Rapid internal acyl migration and protein binding of synthetic probenecid glucuronides

Article abstract of DOI:10.1021/tx010166t

Internal acyl migration reactions of drug 1-O-acyl-β-D-glucopyranuronates (1β-acyl glucuronides) are of interest because of their possible role in covalent binding to proteins and consequent adverse effects. The reactivity of the synthetic probenecid 1β-acyl glucuronide (PRG), the principal metabolite of probenecid (PR) in humans, has been investigated in terms of acyl migration, hydrolysis, and covalent binding to proteins in phosphate buffer (pH 7.4) and human plasma at 37 °C. PRG primarily degraded by acyl migration according to apparent first-order kinetics and the 2-, 3-, and 4-acyl isomers sequentially appeared as both α- and β-anomeric forms. In addition, small amounts of PRG and extremely labile 1α-acyl isomer existed in the equilibrated mixture favoring the 2α/β-acyl isomer, that provided significant information regarding the mechanism of acyl migration. All of the positional isomers and anomers were characterized using preparative HPLC and NMR spectroscopy. Acyl migration was observed to predominate over hydrolysis in both media although the extent of hydrolysis in plasma was larger than that in the buffer. The overall degradation half-lives (h) in the buffer and plasma were 0.27 ± 0.003 and 0.17 ± 0.007, respectively. The covalent binding rapidly proceeded mainly via the Schiff's base mechanism and reached a plateau after 2 h of incubation. The maximal binding was 146 ± 4.8 pmol/mg of protein, and ca. 10% of the initial concentration of PRG. These results indicated that PRG is most labile and susceptible to acyl migration of all the drug acyl glucuronides reported to date in the physiological conditions, and highly reactive to plasma proteins, that could provide a possible explanation for the immunologically based adverse effects of PR.

Full text of DOI:10.1021/tx010166t

J UNE 2002
VOLUME 15, NUMBER 6
© Copyright 2002 by the American Chemical Society
Articles
Ra p id In ter n a l Acyl Migr a tion a n d P r otein Bin d in g of
Syn th etic P r oben ecid Glu cu r on id es
Kazuki Akira,* Takafumi Uchijima, and Takao Hashimoto
School of Pharmacy, University of Pharmacy and Life Science, 1432-1 Horinouchi,
Hachioji, Tokyo 192-0392, J apan
Received October 23, 2001
Internal acyl migration reactions of drug 1-O-acyl-â-D-glucopyranuronates (1â-acyl glu-
curonides) are of interest because of their possible role in covalent binding to proteins and
consequent adverse effects. The reactivity of the synthetic probenecid 1â-acyl glucuronide (PRG),
the principal metabolite of probenecid (PR) in humans, has been investigated in terms of acyl
migration, hydrolysis, and covalent binding to proteins in phosphate buffer (pH 7.4) and human
plasma at 37 °C. PRG primarily degraded by acyl migration according to apparent first-order
kinetics and the 2-, 3-, and 4-acyl isomers sequentially appeared as both R- and â-anomeric
forms. In addition, small amounts of PRG and extremely labile 1R-acyl isomer existed in the
equilibrated mixture favoring the 2R/â-acyl isomer, that provided significant information
regarding the mechanism of acyl migration. All of the positional isomers and anomers were
characterized using preparative HPLC and NMR spectroscopy. Acyl migration was observed
to predominate over hydrolysis in both media although the extent of hydrolysis in plasma was
larger than that in the buffer. The overall degradation half-lives (h) in the buffer and plasma
were 0.27 ( 0.003 and 0.17 ( 0.007, respectively. The covalent binding rapidly proceeded mainly
via the Schiff’s base mechanism and reached a plateau after 2 h of incubation. The maximal
binding was 146 ( 4.8 pmol/mg of protein, and ca. 10% of the initial concentration of PRG.
These results indicated that PRG is most labile and susceptible to acyl migration of all the
drug acyl glucuronides reported to date in the physiological conditions, and highly reactive to
plasma proteins, that could provide a possible explanation for the immunologically based
adverse effects of PR.
transferred to the C-2, C-3, or C-4 position of the
glucuronic acid ring (1) (Figure 1). In general, acyl
migration has been observed to predominate over hy-
drolysis under physiological conditions (2-11). Further-
more, the 1â-acyl glucuronides of many carboxylate drugs
have been shown to form covalent adducts with proteins
(12-16). Currently, there is speculation that the protein
In tr od u ction
Conjugation with glucuronic acid to yield 1-O-acyl-â-
D-glucopyranuronates (1â-acyl glucuronides) is a major
metabolic route for many carboxylate drugs (1). It is well-
known that 1â-acyl glucuronides are labile and reactive.
