The nonenzymatic reactivity of the acyl-linked metabolites of mefenamic acid toward amino and thiol functional group bionucleophiles

Article abstract of DOI:10.1124/dmd.113.053223

Mefenamic acid (MFA), a carboxylic acid-containing nonsteroidal anti-inflammatory drug, is metabolized into the chemically-reactive MFA-1-O-acyl-glucuronide (MFA-1-O-G), MFA-acyl-adenylate (MFAAMP), and the MFA-S-acyl-coenzyme A (MFA-CoA), all of which are electrophilic and capable of acylating nucleophilic sites on biomolecules. In this study, we investigate the nonenzymatic ability of each MFA acyl-linked metabolite to transacylate amino and thiol functional groups on the acceptor biomolecules Gly, Tau,L-glutathione (GSH), and N-acetylcysteine (NAC). In vitro incubations with each of the MFA acyl-linked metabolites (1 mM) in buffer under physiologic conditions with Gly, Tau, GSH, or NAC (10 mM) revealed that MFA-CoA was 11.5- and 19.5-fold more reactive than MFA-AMP toward the acylation of cysteine-sulfhydryl groups of GSH and NAC, respectively. However, MFA-AMP was more reactive toward both Gly and Tau, 17.5-fold more reactive toward the N-acyl-amidation of taurine than its corresponding CoA thioester, while MFA-CoA displayed little reactivity toward glycine. Additionally, mefenamic acid-S-acyl-glutathione (MFA-GSH) was 5.6- and 108-fold more reactive toward NAC than MFA-CoA and MFA-AMP, respectively. In comparison with MFA-AMP and MFA-CoA, MFA-1-O-G was not significantly reactive toward all four bionucleophiles. MFA-AMP, MFA-CoA, MFA-1-O-G, MFA-GSH, and mefenamic acid-taurine were also detected in rat in vitro hepatocyte MFA (100 mM) incubations, while mefenamic acid-glycine was not. These results demonstrate that MFA-AMP selectively reacts with the amino functional groups of glycine and lysine nonenzymatically, MFA-CoA selectively reacts nonenzymatically with the thiol functional groups of GSH and NAC, and MFA-GSH reacts with the thiol functional group of GSH nonenzymatically, all of which may potentially elicit an idiosyncratic toxicity in vivo. Copyright

Full text of DOI:10.1124/dmd.113.053223

1521-009X/41/11/1923–1933$25.00
http://dx.doi.org/10.1124/dmd.113.053223
DRUG METABOLISM AND DISPOSITION
Drug Metab Dispos 41:1923–1933, November 2013
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
The Nonenzymatic Reactivity of the Acyl-Linked Metabolites of
Mefenamic Acid toward Amino and Thiol Functional
Group Bionucleophiles
Howard Horng and Leslie Z. Benet
Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, California
Received June 21, 2013; accepted August 23, 2013
ABSTRACT
Mefenamic acid (MFA), a carboxylic acid–containing nonsteroidal of taurine than its corresponding CoA thioester, while MFA-CoA
anti-inflammatory drug, is metabolized into the chemically-reactive displayed little reactivity toward glycine. Additionally, mefenamic
MFA-1-O-acyl-glucuronide (MFA-1-O-G), MFA-acyl-adenylate (MFA- acid-S-acyl-glutathione (MFA-GSH) was 5.6- and 108-fold more
AMP), and the MFA-S-acyl-coenzyme A (MFA-CoA), all of which are reactive toward NAC than MFA-CoA and MFA-AMP, respectively.
electrophilic and capable of acylating nucleophilic sites on bio- In comparison with MFA-AMP and MFA-CoA, MFA-1-O-G was not
molecules. In this study, we investigate the nonenzymatic ability
of each MFA acyl-linked metabolite to transacylate amino and
thiol functional groups on the acceptor biomolecules Gly, Tau,
significantly reactive toward all four bionucleophiles. MFA-AMP,
MFA-CoA, MFA-1-O-G, MFA-GSH, and mefenamic acid-taurine
were also detected in rat in vitro hepatocyte MFA (100 mM) in-
L-glutathione (GSH), and N-acetylcysteine (NAC). In vitro incuba- cubations, while mefenamic acid-glycine was not. These results
tions with each of the MFA acyl-linked metabolites (1 mM) in buffer demonstrate that MFA-AMP selectively reacts with the amino
under physiologic conditions with Gly, Tau, GSH, or NAC (10 mM)
revealed that MFA-CoA was 11.5- and 19.5-fold more reactive than
MFA-AMP toward the acylation of cysteine-sulfhydryl groups of GSH
functional groups of glycine and lysine nonenzymatically, MFA-CoA
selectively reacts nonenzymatically with the thiol functional groups
of GSH and NAC, and MFA-GSH reacts with the thiol functional group
and NAC, respectively. However, MFA-AMP was more reactive toward of GSH nonenzymatically, all of which may potentially elicit an
both Gly and Tau, 17.5-fold more reactive toward the N-acyl-amidation
idiosyncratic toxicity in vivo.
Introduction
glucuronides of acidic drugs are proposed to bind covalently to protein
via a direct transacylation reaction in which protein nucleophiles react
with the facile carbonyl-carbon of the acyl glucuronide, resulting in the
liberation of the glucuronic acid, and via the formation of a drug-protein
conjugate or a glycation mechanism involving prior acyl migration of
the drug on the glucuronic acid moiety permitting ring opening of the
sugar resulting in an exposed reactive aldehyde group that reversibly
forms an imine (Schiff base) with an amine group on proteins.
