Stereoselective flunoxaprofen-S-acyl-glutathione thioester formation mediated by acyl-CoA formation in rat hepatocytes

Article abstract of DOI:10.1124/dmd.109.029371

Flunoxaprofen (FLX) is a chiral nonsteroidal anti-inflammatory drug that was withdrawn from clinical use because of concerns of potential hepatotoxicity. FLX undergoes highly stereoselective chiral inversion mediated through the FLX-S-acyl-CoA thioester (FLX-CoA) in favor of the (R)-(-)-isomer. Acyl-CoA thioester derivatives of acidic drugs are chemically reactive species that are known to transacylate protein nucleophiles and glutathione (GSH). In this study, we investigated the relationship between the stereo-selective metabolism of (R)-(-)- and (S)-(+)-FLX to FLX-CoA and the subsequent transacylation of GSH forming FLX-S-acyl-glutathione (FLX-SG) in incubations with rat hepatocytes in suspension. Thus, when hepatocytes (2 million cells/ml) were treated with (R)-(-)- or (S)-(+)-FLX (100 μM), both FLX-CoA and FLX-SG were detected by sensitive liquid chromatography-tandem mass spectrometry techniques. However, these derivatives were observed primarily from (R)-(-)-FLX incubation extracts, for which the formation rates of FLX-CoA and FLX-SG were rapid, reaching maximum concentrations of 42 and 2.8 nM, respectively, after 6 min of incubation. Incubations with (S)-(+)-FLX over 60 min displayed 8.1 and 2.7% as much FLX-CoA and FLX-SG area under the concentration versus time curves, respectively, compared with corresponding incubations with (R)-(-)-FLX. Coincubation of lauric acid (1000 μM) with (R)-(-)-FLX (10 μM) led to the complete inhibition of FLX-CoA formation and a 98% inhibition of FLX-SG formation. Reaction of authentic (R,S)-FLX-CoA (2 μM) with GSH (10 mM) in buffer (pH 7.4, 37°C) showed the quantitative formation of FLX-SG after 3 h of incubation. Together, these results demonstrate the stereoselective transacylation of GSH in hepatocyte incubations containing (R)-(-)-FLX, which is consistent with bioactivation by stereoselective (R)-FLX-CoA formation. Copyright

Full text of DOI:10.1124/dmd.109.029371

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0090-9556/10/3801-133–142$20.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
DMD 38:133–142, 2010
Vol. 38, No. 1
29371/3539094
Printed in U.S.A.
Stereoselective Flunoxaprofen-S-acyl-glutathione Thioester
Formation Mediated by Acyl-CoA Formation in Rat Hepatocytes
□
S
Mark P. Grillo, Jill C. M. Wait,1 Michelle Tadano Lohr,2 Smriti Khera, and Leslie Z. Benet
Departments of Pharmacokinetics and Drug Metabolism (M.P.G., J.C.M.W., M.T.L.) and Department of Medicinal Chemistry
(S.K.), Amgen Inc., South San Francisco, California; and Department of Biopharmaceutical Sciences, School of Pharmacy,
University of California, San Francisco, California (L.Z.B.)
Received July 7, 2009; accepted September 25, 2009
ABSTRACT:
Flunoxaprofen (FLX) is a chiral nonsteroidal anti-inflammatory drug primarily from (R)-(؊)-FLX incubation extracts, for which the for-
that was withdrawn from clinical use because of concerns of mation rates of FLX-CoA and FLX-SG were rapid, reaching maxi-
potential hepatotoxicity. FLX undergoes highly stereoselective mum concentrations of 42 and 2.8 nM, respectively, after 6 min of
chiral inversion mediated through the FLX-S-acyl-CoA thioester incubation. Incubations with (S)-(؉)-FLX over 60 min displayed 8.1
(FLX-CoA) in favor of the (R)-(؊)-isomer. Acyl-CoA thioester deriv- and 2.7% as much FLX-CoA and FLX-SG area under the concen-
atives of acidic drugs are chemically reactive species that are tration versus time curves, respectively, compared with corre-
known to transacylate protein nucleophiles and glutathione (GSH). sponding incubations with (R)-(؊)-FLX. Coincubation of lauric acid
In this study, we investigated the relationship between the stereo- (1000 ␮M) with (R)-(؊)-FLX (10 ␮M) led to the complete inhibition of
selective metabolism of (R)-(؊)- and (S)-(؉)-FLX to FLX-CoA and FLX-CoA formation and a 98% inhibition of FLX-SG formation.
the subsequent transacylation of GSH forming FLX-S-acyl-gluta- Reaction of authentic (R,S)-FLX-CoA (2 ␮M) with GSH (10 mM) in
thione (FLX-SG) in incubations with rat hepatocytes in suspension. buffer (pH 7.4, 37°C) showed the quantitative formation of FLX-SG
Thus, when hepatocytes (2 million cells/ml) were treated with (R)- after 3 h of incubation. Together, these results demonstrate the
(؊)- or (S)-(؉)-FLX (100 ␮M), both FLX-CoA and FLX-SG were stereoselective transacylation of GSH in hepatocyte incubations
detected by sensitive liquid chromatography-tandem mass spec- containing (R)-(؊)-FLX, which is consistent with bioactivation by
trometry techniques. However, these derivatives were observed stereoselective (R)-FLX-CoA formation.
Flunoxaprofen, [(S)-(ϩ)-2-[2-(4-fluorophenyl)-1,3-benzoxazol-5- have a chiral center at the carbon alpha to the carboxylic acid, where
the significant anti-inflammatory activity resides primarily in the
(S)-(ϩ)-isomer (Evans, 1992). FLX was marketed only in Italy and
specifically as the (S)-(ϩ)-isomer; however, it was withdrawn from
clinical use because of concerns of potential hepatotoxicity based on
its close-structural similarity to the previously withdrawn hepatotoxic
NSAID, benoxaprofen [(R,S)-2-[2-(4-chlorophenyl)-1,3-benzoxazol-
5-yl]propanoic acid, BNX] (Bakke et al., 1995). Other profen-type
NSAIDs that have been withdrawn from clinical use include indopro-
fen, suprofen, and pirprofen (Fung et al., 2001). The most frequent
types of adverse reactions leading to their discontinuation were idio-
syncratic allergic reactions including hepatotoxicity, nephrotoxicity,
blood dyscrasias, photosensitivities, allergic skin reactions, and ana-
phylaxis, sometimes associated with fever, rash, and eosinophilia
(Boelsterli et al., 1995).
yl]propanoic acid; FLX] (Fig. 1), is a nonsteroidal anti-inflammatory
drug (NSAID) that belongs to the 2-phenylpropionic acid (profen)
structural class of carboxylic acid-containing drugs used for the treat-
ment of inflammatory diseases (Williams et al., 1993). Profen drugs
This work was supported in part by the National Institutes of Health National
Institute of General Medical Sciences [Grant GM36633].
1 Current affiliation: BioMarin Pharmaceutical Inc., Novato, California.
2 Current affiliation: School of Pharmacy, University of California at San Fran-
cisco, San Francisco, California.
Parts of this work were previously presented at the following conference: Wait
JCM, Tadano-Lohr M, and Grillo MP (2005) Stereoselective bioactivation of
flunoxaprofen by Acyl-CoA formation. 13th North American ISSX/20th JSSX
meeting; 2005 Oct 23–27; Maui, Hawaii. International Society for the Study of
Xenobiotics, Washington, DC.
A remarkable metabolic feature of profen-type drugs is that they
can undergo stereoselective chiral inversion resulting in the (R)-
isomer being converted to the (S)-isomer (Caldwell et al., 1988;
Iwakawa et al., 1991). The profen chiral inversion process is known
to be initiated by acyl-CoA ligase-mediated conjugation with CoA,
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.029371.
□S The online version of this article (available at http://dmd.aspetjournals.org)
contains supplemental material.
