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In Vitro Nimesulide Studies toward Understanding Idiosyncratic Hepatotoxicity: Diiminoquinone Formation and Conjugation Fengping Li, Mahendra D. Chordia, Tao Huang, and Timothy L. Macdonald* Chemistry Department, UniVersity of Virginia, McCormick Road, CharlottesVille, Virginia 22901 ReceiVed April 29, 2008
Nimesulide is a nonsteroidal anti-inflammatory drug (NSAID) marketed in more than 50 countries. This drug has caused rare and idiosyncratic but severe hepatotoxicity. The mechanisms associated with and factors responsible for this toxicity remain unknown. One of the nimesulide metabolites identified in human urine is 4-amino-2-phenoxy-methanesulfonanilide (M1). In the current study, we demonstrate that M1 is a stable metabolite that is highly susceptible to facile oxidation by cytochrome P450 enzymes (P450s) to form a reactive diiminoquinone intermediate (M2). Direct detection of M2 was difficult by LC-MS. However, its formation was confirmed indirectly by identification of N-acetyl-cysteine (NAC) adducts of M2. The formation of diiminoquinone M2 was P450 mediated with 2C19 and 1A2 as the two principal P450 enzymes catalyzing M1 oxidation. M1 metabolism irreversibly inhibited 2C19 but activated 1A2 in a time-dependent manner. P450 2C19 exclusively mediated further metabolism of M1 to the amino hydroxynimesulide M3 and its diiminoquinone M4. Similar to M2, M4 is also reactive and can be observed indirectly as its NAC adduct. Nucleophilic addition to diiminoquinone M2 occurs with low regioselectivity, yielding three adducts (the peak area ratio 1:0.08:12). The three regioisomers have the same m/z for [M + H]+, presumably due to nucleophilic addition at the three possible electrophilic sites (C-3, -5, and -6 positions of the sulfonaniline ring). The primary adduct, R, was derived from the attack of the nucleophile at the C-5 position of the sulfonaniline ring and was determined by MS/MS and 1H and 13C NMR analyses. The structural assignments were confirmed by chemical synthesis of the adduct R. M2 demonstrated its electrophilic reactivity by selectively alkylating human serum albumin (HSA) at the only free thiol, Cys-34. This suggests the possibility that other proteins may undergo a similar conjugation to form irreversible adducts. Under oxidizing conditions in the presence of cumene hydroperoxide (CHP), the formation of M2 was enhanced, indicating that oxidative stress may accelerate the production of reactive diiminoquinone species (M2 and M4). Introduction Nimesulide, a classical nonsteroidal anti-inflammatory drug (NSAID),1 is a cyclooxygenase-2 (COX-2) inhibitor (1-3). COX-2 is one of the two isoforms of the COX family of enzymes and has been shown to be stimulated by inflammatory responses. Nimesulide exhibits anti-inflammatory and analgesic effects by inhibiting the formation of prostaglandins through the inhibition of COX-2. Nimesulide was licensed by Helsinn Healthcare SA (Switzerland) in 1980 and first launched in Italy as a therapeutic agent in 1985. Since 1985, it has been marketed in more than 50 countries with the exception of the United States and a few other countries due to safety concerns. After widespread clinical use of nimesulide (2), hepatic toxicities, including both acute hepatitis and the more severe fulminant hepatic failure, were reported (2, 4-7). The relatively high occurrence of these adverse events (9.4 cases per million patients treated) in Finland and Spain caused nimesulide to be withdrawn from the market in those countries (8, 9). * To whom correspondence should be addressed. Tel: 434-924-7718. E-mail:
[email protected]. 1 Abbreviations: NSAID, nonsteroidal anti-inflammatory drug; CHP, cumene hydroperoxide; HSA, human serum albumin; GSH, glutathione; NAC, N-acetyl-cysteine; HLM, human liver microsome; IDR, idiosyncratic drug reaction; COX-2, cyclooxygenase-2; NAPQI, N-acetyl-p-benzoquinone imine; HRP, horseradish peroxidase; TCA, trichloroacetic acid; DTT, dithiothreitol; IAA, iodoacetamide; Cys-34, cysteine-34; ESI-MS, electrospray ionization-mass spectrometry; PBS, phosphate-buffered saline; Rt, retention time.
