Studies on the Metabolism of Troglitazone to Reactive Intermediates in

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Studies on the Metabolism of Troglitazone to Reactive Intermediates in Vitro and in Vivo. Evidence for Novel Biotransformation Pathways Involving Quinone Methide Formation and Thiazolidinedione Ring Scission† Kelem Kassahun,*,‡ Paul G. Pearson,‡ Wei Tang,§ Ian McIntosh,‡ Kwan Leung,§ Charles Elmore,§ Dennis Dean,§ Regina Wang,§ George Doss,§ and Thomas A. Baillie‡ Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania 19486, and Rahway, New Jersey 07065 Received August 17, 2000

Therapy with the oral antidiabetic agent troglitazone (Rezulin) has been associated with cases of severe hepatotoxicity and drug-induced liver failure, which led to the recent withdrawal of the product from the U.S. market. While the mechanism of this toxicity remains unknown, it is possible that chemically reactive metabolites of the drug play a causative role. In an effort to address this possibility, this study was undertaken to determine whether troglitazone undergoes metabolism in human liver microsomal preparations to electrophilic intermediates. Following incubation of troglitazone with human liver microsomes and with cDNA-expressed cytochrome P450 isoforms in the presence of glutathione (GSH), a total of five GSH conjugates (M1-M5) were detected and identified tentatively by LC-MS/MS analysis. In two cases (M1 and M5), the structures of the adducts were confirmed by NMR spectroscopy and/or by comparison with an authentic standard prepared by synthesis. The formation of GSH conjugates M1-M5 revealed the operation of two distinct metabolic activation pathways for troglitazone, one of which involves oxidation of the substituted chromane ring system to a reactive o-quinone methide derivative, while the second involves a novel oxidative cleavage of the thiazolidinedione (TZD) ring, potentially generating highly electrophilic R-ketoisocyanate and sulfenic acid intermediates. When troglitazone was administered orally to a rat, samples of bile were found to contain GSH conjugates which reflected the operation of these same metabolic pathways in vivo. The finding that metabolism of the TZD ring of troglitazone was catalyzed selectively by P450 3A enzymes is significant in light of the recent report that troglitazone is an inducer of this isoform in human hepatocytes. The implications of these results are discussed in the context of the potential for troglitazone to covalently modify hepatic proteins and to cause oxidative stress through redox cycling processes, either of which may play a role in drug-induced liver injury.

Introduction Troglitazone (Rezulin; Figure 1), an oral antidiabetic drug belonging to the 2,4-thiazolidinedione (TZD)1 structural class, acts by decreasing tissue resistance to insulin via agonism of the PPAR receptor (1). Thus, troglitazone enhances insulin-dependent glucose metabolism in muscle and adipose tissue and inhibits hepatic gluconeogenesis. However, despite its effectiveness as an antidiabetic agent, use of troglitazone has been associated with severe hepatotoxicity which, in some cases, has led to hepatic † A preliminary account of this work was presented at the Sixth International Symposium on Biological Reactive Intermediates, July 2000, Paris, France. * To whom correspondence should be addressed: Department of Drug Metabolism, WP75A-203, Merck Research Laboratories, West Point, PA 19486. E-mail: [email protected]. ‡ Department of Drug Metabolism, Merck Research Laboratories, West Point, PA. § Department of Drug Metabolism, Merck Research Laboratories, Rahway, NJ. 1 Abbreviations: TZD, thiazolidinedione; PPAR, peroxisome proliferator activated receptor; P450, cytochrome P450; GSH, glutathione; DTT, dithiothreitol; CID, collision-induced dissociation.

Figure 1. Structures of troglitazone and GSH conjugates M1-M5.

failure and death (2-4). As a consequence of safety concerns over the use of this agent, troglitazone was withdrawn from the U.S. market in March 2000. In the course of our studies on the interaction of several TZD derivatives with cytochrome P450 enzymes in vitro, we found that troglitazone caused preincubation- and

10.1021/tx000180q CCC: $20.00 © 2001 American Chemical Society Published on Web 12/08/2000

Metabolism of Troglitazone to Reactive Intermediates

Chem. Res. Toxicol., Vol. 14, No. 1, 2001 63

Figure 2. Scheme for the synthesis of GSH conjugate M1.