These compounds undergo both hydrolysis and internal
acyl migration. In the acyl migration, the aglycon is
10.1021/tx010166t CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/23/2002

766 Chem. Res. Toxicol., Vol. 15, No. 6, 2002
Akira et al.
albumin has been found to occur also in vitro (35). The
protein adduct formation may be responsible for the
above-mentioned adverse effects of PR.
The reactivity of PRG has been investigated in buffer
solutions with or without serum albumin, urine, and
plasma using high-performance liquid chromatography
(HPLC) (35, 36). However, the reactivity is still unclear,
and the contribution of acyl migration to the lability is
entirely unknown, because PRG seems to not be clearly
discriminated from its isomeric glucuronides in the HPLC
method used in the literature. In this paper, we have
investigated the reactivity of the synthetic PRG in buffer
solutions and human plasma using an HPLC method to
simultaneously determine PR, PRG, and its isomeric
glucuronides.
F igu r e 1. General scheme for the acyl migration of 1â-acyl
glucuronides. R ) drug moiety. Mutarotation is possible in the
isomeric glucuronides.
Ma ter ia ls a n d Meth od s
F igu r e 2. Schiff’s base mechanism proposed for covalent
binding of 1â-acyl glucuronides to proteins.
Ma ter ia ls. Ammonium-2H4 deuterioxide [26% (w/v), >99.0
atom % 2H] and sulfaphenazole were purchased from Sigma-
Aldrich (Tokyo, J apan). â-Glucuronidase (>76 × 104 Fishman
units/g) was purchased from Funakoshi (Tokyo, J apan). Silica
gel (Wakogel C-300) and PR were purchased from Wako Pure
Chemical Industries (Osaka, J apan). Deuterated solvents and
thin-layer chromatography plates (silica gel 60F254) were pur-
chased from Merck (Darmstadt, Germany). Other reagents were
purchased from Kanto Chemical (Tokyo, J apan). Plasma was
obtained by centrifugation of heparinized blood from healthy
male volunteers not on any medication. The plasma was
immediately used for incubation following pH measurements.
Protein was measured with Lowry method using bovine serum
albumin as the standard.
In str u m en ta tion . 1H NMR, 1H-decoupled 13C (13C{1H})
NMR, and two-dimensional NMR spectra [correlated spectros-
copy (COSY)] of 1-O-[p-(dipropylsulfamoyl)benzoyl]-R-D-gluco-
pyranuronic acid (1R-acyl isomer), PRG, and the degradation
products were recorded in 2H2O on a Bruker DPX400 spectrom-
eter (9.4 T) or a Bruker DRX500 spectrometer (11.75 T), and
chemical shifts were referenced to that of sodium 3-(trimethyl-
silyl)-[2,2,3,3-2H4]-propionate (TSP, δ1H 0), unless otherwise
stated. 1H NMR spectra of other compounds were recorded in
C2HCl3 on a Varian GEMINI300 spectrometer, and chemical
shifts were referenced to that of tetramethylsilane (δ1H 0). All
the NMR spectra were obtained at 300 K using 5-mm NMR
tubes. Mass spectra (MS) were recorded on a ThermoQuest (San
J ose, CA) TSQ700 spectrometer in the electron impact ionization
mode (EI) or a Hitachi (Tokyo, J apan) M-1000 spectrometer in
the electron spray ionization mode (ESI). Melting points were
determined on a Yanako (Kyoto, J apan) MP-S3 melting point
apparatus and were uncorrected. HPLC was performed using
a Waters (Milford, MA) M600E multisolvent delivery system, a
Rheodyne (Cotati, CA) 7125 injector, a Shimadzu (Kyoto, J apan)
SPD-6A UV Spectrophotometric Detector set at 254 nm, and a
LiChrospher 100 RP-18 (e) column (250 × 4 mm i.d., 5 µm,
Kanto Chemical, Tokyo, J apan) or an Inertsil PREP-ODS
column (250 × 30 mm i.d., 10 µm, GL Sciences, Tokyo, J apan).
