Introduction

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BOP¹ [2-(4-chlorophenyl)--methyl-5-benzoxazoleacetic acid] is a 2-arylpropionic acid derivative that exhibits anti-inflammatory effects (Cashin et al., 1977; Tsurumi et al., 1980, 1982) and was withdrawn from the market because of unexplained fatal liver toxicity (Duthie et al., 1982; Goudie et al., 1982). The metabolic fate of BOP has been studied in mice, rats, rabbits, dogs, rhesus monkeys, and humans (Chatfield and Green, 1978; Ohtsuki et al., 1981a,b; Furlanut et al., 1985). Results obtained in rats with the ¹⁴C-labeled compound showed that BOP was readily absorbed from the gastrointestinal tract and 11-24% and 79-85% of the dose was excreted in the urine and feces, respectively, within 192 hr (Ohtsuki et al., 1981b). Only BOP-G has been identified as a major metabolite of BOP in mammals (Chatfield and Green, 1978; Ohtsuki et al., 1981b; Spahn et al., 1989). Chatfield and Green (1978) and Ohtsuki et al. (1981b) examined the biliary excretion of BOP using radioactive [¹⁴C]BOP, and they reported the presence of BOP and BOP-G in bile.

In this study, we describe the isolation from rat bile of a new BOP metabolite, BOP-T, the physicochemical characteristics of which were compared with those of the synthesized authentic standard. Furthermore, the biliary excretion of BOP after iv administration of a 10 mg/kg dose to bile duct-cannulated rats was investigated. BOP and its metabolites in plasma and bile were analyzed using reverse-phase analytical and chiral HPLC columns.

	Materials and Methods

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Measurements. Elemental analyses were performed using a Perkin-Elmer model 240B elemental analyzer (Perkin-Elmer Corp., Norwalk, CT). High-resolution mass spectra were obtained with a JEOL JMS-DX302 instrument (JEOL, Tokyo, Japan), using a direct-inlet system. Operating conditions were as follows. The accelerating voltage and ionization current were 3 kV and 300 µA, respectively. Ionization was by electron impact with 70-eV electrons, with a source temperature of 150°C. Perfluorokerosene was used as a standard compound. Positive-ion FAB mass spectra were obtained with an Auto Spec mass spectrometer (Micromass UK Ltd., Manchester, England) equipped with a FAB source using cesium atoms. The instrument was operated with an accelerating voltage of 8 kV, and glycerol was used as the FAB matrix for the acquisition of positive-ion spectra. ¹H, ¹H-¹H, and ¹H-¹³C COSY NMR spectra were obtained using a JEOL JNM-LA500 spectrometer (JEOL), with tetramethylsilane as the IS. Chemical shifts are expressed in ppm downfield from tetramethylsilane, and coupling constants are given in hertz. Column chromatography was carried out with silica gel (230-400 mesh; Kanto Chemical Co., Tokyo, Japan). TLC was undertaken using Kieselgel 60 F₂₅₄ plates (Merck, Darmstadt, Germany). Spots on the TLC plates were detected under UV light (253.6 nm). The HPLC system consisted of a model BIP-I pump (JASCO Co., Tokyo, Japan) equipped with a model 820-FP fluorometer (JASCO), a model SIL-9A autoinjector (Shimadzu Co., Kyoto, Japan), and a model C-R4A Chromatopac integrator (Shimadzu). The excitation and emission wavelengths (xenon lamp) were set at 315 and 365 nm, respectively.

Chemicals. TBA hydrogen sulfate was purchased from Aldrich Chemical Co. (Milwaukee, WI). Naproxen was purchased from Sigma Chemical Co. (St. Louis, MO). BOP was kindly donated by Eli Lilly Co. (Indianapolis, IN) before withdrawal of the drug from the market. MeOH, MeCN, and tetrahydrofuran were HPLC grade (Wako Pure Chemical Industries, Osaka, Japan). All other chemicals were reagent grade. The water used had been double-distilled in a glass still. NAP-Me (IS) and reference BOP-T were synthesized in our laboratory, as described below. Standard BOP-G was obtained biosynthetically, as reported previously (Spahn et al., 1989). Standard (R)- and (S)-BOP enantiomers were obtained by the method described previously (Spahn et al., 1989). Small amounts of standard (R)- and (S)-BOP-T were synthesized from the enantiomers by the Schotten-Baumann method (Idle et al., 1978). Small amounts of (R)- or (S)-BOP enantiomers were administered to rats, and standard (R)- or (S)-BOP-G, respectively, was obtained from rat bile.