However, nimesulide remains on the market in a number of countries where the rate of nimesulide-induced injury is reported to be relatively low (about 1 per million patients treated). The hepatotoxicity of nimesulide is rare, relatively insensitive to accumulated dose, and specific to a patient and is thus considered to be an idiosyncratic toxicity (2, 9, 10). A number of human nimesulide urinary metabolites have been identified (Figure 1). These include hydroxynimesulide (A, ∼18%); amino des-nitro nimesulide (M1, ∼0.72%) in which the nitro group has been reduced; amino hydroxynimesulide (M3, ∼3%); and N-acetylated metabolites (B, < 0.5% and C, ∼19%) (11). The observed idiosyncratic toxicity cannot be explained directly from the known metabolism of nimesulide because most of the metabolites are stable and not electrophilic in nature. However, these known metabolites exhibit structural features that enable them to be oxidatively bioactivated to reactive electrophilic species that may be potentially capable of inducing a toxic response. Of particular interest to us is the amino des-nitro nimesulide metabolite, M1. The formation of a highly electrophilic iminoquinone has been reported previously for several drugs. For example, a well-known reactive intermediate of acetaminophen is N-acetyl-p-benzoquinone imine (NAPQI) (12, 13). Glutathione (GSH) depletion by conjugation with NAPQI has been demonstrated (14, 15). It is believed that NAPQI formation is responsible for the observed acetaminophen hepatotoxicity, which usually occurs at higher doses. Previous work in our laboratory showed that a reactive o-quinone or
10.1021/tx800152r CCC: $40.75 2009 American Chemical Society Published on Web 12/03/2008
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Figure 1. Known metabolism of nimesulide in human.
quinone-imine species produced from tolcapone can form a GSH adduct in a P450-dependent manner (16). It has been proposed that the formation of the quinone-imine species is responsible for tolcapone-related idiosyncratic hepatotoxicity. In the present investigation, we hypothesized that M1 is a stable metabolite that is highly susceptible to oxidation by P450 enzymes to form a reactive diiminoquinone intermediate (M2). Similar to the drugs mentioned above, M2 may be potentially responsible for the covalent modification of biomolecules, impairing their function and changing the redox state of the cell. The objective of this study was to identify and characterize a reactive diiminoquinone species and demonstrate its reactivity toward small nucleophiles, GSH/N-acetyl-cysteine (NAC), and a macromolecule, human serum albumin (HSA).
Experimental Procedures Chemicals and Instruments. Analytical grade nimesulide (>95%) was purchased from Toronto Research Chemicals, Inc. (Toronto, Canada) and was used without further purification. Human liver microsomes (HLMs) were purchased from BD Gentest Corp. (Woburn, MA). cDNA-expressed human P450 enzymes (1A1, 1A2, 3A4, 2C9, 2C19, 2D6, and 2E1) expressed from either baculovirusinfected insect cells or human lymphoblast cells were purchased from BD Gentest Corp. HSA and all other chemicals were purchased from Sigma-Aldrich and were of highest quality available. Reverse phase C18 preparative TLC plates (250 µm, 20 cm × 20 cm, w/UV 254) were obtained from Sorbent Technology (Atlanta, GA). HPLC-MS and HPLC-MS/MS were performed with a Shimadzu CBM-20A HPLC (Columbia, MD) interfaced to a Thermo-Finnigan LTQ ion trap mass spectrometer (San Jose, CA) equipped with an electrospray ionization (ESI) source. Data processing was achieved with the Xcalibur version 1.4 software. 1 H NMR data were collected on a Varian Unity 300 MHz spectrometer. Chemical shifts were expressed in ppm, and coupling constants were expressed in Hz.