NADPH-dependent inhibition of P450 3A4 activity in human liver microsomal preparations. This finding raised the possibility that troglitazone undergoes P450 3A4mediated conversion to a chemically reactive intermediate(s), and that such a species may play a role in troglitazone-induced hepatotoxicity. The studies reported in this communication provide evidence that troglitazone does indeed undergo metabolic activation, both in vitro and in vivo, and point to both the TZD ring and the substituted chromane moiety as sites for reactive intermediate formation through cytochrome P450 catalysis.

stirred at reflux for 1.5 h. The solution then was concentrated to dryness at reduced pressure to afford 99 mg of 2 as a white solid: 1H NMR (CDCl3) δ 1.42 (s, 3H), 1.88 (m, 2H), 1.99 (s, 3H), 2.03 (s, 3H), 2.09 (s, 3H), 2.14 (m, 2H), 2.34 (s, 3H), 2.63 (m, 2H), 3.15 (dd, 1H, J ) 7.3, 14.4 Hz), 3.37 (dd, 1H, J ) 4.3, 14.4 Hz), 3.87 (d, 1H, J ) 9.1 Hz), 3.98 (d, 1H, J ) 9.0 Hz), 4.47 (dd, 1H, J ) 4.5, 7.0 Hz), 5.91 (br s, 1H), 6.36 (br s, 1H), 6.87 (d, 2H, J ) 8.5 Hz), 7.17 (d, 2H, J ) 8.4 Hz); 13C NMR (CDCl3) δ 11.8, 12.1, 12.9, 20.1, 20.5, 22.8, 28.2, 40.3, 61.0, 72.4 (br), 74.5, 114.5, 117.3, 123.1, 125.0, 127.0, 128.2, 130.6, 140.8, 148.9, 158.2, 169.7, 170.9; LC-MS (tR ) 15.53 min) m/z (relative intensity) [M + H]+ 460.2 (100), [M + NH4]+ 477.2 (25), [M + Na]+ 482.2 (20).

Experimental Procedures

2-Chloro-3-{4-[(6-hydroxy-2,5,7,8-tetramethylchroman2-yl)methoxy]phenyl}propanamide (3). A solution of 99 mg (0.21 mmol) of 2 in 19 mL of MeOH was stirred at 0 °C under N2 as 238 mg (9.9 mmol) of NaH was added in four portions over 30 min. After the reaction mixture had been stirred at room temperature for 2 h, the solution was concentrated to 5 mL under a stream of N2, diluted with 10 mL of water, and extracted four times with 10 mL of CH2Cl2. The organic layer was dried (MgSO4), filtered, and concentrated to give 72 mg of 3 as a pale yellow solid: 1H NMR (CDCl3) δ 1.41 (s, 3H), 1.89 (m, 2H), 2.11 (s, 3H), 2.12 (s, 3H), 2.12 (m, 2H), 2.17 (s, 3H), 2.64 (m, 2H), 3.15 (dd, 1H, J ) 7.9, 14.4 Hz), 3.37 (dd, 1H, J ) 4.5, 14.4 Hz), 3.87 (d, 1H, J ) 9.1 Hz), 3.96 (d, 1H, J ) 9.1 Hz), 4.47 (dd, 1H, J ) 4.5, 7.9 Hz), 4.53 (bs s, 1H), 5.97 (bs s, 1H), 6.39 (br s, 1H), 6.87 (d, 2H, J ) 8.6 Hz), 7.16 (d, 2H, J ) 8.6 Hz); 13C NMR (CDCl3) δ 11.3, 11.8, 12.2, 20.3, 22.7, 28.7, 40.3, 61.0, 72.5, 76.7, 114.5, 115.2, 117.3, 18.7, 121.4, 122.6, 128.1, 130.6, 145.0, 158.3, 171.1; LC-MS (tR ) 13.81 min) m/z (relative intensity) [M + H]+ 418.2 (100), [M + NH4]+ 435.2 (30), [M + Na]+ 440.2 (25).

