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`DRUG METABOLISM AND DISPOSITION
`Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics
`
`Vol. 26, No. 7
`Printed in U.S.A.
`
`Short Communication
`
`Isolation and Identification of Bromfenac Glucoside From Rat Bile
`
`(Received August 13, 1997; accepted May 19, 1998)
`
`This paper is available online at http://www.dmd.org
`
`ABSTRACT:
`
`Bromfenac (Duract®), a drug approved for pain, was expected to
`be metabolized by the rat to an acyl glucuronide, a metabolite
`formed with most compounds of similar structure. During the in-
`vestigation of metabolite profiles in rat bile following administra-
`
`tion of 1 mg/kg iv doses of 14C-bromfenac, an acid-labile metab-
`olite was found that degraded to form 14C-bromfenac. Isolation
`and characterization of this metabolite indicated that it is an un-
`usual conjugate, bromfenac N-glucoside.
`
`Bromfenac (Duract®, 2-amino-3-(4-bromobenzoyl)benzene acetic
`acid), a compound approved for analgesia, has both analgesic and
`anti-pyretic properties (Sancilio et al., 1987). It is structurally similar
`to non-steroidal antiinflammatory drugs and thus contains a carboxyl
`group. Metabolism of these compounds generally includes formation
`of an acyl glucuronide at this carboxyl group, as is the case with the
`structurally similar tolmetin (Hyneck et al., 1988) and ketoprofen
`(Upton et al., 1980). Acyl glucuronides are labile compounds subject
`to hydrolysis in solutions of dilute alkali and at physiological pH
`conditions, yielding glucuronic acid and the aglycone (Ruelius et al.,
`1985). In preliminary studies in the rat, bromfenac was observed in
`bile after rigorous base hydrolysis (18 hr, 0.1 N NaOH, 37°C), and it
`was first concluded that bromfenac was formed by hydrolysis of an
`acyl glucuronide, although the glucuronide had not been observed in
`any of the acid-stabilized biological fluids examined. In order to
`determine the stability of the putative bromfenac acyl glucuronide
`metabolite, the present studies were undertaken to isolate and char-
`acterize this putative metabolite. In rat bile, after administration of a
`3 mg/kg iv dose of 14C-bromfenac, only one metabolite was observed,
`which formed bromfenac at acid or basic pH. Isolation and charac-
`terization of this compound indicated that the metabolite was brom-
`fenac N-glucoside.
`
`Materials and Methods
`14C-Bromfenac, labeled in the keto carbon, was obtained from Amersham
`International, Buckinghamshire, England, as the sodium salt. Bromfenac and
`its analogs AHR-10240, (lactam analog), AHR-11665 (benzoic acid analog),
`and AHR-11779 (ethyl ester analog) were obtained from the Wyeth-Ayerst
`Research in-house compound bank. Trimethylsilylglucose (TMS-glucose1)
`and bis(trimethylsilyl)trifluoroacetamide were obtained from Sigma Chemical
`Company, St. Louis, MO. All other reagents and solvents were reagent-grade
`or better.
`
`Dosing and Sample Collection. Three studies were conducted. In the first
`study, male bile-duct cannulated Sprague-Dawley rats (330 –375 g) were
`administered 3 mg/kg iv doses of 14C-bromfenac (as the sodium salt) dissolved
`in sterile distilled water. Bile was collected for 12 hr over wet ice into tubes
`containing nothing or 0.6 ml of 1M citric acid (pH 3.5). In the second study,
`all conditions were similar to the first, except that bile was collected into tubes
`containing 0.5 ml of 1M sodium phosphate buffer, pH 7.5. The third study was
`conducted in the same manner as the second, utilizing nonradioactive brom-
`fenac. All bile was stored frozen until analyzed.
`Hydrolysis. Bile was adjusted to pH 11 with 1 N NaOH and incubated at
`room temperature for 15 min to hydrolyze any acyl glucuronides present. Bile
`was then injected directly onto the HPLC immediately after the incubation
`period.
`HPLC. Bile metabolite profiles were determined by HPLC on a 5-micron
`Brownlee Spheri-5 RP-18 column (Applied Biosystems, Inc., Foster City, CA).
