Introduction

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Indomethacin is a very effective nonsteroidal anti-inflammatory drug, but its use is limited by a high incidence of adverse reactions, including blood disorders. Of a total of 1261 cases of adverse reactions to indomethacin that were reported to the United Kingdom Committee on Safety of Medicines between June 1964 and January 1973, blood disorders were recorded in 157 cases (with 25 fatalities), including thrombocytopenia (35 cases, with 5 fatalities), aplastic anemia (17 cases, with no fatalities), and agranulocytosis or leukopenia (21 cases, with 3 fatalities) (Cuthbert, 1974). Subsequently, the First Report from the International Agranulocytosis and Aplastic Anemia Study confirmed a significant relationship between the use of indomethacin and agranulocytosis and aplastic anemia. In this population-based case-control study conducted in Europe and Israel, the excess risk estimated for agranulocytosis and aplastic anemia was 0.6/10⁶ for indomethacin exposure in a 1-week period (Shapiro, 1986). The mechanism of drug-induced agranulocytosis is unknown; however, it is speculated that reactive metabolites produced by activated neutrophils or neutrophil precursors may be responsible for agranulocytosis, either by direct toxicity or by an immune mechanism. It has been shown that several drugs that are associated with agranulocytosis are metabolized to reactive intermediates by activated neutrophils and monocytes (Uetrecht, 1990, 1992, 1996). When activated, neutrophils release MPO¹ and generate H₂O₂ (Klebanoff, 1968; Weiss, 1989). MPO is the major oxidizing enzyme in neutrophils; HOCl is generated by the MPO system, which uses H₂O₂ to oxidize Cl to HOCl.

A study by Duggan et al. (1972) demonstrated that the dominant pathways of indomethacin metabolism in humans are demethylation and deacylation. The major metabolites are DMI and DMBI (fig. 1). The objectives of this work were to determine whether indomethacin and/or its major metabolites can be metabolized to reactive intermediates by activated neutrophils, HOCl, and the MPO system and, if so, to identify the proposed reactive metabolite and to characterize its chemical reactivity.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Major metabolic pathway of indomethacin.

	Materials and Methods

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Synthesis of DMI. DMI was synthesized by demethylation of indomethacin using a modification of the method of McOmie et al. (McOmie et al., 1968). Indomethacin (0.7 g; Sigma Chemical Co., St. Louis, MO) was dissolved in 200 ml of dichloromethane. To this solution, 10 ml of 1 M BBr₃ was added dropwise. The reaction was protected from moisture by bubbling N₂ through the reaction mixture. The reaction was carried out at room temperature for 3 hr. The reaction mixture was then shaken with water to hydrolyze excess reagent and boron complexes. DMI was obtained by extraction into ether. The mass spectrum of the synthesized DMI included a MH⁺ ion at m/z 344. The synthesized DMI demonstrated the same HPLC retention time as did an authentic sample provided by Merck Research Laboratories, and it was used to synthesize DMBI without further purification.

Synthesis of DMBI. DMI (15 mg) was hydrolyzed in 3 ml of 1 N sodium hydroxide. The reaction was carried out at room temperature for 5 min. The reaction mixture was made acidic with concentrated hydrochloric acid and then extracted with ethyl acetate. The ethyl acetate layer was dried with magnesium sulfate, and the solvent was evaporated under a stream of N₂. The product was then purified by silica gel TLC, using a solvent of dichloromethane/methanol (9:1, v/v). The yield was approximately 10% [m.p. = 151-152°C; R_F = 0.35; MS: m/z 206 (MH⁺) and 160 [(MHHCOOH)⁺]; ¹H NMR (dimethylsulfoxide-d₆):  10.64 ppm (1H, s);  8.50 ppm (1H, bs); H-7,  7.00 ppm (1H, d, J = 8.54 Hz); H-4,  6.72 ppm (1H, d, J = 2.20 Hz); H-6,  6.48 ppm (1H, dd, J = 8.30 and 2.20 Hz); methylene protons,  3.42 ppm (2H, s); methyl protons on the indole ring,  2.28 ppm (3H, s)].

