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Studies on the Metabolism of Tolmetin to the Chemically Reactive Acyl-Coenzyme A Thioester Intermediate in Rats

Department of Pharmaceutics and Analytical Chemistry, the Danish University of Pharmaceutical Sciences, Copenhagen, Denmark (J.O., C.S., U.S., S.H.H.); Department of Drug Metabolism and Pharmacokinetics, Novo Nordisk A/S, Måløv, Denmark (I.B.), and Department of Biopharmaceutical Sciences, University of California, San Francisco, California (C.L., L.Z.B.)

(Received October 28, 2006; accepted February 12, 2007)

	Abstract

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Carboxylic acids may be metabolized to acyl glucuronides and acyl-coenzyme A thioesters (acyl-CoAs), which are reactive metabolites capable of reacting with proteins in vivo. In this study, the metabolic activation of tolmetin (Tol) to reactive metabolites and the subsequent formation of Tol-protein adducts in the liver were studied in rats. Two hours after dose administration (100 mg/kg i.p.), tolmetin acyl-CoA (Tol-CoA) was identified by liquid chromatography-tandem mass spectrometry in liver homogenates. Similarly, the acyl-CoA-dependent metabolites tolmetin-taurine conjugate (Tol-Tau) and tolmetin-acyl carnitine ester (Tol-Car) were identified in rat livers. In a rat bile study (100 mg/kg i.p.), the S-acyl glutathione thioester conjugate was identified, providing further evidence of the formation of reactive metabolites such as Tol-CoA or Tol-acyl glucuronide (Tol-O-G), capable of acylating nucleophilic functional groups. Three rats were treated with clofibric acid (150 mg/kg/day i.p. for 7 days) before dose administration of Tol. This resulted in an increase in covalent binding to liver proteins from 0.9 nmol/g liver in control rats to 4.2 nmol/g liver in clofibric acid-treated rats. Similarly, levels of Tol-CoA increased from 0.6 nmol/g to 4.4 nmol/g liver after pretreatment with clofibric acid, whereas the formation of Tol-O-G and Tol-Tau was unaffected by clofibric acid treatment. However, Tol-Car levels increased from 0.08 to 0.64 nmol/g after clofibric acid treatment. Collectively, these results confirm that Tol-CoA is formed in vivo in the rat and that this metabolite can have important consequences in terms of covalent binding to liver proteins.

Covalent binding of Tol has also been studied in humans, where Tol-protein adducts have been detected in plasma (Hyneck et al., 1988; Zia-Amirhosseini et al., 1994). Tol-protein adducts in plasma were believed to result from reaction of the acyl glucuronide with plasma proteins, since a good correlation between covalent plasma-protein adducts and exposure to Tol-O-G was observed (Hyneck et al., 1988). In the liver, other metabolic pathways may contribute to Tol-protein adduct formation. In a recent study, it has been shown that the pyrrole moiety of Tol may undergo bioactivation to a reactive arene oxide (Chen et al., 2006). Other reports have shown that acyl-coenzyme A thioesters (acyl-CoAs) are also reactive electrophilic metabolites (Sallustio et al., 2000; Grillo and Benet, 2002; Sidenius et al., 2004). Acyl-CoAs are intermediates in amino acid conjugation, carnitine conjugation, fatty acid synthesis, and ß-oxidation. In vitro characterization of the chemical reactivity has shown that the acyl-CoAs are significantly more reactive than the corresponding acyl glucuronides in the spontaneous reaction with glutathione (Grillo and Benet, 2002; Li et al., 2002; Olsen et al., 2002). Therefore, covalent binding of xenobiotic carboxylic acids in vivo may occur via at least two mechanisms, the importance of which depends on the concentration and the relative reactivity of each reactive metabolite.