Chem. Res. Toxicol., Vol. 22, No. 1, 2009 73 Synthesis of 4-Amino-2-phenoxy-methanesulfonanilide (M1). Nimesulide (2.96 g, 10.0 mmol) was dissolved in 20 mL of methanol. Activated Pd/C (5% w/w, 200 mg) was added, and the solution was degassed by repeated cycles (×3) of vacuum and refilling with hydrogen gas. The mixture was finally stirred under hydrogen atmosphere (1 atm) using a balloon at room temperature overnight. The catalyst was removed by filtration through double Whatman qualitative filter paper washed with ethyl acetate. The combined filtrate was concentrated under reduced pressure to yield a yellow solid (2.50 g, 94% yield). The sample was homogeneous on TLC and was sufficiently pure by spectral (LC-MS and NMR) data. LC-MS analysis: retention time (Rt) 12.6 min (using the same HPLC conditions as those in M1 bioactivation studies), m/z 279 for MH+. 1H NMR (CD3OD): 2.88 (s, 3H, SO2CH3), 6.18 (d, 1H, J ) 2, C-3 ArH), 6.41 (dd, J ) 2, 6 Hz, 1H C-5 ArH), 7.05 (d, 2H, J ) 6 Hz, O-ArH), 7.13 (m, 2H, O-ArH and C-6 ArH), 7.37 (dd, J ) 6, 9 Hz, 2H, O-ArH). Synthesis of M2-NAC Adduct. M1 (53.2 mg, 0.2 mmol) and NAC (163.0 mg, 10.0 mmol) were dissolved in CH2Cl2 and DMF (2:1, 3 mL). Lead tetraacetate (LTA, 90.0 mg, 0.203 mmol) (17) was added to the homogeneous solution at room temperature. The color of the mixture immediately turned dark red-orange and then light yellow upon stirring for 3 h at room temperature. The mixture was filtered through filter paper and washed with EtOAc. The combined filtrate was washed copiously with water and finally with brine. The organic layer was concentrated under reduced pressure to yield a thick oily residue. The residue was then dissolved in methanol and further purified by preparative reverse phase C18 TLC using water followed by 50% methanol in water (Rt 0.4). The band of silica gel for the M2-NAC adduct (observable under UV) was collected and eluted with methanol. Evaporation of methanol under reduced pressure afforded an off white solid (30.6 mg, 35.6%). LC-MS analysis: Rt 16.06 min for the major adduct R (using the same HPLC conditions as those in M1 bioactivation studies), m/z 440 (M + H+). 1H NMR (CD3OD): 1.95 (s, 3H, COCH3), 2.94 (s, 3H, SO2CH3), 3.00 (dd, J ) 6, 12 Hz, 1H, CH2-S), 3.22 (dd, J ) 3, 12 Hz, 1H, CH2-S), 4.47 (dd, J ) 3, 6 Hz, 1H, CH-N), 6.21 (s, 1H, C-3 ArH), 7.09 (dd, 2H, J ) 3, 6 Hz, O-ArH), 7.17 (t, 1H, O-ArH), 7.38 (dd, J ) 6, 9 Hz, 2H, O-ArH), 7.44 (s, 1H, C-6 ArH). 13 C NMR (CD3OD): 22.4, 37.2, 40.0, 53.8, 104.0, 111.4, 117.6, 120.4, 121.0, 125.4, 138.0, 151.3, 157.1, 157.7, 173.2, 173.5. Bioactivation of M1/Nimesulide. The microsomal incubations containing 2 mg/mL HLM, 100 µM M1 or nimesulide, 1 mM NADPH, 5 mM MgCl2, and 5 mM NAC or GSH in 10 mM phosphate-buffered saline (PBS) were performed in a dry air bath at 37 °C for 2-18 h. After incubation, the solution was diluted three times with PBS and quenched with 2% trichloroacetic acid (TCA). The reactions were centrifuged at 14000 rpm for 10 min at room temperature. The supernatant was directly injected onto the HPLC-MS for analysis. Negative controls were treated similarly without either HLM, NADPH, or GSH/NAC. A Phenomenex Luna C18 reversed phase column (2.0 mm × 150 mm) was used to separate samples with a flow rate of 0.2 mL/min. The separation was achieved with methanol (solvent A) and 0.1% formic acid (solvent B). The gradient conditions were as follows: 98% solvent B decreasing to 0% B for 20 min, holding at 0% B for 5 more min, and re-equilibrating at 98% B. These HPLC conditions were used throughout with the exception of the HSA studies. ESI-MS parameters were as follows: heated capillary temperature, 200 °C; spray voltage, 5 kV; capillary voltage, 43 V; sheath gas flow, 25 units; and auxiliary gas flow, 3 units. Horseradish peroxidase (HRP) incubations were performed in 10 mM PBS with 5 units/mL HRP, 100 µM M1, 5 mM GSH or NAC, and 1 mM H2O2 in a dry air bath at 37 °C for 5 min. The incubation was initiated by the addition of H2O2. After 5 min, the reaction was quenched with the addition of 5 mg/mL catalase and 1.2% TCA. The supernatant obtained from the centrifugation step was collected and analyzed by LC-MS. Negative controls were treated similarly without either HRP, GSH/NAC, or H2O2. Additional incubations with P450 1A1, 1A2, 3A4, 2C9, 2C19, 2D6, and 2E1 were also performed. Incubation conditions were
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Figure 2. (A) LC-MS chromatogram of M2-NAC adducts with [M + H]+ extracted for m/z 440 in HLM incubations. (B) MS spectrum of the major M2-NAC adduct R. Adducts P and Q had the same MS spectrum. (C) Expanded LC-MS chromatogram of M2-NAC adducts. (D) MS/MS spectrum of the major M2-NAC adduct R. The three peaks (P, Q, and R) had the same fragment ion with m/z 361.