Oxidation of DMBI by HOCl. Sodium hypochlorite (various concentrations) was added to DMBI (final concentration, 0.1 mM) in 100 µl of 0.1 M phosphate buffer at room temperature. The pH of the phosphate buffer was varied from 4.0 to 7.0. Aliquots (20 µl) of the solution were analyzed by HPLC.

Oxidation of Indomethacin and DMI by HOCl. Sodium hypochlorite (50 µl of a 4 mM solution) was added to indomethacin or DMI (final concentration, 1 mM) in 150 µl of 0.1 M phosphate buffer (pH 6) at room temperature. Aliquots (20 µl) of the solution were analyzed by HPLC.

Oxidation of DMBI by the MPO System. DMBI (5 µl of a 10 mM methanolic solution) was added to 50 µl of phosphate buffer (0.1 M, pH 6 or 7) to yield a final concentration of 0.1 mM. MPO (2.5 units/ml) was added, and then the reaction was initiated by the addition of hydrogen peroxide (final concentration, 0.4 mM). Incubations were carried out in the absence or in the presence of added chloride. Aliquots (20 µl) of the solution were analyzed by HPLC.

Oxidation of DMBI, Indomethacin, and DMI by Activated Neutrophils. Blood was drawn from normal volunteers, into heparinized syringes, and the neutrophils were isolated by differential centrifugation on Ficoll-paque (Pharmacia, Uppsala, Sweden) according to a standard procedure (Boyum, 1984). Neutrophils (1 × 10⁶ cells/ml) were suspended in phosphate-buffered saline (pH 7.4, 0.5 ml). Phorbol myristate acetate (40 ng/ml; Sigma) was added, followed by DMBI (5 µl of a 10 mM methanolic solution). The suspension was then incubated at 37°C in a shaking water bath. After incubation for various periods, the cells were centrifuged and 20 µl of the supernatant was analyzed by HPLC. Indomethacin and DMI were also incubated with activated neutrophils under the same conditions, except that their concentrations were twice that of DMBI.

Synthesis of the Reactive Intermediate-NAC Adduct. Sodium hypochlorite (1 ml of a 100 mM aqueous solution) was added to DMBI (50 ml of a 2 mM aqueous solution). The reaction mixture was vortex-mixed for 10 sec, and then NAC (0.5 ml of a 200 mM aqueous solution) was added. The mixture was then concentrated on a rotary evaporator. The NAC adduct was purified by HPLC using a 10- × 150-mm preparative column packed with 3-µm Spherisorb ODS 2 material. The solvent consisted of water, acetonitrile, acetic acid, and triethylamine (89:10:1:0.05, v/v). The flow rate was 3 ml/min, and the retention time of the NAC adduct was 9.4 min.

Reaction Kinetics Determined by Spectrophotometry. A Hewlett-Packard diode-array spectrophotometer (HP8452A; Hewlett-Packard, Palo Alto, CA) was used to determine the rate of oxidation of DMBI by HOCl. Scanning of the reaction mixture was initiated immediately after the addition of sodium hypochlorite (10 µl of a 10 mM aqueous solution) to DMBI (2 ml of a 50 µM aqueous solution), with rapid stirring. The reaction was monitored at 1.0-sec intervals for 15 sec, over a wavelength range of 200-500 nm.

Analytical Methods. HPLC was performed using a 2- × 100-mm Phenomenex column (Phenomenex, Torrance, CA) packed with 5-µm Ultracarb ODS 30 material. The solvent used to analyze the products of DMBI oxidation by HOCl, MPO, and activated neutrophils consisted of water, acetonitrile, and acetic acid (84:15:1, v/v) containing 1 mM ammonium acetate. The solvents used to analyze the products of indomethacin and DMI oxidation by HOCl and activated neutrophils consisted of water, acetonitrile, and acetic acid (49:50:1 and 59:40:1, v/v, respectively). The flow rate was 0.2 ml/min.