ABBREVIATIONS: FLX, flunoxaprofen, (S)-(ϩ)-2-[2-(4-fluorophenyl)-1,3-benzoxazol-5-yl]propanoic acid; NSAID, nonsteroidal anti-inflammatory
drug; BNX, benoxaprofen, (R,S)-2-[2-(4-chlorophenyl)-1,3-benzoxazol-5-yl]propanoic acid; FLX-CoA, FLX-S-acyl-CoA thioester; FLX-1-O-G,
FLX-1-O-acyl glucuronide; GSH, glutathione; FLX-SG, FLX-S-acyl-glutathione thioester; CBZ, carbamazepine; LC, liquid chromatography;
MS/MS, tandem mass spectrometry; MS, mass spectrometry; CID, collision-induced dissociation; HPLC, high-performance liquid chromatog-
raphy; MRM, multiple reaction monitoring; I-CoA, (R,S)-ibuprofen-acyl-CoA; I-SG, ibuprofen-S-acyl-glutathione; P450, cytochrome P-450; AUC,
area under the curve.
133

134
GRILLO ET AL.
cratic immune-mediated hepatotoxic reaction (Benet et al., 1993;
F
Zia-Amirhosseini et al., 1995; Boelsterli, 2002; Dong et al., 2005;
Skonberg et al., 2008).
F
Acyl
glucuronidation
Acyl-CoA
formation
F
O
N
In the present studies, we investigated the ability of the (R)-(Ϫ)-
and (S)-(ϩ)-isomers of FLX to be metabolized stereoselectively to
FLX-CoA in incubations with freshly isolated rat hepatocytes and
subsequently through the transacylation-type reaction of FLX-CoA
with GSH to FLX-S-acyl-GSH (FLX-SG) (Fig. 1). We also performed
experiments examining the chemical reactivity of authentic FLX-CoA
with GSH forming FLX-SG in buffer under physiologic conditions.
In summary, these studies were focused on investigating the con-
tribution of FLX metabolism by acyl-CoA formation in the subse-
quent transacylation of GSH and therefore potentially the transacyla-
tion of hepatocellular protein nucleophiles, in incubations with freshly
isolated rat hepatocytes in suspension.
O
O
H3C
N
O
N
H
OH
(R)-(-)-FLX
H3C
H3C
O
O
GSH
H
H
O
O
CO2H
OH
SCoA
F
(R)-FLX-CoA
HO
OH
O
(R)-FLX-1-O-G
N
Chiral
inversion
H3C
H
O
S
O
H
F
F
HO2C
N
NH2
N
H
GSH
O
CO2H
Materials and Methods
O
O
FLX-SG
N
N
H
Materials. (R,S)-, (R)-(Ϫ)-, and (S)-(ϩ)-FLX were obtained from Ravizza
Laboratories (Milan, Italy) and are the same derivatives that were used in prior
studies (Iwakawa et al., 1991). The (R)-(Ϫ)-isomer contained 9.3% of the
(S)-(ϩ)-antipode, and the (S)-(ϩ)-isomer contained 3.8% of the (R)-(Ϫ)-
antipode. Carbamazepine (CBZ), GSH, lauric acid sodium salt, (Ϫ)-borneol,
and CoA were purchased from Sigma-Aldrich (St. Louis, MO). FLX-CoA and
FLX-SG were synthesized as described below. Ibuprofen-S-acyl-CoA
and ibuprofen-S-acyl-glutathione were available from previous studies (Grillo
and Hua, 2008). All solvents used for liquid chromatography (LC)-tandem
mass spectrometry (MS/MS) analysis were of chromatographic grade. Stock
solutions of (R)-(Ϫ)-FLX, (S)-(ϩ)-FLX, FLX-CoA, and FLX-SG were pre-
pared as solutions in acetonitrile-water (50:50, v/v; pH 5). All FLX-related
solutions were stored frozen (Ϫ20°C) and kept away from light to avoid photo
degradation.
H
O
O
F
H3C
H3C
O
O
CO2H
OH
SCoA
(S)-FLX-CoA
HO
O
N
OH
Acyl
glucuronidation
Acyl-CoA
formation
(S)-FLX-1-O-G
H
H3C
O
OH
(S)-(+)-FLX
FIG. 1. Proposed scheme for the metabolism of FLX by acyl glucuronidation and
acyl-CoA formation, leading to the stereoselective transacylation of GSH to form
FLX-SG.
followed by an epimerase-mediated chiral inversion of the acyl-CoA
intermediate and finally by nonstereoselective hydrolysis to the chiral
inverted free acid (Williams et al., 1993). The stereoselectivity of
profen chiral inversion is believed to be driven by the stereoselectivity
of profen-S-acyl-CoA thioester formation in favor of the (R)-isomer
(Caldwell et al., 1988; Xiaotao and Hall, 1993). Indeed, for FLX, in
vivo studies in rat with the (R)-(Ϫ)- and (S)-(ϩ)-isomers showed that
chiral inversion was highly stereoselective for the (R)-(Ϫ)-isomer
(Iwakawa et al., 1991), and, therefore, FLX was proposed to have
undergone highly stereoselective metabolism to (R)-FLX-S-acyl-CoA
[(R)-FLX-CoA] (Fig. 1). However, studies on the direct identification
of FLX-CoA formed in vitro have not been performed before the
present work. In contrast to undergoing (R)-isomer selective chiral
inversion, FLX undergoes stereoselective acyl glucuronidation to
FLX-1-O-acyl glucuronide (FLX-1-O-G) in vivo in rat by approxi-
mately 2-fold in favor of the (S)-(ϩ)-isomer (Iwakawa et al., 1991).
The FLX-1-O-G metabolite is known to be unstable and chemically
reactive and has been proposed to mediate the covalent binding of
FLX to hepatic and plasma proteins shown to occur in vivo in rat and
human (Dahms and Spahn-Langguth, 1996; Dong et al., 2005). Acyl
glucuronide metabolites of acidic drugs can react with protein by the
Instrumentation and Analytical Methods. The FLX-CoA and FLX-SG
derivatives were characterized by LC-MS/MS on a TSQ Quantum Discovery
Max mass spectrometer (Thermo Fisher Scientific, Waltham, MA) linked to an
Agilent 1100 high-performance liquid chromatograph (Agilent Technologies,
Santa Clara, CA) and a CTC HTS PAL autosampler (Leap Technologies,
Carrboro, NC). LC-MS/MS analysis of FLX-CoA, FLX-SG, and FLX-1-O-G
derivatives was performed with a reverse-phase column [Luna 5 ␮, C18(2),
100 Å, 150 ϫ 2.00 mm; Phenomenex, Torrance, CA] and eluted with a mobile
phase flow rate of 0.3 ml/min. The mobile phase used for the analysis of
FLX-CoA consisted of ammonium acetate (10 mM, pH 6.0) in water (solvent
A) and acetonitrile (solvent B). The mobile phase used for the analysis of
FLX-SG and FLX-1-O-G derivatives consisted of 0.1% formic acid in water
(solvent C) and 0.08% formic acid in acetonitrile (solvent D). For these
LC-MS/MS analyses, gradient elution was achieved by increasing solvent B or
solvent D from 0 to 95% over 13 min. Electrospray ionization was used with
the needle potential held at 4.5 kV. The MS/MS conditions used were 2 mtorr
argon collision gas and a collision potential of 25 eV. Positive ion mode
full-scan (m/z 50 to m/z 1200) LC-MS analysis was conducted with a scan time
of 0.73 s and source collision energy of 10 V. Xcalibur software (version 2.0;
Thermo Fisher Scientific) was used to acquire all data. 1H NMR spectra were
recorded on an Avance II spectrometer (Bruker Deltronics, Billerica, MA)
operating at 500 MHz and using a 5-mm, general-purpose, cryogenically
cooled probe (QNP CryoProbe; Bruker BioSpin Corporation, Fremont, CA).
transacylation of nucleophilic amino acid residues with the 1-O-acyl Chemical shifts are reported in parts per million as referenced to the residual
solvent peak (2.49 ppm for 2H6-dimethyl sulfoxide).