Chemicals. Troglitazone was extracted from Rezulin tablets using acetonitrile, while the quinone metabolite of troglitazone was synthesized at Merck Research Laboratories (5). GSH, NADPH, and DTT were purchased from Sigma Chemical Co. (St. Louis, MO), while ketoconazole and sulfaphenazole were obtained from Research Biologics Inc. (Natick, MA). Quinidine and 13-cis-retinoic acid were supplied by Aldrich Chemical Co. (Milwaukee, WI), and fluvoxamine was obtained from the Merck Compound Repository. Synthesis. The disulfide-linked GSH conjugate of troglitazone (M1) was synthesized as outlined in Figure 2 using the procedure described below. 1H and 13C NMR spectra were recorded on a Varian U-400 spectrometer and are referenced to the residual solvent peak (7.26 and 77.0 ppm for CHCl3). LCMS data were recorded on an HP 1100 MSD instrument with a gradient elution from 5 to 95% acetonitrile and 0.1% aqueous formic acid on a Zorbax XDB C8 column. 2-Chloro-3-{4-[(6-acetoxy-2,5,7,8-tetramethylchroman2-yl)methoxy]phenyl}propanamide (2). A solution of 104 mg (0.213 mmol) of ethyl 3-{4-[(6-acetoxy-2,5,7,8-tetramethylchroman-2-yl)methoxy]phenyl}-2-chloropropionate (1), available in five steps from 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (6), in 10 mL of MeOH and 20 mL of NH3 was

2-Amino-4-(N-{1-[N-(2,2-dihydroxyethyl)carbamoyl]-2[(1-carbamoyl-2-{4-[(6-hydroxy-2,5,7,8-tetramethylchroman2-yl)methoxy]phenyl}ethyl)disulfanyl]ethyl}carbamoyl)butanoic Acid (M1). A modification of the procedure reported by Hu and Fox was followed (7). A solution of 5 mg (12 µmol) of chloride 3 in 1 mL of THF was stirred and cooled at -5 °C as

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20 µL (95 µmol) of hexamethyldisilathiane and 80 µL (80 µmol) of 1 M tetrabutylammonium fluoride in THF were added in two aliquots over the course of 1 h. A third aliquot of 20 µL (95 mol) of hexamethyldisilathiane and 80 µL (80 µmol) of 1 M tetrabutylammonium fluoride in THF were added, and the reaction mixture was heated to 40 °C for 1 h. HPLC analysis showed that the starting material is consumed. The solution was cooled to room temperature and was diluted with 5 mL of saturated aqueous NaCl solution. The resulting solution was extracted with 10 mL of Et2O, and the organic phase was washed twice with 3 mL of water, concentrated to approximately 0.2 mL, diluted with 10 mL of THF, and concentrated to 1 mL. A solution of 10 mg of NaHCO3 in 1 mL of water was added to the THF solution followed by 10 mg (22 µmol) of glutathione thiosulfonate 4 (8). After 12 h at room temperature, the reaction was incomplete as judged by HPLC; 20 mg of 4 was added, and the reaction mixture was heated to 40 °C for 4 h. After 4 h, an additional 30 mg of 4 was added and the heating continued for 4 h. HPLC analysis showed a complex reaction mixture that contained two components with an MH+ ion at m/z 721. The solution was concentrated to near dryness and taken up in 1 mL of THF and 0.5 mL of 0.1% trifluoroacetic acid. Purification was effected by semipreparative HPLC (YMC ODS-AM column, flow rate of 5 mL/min, isocratic at 30% acetonitrile and 0.1% trifluoroacetic acid for 25 min and then 35% acetonitrile and 0.1% trifluoroacetic acid, UV at 230 nm). The column fractions were analyzed by HPLC and LC-MS; the fraction containing the desired product (tR ) 56.3-58.1 min) was concentrated to remove the acetonitrile under reduced pressure and was lyophilized to give 0.5 mg of 1 that was 80% pure (determined by HPLC, via the YMC ODS-AM column, 35% acetonitrile and 0.1% trifluoroacetic acid, 1 mL/min, tR ) 16.0 and 16.4 min): MS m/z 721 (MH+). For the 1H NMR data, see Table 1. M5 was isolated by HPLC (using a Zorbax XDB-C18 4.6 mm × 250 mm column and a gradient containing 0.1% aqueous formic acid and acetonitrile) following incubation of troglitazone (100 µM) with rat liver microsomes (2 mg/mL) in the presence of GSH (5 mM) in a total incubation volume of 50 mL: MS m/z 747 (MH+). For the 1H NMR data, see Table 1. Liver Microsomal Preparations. A human liver microsomal preparation (from a pool of 15 livers) was purchased from Xenotech LLC (Kansas City, KS). The recombinant P450 enzymes and FMO3 that were used were from baculovirusinfected insect cells and were either prepared in-house or purchased from Gentest Corp. (Woburn, MA). Effect of Troglitazone on Human Liver Microsomal Testosterone 6β-Hydroxylase Activity. For non-preincubation-dependent enzyme inhibition, a pooled human liver microsomal preparation was incubated with testosterone (250 µM) and troglitazone at concentrations ranging from 0.1 to 50 µM at 37 °C for 10 min in the presence of an NADPH-generating system. For preincubation-dependent inhibition, microsomes were incubated with troglitazone at concentrations varying from 0.1 to 50 µM in the presence of an NADPH-generating system at 37 °C for 30 min, after which incubation with testosterone (250 µM) was carried out for 10 min. To determine if the preincubation-dependent inhibition also was NADPH-dependent, a third incubation in which the NADPH-generating system was absent during the preincubation period was carried out. Incubations with Human Liver Microsomes. Troglitazone and its quinone metabolite were dissolved in methanol for addition in incubation media such that the volume of methanol in the final incubation mixture was e1%. Incubations were carried out at 37 °C for 60 min in a shaking water bath. The incubation volume was 1 mL and consisted of the following: 0.1 M potassium phosphate buffer (pH 7.4), magnesium chloride (1 mM), microsomal protein (2 mg), substrate (50 µM), and NADPH (1 mM). GSH (5 mM) was added 3 min after initiation of the reaction with NADPH. Incubations that lacked either NADPH or GSH served as negative controls, and reactions were terminated by the addition of ice-cold acetonitrile (1 mL). After centrifugation, the supernatant from each incubation was

