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IDENTIFICATION AND METABOLISM OF A NOVEL DIHYDROHYDROXY-S-GLUTATHIONYL CONJUGATE OF A PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR AGONIST, MK-0767 [(±)-5-[(2,4-DIOXOTHIAZOLIDIN-5-YL)METHYL]-2-METHOXY-N-[[(4-TRIFLUOROMETHYL) PHENYL]METHYL]BENZAMIDE], IN RATS

Department of Drug Metabolism, Merck Research Laboratories, Rahway, New Jersey

(Received April 9, 2004; accepted June 23, 2004)

	Abstract

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MK-0767 [(±)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide] is a novel thiazolidinedione-containing peroxisome proliferator-activated receptor / agonist. In rats dosed orally with [¹⁴C]MK-0767, a dihydrohydroxy-S-glutathionyl conjugate of the parent compound was identified in the bile using liquid chromatography-mass spectometry and ¹H NMR techniques. The formation of the conjugate likely proceeded via an arene oxide intermediate. The corresponding cysteinylglycine and cysteinyl conjugates likely formed from the further metabolism of the dihydrohydroxy-S-glutathionyl conjugate also were detected in rat bile. The dihydrohydroxy-S-glutathionyl conjugate was formed in vitro following the incubation of MK-0767 and glutathione with rat, dog, or monkey liver microsomes, and its formation was NADPH-dependent; however, this conjugate was not detected in human liver microsomal incubations. When incubated with rat intestinal contents, the dihydrohydroxy-S-glutathionyl conjugate was reduced to the parent compound (MK-0767), suggesting the involvement of intestinal microflora in its metabolism. There was no reduction of the conjugate by rat intestinal cytosol.

The in vitro and in vivo metabolism of MK-0767 has been studied extensively in rats, dogs, monkeys, and humans (S. Vincent, M. Creighton, C. Kochansky, B. Karanam, and R. Franklin, unpublished results; Karanam et al., 2004; Liu et al., 2004), and its metabolism was qualitatively similar across the species. The major biotransformation pathway involved the scission of the TZD ring followed by S-methylation to form the methyl mercapto derivative, which was subsequently oxidized to the methyl sulfoxide and methyl sulfone metabolites (Karanam et al., 2004). Following i.v. or p.o. administration of a single dose of MK-0767 to rats, dogs, and monkeys, the compound was eliminated mainly by way of metabolism followed by excretion of the metabolites into urine and feces via the bile (S. Vincent, M. Creighton, C. Kochansky, B. Karanam, and R. Franklin, unpublished results). It was observed, however, that the metabolite profiles in rat bile were different from those in feces from intact rats. The metabolite profile in bile was characterized by multiple polar entities as the major components, with trace amounts of intact parent. The metabolite profile in feces, on the other hand, was distinct in that it showed large amounts of the parent compound as well as the above-mentioned polar metabolites. Furthermore, incubation of the bile samples with intestinal contents generated metabolite profiles similar to the fecal profiles, suggesting that the polar components in bile were most likely conjugates of the parent compound as well as its metabolites (S. Vincent, M. Creighton, C. Kochansky, B. Karanam, and R. Franklin, unpublished results). The present study was undertaken to identify and characterize the polar metabolite(s) in rat bile, and it led to the identification of a novel dihydrohydroxy-S-glutathionyl (DHSG) conjugate of MK-0767. Such conjugates, formed by the nucleophilic addition of glutathione to an arene oxide intermediate, are generally unstable. In most cases, DHSG conjugates undergo dehydration to produce rearomatized glutathione conjugates. In the present study, the DHSG conjugate of MK-0767 was found to be stable, and it was isolated and characterized by LC-MS and ¹H NMR spectroscopic techniques. Furthermore, incubation of the isolated conjugate with rat intestinal contents produced the parent compound, suggesting a novel reduction by gut microflora. The mechanism of formation of the DHSG conjugate and its subsequent reduction to the parent compound are discussed.