Synthesis of (R,S)-FLX-SG Thioester. (R,S)-FLX-SG was synthesized by
a conventional method using ethyl chloroformate, analogous to a procedure
reported previously for the synthesis of clofibryl-S-acyl-glutathione thioester
(Grillo and Benet, 2002). When the synthetic (R,S)-FLX-SG derivative, which
was obtained as a white solid (27% yield), was characterized by LC-MS with
glucuronide isomer and by glycation of lysine amino groups via the
open-chain aldehyde forms of acyl migration glucuronide isomers
(Faed, 1984; Spahn-Langguth and Benet, 1992). Acyl-CoA thioester
derivatives of carboxylic acid-containing drugs are also chemically
reactive species that can transacylate protein nucleophiles as well as
the nucleophilic cysteinyl thiol of GSH (Grillo and Benet, 2002;
use of the gradient elution method described above, it eluted at a retention time
Skonberg et al., 2008). A working hypothesis is that carboxylic acid
of 7.0 min and showed no detectable impurities when analyzed by both
drug-protein adducts, formed from the reaction of acyl glucuronides
and/or acyl-CoA thioester intermediates with protein nucleophiles, are
positive and negative ion scan modes. LC-MS/MS analysis of (R,S)-FLX-SG:
[collision-induced dissociation (CID) of MHϩ ion at m/z 575], m/z (%): m/z
recognized by the immune system as foreign, resulting in an idiosyn- 500 ([M ϩ H Ϫ Gly]ϩ, 32%), m/z 446 ([M ϩ H Ϫ pyroglutamic acid]ϩ, 21%),

STEREOSELECTIVE FLUNOXAPROFEN-S-ACYL-GLUTATHIONE FORMATION
135
m/z 428 ([M ϩ HϪ pyroglutamic acid Ϫ water]ϩ, 60%), m/z 371 ([FLX-S- acid sodium salt was prepared as a 100 mM solution in distilled water
CH2CH(NH2)C ϭ O]ϩ, 100%), m/z 343 ([FLX-S-CH2CH ϭ NH2]ϩ, 95%), (pH 7).
m/z 308 ([glutathione ϩ H]ϩ, 4%), m/z 240 ([2(4-fluorophenyl)-␣-methyl-5-
An experiment was performed to assess the stability of (R,S)-FLX-SG
ethylbenzoxazole]ϩ, 54%). 1H NMR analysis of (R,S)-FLX-SG (2H6-dimethyl
thioester (1 ␮M) in incubations with a rat hepatocytes (2 million cells/ml).
Aliquots (200 ␮l) from hepatocyte incubations (5-ml volume, n ϭ 2 replicates)
were taken at 0, 1, 2, 3, 4, 5, and 7 min, added to quench solution, and
processed as described above for the LC-MS detection of FLX-SG. Analysis
of the amount of FLX-SG remaining in the incubations was performed by
LC-MS (positive ion scan mode) detection and quantified by a linear standard
curve generated from (R,S)-FLX-SG (MHϩ m/z 575)/CBZ (MHϩ m/z 237)
peak area ratios obtained from extracted ion chromatograms.
Identification and Quantification of FLX-SG. Extracts of (R)-(Ϫ)- and
(S)-(ϩ)-FLX-treated rat hepatocyte incubations were analyzed by LC-MS/MS
for FLX-SG and CBZ by using the multiple reaction monitoring (MRM)
transitions MHϩ m/z 575 to m/z 240, for FLX-SG detection, and MHϩ m/z 237
to m/z 194, for CBZ detection, in the positive ion mode with use of the
chromatographic method described above. Authentic FLX-SG standard eluted
at a retention time of 7.0 min, whereas CBZ eluted at 7.6 min. The concen-
tration of FLX-SG thioester was determined from a linear standard curve
generated from FLX-SG/CBZ peak area ratios.
Identification and Quantification of FLX-CoA. Extracts of (R)-(Ϫ)- and
(S)-(ϩ)-FLX-treated rat hepatocyte incubations were analyzed by LC-MS/MS
for FLX-CoA and CBZ by using the MRM transitions MHϩ m/z 1035 to m/z
528 and MHϩ m/z 237 to m/z 194, respectively, in the positive ion mode and
by using the chromatographic method described above. Authentic (R,S)-FLX-
CoA standard eluted at a retention time of 5.5 min, whereas CBZ eluted at 6.2
min. The concentration of FLX-CoA thioester was determined from a linear
standard curve generated from FLX-CoA/CBZ peak area ratios.
Identification of FLX-1-O-G. The (R)- and (S)-FLX-1-O-G derivatives
were not obtained as authentic standards for these studies, but their formation
in incubations with hepatocytes was confirmed by treatment of (R)-(Ϫ)- and
(S)-(ϩ)-FLX (100 ␮M) incubation (2 million cells/ml, 10 min) extracts with
␤-glucuronidase. For carboxylic acid compounds, the ␤-glucuronidase enzyme
is known to specifically cleave 1-O-acyl-linked acyl glucuronides and not the
2-, 3-, or 4-O-acyl glucuronide migration isomers (Faed, 1984). Thus, (R)-(Ϫ)-
and (S)-(ϩ)-FLX hepatocyte incubation extracts (acetonitrile and formic acid,
1:1, 50 ␮l) were incubated with ␤-glucuronidase (1000 units/ml) for 0 and 30
min (2-ml total volume, pH 5.0, and 37°C, as per the manufacturer’s instruc-
tions) and then quenched by the addition of a solution containing acetonitrile,
3% formic acid, and 0.2 ␮M CBZ, followed by centrifugation (14,000 rpm, 10
min). Results from HPLC analysis of the resulting supernatants (reverse-phase
gradient elution as described above with UV analysis at 210 nm) of the 0- and
30-min incubation extracts showed that the HPLC peaks corresponding to
(R)-FLX-1-O-G (retention time ϳ10.6 min) and (S)-FLX-1-O-G (retention
time ϳ10.3 min) detected in the 0-min ␤-glucuronidase-treated extract, were
completely absent in the 30-min ␤-glucuronidase-treated extract (data not
shown). Therefore, these acyl glucuronides were designated as the FLX-1-O-G
metabolites of (R)-(Ϫ)- and (S)-(ϩ)-FLX. LC-MS/MS analysis showed mass
spectra that were nearly identical for the FLX-1-O-G derivatives obtained from
incubations with (R)-(Ϫ)- and (S)-(ϩ)-FLX: (CID of MHϩ ion at m/z 462), m/z
240 ([2(4-fluorophenyl)-␣-methyl-5-ethylbenzoxazole]ϩ, 20%), m/z 286
(FLX ϩ Hϩ, 100%) (Supplemental Fig. 3). Analysis for the formation of
FLX-1-O-G in incubations of (R)-(Ϫ)- or (S)-(ϩ)-FLX with rat hepatocytes
was performed by LC-MS/MS in the positive ion mode with MRM transitions
MHϩ m/z 462 to m/z 286 for FLX-1-O-G detection and MHϩ m/z 237 to m/z
194 for CBZ detection and with the same LC-MS/MS chromatography method
as described above for the analysis of FLX-SG.
sulfoxide): ␦ 1.49–1.50 (d, 3H, ␣-CH3), ␦ 1.75–1.95 (m, 2H, Glu-␤, Glu-␤Ј),
␦ 2.22–2.38 (m, 2H, Glu-␥,␥Ј), ␦ 2.85–2.95 (m, 2H, Cys-␤,␤Ј), ␦ 2.40–2.42,
2H, isopropyl-CH2), ␦ 3.44–3.48 (t, 1H, Glu-␣), ␦ 3.65–3.72 (m, 2H, Gly-
␣,␣Ј), ␦ 4.18–4.22 (d, 1H, FLX ␣-CH—), ␦ 4.30–4.41 (dt, 1H, Cys-␣), ␦
7.35–7.39 (dd, 1H, benzoxazole, para to N), ␦ 7.45–7.51 (m, 2H, fluoroben-
zene, meta to F), ␦ 7.74–7.78 (d, 2H, benzoxazole, meta and ortho to N),
8.24–8.28 (m, 2H, fluorobenzene, ortho to F), ␦ 8.42–8.47 (d, 1H, Cys NH),
␦ 8.64–8.67 (d, 1H, Gly NH).