Animals and Drug Administration. Male Sprague-Dawley rats (Sankyo Labo Service Co., Tokyo, Japan), weighing 300-350 g, were used throughout the study. The rats were housed in stainless steel cages in groups of 10, in a temperature-controlled (20-28°C) room with a 12-hr light/dark cycle. The rats were allowed free access to standard rat chow (Sankyo) and water for 1 week before the experiments. Each animal was anesthetized with 20% (w/v) urethane (1 g/kg body weight, ip). The femoral vein was cannulated with PE-10 tubing (Clay Adams, Parsippany, NJ) for instillation of saline solution. An abdominal incision was made and the common bile duct was cannulated with PE-10 tubing (Clay Adams), for collection of bile samples, and closed with surgical clips. Bile samples were collected in 100-ml plastic tubes and stored at 80°C. During experimental procedures, body temperatures were maintained at 38 ± 0.5°C with a heating lamp, to prevent hypothermic alterations of bile flow. The solution of BOP for injection was prepared by dissolving 500 mg of BOP in a 20-ml mixture of 1 M NaOH, ethanol, and saline (8:8:4, v/v). Fifty rats received 20-50 mg/kg doses of BOP. Saline was injected into the rats through the cannula to supplement body fluids.

Isolation and Purification of a New Metabolite. MeCN (5 ml/1 ml of bile) was added to the collected bile to precipitate protein. The mixture was vigorously vortex-mixed and centrifuged for 20 min at 5000g; the supernatant was transferred into a 300-ml glass bottle and evaporated to dryness under reduced pressure at 30°C. Ten milliliters of 1 M NaOH were added to the residue and incubated at 37°C for 1 hr, after which 1% metaphosphoric acid (50 ml) was added. The acidic solution was saturated with NaCl, 1 g of TBA hydrogen sulfate was added, and the mixture was extracted five times with ethyl acetate (each time, 100 ml). The ethyl acetate layer was dried with anhydrous sodium sulfate and, after filtration, evaporated to dryness under reduced pressure at 30°C. The residue was dissolved in a small amount of MeOH and pipetted onto silica gel TLC plates. The TLC plates were developed with a mixture of CHCl₃/MeOH (4:1, v/v), and the band of approximately R_F 0.2 was scraped off and extracted with MeOH. The MeOH extract was evaporated to dryness under reduced pressure at 30°C. The TLC purification procedure for the new metabolite was repeated several times.

Synthesis of Standard BOP-T. BOP-T was synthesized by the Schotten-Baumann method (Idle et al., 1978), as follows. BOP (3.32 mmol) was refluxed in 10 ml of thionyl chloride for 1 hr, followed by evaporation to dryness. Taurine (5.02 mmol) dissolved in 5 ml of 1 M NaOH was added dropwise to the residue (BOP chloride) dissolved in 10 ml of anhydrous benzene, and the mixture was stirred overnight at room temperature (23°C). The reaction mixture was evaporated to dryness under reduced pressure, and the residue was subjected to silica gel column chromatography. BOP-T was eluted with a mixture of CHCl₃ and MeOH (10:1, v/v). A white powder, recrystallized from hot MeOH, exhibited the following characteristics: TLC: R_F 0.19 (CHCl₃/MeOH, 4:1, v/v); HPLC: R_T 5.9 min; FAB MS: m/z 409 ([M+H]⁺); ¹H NMR (DMSO-d₆):  1.39 (d, 3H, J = 7, CH-CH₃), 2.53 (m, 2H, CH₂-SO₃H), 3.30 (m, 2H, NH-CH₂-), 3.70 (q, 1H, J = 7, CH-CH₃), 7.38 (dd, 1H, J = 1.8 and 8.5, Ar-H), 7.67 (d, 2H, J = 8.5, Ar-H₂), 7.69 (d, 1H, J = 1.8, Ar-H₁), 7.70 (d, 1H, J = 1.8, Ar-H₁), 7.88 (dd, 1H, J = 5.2 and 5.5, NH), and 8.19 (d, 2H, J = 8.5, Ar-H₂).