the same as HLM incubations except that the concentrations of P450 enzymes were 0.1 µM. P450 3A4 was the lymphoblastexpressed enzyme. All other P450s were baculovirus-expressed enzymes. Effect of Cumene Hydroperoxide (CHP) on M1 Bioactivation. The incubation conditions were set the same as in HLM incubation except that 1 mM CHP was used in place of 1 mM NADPH. Two control experiments were performed, HLM with NADPH as the cofactor and only CHP without HLM or NADPH. The amount of the major M2-NAC adduct R generated was measured. P450 Inhibition Studies. P450 inhibition studies were conducted using known inhibitors in HLM. The effects of inhibitors on the formation of M2-NAC/GSH adduct were monitored by LC-MS using the following concentrations: 100 µM furafylline (P450 1A2), 100 µM ticlopidine (P450 2C19), and 100 µM quinidine (P450 2D6). The stock solutions of the inhibitors were prepared by dissolving them in acetonitrile. The inhibitors were then incubated individually or together with HLM and no GSH/NAC for 2 h. Then, 100 µM M1 and 5 mM NAC were added to the reaction mixture, and the solution was incubated for an additional 2 h. The measurement of the M2-NAC adduct R was the same as described for the HLM incubation. Time-Dependent Inactivation of P450 2C19 and Activation of P450 1A2 by M1. Two types of solutions were prepared (18). Solution E included 0.5 µM P450 2C19 or 1A2, 1 mM NADPH, and 100 µM M1 in 10 mM PBS. Solution F included 1 mM NADPH, 200 µM (s)-mephenytoin (a substrate of 2C19), or phenacetin (a substrate of 1A2) in 10 mM PBS. Solution E was first incubated at 37 °C for different time points, and then, solution F was added. Finally, the mixtures were incubated further for 1 h. The incubation was quenched by the addition of 2% TCA. After centrifugation, an internal standard was added to the supernatant. The formation of O-deethylated phenacetin for P450 1A2 or 4′hydroxylated (s)-mephenytoin for P450 2C19 was monitored by LC-MS (19). Effect of Dialysis on the Time-Dependent Inactivation of P450 2C19. For P450 2C19 and 1A2 incubations, solution E was prepared as described above. The control experiment did not include M1. After 30 min of incubation at 37 °C, the reaction mixture was dialyzed against 10 mM PBS using a 0.1-0.5 mL 10 kDa molecular mass cutoff Slide-A-Lyzer dialysis cassette (Pierce, Rockford, IL) at 4 °C overnight. After the dialysis, the remaining solution E in the dialysis cassette was mixed with solution F. Quantitation of
O-deethylphenacetin or hydroxyl (s)-mephenytoin was performed as described above. Alkylation of HSA and Tryptic Digestion. M1 (100 µM) was incubated with HSA (16 mg/mL) and HRP (5 units/mL) in 500 µL of 10 mM PBS solution (pH 7.4) at 37 °C. The control experiment did not contain M1. The reaction was initiated by the addition of 1 mM H2O2. After 5 min, low molecular weight species were removed by centrifugation through a Microcon YM-10 filter. The remaining protein was washed thoroughly (200 µL × 3) with 10 mM Tris-HCl buffer and centrifuged to remove excess M1. HSA was reconstituted in 500 µL of PBS buffer with 6 M guanidineHCl and 20 mM dithiothreitol (DTT) and incubated at 55 °C for 1 h. Upon cooling to room temperature, excess iodoacetamide (IAA, 100 mM) in 0.25 M Tris-HCl buffer was added, and the solution was incubated in the dark at room temperature for 30 min. The protein was then desalted using a PD-10 column (GE Healthcare) and detected with the Biorad assay. Ammonium bicarbonate (w/w 0.4%) was used for column equilibration and protein elution. Fractions containing the protein were collected (total volume, ∼2 mL). The desalted HSA (700 µL) obtained above was digested by trypsin (20 µg, Promega, w/w 50:1) overnight at 37 °C. The digestion was quenched by the addition of 20% acetic acid. Before MS analysis, samples were diluted (1:3) with 5% acetic acid, and 15 µL of the diluted solution was injected onto a Phenomenex Jupiter C18 reversed phase column (2.0 mm × 150 mm). HPLC solvents were the same as those used in the M1 bioactivation