Synthesis of (R,S)-FLX-CoA Thioester. (R,S)-FLX-CoA thioester was
obtained by a synthetic procedure using ethyl chloroformate and analogous to
that previously reported for the synthesis and purification of clofibryl-S-acyl-
CoA thioester (Grillo and Benet, 2002). The (R,S)-FLX-CoA thioester eluted
at a retention time of 5.5 min and showed no detectable impurities when
analyzed by both positive and negative ion LC-MS scan modes via reverse-
phase gradient elution as described above. LC-MS/MS analysis of synthetic
(R,S)-FLX-CoA by CID of the protonated molecular ion at MHϩ m/z 1035
yielded a product ion mass spectrum, m/z (%): m/z 608 ([M ϩ H Ϫ 427]ϩ,
13%), m/z 528 ([M ϩ H Ϫ 507]ϩ, 100%), m/z 428 ([adenosine diphosphate ϩ
2H]ϩ, 5%), m/z 426 ([M ϩ H Ϫ 609]ϩ, 5%), m/z 240 ([2(4-fluorophenyl)-␣-
methyl-5-ethylbenzoxazole]ϩ, 45%).
In Vitro Studies with Rat Hepatocytes. Freshly isolated hepatocytes were
prepared and incubated according to the method of Molde´us et al. (1978).
Hepatocytes were isolated from Sprague-Dawley rats (250–300 g, male;
Charles River Laboratories, Worcester, MA) and Ͼ95% viability was achieved
as assessed by trypan blue exclusion testing. Hepatocytes were warmed to
37°C in a water bath under an atmosphere of 95% O2 and 5% CO2 for 15 min
before the initiation of metabolism experiments. Incubations of hepatocytes (2
million viable cells/ml; 4–10 ml total volume; n ϭ 3) with (R)-(Ϫ)- and/or
(S)-(ϩ)-FLX isomers were performed in Krebs-Henseleit buffer (pH 7.4) in
20-ml glass vials and with continuous rotation under an atmosphere of 95% O2
and 5% CO2 at 37°C in a model 1927 humidified cell culture incubator (VWR,
Willard, OH).
For time-dependent studies, freshly isolated hepatocytes were incubated
with (R)-(Ϫ)- or (S)-(ϩ)-FLX (100 ␮M) and analyzed for FLX-CoA, FLX-SG,
and FLX-1-O-G formation over a 60-min time period. For the analysis of
FLX-SG and FLX-1-O-G derivatives, aliquots (200 ␮l) of the incubation
mixture were taken at 0.2, 4, 6, 8, 10, 20, 30, and 60 min and added directly
to microcentrifuge tubes (2 ml) containing a quench solution (200 ␮l) con-
sisting of acetonitrile, 3% formic acid, and 2 ␮M CBZ internal standard.
Samples then were centrifuged (14,000g, 5 min), and aliquots (300 ␮l) of the
supernatants were transferred to HPLC autosampler vials (0.4-ml polypro-
pylene; Sun International, Wilmington, NC) before LC-MS/MS analysis.
For the analysis of FLX-CoA formation, aliquots (200 ␮l) from the same
incubations as described above were taken and added to microcentrifuge tubes
(2 ml) containing a quench solution consisting of acetonitrile (without formic
acid) and CBZ (2 ␮M, 400 ␮l), followed by the addition of hexane (600 ␮l).
The samples then were vortex-mixed (1 min) and centrifuged (14,000g, 10
min), and aliquots (300 ␮l) of the aqueous layer were transferred to HPLC
autosampler vials for LC-MS/MS analysis of FLX-CoA.
Concentration-dependent experiments were performed with increasing con-
centrations of (R)-(Ϫ)-FLX or (S)-(ϩ)-FLX (3.90, 7.81, 15.6, 31.3, 62.5, 125,
and 250 ␮M) incubated with rat hepatocytes (2 million cells/ml) for 6 min and
processed as described above for LC-MS/MS analysis of FLX-SG and FLX-
CoA derivatives.
An inhibition experiment was performed with (R)-(Ϫ)-FLX (10 ␮M)
incubated with rat hepatocytes (2 million cells/ml) in the presence or
absence of (Ϫ)-borneol (100 ␮M), for the inhibition of FLX-1-O-G for-
mation, or lauric acid (1000 ␮M), for the inhibition of FLX-CoA formation.
Hepatocyte incubations (n ϭ 3) were performed for 6 min, and aliquots
were taken and processed as described above for the analysis of FLX-SG,
FLX-1-O-G, and FLX-CoA derivatives. A stock solution of (Ϫ)-borneol
(100 mM) was prepared in ethanol, and control incubations included the
same final concentration of ethanol (0.1%, v/v). A stock solution of lauric
Reactions of (R,S)-FLX-CoA with GSH in Buffer. Incubations (4 ml, n ϭ
3) containing both (R,S)-FLX-CoA (2 ␮M) and (R,S)-ibuprofen-acyl-CoA
(I-CoA, 2 ␮M, used as a direct comparator) thioesters were performed in
phosphate buffer (0.1 M, pH 7.4) at 37°C with 10 mM GSH in 20-ml glass
vials. Aliquots (200 ␮l) were removed from the incubations at 0, 5, 10, 20, 30,
60, 120, and 180 min and added to a quench solution (acetonitrile containing
3% formic acid and 2 ␮M CBZ, 200 ␮l) in a 96-well plate. The quenched
mixtures then were analyzed for FLX-SG (as described above) and ibuprofen-
S-acyl-glutathione (I-SG) concentrations by LC-MS/MS. The I-SG derivative
was detected using the MRM transition MHϩ m/z 496 to the major product ion
m/z 349 (Grillo and Hua, 2008).

136
GRILLO ET AL.
100
90
80
70
60
50
40
30
20
10
0
F
O
N
NH2
H
H3C
O
CH3
N
N
O
O
N
H3C
S
O
O
O
N
N
P
P
N
FIG. 2. Representative reverse-phase gradient LC-MS/MS MRM
chromatograms of FLX-CoA from the analysis of extracts from
untreated rat hepatocytes (A), rat hepatocytes incubated for 6 min
with 100 ␮M (S)-(ϩ)-FLX (B), rat hepatocytes incubated for 6-min
with 100 ␮M (R)-(Ϫ)-FLX (C), and extract from rat hepatocytes
spiked with (R,S)-FLX-CoA authentic standard (10 nM) (D). The
LC-MS/MS MRM transition used for the analysis of FLX-CoA was
MHϩ m/z 1035 to m/z 528. The relative abundances for each trace
on the y-axes are equal.
H
H
O
OH
H
O
O OH
OH
[M+H-507]+,528
O
OH
OH
FLX-CoA
O P
MH+ m/z 1035
OH
D)
C)
B)
A)
0
2
4
6
8
10
12
14
16
Retention Time (min)
Identification of Thioether-Linked FLX-GSH Adducts. Extracts of (R)-
(Ϫ)- and (S)-(ϩ)-FLX-treated rat hepatocyte incubations (60 min) were ana-
lyzed by LC-MS/MS for proposed GSH adducts potentially formed through
common cytochrome P450 (P450)-mediated bioactivation reactions by search-
ing for the respective mass changes of the drug moiety (Zheng et al., 2007).
