Thiazolidinedione Bioactivation: A Comparison of ... - ACS Publications

1106

Chem. Res. Toxicol. 2006, 19, 1106-1116

Thiazolidinedione Bioactivation: A Comparison of the Bioactivation Potentials of Troglitazone, Rosiglitazone, and Pioglitazone Using Stable Isotope-Labeled Analogues and Liquid Chromatography Tandem Mass Spectrometry† Rubeń Alvarez-Sańchez,‡ Franç ois Montavon,§ Thomas Hartung,§ and Axel Pa¨hler*,‡ Drug Metabolism & Pharmacokinetics and Chemical Synthesis, Safety and Technical Sciences, F. Hoffmann-La Roche Ltd., 4070 Basel, Switzerland ReceiVed December 19, 2005

Troglitazone, a thiazolidinedione (TZD) type insulin sensitizer for the treatment of diabetes, was withdrawn from the U.S. market after several fatal cases of hepatotoxicity. Although the mechanism(s) of these idiosyncratic adverse reactions are not completely understood, circumstantial evidence suggests at least a partial contribution of reactive metabolite formation. Despite isolated case reports of hepatotoxicity, the other TZD derivatives pioglitazone and rosiglitazone are comparatively safe. Herein, we report on the bioactivation potential of these drugs and their TZD ring isotope-labeled 2-15N-3,4,513C analogues in rat and human liver microsomes supplemented with glutathione (GSH). Screening for 3 GSH adducts as surrogate markers for reactive intermediate formation was performed by liquid chromatography tandem mass spectrometry. Chemical characterization of the GSH conjugates was conducted by acquisition of their respective product ion spectra and the comparison between unlabeled and stable isotope-labeled TZD derivatives. The data suggest that all drugs undergo bioactivation processes via a common metabolic activation on the TZD ring, yielding disulfide type GSH conjugates as evidenced by the loss of labeled positions in the TZD moiety. Additional bioactivation processes leading to GSH adducts not involving TZD ring scission were evident for troglitazone. In human liver microsomes at low substrate concentrations, only troglitazone yielded a predominant GSH adduct not involving TZD ring scission. This property may contribute, together with other factors such as the relatively high dose administered as well as its potential to induce hepatic cholestasis and oxidative stress, to the hepatotoxicity of this drug. Introduction The thiazolidinedione (TZD)1 class of compounds represents a family of drugs used for the treatment of noninsulin-dependent type 2 diabetes mellitus. These compounds act as ligands for the γ-subtype peroxisome proliferator acceptor receptor, involved in the regulation of glucose and lipid metabolism (1). The first commercialized TZD drug, troglitazone (Rezulin, TGZ; Figure 1), was withdrawn from the U.S. market after numerous reported cases of severe liver failures leading to liver transplantation or death (2, 3). The mechanisms of TGZ-induced hepatotoxicity are not fully understood and seem to be multifactorial (4), one of the potential factors being the formation of reactive metabolites after enzyme-mediated bioactivation. The major pathway for TGZ metabolism is P450 3A4-mediated oxidation of the chromane moiety to a quinone, followed by reduction and sulfation (5, 6). TZD ring scission represents a minor metabolic pathway for TGZ and pioglitazone (PGZ) (7, 8). Kassahun et al. described the formation of several reactive †

A preliminary account of this work was presented at the 7th International ISSX Meeting, September 2004, in Vancouver, Canada. * To whom correspondence should be addressed. Tel: +41(61)6889920. Fax: +41(61)6882908. E-mail: [email protected]. ‡ Drug Metabolism & Pharmacokinetics. § Chemical Synthesis. 1 Abbreviations: CID, collision-induced dissociation; CNL, constant neutral loss; LC-ESI/MS/MS, liquid chromatography coupled to electrospray ionization tandem mass spectrometry; PGZ, pioglitazone; RGZ, rosiglitazone; TGZ, troglitazone; TZD, thiazolidinedione.

Figure 1. Structures of the TZD drugs TGZ, RGZ, and PGZ. Asterisks denote the labeled positions of the corresponding 2-15N-3,4,5-13C3labeled analogues.

intermediates that were trapped as GSH adducts in vitro and in rat bile in vivo (8) involving bioactivation of either the TZD ring or the chromane moiety. Others reported the detection of TGZ-derived GSH adducts in human hepatocytes (9) as well as in rats in vivo (5, 10). Clinical and postmarketing surveillance

10.1021/tx050353h CCC: $33.50 © 2006 American Chemical Society Published on Web 07/14/2006

ComparatiVe BioactiVation of Thiazolidinediones

indicated that TGZ-induced hepatotoxicity was not a characteristic of the TZD drugs in general, although isolated case reports of hepatotoxic events for rosiglitazone (RGZ) and PGZ exist (11, 12). PGZ undergoes predominantly hydroxylation of the ethyl side chain by P450 3A4 and 2C8 followed by sulfation, glucuronidation, or taurine conjugation (13, 14). Similar to TGZ, TZD ringopening biotransformation reactions have been described for PGZ in dog liver microsomes (7). Baughman et al. (15) reported on the formation of PGZ-derived TZD ring-opened GSH adducts similar to the mixed disulfide amide and carboxylic acid derivatives described for TGZ. RGZ is metabolized predominantly via P450-mediated N-demethylation and hydroxylation followed by glucuronidation (16). A RGZ-derived GSH adduct in human liver microsomes fortified with GSH was reported by Soglia et al. (10) using liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI/MS/ MS) and targeted multiple reaction monitoring method. However, structural characterization of this conjugate has not yet been reported. Here, we report on the different bioactivation potentials of the three TZD drugs TGZ, PGZ, and RGZ to determine whether the safe analogues PGZ and RGZ are also prone to reactive metabolite formation via TZD ring-opening processes. In vitro studies using either nonlabeled or stable isotope-labeled 2-15N3,4,5-13C3 TZD analogues of PGZ, RGZ, and TGZ were conducted for the detection of TZD-derived GSH conjugates. The use of different isotopes combined with LC-ESI/MS/MS techniques facilitated the detection and characterization of the drug GSH conjugates due to the shift in m/z values of the labeled and unlabeled GSH conjugates. This approach using stable isotope-labeling strategies (17, 18) gave valuable support for the identification and structural elucidation of TZD-related bioactivation pathways.