	Materials and Methods

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Materials. MK-0767 was synthesized at the Kyorin Pharmaceutical Co., Ltd. (Tokyo, Japan). [¹⁴C]MK-0767 was synthesized by the Labeled Compound Synthesis Group at Merck Research Laboratories in Rahway, NJ. The specific activity was 50 Ci/mmol, and the radiochemical purity, as determined by HPLC, was 99.5%. Acetonitrile and methanol (HPLC grade) were purchased from Fisher Scientific Co. (Pittsburgh, PA). NADPH, ammonium acetate, reduced glutathione, and glutathione S-transferase (from rat liver) were obtained from Sigma-Aldrich (St. Louis, MO). Rat, dog, monkey, and human liver microsomes were prepared using literature procedures. The amount of P450 enzyme present in rat, dog, and human liver microsomal protein was 0.21, 0.43, and 0.60 nmol/mg, respectively. The amount of P450 enzyme present in monkey liver microsomes was not estimated.

Animal Experiments. Experiments were performed according to procedures approved by the Merck Institutional Animal Care and Use Committee. Bile duct-cannulated male Sprague-Dawley rats, male beagles, and male rhesus monkeys were dosed orally at 2 or 100 mg/kg [¹⁴C]MK-0767 (2 or 25 mg/ml, n = 3/species). The dose was formulated as a solution in ethanol: 0.5% aqueous methylcellulose (10:90 v/v) with 0.02% SDS (w/v). Bile was collected over cold packs, urine was collected over dry ice, and fecal samples were collected at room temperature for up to 120 h postdose and stored at -80°C until analysis.

Analysis of Bile and Isolation of the DHSG Conjugate. The pH of an aliquot of bile (0-72 h) from rat, dog, or monkey was adjusted to 6 with 50% acetic acid, and an equal volume of acetonitrile was added. The mixture was vortex-mixed and spun in a centrifuge at 14,000g for 10 min. The supernatant was used for the metabolite analysis by LC-MS/MS. For the isolation of the DHSG conjugate, 10 ml of rat bile (8-24 h) was concentrated to about 1 ml under a stream of nitrogen, an equal volume of acetonitrile was added, and the mixture was spun in a centrifuge as described above. The conjugate was isolated from the supernatant by preparative HPLC.

Incubation of the DHSG Conjugate with Intestinal Contents. The following experiments were conducted under an atmosphere of nitrogen. The fresh contents of the cecum (6 g) from a male Sprague-Dawley rat were suspended in 20 ml of 0.1 M phosphate buffer, pH 7.4, at 4°C. The suspension was vortex-mixed briefly and spun in a centrifuge at 4°C (2000 rpm, 5 min). The pellet was discarded, and the supernatant, termed intestinal contents, was used for the experiments. The purified conjugate (2 µg, 40,000 dpm, 50 µl) was incubated with 1 ml of intestinal contents for 2 h at 37°C in a water bath. At the end of the incubation period, 2 ml of acetonitrile was added; the suspension was vortex-mixed and spun in a centrifuge at 3,000 rpm for 15 min. The supernatant was concentrated to dryness under a stream of nitrogen. The residue was reconstituted in 180 µl of 25% acetonitrile in water, and 50-µl portions were analyzed by LC-MS/MS. Control experiments were run in parallel with heat-inactivated intestinal contents suspended in phosphate buffer.

Incubation of the DHSG Conjugate with Intestinal Cytosol. Small intestines from three rats were rinsed with 10 mM phospate buffer, pH 7.4, containing 0.5% NaCl (phosphate-buffered saline). Intestines were cut longitudinally, and the surface was scraped gently with a cell policeman. The mucosal scrapings were mixed with 2 volumes (w/v) of 10 mM phosphate buffer containing 5 mM EDTA, pH 7.4, and homogenized for 15 s at 600 rpm using a Con-Torque homogenizer (Eberbach Corp., Ann Arbor, MI). The homogenate was subjected to differential centrifugation to isolate cytosol and microsomes. The DHSG conjugate (2 µg, 40,000 dpm, 50 µl) was incubated with cytosol at a protein concentration of 4 mg/ml in 0.1 M KH₂PO₄ buffer (pH 6.5) containing 1 mM glutathione for 60 min at 37°C in a shaking water bath. The reaction was quenched with 2 volumes of acetonitrile and analyzed by LC-MS/MS as described below.