	Results

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Oxidation of DMBI by Activated Neutrophils. DMBI was metabolized by neutrophils to a major metabolite with a MH⁺ ion at m/z 409 and a minor metabolite with a MH⁺ ion at m/z 407. The retention times of the products with MH⁺ ions at m/z 409 and 407 were 5.8 and 24.6 min, respectively. No significant metabolism occurred in the absence of activation of the cells. The metabolism increased with time, initial DMBI concentration, and cell number (fig. 2), and it was inhibited by azide and catalase. The formation of the product with a MH⁺ ion at m/z 409 was decreased by a factor of 2 or 10 when catalase (200 units/ml) or azide (0.2 mM), respectively, was included in the incubation mixture.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Metabolism of DMBI by activated neutrophils as a function of time (A), initial DMBI concentration (B), and neutrophil concentration (C), showing decreases in the DMBI concentration () and the production of the major product (), which has a MH+ ion at m/z 409 by MS. Standard conditions were as follows: neutrophil concentration, 1 × 106 cells/ml; DMBI concentration, 40 µM; phorbol myristate acetate concentration, 40 ng/ml; incubation time, 30 min. The peak areas are in arbitrary units, because we did not have enough material to determine a standard curve. The values are the mean ± SD from three experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   MS/MS spectra of the metabolites with MH+ ions at m/z 409 (A) and 407 (B), formed during DMBI oxidation by activated neutrophils.

View larger version (10K): [in this window] [in a new window]   Fig. 4.   UV absorption spectrum of the metabolite with a MH+ ion at m/z 407, during DMBI oxidation by activated neutrophils.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Proposed pathway for the formation of the two stable metabolites during DMBI oxidation by activated neutrophils.

Oxidation of DMBI by HOCl. DMBI was readily oxidized by HOCl. Scanning of the UV absorption spectrum of this mixture at 1-sec intervals resulted in the pattern shown in fig. 6. The absorbance of DMBI (_max = 280 nm) decreased rapidly and a new species was generated, with increased absorption at longer wavelengths.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Repetitive absorption spectra from the reaction of DMBI with HOCl. The concentrations of both DMBI and HOCl were 50 µM.

View larger version (10K): [in this window] [in a new window]   Fig. 7.   Proton NMR spectrum of the iminoquinone-NAC conjugate. The integrals are shown at the bottom. The numbering of the molecule is shown in fig. 8.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Proposed scheme for DMBI metabolism by activated neutrophils and trapping of the reactive intermediate by NAC.

Oxidation of DMBI by the MPO System. Compared with oxidation by neutrophils, a similar pattern of products was obtained when DMBI was oxidized by the MPO/H₂O₂ system without added Cl. The major product showed a MH⁺ ion at m/z 409. When GSH was included in the incubation mixture, the same GSH conjugate, with a MH⁺ ion at m/z 511, was observed. When 0.15 mM Cl was added to the phosphate buffer, the reaction was more rapid and the major product observed had a MH⁺ ion at m/z 238. This is the same product as that observed when DMBI was oxidized by higher concentrations of HOCl.

Oxidation of Indomethacin by HOCl and Activated Neutrophils. Indomethacin was oxidized by HOCl to a major product with a molecular weight of 391, corresponding to indomethacin plus chlorine minus hydrogen. However, no GSH conjugates were observed when GSH was added to the reaction mixture of indomethacin and HOCl. When indomethacin was incubated with activated neutrophils under the same conditions as those used for DMBI, no oxidation was observed.

Oxidation of DMI by HOCl and Activated Neutrophils. DMI was oxidized by HOCl to two major products, with retention times of 16 and 28 min. The molecular weight of the product with a retention time of 16 min was 2 mass units less than that of DMI. The other product, with a retention time of 28 min, showed a molecular weight of 378, corresponding to DMI plus chlorine minus hydrogen. No GSH conjugates were observed when GSH was added to the reaction mixture of DMI and HOCl. When DMI was incubated with activated neutrophils, only the product with a retention time of 16 min was observed. When GSH was included in the incubation mixture, no GSH conjugates were detected.

	Discussion

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DMBI was oxidized by neutrophils to a reactive intermediate that could be trapped by GSH. MPO appears to be the enzyme in activated neutrophils responsible for this oxidation. This is supported by our observations that the MPO/H₂O₂ system generated the same metabolites as did neutrophils, that the metabolism by neutrophils required activation of the cells (resulting in the release of MPO and the generation of H₂O₂), and that the metabolism by neutrophils was inhibited by azide (which inhibits MPO) and catalase (which catalyzes the breakdown of H₂O₂).