Therefore, we examined extracts by LC-MS, using the same chromatography
methods as those used for the detection of FLX-SG, by positive ion scanning
for MHϩ ions at m/z 589, m/z 591, and m/z 609, which represent GSH adduct
compositions of FLX ϩ GSH ϩ oxygen Ϫ F, FLX ϩ GSH, and FLX ϩ
GSH ϩ oxygen, respectively. LC-MS/MS analysis of these extracts was
performed on these ions as described above for the analysis of FLX-SG.
formed in rat hepatocyte incubations (Fig. 2). The transition used for
this analysis was MHϩ m/z 1035 to m/z 528, which was chosen
because it is a major fragmentation pathway for authentic (R,S)-FLX-
CoA as assessed by positive ion LC-MS/MS CID of the MHϩ ion
(Fig. 3A). Reverse-phase LC-MS/MS analysis showed the presence of
FLX-CoA in incubations of (R)-(Ϫ)- and (S)-(ϩ)-FLX (100 ␮M) with
rat hepatocytes, which coeluted with authentic (R,S)-FLX-CoA stan-
dard at a retention time of 5.5 min (Fig. 2). FLX-CoA was also
detected in extracts from incubations with (S)-(ϩ)-FLX (Fig. 3B), but
at a level that was ϳ8% (relative LC-MS/MS peak area) as much as
detected at the 6-min time point compared with that detected in
extracts from incubations with (R)-(Ϫ)-FLX. LC-MS/MS analysis of
Results
Identification of FLX-CoA. Analysis of incubation extracts by FLX-CoA formed in hepatocytes provided a product ion spectrum
LC-MS/MS MRM detection allowed identification of FLX-CoA from CID of the MHϩ ion at m/z 1035 that was identical to the
528
528
100
90
80
70
60
50
40
30
20
10
100
90
80
70
60
50
40
30
20
10
B
A
F
O
240
O
N
608
NH2
N
428
H
H3C
CH3
O
N
N
O
O
H3C
S
O
P
O
P
N
N
N
H
H
O
OH
H
O
O OH
OH
[M+H-609]+, 426
[M+H-507]+,528
[M+H-409]+, 626
O
OH
OH
O P
OH
FLX-CoA
MH+ m/z 1035
1035
426
608
1035
426
428
608
240
240
341
428
158
626
909
0
0
100 200 300 400 500 600 700 800 900 1000
100 200 300 400 500 600 700 800 900 1000
m/z
m/z
FIG. 3. Tandem mass spectra of authentic (R,S)-FLX-CoA standard (A) and FLX-CoA (B) formed in rat hepatocyte incubations with (R)-(Ϫ)-FLX (100 ␮M, 6 min)
obtained by CID of the protonated molecular ion at m/z 1035. The origins of the characteristic fragment ions are as shown.

STEREOSELECTIVE FLUNOXAPROFEN-S-ACYL-GLUTATHIONE FORMATION
137
cytes, which coeluted with authentic (R,S)-FLX-SG standard at a
retention time of 7.0 min (Fig. 4). FLX-SG was also detected in
extracts from incubations with (S)-(ϩ)-FLX (Fig. 4B) but at a level
that was ϳ3% (relative LC-MS/MS peak area) as much as detected at
the 6-min time point compared with that detected in extracts from
incubations with (R)-(Ϫ)-FLX. LC-MS/MS analysis of FLX-SG
formed in hepatocytes provided a product ion spectrum that produced
fragment ions identical to the authentic (R,S)-FLX-SG standard and
also consistent with its chemical structure (Fig. 5) [see Baillie and
Davis (1993) for characteristic GSH adduct product ions from positive
ion electrospray LC-MS/MS analysis].
LC-MS/MS Detection of FLX-1-O-G. The 1-O-acyl glucuronides
of (R)-(Ϫ)- and (S)-(ϩ)-FLX were detected by positive ion LC-
MS/MS analysis of (R)-(Ϫ)- and (S)-(ϩ)-FLX rat hepatocyte (2
million cells/ml) incubation extracts. Results from these analyses
showed FLX-1-O-G eluting at retention times 10.6 and 10.3 min from
incubations with (R)-(Ϫ)- and (S)-(ϩ)-FLX, respectively (Supple-
mental Figs. 1 and 2). The order of elution of the FLX-1-O-G isomers,
for which the glucuronide formed from the (S)-(ϩ)-isomer eluted
earlier than the glucuronide of the (R)-(Ϫ)-isomer, is consistent with
the elution order shown in previous reports for other profen-1-O-acyl
glucuronides (el Mouelhi et al., 1987). LC-MS/MS analysis of FLX-
1-O-G metabolites formed in hepatocytes incubated with (R)-(Ϫ)- or
(S)-(ϩ)-FLX provided product ion spectra containing fragment ions
that were consistent with their chemical structures (Supplemental Fig.
3). The product ion spectrum for both derivatives showed that the
major fragment ion (100% relative abundance) upon CID of the MHϩ
ion at m/z 462 was m/z 286. Evidence of acyl migration isomers was
detected during the analysis of both (R)-(Ϫ)- and (S)-(ϩ)-FLX incu-
bation extracts (30- and 60-min time points) (Supplemental Figs. 1
and 2). For the (R)-(Ϫ)-FLX incubation extracts, the formation of at
least two acyl migration isomers was detected; they eluted before and
after the 1-O-acyl isomer at 9.9 and 11.2 min, respectively (Supple-
mental Fig. 1). Acyl migration seemed to occur less for the (S)-FLX-
1-O-acyl glucuronide, for which only one acyl migration isomer was
detected, eluting after (S)-FLX-1-O-G at 11.3 min (Supplemental Fig.
2). The increased instability of the (R)-FLX-1-O-acyl isomer com-
100
F
90
80
70
60
50
40
30
20
10
0
O
N
H
H3C
O
S
O
240
N
CO2H
O
H
NH
H2N
CO2H
FLX-SG
MH+ m/z 575
D)
C)
B)
A)
0
2
4
6
8
10
12
14
16
Retention Time (min)
FIG. 4. Representative reverse-phase gradient LC-MS/MS MRM chromatograms
of FLX-SG from the analysis of untreated rat hepatocytes (A), extract from rat
hepatocytes incubated for 6 min with 100 ␮M (S)-(ϩ)-FLX (B), extract from rat
hepatocytes incubated for 6 min with 100 ␮M (R)-(Ϫ)-FLX (C), and extract from
rat hepatocytes spiked with (R,S)-FLX-SG authentic standard (2 nM) (D). The
transition used for the LC-MS/MS MRM analysis of FLX-SG was MHϩ m/z 575 to
m/z 240. The relative abundances for each trace on the y-axes are equal.
authentic (R,S)-FLX-CoA standard and consistent with its chemical
structure (Fig. 3).
Identification of FLX-SG. Analysis of extracts by a sensitive
LC-MS/MS MRM detection technique allowed identification of
FLX-SG formed in rat hepatocyte incubations (Fig. 4). The transition
used for this analysis was MHϩ m/z 575 to m/z 240, which was chosen
because it is the major fragmentation pathway for FLX-SG as as-
sessed by positive ion LC-MS/MS CID of the MHϩ ion of authentic
(R,S)-FLX-SG (Fig. 5A). LC-MS/MS analysis showed the presence of
FLX-SG in incubations of (R)-(Ϫ)-FLX (100 ␮M) with rat hepato- pared with that of the (S)-antipode is consistent with our chemical
240
240
F
F
B
100
90
80
70
60
50
40
30
20
10
100
90
80
70
60
50
40
30
20
10
0
A
O
O
343
N
N
268
500
H
H3C
H
O
O
268
240H C
3
S
S
O
N
H
CO2H
O
343
NH
H2N
446
m/z 343
129
136
H2N
(-H2O, 428)
CO2H
FLX-SG
MH+ m/z 575
224
268
370
435
428
428
370
535
488
481
298
224
573
500
129
0
50 100 150 200 250 300 350 400 450 500 550 600
50 100 150 200 250 300 350 400 450 500 550 600
m/z
m/z
FIG. 5. Tandem mass spectra of authentic (R,S)-FLX-SG standard (A) and FLX-SG formed in rat hepatocyte incubations with (R)-(Ϫ)-FLX (100 ␮M, 6 min) obtained by
CID of the protonated molecular ion at m/z 575 (B). The origins of the characteristic fragment ions are as shown.