Kassahun et al. Table 1. 1H NMR Data for Synthetic M1 and for M5 Isolated from Liver Microsomal Incubations of Troglitazone

assignmenta

chemical shift (ppm)

assignmentb

a b Gluβ Gluβ c d e f

1.25 (s) 1.76 (m) 1.78 (m) 1.89 (m) 1.92 (s) 1.94 (m) 1.98 (s) 2.00 (s)

a b+d c Gluβ e Gluγ g Cysβ

Gluγ

2.26 (m)

h

Gluγ g

i k

h

2.31 (m) 2.52 (t, J ) 6.6 Hz) 2.79 (m)

Cysβ

2.83 (m)

j

i Cysβ GluR j GlyR k CysR l

2.98 (m) m 3.19 (m) l 3.22 (m) 3.51 (m) 3.61 (m) 3.87 (AB) 4.47 (m) 6.82 (d, J ) 8.4 Hz) 7.05 (d, J ) 8.4 Hz) 7.16, 6.61 (br s) 8.46 (br d) 8.66 (br)

m NH2 Cys NH Gly NH

CysR

chemical shift (ppm) 1.33 (s) 1.85 (m) 1.98 (s) 2.03 (m) 2.07 (s) 2.46 (m) 2.70 (m) 2.91 (dd, J ) 5.4, 4.2 Hz) 3.08 (dd, J ) 9.1, 14.1 Hz) 3.32 (m) 3.95 (AB) 4.48 (dd, J ) 5.4, 8.6 Hz) 4.58 (dd, J ) 4.3, 8.6 Hz) 6.86 (d, J ) 8.6 Hz) 7.12 (d, J ) 8.6 Hz)

a Obtained at 600 MHz in DMSO-d . b Obtained at 500 MHz in 6 CD3CN/17% D2O.

removed and evaporated to dryness. The residue was reconstituted in 30% acetonitrile in water (200 µL), vortex-mixed, and centrifuged. Aliquots (10 µL) of the final supernatant were analyzed by LC-MS and LC-MS/MS. Incubations with Microsomes Containing cDNAExpressed Enzymes. Incubations with recombinant enzymes were conducted as described above for native liver microsomes. The expressed enzymes that were used, and the quantities added to incubation mixtures (final volume of 1 mL), were as follows: 300 pmol of P450 1A2, 300 pmol of P450 2A6, 500 pmol of P450 2C8, 400 pmol of P450 2C9, 200 pmol of P450 2C19, 300 pmol of P450 2D6, 500 pmol of P450 3A4, and 200 µL of FMO3 from a preparation having a protein concentration of 5 mg/mL. Inhibition Studies. The effect of P450 form-selective chemical inhibitors on the formation of troglitazone metabolites in human liver microsomes was investigated using the following inhibitors at the indicated concentration: fluvoxamine (10 µM; P450 1A2), ketoconazole (2 µM; P450 3A), sulfaphenazole (5 µM; P450 2C9), quinidine (10 µM; P450 2D6), and 13-cis-retinoic acid (150 µM; P450 2C8). The inhibitors, which were dissolved in 50% acetonitrile in water, were incubated individually with human liver microsomes at troglitazone concentrations of 5 and 50 µM. Reduction of Disulfide-Linked GSH Conjugates. GSH adducts generated by human liver microsomes or recombinant P450 3A4 were treated with DTT to explore the nature of the