Experimental Procedures Chemicals and Standards. GSH and TGZ were obtained from Merck and Sigma, respectively. Labeled TZD derivatives, unlabeled RGZ, and PGZ were synthesized according to methods described elsewhere (19-21) using 3,4,5-13C3-2-15N-2,4-TZD as a common labeled precursor (see synthesis section). NMR Analysis. All NMR experiments were performed on a Bruker AV 600 spectrometer operating at 600.13 MHz for 1H. The spectrometer was equipped with a 5 mm TCI cryoprobe with a z-gradient. The NMR experiments were measured at 27 °C, and TMS was used as an internal standard for referencing chemical shifts. Multiplicities are indicated by s (singlet), d (doublet), t (triplet), and m (multiplet). Coupling constants are reported where heteronuclear coupling was observed due to the isotopic enrichment. Assignment of coupling constants established between labeled positions was accomplished by performing additional 13C {1H, 15N} experiments where the 15N decoupling was applied during the acquisition time and performed by composite pulse decoupling at the resonance frequency characteristic of the imide group (169 ppm). Air- or moisture-sensitive reactions were conducted in dried glassware under an atmosphere of dry nitrogen or argon using dried solvents. Synthesis of 3,4,5-[13C3]-2-[15N]-2,4-TZD (3). 13C-15N2-thiourea (1.01 g, 12.6 mmol) and 13C2-bromoacetic acid (1.95 g, 13.7 mmol) were suspended in 2 mL of water and stirred at 96 °C under reflux for 70 h. After the mixture was cooled, the yellowish suspension gave a solid, which was fragmented and homogenized by sonication. The suspension was stirred in an ice bath. The solid was filtered and washed with water (3 mL), 2-propanol/hexane (40:60, 3 mL), and hexane (5 mL). After it was dried at 50°C, the labeled TZD was obtained as a yellowish crystalline solid (1.01 g, 66%). 1H

Chem. Res. Toxicol., Vol. 19, No. 8, 2006 1107 NMR (d6-DMSO): δ 11.99 (s, 1H), 4.14 (ddd, 1JCH ) 148.0 Hz, 3J 2 NH ) 6.2 Hz, JCH ) 2.5 Hz, 2H). LC-ESI/MS showed [M + H]+ at m/z 121. Synthesis of 3,4,5-[13C3]-2-[15N]-5-[4-[2-(5-Ethyl-2-pyridyl)ethoxy]benzylidene]-2,4-TZD (2a). 4-[2-(5-Ethyl-2-pyridyl)ethoxy]benzaldehyde (600 mg, 2.32 mmol) and the labeled TZD 3 (299 mg, 2.55 mmol) were dissolved in methanol (7 mL) together with a catalytic amount of piperidine (1.85 mmol). The yellow mixture was heated under reflux overnight. The suspension was acidified with acetic acid (140 mg, 2.3 mmol) and stirred for one additional hour after the addition of methanol (5 mL). The mixture was cooled in an ice bath, and the resulting solid was filtered and washed with methanol to yield 2a as yellowish crystals (557 mg, 67%) after drying under vacuum (2 h at 50 °C); mp 171.5 °C. 1H NMR (d6DMSO): δ 1.18 (t, J ) 7.8 Hz, 3H), 2.59 (q, J ) 7.8 Hz, 2H), 3.17 (t, J ) 6.6 Hz, 2H), 4.42 (t, J ) 6.6 Hz, 2H), 7.09 (d, J ) 8.9 Hz, 2H), 7.28 (d, J ) 8.1 Hz, 1H), 7.54 (d, J ) 8.9 Hz, 2H), 7.58 (dd, J ) 8.1, 2.4 Hz, 1H), 7.74 (d, J ) 6.5 Hz, 1H), 8.37 (d, J ) 2.4 Hz, 1H), 12.51 (s, 1H). LC-ESI/MS showed [M - H]- at m/z 357. Synthesis of 3,4,5-[13C3]-2-[15N]-5-[4-[2-(5-Ethyl-2-pyridyl)ethoxy]benzyl]-2,4-TZD (PGZ, 1a). Compound 2a (520 mg, 1.45 mmol) dissolved in THF (6 mL) was mixed with water (15 mL) and 1.4 mL of 1 N sodium hydroxide under stirring. The catalyst solution made of cobalt(II)-chloride hexahydrate (17.6 mg, 0.145 mmol) and dimethylglyoxime (84.9 mg, 0.724 mmol) in 1.6 mL of DMF was added dropwise followed by addition of an aqueous 0.25 N sodium hydroxide solution (1.3 mL containing 427.7 mg, 10.85 mmol of sodium borohydride) at a rate to maintain a basic reaction medium (pH 8.5-9.5). The resulting mixture was stirred at room temperature for 5 h, and additional amounts of cobalt chloride (17.6 mg) and sodium borohydride (142 mg) were added. The mixture was stirred overnight before the reaction was quenched with acetone (1 mL) and brought to pH 3.2 with 1 N HCl, leading to the formation of a precipitate. The addition of water (20 mL) and stirring at 0-5 °C for 3 h was followed by filtration of the purplish solid and was subsequently washed with water, methanol, and pentane. The mother liquor was extracted with ethyl acetate (3 × 50 mL). The organic phase was evaporated to give purplish and yellow solids that were further purified after dissolution in THF (100 mL) and heating to 60 °C under stirring for 1 h. The resulting solution containing insoluble purple particles was filtered on silica using warm THF (100 mL). The solvent was evaporated under reduced pressure, and the resulting solid was resuspended in methanol (25 mL) and stirred under reflux for 2 h. After it was cooled, the solid was recovered by filtration, washed with methanol, and dried under vacuum at 50 °C to yield 1a as a white solid (390 mg, 74%) whose purity (99.4%) was assessed by HPLC; mp 183 °C. 1H NMR (d6-DMSO): δ 1.19 (t, J ) 7.5 Hz, 3), 3.05 (m, 1H), 3.14 (t, J ) 6.7 Hz, 2H), 3.30 (m, 1H), 4.31 (t, J ) 6.7 Hz, 2H), 4.84 (m, JHC ) 148 Hz, 1H), 6.87 (m, 2H), 7.15 (m, 2H), 7.28 (d, J ) 7.8 Hz, 1H), 7.58 (dd, J ) 7.8, 6.4 Hz, 1H), 7.60 (q, J ) 7.5 Hz, 2H), 8.38 (d, J ) 2.2 Hz, 1H). 13C NMR: δ 15.8, 25.4, 36.6, 37.1, 53.4 [13C(5), 1JCC ) 45.6 Hz, 2JNC ) 5.9 Hz], 67.1, 114.8, 120.8, 123.5, 129.1, 136.2, 137.1, 149.0, 155.8, 157.9, 172.4 [13C(2), 1J 2 13 1 NC ) 14.6 Hz, JCC ) 10.8 Hz], 176.4 [ C(4), JCC ) 45.6 Hz, 1J 2J ) 14.3 Hz, ) 10.8 Hz]. LC-ESI/MS showed [M + H]+ NC CC at m/z 361.5. Synthesis of 3,4,5-[13C3]-2-[15N]-(Z)-5-[[4-[2-(Methyl-2-pyridinylamino)ethoxy]phenyl]methylene]-2,4-TZD (2b). 4-[2-(Methylpyridin-2-yl-amino)ethoxy]benzaldehyde (667 mg, 2.60 mmol) and the labeled TZD 3 (350 mg, 2.88 mmol) were dissolved in toluene (15 mL) together with a catalytic amount of piperidinium acetate (0.72 mmol). The mixture was heated for 2 h under reflux using a Dean-Stark water trap. After the mixture was cooled to room temperature, a solid crystallized. The toluene was removed by evaporation under vacuum, and the remainder was stirred with methanol (7 mL) at 40 °C. The crystals obtained were filtered, washed with methanol and diethyl ether, and dried under vacuum at 50 °C for 2 h to afford a yellowish crystalline solid (0.78 g, 82%); mp 190-191 °C. 1H NMR (d6-DMSO): δ 3.07 (s, 3H), 3.93