Subsequent Amadori rearrangement results in a stable ketoamine
Mefenamic acid (MFA), (29,39)-dimethyl-N-phenyl-anthranilic acid,
is a carboxylic acid-containing nonsterodial anti-inflammatory drug
associated with a rare, but sometimes serious idiosyncratic nephrotox-
icity (Robertson et al., 1980; Drury et al., 1981; Woods and Michael,
1981; Taha et al., 1985) and possibly hepatotoxicity (Somchit et al.,
2004). A proposed mechanism for the occurrence of these MFA-induced
toxicities suggests that MFA undergoes bioactivation into chemically-
reactive acyl-linked metabolites that ultimately become covalently bound
to tissue proteins, resulting in adverse immunologic responses (Fig. 1). derivative in which both the drug and the glucuronic acid moiety
MFA is metabolized to 3-hydroxy-MFA (Glazko, 1966) and 3-carboxy-
MFA (Sato et al., 1993) via CYP 2C9. MFA also undergoes glucuronidation
via uridine 59-diphospho-glucuronosyltransferase (UGT) into the un-
stable, reactive acyl glucuronide metabolite MFA-1-O-acyl-glucuronide
(MFA-1-O-G) (McGurk et al., 1996; Somchit et al., 2004). Acyl
become covalently bound onto the protein (Benet et al., 1993). MFA is
also metabolized into the reactive MFA-acyl-adenylate (MFA-AMP)
and MFA-S-acyl-coenzyme A (MFA-CoA) via acyl-CoA synthetase
(ACS), both of which have been demonstrated to form L-glutathione
(GSH)-adducts and are proposed to play a role in MFA-mediated
idiosyncratic toxicity (Grillo et al., 2012; Horng and Benet, 2013). This
pathway occurs when the AMP moiety of ATP is covalently
transferred to the carboxyl group of MFA to form MFA-AMP,
followed by the displacement of the AMP with coenzyme A (CoA) to
form MFA-CoA thioesters (Kelley and Vessey, 1994; Mano et al., 2001).
Upon their formation, the carbonyl carbons of both MFA-AMP and
This work was supported in part by the National Institutes of Health [Grant
GM36633]; and by the University of California, San Francisco, Liver Center Cell
Biology Core through the National Institutes of Health [Grant P30 DK026743]. The
authors declare no competing financial interests.
dx.doi.org/10.1124/dmd.113.053223.
ABBREVIATIONS: ACN, acetonitrile; CA, cholic acid; CBZ, carbamazepine; CID, collision-induced dissociation; CoA, coenzyme A; DMSO,
dimethylsulfoxide; ECF, ethyl chloroformate; ESI, electrospray ionization; GSH, L-glutathione; HPLC, high-performance liquid chromatography; Kpi,
potassium phosphate buffer; LC-MS/MS, liquid chromatography–tandem mass spectrometry; MFA, mefenamic acid; MFA-1-O-G, mefenamic acid-
1-O-acyl-glucuronide; MFA-AMP, mefenamic acid-acyl-adenylate; MFA-CoA, mefenamic acid-S-acyl-coenzyme A; MFA-Gly, mefenamic acid-
glycine; MFA-GSH, mefenamic acid-S-acyl-glutathione; MFA-NAC, mefenamic acid-N-acetylcysteine; MFA-Tau, mefenamic acid-taurine; NAC,
N-acetylcysteine; SRM, single-reaction monitoring; THF, tetrahydrofuran; Tol, tolmetin; ZP, zomepirac.
1923

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Horng and Benet
Fig. 1. Proposed conjugative bioactivation pathways of mefenamic acid.
MFA-CoA increase in electrophilicity, enabling them to transacylate MFA-1-O-G, and MFA-GSH (NAC only), as well as the detection of
the biologic nucleophile GSH (Horng and Benet, 2013). It is pro- these adducts in rat hepatocyte in vitro incubations. We propose that
posed that these drug-protein adducts could act as haptens and are MFA-AMP, MFA-CoA, and MFA-1-O-G are all selective toward their
recognized by the immune system as foreign, eliciting an autoimmune- acylation of nucleophilic functional groups on biologic molecules, all of
type response resulting in the associated idiosyncratic toxicity (Uetrecht, which can contribute to the formation of MFA adducts with amino acids,
2007). Previous in vitro incubations with the model nucleophile GSH peptides, and proteins. We also hypothesize that MFA-GSH is reactive in
under physiologic conditions showed MFA-AMP to be reactive toward its own right due to its structural similarity to MFA-CoA via the thioester
GSH, but 11-fold less reactive than MFA-CoA, while MFA-1-O-G bond. If this proposal is correct, the reactive acyl-linked metabolites of
exhibited little GSH reactivity (Horng and Benet, 2013). In vitro rat MFA would be anticipated to be selective in their formation of drug-
hepatocyte incubations have also resulted in the detection of MFA- protein adducts in vivo, which may potentially mediate the idiosyncratic
AMP, MFA-CoA, MFA-1-O-G, and mefenamic acid-S-acyl-glutathione toxicities associated with MFA and other carboxylic acid-containing
(MFA-GSH) (Horng and Benet, 2013), all of which could be more drugs.
reactive than MFA per se and potentially involved in the formation of
drug-protein adducts. Additionally, studies involving the acyl-linked
Materials and Methods
metabolites of the bile acid cholic acid (CA) revealed that CA-AMP
MFA, AMP, CoA, anhydrous tetrahydrofuran (THF), triethylamine, ethyl
selectively reacts nonenzymatically with amino functional groups while
chloroformate (ECF), N,N9-dicyclohexylcarbodiimide, pyridine, potassium
CA-CoA preferentially reacts nonenzymatically with thiol functional
phosphate, potassium carbonate, dimethylsulfoxide (DMSO), carbamazepine
(CBZ), GSH, Gly, NAC, and Tau were all purchased from Sigma-Aldrich (St.
Louis, MO). Acetonitrile (ACN), methanol, acetone, ammonium acetate, and
groups (Mitamura et al., 2011).