In a recent study, the reactivity of tolmetin-acyl-CoA (Tol-CoA) was examined, and the Tol-CoA-dependent metabolites tolmetinglycine (Tol-Gly) and tolmetin-taurine conjugates (Tol-Tau) were identified in rat urine (Olsen et al., 2003a). The aim of the present study was to investigate the acyl-CoA-dependent metabolism of Tol, and to examine the significance with respect to drug-protein adduct formation in vivo. Therefore, acyl-CoA formation was induced with clofibric acid to test the hypothesis that increased levels of acyl-CoA would result in a corresponding increase in the levels of drug-protein adducts in the liver.

	Materials and Methods

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Chemicals. Tol (sodium, dihydrate salt), zomepirac (5-[4'-chlorobenzoyl]-1,4-dimethylpyrrole-2-acetic acid, sodium salt), glycine, taurine, DL-carnitine, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO). N,N-Carbonyldiimidazole was obtained from Fluka Chemie AG (Buchs, Switzerland). N-Hydroxysuccinimide and anhydrous tetrahydrofuran were purchased from Aldrich Chemical Co. (Milwaukee, WI). CoA (trilithium, dihydrate salt) was purchased from AppliChem (Darmstadt, Germany). Tol-CoA, Tol-Tau, Tol-Gly, Tol-Car, and Tol-SG were synthesized as described below.

Animal Experiments and Sample Preparation Procedures. Male Sprague-Dawley rats (250–300 g) were purchased from Bantan and Kingman Universal (Livermore, CA), maintained in a controlled housing environment with a 12-h light/dark cycle, and received standard laboratory chow and water ad libitum. Rats were acclimated to the housing conditions for at least 3 days before use in the experiments. All animal use was approved by the University of California San Francisco Committee on Animal Research.

Three rats were treated with clofibric acid (150 mg/kg/day i.p.) and three rats received blank vehicle for 7 days. The dose solution of Tol was prepared in a 0.05 M phosphate buffer, pH 7.4 (40 mg/ml). The dose solution of clofibric acid (40 mg/ml) was made in a 0.1 M bicarbonate buffer. On day 7, 2 h after the last dose of clofibric acid or blank vehicle, the rats received 100 mg/kg tolmetin (i.p.). Two hours after administration of Tol, the rats were sacrificed by cervical dislocation, and the livers were removed and snap-frozen in liquid nitrogen. The livers were processed essentially as described by Li et al. (2003). In brief, 1.0 g of liver was homogenized in 3.0 ml of 0.05 M KH₂PO₄ (pH 4.5, 0°C) to which 200 µl of 20% phosphoric acid was added. Proteins in the liver homogenates were precipitated by addition of 1 volume of ice-cold acetonitrile, and the samples were then centrifuged at 4°C (10,000g for 2 min). The supernatant was stored at –80°C until analysis for Tol metabolites, and the pellet was assayed for covalent binding of Tol to proteins.

One rat (250 g) was given a dose of Tol (100 mg/kg i.p.), and bile, including predose bile, was collected over a 4-h period at 1-h intervals. Bile was collected over 21% phosphoric acid (200 µl to each container). Bile (100 µl) was diluted with 400 µl of 50% acetonitrile containing 1% formic acid and centrifuged (10,000g for 2 min) before analysis of the supernatant.