1108 Chem. Res. Toxicol., Vol. 19, No. 8, 2006 (t, J ) 5.8 Hz, 2H), 4.22 (t, J ) 5.8 Hz, 2H), 6.57 (ddd, J ) 7.1, 5.0, 0.7 Hz, 1H), 6.65 (ddd, J ) 8.7, 0.9, 0.7 Hz, 1H), 7.10 (d, J ) 8.9 Hz, 2H), 7.50 (ddd, J ) 2.1, 7.1, 8.7 Hz, 1H), 7.54 (d, J ) 8.9 Hz, 2H), 7.73 (d, J ) 6.64 Hz, 1H), 8.08 (ddd, J ) 5.0, 2.1, 0.9, 1H), 12.50 (s, 1H). LC/MS showed [M + H]+ at m/z 360.2. Synthesis of 3,4,5-[13C3]-2-[15N]-5-[[4-[2-(Methyl-2-pyridinylamino)ethoxy]phenyl]methyl]-2,4-TZD (RGZ, 1b). After three vacuum/H2 purging cycles to remove air from the hydrogenation reactor, a stirred mixture of 3,4,5-[13C3]-2-[15N]-(Z)-5-[[4-[2(methyl-2-pyridinylamino)ethoxy]phenyl]methylene]-2,4-TZD (750 mg, 2.09 mmol) and 10% Pd/C (1.2 g) in 50 mL of THF was hydrogenated at 110 kPa at room temperature for 20 h. The catalyst was removed by filtration, and the THF was evaporated under reduced pressure to afford 0.7 g of crude product, which was purified by chromatography on silica (2% CH2Cl2-methanol) and then crystallized from diethyl ether to give 1b as a yellowish solid (0.45 g, 57%) whose purity (96.8%) was assessed by HPLC; mp 152-153 °C. 1H NMR (d6-DMSO): δ 3.05 (m, 1H), 3.08 (s, 3H), 3.29 (m, 1H), 3.90 (t, J ) 6.0 Hz, 2H), 4.12 (t, J ) 6.0 Hz, 2H), 4.85 (m, JHC ) 148 Hz, 1H), 6.65 (ddd, J ) 8.6, 1.0, 1.0 Hz, 1H), 6.75 (ddd, J ) 7.1, 5.0, 1.0 Hz, 1H), 6.89 (m, 2H), 7.15 (m, 2H), 7.50 (ddd, J ) 8.6, 7.1, 2.0 Hz, 1H), 8.09 (ddd, J ) 5.0, 1.0, 1.0, 1H). 13C NMR: δ 36.7, 49.9, 53.4 [13C(5), 1JCC ) 45.6 Hz, 2JNC ) 5.9 Hz], 65.8, 106.1, 112.0, 114.7, 130.8, 137.1, 147.9, 157.9, 158.5, 172.3 [13C(2), 1JNC ) 14.6 Hz, 2JCC ) 10.8 Hz], 176.4 [13C(4), 1JCC ) 45.6 Hz, 1JNC ) 14.3 Hz, 2JCC ) 10.8 Hz]. LCESI/MS showed [M + H]+ at m/z 362.2. Synthesis of 3,4,5-[13C3]-2-[15N]-5-[4-(6-Benzyloxy-2,5,7,8-tetramethyl-chroman-2-ylmethyloxy)benzylidene]-2,4-TZD (2c). 4-(6Benzyloxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)benzaldehyde (1.24 g, 2.88 mmol), 4,5-[13C3]-2-[15N]-2,4-TZD (3) (520 mg, 4.29 mmol), and water-free sodium acetate (245 mg, 2.99 mmol) were mixed and melted at 150 °C for 2 h under stirring. The resulting solid was dissolved at room temperature in a mixture of ethyl acetate (50 mL) and AcOH (0.5 mL) using ultrasound and then filtered on silica gel using ethyl acetate (20 mL). The solvent was evaporated under reduced pressure. The crude product was dissolved in dichloromethane (20 mL) that was exchanged with methanol (30 mL) by evaporation under reduced pressure. The suspension obtained was concentrated to a final volume of 15 mL and stirred for 30 min in an ice bath. The crystals were filtered, washed with methanol, and dried under vacuum at 55 °C until a constant weight to afford 2c as yellow crystals (1.17 g, 76%). LC/ MS showed [M - H]- at m/z 532 with an identical retention time as the unlabeled analogue. Synthesis of 3,4,5-[13C3]-2-[15N]-5-[4-(6-Benzyloxy-2,5,7,8-tetramethyl-chroman-2-ylmethyloxy)benzyl]-2,4-TZD (1c). Magnesium turnings (575 mg, 23.42 mmol) were added to 2c (1.15 g, 2.16 mmol) dissolved in THF (11.5 mL) and methanol (11.5 mL). The reaction was started by addition of a crystal of iodine and heating at 35 °C. After it was stirred for 90 min at 35 °C and 30 min at room temperature, the mixture was poured into a 10% citric acid solution. The resulting solution was extracted with MTBE (3 × 30 mL), and the organic phase was washed with brine (20 mL). After this was stirred with sodium sulfate and silica (2 g) for 5 min, filtered, and evaporated, 1.4 g of a yellow oil was afforded. Further purification by chromatography on silica (heptane/ethyl acetate 65:35) gave 1c as a white solid (820 mg, 71%) whose purity (95%) was assessed by HPLC. The retention time in HPLC analysis matched the one observed for the previously synthesized unlabeled analogue. Synthesis of 3,4,5-[13C3]-2-[15N]-5-[4-(6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-ylmethyloxy)benzyl]-2,4-TZD (TGZ, 1d). Compound 1c (800 mg, 1.49 mmol) and pyridine hydrochloride (4 g, 33.9 mmol) were mixed in CH2Cl2 (5 mL), and the solvent was removed under reduce pressure. The mixture was melted at 152 °C for 1 h and cooled to room temperature. The resulting yellow solid was dissolved in a mixture of water (10 mL) and ethyl acetate (10 mL) using ultrasounds, acidified with 2 N HCl (5 mL), and extracted with ethyl acetate (3 × 30 mL). The organic phase was washed with brine (20 mL), dried with sodium sulfate, filtered,