The following experiments were designed to characterize the non-
enzymatic acylation of the nucleophilic biomolecules containing the
amino functional groups of Gly and Tau and the thiol functional groups
ethyl acetate were all purchased from Fisher Scientific (Pittsburgh, PA). MFA-1-
O-G was purchased from Toronto Research Chemicals (TRC), Inc. (North York,
of GSH and N-acetylcysteine (NAC) by MFA-AMP, MFA-CoA, ON, Canada). All solvents used for high-performance liquid chromatography

Reactivity of the Acyl-Linked Metabolites of Mefenamic Acid
1925
(HPLC) and liquid chromatography–tandem mass spectrometry (LC-MS/MS) H – Tau]+, 99%), m/z 209 ([M + H – 140]+, 25%), m/z 180 ([M + H – 169]+,
analysis were of chromatographic grade. Williams’ Medium E and L-glutamine
16%), m/z 152 ([M + H – 197]+, 4%), and m/z 126 ([Tau + H+]+, 2%) (Fig. 3, A
were purchased from Gibco (Grand Island, NY). Male Sprague-Dawley rats were and B). MFA-NAC eluted at a retention time of 9.3 minutes (Fig. 4C) and
purchased from Charles River (Wilmington, MA). Stock solutions of MFA- showed no impurities when analyzed by HPLC/UV (wavelengths: 220, 254, 262,
AMP, MFA-CoA, MFA-GSH, MFA-1-O-G, mefenamic acid-glycine (MFA- and 280 nm) and LC-MS via reverse-phase gradient elution (as described above).
Gly), mefenamic acid-taurine (MFA-Tau), and mefenamic acid-N-acetylcysteine LC-MS/MS analysis of MFA-NAC (CID of MH+ ion at m/z 387), m/z (%): m/z
(MFA-NAC) were all prepared as 1 mM solutions in DMSO.
Instrumentation and Analytical Methods. HPLC/UV analysis was per-
309 ([M + H – 78]+, 30%), m/z 224 ([M + H – NAC]+, 99%), m/z 209 ([M + H –
178]+, 18%), m/z 180 ([M + H – 207]+, 13%), and m/z 165 ([NAC + H]+, 3%)
formed on a Hewlett Packard 1100 series binary pump HPLC system (Santa (Fig. 4, A and B).
Clara, CA) coupled to a Hewlett Packard 1100 UV-Vis detector, utilizing HP
Chemstation software for the acquisition of all HPLC/UV data. LC-MS/MS
Synthesis of MFA-CoA and MFA-GSH Thioester Derivatives. The
synthesis of MFA-CoA and MFA-GSH thioesters was accomplished by
analyses of synthetic standards and in vitro samples were performed on a method employing ECF as described previously (Stadtman and Elliott, 1957;
a Shimadzu LC-20AD (Kyoto, Japan) HPLC system coupled to an Applied Grillo and Benet, 2002; Horng and Benet, 2013). Briefly, MFA (1.6 mmol) was
Biosystem/MDS Sciex API (Framingham, MA) 4000 triple quadrupole mass dissolved in anhydrous THF (25 ml). While stirring at room temperature,
spectrometer outfitted with a Turbo V ion source using positive ionization triethylamine (1.6 mmol) was added to the solution followed by the addition of
mode. All LC-MS/MS analyses were performed on a reverse phase column
ECF (1.6 mmol). After 30 minutes, the resulting triethylamine hydrochloride
was removed by passing the reaction mixture through a glass funnel fitted with
(XTerra C-18, 5.0 mm, 4.6 Â 150 mm; Waters, Milford, MA). The detection of
MFA, MFA-AMP, MFA-CoA, MFA-1-O-G, MFA-GSH, MFA-Gly, MFA- a glass wool plug. The filtered solution was then added to a solution containing
Tau, and MFA-NAC were obtained using electrospray ionization (ESI) in
positive mode, and a gradient system of either aqueous ammonium acetate
(10 mM, pH 5.6) and acetonitrile (MFA-CoA) or aqueous solution (0.1%
CoA (0.13 mmol, 100 mg) or GSH (1 g) and KHCO3 (1.6 mmol) in nanopure
water (10 ml) and THF (15 ml). The solution was stirred continuously at room
temperature for 2 hours, after which the reaction was terminated by acidification
formic acid) and acetonitrile (0.1% formic acid) (MFA, MFA-AMP, MFA-1-O-G, (pH 4–5) through the addition of 1 M HCl. THF was then removed by
MFA-GSH, MFA-Gly, MFA-Tau, and MFA-NAC), 5% ACN to 100%, over
15 minutes at a flow rate of 0.5 ml/min. The high pH and ion strength afforded
by the aqueous ammonium acetate is necessary to elute MFA-CoA from the
column. Electrospray positive ionization was employed with a needle potential
held at 5.5 kV. Tandem mass spectrometry conditions used 2 mTorr argon
collision gas and a collision potential of 89 eV. Mass spectral data were
acquired with Analyst software (version 1.5.2; AB Sciex, Framingham, MA).