`The mobile phase was a linear gradient of 10% to 45% acetonitrile in ammo-
`nium acetate, pH 5, or sodium phosphate, pH 5. Detection of radioactivity
`utilized a Radiomatic Flo One b Model A500 flow detector and Ultima Flo M
`as scintillant (both from Packard Instrument Co., Meridan, CT). Ultraviolet
`absorbance was detected at 270 nm. A conjugate was isolated by HPLC using
`both small (2.1 mm) and standard (4.6 mm) bore columns. Conjugate peaks
`were collected directly from the ultraviolet detector and stored in mobile phase
`at 220°C until used.
`pH Stability of Conjugate. After adjustment of pH to 4, 5, 6, 7, and 8 with
`sodium phosphate buffer, aliquots of the isolated conjugate were incubated for
`2 hr at 37°C in a shaking water bath to assess stability. Samples were injected
`directly onto the HPLC system at the end of the incubation period.
`Preparation of Bromfenac Glucoside Ethyl Ester. Approximately 0.04
`mmol of isolated conjugate (97% pure, based on HPLC and detection at 270
`nm) was evaporated to dryness and redissolved in 100 ml of dimethylform-
`amide. After the addition of 20 ml of triethylamine and 0.5ml (;100-fold molar
`excess) of ethyl iodide, the reaction was stirred for 2 hr at room temperature.
`The reaction mixture was evaporated to dryness and the residue was redis-
`solved in acetonitrile in ammonium acetate, pH 7.5 (50:50). Yield was 72%,
`based on HPLC analysis and detection at 270 nm, with 23% remaining as
`unreacted conjugate. The ester was isolated by HPLC as described above,
`using a linear gradient of 10 to 80% acetonitrile in ammonium acetate, pH 7.5,
`over 40 min.
`Preparation of Isolated Conjugate for Mass Spectrometry. In addition to
`LC/MS analysis of the intact conjugate, the isolated conjugate was hydrolyzed
`to determine molecular weights of the aglycone and the conjugating moiety by
`mass spectrometry. For analysis of the aglycone, the conjugate solution in
`mobile phase was hydrolyzed by incubation for 15 min at pH 4 and 37°C. The
`resulting solution was adjusted to pH 7.5, desalted, concentrated, and analyzed
`720
`
`1 Abbreviations used are: TMS-glucose, trimethylsilylglucose; HPLC, high-
`performance liquid chromatography; LC/MS, liquid chromatography/mass spec-
`trometry; GC/MS, gas chromatography/mass spectrometry; ESI, electrospray
`ionization; CI, chemical
`ionization; CID, collision-induced dissociation; 5-ASA,
`5-aminosalicylic acid.
`
`Send reprint requests to: Sandra Kirkman Gonzales, Wyeth-Ayerst Research,
`CN 8000, Princeton, NJ 08543.
`
`

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`BROMFENAC GLUCOSIDE
`
`721
`
`by LC/MS. For analysis of the conjugating moiety, the conjugate solution in
`mobile phase was adjusted to pH 4 and incubated at 37°C for 30 min. The
`solution was evaporated to dryness and the residue redissolved in pyridine
`(dried over KOH pellets), silylated by the addition of freshly opened bis(tri-
`methylsilyl)trifluoroacetamide, and analyzed by GC/MS. Mass spectra were
`compared with those of standards of bromfenac and TMS-glucose.
`Mass Spectrometry. The intact conjugate and the aglycone were analyzed
`by ESI LC/MS on a Finnigan MAT TSQ-700 mass spectrometer (Finnigan
`Corp., San Jose, CA). HPLC was performed on a 5-micron Supelco LC-18-DB
`column (Supelco, Inc., Bellefonte, PA). Mobile phase was a linear gradient of
`acetonitrile:ammonium acetate, pH 7.5. Mass spectra were obtained at a scan
`rate of 1 sec/scan over a range of m/z 250 –750 and m/z 150 –500 for the
`bromfenac conjugate and the aglycone, respectively. The heated capillary
`temperature in the ion source was 225°C. The conjugating moiety (glucose)
`was analyzed by CI GC/MS on a Finnigan MAT SSQ-70 mass spectrometer.