AlVarez-Sa´ nchez et al. and evaporated to afford 770 mg of a brown resin that was further purified by chromatography on silica (heptane/ethyl acetate/AcOH 65:35:0.2) and recrystallization (acetone/heptane) to yield 1d as an off-white solid (580 mg, 87%) whose purity (99.6%) was assessed by HPLC. 1H NMR (d6-DMSO): δ 1.31 (s, 3H), 1.96 (s, 3H), 1.82 (m, 1H), 2.00 (m, 1H), 2.04 (s, 3H), 2.06 (s, 3H), 2.58 (t, J ) 6.7 Hz, 2H), 3.06 (m, 1H), 3.30 (m, 1H), 3.95 (m, 2H), 4.85 (m, JHC ) 148 Hz, 1H), 6.15 (m, 2H), 6.92 (m, 2H), 7.41 (s, 1H). 13C NMR: δ 12.2, 12.2, 13.0, 20.1, 22.2, 28.7, 36.6, 53.4 [13C(5), 1JCC ) 45.6 Hz, 2JNC ) 5.9 Hz], 72.8, 74.2, 115.1, 117.2, 120.7, 121.5, 123.2, 129.2, 130.7, 138.3, 144.6, 145.9, 172.3 [13C(2), 1J 2 13 1 NC ) 14.6 Hz, JCC ) 10.8 Hz], 176.5 [ C(4), JCC ) 45.6 Hz, 1J 2 NC ) 14.3 Hz, JCC ) 10.8 Hz]. LC/MS showed [M - H] at m/z 444.1. Hepatic Microsomal Preparations. Microsomes were prepared from fresh rat liver. Human liver microsomes (pool of 10 donors) were obtained from Gentest (Woburn, MA). Tissue homogenization of rat livers and differential centrifugation were performed as described elsewhere (22), and the resuspended microsomes were stored frozen at -80 °C. Protein concentrations were determined by a bicinchoninic acid method (23). The cytochrome P450 content was determined according to published procedures (24). Microsomal Incubations. Microsomal incubations (1 mg/mL of protein) were performed with 10 or 100 µM substrate and 5 mM GSH. All experiments were conducted in potassium phosphate buffer (100 mM, pH 7.4) at a final volume of 500 µL containing 1 mM NADPH. Incubations without substrate were used as controls. Incubations were performed at 37 °C for 1 h and were stopped by the addition of cold acetonitrile (0.5 mL). After centrifugation (15 min at 14000g), the supernatants were evaporated under vacuum to an approximate final volume of 400 µL. LC-ESI/MS/MS Analysis. Samples were analyzed using an ABS/MDS Sciex 4000Qtrap mass spectrometer coupled to an Agilent 1100 series pump equipped with a Pal CTC autosampler. GSH adducts were extracted on-line using a column switching SPE system. The sample was delivered to the trapping column (Develosil C30, 10 mm × 2 mm) using 0.1% aqueous ammonium formate (solvent A) at 0.3 mL/min for 1 min followed by a washing step at 90% A and 10% B (ACN with formic acid 0.1%) for 2 min. Analyte elution in backflush mode was directed into the analytical column (Phenomenex Synergi 4 µm Hydro-RP 80A, 150 mm × 2 mm) by means of a gradient from 2% B for 2 min, increased up to 20% B in 1 min, and linearly increased to 70% in 11 min. The column was then washed with 95% B for 3 min and equilibrated with 2% B for 5 min. Two different survey scans were performed from m/z 400 to 800, both linked by information-dependent acquisition criteria to an enhanced product ion scan (4000 amu/s; collision energy, 30 eV; and linear ion trap with fill time of 10 ms), directed to the two main ions detected. Mass confirmations of the parent ions were obtained by an enhanced resolution scan prior to the acquisition of the enhanced product ion spectrum. The first survey scan experiment, a constant neutral loss (CNL) scan of 129 amu in positive ion mode, was performed at an ionization potential of 5 kV and a source temperature of 300 °C, and the declustering potential and collision energy were set at 70 and 23 eV, respectively. In the second survey scan experiment, a precursor ion scan of m/z 272 in negative ion mode was carried out at an ionization potential of -4 kV and at a source temperature of 300 °C, and the declustering potential and collision energy were set at -90 V and -24 eV, respectively.

Results Synthesis. To demonstrate metabolic modifications of PGZ, RGZ, and TGZ on the TZD ring, a synthesis of these three compounds 13C3-15N-labeled on the heterocyclic positions has been developed using 3,4,5-[13C3]-2-[15N]-2,4-TZD 3 as a


Chem. Res. Toxicol., Vol. 19, No. 8, 2006 1109

Scheme 1. Synthesis of the 13C3-15N-Labeled TZD Derivatives PGZ, RGZ, and TGZ (1a, 1b, and 1d, Respectively)a

a The labeled TZD moiety was introduced by coupling of the corresponding benzaldehyde derivatives with the common TZD-labeled precursor 3. Reagents and conditions: (a) water, 96 °C, 70 h, 66%. (b) Compound 3, piperidine, MeOH, reflux, 24 h, 67%. (c) CoCl2, dimethylglyoxime, NaBH4, THF/aqueous NaOH, room temperature, 24 h, 74%. (d) Compound 3, Pyr-AcOH, toluene, reflux, 2 h, 82%. (e) 10% Pd/C, H2 (0.1 barg), THF, room temperature, 20 h, 57%. (f) Compound 3, NaOAc, 150 °C, 2 h, 76%. (g) Mg, I2, THF/MeOH, 35 °C, 2 h, 71%. (h) Pyr-HCl, 152 °C, 1 h, 87%.

common synthetic precursor, directly obtained by reaction of 13C-15N -thiourea with 13C -bromoacetic acid (Scheme 1). The 2 2 general coupling step was a Knoëvenagel condensation, carried out between the labeled TZD 3 and the corresponding benzaldehydes, synthesized according to procedures described in the literature (19-21). Labeled PGZ 1a was directly obtained by reduction of the resulting benzylidene-TZD 2a with sodium borohydride in the presence of catalytic cobalt chloride and dimethylglyoxime. Similarly, the hydrogenation of the analogue 2b using 10% Pd/C as catalyst afforded the isotopically enriched RGZ derivative 1b. The synthesis of labeled TGZ 1d involved a condensation step of 3 with the corresponding benzyl-protected benzaldehyde. The reaction took place in the presence of melted sodium acetate leading to the product 2c. The reduction of this intermediate was achieved by magnesium-methanol electron transfer to yield 1c and was followed by deprotection of the benzyl ether by treatment with pyridine hydrochloride. Identification of PGZ-Derived Glutathione Conjugates in Hepatic Microsomes. Rat liver microsomal incubations of PGZ or its stable-labeled isotope supplemented with GSH were analyzed by LC-ESI/MS/MS for the detection of PGZ-derived GSH adducts. Incubations in the absence of substrate served as controls. A comparison of the survey scan MS data led to the detection of different chromatographic peaks showing a shift in the m/z value of the precursor ions, which were not observed in the control incubation. Two different putative PGZ GSH conjugates could be identified (Figure 2A) in positive ion mode, using a CNL of 129 amu. The predominant PGZ GSH adduct observed (P1, m/z 637, positive ion mode) showed a shift of 2 amu for the molecular ion when compared to the corresponding labeled analogue P1′ (m/z 639), indicating the loss of two out of four labeled atoms on the TZD ring. The product ion spectra of both compounds revealed key fragments (Figure 3, top) accounting for the loss of pyroglutamate (loss of 129 amu, m/z 508/510 for P1/P1′) and S-C bond cleavage of GSH (loss of 273, m/z 364/366 for P1/P1′) commonly observed in GSH adducts. In addition, fragments that can be explained by the sequential loss of two