Synthesis of NAP-Me. Diazomethane (0.3 g) in ether (7 ml) was added to naproxen (1 g) in ether (20 ml), and the reaction mixture was maintained for 1 hr at room temperature (23°C). After evaporation of the ether, the residue was subjected to silica gel column chromatography. NAP-Me was eluted with a mixture of n-hexane and ethyl acetate (30:1, v/v).

HPLC Conditions. The analysis was performed at room temperature (23°C) with a reverse-phase analytical Capcell Pak C₁₈ column (4.6 mm i.d. × 25 cm; particle size, 5 µm; Shiseido Co., Tokyo, Japan) equipped with a guard column packed with Capcell Pak C₁₈ material (4.6 mm i.d. × 1 cm; particle size, 5 µm; Shiseido). Mobile phase, consisting of 10 mM TBA buffer/MeCN/tetrahydrofuran (100:35:35, v/v), was pumped through the column at a rate of 1.3 ml/min and was degassed with an ERC-3322 degasser (Erma Co., Saitama, Japan) under reduced pressure. For the separation of optical isomers of BOP and its metabolites, a chiral HPLC column (SUMICHIRAL OA 2500, 4.6 mm i.d. × 25 cm; Sumika Chemical Analysis Service, Osaka, Japan) equipped with a guard column packed with SUMICHIRAL OA 2500 material (4.6 mm i.d. × 1 cm; Sumika) was used. The mobile phases used for the separation of the optical isomers were 0.04 M ammonium acetate in MeOH for BOP enantiomers in plasma, 0.06 M ammonium acetate in MeOH for BOP-G enantiomers in bile, and 0.01 M ammonium acetate in MeOH/MeCN/H₂O (85:15:5, v/v) for BOP and BOP-T enantiomers in bile. The flow rate was 1.0 ml/min.

Pretreatment of Biological Samples for HPLC. To 10 µl of plasma, bile, or urine samples in 2-ml plastic tubes, 180 µl of MeCN, 30 µl of IS (100 µl/ml in DMSO), and 30 µl of distilled water were added, with vigorous mixing. After centrifugation of the sample for 10 min at 15,000g at 4°C, 10 µl of the supernatant was injected directly into the HPLC system.

Analytical Methods. Calibration curves (1-100 µg/ml) were established, using linear least-squares regression analyses, from BOP/IS peak area ratios vs. various concentrations of BOP in drug-free plasma, bile, or urine samples to which aliquots of standard BOP had been added. In the case of BOP enantiomers, the calibration curves involve 0.5-50 µg/ml ranges for the (R)- and (S)-BOP enantiomers. The BOP-G and BOP-T concentrations in bile and urine were calculated from the BOP calibration curve as BOP equivalents. However, because the molar absorptivities for BOP-G and BOP-T in the mobile phase were different from that of BOP, the BOP-T concentrations obtained from the BOP calibration curve were corrected using the following equation: BOP-T concentration = (1 mM BOP peak area/1 mM BOP-T peak area) × peak concentration of BOP-T calculated from the BOP calibration curve. BOP-G was hydrolyzed by incubation in 1 M NaOH for 30 min at 30°C, and the peak areas measured before and after hydrolysis were compared. The concentration was calculated as follows: BOP-G concentration = (BOP peak area after alkaline hydrolysis/BOP-G peak area before alkaline hydrolysis) × peak concentration of BOP-G calculated from the BOP calibration curve. All samples were analyzed in duplicate.