Incubation with Liver Microsomes. [¹⁴C]MK-0767 (2 µM) was incubated with rat, dog, monkey or human hepatic microsomes at a protein concentration of 2 mg/ml in 0.1 M KH₂PO₄ buffer containing 0.1 mM MgCl₂, with or without 1 mM glutathione and 100 units of glutathione S-transferase. Incubations were initiated by the addition of 1 mM NADPH, and [¹⁴C]MK-0767 was added as an acetonitrile solution. The final concentration of acetonitrile in the assay mixture was 1% (by volume). Testosterone was used as a positive control. The reactions were carried out for 60 min at 37°C in a shaking water bath. Reaction mixtures were quenched with the addition of 2 volumes of acetonitrile. Following centrifugation, the supernatant was concentrated to dryness under a stream of nitrogen, and the residue was reconstituted in 180 µl of water/ethanol/acetonitrile (1:1:1 by volume). Aliquots of 50 µl were analyzed by LC-MS/MS. The glutathione conjugate (m/z 762) and parent compound (m/z 439) were identified by single ion monitoring, and the product ion spectra were obtained upon collision-induced dissociation (CID) of the [M + H]⁺ ions.

Instrumentation. Metabolites were identified by electrospray LC-MS/MS analysis using a Finnigan LCQ mass spectrometer (Thermo Finnigan, San Jose, CA), which was interfaced with a Shimadzu HPLC system equipped with two Series LC-10ADVP micro pumps and a Series SIL-10ADVP auto sampler (Shimadzu, Kyoto, Japan). The spray voltage was maintained at 4.1 kV, and the capillary temperature was set at 250°C. Full scan spectra, from m/z 400 to 1000, were obtained in the positive or negative ion mode, and product ion spectra were generated by CID of MH⁺ ions of interest. The CID of MH⁺ was achieved with nitrogen as the collision gas at the collision energy of 35 eV. Separation of metabolites was achieved on a 5-µm Zorbax SB C8 column (4.6 x 250 mm) (Agilent, Wilmington, DE) at a flow rate of 1 ml/min. The mobile phase consisted of 10 mM ammonium acetate in water (A) and 7 mM ammonium acetate in acetonitrile/methanol (93:7 v/v; B). Mobile phase A and B contained 0.1% acetic acid. The column was eluted with a linear gradient from 25 to 90% B over 40 min. One-fourth of the column eluate was directed into the mass spectrometer; the remaining column eluate was directed into a ß-Ram radiometric detector (IN/US Systems, Inc., Pine Brook, NJ) for online radio-profiling. For the purification of the glutathione conjugate, the column was eluted at a linear gradient from 20 to 30% over 30 min followed by a wash with 90% B for 10 min at a flow rate of 1 ml/min.

NMR Analysis. Proton NMR spectra were acquired on a Varian Inova 600 MHz spectrometer (Varian Inc., Palo Alto, CA) using a 3-mm probe. Authentic standards of MK-0767 and the purified metabolite were dissolved in CD₃CN/D₂O (2:1 v/v). The chemical shifts are expressed as parts per million relative to tetramethylsilane.

	Results

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Identification of the DHSG Conjugate in Rat Bile. A representative HPLC radiochromatogram of the bile from rats dosed orally with [¹⁴C]MK-0767 is shown in Fig. 1. A polar peak eluting at 7.3 min constituted about 30% of the radioactivity in bile (19% of the dose). This peak was not present in the bile of dogs and monkeys analyzed under the same HPLC and LC-MS/MS conditions as the rat bile (data not shown). LC-MS analysis of the purified metabolite from rat bile indicated an MH⁺ ion at m/z 762 (or an MH^- ion at m/z 760), which was 323 Da higher than that of the parent compound (MK-0767, MH⁺ 439 Da).

View larger version (10K): [in this window] [in a new window]   FIG. 1. Representative HPLC radiochromatogram of bile from rats dosed with [14C]MK-0767. Bile duct-cannulated rats were dosed orally at 2 mg/kg formulated in ethanol: 0.5% methylcellulose (10:90 v/v). Bile was collected and analyzed as described in the text.

