Introduction

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Flunixin [2-(2'-methyl-3'-trifluoromethylanilino)nicotinic acid] (fig. 1) is a nonsteroidal anti-inflammatory drug. This drug suppresses prostaglandin synthesis through its inhibitory effect on cyclooxygenase activity. This results in analgesia and suppression of the inflammatory response (Ciofalo et al., 1977). Flunixin is used in veterinary medicine as an alternative to narcotic analgesics and as an anti-inflammatory agent. Flunixin is used to treat colic (Vernib and Hennessey, 1977), lameness (Houdeshell and Hennessey, 1977), and endotoxemia (Moore et al., 1981) in horses and septic peritonitis in dogs (Hardee et al., 1983). The pharmacokinetics of flunixin have been investigated in mixed-breed dogs (Chay et al., 1982) and in horses (Hardee et al., 1985). Those studies show that flunixin is rapidly and almost completely absorbed after oral administration in those animals. The drug has a half-life of 0.6-4.2 hr in horses and 2.5-4.9 hr in dogs. These are very short half-lives for a drug that may exhibit pharmacological effects for up to 30 hr after a single dose (Tobin, 1981). Flunixin has been shown to be excreted in the urine of horses as free flunixin (Johansson and Anler, 1988), 5-hydroxyflunixin (Jaussad et al., 1987), and a major metabolite that releases free flunixin after treatment with strong base (Johansson and Anler, 1988). There have been no reports of the identification of this base-sensitive metabolite in horses. Unpublished work performed in our laboratory, employing a method used successfully with horse urine (Jaussad et al., 1987), failed to demonstrate the presence of an hydroxylated metabolite of flunixin in the urine of greyhounds administered the drug. Additionally, work performed previously in our laboratory demonstrated the presence of a quantitatively significant urinary metabolite of flunixin in greyhound dogs that, upon treatment with strong base or -glucuronidase, was hydrolyzed to free flunixin (Hill et al., 1994). This suggests the presence of an acyl glucuronide metabolite of flunixin. There have been no reports of the identification of a strong base/-glucuronidase-hydrolyzable metabolite or the identification of any metabolite of flunixin in dogs.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Structures of flunixin (A) and flunixin C1--glucuronide (B).

Acyl glucuronide metabolites of some organic acid drugs have been shown to act as chemically reactive intermediates in the formation of covalent bonds between the parent drug and tissue proteins (Smith et al., 1990, 1992; Williams et al., 1992; Kauffman, 1994; McGurk et al., 1996). This covalent binding has, in some cases, resulted in altered pharmacodynamics and/or pharmacokinetics of the drug in question (Faed, 1984). Covalent binding of drug to tissue proteins has, in some cases, resulted in drug hypersensitivity resulting from an immunological response to the drug-carrier complex (Pohl et al., 1988). This study describes the techniques used in the isolation, purification, and structural characterization of the acyl glucuronide of flunixin, the major urinary metabolite of flunixin in greyhound dogs.

	Materials and Methods

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Chemicals and Reagents. Solvents used in extractions and chromatography were of HPLC grade and were obtained from EM Sciences (Gibbstown, NJ). These included acetonitrile, methanol, ethyl acetate, and chloroform. HPLC-grade phosphoric acid, reagent grade glacial acetic acid, and reagent-grade sodium hydroxide were obtained from Fisher Scientific. TEA² was obtained from Acros Organics, Fisher Scientific (Springfield, NJ). -Glucuronidase (EC 3.2.1.31, 1-3 × 10⁶ units/g) was obtained from Sigma Chemical Co. (St. Louis, MO). The C₁₈ bonded-phase column used for the SPE was obtained from J.T. Baker, Inc. (Phillipsburg, NJ). The Zorbax Rx C₈ HPLC column (4.6 mm × 25 cm, packed with 5-µm particles) was obtained from MAC-MOD Analytical Inc. (Chadds Ford, PA). Flunixin, as the meglumine salt, was a gift from Schering Corp. (Union, NJ). Flunixin meglumine paste (Banamine), used in the greyhound administration studies, was obtained from Schering.

Instrumentation. HPLC analyses were performed with a Hewlett-Packard (Palo Alto, CA) HP1090 system with an HP1040 diode-array detector. Solvent A was 0.15 M phosphoric acid/0.05 M TEA in water and solvent B was 0.15 M phosphoric acid/0.05 M TEA/20% water in acetonitrile. The flow rate was 2.0 ml/min. The detector was set to monitor absorbance at 210 nm, and UV spectral data were collected from 195 to 405 nm at 640-msec intervals. In the gradient-elution program, the solvent composition was held at 100% solvent A for 2.2 min and then changed linearly to 100% solvent B in 20 min. The column was a C₈ Zorbax Rx column (4.6 mm × 25 cm) packed with 5-µm spherical particles. The column temperature was maintained at 30°C. Ten microliters of sample were injected onto the column by the HP1090 autosampler.