`GC was performed on a DB5-MS column, 0.25 mm x 15 m, coated as 0.25 m
`film (J and W Scientific, Folsom, CA). GC injector temperature was 250°C.
`The column temperature program started at 80°C for 1 min, rose 20°C per min
`for 8.5 min, then remained constant at 250°C for 3 min, for a total run time of
`12.5 min. Mass spectra were obtained in positive-ion chemical ionization mode
`using ammonia gas. Source temperature was 148°C.
`The conjugate and its ethyl ester were analyzed by atmospheric pressure CI
`mass spectrometry on a Finnigan SSQ 710c mass spectrometer. A Michrom
`HPLC system (Michrom BioResources, Inc., Pleasanton, CA) was interfaced
`to the mass spectrometer with a mobile phase of methanol and water (80:20)
`pumped at a flow of 100 ml/min. The spectra were acquired at unit mass
`resolution by flow injection of sample in a solution of acetonitrile:water, 50:50
`(500 ng in 5 ml). The sample was sprayed into the mass spectrometer at
`vaporizer and heated capillary temperatures of 450 and 150 oC, respectively.
`Spectra were obtained at a scan rate of 2 sec/scan over a range of m/z 200 – 800.
`The electron multiplier and the conversion dynode were set at 1.0 kV and 215
`kV, respectively. The fragment ions were obtained using collision energy of 20
`or 30 eV in the ion source.
`
`Results
`All attempts at preparation of a standard of bromfenac acyl gluc-
`uronide using rat, beagle dog, cynomolgus monkey, and human he-
`patic microsomes supplemented with uridine 59-diphosphoglucuronic
`acid were unsuccessful, suggesting that the glucuronide does not form
`under physiological conditions.
`Biliary Metabolite Profiles of Bromfenac and its Metabolites.
`HPLC metabolite profiles of bile collected in citric acid exhibited a
`large peak at the retention time of the lactam analog of bromfenac
`(AHR-10240), but no bromfenac. Bromfenac has been shown to
`degrade to the lactam under conditions of acidic pH but was demon-
`strated to be stable at pH 9 to 11 under conditions similar to those
`required for base hydrolysis of acyl glucuronides. A peak correspond-
`ing to a bromfenac conjugate and exhibiting a retention time of
`approximately 26 –28 min was prominent in bile collected without pH
`adjustment at approximately pH 7 but was not present or present in
`small amounts in bile collected in citric acid at pH 3.5. In bile
`collected in phosphate buffer at pH 7.5, this peak at 26 –28 min was
`a major component of the profiles.
`Hydrolysis Studies. No bromfenac was observed after mild base
`hydrolysis of bile collected without pH adjustment, suggesting that an
`acyl glucuronide of bromfenac was not present. The peak at 26 –28
`min observed in bile collected without pH adjustment was not affected
`by these mild hydrolysis conditions. However, mild alkaline treatment
`of bile resulted in generation of large quantities of AHR-11665 in both
`acidified and non-acidified bile, suggesting the presence of an acyl
`glucuronide of AHR-11665, a benzoic acid metabolite of bromfenac
`(fig. 2). This AHR-11665 conjugate appeared to be at least moderately
`stable, as no AHR-11665 was apparent in bile collected without pH
`adjustment.
`Characterization of Bromfenac Glucoside: pH Stability Profile.
`
`Stability of the isolated 14C-bromfenac glucoside incubated at various
`pH, from 4 to 8, for 2 hr at 37°C was investigated. At pH 4, the
`compound was almost completely degraded to bromfenac, with some
`lactam also evident. At pH 6, approximately 50% of the conjugate was
`degraded to bromfenac, while 50% remained unchanged. At pH 8,
`very little degradation was noted; the profile is similar to that of the
`untreated control.