sulfur atoms, indicative of a disulfide type structure, could also be observed (fragment ions m/z 364, 330, and 298 for P1). The differences in m/z values for these fragment ions imply losses of 34 and 32 amu and may correspond to hydrogen sulfide and atomic sulfur losses, respectively. The difference of 44 amu between the fragment ions at m/z 298 and 254 for P1, indicative of a decarboxylation process, translated into a 45 amu difference in the case of P1′. This observation suggests that the carbon atom that is lost during this fragmentation process corresponds to a 13C-labeled position of the TZD ring. The second PGZ-derived GSH adduct P2 (m/z 636) and P2′ (m/z 639) showed a shift of the molecular ion by 3 amu and similar fragmentation patterns as to P1 and P1′ (Figure 3), namely, loss of pyroglutamate (m/z 507/510 for P2/P2′) and cleavage of the S-C bond of GSH (m/z 363/366 for P2/P2′) followed by loss of sulfhydric acid (m/z 329/332 for P2/P2′). Differences of 45 and 47 amu between certain fragment ions were observed for P2 (m/z 329 and 284) and P2′ (m/z 332 and 285), respectively. These differences are indicative of a loss of formamide involving two labeled positions for P2′. The molecular weights and fragmentation patterns of these two pairs of adducts and the retention of the three-labeled atoms suggest that these conjugates may be the disulfide carboxylic acid (P1 and P1′ retaining two-labeled atoms) and amide analogues (P2 and P2′ retaining three-labeled atoms) as depicted in Figure 3. Confirmatory analyses were carried out in negative ion mode employing the characteristic precursor ion scan of m/z 272, used for the detection of GSH conjugates (25). Although the sensitivity in this ionization mode was lower, both conjugates were detected and product ion spectra were acquired showing ions characteristic of the GSH moiety (data not shown). These results served as a further confirmation of the molecular weight of the conjugates but did not provide any additional structural information. Human microsomal incubations performed under the same conditions led to the formation of both conjugates to a relatively lower extent when compared to rat liver microsomes. The amide derivative P2 was the predominant GSH adduct detected in

1110 Chem. Res. Toxicol., Vol. 19, No. 8, 2006

Figure 2. Extracted ion chromatograms (XIC) from rat liver microsomal incubations of TZD drugs in the presence of GSH (solid line) and blank incubations performed in the absence of substrate (dotted line). (A) PGZ (CNL scan 129 amu, positive ion mode, XIC m/z 600700); (B) RGZ (CNL scan 129 amu, positive ion mode, XIC m/z 600700); and (C) TGZ (CNL scan 129 amu, positive ion mode, XIC m/z 700-800).

comparison to the carboxylic acid derivative P1. At lower substrate concentrations (10 µM), only traces of P2 could be detected (data not shown). Identification of RGZ-Derived Glutathione Conjugates in Hepatic Microsomes. RGZ and its labeled analogue were incubated in rat and human liver microsomes in the presence of GSH. The evaluation of the MS data obtained from the two different RGZ isotopes allowed the detection of up to five different RGZ-derived GSH conjugates (Figure 2B). The molecular ions of the adducts and the increments in molecular weight due to the labeling effect are summarized in Table 1. The predominant RGZ-derived GSH adduct R1 and its isotope R1′ (Figure 4, top) revealed a difference of 2 amu in molecular weight indicating the loss of two labeled atoms from the TZD ring during metabolic activation and adduction. Their product ion spectra showed the loss of pyroglutamic acid (m/z 509/511 for R1/R1′) as well as the sequential loss of two sulfur atoms (fragments m/z 365, 331, and 299 for R1) upon collision-induced dissociation (CID) of the molecular ions (R1 m/z 638 and R1′ m/z 640). Additionally, losses of 44 and 45 amu (from m/z 299 to 255 for R1 and from m/z 301 to 256 for R1′) suggest a

AlVarez-Sa´ nchez et al.

decarboxylation step for which a 13C-labeled position is lost in the case of R1′. Another key fragment at m/z 135 detected in both compounds further confirms the formation of RGZ-derived GSH adducts since this fragment is characteristic of RGZ and it is also detected in the product ion spectrum of the parent drug. These observations led to the proposal of their chemical structures as being the RGZ-disulfide type derivative containing a carboxylic acid moiety. The GSH adduct R2 (m/z 637) and its labeled analogue R2′ (m/z 640) showed similar fragmentation patterns as compared to R1 and R1′ (Figure 4) and revealed a shift of 3 amu of their corresponding molecular ions indicating the conservation of three out of four labeled atoms. In addition to the characteristic losses of pyroglutamate and sulfur observed for the previous pair of metabolites, R2 showed differences in product ions of 43 (fragment ions m/z 298 and 255) and 45 amu (fragment ions m/z 330 and 285) that translated into differences of 45 and 47 amu for the labeled derivative R2′ (m/z 301 and 256; m/z 333 and 286). In agreement with a fragmentation process leading to deamidation, these data suggest that R2 is the disulfide type amide conjugate (Figure 4, bottom). The structurally related pairs of GSH adducts R3/R3′ and R4/R4′ revealed molecular ions at m/z 624/626 and m/z 623/ 626, respectively, and a difference of 14 amu in molecular weight relative to R1/R1′ and R2/R2′. Additionally, both compounds showed a fragmentation pattern identical to that of R1 and R2, namely, a desulfuration sequence followed by decarboxylation or deamidation processes involving labeled positions (Figure 4). On the basis of these results and in agreement with the reported N-desmethylation process observed for this substrate (16), this pair of compounds was assigned as the corresponding N-desmethylated analogues of R1 and R2 (Figure 4, bottom). In addition to the disulfide type conjugates, the presence of another GSH adduct R5 was revealed in the CNL scan. The 4 amu labeling shift observed between R5 and R5′ (m/z 665 and 669) indicates that all labeled atoms of the TZD ring remain in the molecule. Additionally, their molecular weights correspond to the sum of both GSH and RGZ moieties. Their corresponding MS/MS spectra (data not shown) reveal only fragments accounting for characteristic losses of GSH adducts, namely, loss of pyroglutamate (m/z 536 for R5) and thioether cleavage (m/z 392 for R5). Although indicative of a GSH adduct, these product ion spectra do not provide any additional structural information regarding the regioselectivity of the adduction process. Incubations of RGZ in human microsomes showed a less pronounced formation of GSH conjugates as compared to rat liver microsomes. The amide derivatives R2 and R4 appeared predominant as compared to the carboxylic derivatives R1 and R3 and to R5. At lower substrate concentrations (10 µM), the only GSH adduct of RGZ detected in traces was R5 (data not shown). Identification of TGZ-Derived Glutathione Conjugates in Hepatic Microsomes. Rat and human liver microsomes were incubated with TGZ and its corresponding labeled analogue, and the samples were analyzed for the formation of GSH adducts. The chromatograms obtained (Figure 2C) showed formation of the predominant GSH conjugates T1 (m/z 747) and of T2 (m/z 781). The labeling shifts of 4 amu observed in these two compounds were in agreement with the structures proposed by Kassahun et al. (8) (Figure 5). In addition to these two adducts, the m/z 272 precursor ion scan performed in negative ion mode revealed the presence of a third conjugate T3 (m/z 749, data not shown), showing a 4



Figure 3. (Top) Enhanced product ion spectra (positive ion mode, collision energy 35) of the PGZ GSH conjugates detected in liver microsomes. P1 ([M + H]+ at m/z 637, nonlabeled) and P1′ ([M + H]+ at m/z 639, labeled) and P2 ([M + H]+ at m/z 636, nonlabeled) and P2′ ([M + H]+ at m/z 639, labeled). (Bottom) Structures proposed for these conjugates and their main characteristic MS/MS fragments. Labeled positions are indicated with asterisks. Letters in bold italic on the structure refer to the corresponding fragmentation in the MS/MS spectra. Table 1. GSH Conjugates Detected in Human and Rat Liver Microsomes at 100 µM Substrate Concentration Using Either Unlabeled or 15N-3,4,5-13C3 TZD Derivatives with a 4 Amu Shift in Molecular Weight as Compared to the Unlabeled Analogues

substrate

adduct

molecular ion [M + H]+

PGZ

P1 P2 R1 R2 R3 R4 R5 T1 T2 T3 T4a T5a

637 636 638 637 624 623 665 747 781 751 722 721

RGZ

TGZ

a

shift in molecular weight (amu) as compared to the unlabeled analogue +2 +3 +2 +3 +2 +3 +4 +4 +4 +4 +2 +3