The CID spectrum of the MH⁺ ion m/z 762 yielded fragment ions m/z 633 corresponding to the loss of 129 Da, m/z 615 due to the loss of 147 Da (129 Da + water), and m/z 669 corresponding to the loss of 93 Da (75 Da + water) (Fig. 2). These are the characteristic losses of glutathione-conjugated metabolites—129 Da due to glutamate and 75 Da due to glycine (Murphy et al., 1992; Baillie and Davis, 1993). The product ion spectrum also showed the deconjugation process generating fragment ions at m/z 439 due to the MK-0767 ion and the ion at m/z 306 attributed to the glutathionyl group. Based on the above data, a tentative structure for the metabolite was assigned, as shown in Fig. 2. A possible fragmentation pattern yielding the parent ion m/z 439 also is shown in Fig. 2, which may explain the origin of the fragment ion at m/z 306 as well due to the glutathionyl group.

View larger version (21K): [in this window] [in a new window]   FIG. 2. Product ion spectra of the dihydrohydroxy-S-glutathionyl conjugate of MK-0767 (m/z 762, +c electrospray ionization).

Confirmation of the structure of the DHSG conjugate was obtained by ¹H NMR analysis of the purified metabolite. The peak assignments are summarized in Table 1. The downfield regions of the ¹H NMR spectra of the metabolite and the parent compound MK-0767 are shown in Fig. 3. The two doublets between 7.4 and 7.6 ppm arise from the four aromatic protons on the terminal phenyl ring of the parent compound MK-0767. This part of the spectrum is absent from the corresponding region of the NMR spectrum of the metabolite, suggesting that the terminal phenyl ring, and not the methoxyphenyl ring, had undergone metabolism. The two doublets at 6.62 and 6.00 ppm are due to two new olefinic protons (a and b) on the trifluoromethyl-cyclohexadienyl ring (Fig. 3), also suggesting that the hydroxyl and glutathionyl groups are located at the positions a' and b'. Other positions were ruled out since they would have led to conjugates showing three olefinic protons.

View larger version (17K): [in this window] [in a new window]   FIG. 3. 1H NMR spectrum (downfield region) of the dihydrohydroxy-S-glutathionyl conjugate (A) compared with the parent compound MK-0767 (B).

The exact positions of the hydroxyl and glutathionyl groups were further distinguished based on the relative chemical shifts of the attached methine protons, CH-O being more downfield than CH-SG. The two methine protons (H-a' versus H-b') were unambiguously assigned by a heteronuclear ¹H {¹⁹F} NOE experiment, wherein the CF₃ group was irradiated and NOE's at adjacent protons (H-a and H-a') were detected (Fig. 4). Thus, the chemical shift of H-a' was found to be downfield compared with H-b', indicating that it is the CH-OH rather than the CH-SG; hence, the positions of the -OH and glutathionyl groups were assigned as shown in Fig. 3.

View larger version (11K): [in this window] [in a new window]   FIG. 4. 1D 1H {19F} NOE experiment irradiating the CF3 group of the dihydrohydroxy-S-glutathionyl conjugate of MK-0767.

Identification of DHSCG and DHSC Conjugates. Further LC-MS analysis of the rat bile indicated the presence of MH⁺ ions of m/z 633 and 576. The CID of the ion at m/z 633 produced fragments at m/z 615 due to the loss of water and m/z 439 likely due to the loss of cysteinylglycine and water. Similarly, CID of the m/z 576 ion yielded fragments at m/z 558 due to the loss of water and m/z 439 likely due to the loss of cysteine and water. Based on this data, m/z 633 and 576 were identified as DHSCG and DHSC conjugates of MK-0767, respectively. These conjugates were identified also in bile from dogs but not from rhesus monkeys (data not shown).

In Vitro Formation of the DHSG Conjugate of MK-0767. The DHSG conjugate of MK-0767 was identified in rat, dog, and monkey hepatic microsomal incubations of MK-0767 in the presence of NADPH and reduced glutathione, as confirmed by the product ion spectra of the selected ions m/z 762 and 439 (Fig. 5). Supplementing the incubation mixtures with commercially available rat hepatic glutathione transferase did not have any effect on the formation of glutathione conjugate (data not shown). The peak area of the conjugate formed in the microsomal incubations followed the rank order dog >> rat monkey (Fig. 5). The conjugate was undetectable in human hepatic microsomal incubations. When the dog hepatic microsomal incubations were supplemented with cysteine instead of reduced glutathione, a cysteine conjugate of MK-0767 was identified. The CID of the selected ion of this conjugate at m/z 576 was identical to that observed in rat bile.