Administration of Flunixin to Greyhounds. Flunixin meglumine (Banamine) was administered orally to four greyhound dogs, two with a dose of 0.55 mg/kg flunixin and two with a dose of 1.0 mg/kg flunixin. Urine samples were collected before administration (0 hr) and 2, 4, 8, 24, 48, and 72 hr after administration for all four dogs. A 96-hr urine sample was collected for each dog treated at 1.0 mg/kg. Analysis of the urine samples by HPLC before and after base hydrolysis showed that the 8-hr urine samples contained an appreciable concentration of flunixin metabolite (Brady et al., 1997). The 8-hr urine samples were used as the source of metabolite in this study. The 0-hr samples were used as control DFU samples.

Isolation and Purification of Flunixin Glucuronide from Greyhound Dog Urine. The metabolite of flunixin was isolated from greyhound urine by gradient elution from a C₁₈ SPE column. Five milliliters of an 8-hr urine sample were adjusted to pH 4.5 with 1.0 N acetic acid and exhaustively extracted with three 10-ml volumes of chloroform to remove free flunixin from the sample. The extracted urine sample was applied to a chromatographic column containing 0.5 g of C₁₈ bonded-phase packing material. The column was step-eluted with 5-ml portions of CH₃CN/0.1 N acetic acid (pH 4.5), starting with 100% 0.1 N acetic acid and progressing, in 5% CH₃CN/0.1 N acetic acid increments, to 15% CH₃CN/0.1 N acetic acid. From 16 to 20% CH₃CN/0.1 N acetic acid, the column was eluted in 1% CH₃CN/0.1 N acetic acid increments. All eluates were diluted 1/100 with 0.01 M phosphate-buffered saline (pH 7.4) and were assayed for flunixin reactivity using a flunixin ELISA (Brady et al., 1997). A quantitatively significant flunixin ELISA-reactive substance was detected in the 16% CH₃CN/0.1 N acetic acid eluate. The CH₃CN was evaporated from this eluate in a water bath at 65°C under nitrogen, and the remaining aqueous solution was passed through a second C₁₈ column. After the column was washed with 0.1 N acetic acid (pH 4.5), it was eluted with 1 ml of 30% CH₃CN/0.1 N acetic acid. A flunixin ELISA-reactive substance was shown to be present in this eluate. The CH₃CN was evaporated from this eluate, and the remaining aqueous solution was analyzed by reverse-phase HPLC/DAD. The 0-hr DFU sample (negative control) and a 0-hr DFU sample spiked with 10,000 ng/ml flunixin (DFU_10,000ng/ml, positive free flunixin control) were subjected to the same SPE procedure as was the 8-hr urine sample. The 0-hr DFU and DFU_10,000ng/ml samples were not extracted with chloroform before SPE, as was the 8-hr urine specimen.

Identification and Characterization of the Flunixin Metabolite. The eluate containing the ELISA-reactive metabolite was analyzed by reverse-phase HPLC/DAD, along with the corresponding DFU eluate and the ELISA-reactive DFU_10,000ng/ml eluate. HPLC R_T values and UV spectral data for the metabolite and free flunixin were obtained. The SPE eluate containing the metabolite was treated, in separate experiments, with strong base and -glucuronidase. Fifty microliters of the SPE eluate containing the metabolite were mixed with 10 µl of 18 M NaOH and allowed to incubate for 1 hr at room temperature. After incubation, 40 µl of 0.1 N acetic acid (pH 4.5) was added to bring the final volume to 0.1 ml. For the -glucuronidase treatment, 50 µl of the metabolite eluate was mixed with 10 µl of 10,000 units/ml -glucuronidase in 0.1 N acetic acid (pH 4.5) and was allowed to incubate for 18 hr at 37°C. After the incubation period, 40 µl of 0.1 N acetic acid was added to bring the total volume to 0.1 ml. For comparison, 50 µl of the metabolite eluate sample was diluted with 50 µl of 0.1 N acetic acid to a final volume of 0.1 ml. All three samples were analyzed by reverse-phase HPLC/DAD.