Not detected in survey scans but using targeted MS/MS scans.

amu less in its m/z value when compared to its labeled isotope T3′ (m/z 753). The molecular weight of T3 (750 amu) suggests the addition of GSH accompanied by a reductive process. Product ion spectra of the two isotopic forms in negative ion mode (Figure 6, top) show fragment ions in agreement with losses of 129 (m/z 620 for T3) and 273 amu (m/z 476 for T3), presumably arising from glutamate and thioether cleavages. Furthermore, fragments characteristic of the GSH moiety (m/z

306, 272 and 254) were also observed in both isotopes. Product ion spectra of T3 and T3′ in positive ion mode (Figure 6) revealed fragments characteristic of glycine and pyroglutamate loss (m/z 676 and 622), further corroborating the GSH nature of the conjugate. A common fragment for T3 and T3′ was present at m/z 510. The absence of a shift in molecular weight for this fragment points to a cleavage of the TZD moiety. Moreover, this fragment showed differences of 129 and 275 amu to other fragments observed in the spectra (m/z 381 and 235). These differences may account for a loss of pyroglutamate and a cleavage of the GSH on the C-S bond in a way frequently observed in aromatic GSH thioethers (26). Interestingly, this loss of 275 amu, masked by a concomitant loss of water from the molecular ion, was also observed from the parent ion (m/z 458/462 for T3/T3′). On the basis of these observations, the structures of the conjugate and the corresponding fragments were proposed as depicted in Figure 6. These structure proposals are in agreement with the conservation of the labeled atoms and are indirectly supported by the presence of a hydroxy group prone to undergo cleavage by dehydration. In regard to the proposed hydrogenation taking place in the TZD moiety, no indication about the plausible chemical structure involved could be obtained from these data. GSH conjugates representing the TZD ring-opened carboxylic acid and amide derivatives observed for the other two TZDs were not detected in these survey scan experiments. Targeted product ion experiments of the postulated molecular ions of these



Figure 4. (Top) Enhanced product ion spectra (positive ion mode, collision energy 35) of the GSH conjugates of RGZ: R1 ([M + H]+ at m/z 638, nonlabeled), R1′ ([M + H]+ at m/z 640, labeled), R2 ([M + H]+ at m/z 637, nonlabeled), R2′ ([M + H]+ at m/z 640, labeled), R3 ([M + H]+ at m/z 624, nonlabeled), R3′ ([M + H]+ at m/z 626, labeled), R4 ([M + H]+ at m/z 623, nonlabeled), and R4′ ([M + H]+ at m/z 626, labeled). (Bottom) Structures proposed for these conjugates and their main characteristic MS/MS fragments. Labeled positions are indicated with asterisks. Letters in bold italic on the structure refer to the corresponding fragmentation in the MS/MS spectra.

previously reported ring-opened products (8) (m/z 721 and 722 in positive mode, Figure 5) were performed. As a result of these experiments, two isotopic pairs of conjugates were detected. The first pair T4 and T4′ exhibited the retention of two labeled

positions (m/z 722 and 724, respectively), in agreement with their proposed structures. Likewise, the pair T5 and T5′ (m/z 721 and 724, respectively) showed a mass difference of 3 amu, supporting the amide type structure.



Figure 5. Structures proposed by Kassahun et al. for T1 ([M + H]+ at m/z 747), T2 ([M + H]+ at m/z 781), T4 ([M + H]+ at m/z 722), and T5 ([M + H]+ at m/z 721). Experiments performed with the labeled TGZ derivative show a 4 amu increase in mass for T1′ ([M + H]+ at m/z 751) and for T2′ ([M + H]+ at m/z 785), 2 amu for T4′ ([M + H]+ at m/z 724), and 3 amu for T5′ ([M + H]+ at m/z 724), in agreement with the proposed structures. Stable isotope-labeled atoms are indicated with asterisks.

Figure 6. Enhanced product ion spectra in negative (top) and positive (bottom) ion modes of the TGZ GSH conjugates T3 (750 amu, left) and the labeled analogue T3′ (754 amu, right) detected in human and rat liver microsomal incubations. The structure proposed for T3 and assignment of main fragments observed in the positive ion mode product ion spectrum are depicted on the bottom. Mass shifts observed for these fragments for the labeled analogue are in agreement with the proposed structures.

Incubations in human liver microsomes led to similar patterns as observed for rat liver microsomes (Figure 2C). The quinone-

methide-derived GSH adduct T1 was the predominant peak detected as well at lower substrate concentrations (10 µM); T2



Figure 7. Proposed mechanism for the CID fragmentation of the disulfide type derivatives detected in incubations of PGZ and RGZ. The m/z values of the corresponding fragments detected in the MS/MS spectra of the derivatives R1 and R2 are depicted as example. Numbers in parentheses indicate the difference in m/z values observed for the respective labeled derivatives, R1′ and R2′. Labeled positions (13C or 15N) are indicated with an asterisk.

and T3 were much less abundant. The TZD ring-opened products T4 and T5 were not formed in detectable amounts (data not shown).

Discussion The bioactivation of the TZD drugs TGZ, RGZ, and PGZ was studied in rat and human liver microsomes. The detection of GSH conjugates formed during phase I oxidative metabolism was achieved by LC-ESI/MS/MS analysis of the incubates for the common neutral loss of 129 amu in positive ion mode (2729) or by a precursor ion scan for the fragment ion m/z 272 in negative ion mode (25). Stable isotope-labeled TZD derivatives were employed to demonstrate that bioactivation processes of the drugs occur in the TZD moiety that involved the loss of labeled positions in the TZD ring during reactive metabolite formation. All three drugs studied in this report exhibited GSH adduct formation related to TZD bioactivation processes. These data are in agreement with previously reported studies on GSH conjugates of TGZ (5, 8, 30) and, lately, PGZ (7, 15). The use of stable isotope labeling at metabolically labile positions combined with LC-MS/MS techniques allows for a simple detection of the drug conjugates due to the shift in m/z values of the labeled and unlabeled GSH conjugates. Additionally, this approach gives valuable support in the structural elucidation work (17, 18). P450-mediated S-oxidation is suggested to lead to an unstable TZD-sulfoxide, which undergoes spontaneous ring opening to form a reactive sulfenic acid-R-keto-isocyanate (8, 31). This mechanism appears to be common for the series of TZD drugs investigated. Mixed disulfide type GSH conjugates were detected for TGZ, RGZ, and PGZ. The loss of carbon dioxide as a result of bioactivation on the TZD moiety during GSH adduct formation resulted in a characteristic change of the isotopic difference between the nonlabeled and the labeled analogues of TGZ, RGZ, and PGZ. The mixed disulfide GSH conjugates of the amide type derived from PGZ (P2, m/z 636, and P2′, m/z 639), RGZ (R2, R4, m/z 637, 623, and R2′, R4′, m/z 640, 626), and TGZ (T5, m/z 721, and T5′, m/z 724) exhibited a 3 amu difference in molecular weight between the nonlabeled and the labeled derivatives due to a loss of 13CO2 from the TZD ring. Subsequent oxidative deamination resulted