View larger version (31K): [in this window] [in a new window]   FIG. 5. In vitro formation of dihydrohydroxy-S-glutathionyl conjugate of MK-0767 by liver microsomes from rat, dog, and monkey. MK-0767 was incubated with liver microsomes in the presence of NADPH and glutathione, and samples were analyzed by LC-MS/MS as described in the text.

Reduction of the DHSG Conjugate by Intestinal Contents. When the purified DHSG conjugate of MK-0767 was incubated with fresh rat intestinal contents, the conjugate was converted to the parent compound (Fig. 6). The identity of the parent compound was confirmed by comparing the HPLC retention time and CID of the MH⁺ ion m/z 439 to that of the authentic standard of MK-0767. The conjugate was unchanged when incubated with heat-inactivated intestinal contents or intestinal cytosol in the presence of glutathione as a cofactor (data not shown).

View larger version (20K): [in this window] [in a new window]   FIG. 6. Reduction of the dihydrohydroxy-S-glutathionyl conjugate of MK-0767 by rat intestinal contents. The conjugate was incubated (A, control; B, 2 h) with rat intestinal contents under an atmosphere of nitrogen, and the samples were extracted with acetonitrile and analyzed by LC-MS/MS as described in the text.

	Discussion

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MK-0767, a PPAR / agonist, is metabolized in vitro mainly via CYP3A4-mediated oxidation of the TZD ring. The resulting TZD ring-opened product, a mercapto metabolite, undergoes S-methylation and further oxidation to the sulfoxide and sulfone derivatives (Karanam et al., 2004). In rats, dogs, and monkeys, MK-0767 is eliminated primarily through metabolism followed by excretion of the metabolites into bile (50-65%) and urine (15-35%). In the present study, a DHSG conjugate of MK-0767 was identified in the bile from rats dosed with MK-0767. Both LC-MS/MS and NMR analyses indicated the presence of an intact cyclohexadiene moiety having hydroxyl and glutathionyl groups ortho- and meta-, respectively, to the -CF₃ group in the conjugate. A mechanism is proposed for the formation of this metabolite by way of epoxidation of the terminal phenyl ring across the positions C2-C3, followed by nucleophilic addition of glutathione (Fig. 7). Theoretically, the addition of glutathione to an epoxide should produce two regioisomers, with a glutathionyl group either ortho- or meta- to the -CF₃ moiety. Indeed, two peaks were present in the m/z 762-extracted ion chromatogram; however, the other isomer was found to be present in trace amounts only (data not shown). Epoxidation of the methoxyphenyl ring, instead of the trifluoromethylphenyl ring, followed by glutathione addition may also produce similar mass fragmentation as shown in Fig. 2; however, this possibility was ruled out based on the data obtained from the NMR spectra (see Results). Examination of the bile from dogs and monkeys dosed with [¹⁴C]MK-0767 indicated that this conjugate was not present in these species; however, dog bile showed significant amounts of the DHSC and DHSCG conjugates of MK-0767, most likely formed as a result of hydrolysis of the corresponding DHSG conjugate. These conjugates were not detected in bile of monkeys (data not shown). It is well known that glutathione conjugates are further processed by -glutamyltransferase, aminopeptidase M, and cysteinylglycine dipeptidase to cysteinylglycine and cysteine conjugates (Tate, 1980, 1985; Stevens and Jones, 1989).

View larger version (17K): [in this window] [in a new window]   FIG. 7. Proposed pathway for the formation and metabolism of dihydrohydroxy-S-glutathionyl conjugate of MK-0767.

The in vitro formation of the DHSG conjugate was investigated in rat, dog, monkey, and human liver microsomes fortified with NADPH and glutathione. The DHSG conjugate was identified in the rat, dog, and monkey liver microsomes but not in human liver microsomes (Fig. 5). When cysteine was added instead of glutathione in the dog liver microsomal incubations, the DHSC conjugate was identified as a product. These data suggest the involvement of an arene oxide intermediate. Nucleophilic attack of the putative arene oxide intermediate by glutathione (Fig. 7) could be either a nonenzymatic reaction or catalyzed by a glutathione S-transferase. Supplementing the microsomal incubation mixtures with rat hepatic glutathione S-transferase did not have any effect on the extent of conjugate formation. Microsomal glutathione S-transferase activity is high in the liver, although low activities are found in extrahepatic sites (DePierre and Morgenstern, 1983; Morgenstern et al., 1984). In this case, there seems to be species difference in the epoxidation of MK-0767. Dog liver microsomes had higher activity in the formation of the DHSG conjugate when compared with that of rat or monkey.