The mechanism presumably involved N-chlorination, followed by the loss of HCl to form an iminoquinone, as shown in fig. 8. N-Chloro-DMBI was not directly observed. However, strong evidence for the identity of the reactive iminoquinone intermediate was obtained. The diode-array UV spectrophotometric data showed that DMBI absorbance (_max = 280 nm) decreased rapidly upon addition of HOCl and a new species, with absorbance at longer wavelengths (suggesting a quinone-like structure), was formed within seconds. When the products of the reaction between DMBI and HOCl were analyzed by MS in the flow system, the iminoquinone intermediate, with a MH⁺ ion at m/z 204, was observed. This iminoquinone intermediate was reactive with sulfhydryl-containing molecules, such as GSH and NAC. The proton NMR data confirmed the structure of the iminoquinone-NAC conjugate, in which the sulfur was substituted in the ortho-position (relative to the hydroxyl group) on the aromatic ring.

When DMBI was oxidized by activated neutrophils, the final stable metabolites, with MH⁺ ions at m/z 409 and 407, were formed. Both products were generated from the iminoquinone intermediate reacting with another molecule of DMBI. In the case of the product with a MH⁺ ion at m/z 409, the iminoquinone intermediate reacted with another DMBI molecule in the 4-position, which is ortho to the hydroxyl group. The aromatic carbon ortho to the hydroxyl group of DMBI molecule is expected to be electron-rich and could act as a nucleophile to attack carbon-4 of the iminoquinone intermediate (fig. 5). Another possible mechanism for the formation of the product with a MH⁺ ion of m/z 409 involves a coproportionation reaction between DMBI and the iminoquinone, to form two radicals that could dimerize. However, because of the solvent cage effect, the free radical might not be able to react with other molecules and contribute to the toxicity; even if a free radical is formed, the major reaction with GSH involves the electrophilic attack of the iminoquinone, rather than the abstraction of a hydrogen atom by a free radical. Presumably the same is true of the reaction between the reactive metabolite and other biological molecules. In the case of the minor product with a MH⁺ ion at m/z 407, the iminoquinone intermediate reacted with another molecule of DMBI, on the nitrogen of the indole ring, to form a dimer. This is consistent with our observation that the iminoquinone intermediate can react with nitrogen-containing nucleophiles (e.g. n-butylamine), in addition to sulfhydryl-containing nucleophiles. The initially formed dimer can be further oxidized to yield the product with a MH⁺ ion at m/z 407 (fig. 5).

A product with a MH⁺ ion at m/z 238 was observed only when DMBI was oxidized by HOCl at high concentrations (presumably yielding an excess of chlorine). When GSH or NAC was added immediately to the reaction mixture of DMBI and HOCl, this product with a MH⁺ ion at m/z 238 was not observed, which suggested that it was a downstream product when the iminoquinone was the initial reactive metabolite. This product, with a MH⁺ ion at m/z 238, was not observed when DMBI was oxidized by activated neutrophils or by purified MPO in the absence of added Cl. This product is probably not a significant in vivo metabolite.

The formation of reactive metabolites is central to most mechanistic theories of drug hypersensitivity reactions (Uetrecht, 1990). Studies from our laboratory have found that several drugs that are associated with a relatively high incidence of hypersensitivity reactions (especially agranulocytosis and lupus) are metabolized to reactive metabolites by activated leukocytes. Similarly, we propose that oxidation of DMBI to the iminoquinone reactive intermediate by activated neutrophils is responsible for hypersensitivity reactions to indomethacin (i.e. agranulocytosis). There is evidence to suggest that other iminoquinones generated by the MPO system or activated neutrophils may cause hypersensitivity reactions (Maggs et al., 1987; Miyamoto et al., 1997; Parrish et al., 1997; Smith et al., 1989; Uetrecht et al., 1994).

In summary, DMBI, a major metabolite of indomethacin in the liver, is metabolized by the MPO enzyme system in activated neutrophils, forming a reactive iminoquinone intermediate. We propose that the iminoquinone intermediate is responsible for some of the idiosyncratic reactions associated with indomethacin, especially agranulocytosis.