138
GRILLO ET AL.
stability reports on a range of profen 1-O-acyl glucuronides (Benet et for diclofenac-S-acyl-glutathione (Grillo et al., 2003) and not due to
al., 1993; Li et al., 2002a). ␥-glutamyltranspeptidase-mediated degradation, whereas the activity
Time Course of FLX-CoA, FLX-SG, and FLX-1-O-G Forma- of this enzyme is known to be negligible in rat liver tissue (Hinchman
tion in Incubations with Rat Hepatocytes. When (R)-(Ϫ)-FLX (100 and Ballatori, 1990). This rapid degradation is similar to that shown in
␮M) was incubated with hepatocytes, the formation of FLX-CoA was published reports with zomepirac-SG, diclofenac-SG, and ibupro-
rapid and reached a maximum concentration of 42 nM after only 6 fen-SG thioesters, for which the degradation rates were also rapid
min of incubation (Fig. 6A). The formation of FLX-CoA in incuba- (measured t1/2 values of 0.8, 1.0, and 4.0 min, respectively) (Grillo
tions with (S)-(ϩ)-FLX (100 ␮M) was much lower, whereas area and Hua, 2003, 2008; Grillo et al., 2003). More importantly, S-acyl-
under the curve (AUC0360 min) was 8.1% compared with the forma- glutathione thioesters are themselves chemically reactive species that
tion of FLX-CoA occurring in corresponding incubations with (R)- can transacylate nucleophiles such as N-acetylcysteine and therefore
(Ϫ)-FLX. Formation of FLX-SG in hepatocyte incubations with (R)- might also contribute to the transacylation of protein nucleophiles
(Ϫ)-FLX was also rapid and reached a maximum concentration of (Grillo and Benet, 2002); however, we did not characterize the chem-
ϳ2.8 nM at the 6-min time point (Fig. 6B). In contrast, incubations ical reactivity of FLX-SG in the present studies.
with (S)-(ϩ)-FLX showed significantly less AUC0360 min of FLX-
Concentration-Dependent Formation of FLX-CoA and FLX-SG
SG, forming ϳ2.7% of that produced in corresponding incubations in Incubations with Rat Hepatocytes. When rat hepatocytes (2
with (R)-(Ϫ)-FLX. For both (R)-(Ϫ)- and (S)-(ϩ)-FLX isomers in million cells/ml) were incubated for 6 min with increasing concen-
these hepatocyte incubations, no significant change in the initial trations of (R)-(Ϫ)-FLX, results showed a sharp concentration-depen-
substrate concentration was observed over the 60-min time period; dent formation of FLX-CoA from 3.90 to 62.5 ␮M (R)-(Ϫ)-FLX,
however, we were not able to distinguish between the (R)-(Ϫ)- and reaching 39.5 nM FLX-CoA (Fig. 7A). No statistically significant
(S)-(ϩ)-FLX isomers and the potential for chiral inversion in the increase in FLX-CoA formation was observed at concentrations of
present work (Fig. 6D). The time course of FLX-1-O-G formation (R)-(Ϫ)-FLX greater than 62.5 ␮M. From these same incubations,
observed from the analysis of extracts from hepatocyte incubations FLX-SG formation was also found to increase sharply with increasing
with (R)-(Ϫ)- and (S)-(ϩ)-FLX and based on FLX-1-O-G/CBZ peak concentrations of (R)-(Ϫ)-FLX (Fig. 7B). The concentration of
area ratios showed that the FLX-1-O-G formation rates were linear FLX-SG reached a maximum of ϳ2.6 nM at 62.5 ␮M (R)-(Ϫ)-FLX.
and identical over the first 20 min of incubation for these two isomers At greater than 62.5 ␮M (R)-(Ϫ)-FLX, the FLX-SG concentration did
(Fig. 6C). After 60 min of incubation, based on LC-MS/MS peak area not change significantly. A similar trend was observed for incubations
ratios, the relative amount of (R)-FLX-1-O-G formed in incubations conducted with the (S)-(ϩ)-FLX isomer, except that there was much
with (R)-(Ϫ)-FLX was ϳ60% of the (S)-FLX-1-O-G formed in incu- less FLX-CoA and FLX-SG detected. Based on the R/S ratio of
bations with (S)-(ϩ)-FLX. In addition to these time course data, the calculated AUC3.93250 ␮M values, the average amounts of FLX-CoA
time course of degradation of authentic (R,S)-FLX-SG (1 ␮M) in and FLX-SG formed in incubations with (S)-(ϩ)-FLX were 12.5 and
incubations with rat hepatocytes showed the derivative to be degraded 6.2%, respectively, as much as that formed in incubations with (R)-
in a very rapid fashion (t1/2 ϳ1.5 min) (Supplemental Fig. 4) and (Ϫ)-FLX. More importantly, a significant portion or all of the FLX-
almost completely cleared from the incubation by the 7-min time CoA and FLX-SG formed in incubations with the (S)-(ϩ)-FLX
point. The FLX-SG derivative was found to be hydrolyzed to FLX isomer may have come from the 3.8% (R)-(Ϫ)-FLX isomer con-
free acid in an almost quantitative fashion, similar to that determined tamination. For instance, hepatocyte incubations containing 100 ␮M
A
B
45
40
35
30
25
20
15
10
5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
(R)-(-)-FLX
(S)-(+)-FLX
(R)-(-)-FLX
(S)-(+)-FLX
FIG. 6. Time course for the formation of FLX-CoA (A), FLX-SG
(B), and FLX-1-O-G in rat hepatocytes (2 million cells/ml) incu-
bated with 100 ␮M (R)-(Ϫ)- or (S)-(ϩ)-FLX (C). D, concentration
of FLX free acid [(R)-(Ϫ)- and/or (S)-(ϩ)-FLX] over the 60-min
incubation. Values are expressed as the mean Ϯ S.D. from three
incubations.
0
0
10 20 30 40 50 60
Incubation Time (min)
0
10 20 30 40 50 60
Incubation Time (min)
C
D
0.5
110
100
90
80
70
60
50
40
30
20
10
0
0.4
0.3
0.2
0.1
0.0
(R)-(-)-FLX
(S)-(+)-FLX
(R)-FLX-1-O-G
(S)-FLX-1-O-G
0
10
20
30
40
50
60
0
10
20
30
40
50
60
Incubation Time (min)
Incubation Time (min)

STEREOSELECTIVE FLUNOXAPROFEN-S-ACYL-GLUTATHIONE FORMATION
139
A
B
45
40
35
30
25
20
15
10
5
3.5
3.0
2.5
2.0
FIG. 7. Concentration-dependent formation of FLX-CoA (A)
and FLX-SG (B) in rat hepatocytes (2 million cells/ml) incu-
bated with increasing concentrations of (R)-(Ϫ)- or (S)-(ϩ)-
FLX for 6 min. Values are expressed as the mean Ϯ S.D. from
three incubations.
(R)-FLX
(S)-FLX
1.5
1.0
0.5
0.0
(R)-FLX
(S)-FLX
0
µM
[(R)- or (S)-FLX], µM
[(R)- or (S)-FLX],
TABLE 1
Effect of inhibitors of FLX-1-O-G and FLX-CoA formation on the production of FLX-SG in incubations of (R)-(Ϫ)-FLX (10 ␮M) with rat hepatocytes (2 million cells/
ml, 6-min incubation time)
Values are expressed as the mean Ϯ S.D. from three incubations.
FLX-1-O-G
%Control
FLX-CoA
FLX-SG
Inhibitor
Conc.
Conc.
% Control
Conc.
%Control
nM
nM
nM
Control
(Ϫ)-Borneol (100 ␮M)
Lauric acid (1000 ␮M)
N.D.
N.D.
N.D.
24.0 Ϯ 1.20
24.1 Ϯ 4.60
0.00
1.60 Ϯ 0.50
1.57 Ϯ 0.26
0.04 Ϯ 0.06
2.50 Ϯ 0.50
12.5 Ϯ 0.70
100 Ϯ 19.2
98.4 Ϯ 16.1
2.3 Ϯ 4.1
0.00
N.D., not determined.
F
2.00
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
O
N
H
H
O
O
H3C
H3C
S-CoA
FLX-CoA
S-CoA
Ibuprofen-CoA
FIG. 8. Time-dependent formation of the respective S-acyl-
glutathione adducts obtained from the coincubation of (R,S)-
FLX-CoA (2 ␮M) and (R,S)-I-CoA (2 ␮M) with GSH (10 mM)
in potassium phosphate buffer (0.1 M, pH 7.4, 37°C). Values
are expressed as the mean Ϯ S.D. from three incubations.