It is feasible that the precursor of the DHSG conjugate is an arene oxide. There are several reports on the bioactivation of xenobiotics by P450 enzymes that suggest the involvement of arene oxide intermediates. The stability and reactivity of the arene oxide intermediates vary considerably (Guengerich, 2003). Epoxide hydrolases further metabolize arene oxides to dihydrodiols (Fretland and Omiecinski, 2000). On the other hand, in the presence of reduced glutathione, glutathione-S-transferases metabolize arene oxides to produce thioether adducts. These products, depending on their stability, may or may not undergo subsequent dehydration to form rearomatized products. For example, in the rat, lamotrigine, a drug used in the management of epilepsy syndromes, was metabolized primarily by thioether addition to a reactive intermediate arene oxide, yielding the glutathione conjugate of dihydrohydroxylamotrigine. Unlike the DHSG conjugate of MK-0767, the DHSG conjugate of lamotrigine is unstable and spontaneously dehydrates and rearomatizes at room temperature or upon freezing and thawing (Maggs et al., 2000). Similarly, 1,2-dichlorobenzene was metabolized to dichlorocyclohexadiene epoxide, which underwent glutathione addition followed by the spontaneous loss of water to generate the glutathione conjugate of dichlorobenzene (Hissink et al., 1996). DFU, a selective cyclooxygenase 2 inhibitor, and raloxifene were shown also to undergo adduction with glutathione, possibly via the arene oxide intermediate and rearomatization (Yergey et al., 2001; Chen et al., 2002). In the present study, data shows that an intact, stable DHSG conjugate of MK-0767 was formed in rat, which facilitated its isolation and characterization by LC-MS and ¹H NMR techniques. Furthermore, evidence suggests that the DHSG conjugate of MK-0767 underwent further in vivo metabolism to DHSCG and DHSC conjugates with an intact cyclohexadiene moiety, although trace amounts of the corresponding rearomatized conjugates were identified in rat bile (data not shown). Carbamazepine has been reported also to form glutathione conjugates derived from the carbamazepine arene oxide intermediate, similar to MK-0767 (Madden et al., 1996). The only reported NMR and MS characterization of the DHSG conjugate seems to be that of verlukast, a leukotriene D₄ antagonist. When incubated with ß-naphthoflavone-induced rat liver microsomes in vitro, verlukast produced an epoxide that underwent addition with glutathione to produce the DHSG conjugate; however, this conjugate was not identified in vivo (Nicoll-Griffith et al., 1993).

Following the incubation of rat bile containing the DHSG conjugate of MK-0767 with rat intestinal contents, there was a time-dependent decrease in the conjugate peak with an increase in the peak corresponding to the parent compound (MK-0767) (data not shown). Further experiments with the purified DHSG conjugate with intestinal contents confirmed this observation. LC-MS/MS analysis of the reaction mixture showed the conversion of the DHSG conjugate to the parent compound MK-0767 (Fig. 6), suggesting that the reduction of the conjugate to parent compound MK-0767 was catalyzed by intestinal bacteria. Furthermore, when the conjugate was incubated with heat-inactivated intestinal contents, there was no reduction, suggesting that the reduction was an enzymatic reaction. To our knowledge, this is the first report on the characterization of the DHSG conjugate isolated from in vivo samples and its reduction to the parent compound by intestinal contents. The reduction is possibly catalyzed by an enzyme having cysteine-thiol at the active site. A nucleophilic attack of this cysteine-thiol on the sulfur atom of the DHSG conjugate may result in the cleavage of C-S bond, rearomatization, and elimination of the -OH group as shown (Fig. 8).

View larger version (7K): [in this window] [in a new window]   FIG. 8. Proposed mechanism for the reduction of the dihydrohydroxy-S-glutathionyl conjugate to MK-0767.