in the generation of carboxylic acid type mixed disulfide GSH conjugates, revealing loss of the 15N-position. The resulting GSH conjugates P1, R1, R3 (the N-desmethylated analogue of R1), and T4 showed a 2 amu difference in molecular weight to their corresponding labeled analogues (Table 1). Fragmentation of these disulfide type conjugates upon CID of the molecular ions shows the cleavage of the C-S bond of the GSH moiety followed by loss of hydrogen sulfide. This process would give rise to an intermediate positively charged on the sulfur atom that may undergo desulfuration to generate the corresponding carbocation (Figure 7). Another fragmentation pathway would involve the TZD ring where the carboxylic acid or the amide group may undergo decarboxylation/deamidation, resulting in the loss of 44 or 43 amu, respectively. In addition, the amide derivatives may be cleaved via a pathway leading to the loss of the amide group as formamide (45 amu). In this case, desulfuration does not seem to take place, probably due to the nature of the C-S multiple bond established. The predominant GSH adduct of TGZ T1 in rat and human liver microsomes showed the conservation of all four labeled positions of the TZD moiety. Product ion spectra of this adduct (T1, m/z 747 in positive ion mode) were consistent with the structure of the quinone-methide-derived adduct reported previously (8). T1 dominated the survey scan chromatograms obtained in positive ion mode (Figure 2C) in contrast to the less abundant GSH adducts derived from TZD ring scission (T2, T4, and T5). Here, we demonstrated that TZD-dependent bioactivation occurs for all TZD drugs in rat and human liver microsomes. These studies reveal that RGZ and PGZ are prone to form reactive intermediates at higher substrate concentrations, suggesting that covalent binding to liver proteins may also be expected for these two compounds. In human liver microsomes at low drug concentrations, the formation of GSH adducts was evident for TGZ but almost not detectable for RGZ and PGZ. For TGZ, the non-TZD-dependent GSH adduct T1 was the predominant product detected. This observation suggests that bioactivation via TZD ring scission does not contribute significantly to the extent of GSH adduct formation in the case of TGZ. Although the relative contribution of less abundant reactive intermediates to hepatotoxic events remains unknown,


these findings suggest that the observed hepatotoxic potential of TGZ might be associated with the formation of reactive intermediates not related to the TZD moiety. Formation of quinone and quinone-methide type reactive intermediates has been linked to the pro-oxidant activity of TGZ in rat primary hepatocytes on cumene-hydroperoxide-induced lipid peroxidation and cytotoxicity. This effect was significantly higher for TGZ as compared to other vitamin E analogues (32). Cytotoxicity in hepatic cells and oxidative stress-inducing properties for TZD derivatives were dependent on the presence of the 6-hydroxychromane moiety, suggesting a link to this specific molecular structure of TGZ. In addition, N1S1 rat hepatoma cells were more sensitive to TGZ and TGZ-quinone than to other TZD derivatives lacking the 6-hydroxychromane moiety (33). Besides the potential direct cytotoxic effect of TGZderived reactive metabolites on cellular structures, the drug was demonstrated to disrupt the mitochondrial function in rat hepatocytes (33, 34). Funk and others have demonstrated that TGZ and its major hepatic metabolite TGZ-sulfate competitively inhibit the cannalicular bile salt export pump. This effect has been associated with the induction of intrahepatic cholestasis in rats (35). Although these molecular properties of TGZ do not represent a cause for hepatotoxic events per se, it seems likely that certain individuals are more susceptible to TGZ-induced hepatotoxicity, especially in disease populations. Diabetic patients with a history of chloestasis are especially at risk from TGZ (3-5). In conclusion, TZD-related bioactivation seems to represent a common property for these antidiabetic drugs. For TGZ, the predominant GSH adduct at lower substrate concentrations in human liver microsomes was not TZD-dependent. This TGZspecific biactivation potential, its relative higher clinical dose, and other risk factors such as hepatic oxidative stress, cholestatic and P450 inhibition, and induction potentials (13) might contribute to the hepatotoxicity of this compound. Acknowledgment. We are grateful to Jean-Claude Alt, Philipp Cueni, Fritz Koch, Roland Simon, and Martin Binder for their contributions to the synthesis and NMR analysis of the labeled TZD derivatives. We thank Drs. Christoph Funk and Stephen Fowler for their comments and critical review of the manuscript.

References (1) Saltiel, A. R., and Olefsky, J. M. (1996) Thiazolidinediones in the treatment of insulin resistance and type II diabetes. Diabetes 45, 16611669. (2) Kohlroser, J., Mathai, J., Reichheld, J., Banner, B. F., and Bonkovsky, H. L. (2000) Hepatotoxicity due to troglitazone: Report of two cases and review of adverse events reported to the United States Food and Drug Administration. Am. J. Gastroenterol. 95, 272-276. (3) Menon, K. V. N., Angulo, P., and Lindor, K. D. (2001) Severe cholestatic hepatitis from troglitazone in a patient with nonalcoholic steatohepatitis and diabetes mellitus. Am. J. Gastroenterol. 96, 16311634. (4) Smith, M. T. (2003) Mechanisms of troglitazone hepatotoxicity. Chem. Res. Toxicol. 16, 679-687. (5) Tettey, J. N., Maggs, J. L., Rapeport, W. G., Pirmohamed, M., and Park, B. K. (2001) Enzyme-induction dependent bioactivation of troglitazone and troglitazone quinone in vivo. Chem. Res. Toxicol. 14, 965-974. (6) Kawai, K., Kawasaki-Tokui, Y., Odaka, T., Tsuruta, F., Kazui, M., Iwabuchi, H., Nakamura, T., Kinoshita, T., Ikeda, T., Yoshioka, T., Komai, T., and Nakamura, K. (1997) Disposition and metabolism of the new oral antidiabetic drug troglitazone in rats, mice and dogs. Arzneimittelforschung 47, 356-368. (7) Shen, Z., Reed, J. R., Creighton, M., Liu, D. Q., Tang, Y. S., Hora, D. F., Feeney, W., Szewczyk, J., Bakhtiar, R., Franklin, R. B., and Vincent, S. H. (2003) Identification of novel metabolites of pioglitazone in rat and dog. Xenobiotica 33, 499-509.

