mg of protein/ml) in a total volume of 200 l, for 60 min. Reactions were carried out in the presence and absence of 1 mM NADPH plus or minus 100 M SAM. Typically, the total volume of the incubations was 200 l and reactions were quenched with 50 l of acetonitrile. Incubations of MK-0767 and M22 with Rat, Dog, and Human Liver Cytosol. The compounds (1–10 M) were incubated with 2 to 5 mg/ml cytosolic protein in a total volume of 0.5 ml in the presence of [H]SAM for 60 min. Typically, the total volume of the incubations was 200 l and reactions were quenched with 50 l of acetonitrile. Metabolism of MK-0767, M25, and M16 by Heterologously Expressed P450 Isoforms and FMO3. [C]MK-0767 (5 M) was incubated with microsomes from cells containing recombinant P450 isoforms (80 pmol/ml CYP3A4, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP1A2) in the presence of an NADPH-regenerating system at 37°C for 60 min. Both M25 and M16 (10 M) were incubated with recombinant CYP3A4 microsomes for 60 min. MK-0767, M25, and M16 (10 M) were incubated with microsomes (1 mg/ml) containing baculovirus-expressed human FMO3 for 60 min in the presence of an NADPH-regenerating system. Typically, the total volume of the incubations was 200 l and reactions were quenched with 50 l of acetonitrile. Effect of Inhibitory Monoclonal Antibodies on Metabolism of MK0767, M25, and M16. To determine the contribution of specific P450 isozymes to the metabolism of MK-0767, human liver microsomes (2 mg/ml) were preincubated with anti-CYP3A4, CYP2C8, CYP2C9, or CYP2C19 (0–25 l/mg microsomal protein) monoclonal antibody-containing ascites fluid at room temperature for 10 min. Reactions were initiated with the addition of 5 M [C]MK-0767 and an NADPH-regenerating system, and were carried out in the absence or presence of SAM. The effect of CYP3A4 monoclonal antibody on the metabolism of M25 and M16 in human liver microsomes (1 mg/ml) was determined in a similar manner. Reactions were initiated by the addition of 10 M substrate and incubations were carried out for 30 min at 37°C. Typically, the total volume of the incubations was 200 l and reactions were quenched with 50 l of acetonitrile. Chemical Inhibition Studies. To establish the role of enzymes other than cytochrome P450 and FMOs in the metabolism of MK-0767, specific enzyme inhibitors were used. The effect of the following inhibitors was studied in microsomal incubations of MK-0767 and M22: ketoconazole (CYP3A4), DCMB [microsomal thiol methyltransferase (TMT)], and SAH (microsomal and cytosolic methyltransferase). Solutions of these compounds were prepared FIG. 1. Biotransformation pathways for MK-0767. 1016 KARANAM ET AL. at A PE T Jornals on O cber 9, 2017 dm d.aspurnals.org D ow nladed from in acetonitrile, water, and 0.1 M potassium phosphate, pH 7.4, respectively. Final concentrations in the incubations ranged from 0.1 to 1000 M. The incubations contained 2 mg/ml of microsomal protein in 0.1 M potassium phosphate, pH 7.4, with 1 mM EDTA, 5 mM MgCl2, 100 M SAM, and 5 M [C]MK-0767 or 10 M M22. Reactions were initiated with an NADPHregenerating system. Also, the effect of M22 and ketoconazole on the metabolism of MK-0767 was studied in the absence of SAM under similar conditions. The effect of the following inhibitors was studied in incubations of MK0767 and M22 with rat and human hepatocyte suspensions: BNPP and paraoxon (esterase), anisic acid (cytosolic methyltransferase), and SAH (microsomal and cytosolic methyltransferase). Solutions of BNPP, paraoxon, and anisic acid were prepared in methanol, and SAH was dissolved in 0.1 M potassium phosphate, pH 7.4. Final concentrations of the inhibitors ranged from 1 to 1000 M. The incubations consisted of 1 to 2 million hepatocyte cells/ml and substrate concentrations ranging from 1 to 5 M. The effect of the esterase inhibitors, BNPP and paraoxon, on testosterone 6 -hydroxylation (a CYP3A4-mediated reaction) was studied in human liver microsomal incubations. Testosterone (100 M) was incubated with 0.5 mg/ml microsomal protein in the presence of BNPP and paraoxon at concentrations ranging from 0.1 to 1 mM. Incubations were carried out in the presence of an NADPH-regenerating system for 20 min at 37°C in a shaking water bath. To study the inhibitory effect of M22, the compound was incubated at concentrations ranging from 0.01 to 100 M with 2 M MK-0767 or 50 M testosterone in the presence of an NADPH-regenerating system. Reactions were terminated with 5 volumes of acetonitrile. Incubations of M16 and M25 with Heat-Treated Human Liver Microsomes. Microsomes were incubated first with 50 mM sodium pyrophosphate, pH 9, at 55°C for 55 s in the absence of NADPH, and then cooled over ice. Reactions were initiated by the addition of the substrates, M25 or M16, and an NADPH-regenerating system. Data Analysis. The IC50 values were calculated by nonlinear regression analysis using KaleidaGraph software (Synergy Software, Reading, PA). Analytical Methods. HPLC. In vitro incubations of MK-0767 quenched with acetonitrile were centrifuged at 14,000 rpm at 4°C to precipitate proteins. The acetonitrile supernatants were analyzed on a Zorbax XDB-C8 column (3 150 mm, 3.5 m; MAC-MOD Analytical, Chadds Ford, PA) at ambient temperature. The column was eluted at a flow rate of 0.5 ml/min with a 30-min linear gradient from 73% A (10 mM ammonium acetate) to 50% B (93% acetonitrile and 7% methanol in 1 mM ammonium acetate). The HPLC system included a Shimadzu 10A system controller, two LC-10 AD pumps, an SIL 10A automatic sample, an SPD 10A UV-visible spectrophotometric detector, and an on-line radiometric detector (PerkinElmer Life and Analytical Sciences, Boston, MA). The wavelength for UV detection was 220 nm. Mass Spectrometry. LC-MS analyses were conducted on a PE Sciex API 3000 mass spectrometer (PerkinElmerSciex Instruments, Boston, MA) using the Turbo-Ionspray interface operated in the positive ion mode, which was interfaced with a PerkinElmer HPLC system. The temperature of the auxiliary gas was 350°C and the ionization voltage was 4500 V. The orifice and ring voltages were set to 48 V and 240 V, respectively. Tandem mass spectrometry experiments were performed using a collision energy of 42 eV (laboratory frame) and a collision cell gas pressure of 4 (N2). A Zorbax XDB-C8 column (3.0 150 mm, 3.5 m) was used for chromatographic separation with a gradient of 10 mM ammonium acetate in water (A) and 7 mM ammonium acetate in acetonitrile with 7% methanol (B) for elution at a flow rate of 0.5 ml/min. The gradient was started at 26% B, and increased linearly to 30% B in 5 min, followed by a hold at 30% B for 7 min. It was then increased linearly to 55% B in 20 min, followed by a linear ramp to 90% B in 1 min. Accurate mass measurements were conducted on a Micromass Q-ToF II mass spectrometer (Waters, Milford, MA) operated in the positive electrospray ionization mode. The desolvation gas temperature was 80°C and the capillary voltage was 3000 V. The cone voltage and collision energy were 48 V and 10 V, respectively.