evaporation under N2 gas, followed by further solvent washes: acidified water
(pH 5) (3 Â 10 ml) and ethyl acetate (3 Â 10 ml) for MFA-CoA or acetone (3 Â
10 ml) for MFA-GSH. MFA-CoA and MFA-GSH precipitate was blown down to
dryness using N2 gas and then weighed out for preparation of a 1-mM MFA-CoA
or 1-mM MFA-GSH solution in DMSO. HPLC analysis of MFA-CoA thioester
resulted in an elution time of 7.3 minutes and showed no impurities when
analyzed by HPLC/UV (wavelengths: 220, 254, 262, and 280 nm) and LC-MS
Synthesis of MFA-AMP, MFA-Gly, MFA-NAC, and MFA-Tau Deriva- via reverse-phase gradient elution (as described above). LC-MS/MS analysis of
tives. The synthesis of MFA-AMP, MFA-Gly, MFA-NAC, and MFA-Tau was MFA-CoA standard yielded (CID of MH+ ion at m/z 991), m/z (%): m/z 582 ([M
carried out with a solution consisting of 110 mg N,N9-dicyclohexylcarbodiimide + H – adenosine diphosphate – H2O]+, 20%), m/z 484 ([M + H – adenosine
in 0.4 ml pyridine (Ikegawa et al., 1999; Horng and Benet, 2013). Briefly, an N, triphosphate]+, 94%), m/z 428 ([adenosine diphosphate + H+]+, 40%), m/z 382
N9-dicyclohexylcarbodiimide solution was added to a solution containing MFA ([M + H – 609]+, 25%), m/z 330 ([adenosine monophosphate + H – H2O]+, 3%),
(0.49 mmol), and either AMP, Gly, Tau, or NAC (0.49 mmol) separately in 75% m/z 224 ([M + H – CoA]+, 99%). Synthetic MFA-GSH eluted at a retention time
pyridine/25% water. The reaction mixture was stirred at 4°C for 7 hours and then of 7.7 minutes and showed no detectable impurities when analyzed by HPLC/UV
centrifuged at 3000g for 5 minutes to remove any N-acylurea derivatives. The (wavelengths: 220, 254, 262, and 280 nm) and LC-MS via reverse-phase gradient
supernatant was transferred to another culture tube for precipitation by the elution (as described above). LC-MS/MS analysis of MFA-GSH standard yielded
addition of acetone (10 ml). The resulting precipitate was isolated by cen- product in mass spectrum under CID of the protonated molecular ion at MH+ m/z
trifugation at 3000g for 5 minutes followed by further washes with acetone (10 Â 531, m/z (%): m/z 456 ([M + H – GSH]+, 10%), m/z 384 ([M + H – pyroglutamic
10 ml) and acidified water (pH 4–5) (10 Â 10 ml). For MFA-AMP, the acid – water]+, 82%), m/z 224 ([MFA + H – H2O]+, 73%).
precipitate was dissolved in 0.1 M potassium phosphate buffer (pH 6) and
Stability and Reactivity Incubation Conditions and Quantitative
underwent continued liquid-liquid washes with ethyl acetate (10 Â 10 ml). Analysis of Reaction Products. Chemical stability was assessed by incubating
Following precipitation via 1 M HCl, the MFA-AMP was further washed with MFA-AMP, MFA-CoA, MFA-GSH, MFA-Gly, MFA-Tau, and MFA-NAC
acetone (10 Â 10 ml). The MFA-AMP precipitate was -down to dryness using (1 mM) with CBZ (internal standard) in 0.1 M potassium phosphate buffer
N2 gas and weighed out for preparation of a 1 mM MFA-AMP solution in (Kpi) (pH 7.4) in 2-ml HPLC vials (n = 3). Each solution was then placed into
DMSO. For MFA-Gly, MFA-NAC, and MFA-Tau, the initial acetone-derived an HPLC autosampler warmed to 37°C and injections were taken every 15
precipitate was dissolved in DMSO and subjected to purification via HPLC/UV- minutes for 3 or 24 hours for LC-MS/MS analysis to determine each metabolite’s
mass spectrometry. The correct HPLC eluent fractions, as determined by chemical stability. The stability of each sample was determined by comparing the
UV-MS, of each acyl-linked metabolite were collected, blown down to dryness, analyte peak-area-to-peak-area ratios of CBZ, which we previously found to be
weighed, and then prepared as 1-mM solutions in DMSO. MFA-AMP eluted at stable for at least 72 hours (data not shown). Chemical reactivity experiments for
a retention time of 7.6 minutes and showed no impurities when analyzed by MFA-AMP, MFA-CoA, and MFA-1-O-G were performed by incubating each
HPLC/UV (wavelengths: 220, 254, 262, and 280 nm) and LC-MS via reverse- acyl-linked metabolite (1 mM) separately in 0.1 M Kpi (pH 7.4) containing Gly,
phase gradient elution (as described above), and 1H-NMR (Horng and Benet, Tau, GSH, or NAC and MFA-GSH with NAC (10 mM) (n = 3) at 37°C in
2013). LC-MS/MS analysis of MFA-AMP revealed collision-induced dissoci- screw-capped glass vials in a shaking incubator (Fig. 5). Aliquots (100 ml) of the
ation (CID) of MH+ ion at m/z 571, m/z (%) yielded: m/z 224 ([M + H – AMP]+, incubation mixture were taken at 0, 2, 5, 10, 30, and 60 minutes and quenched
100%), m/z 207 ([M + H – 364]+, 25%), and m/z 136 ([M + H – adenine]+, 28%). with 1 mM CBZ/ACN solution and then injected onto the column for LC-MS/
MFA-Gly eluted at a retention time of 8.7 minutes (Fig. 2C) and showed no MS analyses. Quantitative measurements were performed by plotting peak area
impurities when analyzed by HPLC/UV (wavelengths: 220, 254, 262, and 280 ratios of MFA-GSH, MFA-Gly, MFA-Tau, or MFA-NAC to CBZ versus the
nm) and LC-MS via reverse-phase gradient elution (as described above). LC-MS/ concentration of each acyl-linked MFA metabolite.
MS analysis of MFA-Gly (CID of MH+ ion at m/z 299), m/z (%): m/z 224 ([M +
In Vitro Studies with Rat Hepatocytes. Freshly isolated rat (250–300 g,
H – Gly]+, 99%), m/z 209 ([M + H – 90]+, 20%), m/z 180 ([M + H – 119]+, male Sprague-Dawley) hepatocytes were prepared according to the method of
18%), m/z 152 ([M + H – 147]+, 4%), m/z 127 ([M + H – 172]+, 2%), m/z 77 Irving et al. (1984) and greater than 85% viability was achieved routinely as
([Gly + H]+, 1%) (Fig. 2, A and B). MFA-Tau eluted at a retention time of 9.1 determined by trypan blue exclusion testing. Incubations of hepatocytes (2
minutes (Fig. 3C) and showed no impurities when analyzed by HPLC/UV million viable cells/ml) with MFA (100 mM) were performed in Williams’
(wavelengths: 220, 254, 262, and 280 nm) and LC-MS via reverse-phase Medium E fortified with L-glutamine (4 mM) in a 50-ml round bottom flask.
gradient elution (as described above). LC-MS/MS analysis of MFA-Tau (CID of Incubations (n = 3) were performed with continuous rotation and gassed with
MH+ ion at m/z 349), m/z (%): m/z 332 ([M + H – H2O]+, 10%), m/z 224 ([M +
95% O2/5% CO2 at 37°C. Aliquots were taken at 0, 0.2, 0.5, 1, 2, 4, 8, 10, 20,

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Horng and Benet
Fig. 2. Proposed identities of the fragment ions of MFA-Gly (A), tandem mass spectrum (B), and representative reverse-phase gradient LC-MS/MS SRM (m/z 299 to m/z
224) (C) of MFA-Gly authentic standard.