Covalent Binding of Tol to Liver Proteins. The protein pellets were washed seven times with 6 ml of methanol/ether (3:1, v/v) following the procedure reported previously (Smith et al., 1986). Subsequently, the pellets were washed as described by Bailey and Dickinson (1996) with slight modifications. Briefly, the pellet was resuspended in 1 ml of 10 mM KH₂PO₄ before re-precipitation with 4 ml of acetonitrile followed by centrifugation. This step was repeated nine times. The washed protein pellets were dried overnight and dissolved in 2 ml of 1 N NaOH at 60°C for 12 h. The protein hydrolysates were cooled to room temperature, and 0.2 ml of 85% H₃PO₄ was added before the internal standard was added (1 µg of zomepirac). To extract hydrolyzed Tol and internal standard, 8 ml of dichloromethane was added and the solution was shaken gently for 10 min. After centrifugation to separate the layers, the organic phase was evaporated gently under nitrogen and reconstituted in 200 µl of 10% acetonitrile containing 0.1% trifluoroacetic acid. From this mixture, 50 µl was injected onto the HPLC system to quantify the amount of Tol covalently bound to liver proteins. For construction of standard curves, blank pellets were spiked with Tol and internal standard. Duplicate standards at six levels, covering the concentration range of the samples, underwent the same sample preparation procedure as the samples. Recoveries were 80 to 100% and the correlation coefficient was 0.997. The repeatability of the extraction procedure, expressed as the relative standard deviation, was 3.9% (1.2 nmol tolmetin/g liver, n = 6). The adequacy of the washing procedure was assessed from a control experiment in which 5 mg of Tol was added to 1 g of liver that was homogenized in 3 ml of 50 mM phosphate buffer (pH 4.5). The control samples (n = 3) were precipitated with 1 volume of cold acetonitrile and subsequently underwent the same washing, hydrolysis, and extraction procedures as described above for the samples. No binding could be detected in the control samples. HPLC conditions for quantification of extracted tolmetin were as follows: mobile phases A consisted of 10% acetonitrile containing 0.1% trifluoroacetic acid, whereas mobile phase B was 50% acetonitrile containing 0.1% trifluoroacetic acid. A linear gradient from 0% B to 100% B over 20 min was used at a flow rate of 1 ml/min. A Luna C-18 column (15 cm x 4.6 mm i.d.) from Phenomenex (Torrance, CA) was used with UV detection at 320 nm.

LC-MS Analysis. Unless otherwise stated, LC/MS was performed on an Agilent 1100 LC system (Agilent Technologies, Palo Alto, CA) connected to an ion trap mass spectrometer (MSD trap from Agilent Technologies) equipped with an electrospray ionization (ESI) interface. LC/ESI/MS with accurate mass measurements was performed on a Surveyor HPLC system (Thermo Fisher Scientific, San Jose, CA) that was connected to a triple quadrupole mass spectrometer (TSQ Quantum Ultra AM from Thermo Fisher Scientific).

Analysis of Tol-CoA was conducted as described recently for analysis of zomepirac acyl-CoA (Olsen et al., 2005). In brief, HPLC was performed on a Waters (Milford, MA) Xterra column (3 µ, 100 mm x 2.0 mm i.d.), which was maintained at 40°C and operated at a flow rate of 0.3 ml/min. Mobile phase A consisted of 10 mM ammonium acetate and mobile phase B consisted of 10 mM ammonium acetate in 80% acetonitrile. Gradient elution was performed from 5 to 100% B in 20 min. MS/MS fragmentation was performed in positive ionization mode with a spray voltage of 3500 V. Quantification of Tol-CoA was performed by LC/MS/MS of [M + H]⁺ at m/z 1007 with extraction of fragment ions at m/z 500, m/z 580, and m/z 598. Calibration standards were constructed in blank matrix by addition of synthetic Tol-CoA, and the standards then underwent the same preparation as the samples. Determination of Tol-CoA in liver samples was performed in duplicate.

Tol and Tol-O-G were analyzed using the same HPLC conditions as described for analysis of Tol-CoA. Identification and quantification of Tol-O-G were performed as described by Olsen et al. (2003a). In brief, Tol-O-G was identified by LC/MS/MS in positive and negative ionization modes. Both Tol and Tol-O-G were quantified by UV detection at 320 nm using Tol as the calibration standard assuming that the molar absorptivities of Tol and Tol-O-G were the same.

HPLC analysis of Tol-Tau and Tol-Gly was performed on a Luna Phenylhexyl column (5 µ, 150 x 2.0 mm i.d.) from Phenomenex, which was maintained at 40°C. The composition of mobile phases A and B was: A, 10 mM ammonium formate (pH 3.5), and B, 10 mM ammonium formate (pH 3.5) in 80% acetonitrile. The pH of the ammonium formate solution was adjusted to pH 3.5 with concentrated formic acid. Gradient elution from 30 to 100% B in 10 min was performed at a flow rate of 0.3 ml/min. The mass spectrometer was operated in both positive and negative ionization mode for identification of the conjugates. Quantification was performed in negative ionization mode where the m/z 124 fragment of Tol-Tau ([M – H]^– at m/z 363) and the m/z 238 fragment of Tol-Gly ([M – H]^– at m/z 313) were extracted. Calibration standards were constructed in the blank matrix using the preparation procedure described for the samples. Analyses of the liver samples were performed in duplicate.