Figure 3. Preincubation-dependent inhibition of P450 3A activity (testosterone 6β-hydroxylase) in human liver microsomal preparations incubated with troglitazone. linkage between the drug residue and GSH. Thus, following incubation with microsomal preparations, termination of the reaction, and evaporation of the supernatant as outlined above, the residue was dissolved in 30% acetonitrile in water and treated with a solution of DTT (100 µL of a 20 mM solution in phosphate buffer). The resulting mixture was held at 37 °C for 60 min, and the products were analyzed directly by LC-MS/MS. In Vivo Studies. A fasted male Sprague-Dawley rat, which had been cannulated at the common bile duct, was dosed intraperitoneally with a solution of troglitazone in DMSO (100 mg/mL) such that the administered dose was 100 mg/kg. Bile was collected over ice during the following time intervals: 0-1, 1-2, 2-4, 4-6, 6-8, and 8-24 h after administration. A sample of pooled bile (0-8 h) was analyzed by LC-MS/MS for the presence of GSH conjugates. LC-MS/MS Analysis. Metabolites were identified by electrospray LC-MS and LC-MS/MS analysis using a Finnigan LCQ mass spectrometer (San Jose, CA). The spray voltage was kept at 4.1 kV, and the capillary temperature was set at 200 °C. Full scan spectra (from m/z 400 to 850) were obtained in the positive ion mode, and product ion spectra were generated by CID of MH+ ions of interest. Analytes were introduced into the electrospray source via an Inertsil C18 column (2 mm × 150 mm, 5 µm; MetaChem Technologies Inc., Torrance, CA) at a flow rate of 200 µL/min. Separation of analytes was achieved using a gradient consisting of 0.1% aqueous formic acid and acetonitrile. The initial solvent composition was 10% acetonitrile and 90% aqueous, and after 2 min, the proportion of acetonitrile was increased at a rate of 2 mL/min. A modification of the above gradient (5% acetonitrile from 0 to 5 min, 15% acetonitrile at 10 min, and 80% acetonitrile at 30 min) was used for the analysis of bile samples from troglitazone-treated rats.

Results Effect of Troglitazone on Human Liver Microsomal Testosterone 6β-Hydroxylase Activity. Figure 3 shows the effect of varying concentrations of troglitazone on P450 3A-mediated testosterone 6β-hydroxylation in human liver microsomes both with and without preincubation of microsomes with troglitazone. The magnitude of enzyme inhibition was higher when the experiment was carried out with preincubation in the presence of NADPH, indicating that troglitazone is a preincubation-

Figure 4. LC-MS/MS chromatogram (A) and product ion spectrum (B) obtained by CID of the MH+ ion (m/z 721) of M1 detected (retention time of 21.1 min) in human liver microsomal extracts. The origins of characteristic ions are as indicated.

and NADPH-dependent inhibitor of P450 3A activity in human liver microsomes. Identification of Troglitazone Metabolites. LCMS/MS analysis of extracts of human liver microsomal incubations containing troglitazone, NADPH, and GSH revealed the presence of five GSH adducts (designated M1-M5), none of which were evident in control incubations that lacked one or both cofactors. M1 exhibited an MH+ ion at m/z 721 and yielded the product ion spectrum shown in Figure 4B. A notable feature of this spectrum was the presence of ions resulting from neutral losses characteristic of GSH adducts (9), namely, m/z 646 [(MH - 75)+] and 592 [(MH - 129)+]. When the conjugate was treated with excess DTT, reaction occurred to yield a product (MH+ at m/z 416) whose molecular mass was consistent with an R-thiolamide derivative resulting from TZD ring cleavage (data not shown). This observation suggested that M1 was a disulfide-linked GSH conjugate, with the structure depicted in Figure 4B. Subsequently, an authentic sample of this conjugate was prepared by chemical synthesis and, upon LC-MS/MS analysis, yielded an HPLC retention time and product ion mass spectrum identical to those obtained from the isolated metabolite. Although several diastereomers of M1 are possible, both the isolated metabolite and the authentic standard eluted as a single chromatographic peak (Figure 4A) under the LC-MS conditions used in these studies. M2 (which appeared to be a mixture of two isomers as shown in Figure 5A) exhibited an MH+ ion at m/z 781, the product ion spectrum of which (Figure 5B) contained fragment ions at m/z 706 [(MH - 75)+], 652 [(MH 129)+], and 308 [(GSH2)+]. On the basis of the molecular mass of 780 Da and the appearance in the mass spectrum of the weak, but structurally informative, ion at m/z 308, it appears that this metabolite corresponds to the carbamate thioester GSH adduct of the putative isocyanate derivative of troglitazone in which the sulfur atom of the