	Results

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HPLC. Under the HPLC conditions described above, BOP, BOP-G, BOP-T, IS, and their enantiomers exhibited symmetrical peaks with baseline resolution, with no interfering peaks observed for endogenous components of rat plasma and bile (figs. 1 and 2). Fig. 1A shows a chromatogram of a drug-free bile sample, and fig. 1B shows a chromatogram of a drug-free bile sample to which BOP and IS had been added at concentrations of 50 and 300 µg/ml, respectively. Fig. 1C illustrates the analysis of a bile sample obtained 30 min after BOP administration. The retention times, with the ODS column, for BOP-T, BOP-G, IS, and BOP were 5.9, 7.2, 13.2, and 17.5 min, respectively. The elution orders for the enantiomers of BOP, BOP-T, and BOP-G in the HPLC chromatograms were determined by direct injection of authentic compounds into the HPLC system. The retention times, with the chiral column, for (R)-BOP, (S)-BOP, and IS in plasma were 10.3, 11.8, and 4.4 min, respectively. The retention times for (R)-BOP-G, (S)-BOP-G, and IS in bile were 17.3, 21.3, and 4.4 min and for (R)-BOP-T, (S)-BOP-T, IS, (R)-BOP, and (S)-BOP in bile were 16.7, 16.1, 4.3, 22.5, and 26.0, respectively. The mobile phases used for the separation of enantiomers are described in HPLC Conditions.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   HPLC chromatograms of BOP, BOP metabolites (BOP-T and BOP-G), and NAP-Me (IS). A, Control bile. B, Control bile supplemented with BOP (50 µg/ml) and IS (300 µg/ml). C, Bile sample obtained 30 min after BOP administration (10 mg/kg, iv).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   HPLC chromatograms of BOP enantiomers, BOP-G enantiomers, BOP-T enantiomers, and NAP-Me (IS). A, Plasma sample obtained 5 min after BOP administration (10 mg/kg, iv). B, Bile sample obtained 30 min after BOP administration. C, Control bile supplemented with standard BOP (50 µg/ml) and BOP-T (50 µg/ml). The solvent systems used were 0.04 M ammonium acetate in MeOH for BOP enantiomers in plasma, 0.06 M ammonium acetate in MeOH for BOP-G enantiomers in bile, and 0.01 M ammonium acetate in MeOH/MeCN/H2O (85:15:5, v/v) for BOP and BOP-T enantiomers in bile.

Quantification and Analytical Variables. The BOP concentration in urine was below the detection limit of this HPLC assay method. For the ODS column, the correction factors to calculate BOP-T and BOP-G concentrations in bile were 1.14 ± 0.01 and 0.97 ± 0.02, respectively; for the chiral column, the correction factors to calculate BOP-T and BOP-G concentrations in bile were 0.24 ± 0.002 and 0.21 ± 0.004, respectively. The calibration curves for BOP (1-100 µg/ml), prepared as described above, were linear and apparently passed through the origin, as follows: y = 0.066x  0.001 (r = 1.000) for BOP in bile and y = 0.065x  0.002 (r = 1.000) for BOP in plasma. The lower detection limit for BOP was 10 pg/ml (with a signal/noise ratio of 10). Analytical reproducibility (per cent coefficient of variation) values for intra- and interday assay variations were 0.34 and 1.80%, respectively, for 50 µg/ml BOP in bile.

Characterization of a New BOP Metabolite. The compound was recrystallized from hot MeOH as a white powder [TLC: R_F 0.19 (CHCl₃/MeOH, 4:1, v/v); HPLC: R_T 5.9 min (fig. 1C, first peak); FAB MS: m/z 409 ([M+H]⁺); ¹H NMR (DMSO-d₆):  1.39 (d, 3H, J = 7, CH-CH₃), 2.53 (m, 2H, CH₂-SO₃H), 3.30 (m, 2H, NH-CH₂-), 3.70 (q, 1H, J = 7, CH-CH₃), 7.38 (dd, 1H, J = 1.8 and 8.5, Ar-H), 7.67 (d, 2H, J = 8.5, Ar-H₂), 7.69 (d, 1H, J = 1.8, Ar-H₁), 7.70 (d, 1H, J = 1.8, Ar-H₁), 7.88 (dd, 1H, J = 5.2 and 5.5, NH), and 8.19 (d, 2H, J = 8.5, Ar-H₂)].