Gut microflora are known to catalyze reductive reactions and play a role in drug absorption kinetics. Reduction of sulfoxides due to intestinal bacteria have been reported [e.g., sulfinpyrazone, a uricosuric agent; and sulindac, a nonsteroidal anti-inflammatory antipyretic and analgesic agent (Strong et al., 1984, 1985, 1987)]. Gut flora also reduced cyclamate to cyclohexylamine in human, guinea pig, rabbit, and rat, allowing further metabolism of cyclohexylamine to occur in tissues of the body (Drasar et al., 1972). Furthermore, in the present study, the DHSG conjugate of MK-0767 was not reduced by rat intestinal cytosol. Cytosolic enzymes involved in the reduction of C-S bond have been reported (Brundin et al., 1982; Reddy and Gold, 2001). In such instances where glutathione conjugates of compounds are reduced to the parent compound, subsequent excretion of the parent compound in the feces may be mistaken for the unabsorbed drug. It is also possible that such reversible glutathione conjugation, which results in the generation of the parent compound, could play a role in enterohepatic recirculation.

In conclusion, a DHSG conjugate of MK-0767 was isolated from rat bile and identified using LC-MS/MS and ¹H NMR techniques. This conjugate was further metabolized in vivo to cysteinylglycine and cysteine conjugates with intact cyclohexadiene moiety. Upon incubation with rat intestinal contents, the DHSG conjugate was reduced to the parent compound, suggesting the involvement of intestinal bacteria, and provided an explanation for the prevalent presence of parent drug in feces from intact rats. Further studies on the mechanism of reduction are in progress.

	Acknowledgments

 
We thank Dr. Ashok Chowdary for the synthesis of [¹⁴C]MK-0767 and Drs. Thomas A. Baillie and David C. Evans (Merck Research Laboratories) for reviewing the manuscript.

	Footnotes

 
doi:10.1124/dmd.104.000240.

ABBREVIATIONS: MK-0767, (±)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide; TZD, thiazolidinedione; PPAR, peroxisome proliferator-activated receptor; DHSG, dihydrohydroxy-S-glutathionyl; LC, liquid chromatography; MS, mass spectometry; MS/MS, tandem mass spectometry; HPLC, high-performance liquid chromatography; P450, cytochrome P450; CID, collision-induced dissociation; NOE, nuclear Overhauser effect; DHSC, dihydrohydroxy-S-cysteinyl; DHSCG, dihydrohydroxy-S-cysteinylglycinyl.

Address correspondence to: Dr. Vijay Bhasker G. Reddy, RY 80L-109, Merck Research Laboratories, 126 East Lincoln Avenue, Rahway, NJ 07065. E-mail: g_vijay_reddy{at}merck.com

	References

Top Abstract Materials and Methods Results Discussion References

 


Baillie TA and Davis MR (1993) Mass spectrometry in the analysis of glutathione conjugates. Biol Mass Spectrom 22: 319-325.[CrossRef][Medline]

Brundin A, Ratnayake JH, Sunram JM, and Anders MW (1982) Glutathione-dependent reductive dehalogenation of 2,2',4'-trichloroacetophenone to 2',4'-dichloroacetophenone. Biochem Pharmacol 31: 3885-3890.[CrossRef][Medline]

Chen Q, Ngui JS, Doss GA, Wang R, Cai X, DiNinno FP, Blizzard TA, Hammond ML, Stearns RA, Evans DC, et al. (2002) Cytochrome P450 3A4-mediated bioactivation of raloxifene: irreversible enzyme inhibition and thiol adduct formation. Chem Res Toxicol 15: 907-914.[CrossRef][Medline]

DePierre JW and Morgenstern R (1983) Comparison of the distribution of microsomal and cytosolic glutathione S-transferase activities in different organs of the rat. Biochem Pharmacol 32: 721-723.[CrossRef][Medline]

Drasar BS, Renwick AG, and Williams RT (1972) The role of gut flora in the metabolism of cyclamate. Biochem J 129: 881-890.[Medline]

Fretland AJ and Omiecinski CJ (2000) Epoxide hydrolases: biochemistry and molecular biology. Chem-Biol Interact 129: 41-59.[CrossRef][Medline]

Guengerich FP (2003) Cytochrome P450 oxidations in the generation of reactive electrophiles: epoxidation and related reactions. Arch Biochem Biophys 409: 59-71.[CrossRef][Medline]