Acyl-CoA (2
GSH (10 mM)
(pH 7.4, 37οC)
µ
M)
(R,S)-FLX-CoA
(R,S)-Ibuprofen-CoA
F
O
N
H
H3C
H
H3C
O
O
0
30 60 90 120 150 180
Incubation Time (min)
S-glutathione
S-glutathione
Ibuprofen-SG
FLX-SG
(S)-(ϩ)-FLX would have been contaminated with 3.8 ␮M (R)-(Ϫ)- each) with GSH (10 mM) in buffer (0.1 M potassium phosphate, pH
FLX isomer. Results from these incubations showed that the concen- 7.4, 37°C) resulted in a rapid transacylation of GSH forming ϳ1.0
trations of FLX-CoA and FLX-SG formed after 6 min of incubation ␮M FLX-SG and ϳ0.7 ␮M I-SG, respectively, after 20 min of
were ϳ50% of those formed in incubations with 3.9 ␮M (R)-(Ϫ)-FLX incubation (Fig. 8). The reaction of FLX-CoA with GSH forming
(Fig. 7).
FLX-SG occurred at a rate of 0.21 nmol/min over the first 20 min of
Inhibition Study. An inhibition experiment was performed with incubation, which was 1.5-fold more rapid than the reaction of (R,S)-
(R)-(Ϫ)-FLX (10 ␮M) in incubations with rat hepatocytes (2 million I-CoA forming I-SG over the same incubation time period. Both
cells/ml, 6 min) in the presence or absence of (Ϫ)-borneol (100 ␮M), (R,S)-FLX-CoA and (R,S)-I-CoA reacted quantitatively with GSH to
for the inhibition of FLX-1-O-G formation (Watkins and Klaassen, form the respective GSH thioesters by the 180-min incubation time
1982), or lauric acid (1000 ␮M), for the inhibition of FLX-CoA point.
formation (Xiaotao and Hall, 1993). Results showed that (Ϫ)-borneol
Identification of Thioether-Linked FLX-GSH Adducts. LC-
inhibited FLX-1-O-G formation by 98%; however, no significant MS/MS analysis of extracts from (R)-(Ϫ)- and (S)-(ϩ)-FLX-treated
inhibition of FLX-SG production was observed (Table 1). In contrast, rat hepatocyte incubations for thioether-linked GSH adducts poten-
coincubation of (R)-(Ϫ)-FLX with lauric acid led to a complete tially formed through common P450-mediated bioactivation pathways
inhibition of FLX-CoA formation and also to 98 Ϯ 4% inhibition of showed the presence of two isobaric GSH adducts, both having MHϩ
FLX-SG formation (Table 1).
ions at m/z 589, which is consistent with a GSH adduct composition
Reaction of GSH with (R,S)-FLX-CoA and (R,S)-I-CoA in consisting of FLX ϩ GSH (306 Da) ϩ oxygen (16 Da) Ϫ F (19 Da).
Buffer. Coincubation of (R,S)-FLX-CoA and (R,S)-I-CoA (2 ␮M LC-MS/MS analysis by CID of the MHϩ m/z 589 ion provided

140
GRILLO ET AL.
tandem mass spectra that were similar for both conjugates and con- their stereoselective metabolism to chemically reactive acyl-CoA
sistent with product ions usually observed for GSH adducts (Supple- thioester derivatives.
mental Fig. 5) (Baillie and Davis, 1993). The major fragment ion for
Another significant difference between these drugs was the dose
both conjugates was m/z 316 because of the loss of GSH (except for used clinically. Although we were unable to locate the dose of
the sulfur atom) by fragmentation between the cysteinyl-carbon-sulfur (S)-(ϩ)-FLX used before its withdrawal, the oral dose used in pub-
bond. The extracts from the(R)-(Ϫ)- and (S)-(ϩ)-FLX-treated rat lished human pharmacokinetic studies was 100 mg (Furlanut et al.,
hepatocyte incubations were then analyzed by LC-MS/MS MRM 1985; Segre et al., 1987; Dahms and Spahn-Langguth, 1996). In one
detection using the transition MHϩ m/z 589 to m/z 316 and provided pharmacokinetic study, the Cmax of (S)-(ϩ)-FLX (Priaxim, 100-mg
qualitative evidence showing that the adducts were ϳ10-fold more tablet) was ϳ7.3 ␮g/ml (Dahms and Spahn-Langguth, 1996), which
abundant (peak height comparison) in the (R)-(Ϫ)- compared with the was consistent with the Cmax of (S)-(ϩ)-FLX measured in elderly
(S)-(ϩ)-FLX hepatocyte incubation extracts (Fig. 9). Therefore, as subjects (Segre et al., 1987). In that same study, the Cmax values of
was the case for FLX-SG, thioether-linked GSH adduct formation was (R)-(Ϫ)- and (S)-(ϩ)-BNX after a single racemic 600-mg dose
also highly stereoselective for the (R)-FLX-(Ϫ)-isomer.
(coated tablet) were ϳ25 and 30 ␮g/ml, respectively (Dahms and
Spahn-Langguth, 1996). Therefore, the Cmax of (S)-(ϩ)-FLX was
ϳ13% as much as the combined Cmax values of (R)-(Ϫ)- and (S)-
(ϩ)-BNX. The hepatotoxicity caused by BNX was shown to occur in
patients taking 600 mg daily for 1 to 12 months (Lewis, 2003).
Therefore, the lower concentration of (S)-(ϩ)-FLX relative to that of
BNX under such dosing regimens may have contributed to the lack
of reported (S)-(ϩ)-FLX idiosyncratic toxicity compared with
BNX (Uetrecht, 2001).
The metabolism of FLX has been characterized in rat and in
humans; the major route of metabolism is acyl glucuronidation, which
exhibits a preference for the (S)-(ϩ)-FLX isomer in both rat and
human (Palatini et al., 1988; Iwakawa et al., 1991). In rats, FLX
undergoes extensive stereoselective chiral inversion in favor of the
(R)-(Ϫ)-FLX isomer being converted to the (S)-(ϩ)-FLX isomer.
After administration of (R)-(Ϫ)-FLX to rats, the exposure to the
(S)-(ϩ)-FLX isomer was ϳ1.5-fold greater than exposure to the
(R)-(Ϫ)-FLX-isomer, whereas after administration of the (S)-(ϩ)-
FLX isomer to rats, the exposure to the (R)-(Ϫ)-FLX isomer was only
ϳ2% compared with that of its antipode (Iwakawa et al., 1991). The
proposed stereoselective step for the overall chiral inversion process
occurs via the stereoselective formation of an (R)-FLX-CoA thioester
intermediate (Fig. 1); however, the formation of FLX-CoA has not
been directly identified before these studies.
Discussion
One hypothesis used to explain toxicities mediated by some car-
boxylic acid-containing NSAIDs is that the covalent modification of
tissue proteins by chemically reactive metabolites leads to the gener-
ation of immunogenic drug-protein adducts (Boelsterli, 2002). Reac-
tive metabolites in common for profen drugs include acyl glucu-
ronides and acyl-CoA thioesters (Skonberg et al., 2008). Studies have
shown that both of these derivatives are able to react with GSH and
protein, leading to thioester-, ester- and/or amide-linked covalent
adducts (Benet et al., 1993; Sallustio et al., 2000; Grillo and Benet,
2002; Grillo et al., 2003, Sidenius et al., 2004).