Characterization of NAP-Me. The compound was recrystallized from MeOH [TLC: R_F 0.27 (n-hexane/ethyl acetate, 20:1, v/v); HPLC: R_T 13.2 min (fig. 1C, third peak); elemental analysis for NAP-Me: C, 73.75; H, 6.63; calculated for C₁₅H₁₆O₃ (molecular weight, 244.28): C, 73.75; H, 6.60; high-resolution MS: m/z 244.1106 (M⁺, 45.6% relative intensity), 229 (M⁺15), 213 (M⁺31), and 185 (M⁺59); ¹H NMR (CDCl₃):  1.56 (d, 3H, J = 7.0, CH-CH₃), 3.65 (s, 3H, COOCH₃), 3.84 (q, 1H, J = 7, CH-CH₃), 3.89 (s, 3H, Ar-OCH₃), 7.09 (d, 1H, J = 2.4, Ar-H), 7.12 (dd, 1H, J = 2.4 and 8.5, Ar-H), 7.38 (dd, 1H, J = 1.5 and 8.5, Ar-H), 7.64 (d, 1H, J = 1.5, Ar-H), and 7.68 (d, 2H, J = 8.5, Ar-H₂)].

Biliary Excretion of BOP and Its Metabolites. Characterization of the biliary metabolites of BOP was performed by subjecting bile samples to HPLC analysis, as described in Materials and Methods. The plasma concentration-time curve for BOP after bolus iv administration and cumulative curves for BOP and its metabolites excreted into rat bile are illustrated in fig. 3. The results analyzed with a chiral column are shown in figs. 4 and 5.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   BOP levels in plasma (left ordinate) and cumulative excretion of BOP and its metabolites in bile (right ordinate) after a single iv administration of BOP. The dose was 10 mg/kg. Each point and vertical bar represent the mean and SD (N = 5). , BOP; , BOP-G; , BOP-T.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Plasma concentrations of BOP enantiomers (left ordinate) and cumulative excretion of BOP, BOP-G, and BOP-T enantiomers in bile (right ordinate) after administration of racemic BOP to rats. The dose was 10 mg/kg body weight.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Cumulative excretion (percentage of dose) of BOP, BOP-G, and BOP-T enantiomers in bile after administration of racemic BOP to rats.

Pharmacokinetic Data. Twelve-hour cumulative amounts of BOP, BOP-G, and BOP-T (as percentages of the dose) excreted into bile were 0.33 ± 0.09, 13.22 ± 2.28, and 2.54 ± 0.80%, respectively. It is apparent from fig. 3 that BOP exhibits a long-half in rat plasma. If the estimated 31-hr terminal half-life (determined from 12-hr sampling only) is correct, >78.3 ± 13.0% of the BOP dose would remain to be excreted from the body after 12 hr, as estimated from a preliminary fit of the data representing the solid line drawn through the plasma concentration measurements. The same plasma and bile samples were analyzed using a chiral column (figs. 4 and 5). (R)-BOP showed rapid plasma elimination, whereas the plasma elimination of (S)-BOP was very slow. The amounts of BOP, BOP-G, and BOP-T enantiomers excreted into bile were as follows: (R)-BOP-G and (S)-BOP-G, 2.1 ± 0.6 and 12.5 ± 1.8% of the dose; (R)-BOP-T and (S)-BOP-T, 2.0 ± 0.6 and 0.3 ± 0.05% of the dose; (R)-BOP and (S)-BOP, 0.02 ± 0.03 and 0.2 ± 0.1% of the dose, respectively.

	Discussion

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After observing an unidentified peak in the HPLC chromatograms (fig. 1C, first peak) for bile samples from rats administered BOP, we isolated (from bile) the compound corresponding to the peak and we studied its chemical structure. When the bile samples (1 ml) were incubated with 1 M HCl (1 ml) at 37°C for 1 hr, no change was observed in the HPLC chromatograms of the samples. When the bile samples (1 ml) were incubated with 1 M NaOH (1 ml), the second peak (fig. 1C) in the HPLC chromatograms disappeared and the BOP peak (fig. 1C, fourth peak) increased. The retention time of the second peak in the HPLC chromatograms was identical to that of standard BOP-G. Thus, the second peak was identified as BOP-G. The first peak in the chromatogram (fig. 1C) was stable under not only acidic but also alkaline conditions.