The mobile phase for the LiChrospher column was 10 mM
sodium phosphate buffer (pH 5.0)-acetonitrile (72/28, v/v) at a
flow rate of 1 mL/min. The mobile phase for the Inertsil column
was 10 mM ammonium acetate buffer (pH 5.0)-acetonitrile (72/
28, v/v) at a flow rate of 15 mL/min. Unless otherwise noted,
the former analytical column (LiChrospher) was used.
adducts are at least partially responsible for immunologi-
cal side effects of carboxylate drugs (1, 17, 18). Recently,
a number of NSAIDs (e.g., benoxaprofen, indoprofen,
tolmetin, zomepirac) have been withdrawn from clinical
use because of a high incidence of allergic reactions, and
this may have some relation to the immunotoxicity
induced by the protein adducts. Toxicity mechanisms
involving modification of active sites of enzymes (19) and
interaction with structural proteins (20) have been also
suggested. It is now accepted that the covalent binding
can occur by either transacylation or Schiff’s base mech-
anisms shown in Figure 2 (1). The covalent binding
appears to occur mainly by the Schiff’s base mechanism,
in the drug acyl glucuronides which readily undergo acyl
migration (12, 15, 21, 22).
p-(Dipropylsulfamoyl)benzoic acid (probenecid, PR) has
been used for many years as a therapeutic agent for
chronic gout. PR inhibits glucuronidation of some drugs
and the active transport of organic acids in the renal
tubules (23-25). Recently, these effects have therapeutic
advantages in the medication of patients with acquired
immunodeficiency syndrome; PR prolongs the duration
of serum levels of zidovudin (26, 27) and zalcitabine (28)
by decreasing the glucuronidation and the renal tubular
excretion, respectively, and reduces the cidofovir-induced
nephrotoxicity by decreasing its renal tubular concentra-
tion (29, 30). However, PR causes a high frequency (11-
25%) of hypersensitivity reactions among patients with
human immunodeficiency virus whereas in other patients
the incidence is low (2-4%) (30). It has been suggested
that changes in drug metabolism and responsiveness to
some reactive metabolites of PR could explain the high
rate of adverse effects (31). In addition, PR-induced
immune hemolytic anemia has been reported, that is
speculated to be mediated by an immune complex with
some PR metabolites (32, 33).
PR is metabolized to form an acyl glucuronide, 1-O-
[p-(dipropylsulfamoyl)benzoyl]-â-D-glucopyranuronic acid
(PRG), and several phase-I metabolites, which are ex-
creted in the urine together with a small amount of the
parent compound in humans (34). Of the dose, 25-70%
has been reported to be excreted as PRG (34-37). Protein
adducts of PR have been detected in plasma from human
subjects after dosing PR and diflunisal, that is formed
probably through the acyl migaration of PRG (38),
although the presence of PRG in plasma has not been
demonstrated (36, 38). The protein binding to serum
Syn th esis of 1-O-[p-(Dip r op ylsu lfa m oyl)ben zoyl]-r-D-
glu cop yr a n u r on ic Acid (1r-Acyl Isom er ) (4) a n d 1-O-[p-
(Dip r op ylsu lfa m oyl)ben zoyl]-â-D-glu cop yr a n u r on ic Acid
(P RG) (5) (See F igu r e 3). PR (202 mg) and benzyl 2,3,4-tri-
O-benzyl-D-glucopyranuronate 1 (39) (444 mg) were condensed
using the trichloroacetimidate method (40) according to the
previously reported manner (9). The product, which showed a
single spot (Rf 0.5) on thin-layer chromatography [hexane/ethyl
acetate ) 3/1 (v/v)], was isolated from the reaction mixture by

Acyl Migration of Probenecid Glucuronide
Chem. Res. Toxicol., Vol. 15, No. 6, 2002 767
In cu ba tion of Glu cu r on id es in P h osp h a te Bu ffer . A
stock solution (100 µL) of PRG, 1R-acyl isomer or 3-acyl isomer
in acetonitrile (each 200 µM), was placed in a test tube and
evaporated to dryness by a stream of dry nitrogen. To the
residue was added 1 mL of 100 mM sodium phosphate buffer
(pH 7.4) followed by mixing. Triplicate incubations were per-
formed at 37 °C. The reaction was monitored by analyzing 10
µL portions of the solution using HPLC. The ratio of each peak
area to the total area of peaks due to PR, PRG, and its isomeric
glucuronides was calculated. The reactivity of PRG was also
examined in 10 mM sodium phosphate buffer (pH 3.0 and 5.0)
at 37 °C.