studies. The HPLC flow rate was 0.2 mL/min. The gradient conditions were as follows: 98% solvent B decreasing to 80% B for 40 min, to 40% B for 40 min, and to 0% B for 15 more min. LC-MS and MS/MS Analysis of HSA Peptides. The detailed procedure for the LC-MS analysis of peptides was described previously (20). Full parameters for ESI-MS were set as follows: heated capillary temperature, 200 °C; spray voltage, 5 kV; capillary voltage, 25 V; sheath gas flow, 25 units; and auxiliary gas flow, 3 units. In the MS/MS experiments, the parent ion was set at m/z 904 for the M2 alkylated peptide. The collision energy was set at 30% with an isolation width of 2 Da.
Results Bioactivation of M1/Nimesulide. Nimesulide incubation with HLM exhibited a similar pattern of metabolism as observed in
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Figure 3. (A) Comparison of 1H NMR spectra of M1 and M2-NAC adduct R and (B) expanded 1H NMR spectra of the aromatic regions of M1 and R.
Figure 4. Formation of NAC adduct R under different conditions. The side panel is the expanded y-axis version of the formation of R with only CHP or HLM with NADPH incubation.
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Figure 5. (A) Oxidation of M1 by P450 enzymes. The adduct R (∼1.8 µM) was produced by P450 2C19 from 100 µM M1 and (B) the effect of P450 inhibitors on the formation of the adduct R in HLM.
Figure 6. Inhibition of P450 2C19 activity after preincubation with M1. The control did not contain M1. The activity of P450 2C19 was measured by hydroxylation of (s)-mephenytoin.
Figure 7. Activation of P450 1A2 activity after preincubation with M1. The control did not contain M1. The activity of P450 1A2 was measured by deethylation of phenacetin.
human urine (11). Two major reported metabolites, M1 and hydroxynimesulide A, were monitored (Figure 1). Incubating M1 with HLM revealed its hepatic metabolism pattern. M1 was consumed faster with HLM than in the control experiment (no HLM). However, the formation of a comparable amount of new species was not observed by the LC-MS method. We presumed that the reactive diiminoquinone species, if formed, might not survive under the experimental conditions. All attempts to detect
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such reactive species without trapping agents failed. When trapping agents such as GSH and NAC were included in the incubations, the indirect detection of diiminoquinone was observed. Along with the reactive diiminoquinone, other minor metabolites were detected, such as reduced hydroxynimesulide (M3). LC-MS analysis of the M2-NAC adduct (Figure 2A) showed three peaks (1:0.08:12) from the [M + H]+ ions with m/z 440 at Rt around 16 min. The MS/MS analysis (Figure 2B) showed that these peaks had similar fragmentation patterns. The base ion from all three adducts (m/z 361) was generated from the methanesulfoxide cleavage. Similar to the NAC adducts, three GSH adducts (1:0.05:9) for the [M + H]+ ions, with m/z 584, were also observed at Rt around 13.9 min under the same HPLC conditions. The typical fragments expected from GSH conjugation such as the loss of γ-glutamine [(M - 129) + H]+ (m/z 455) and glycine [(M - 75) + H]+ (m/z 509) were confirmed by the MS/MS analysis (shown in the Supporting Information). HRP provides an alternative to hepatic P450-mediated oxidations, in which two sequential single electron transfer oxidation occurs (21). HRP rapidly oxidized M1 to diiminoquinone M2 (10 min with ∼95% of M1 conversion as compared to 12 h with ∼5% conversion for HLM). Three GSH/NAC adducts were observed similar to those in the HLM incubations. 