30, and 60 minutes and analyzed for MFA-AMP, MFA-CoA, MFA-GSH,
MFA-1-O-G, MFA-Gly, MFA-Tau, and MFA-NAC by LC-MS/MS.
For the analyses of MFA-AMP, MFA-GSH, MFA-1-O-G, MFA-Gly, MFA-
directly into microcentrifuge tubes (2 ml) followed by quenching with a
solution of ACN containing 3% formic acid/2 mM CBZ (200 ml). Samples were
then centrifuged (14,000g, 5 minutes) and the supernatant fractions (200 ml)
Tau, and MFA-NAC, aliquots (200 ml) of the incubation mixture were added were transferred to HPLC autosampler vials for LC-MS/MS analysis.

Reactivity of the Acyl-Linked Metabolites of Mefenamic Acid
1927
Fig. 3. Proposed identities of the fragment ion of MFA-Tau (A), tandem mass spectrum (B), and representative reverse-phase gradient LC-MS/MS SRM (m/z 349 to m/z
224) (C) of MFA-Tau authentic standard.
For the analyses of MFA-CoA formation, aliquots (200 ml) from the same
incubations were transferred directly into microcentrifuge tubes and quenched
with a solution of ACN/2 mM CBZ (400 ml) followed by the addition
of hexane (600 ml). The samples were vortexed (1 minute), centrifuged
(14,000g, 5 minutes), and aliquots (300 ml) of the aqueous layer were
transferred to an HPLC autosampler vial followed by a 1-hour evaporation of
residual hexane under the fume hood. Samples were then analyzed by LC-
MS/MS.

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Horng and Benet
Fig. 4. Proposed identities of the fragment
ions of MFA-NAC (A), tandem mass spectrum
(B), and representative reverse-phase gradient
LC-MS/MS SRM (m/z 387 to m/z 224) (C) of
MFA-NAC authentic standard.
Identification and Quantification of MFA-AMP, MFA-CoA, MFA- transitions: MH+ m/z 571 to m/z 224 (MFA-AMP), MH+ m/z 991 to m/z 224
GSH, and MFA-1-O-G. MFA-treated rat hepatocyte extracts were analyzed by (MFA-CoA), MH+ m/z 531 to m/z 224 (MFA-GSH), MH+ m/z 418 to m/z 224
LC-MS/MS for MFA-AMP, MFA-CoA, MFA-GSH, and MFA-1-O-G as (MFA-1-O-G), and MH+ m/z 237 to m/z 194 for CBZ. The elution times of
previously described (Horng and Benet, 2013). Single-reaction monitoring
(SRM) in positive ionization mode with the chromatographic conditions
described above were used for the quantitation with the following mass
each acyl-linked metabolite were as follows: 7.6 minutes (MFA-AMP), 7.3
minutes (MFA-CoA), 7.7 minutes (MFA-GSH), 7.9 minutes (MFA-1-O-G),
and 9.3 minutes (CBZ). No chromatographic peaks corresponding to each acyl-

Reactivity of the Acyl-Linked Metabolites of Mefenamic Acid
1929
Fig. 5. Scheme of the transacylation of MFA-AMP, MFA-CoA, and MFA-1-O-G with the bionucleophiles Gly, Tau, GSH, and NAC in buffer (0.1 M Kpi; pH 7.4, 37°C).
linked metabolite were detected in blank incubation extracts lacking MFA. The
were obtained for authentic MFA-NAC and CBZ, respectively. No chromato-
concentration of each MFA-acyl–linked metabolite was determined by plotting graphic peaks corresponding to MFA-NAC were detected in blank incubation
peak area ratios of each metabolite to CBZ versus the concentration.
Identification and Quantification of MFA-Gly. The identification and
extracts lacking MFA. The concentration of MFA-NAC was determined by
plotting peak area ratios of MFA-NAC to CBZ versus the concentration of
quantitation of MFA-Gly by LC-MS/MS was carried out by SRM using the MFA-NAC.
mass transitions MH+ m/z 299 to m/z 224 and MH+ m/z 237 to m/z 194 for CBZ
detection using ESI in positive mode and the chromatographic methods
described above. The elution times of 8.7 minutes (Fig. 2C) and 9.3 minutes
were obtained for authentic MFA-Gly and CBZ, respectively. No chromato-
Results
Identification of MFA-AMP, MFA-CoA, MFA-1-O-G, and
MFA-GSH. Analysis of rat hepatocyte extracts incubated with MFA
by LC-MS/MS detection allowed for the identification of MFA-AMP,
MFA-CoA, MFA-1-O-G, and MFA-GSH formed in incubations with
MFA, as previously described (Horng and Benet, 2013). MFA-AMP,
MFA-CoA, MFA-GSH, and MFA-1-O-G formed in rat hepatocyte
extracts and authentic standards coeluted at retention times of 7.6, 7.3,
graphic peaks corresponding to MFA-Gly were detected in blank incubation
extracts lacking MFA. The concentration of MFA-Gly was determined by
plotting peak area ratios of MFA-Gly to CBZ versus the concentration of MFA-
Gly.