`Mass Spectrometry of Bromfenac Conjugate. In the ESI LC/MS
`spectrum of the 14C-bromfenac conjugate, the base peak was the
`molecular ion and was observed at m/z 496/498, [M-H]2. In the
`spectrum of the 14C-aglycone resulting from hydrolysis of the conju-
`gate, the molecular ion was also the base peak and was observed at
`m/z 334/336, [M-H]2. The only other prominent ion pair in the
`spectrum, resulting from loss of the carboxyl group, was observed at
`m/z 290/292, [M-COOH]2. The ESI LC/MS spectrum of a bromfenac
`standard was similar to that of the 14C-aglycone; the base peak was
`the molecular ion, which was observed at m/z 332/334, [M-H]2, and
`the loss of the carboxyl group was observed at m/z 288/290, [M-
`COOH]2. As the specific activity of the 14C-bromfenac utilized for
`this study was approximately 85%–90% of the theoretical maximum,
`an isotope effect was observed, resulting in a molecular ion for the
`radioactive compound 2 Da higher than that observed with the non-
`radioactive standard. With the exception of the 2-Da shift, the two
`spectra were identical, indicating that the aglycone from the isolated
`conjugate was 14C-bromfenac. The GC retention times of the TMS-
`glucose standard and the silylated conjugating moiety after hydrolysis
`of isolated conjugate were similar at 10.4 and 10.3 min, respectively.
`The CI GC/MS spectra of the silylated standard and hydrolyzed
`conjugate were nearly identical, with the molecular ion observed in
`both spectra at m/z 558, [M1NH4]1, representing the ammonia ad-
`duct of fully silylated glucose. Ions were also observed in both spectra
`at m/z 468, m/z 378, m/z 288, and m/z 198, representing loss of 1, 2,
`3, and 4 O-trimethylsilyl groups, respectively.
`Mass Spectrometry of Ethyl Esters. The atmospheric pressure CI
`mass spectra of bromfenac, AHR-11779, the non-radioactive isolated
`conjugate (purity 97%, based on HPLC analysis with detection at 270
`nm), and the ethyl ester of the isolated conjugate (purity .98%) are
`shown in fig. 1, panels A-D, respectively. In spectra (not shown) of
`the conjugate and its ethyl ester obtained without CID, the base peak
`in both spectra was the molecular ion, [M1H]1, at m/z 496/498 and
`m/z 524/526, respectively, with very weak fragment ions. In the CID
`spectra of bromfenac standard (fig. 1, panel A), fragments were
`observed at m/z 316/318 and m/z 288/290, representing loss of water
`and loss of the carboxyl moiety, respectively. In the CID spectra of
`AHR-11779, (fig. 1, panel B), fragments were again observed at m/z
`316/318 and m/z 288/290, representing loss of ethanol and loss of the
`ethyl ester moiety, respectively. The most prominent fragments in the
`CID spectra of the conjugate and its ethyl ester were observed at m/z
`334/336 and m/z 362/364, respectively, representing loss of the glu-
`1H]1. This fragmentation pattern indi-
`cose moiety, [MH-C6H11O5
`cated that conjugation is through an N-linkage, as it is expected that
`loss of the glucose moiety in an O-linked glucoside would result in
`loss of an additional oxygen from the carboxyl moiety, [MH-
`HOC6H11O5]1 at m/z 320/322 and m/z 348/350, respectively, as was
`observed in the loss of ethanol from the AHR-11779 (fig. 1, panel B).
`The fragmentation patterns of the conjugate and its ethyl ester (fig. 1,
`panels C, D) indicate that in the reaction of the conjugate with ethyl
`iodide, the ethyl group was added to the carboxyl moiety and not to
`the amine, as the CID mass spectrum of the ethyl ester of the
`conjugate is nearly identical to that of the authentic standard AHR-
`11779, the ethyl ester of bromfenac (fig. 1, panel B), providing further
`supporting evidence that the glucoside is N-linked.
`
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`FIG. 2. Metabolic pathways for bromfenac in rat bile.
`
`purify the conjugate. The lack of purity is evident
`spectrum of the conjugate (fig. 1, panel C).