Analysis of Tol-Car was performed on a Luna C-18 column (3 µ, 100 mm x 2.0 mm i.d.) from Phenomenex. Mobile phase A consisted of 0.2% formic acid and mobile phase B consisted of 0.2% formic acid in acetonitrile. A gradient was performed from 10 to 50% B in 20 min at a flow rate of 0.3 ml/min. MS detection was performed using ESI in positive ionization mode with a spray voltage of 4000 V. Tol-Car was quantified by UV detection at 320 nm using Tol as the calibration standard assuming the UV contribution from the carnitine moiety was negligible. When blank liver extracts treated as the sample were analyzed, no chromatographic peaks at 320 nm were observed at the retention time observed for Tol-Car. For LC/ESI/MS with accurate mass measurements, gradient elution was performed from 10 to 100% B in 10 min at a flow rate of 0.2 ml/min. Accurate mass measurements were performed in selected ion monitoring mode at m/z 401.2. A solution of lock masses (m/z 520.3328 and m/z 564.3590 from a solution of polyethylene glycol 200, 400, and 600) was infused into the HPLC eluent via a T-piece at a flow rate of 5 µl/min.

Identification of Tol metabolites in rat bile was performed by LC-MS(/MS) on a Luna Phenylhexyl column (5 µ, 150 mm x 2.0 mm i.d.) from Phenomenex. The temperature of the HPLC column was maintained at 40°C and the flow rate was 0.3 ml/min. Mobile phase A consisted of 0.2% formic acid and mobile phase B consisted of 0.2% formic acid in acetonitrile. Gradient elution was performed from 10 to 100% B in 30 min. Tol-O-G, Tol-Tau, and Tol-SG were identified based upon 1) MS/MS fragmentation as described above and 2) comparison of HPLC retention times and MS/MS spectra with those of authentic standards when available. Accurate mass measurement of Tol-SG was performed as described above for analysis of Tol-Car with the exception that Tyr-Tyr-Tyr (m/z 508.2080) and reserpine (m/z 609.2812) were used as lock masses. Identification of hydroxy-Tol (Tol-OH) and carboxyl-Tol (Tol-COOH) was performed as described previously (Olsen et al., 2003a).

View larger version (14K): [in this window] [in a new window]   FIG. 1. LC/MS/MS analysis of Tol-CoA ([M + H]+ at m/z 1007) in a liver sample from a rat pretreated with clofibric acid and administered Tol (100 mg/kg i.p.). See Fig. 2 for proposed identities of fragment ions.

View larger version (9K): [in this window] [in a new window]   FIG. 2. Proposed identities of the fragment ions of Tol-CoA.

Tol-Gly and Tol-Tau were synthesized as described previously (Olsen et al., 2005). ESI/MS/MS: Tol-Tau, [M + H]⁺ at m/z 365: m/z 240 (100%, [Tol + H – H₂O]⁺), m/z 212 (35%, [Tol-COOH]⁺), m/z 119 (18%, [CH₃(C₆H₄)CO⁺]). [M – H]^– at m/z 363: m/z 124 (100%, [Taurine – H]^–). Tol-Gly: [M + H]⁺ at m/z 315: m/z 240.1 (100%, [Tol + H – H₂O]⁺), m/z 212 (30%, [Tol-COOH]⁺), m/z 179 (9%, [Tol-Gly + 2H – CH₃(C₆H₄)CO – H₂O]⁺), m/z 151 (5%, [m/z 179 – CO]⁺) and m/z 119 (23%), [CH₃(C₆ H₄)CO⁺]). [M – H]^– at m/z 313: m/z 238 (100%, [Tol – H – H₂O]^–).