1 H NMR of M2-NAC Adduct. The major M2-NAC adduct R was further characterized by 1H NMR analysis. The 1H NMR spectrum for the chemically synthesized R is shown in Figure 3. Characteristic peaks for the NAC component “a”, “b”, and “c” protons were identified at 1.95 (s, COCH3), 3.00 (dd, CH2S), 3.22 (dd, CH2-S), and 4.47 (dd, CH-N) ppm, respectively (Figure 3). In addition, the aromatic region for R has important differences when compared to M1. The disappearance of the doublet at 6.4 ppm for C-5 H and the appearance of two new singlets at 6.21 and 7.44 ppm confirmed the addition of NAC to the C-5 position. Effect of CHP on the Formation of the NAC Adduct R. It is reported that NADPH reduces NAPQI to acetaminophen (12). To evaluate the effect of oxidation conditions on the formation of diiminoquinone M2, CHP was used in the HLM incubation. When M1 was incubated with HLM and CHP, the rapid formation (∼1 h) of the NAC adduct R was observed and was saturated thereafter (∼100%) (Figure 4). More than 95% of M1 was consumed after 4 h of incubation. It should be noted that in the negative controls (HLMs with NADPH and only CHP incubations), a very slow and steady increase of the adduct R was observed over a 4 h incubation period. The maximum adduct R formed was ∼2% (Figure 4), and less than 5% of M1 was consumed after 4 h of incubation. Incubations with Human cDNA-Expressed P450 Enzymes. To determine which P450 enzymes preferentially oxidize M1 to M2, cDNA-expressed human P450 enzymes (1A1, 1A2, 3A4, 2C9, 2C19, 2D6, and 2E1) were incubated with M1, and the formation of the adduct R was monitored by LC-MS. Each P450 enzyme tested had some capability of bioactivating M1 (Figure 5A). However, P450 2C19 and 1A2 were the two major enzymes exhibiting the high efficiency. For P450 2C19, a ∼1.8 µM concentration of the adduct R was generated. Furafylline (P450 1A2 inhibitor), ticlopidine (P450 2C19 inhibitor), and quinidine (P450 2D6 inhibitor) (19) were used in the inhibition studies to monitor the formation of the adduct R in HLM. As illustrated in Figure 5B, the percentage of R formed in HLM was inhibited up to 50% by furafylline, 60% by ticlopidine, and 25% by quinidine as compared to the control
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Figure 8. LC-MS chromatogram of trypsin digested HSA peptide with [M + 3H] 3+ extracted for m/z 904 from (A) M1, HRP, and HSA incubation; (B) control without M1; and (C) MS/MS spectrum of the M2-HSA peptide.
(no inhibitor). Furafylline and ticlopidine together inhibited the formation of R up to 70%. The data confirmed that 2C19 and 1A2 were the two key P450 enzymes oxidizing M1. Time-Dependent Inactivation of P450 2C19 and Activation of P450 1A2 by M1. When P450 2C19 was preincubated with M1, and its oxidizing capability to metabolize (s)-mephenytoin was decreased as compared to the control (without M1). The maximum inactivation of P450 2C19 (∼ 36%) was achieved at the 30 min time point (Figure 6). A dialysis experiment (data not shown) concluded that the observed inhibition was irreversible. The oxidation ability of P450 1A2 to metabolize phenacetin appeared to be increased after its preincubation with M1. The activity increase of P450 1A2 was about 25% at the 30 min time point (Figure 7). A dialysis experiment suggested that the observed activation was irreversible. Alkylation of HSA by M2. HRP can also oxidize M1 to M2 as shown above. HRP was used to study the conjugation
of HSA by M2 because of its high oxidizing efficiency. The oxidation of M1 to M2 by HRP in the presence of HSA afforded modified HSA protein. After trypsin digestion, HSA peptides were analyzed by LC-MS. A new peak with m/z 904 was identified at 87.4 min by manually searching peaks in the LCMS chromatogram (Figure 8A) when compared to the control experiment without M1 (Figure 8B). The amino acid sequence for this peptide was assigned as Ala-21 to Lys-41 (ALVLIAFAQYLQQCPFEDHVK). The M2 conjugated peptide was analyzed using LC-MS/MS. Relevant b and y ions were identified (Figure 8C). In the b series, fragments b3+ to b8+ and b12+ and b13+ were from the original peptide. In the y series, natural y4+ was observed. However, fragments y82+ to y192+ all had mass increases of 276 Da from M2 addition. Among the four amino acids (Cys, Pro, Phe, and Glu), cysteine-34 (Cys-34) with a free sulfhydryl group was the most nucleophilic amino acid. Therefore, it was concluded that Cys-34 was alkylated by M2.