Hissink AM, Oudshoorn MJ, Ommen BV, Haenen GR, and Van Bladeren PJ (1996) Differences in cytochrome P450 mediated biotransformation of 1,2-dichlorobenzene by rat and man: implications for human risk assessment. Chem Res Toxicol 9: 1249-1256.[CrossRef][Medline]

Karanam BV, Hop CECA, Liu D, Wallace M, Dean D, Toriumi C, Komuro M, Taga F, Kusajima H, and Vincent S (2004) 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. Drug Metab Dispos 32: 1015-1022.[Abstract/Free Full Text]

Liu DQ, Karanam BV, Doss GA, Sidler RR, Vincent SH, and Hop CECA (2004) 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. II. Identification of metabolites by liquid chromatography-tandem mass spectrometry. Drug Metab Dispos 32: 1023-1031.[Abstract/Free Full Text]

Madden S, Maggs JL, and Park BK (1996) Bioactivation of carbamazepine in the rat in vivo: evidence for the formation of reactive arene oxide(s). Drug Metab Dispos 24: 469-479.[Abstract]

Maggs JL, Naisbitt DJ, Tetty JNA, Pirmohamed M, and Park BK (2000) Metabolism of lamotrigine to a reactive arene oxide intermediate. Chem Res Toxicol 13: 1075-1081.[CrossRef][Medline]

Morgenstern R, Lunqvist G, Andersson G, Balk L, and DePierre JW (1984) The distribution of microsomal glutathione transferase among different organelles, different organs and different organisms. Biochem Pharmacol 33: 3609-3614.[CrossRef][Medline]

Mudaliar S and Henry RR (2001) New oral therapies for type 2 diabetes mellitus: the glitazones or insulin sensitizers. Annu Rev Med 52: 239-257.[CrossRef][Medline]

Murphy CM, Fenselau C, and Gutierrez PL (1992) Fragmentation characteristics of glutathione conjugates activated by high-energy collisions. J Am Soc Mass Spectrom 3: 815-822.[CrossRef]

Nicoll-Griffith DA, Chauret N, Yergey JA, Trimble LA, Favreau L, Zamboni R, Grossman SJ, Drey J, and Herold E (1993) Characterization of verlukast metabolites arising from an epoxide intermediate produced with hepatic microsomes from ß-naphthoflavone-treated rodents (P-450 1A1). Drug Metab Dispos 21: 861-867.[Abstract]

Reddy GV and Gold MH (2001) Purification and characterization of glutathione conjugate reductase: a component of the tetrachlorohydroquinone reductive dehalogenase system from Phanerochaete chrysosporium. Arch Biochem Biophys 391: 271-277.[CrossRef][Medline]

Stevens JL and Jones DP (1989) The mercapturic acid pathway: biosynthesis, intermediary metabolism, and physiological disposition, in Glutathione: Chemical, Biochemical, and Medical Aspects (Dolphin D, Poulson R, and Avramovic O eds) part B, pp 46-84, Wiley, New York.

Strong HA, Oates J, Sembi J, Renwick AG, and George CF (1984) Role of the gut flora in the reduction of sulfinpyrazone in humans. J Pharmacol Exp Ther 230: 726-732.[Abstract/Free Full Text]

Strong HA, Renwick AG, George CF, Liu YF, and Hill MJ (1987) The reduction of sulphinpyrazone and sulindac by intestinal bacteria. Xenobiotica 17: 685-696.[Medline]

Strong HA, Warner NJ, Renwick AG, and George CF (1985) Sulindac metabolism: the importance of an intact colon. Clin Pharmacol Ther 38: 387-393.[Medline]

Tate SS (1980) Enzymes of mercapturic acid formation, in Enzymatic Basis of Detoxication (WB Jokoby ed) vol 2, pp 95-120, Academic Press, New York.

Tate SS (1985) Microvillus membrane peptidases that catalyze hydrolysis of cysteinylglycine and its derivatives, in Methods in Enzymology (A Meister ed) vol 113, pp 471-484, Academic Press, Orlando.[Medline]

Yergey JA, Trimble LA, Silva J, Chauret N, Li C, Therien M, Grimm E, and Nicoll-Griffith DA (2001) In vitro metabolism of the COX-2 inhibitor DFU, including a novel glutathione adduct rearomatization. Drug Metab Dispos 29: 638-644.[Abstract/Free Full Text]

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