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original TZD ring is in the form of a sulfinic acid (Figure 5B). In support of this tentative structural assignment, the MS/MS behavior of M2 was consistent with that exhibited by GSH conjugates of isocyanates and isothiocyanates (10-12). Metabolite M3, which displayed an MH+ ion at m/z 763 and afforded a product ion spectrum (Figure 6B) closely similar to that of M2, appeared to differ from M2 by the elements of H2O. While the MS/ MS characteristics of M3 are consistent with the sulfine structure proposed in Figure 6B, the identity of this conjugate remains speculative. M4 exhibited an MH+ ion at m/z 737 and prominent fragments at m/z 719 [(MH - 18)+], 662 [(MH - 75)+], and 608 [(MH - 129)+] (Figure 7B). This conjugate was suspected to be related to the known quinone metabolite of troglitazone since its molecular mass was 16 Da higher than that of the adduct (M1) obtained from the parent compound, and since the facile loss of water in the MS/ MS spectrum suggested the presence of a free -OH group. This conclusion was supported by the observation that incubations of the authentic quinone derivative with human liver microsomes fortified with GSH and NADPH resulted in a metabolite with an HPLC retention time and an electrospray MS/MS spectrum identical to those of M4. In addition, treatment of M4 with DTT yielded the corresponding thiol derivative, as evidenced by LCMS/MS analysis (MH+ at m/z 432). The protonated molecular ion of M5 appeared at m/z 747, the product ion spectrum of which exhibited the characteristic losses of 75 and 129 Da (to give ions at m/z 672 and 618, respectively) of a GSH adduct (Figure 8B). The MS/MS spectrum of this conjugate, however, differed from that of the adducts described above in that the spectrum contained an intense ion at m/z 308 [(GSH2)+]. This MS/MS behavior frequently is exhibited by GSH adducts resulting from Michael addition reactions, and corresponds formally to a retro-Michael cleav-

Kassahun et al.



age in the gas phase (13). On the basis of these MS/MS data, the structure shown in Figure 8B, which would result from addition of GSH to an o-quinone methide derivative of troglitazone, is proposed for M5. Following isolation of a specimen of M5, a proton NMR spectrum was obtained which was fully consistent with this structural hypothesis (Table 1).



Figure 9. LC-MS/MS chromatogram obtained by CID of m/z 721 to detect M1 in extracts from a human liver microsomal incubation containing troglitazone and ketoconazole. The arrow indicates the trace amount of M1 formed in comparison to an incubation without ketoconazole (Figure 4A).

Enzymes Involved in the Formation of GSH Adducts of Troglitazone. When troglitazone (5 or 50 µM) was incubated with human liver microsomes fortified with NADPH and GSH, ketoconazole (2 µM) strongly inhibited the formation of M1 (Figure 9), whereas other P450 isoform-selective inhibitors had little effect. Similarly, ketoconazole inhibited the formation of conjugates M2 and M3 to a comparable extent. The formation of both the quinone metabolite of troglitazone and its GSH adduct (M4) also was inhibited only by ketoconazole. Thus, while recombinant P450 1A2, 2C9, 2C19, 2D6, and 3A4 were able to catalyze the formation of M1 from troglitazone, it may be concluded that P450 3A4 is the dominant enzyme in this regard. None of the isoformselective chemical inhibitors suppressed the formation of M5 by >30%, suggesting that multiple P450 isoforms contribute to the generation of this product. Indeed, the