Positive-ion FAB MS analysis of the compound showed a [M+H]⁺ ion at m/z 409, i.e. 108 mass units greater than the molecular weight of BOP (301 mass units), suggesting that the unknown compound might be a taurine conjugate of BOP [from the molecular weight of taurine (125 mass units), with an assumed loss of water]. In the ¹H-¹³C COSY NMR spectrum of the compound in DMSO-d₆, signals assigned to two methylene groups appeared at 2.53 ppm (two protons) and 3.30 ppm (two protons), in addition to BOP signals. The chemical shifts of these two signals were very similar to those of standard taurine in DMSO-d₆ (2.80 and 3.13 ppm, each two protons). The doublet of doublets signal at 7.88 ppm (corresponding to one proton) was coupled with the methylene group at 3.30 ppm and was assigned to the proton of the amide moiety (-CONH-CH₂CH₂-). Because the compound eluting as the first peak in the chromatogram shown in fig. 1C was suspected to be BOP-T, standard BOP-T was chemically synthesized by the Schotten-Baumann method (see Synthesis of Standard BOP-T) (Idle et al., 1978). The TLC R_F and HPLC R_T values for the unknown compound were identical to those for synthesized BOP-T. Furthermore, positive-ion FAB MS and ¹H-¹³C COSY NMR findings for the metabolite were also identical to those for synthesized BOP-T.

To investigate the biliary excretion of BOP and its metabolites, especially BOP-T, the parent drug was administered iv to male Sprague-Dawley rats at a 10 mg/kg dose. The plasma elimination profile (fig. 3) and the half-life (approximately 31 hr) for BOP were almost identical to previously reported results (Ohtsuki et al., 1981b). Ohtsuki et al. (1981b) reported that a large amount of BOP-G and a small amount of BOP were excreted in rat bile. We showed here that BOP-T was also excreted in rat bile. Because no measurable levels of BOP or its metabolites were excreted in urine, it is clear that the disposition of BOP in rats is the result of mainly biliary clearance.

Initially, in this preliminary animal study, we analyzed BOP and its metabolites as racemates, by HPLC with a reverse-phase ODS column (fig. 3). Interestingly, BOP-T excretion in rat bile did not appear to continue beyond 4 hr after BOP administration. In contrast, the glucuronic acid conjugation of BOP proceeded continuously after 4 hr. To test whether this might be the result of depletion of substrates for BOP-T conjugation, taurine (100, 250, 500, and 1000 mg/kg body weight, N = 3 for each dose) was injected into rats through the femoral cannula 4 hr after BOP administration. However, the amount of BOP-T excreted into the bile was not altered (data not shown).

Bopp et al. (1979) and Simmonds et al. (1980) reported that BOP exhibits stereoselective plasma disposition and (R)-BOP is converted unidirectionally to (S)-BOP in the body. Thus, to investigate the changes with time for each BOP enantiomer, as well as their metabolites, in plasma and bile, we reanalyzed the plasma and bile samples using a chiral HPLC column (figs. 4 and 5). (R)-BOP showed rapid plasma elimination, whereas the plasma elimination of (S)-BOP was very slow. These results are similar to those observed for many 2-arylpropionic acid derivatives (Jamali, 1988).

Analysis of BOP conjugates in bile yielded interesting results with respect to the configuration of the propionic acid moiety of the conjugates. Of the glucuronide excreted into the bile, 85.6% had the S-configuration at the propionic acid moiety (figs. 4 and 5). This finding is quite similar to the results reported for humans by Spahn et al. (1989). Those workers reported that the cumulative excretion of the (S)-glucuronide in urine over 96 hr after administration of 300 mg of racemic BOP was about twice that of the (R)-glucuronide. Iwakawa et al. (1991) reported stereoselective disposition of caprofen, flunoxaprofen, and naproxen in rats, whereas the cumulative excretion of the (S)-glucuronide in bile over 8 hr was greater than that of the (R)-glucuronide after administration of racemic flunoxaprofen (a BOP derivative) and naproxen. Excretion of flunoxaprofen (S)-glucuronide was about 2.5-fold greater than that of the (R)-glucuronide, whereas the excretion of naproxen (S)-glucuronide was 3.4-fold greater than that of the (R)-glucuronide. Cumulative excretion of (R)-BOP-T into bile over 12 hr was about 6.7-fold greater than that of (S)-BOP-T (figs. 4 and 5).