Chem. Res. Toxicol., Vol. 19, No. 8, 2006 1115 (8) Kassahun, K., Pearson, P. G., Tang, W., McIntosh, I., Leung, K., Elmore, C., Dean, D., Wang, R., Doss, G., and Baillie, T. A. (2001) 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. Chem. Res. Toxicol. 14, 62-70. (9) Prabhu, S., Fackett, A., Lloyd, S., McClellan, H. A., Terrell, C. M., Silber, P. M., and Li, A. P. (2002) Identification of glutathione conjugates of troglitazone in human hepatocytes. Chem.-Biol. Interact. 142, 83-97. (10) Soglia, J. R., Harriman, S. P., Zhao, S., Barberia, J., Cole, M. J., Boyd, J. G., and Contillo, L. G. (2004) The development of a higher throughput reactive intermediate screening assay incorporating microbore liquid chromatography-micro-electrospray ionization-tandem mass spectrometry and glutathione ethyl ester as an in vitro conjugating agent. J. Pharm. Biomed. Anal. 36, 105-116. (11) Forman, L. M., Simmons, D. A., and Diamond, R. H. (2000) Hepatic failure in a patient taking rosiglitazone. Ann. Intern. Med. 132, 118121. (12) May, L. D., Lefkowitch, J. H., Kram, M. T., and Rubin, D. E. (2002) Mixed hepatocellular-cholestatic liver injury after pioglitazone therapy. Ann. Intern. Med. 136, 449-452. (13) Sahi, J., Black, C. B., Hamilton, G. A., Zheng, X., Jolley, S., Rose, K. A., Gilbert, D., LeCluyse, E. L., and Sinz, M. W. (2003) Comparative effects of thiazolidinediones on in vitro P450 enzyme induction and inhibition. Drug Metab. Dispos. 31, 439-446. (14) Kiyota, Y., Kondo, T., Maeshiba, Y., Hashimoto, A., Yamashita, K., Yoshimura, Y., Motohashi, M., and Tanayama, S. (1997) Studies on the metabolism of the new antidiabetic agent pioglitazone. Identification of metabolites in rats and dogs. Arzneimittelforschung 47, 2228. (15) Baughman, T. M., Graham, R. A., Wells-Knecht, K., Silver, I. S., Tyler, L. O., Wells-Knecht, M., and Zhao, Z. (2005) Metabolic activation of pioglitazone identified from rat and human liver microsomes and freshly isolated hepatocytes. Drug Metab. Dispos. 33, 733-738. (16) Cox, P. J., Ryan, D. A., Hollis, F. J., Harris, A. M., Miller, A. K., Vousden, M., and Cowley, H. (2000) Absorption, disposition, and metabolism of rosiglitazone, a potent thiazolidinedione insulin sensitizer, in humans. Drug Metab. Dispos. 28, 772-780. (17) Rettenmeier, A. W., Gordon, W. P., Barnes, H., and Baillie, T. A. (1987) Studies on the metabolic fate of valproic acid in the rat using stable isotope techniques. Xenobiotica 17, 1147-1157. (18) Taylor, E. W., Jia, W., Bush, M., and Dollinger, G. D. (2002) Accelerating the drug optimization process: Identification, structure elucidation, and quantification of in vivo metabolites using stable isotopes with LC/MSn and the chemiluminescent nitrogen detector. Anal. Chem. 74, 3232-3238. (19) Wolff, H.-P., and Witte, E.-C. (1997) New process for preparing troglitazone. International patent application, publication no. WO 97/ 43283. (20) Adiyaman, M., Guner, D., Yurdakul, A., and Ridvanoglu, N. (2003) A process for the production of phenyl ethers. International patent application, publication no. WO 2004/000810 A1. (21) Cantello, B. C., Cawthorne, M. A., Cottam, G. P., Duff, P. T., Haigh, D., Hindley, R. M., Lister, C. A., Smith, S. A., and Thurlby, P. L. (1994) [[omega-(Heterocyclylamino)alkoxy]benzyl]-2,4-thiazolidinediones as potent antihyperglycemic agents. J. Med. Chem. 37, 39773985. (22) Van der Hoeven, T. A., and Coon, M. (1974) Preparation and properties of partially purified cytochrome P-450 and reduced nicotinamide adenine dinucleotide phosphate P-450 reductase from rabbit liver microsomes. J. Biol. Chem. 249, 6302-6310. (23) Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76-85. (24) Omura, T., and Sato, R. (1964) The carbon monoxide-binding pigment of liver microsomes. II. Solubilization, purification, and properties. J. Biol. Chem. 239, 2379-2385. (25) Dieckhaus, C. M., Fernandez-Metzler, C. L., King, R., Krolikowski, P. H., and Baillie, T. A. (2005) Negative ion tandem mass spectrometry for the detection of glutathione conjugates. Chem. Res. Toxicol. 18, 630-638. (26) Smith, K. S., Smith, P. L., Heady, T. N., Trugman, J. M., Harman, W. D., and Macdonald, T. L. (2003) In vitro metabolism of tolcapone to reactive intermediates: relevance to tolcapone liver toxicity. Chem. Res. Toxicol. 16, 123-128. (27) Baillie, T. A., and Davis, M. R. (1993) Mass spectrometry in the analysis of glutathione conjugates. Biol. Mass Spectrom. 22, 319325. (28) Baillie, T. A., Pearson, P. G., Rashed, M. S., and Howald, W. N. (1989) The use of mass spectrometry in the study of chemically


(29) (30)

(31)

(32)

reactive drug metabolites. Application of MS/MS and LC/MS to the analysis of glutathione- and related S-linked conjugates of Nmethylformamide. J. Pharm. Biomed. Anal. 7, 1351-1360. Chen, W. G., Zhang, C., Avery, M. J., and Fouda, H. G. (2001) Reactive metabolite screen for reducing candidate attrition in drug discovery. AdV. Exp. Med. Biol. 500, 521-524. He, K., Talaat, R. E., Pool, W. F., Reily, M. D., Reed, J. E., Bridges, A. J. and Woolf, T. F. (2004) Metabolic activation of troglitazone: Identification of a reactive metabolite and mechanisms involved. Drug Metab. Dispos. 32, 639-646. Reddy, V. B., Karanam, B. V., Gruber, W. L., Wallace, M. A., Vincent, S. H., Franklin, R. B., and Baillie, T. A. (2005) Mechanistic studies on the metabolic scission of thiazolidinedione derivatives to acyclic thiols. Chem. Res. Toxicol. 18, 880-888. Tafazoli, S., Spehar, D. D., and O’Brien, P. J. (2005) Oxidative stress mediated idiosyncratic drug toxicity. Drug Metab. ReV. 37, 311-325.

AlVarez-Sa´ nchez et al. (33) Narayanan, P. K., Hart, T., Elcock, F., Zhang, C., Hahn, L., McFarland, D., Schwartz, L., Morgan, D. G., and Bugelski, P. (2003) Troglitazoneinduced intracellular oxidative stress in rat hepatoma cells: A flow cytometric assessment. Cytometry A 52, 28-35. (34) Tirmenstein, M. A., Hu, C. X., Gales, T. L., Maleeff, B. E., Narayanan, P. K., Kurali, E., Hart, T. K., Thomas, H. C., and Schwartz, L. W. (2002) Effects of troglitazone on HepG2 viability and mitochondrial function. Toxicol. Sci. 69, 131-138. (35) Funk, C., Ponelle, C., Scheuermann, G., and Pantze, M. (2001) Cholestatic potential of troglitazone as a possible factor contributing to troglitazone-induced hepatotoxicity: In vivo and in vitro interaction at the canalicular bile salt export pump (Bsep) in the rat. Mol. Pharmacol. 59, 627-635.

TX050353H

Thiazolidinedione Bioactivation: A Comparison of ... - ACS Publications

Recommend Documents