Identification and Quantification of MFA-Tau. The identification and
quantitation of MFA-Tau by LC-MS/MS was carried out by SRM using the
mass transitions MH+ m/z 349 to m/z 224 and MH+ m/z 237 to m/z 194 for CBZ
detection using ESI in positive mode and the chromatographic methods 7.7, and 7.9 minutes, respectively, and the product ion spectrum of each
described above. The elution times of 9.1 minutes (Fig. 3C) and 9.3 minutes
were obtained for authentic MFA-Tau and CBZ, respectively. No chromato-
graphic peaks corresponding to MFA-Tau were detected in blank incubation
extracts lacking MFA. The concentration of MFA-Tau was determined by
plotting peak area ratios of MFA-Tau to CBZ versus the concentration of MFA-
Tau.
Identification and Quantification of MFA-NAC. The identification and
quantitation of MFA-NAC by LC-MS/MS was carried out by SRM using the
mass transitions MH+ m/z 387 to m/z 224 and MH+ m/z 237 to m/z 194 for CBZ
detection using ESI in positive mode and the chromatographic methods
conjugate was consistent with its chemical structure and identical to its
corresponding authentic standard.
Identification of MFA-Tau. Analysis of rat hepatocyte extracts
incubated with MFA by LC-MS/MS detection allowed for the iden-
tification of MFA-Tau formed in incubations with MFA. MFA-Tau
formed in rat hepatocyte extracts and authentic standard coeluted at
a retention time of 9.1 minutes (Fig. 3C) and the product ion spectrum
of the conjugate was consistent with its chemical structure and
identical to its corresponding authentic standard. MFA-Gly and MFA-
described above. The elution times of 9.25 minutes (Fig. 4C) and 9.3 minutes NAC were not detected in MFA rat hepatocyte incubations.

1930
Horng and Benet
Chemical Stability of MFA-CoA, MFA-AMP, MFA-GSH,
MFA-NAC, MFA-Gly, and MFA-Tau in Buffer. In vitro incubation
of each acyl-linked metabolite of MFA in Kpi under physiologic
conditions (pH 7.4, 37°C) revealed that MFA-AMP, MFA-CoA, and
MFA-GSH were chemically stable for at least 24 hours, while MFA-
NAC, MFA-Gly, and MFA-Tau were chemically stable with no
detectable hydrolysis for at least 3 hours of incubation (data not
shown). Previous studies carried out under the same conditions have
shown MFA-1-O-G possesses a half-life of degradation of ;16 hours
in buffer under physiologic conditions (McGurk et al., 1996; Grillo
et al., 2012).
Chemical Reactivity of MFA-CoA, MFA-AMP, MFA-1-O-G,
and MFA-GSH with Gly, Tau, GSH, and NAC. Incubation of
MFA-AMP (1 mM) with Gly and Tau (10 mM) in Kpi (0.1 M) under
physiologic conditions (i.e., 37°C, pH 7.4) resulted in the N-amidation
of both glycine and taurine, producing 23.2 6 4.2 nM and 20.1 6 1.8
nM of MFA-Gly and MFA-Tau conjugates, respectively, after 60
minutes of incubation (Fig. 6, A and B). Incubations of MFA-CoA
(1 mM) with glycine and taurine (10 mM) resulted in minimal
N-amidation, producing no MFA-Gly (limit of detection ;0.5 nM for
all MFA-conjugates) and 0.93 6 0.65 nM of MFA-Tau at the
60-minute time-point. MFA-1-O-G (1 mM) exhibited no reactivity
toward both Gly and Tau. In vitro GSH (10 mM) reactivity studies
with MFA-AMP, MFA-CoA, and MFA-1-O-G (1 mM) resulted in
12.8 6 1.0 nM, 145 6 40 nM, and 1.3 6 0.97 nM of MFA-GSH
formation, respectively, at the 60-minute time-point (Fig. 7A). The
incubation of NAC (10 mM) with MFA-AMP, MFA-CoA, and MFA-
GSH (1 mM) under physiologic conditions resulted in the formation of
7.45 6 4.8 nM, 141 6 4.9 nM, and 780 6 26 nM of MFA-NAC
conjugates, respectively, at 60 minutes (Fig. 7B). While MFA-1-O-G
continued to show no reactivity toward the nucleophile NAC.
Time-Course of Formation of MFA-AMP, MFA-Tau, and
MFA-Gly in Rat Hepatocyte Incubations. The incubation of MFA
(100 mM) in rat hepatocytes under physiologic conditions (37°C, pH
7.4) resulted in the detection of MFA-AMP, MFA-CoA, MFA-GSH,
MFA-1-O-G, and MFA-Tau. The initial rise of MFA-AMP formation
was very rapid attaining a concentration of 107.7 nM at ;30 seconds
Fig. 6. Mean reactivity assessment 6 S.D. at 60 minutes of MFA-AMP, MFA-
CoA, and MFA-1-O-G toward Gly (A) and Tau (B) in buffer (0.1 M Kpi; pH 7.4,
37°C).
(Fig. 8A). MFA-CoA was not detectable until the 4-minute time-point, undergoes further conjugation into MFA-AMP, MFA-CoA, and MFA-
reaching a concentration of 45.6 nM at 60 minutes. MFA-Tau levels GSH (Grillo et al., 2012; Horng and Benet, 2013) in rat hepatocytes, all
were undetectable until 4 minutes, reaching a concentration of 15.7 of which possess an increased chemical electrophilicity and are reactive
nM at 60 minutes. The formation of MFA-GSH was linear, reaching toward protein nucleophiles.
a concentration of 2.1 mM at 60 minutes, while the formation of MFA-
Electrophilicity assessment of metabolites in drug development
1-O-G increased to a concentration of 30.0 mM at 60 minutes (Fig. involve screens utilizing nucleophilic trapping agents. Glutathione is
8B). MFA-Gly and MFA-NAC conjugates were undetectable during a commonly used biomarker of reactivity for bioactivation studies.
the 60 minutes incubation period.