In recent years, stereoselective amino acid conjugation of 2-arylpropionic acid derivatives (profens) has been considered together with the metabolic chiral inversion mechanism, because both reactions involve drug-CoA thioester formation. Hutt and Caldwell (1983) have reviewed the metabolic chiral inversion of 2-arylpropionic acids. The chiral inversion of BOP occurs unidirectionally, from the R-enantiomer to the S-enantiomer, in the body, as described previously (Bopp et al., 1979; Simmonds et al., 1980). This is hypothesized to occur as a result of the R-enantiomer of BOP serving as a substrate for the enzyme "acyl-CoA ligase" during acyl-CoA thioester formation, whereas the S-enantiomer is not a substrate for the enzyme (Hutt and Caldwell, 1983; Nakamura et al., 1984).

In the process of conjugation of BOP with taurine, BOP-CoA thioester formation might be required as the first step, with BOP-T being produced from BOP-CoA thioester by taurine N-acyltransferase as a second process. This could explain why taurine conjugation of BOP stopped approximately 4 hr after BOP administration. This might be the result of substrate depletion. However, the substrate is not taurine but (R)-BOP, which is the precursor for BOP-CoA thioester formation in the first step. It is clear from fig. 4 that about 95% of (R)-BOP was converted to (S)-BOP by metabolic chiral inversion within approximately 4 hr after BOP administration.

A small amount of (S)-BOP-T was identified in rat bile. This may be the result of conversion of (R)-BOP-CoA thioester to (S)-BOP-CoA thioester by epimerase, followed by metabolism to (S)-BOP-T by taurine N-acyltransferase.

Although a variety of 2-arylpropionic acid derivatives are widely used in clinical practice, only a few reports of taurine conjugation of these drugs have been published. Taurine conjugates of suprofen (Sakai et al., 1984; Mori et al., 1985), trans-hydroxyloxoprofen (Tanaka et al., 1983), trans-hydroxy-CS-670 (Asami et al., 1995), and M-5011 (Kitamura et al., 1996) were found in dog urine, and a taurine conjugate of ibuprofen was identified in human urine (Shirley et al., 1994). The only taurine conjugate reported in rat and mouse urine was that of pirprofen pyrrole (Egger et al., 1982).

Similarly, only a few reports of stereoselective amino acid conjugation of 2-arylpropionic acid derivatives have appeared in the literature. Shirley et al. (1994) reported that the taurine conjugate of ibuprofen excreted in human urine exhibits the S-configuration at the propionic acid moiety (approximately 87% of the dose after oral administration of the racemate). Asami et al. (1995) reported that trans-hydroxy-CS-670 excreted in dog urine mainly has the S-configuration. The S/R ratios of taurine conjugates after administration of (2R)-CS-670 and (2S)-CS-670 were 75.9:1 and 249:1, respectively. Recently, Kitamura et al. (1996) noted that the taurine conjugate of M-5011 excreted in dog urine and feces exhibited only the S-configuration (15.4 and 13.5% of the dose was excreted in dog urine and feces, respectively). Tanaka et al. (1992) reported that a 2-arylpropionic acid glycine conjugate excreted in dog urine has the S-configuration. All of the aforementioned authors suggested that amino acid N-acyltransferase, rather than acyl-CoA ligase, was responsible for the stereoselective taurine and glycine conjugation of 2-arylpropionic acid derivatives. BOP-T excreted into rat bile, as reported here, mainly had the R-configuration. This is the first report of stereoselective taurine conjugation of 2-arylpropionic acid derivatives in rats, although, as noted above, such results have been described for dog and human urine. Although BOP was withdrawn from the market, we believe it to be a useful compound for the study of stereoselective amino acid conjugation mechanisms.

In conclusion, we identified BOP-T in rat bile and showed that approximately 2.5% of the BOP dose was excreted in bile, over a 4-hr period, as this newly confirmed metabolite. The configuration at the propionic acid moiety of BOP-T was mainly the R-configuration.