`
`in the mass
`
`Discussion
`A survey of the literature indicated that approximately a dozen
`xenobiotics are known to form conjugates with glucose in mammalian
`systems (or models for mammalian systems), exhibiting both N- and
`O- linkages. The N-linked glucosides of 5-ASA (Tjørnelund et al.,
`1989), amobarbital (Tang and Kalow, 1978), and phenobarbital (Tang
`et al., 1979) were identified as major metabolites of these drugs in
`man. N-linkages were formed through primary and secondary amines,
`while O-linkages were formed through aromatic and aliphatic hy-
`droxyl groups, as well as carboxyl groups. The N-linked glucosides of
`sulfamethazine (Paulson et al., 1981), 4,49-methylenebis(2-chloroani-
`line) (Duggan et al., 1974), 3-(4-pyrimidinyl)-5-(4-pyridyl)-1,2,4-
`triazole (3,5 PPT; Manis and Braselton, 1986), and 5-ASA
`(Tjørnelund et al., 1989) were all found to be unstable in acid, and
`those of 3,5 PPT and 5-ASA were stable in base. The O-linked
`glucosides of hopantenic acid (Nakano et al., 1986) and furosemide
`(Hezari and Davis, 1993) were found to be stable in acid and that of
`furosemide was unstable in base. As has previously been noted
`(Tjørnelund et al., 1989), N-glucosides, as well as N-glucuronides,
`exhibit varying degrees of instability under acidic conditions and
`stability under basic conditions. Bromfenac glucoside was shown to
`be unstable under acidic conditions, suggesting that the glucoside
`linkage is through the amine group rather than the carboxyl group. A
`carboxyl O-linkage would be expected to result in a pH stability
`profile for the compound similar to those observed with acyl glucu-
`ronides, i.e. stable in acid, unstable in base. The formation of the ethyl
`ester of bromfenac glucoside by reaction with ethyl iodide, as evi-
`denced by a CID mass spectrum nearly identical to that of AHR-
`11779, the ethyl ester of bromfenac, provides additional evidence of
`the N-linkage for the glucose moiety. Proposed biotransformation
`pathways of bromfenac in rat bile are shown in fig. 2, although the
`metabolites presented are only a few of those observed in rat bile after
`administration of 14C-bromfenac, as the purpose of the experiments
`was to identify the bromfenac conjugate.
`The finding of a glucose conjugate of bromfenac, rather than the
`more common glucuronide conjugate, is unusual in two respects.
`First, compounds in this class are generally metabolized to glucu-
`ronides, and second, the conjugation is probably not linked to the
`carboxyl moiety. It is interesting to note that the configuration of the
`
`FIG. 1. Mass spectra of bromfenac (A), AHR-11779, the ethyl ester of bromfenac
`(B), bromfenac glucoside (C), and the ethyl ester of bromfenac glucoside (D).
`
`Attempts to obtain supporting nuclear magnetic resonance data for
`the N-linkage of the glucose moiety were unsuccessful, as a result of
`large amounts of lipid-like material present in the HPLC isolate.
`Because of the instability of the compound, we were unable to further
`
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`BROMFENAC GLUCOSIDE
`
`723
`
`only nonsteroidal antiinflammatory drug found to form a glucoside
`conjugate, 5-ASA, is structurally similar to bromfenac in that both
`compounds contain a primary amine group near the carboxyl group.
`Many other compounds in this class contain nitrogen, but no others
`containing a primary amine moiety were noted in the literature.
`Interestingly, AHR-11665 is structurally more similar to 5-ASA than
`is bromfenac, as the carboxyl groups in both AHR-11665 and 5-ASA
`are linked directly to the phenyl ring, but AHR-11665 apparently
`forms an acyl glucuronide (while the other compounds do not) and not
`an N-glucoside.
`
`Acknowledgments. We would like to thank William DeMaio,
`Robin Moore, Teresa Cinque, and Brent Kleintop for the mass spec-
`trometric analyses, and Jean Schmid for helpful discussions of brom-
`fenac derivatives.
`
`Wyeth-Ayerst Research
`Drug Safety and Metabolism Division
`
`SANDRA K. KIRKMAN
`MEI-YI ZHANG
`PETER M. HORWATT
`JOANN SCATINA
`
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