	Results

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The SPE eluate from the 8-hr urine sample, from which free flunixin had been exhaustively extracted, contained an ELISA-reactive compound that eluted early in the gradient elution system, relative to flunixin. This ELISA-reactive compound eluted in the 16% (v/v) CH₃CN/0.1 N acetic acid eluate (retention volume, 95 ml). Analysis by HPLC/DAD of the 16% SPE eluate from the 8-hr urine sample showed the presence of only one flunixin metabolite, compared with the 16% eluate from the 0-hr control urine sample. Flunixin in the spiked DFU sample (DFU_10,000ng/ml) eluted in the 18% (v/v) CH₃CN/0.1 N acetic acid eluate (retention volume, 130 ml). Eluates from the 0-hr DFU sample showed no reactivity in the ELISA.

HPLC R_T data for free flunixin and the ELISA-reactive metabolite before and after treatment with sodium hydroxide or -glucuronidase (fig. 2) showed that the metabolite eluted several minutes earlier (R_T = 13.4 min) than flunixin (R_T = 15.2 min). The UV absorption spectra were similar for the two compounds, with the metabolite showing a shift of the 256-nm band of flunixin to 260 nm, with a more pronounced band at 274 nm. Additionally, the spectrum for the metabolite exhibited a red shift of the 326-nm band of flunixin to 340 nm. Treatment of the metabolite with sodium hydroxide and -glucuronidase, in separate experiments, resulted in the disappearance of the metabolite peak, with the concomitant emergence of a peak with the R_T and UV spectral characteristics of flunixin.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Solvent-gradient HPLC chromatograms at 340 nm (1) and UV spectral profiles (2) of flunixin (A), flunixin metabolite (B), flunixin metabolite treated with NaOH (C), and flunixin metabolite treated with -glucuronidase (D).

The positive-ion, electrospray mass spectrum of the flunixin metabolite is shown in fig. 3A. This spectrum showed the presence of an apparent molecular ion (M+1) at m/z 473 and a product ion at m/z 297. CID MS analysis of the m/z 297 fragment ion (fig. 3) showed a fragmentation pattern similar to that of standard flunixin (fig. 3, C and D) analyzed under identical conditions. MS analysis of the SPE eluate from the 0-hr urine sample, under identical conditions, showed an absence of the m/z 473 and m/z 297 ions.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Positive-ion, electrospray mass spectrum of flunixin metabolite (A), the CID spectrum of its m/z 297 product (B), the positive-ion, electrospray mass spectrum of standard flunixin (C), and the CID spectrum of its m/z 297 ion (D).

	Discussion

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A metabolite of flunixin was isolated from greyhound dog urine by SPE. This metabolite was shown to be hydrolyzed to free flunixin when treated with strong base or when incubated with -glucuronidase. The results of these experiments provided preliminary evidence that the isolated compound was the C1--glucuronide of flunixin. Configurations of glucuronide esters at positions other than the C1--position have been reported to be resistant to hydrolysis by -glucuronidase (Dutton, 1966; La Du et al., 1971). Also, N-linked glucuronides are stable and are very resistant to hydrolysis by -glucuronidase under the stated conditions (La Du et al., 1971).

Positive-ion, electrospray MS of the isolated flunixin metabolite showed a protonated molecular ion at m/z 473, which is consistent with the molecular weight of 472 for flunixin glucuronide. The electrospray spectrum of the flunixin metabolite also demonstrated a protonated product ion at m/z 297, which is consistent with the molecular weight of protonated flunixin. CID MS analysis of the m/z 297 product ion produced a fragmentation spectrum consistent with that of flunixin, indicating that flunixin is a component of the m/z 473 molecular ion. These data collectively indicate that the metabolite of flunixin isolated from greyhound dog urine was the C1--glucuronide of flunixin (fig. 1B).

The possibility exists that flunixin metabolites in greyhound urine other than the C1--glucuronide reacted in the ELISA detection system. It is also possible that metabolites of flunixin were nonreactive in the ELISA and, therefore, not detected.

The SPE eluate containing the isolated flunixin metabolite was treated with strong base and -glucuronidase, in separate experiments, and analyzed by HPLC/DAD (fig. 2). The conversion from metabolite to free flunixin was complete and quantitative, indicating the presence of a single metabolite.

These hydrolysis experiments were also performed with postadministration greyhound urine samples that had been exhaustively extracted with chloroform to remove all traces of free flunixin. The results again showed that hydrolysis resulted in complete and quantitative conversion to free flunixin (Brady et al., 1997), with no trace of another metabolite, compared with the 0-hr urine sample. The chloroform fraction from the initial exhaustive extraction contained only free flunixin, as determined by R_T and UV spectral data from HPLC/DAD. These experiments provide evidence that the major metabolite of flunixin in the urine of greyhound dogs is the acyl glucuronide, and they suggest that other metabolites, if present, are minor components.