F igu r e 3. Probenecid glucuronides and the synthetic precur-
sors. 1 ) benzyl 2,3,4-tri-O-benzyl-D-glucopyranuronate; 2 )
benzyl 2,3,4-tri-O-benzyl-1-O-[p-(dipropylsulfamoyl)benzoyl]-R-
D-glucopyranuronate; 3 ) benzyl 2,3,4-tri-O-benzyl-1-O-[p-
(dipropylsulfamoyl)benzoyl]-â-D-glucopyranuronate; 4 ) 1-O-[p-
(dipropylsulfamoyl)benzoyl]-R-D-glucopyranuronic acid (1R-acyl
isomer); 5 ) 1-O-[p-(dipropylsulfamoyl)benzoyl]-â-D-glucopyran-
uronic acid (PRG).
Id en tifica tion of Rea ction P r od u cts fr om P RG. To a
solution of PRG (20 mg) dissolved in H2O (1 mL) was added 1%
NH3 in H2O so that the pH value of the solution becomes
approximately 7.4. The solution was incubated at 37 °C for 45
min, and then acidified to pH 3-4 with 10% acetic acid in H2O
in order to stabilize the products, followed by freeze-drying to
give a white curdy solid. The solid was redissolved in methanol
(1 mL), and 50-100 µL portions of the solution were injected
onto the HPLC column (Inertsil PREP-ODS). Four major peaks
at tR 12.1, 19.9, 22.9, and 26.6 min (PR) and two minor peaks
at tR 16.0 and 17.5 min (PRG) were observed after injection.
The eluates corresponding to the peaks at tR 12.1, 16.0, 19.9,
and 22.9 min were collected (fractions 1, 2, 3, and 4, respectively)
followed by immediate acidification (pH ∼3) with 10% acetic
acid in H2O to stabilize the products. The eluates were evapo-
rated by a rotary evaporator to remove acetonitrile and then
flash column chromatography over 200 g of silica gel with
hexanes/ethyl acetate (5/1, v/v) as the eluent to give an oil (267
mg). The oily product was found to be a mixture of one minor
(Rf 0.2) and one major (Rf 0.15) component by further thin-layer
chromatographic analyses [benzene/diethyl ether ) 50/1 (v/v)].
The minor and major compounds were suggested to be the
benzylated derivatives of the title compounds 2 and 3, respec-
tively, by 1H NMR spectroscopy (data are not shown). Thus, the
oily product was subjected to flash column chromatography over
40 g of silica gel with benzene/diethyl ether (100/1) as the eluent
to give a minor benzylated compound (43 mg) and then a major
benzylated compound (160 mg) as colorless oils. The benzylated
compounds were deprotected by catalytic dehydrogenation as
described previously (9) to give 4 (8.5 mg) and 5 (69 mg) as pale
yellow amorphous powders. 4: 1H NMR δ 0.82 (6H, t, J ) 7.4
Hz, CH3), 1.52 (4H, m, CH2), 3.18 (4H, t, J ) 7.5 Hz, CH2), 3.67
(1H, dd, J ) 10.1 and 9.0 Hz, H4), 3.91 (1H, dd, J ) 9.8 and 3.6
Hz, H2), 3.98 (1H, t, J ) ∼9 Hz, H3), 4.19 (1H, d, J ) 10.1 Hz,
H5), 6.45 (1H, d, J ) 3.6 Hz, H1), 7.99 (2H, d, J ) 8.6 Hz,
aromatic protons), 8.28 (2H, d, J ) 8.6 Hz, aromatic protons).
MS (ESI): m/z 462 [M + H]+. Anal. Calcd for C19H27O10NS: C,
49.45; H, 5.90; N, 3.04. Found: C, 49.34; H, 5.99; N, 3.01. 5:
1H NMR δ 0.81 (6H, t, J ) 7.4 Hz, CH3), 1.52 (4H, m, CH2),
3.18 (4H, t, J ) 7.5 Hz, CH2), 3.6-3.8 (3H, m, H2, H3, H4),
4.11 (1H, d, J ) 9.3 Hz, H5), 5.88 (1H, d, J ) 7.7 Hz, H1), 7.99
(2H, d, J ) 8.6 Hz, aromatic protons), 8.29 (2H, d, J ) 8.6 Hz,
aromatic protons). 13C NMR [(CH3)2SO]: δ 11.1 (CH3), 21.7
(CH2), 49.7 (CH2), 71.5, 72.4, 75.7, 76.3 (C2, C3, C4, C5), 95.4
(C1), 127.4, 130.8 (protonated aromatic carbons), 132.4, 144.3
(quaternary aromatic carbons), 163.7 (CO), 170.0 (CO). The
chemical shifts of 13C resonances were referenced to that of
dimethyl sulfoxide. These 13C chemical shifts of PRG were iden-
tical to those reported for PRG isolated from human urine (41).