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Figure 9. LC-MS chromatogram of M3 with [M + H]+ extracted for m/z 295 from (A) M1 and P450 2C19, (B) M1 and P450 1A2, (C) nimesulide and HLMs, and (D) MS/MS spectrum of M3.
Figure 10. (A) LC-MS chromatogram of M4-NAC adduct T with [M + H]+ extracted for m/z 456 from P450 2C19 incubation and (B) MS/MS spectrum of M4-NAC adduct T.
Identification of Hydroxylated Metabolite M3. The metabolite observed from the nimesulide-HLM experiment (Rt 10.0 min) (Figure 9C) was found to be the same as the one from the metabolism of M1 with P450 2C19 (Figure 9A). This was characterized as a hydroxylated phenoxy ring (M1 + 16) metabolite with m/z 295 for a [M + H]+ ion. M3 is a reported metabolite, and its structure has been assigned. Interestingly, P450 1A2 did not follow this pathway of oxidation for M1 (Figure 9B). The LC-MS/MS spectrum of M3 acquired in the current investigation (Figure 9D) exhibited a typical loss of m/z 79 corresponding to the methanesulfoxide group. Additionally, it exhibited a weak fragment ion at m/z 187, indicating the further loss of 108 Da from a para-hydroxyphenoxy group. This confirms that M3 was oxidized at the phenoxy ring. Such hydroxylation at the para-position of a phenoxy ring is wellknown to be P450 2C19 transformation (19). M3 was prone to oxidation to form M4. Similar to reactive M2, M4 cannot be directly observed and only its NAC adduct was detected (Rt of 15.1 min) with an m/z 456 of a [M + H]+ ion (Figure 10A). The MS/MS spectrum showed a strong fragment with m/z 377 generated from the cleavage of methanesulfoxide (Figure 10B).
The formation of diiminoquinones as NAC adducts accounts for ∼0.05% of the overall nimesulide metabolism by HLM.
Discussion The amino des-nitro nimesulide metabolite, M1, is detected in human urine up to ∼0.75% of the prescribed dose (11). M1 was also detected in the current studies in the incubations with nimesulide and HLM. The principal objective of the investigation was to determine if M1 is metabolically activated to a reactive species and to elucidate the reactivity of the bioactivated species to determine if this species could rationalize the hepatotoxicity associated with nimesulide. The data obtained from the LC-MS/MS and NMR studies illustrated that the oxidation of the sulfonanilide ring to the reactive diiminoquinone intermediate is indeed a feasible metabolic pathway leading to the formation of adducts with NAC/GSH (Figure 2) and with HSA (Figure 8). The diiminoquinone species M2 was difficult to observe directly under a variety of enzymatic incubation conditions in the absence of nucleophilic trapping agents such as GSH or
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Figure 11. Detailed pathway of M1 metabolism to reactive diiminoquinone intermediates and their major adducts.
NAC. A few possibilities were considered as follows: (i) M2 alkylates P450, (ii) M2 reacts with M1 to form a dimer, (iii) M2 reacts with surrounding medium such as water, and, finally, (iv) M2 is reduced back to M1 by NADPH. In scenario i, the amount of P450s used for incubation was miniscule (catalytic) as compared to M1; hence, the extra M2 produced after reacting with P450s should be detectable. Scenario iii is ruled out since the desired water addition product was not observed in LC-MS analysis. Scenario ii was confirmed because LC-MS analysis provided ample evidence that the dimer of M1 + M2 (m/z 555) was formed along with the depletion of M1 with time. Considering scenario iv, it is reported that NAPQI can be detected directly from the acetaminophen incubation with HLMs and CHP instead of NADPH (12). In this paper, evidence is provided for the reduction of NAPQI by NADPH. Thus, it is likely that part of M2 is reduced by NADPH to M1 as soon as it is formed. Figure 4 shows that HLM catalyzed the oxidation of M1. Under enhanced oxidizing conditions (HLM + CHP), the formation of M2 (detected as its major adduct R) was faster (>95% after 1 h; > 95% M1 consumed) than under other conditions (i, HLM + NADPH; ii, CHP) (