Presumably, the greater the in vitro nucleophilic adduct formation, the
greater the probability it will covalently bind onto proteins and elicit
a toxic reaction. However, not all protein covalent bindings result in the
onset of toxicity, and thus the challenge is to identify those protein
Discussion
Carboxylic acid-containing drugs are metabolized into reactive targets that are critical for the onset of a drug-induced toxicity. In
electrophilic acyl-linked metabolites that can form irreversible adducts addition to thioesters (Van Breemen and Fenselau, 1985), acyl-linked
with proteins, potentially causing an allergic reaction in hypersensitive metabolites have also been shown to react with proteins via oxygen ester
individuals (Stogniew and Fenselau, 1982). MFA is a nonsterodial anti- (Wells et al., 1987) and amide-linkage (van Breemen and Fenselau,
inflammatory drug prescribed for its analgesic and anti-inflammatory 1986; Mitamura et al., 2011). In the present study, we investigate the
activity via inhibition of cyclooxygenase-dependent prostanoid forma- selective nonenzymatic acylation of amino and thiol functional groups of
tion (Hawkey, 1999). Commonly used to treat pain, MFA has been four biologic nucleophiles: Gly, Tau, GSH, and NAC.
implicated in several cases of hepatic and renal disturbances and
In vitro GSH reactivity assessment of MFA-AMP, MFA-CoA, and
hypersensitivity reactions (Handisurya et al., 2011). These toxicities are MFA-1-O-G revealed MFA-CoA to be 11.5-fold more reactive than
proposed to occur via bioactivation of MFA into reactive acyl-linked MFA-AMP toward the thiol functional group of GSH (Fig. 7A),
metabolites covalently binding onto macromolecules. MFA undergoes consistent with our previous studies (Horng and Benet, 2013), while
conjugation via the free carboxyl group into the 1-O-acyl glucuronide, MFA-CoA was 19.5-fold more reactive toward the thiol groups of
MFA-1-O-G (Glazko, 1966; Sato et al., 1993), which has been shown NAC than its corresponding MFA-AMP (Fig. 7B). Alternatively,
to irreversibly bind to albumin (McGurk et al., 1996). MFA also incubations with Gly and Tau revealed that MFA-AMP is more

Reactivity of the Acyl-Linked Metabolites of Mefenamic Acid
1931
Fig. 8. Mean time-dependent formation 6 S.D. of MFA-AMP, MFA-CoA, and
MFA-Tau (A), MFA-1-O-G (right axis units) and MFA-GSH (left axis units) (B) in
rat hepatocyte incubations.
derivative. NAC reactivity assessments show that MFA-GSH is
indeed highly reactive, exhibiting a 5.5- and 108-fold greater reactivity
Fig. 7. Mean reactivity assessment 6 S.D. at 60 minutes of MFA-AMP, MFA-
CoA, MFA-1-O-G, and MFA-NAC toward GSH (A) and NAC (B) in buffer (0.1 M toward NAC than MFA-CoA and MFA-AMP, respectively (Fig. 7B).
Kpi; pH 7.4, 37°C).
Grillo and Benet (2002) have demonstrated that the reactive potential
of S-acyl-glutathione conjugates with NAC correlates to the degree
of a-carbon substitution of the acyl-linkage. Increasing a-carbon
reactive toward N-acyl-amidation than its corresponding MFA-CoA,
producing a significant amount of MFA-Gly, while MFA-CoA did not
react with Gly (Fig. 6A). The amidation of Tau by MFA-AMP was
also 17.5-fold greater than by MFA-CoA (Fig. 6B). MFA-1-O-G
exhibited little to no reactivity toward all four bionucleophiles.
Reactivity data were linear during the incubation and therefore the 60-
minute time-point was used to calculate the slope of conjugate
formation (Table 1). Reactivity studies utilizing cholic acid have also
demonstrated a greater reactivity of CA-AMP compared with CA-CoA
toward the N-acyl-amidation of glycine and taurine, while CA-CoA
exhibited a greater reactivity toward the acylation of the cysteine-
sulfhydryl group of GSH and NAC (Mitamura et al., 2011). Studies in
buffer have also shown that acyl-adenylates can spontaneously react
with the amino groups of substance P (Goto et al., 2001), lysosomes
(Goto et al., 2005), and histones (Mano et al., 2004), further suggesting
that acyl-adenylates have a high reactive affinity toward amino
functional groups, and that acyl-AMPs and acyl-CoAs are selective in
their nonenzymatic acylation of amino-versus-thiol functional groups.
This selectivity may be attributed to differences in the degree of
electrophilicity of MFA-AMP versus MFA-CoA and the nucleophilicity
of the amino versus the thiol functional groups, suggesting different
reactivity mechanisms toward protein nucleophilic sites.
substitution results in decreasing reactivity, which is in agreement with
the relative degradation rates associated with acyl glucuronides and
assumed to be identical for that of their respective S-acyl-CoA
thioesters. Therefore, if MFA toxicity is the result of covalent binding
by reactive intermediates, then it is conceivable that the unusually high
formation of MFA-GSH in rat hepatocytes (Grillo et al., 2012; Horng
and Benet, 2013), compared with diclofenac (Grillo et al., 2003),
zomepirac (Olsen et al., 2005), (R)-ibuprofen (Grillo and Hua, 2008),
and (R)-flunoxaprofen (Grillo et al., 2010), may be responsible for the
tissue injury associated with MFA. This assumption is based on the
high covalent binding values in animals dosed with drugs known to
cause hepatotoxicity in humans, such as isoniazid (Nelson et al., 1978)
and acetaminophen (Matthews et al., 1997).
TABLE 1
Mean rates 6 S.D.s of formation of MFA conjugates incubated with Gly, Tau, GSH,
and NAC (10 mM) in physiologic buffer (pH 7.4, 37°C) (n = 3)
Conjugate
MFA-Gly
MFA-Tau
MFA-GSH
MFA-NAC
1 mM
nM/min
MFA-AMP
MFA-CoA
MFA-GSH
MFA-1-O-G
0.39 6 0.07
N.D.