The synthesis of Tol-Car was performed following a method described previously (Stadtmann, 1957). To a solution of Tol (1.6 mmol as protonated carboxylic acid dissolved in 30 ml of anhydrous tetrahydrofuran), 220 µl of triethylamine and 160 µl of ethylchlorformate were added. After 30 min, the reaction mixture was filtered through a paper filter into a mixture of 750 mg of DL-carnitine hydrochloride dissolved in a solution of NaHCO₃ (5.3 g/l with pH adjusted to pH 10 with NaOH). The mixture was allowed to react for 90 min, after which the reaction was stopped by addition of 0.5 ml of 30% HCl. Tetrahydrofuran was removed using a rotary evaporator, and the remaining Tol was removed by extraction with ethyl acetate. The aqueous phase was lyophilized, redissolved in 10% acetonitrile, and further purified by solid-phase extraction on Oasis cartridges (1cc, 30 mg) obtained from Waters. The eluate was dried under a stream of nitrogen, and for NMR analysis, the eluate was reconstituted in CD₃CN. Proton NMR spectra were acquired on a Bruker (Newark, DE) Avance (600 MHz) spectrometer at 25°C using a dual 1H-13C 3-mm cryoprobe. The chemical shifts are expressed as parts per million relative to tetramethylsilane. ¹H NMR. 7.70 (d, J = 8.0 Hz, CH₃(C₆H₂H₂)CO–), 7.33 (d, J = 7.8 Hz, CH₃(C₆H₂H₂)CO–), 6.64 ppm (d, J = 4.0, –1H-pyrrole), 6.15 ppm (d, J = 4.0, –1H-pyrrole), 5.55 ppm (s, T-O-CH–), 3.89 ppm (m, pyrrole-CH₂CO–), 3.89 ppm (m, pyrrole-NCH₃), 3.70 ppm (m, –CH₂N⁺(CH₃)₃), 3.06 ppm (s, –N⁺(CH₃)₃), 2.77 ppm (dd, J = 4.0, 16.5 Hz, –CHCH₂COOH), 2.60 ppm (dd, J = 9.2, 16.5 Hz, –CHCH₂COOH), 2.44 ppm (s, CH₃(C₆H₄)–). ESI/MS/MS of [M]⁺ at m/z 401: m/z 119 (100%), m/z 144 (7%), m/z 254 (40%), m/z 258 (5%), and m/z 342 (69%). Assignment of fragment ions is given under Results.

	Results

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Identification of Tol-CoA. Tol-CoA was identified in vivo in rat livers using the method described for identification of the zomepirac acyl coenzyme A thioester (Olsen et al., 2005). Tol-CoA eluted at 12.1 min and MS/MS fragmentation of [M + H]⁺ at m/z 1007 resulted in an MS/MS spectrum with characteristic fragment ions at m/z 598, 580, 500, 428, and 398 (Fig. 1). Assignment of the fragment ions is shown in Fig. 2. This fragmentation pattern is similar to that described for other acyl-CoAs (Grillo and Benet, 2002; Li et al., 2002; Olsen et al., 2003b; Sidenius et al., 2004), including that of the structurally related nonsteroidal anti-inflammatory drug, zomepirac (Olsen et al., 2005). The retention time and MS/MS spectrum of a synthetic standard were similar to those seen in liver samples from rats that had received a dose of 100 mg/kg.

Identification of Tol-Car. Tol-Car was identified in rat livers after administration of Tol. LC-MS/MS of Tol-Car ([M]⁺ at m/z 401) eluting at 15.5 min gave fragment ions at m/z 342, 258, 254, 144, and 119 as shown in Fig. 3, in which assignment of the fragment ions is also shown. Further evidence for the proposed fragmentation pattern came from LC-MS³ analysis of m/z 401342 (Fig. 4), which gave fragment ions at m/z 258 ([Tol + H]⁺), m/z 240 ([Tol – H₂O + H]⁺), and m/z 119 ([CH₃(C₆H₄)CO⁺]). This MS³ spectrum was similar to an MS/MS spectrum of Tol. Accurate mass measurements gave an [M]⁺ at m/z 401.2087 consistent with an elemental composition corresponding to that of Tol-Car (+2.7 ppm). Finally, a synthetic standard of Tol-Car coeluted and showed the same MS/MS characteristics as that observed from analysis of the liver extracts.