Figure 10. LC-MS/MS analysis of extracts of bile from a rat treated with troglitazone. Product ion spectrum of m/z 722 (B) and the corresponding product ion chromatogram (A) indicating the detection (retention time of 25.2 min) of the proposed carboxyl derivative of M1 (MH+ at m/z 722).

formation of M5 was found to be catalyzed by recombinant P450 2C8, 2C9, 2C19, 2D6, and 3A4. In Vivo Study. A targeted search (based on LC-MS/ MS analysis) for M1 in the bile from troglitazone-treated rats resulted not in the detection of M1 itself but of a metabolite whose molecular mass (721 Da) was one mass unit higher than that of M1 (Figure 10). This biliary metabolite most likely corresponds to the carboxylic acid derivative of M1, formed by hydrolysis of the primary amide group of the latter. The fact that the MS/MS spectrum (Figure 10B) of this in vivo conjugate was almost identical to that of M1 (with the appropriate fragment ions shifted 1 Da to higher masses), coupled with the observation that the metabolite eluted at a later HPLC retention time than M1 with the acidic mobile phase used in this work, further supported the proposal that the biliary metabolite was the free acid derivative of M1. Conjugates M2, M3, and M5 also were detected as metabolites of troglitazone in rat bile.

Discussion The study presented here, which focused on the biotransformation of troglitazone to reactive metabolites, was prompted by our initial observation that the drug inhibits P450 3A4 activity in vitro in an NADPH- and preincubation-dependent fashion. In previous metabolism studies, troglitazone has been shown to undergo biotransformation via conjugation of the phenolic -OH moiety to yield the sulfate and glucuronide derivatives, as well as oxidation of the phenol to generate a p-benzoquinone species (14, 15). In the current study, we employed GSH as a nucleophilic trapping agent and identified two distinct pathways of metabolic activation of troglitazone in liver microsomal preparations, one of which involves

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Figure 11. Proposed scheme for the metabolic activation of troglitazone through P450 3A4-mediated oxidation of the TZD ring. Intermediates I-III have not been identified, but are proposed on the basis of the structures of GSH conjugates M1M3.

a novel oxidative scission of the TZD ring system (Figure 11), while the second entails oxidation of the substituted chromane moiety to an o-quinone methide (Figure 12). Three GSH adducts (M1-M3), whose formation in human liver microsomes was shown to be mediated by P450 3A enzymes, were detected as products of the former pathway, and their genesis is rationalized by the scheme shown in Figure 11. Thus, it is proposed that P450 3Amediated oxidation of the TZD sulfur atom affords a reactive sulfoxide intermediate (I) which undergoes spontaneous ring opening to a highly electrophilic R-keto iosocyanate derivative (II). Hydrolysis of the isocyanate followed by decarboxylation to the amide, accompanied by attack of GSH on the reactive sulfenic acid, would afford M1. Alternatively, conjugation of the isocyanate with GSH to yield intermediate III, followed by oxidation to the corresponding sulfinic acid derivative, provides a route to M2. Although speculative at present, loss of the elements of water from the sulfinic acid moiety of M2 may lead to the formation of the sulfine-containing GSH conjugate, M3 (Figure 11). While these mechanisms account satisfactorily for the formation of the observed GSH adducts M1-M3, it should be noted that a direct attack of GSH on either the sulfur atom or the adjacent carbonyl group of the putative TZD sulfoxide (I) also would produce conjugates M1 and M2, respectively, without the intermediacy of the ring-opened isocyanate II. Regardless of the precise mechanism, it is evident that the TZD ring of troglitazone is subject to a novel pathway of metabolic activation, which has not been described previously, and which leads to products of heterocyclic ring cleavage. It is noteworthy that the three GSH conjugates M1-M3, which were generated in vitro using either native human liver microsomes or expressed P450 3A4, were detected in the bile of troglitazone-treated rats,

Figure 12. Proposed scheme for the metabolic activation of troglitazone through cytochrome P450-mediated oxidation of the substituted chromane ring. Intermediates shown in brackets have not been identified. The hydroxymethyl and quinone metabolites were not characterized in this study, but are known to be metabolites of troglitazone.