0.35 6 0.01
0.02 6 0.01
N.A.
0.21 6 0.02
2.42 6 0.67
N.A.
0.12 6 0.08
2.34 6 0.08
13.0 6 0.44
N.D.
N.A.
N.D.
Acyl-GSH conjugates share a common structural moiety, thioester,
to that of the acyl-CoA. Therefore, it is conceivable that MFA-GSH
is just as, if not more, reactive than its corresponding acyl-CoA
N.D.
N.D.
N.A., not applicable; N.D., not detected.

1932
Horng and Benet
Acyl-CoA thioester synthesis is a two-step reaction. This reaction be of toxicological significance considering that MFA produces a high
occurs when the AMP moiety of ATP is transferred to the acyl group amount of GSH adducts in hepatocytes. MFA-1-O-G was not che-
of the carboxylic acid via acyl-CoA synthetase, forming an acyl- mically reactive toward both amino and thiol functional groups. This
adenylate intermediate. The enzyme-bound activated intermediate is preferential reactivity between MFA-AMP and MFA-CoA provides
then displaced by coenzyme A to yield the associated acyl-CoA product MFA the ability to covalently bind onto a broad range of nucleophilic
and free AMP (Vlahcevic et al., 1999). Acyl-CoA and acyl-AMP sites on proteins, which increases its probability of covalently modifying
metabolites both spontaneously and enzymatically, via glutathione-S- critical protein targets and inducing a toxic reaction. Therefore, acyl-
transferase, form GSH-conjugates (Li et al., 2002; Grillo et al., 2012; linked metabolites may be important in the formation of drug-protein
Horng and Benet, 2013). In addition to GSH, glycine (Keller, 1842) adducts and the onset of an idiosyncratic toxicity, suggesting that the
and, taurine (James et al., 1971; Hutson and Casida, 1978) are two of bioactivation of carboxylic acid-containing drugs into acyl-linked me-
the most commonly cited metabolic amino acid conjugation reactions. tabolites should be further evaluated to allow for structural modification
Amino acid conjugation occurs through the transfer of the acyl group of drug candidates, thereby reducing bioactivation and improving drug
from the acyl-CoA to an amino acid via N-acetyltransferase. However, safety.
previous and our current studies have shown that acyl-adenylates are
capable of spontaneously reacting to both Gly and Tau (Mitamura et al.,
2011), suggesting that acyl-adenylate intermediates have a greater
inherent chemical affinity toward amino groups than both the acyl-
Drug Metabolism, Amgen, Inc., South San Francisco, CA) for many helpful
CoAs and acyl-1-O-G. Rat hepatocyte MFA incubation resulted in
Acknowledgments
The authors thank Dr. Mark Grillo (Department of Pharmacokinetics and
discussions during the course of this work, and Chris Her (UCSF Liver Center
rapid MFA-AMP formation (Cmax 107 nM at ;30 seconds), while
MFA-CoA was undetectable until 4 minutes, achieving a concentration
of 45.6 nM at 60 minutes (Fig. 8A). This sequence is in agreement
with the acyl-CoA biosynthetic pathway. MFA-1-O-G and MFA-GSH
were also shown to increase linearly, achieving concentrations of
30.0 mM and 2.1 mM at 60 minutes, respectively (Fig. 8B). Our
experiments also revealed the presence of MFA-Tau, undetectable
until 4 minutes, reaching a concentration of 15.7 nM at 60 minutes. It
was not determined if the formed MFA-Tau primarily occurs
nonenzymatically from MFA-AMP or enzymatically via the MFA-
CoA thioester. MFA-Gly was not detected in these incubations,
possibly due to an insufficiency in analytical sensitivity (limit of
detection for all MFA-conjugates was ;0.5 nM). Previous zomepirac
(ZP) rat hepatocyte incubations revealed the presence of ZP-CoA,
ZP-1-O-G, ZP-Tau, and ZP-Gly (Olsen et al., 2005). The high
concentration of ZP-1-O-G compared with ZP-Gly and ZP-Tau may
be reflective of dose, a determinant of whether a drug undergoes
glucuronidation or amino acid conjugation (Hutt and Caldwell, 1990).
At lower doses, carboxylic acids tend to undergo amino acid
conjugation, while at higher doses, glucuronidation dominates. Amino
acid conjugation is a high-affinity, low-capacity system, while
glucuronidation is a high-capacity pathway with a broader substrate
selectivity. Dose also influences the type of amino acid conjugate
formed (Hirom et al., 1977), with dosage increases of phenylacetic
acid resulting in decreases in glycine/taurine conjugate ratios. Rat liver
experiments with tolmetin (Tol) also identified the acyl-CoA–derived
conjugates, Tol-Gly and Tol-Tau conjugates in rat urine (Olsen et al.,
2003), whose concentrations were unaffected by clofibric acid,
confirming a high-affinity/low-capacity metabolic pathway (Hutt and
Caldwell, 1990). Oral administration of RS-ibuprofen to humans also
led to the detection of ibuprofen-Tau but not the glycine conjugate
(Shirley et al., 1994), signifying a minor biotransformation pathway.
Gly and Tau conjugates are believed to be formed enzymatically (N-
acyltransferases) via the acyl-CoA; however, we have demonstrated
that Gly and Tau conjugates also form spontaneously via acyl-AMP
conjugates. MFA-NAC conjugates were not detected in rat hepatocyte
incubations, due to the lack of detectable N-acylcysteinylglycine and
glutamyl transpeptidase (g-GT) activity in rat livers (Hinchman and
Ballatori, 1990). However, in humans, NAC formation would be
expected to occur.
Cell Biology Core) for assistance in the rat hepatocyte preparations.
Authorship Contributions
Participated in research design: Horng, Benet.
Conducted experiments: Horng.
Performed data analysis: Horng, Benet.
Wrote or contributed to the writing of the manuscript: Horng, Benet.
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