View larger version (14K): [in this window] [in a new window]   FIG. 3. MS/MS spectrum of Tol-Car ([M]+ at m/z 401) obtained from LC/MS/MS analysis of a liver sample from a rat pretreated with clofibric acid and administered Tol.

View larger version (20K): [in this window] [in a new window]   FIG. 4. LC/MS/MS of Tol-SG (m/z 547) in a bile sample from a rat that received Tol (100 mg/kg). A, UV trace at 320 nm showing elution of 1, Tol-OH; 2, Tol-COOH; 3, Tol-O-G; 4, sulfate conjugate of Tol-OH; 5, Tol; 6, Tol-Tau. B, extracted ion chromatogram of m/z 418. C, MS/MS spectrum of m/z 547 from 13.6 to 13.9 min. Fragmentation patterns are discussed in the text.

Identification of Tol-O-G, Tol-Tau, and Tol-Gly. Tol-O-G and Tol-Tau were identified as described previously (Olsen et al., 2003a). In brief, for the most abundant Tol-O-G isomer, one intense fragment ion of Tol-O-G ([M + H]⁺ at m/z 434) was observed at m/z 258 ([Tol + H]⁺) which, by MS³ (m/z 434 258), gave one intense fragment at m/z 119 ([CH₃(C₆H₄)CO]⁺). This isomer was assumed to be the 1-O-isomer. The most intense MS/MS fragment for the other isomers was a loss of water in positive and negative ionization mode. In negative ionization mode, MS/MS of the most abundant isomer ([M – H]^– at m/z 432) gave fragment ions at m/z 175 (19%, [glucuronic acid – H₂O – H]^–) and m/z 193 (100%, [glucuronic acid – H]^–). Accurate mass measurements showed that the [M + H]⁺ was 434.1454 Da, which is consistent with the m/z value of protonated Tol-O-G with an accuracy of 1.8 ppm.

Tol-Tau was identified in rat livers by LC-MS/MS. The retention time and MS/MS fragmentation patterns in positive and negative ionization modes of a synthetic standard were in accordance with that obtained from analysis of liver samples. The formation of Tol-Gly was also studied, but this metabolite was only observed at levels below the limit of quantification (0.6 nmol/g liver).

In Vivo Formation of Tol Metabolites and Covalent Binding to Liver Proteins. Formation of Tol-O-G, Tol-CoA, Tol-Tau, Tol-Car, and covalent binding of Tol to liver proteins were determined in liver homogenates of rats (either pretreated with clofibric acid or control rats) that had received a dose of 100 mg/kg Tol as shown in Table 1. In control rats, the level of Tol-CoA was 0.6 nmol/g liver, whereas the level of Tol-O-G was approximately 40 times higher. Formation of Tol-O-G was unaffected by the clofibric acid pretreatment, whereas the concentration of Tol-CoA in liver homogenates increased significantly to 4.4 nmol/g. Formation of Tol-Car also increased upon treatment with clofibric acid from 0.08 to 0.64 nmol/g liver, whereas the levels of Tol and Tol-Tau were unaffected by the treatment with clofibric acid. Covalent binding of Tol to liver proteins was determined to be 0.9 nmol/g liver in control rats, whereas covalent binding increased to 4.2 nmol/g liver in rats pretreated with clofibric acid.