It is interesting to note that FLX was withdrawn from clinical
use because of its close structural similarity to BNX, even though
it was never shown to cause toxicity in humans (Bakke et al.,
1995). Major differences between these drugs are that BNX con-
tains a 4-chlorine atom instead of the 4-fluorine atom found in FLX
but also, more importantly, that BNX was dosed as the racemate,
unlike FLX, which was given only as the pharmacologically active
(S)-(ϩ)-isomer. We propose that the differences in hepatotoxic
potential between these two drugs may be related to differences in
In addition to the acyl glucuronidation pathway, bioactivation of
FLX by acyl-CoA formation has also been proposed to contribute to
the covalent binding of FLX to hepatocellular proteins, because it was
shown that upon inhibition of FLX acyl glucuronidation by coincu-
bation with (Ϫ)-borneol in rat hepatocyte cultures, covalent binding to
protein was inhibited by only 50% (Dong et al., 2005). The authors of
that study proposed that the FLX-CoA thioester might be respon-
sible for the covalent binding of FLX to hepatic proteins that was
not accounted for by the FLX acyl glucuronide metabolite. Be-
cause stereoselective differences in the bioactivation of (R)-(Ϫ)-
and (S)-(ϩ)-FLX isomers occur, we predicted that these differ-
ences might lead to stereoselective differences in transacylation of
GSH in studies with rat hepatocyte preparations. Therefore, we
proposed that if the formation of FLX-CoA represents an important
bioactivation route leading to the formation of a chemically reactive
acyl-linked intermediate, then results from in vitro GSH conjugate
detection studies in hepatocytes would show preferential FLX-SG
adduct formation in incubations containing the (R)-(Ϫ)-FLX isomer.
Results from the present studies showed that FLX (100 ␮M) was
metabolized stereoselectively in incubations with rat hepatocytes in
favor of the (R)-(Ϫ)-isomer to FLX-SG, which corresponded to the
high stereoselectivity of FLX-CoA formation in favor of the (R)-(Ϫ)-
isomer (Fig. 6). We also determined that (R,S)-FLX-CoA reacted
readily with GSH in phosphate buffer (pH 7.4 and 37°C), leading to
the formation of the FLX-SG thioester derivative (Fig. 8). We did not
perform experiments to characterize the relative rates of reaction of
100
HO2C
HN
NH2
90
80
70
60
50
40
30
20
10
O
O
316
S
NH
HO
CO2H
O
N
H
O
H3C
OH
Proposed FLX GSH adduct
MH+ m/ z 589
C)
B)
A)
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16
Retention Time (min)
FIG. 9. Representative reverse-phase gradient LC-MS/MS MRM chromatograms
of FLX-GSH thioether-linked adducts from the analysis of untreated rat hepatocytes
(A), extract from rat hepatocytes incubated for 60 min with 50 ␮M (S)-(ϩ)-FLX
(B), and extract from rat hepatocytes incubated for 60 min with 50 ␮M (R)-(Ϫ)-FLX
(C). The transition used for the LC-MS/MS MRM analysis of the thioether linked
FLX-GSH adducts (proposed structures shown) was MHϩ m/z 589 to m/z 316. The
relative abundances for each trace on the y-axes are equal.

STEREOSELECTIVE FLUNOXAPROFEN-S-ACYL-GLUTATHIONE FORMATION
141
the separate (R)-FLX- and (S)-FLX-CoA isomers in the present work; binding studies with 2-phenylpropionic acid, in which inhibition of
however, such studies with 2-phenylpropionic acid showed no differ- acyl glucuronidation had only a minor effect on covalent binding to
ences in the reactivity of the (R)- or (S)-2-phenylpropionic acid-S- protein (Li et al., 2002b). We are currently investigating the metab-
acyl-CoA derivatives with GSH (Li et al., 2002a).
olism of BNX to BNX-S-acyl-CoA and the ability to form varied
types of GSH adducts (no literature reports to date) in vitro in rat and
human hepatocytes and in vivo in rat.
In the present work, the importance of FLX-CoA formation leading
to the transacylation of GSH in incubations with hepatocytes was
clearly demonstrated from time- and concentration-dependent exper-
iments with the separate isomers in which a strong correlation be-
tween FLX-CoA and FLX-SG formation was observed (Figs. 6 and
7). The importance of the acyl-CoA pathway was confirmed further
when results from inhibition studies with lauric acid showed a com-
plete inhibition of both FLX-CoA and FLX-SG formation in incuba-
tions with rat hepatocytes (Table 1). We also determined that the
FLX-1-O-G metabolite, the formation of which was almost com-
pletely inhibited by coincubation with (Ϫ)-borneol, did not contribute
to the transacylation of GSH because there was no significant inhibi-
tion of FLX-SG formation. These results are consistent with analo-
gous experiments performed with (R)-(Ϫ)-ibuprofen (Grillo and Hua,
2008).
In summary, results from the present studies showed that the
metabolic activation of FLX is mediated by acyl-CoA formation and
not by acyl glucuronidation, leading to the transacylation of GSH to
form FLX-SG. Finally, from the results presented here, we propose
that metabolic activation of (R)-(Ϫ)-FLX by acyl-CoA formation may
also lead to covalent binding of the isomer to protein nucleophiles.
Unlike its structural congener, namely racemic BNX, we propose that
the lack of reported FLX toxicity in humans may have been due to the
clinical use of the (S)-(ϩ)-isomer and the lack of significant (S)-FLX-
CoA formation.
Acknowledgments. We thank Craig Uyeda, Stacy Fide, and Mat-
thew Mangels for assistance in the preparation of rat hepatocytes and
Robert Cho for assistance in mass spectrometry.
In addition to FLX-SG thioester detection, analysis of rat hepato-
cyte extracts from incubations with (R)-(Ϫ)-FLX and (S)-(ϩ)-FLX by
LC/MS led to the detection of two thioether-linked FLX-GSH adducts
(Fig. 9). These GSH adducts were isobaric, having MHϩ ions at m/z
589, which is consistent with the loss of the fluorine atom and the
addition of an oxygen atom and the elements of GSH to FLX.
Preliminary qualitative results indicated stereoselective bioactivation,
whereas an apparent ϳ10-fold increased abundance of the thioether-
linked adducts was detected in the (R)-(Ϫ)- compared with the (S)-
(ϩ)-FLX isomer incubation extracts (Fig. 9). We propose that these
adducts may be formed via an unstable epoxide intermediate that
reacts with GSH followed by elimination of HF (Tang et al., 2005;
Zheng et al., 2007). We have not confirmed the identity of these GSH
adducts; however, LC-MS/MS analysis (CID MHϩ m/z 589) provided
product ions consistent with their structures (Supplemental Fig. 5).
From these results, a proposal could be made that similar bioactivation
might be occurring for BNX and that covalent binding to protein in
the liver might occur by a combination of multiple bioactivation
pathways including P450-mediated oxidation, acyl glucuronidation,
and/or S-acyl-CoA-formation. However, in a recent report that
included studies on the covalent binding of [14C]BNX to protein in
NADPH-fortified human liver microsomes, results showed no detect-
able covalent binding to protein (Obach et al., 2008). In that same
report, two other carboxylic acid-containing drugs known to form
cytochrome P450-mediated reactive intermediates and corresponding
GSH adducts, namely diclofenac and tienilic acid, both showed
NADPH-dependent covalent binding to protein (Valadon et al., 1996;
Tang, 2003). Such results suggest that BNX may not be metabolized
by cytochrome P-450 to reactive intermediates (e.g., epoxides) that
react with hepatocyte protein. Further covalent binding studies per-
formed in incubations of BNX with human hepatocytes showed
measurable covalent binding of the drug to protein; however, neither
metabolism nor consumption of BNX was detected (Bauman et al.,
2009). We propose that the observed in vitro covalent binding to
human hepatocyte protein may have been mediated by BNX-S-acyl-
CoA; however, the S-acyl-CoA derivative was not searched for in by
Bauman et al. (2009) in their report. BNX acyl glucuronide is known
to be the major metabolite of BNX formed in humans; however, it was
shown not to be an important reactive intermediate when metabolic
activation experiments conducted in UDP-glucuronic acid-fortified
human liver S-9 fraction showed a complete blockage of covalent
binding to protein (Bauman et al., 2009). Such results are consistent
with observations obtained from mechanistic hepatocyte covalent
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Address correspondence to: Dr. Mark P. Grillo, Pharmacokinetics and Drug
Metabolism, Amgen Inc., South San Francisco, CA 94080. E-mail: grillo@amgen.com