and therefore, the TZD bioactivation pathway also operates in vivo in rats. The second route for the formation of reactive metabolites from troglitazone is depicted in Figure 12 and involves oxidation of the substituted chromane moiety. Thus, cytochrome P450-mediated one-electron oxidation of the phenolic hydroxyl group would result in the phenoxy radical, a resonance form of which (the carboncentered radical shown in Figure 12) undergoes oxygen rebound at the ipso position to form an unstable hemiketal. Spontaneous ring opening of this latter species would afford the known quinone metabolite of troglitazone. A similar mechanism has been proposed for the P450mediated oxidation of a number of aryl ethers (16) and para-substituted phenols (17, 18). Once formed, the quinone metabolite appears to undergo TZD ring oxidation, in a fashion similar to that followed by the parent compound, leading ultimately to GSH conjugate M4. The formation of both the quinone metabolite of troglitazone and GSH adduct M4 was found to be dependent on P450 3A isoforms, consistent with recent reports that metabolism of troglitazone to the quinone is catalyzed by P450 3A (19, 20). An alternative fate for the phenoxy radical intermediate is that it undergoes P450-mediated hydrogen atom abstraction from the 5-methyl group of the substituted chromane ring, leading to the o-quinone methide derivative IV depicted in Figure 12. Capture of the latter intermediate by GSH provides a route to the


observed conjugate M5, while a reaction with water would afford the known benzylic alcohol metabolite of troglitazone (19), although the alcohol metabolite also could be formed by a conventional P450 reaction involving hydrogen atom abstraction from the 5-methyl group followed by oxygen rebound. Unlike the metabolites derived through bioactivation of the TZD ring, the formation of M5 was not specifically catalyzed by P450 3A enzymes, although it was found to be NADPHdependent. Experience to date indicates that troglitazone therapy is associated with hepatotoxicity in diabetic patients. During clinical trials of the drug, 1.9% of the troglitazonetreated patients had liver enzyme level increases 3 times the upper limit of normal compared to 0.6% of the patients on placebo (4). Evaluations of liver biopsies from patients that developed hepatotoxicity were consistent with an idiosyncratic drug reaction (2, 3). Since its introduction to the U.S. market in 1997 until its recent withdrawal, troglitazone has been linked to 26 deaths, and several patients required liver transplants following troglitazone-induced hepatic injury (21, 22). While the mechanism of this serious toxicity remains unknown, it is possible that reactive metabolites of troglitazone play a causative role. The results of this investigation provide support for this hypothesis, inasmuch as indirect evidence was obtained for the cytochrome P450-mediated generation of several electrophilic intermediates of troglitazone in vitro and in vivo. Although not investigated in this preliminary study, it is reasonable to expect that these reactive metabolites will form covalent adducts to hepatic proteins, with potentially deleterious consequences to the liver. Moreover, while the quinone metabolite of troglitazone (which cannot form adducts to proteins directly due to the substitution pattern) may undergo redox cycling and induce oxidative stress, the detection of GSH conjugate M4 raises the intriguing possibility of a protein-bound quinone metabolite, attached to macromolecules through a disulfide bond to the residue of the TZD ring. By this means, the benzoquinone moiety would be “anchored” to hepatocellular proteins, providing for potentially enhanced redox cycling activity. Experiments for testing this hypothesis currently are in progress. Troglitazone has been shown to act as an inducer of P450 3A enzymes in human hepatocytes (23), and it may be significant that the pathways for metabolic activation of troglitazone leading to GSH adducts M1-M4 are catalyzed by P450 3A enzymes. Hence, troglitazone acts as an inducer of the enzymes that catalyze its biotransformation to chemically reactive intermediates, and such autoinduction of metabolism might well be a crucial factor in the etiology of troglitazone-mediated liver injury. Finally, it may be noted that this study is the first to report on the metabolic activation of the TZD ring system, a key structural element of many PPAR agonists marketed, or in development, as antidiabetic agents. While the precise details of the underlying mechanism remain to be established, it appears that oxidation of the sulfur atom activates the TZD ring, in a fashion analogous to that described for thiophene derivatives (24). Whether other sulfur-containing heterocycles undergo analogous metabolic activation processes remains to be studied.

Acknowledgment. We thank Dr. Raju Subramanian (Merck Research Laboratories, West Point, PA) for providing the NMR spectrum of metabolite M5 and Dr.


Derek Von Langen (Merck Research Laboratories, Rahway, NJ) for the synthesis of the quinone metabolite of troglitazone.

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Studies on the Metabolism of Troglitazone to Reactive Intermediates in

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