Identification of Tol Metabolites in Rat Bile. Identification of Tol-SG, resulting from transacylation of Tol-CoA or Tol-O-G, was performed by LC-MS/MS ([M + H]⁺ at m/z 547), which resulted in the characteristic loss of m/z 129 (pyroglutamyl moiety – H) observed at m/z 418 (see Fig. 4). Other fragment ions were produced at m/z 529 ([M + H – H₂O]⁺), m/z 472 ([M + H – glycine]⁺), m/z 411 ([M + H – glycine – COOH – NH₂]⁺), m/z 400 ([M + H – pyroglutamic acid]⁺), m/z 382 ([M + H – pyroglutamic acid – H₂O]⁺), m/z 343 ([Tol-S-CH₂(CO)CHNH₂]⁺), and m/z 240 ([Tol + H – H₂O]⁺). This fragmentation pattern and the retention time were identical to those observed for a synthetic standard. Tol-SG was not quantified in bile, but based on the UV chromatogram at 320 nm (Fig. 4), which is the UV absorption maximum of Tol, Tol-SG (t_R at 13.8 min) was a minor metabolite in bile compared with Tol-O-G (t_R at 15.6 min). In addition, the presence of Tol-SG in rat liver homogenates was investigated, but the metabolite could not be detected. The only acyl-CoA-dependent metabolite that was detected in bile was Tol-Tau (t_R 19.1 min), which was formed at relatively low levels as assessed from the UV chromatogram at 320 nm. Other metabolites that were identified in rat bile were Tol-OH (t_R at 12.1 min) and Tol-COOH (t_R at 13.1 min) for which oxidation occurred on the methyl group on the phenyl-ring, and the sulfate conjugate of Tol-OH (t_R at 16.2 min) as described previously (Olsen et al., 2003a).

View larger version (20K): [in this window] [in a new window]   FIG. 5. Scheme showing the metabolic activation of tolmetin to reactive metabolites.

	Discussion

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It has recently been reported that the pyrrole moiety of Tol undergoes bioactivation to a reactive arene oxide (Chen et al., 2006), which may also contribute to covalent binding to liver proteins. However, the present study focused on reactive phase 2 metabolites, capable of trans-acylating proteins. Covalent binding was determined as the release of Tol by alkaline hydrolysis of Tol-protein adducts, and therefore, the metabolism studies focused on addressing metabolic pathways involved in the formation of these protein adducts.

Three novel Tol metabolites, Tol-CoA, Tol-Car and Tol-SG, were identified in this study. acyl-CoA-dependent metabolism of Tol is important to identify, because studies have shown that Tol-CoA reacts spontaneously with the thiol of glutathione, indicating that trans-acylating reactions may also occur in liver cells in vivo (Olsen et al., 2003a; Sidenius et al., 2004). Tol-Car is formed via the reactive Tol-CoA intermediate by carnitine acyl transferases, and its formation is therefore of mechanistic significance showing the formation of the corresponding reactive acyl-CoA. Although Tol-SG could not be detected in rat liver homogenates and appeared to be a quantitatively minor metabolite in bile, it was also important to identify Tol-SG because the formation of this metabolite indicates that acylating Tol metabolites, e.g., Tol-CoA or Tol-O-G, capable of trans-acylating glutathione to form acyl-S-Tol glutathione conjugate were formed in the liver. It is, however, also interesting to note that Tol-SG is a thioester and, therefore, Tol-SG is also a reactive metabolite that is probably as reactive as Tol-CoA, i.e., significantly more reactive than Tol-O-G. A summary of the pathways leading to metabolic activation of Tol is shown in Fig. 5.

The levels of Tol metabolites were investigated in liver homogenates in an in vivo study. These results were in accordance with observations from a study on zomepirac in which the acyl-glucuronide was identified as the major metabolite and the acyl-CoA and the amino acid conjugates were formed at lower levels (Olsen et al., 2005). In the current study, a group of rats was pretreated with clofibric acid to induce acyl-CoA formation. As shown for palmitic acid (Knights et al., 1991) and ibuprofen (Shirley et al., 1994), such pretreatment leads to an increased formation of acyl-CoA, which was also observed for Tol in this study. Pretreatment with clofibric acid led to an increase in Tol-CoA from 0.6 nmol/g liver to 4.4 nmol/g liver (see Table 1), but had no apparent effect on the concentration of Tol-O-G in liver homogenates compared with control rats. This is in accordance with reports showing that clofibrate induces UGT1A1 and 1A5, but not the isoforms UGT2B1 (in rat) and 2B7 (in human), which generally catalyze the formation of acyl glucuronides of nonsteroidal anti-inflammatory drugs (Ritter, 2000). In general, covalent binding may have been underestimated for both pretreated animals and control rats since imine-protein adducts are chemically unstable and may have been lost during sample processing. However, this error would be approximately the same for both groups of animals, since Tol-O-G levels were similar. In addition, mainly one isomer, presumably the 1-O-isomer, was detected in the liver homogenates, which were treated with acid to avoid isomerization. Therefore, as the covalent binding to proteins increased upon pretreatment of rats with clofibric acid, the levels of Tol-CoA appeared to correlate with the amount of covalent binding of Tol to liver proteins, which supports the hypothesis that the reactive Tol-CoA reacted with hepatic proteins and thus contributed to covalent modification of liver proteins in vivo.

As seen for Tol-CoA formation and covalent binding, clofibric acid pretreatment also led to an increase in Tol-Car. This increase of Tol-Car is in accordance with the reported induction of carnitine acyl transferases (Katsutani et al., 2000) and increases in free carnitine and CoA levels after pretreatment with fibrates (Gregus et al., 1998). Acyl carnitine esters are rarely observed metabolites of xenobiotic carboxylic acids, and to our knowledge, the only xenobiotic carnitine esters that have been previously identified in vivo are the pivaloyl (Vickers et al., 1985; Totsuka et al., 1992), valproyl (Melegh et al., 1990), and zomepirac carnitine esters (Olsen et al., 2005).

As seen in Table 1, conjugation of tolmetin with glycine and taurine appeared to be unaffected by clofibric acid pretreatment, which is consistent with a report on taurine conjugation of ibuprofen in hepatocytes (Shirley et al., 1994). In general, amino acid conjugation is a high-affinity/low-capacity metabolic pathway; i.e., xenobiotic carboxylic acids tend to quantitatively form amino acid conjugates at low doses, but as the dose increases, other metabolic pathways, e.g., glucuronidation, become more important (Hutt, 1990).

In conclusion, Tol-CoA and Tol-Car were identified in this study. The presence of Tol-CoA shows that alternative pathways to glucuronidation may lead to formation of other reactive carboxylic acid metabolites capable of reacting with protein nucleophiles or trans-acylating GSH to form Tol-SG. Mechanistically, identification of Tol-Car is important, since this metabolic route is Tol-CoA dependent.

	Acknowledgments

 
We thank Gunnar Grönberg (AstraZeneca, Mölndal, Sweden) for obtaining NMR spectra of Tol-Car.

	Footnotes

 
This work was supported in part by National Institutes of Health Grant GM 36633, The Lundbeck Foundation, and grants from The Danish Technical Research Council (56-01-0014) and The Danish Medical Research Council (22-02-340).

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.106.013334.

ABBREVIATIONS: Tol, tolmetin; Tol-O-G, tolmetin acyl glucuronide; Tol-CoA, tolmetin acyl CoA thioester; Tol-Gly, tolmetin-glycine conjugate; Tol-Tau, tolmetin-taurine conjugate; ESI, electrospray ionization; Tol-Car, tolmetin acyl carnitine ester; Tol-SG, tolmetin acyl-S-glutathione conjugate; Tol-OH, tolmetin hydroxy metabolite; Tol-COOH, tolmetin carboxyl metabolite; HPLC, high-performance liquid chromatography; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry.

¹ Current affiliation: Department of Drug Metabolism, Merck Research Labs, West Point, Pennsylvania.

Address correspondence to: Dr. Jørgen Olsen, Novo Nordisk A/S, Exploratory ADME, Diabetes Pharmacology, DK-2760 Måløv, Denmark. E-mail: jqgo{at}novonordisk.com

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