MS (ESI): m/z 462 [M + H]+. Anal. Calcd for C19H27O10NS‚2/
3H2O: C, 48.21; H, 6.03; N, 2.95. Found: C, 48.21; H, 6.31; N,
2.76.
2
1
freeze-dried. The residue was redissolved in H2O, and H NMR
and COSY spectra were obtained. Fractions 1, 3, and 4 were
confirmed to correspond to the peaks at tR 3.9, 6.9, and 7.9 min,
respectively, in the analytical HPLC.
Dir ect 1H NMR Sp ect r oscop ic Det ect ion of R ea ct ion
Mixtu r e. PRG (3 mg) dissolved in 2H2O (ca. 5 mL) was
lyophilized to replace the protons of hydroxyl and carboxyl
groups by deuterons. The residue was redissolved in 500 µL of
2H2O and transferred to an NMR tube following filtration. To
the solution was added 2.6 M N2H3 in 2H2O (∼2 µL) to
decompose PRG, monitoring the reaction with 1H NMR spec-
troscopy. The resulting mixture was measured by 1H NMR
spectroscopy for 2.5 h.
In cu ba tion of P RG in P la sm a . A stock solution (0.5 mL)
of PRG in acetonitrile (218 µM) was placed in a test tube and
evaporated to dryness by a stream of dry nitrogen. To the
residue was added 1 mL of freshly collected human plasma (pH
7.8, 7.5 g of protein/dL) prewarmed at 37 °C for 5 min. The
solution (PRG, 109 µM) was incubated triplicately at 37 °C. Fifty
microliter portions were taken with time, and immediately
mixed with 50 µL of acetonitrile/85% phosphoric acid in H2O
(5/0.02, v/v) (14) containing 12.8 µM sulfaphenazole as an
internal standard in order to stop the reaction and deproteinize.
The mixture was centrifuged (3000g × 10 min), and the
supernatant and the precipitate were separated. The recovery
of protein was 95 ( 5.1%. The supernatant was subjected to
HPLC, and the precipitates was stored at -20 °C until analyzed.
Linear calibration curves with correlation coefficients of 0.999
were obtained using blank plasma (50 µL) containing known
amounts of PR (10-2083 ng) and PRG (10-2518 ng). The
isomers of PRG were assumed to have the same molar extinction
coefficients as that of PRG and were quantitated using the
calibration curve for PRG.
The protons of the glucuronide ring of â-anomer 5 resonated
at a higher field than those of R-anomer 4 in the 1H NMR
spectra as described in the literature (42). Compounds 4 and 5
showed a single peak in HPLC. The â-anomer 5 contained no
1
appreciable amount of R-anomer 4 in the H NMR spectra and
vice versa. When compound 5 was treated with â-glucuronidase,
the peak of 5 disappeared and that of PR emerged, which
showed that compound 5 was the natural, biosynthetic 1â-acyl
glucuronide.
An a lysis of Cova len tly Bou n d P R. The precipitates from
the plasma incubated with PRG were washed 7 times with 300
µL of methanol/diethyl ether (3/1, v/v) in order to remove PR,
PRG, and isomeric glucuronides irreversibly unbound to protein,
and then dried by a stream of dry nitrogen. At the last washing
step, the supernatant was evaporated under nitrogen, and the
residue was analyzed by HPLC to determine the effective-
ness of the washing procedures. No PR, PRG, and isomeric
glucuronides were detected in the last wash solution. The loss
of protein by washing was negligible. The washed precipitates
were suspended to 200 µL of 1 M sodium hydroxide by vortexing
â-Glu cu r on id a se Tr ea tm en t. A stock solution (300 µL) of
PRG in acetonitrile (200 µM) was placed in a test tube and
evaporated to dryness by a stream of dry nitrogen. To the
residue was added 1 mL of 10 mM ammonium acetate buffer
(pH 5.0) followed by mixing. The solution was incubated at 37
°C for 45 min with or without â-glucuronidase (1 mg, 760
Fishman units). Ten milliliter aliquots of the solution were
analyzed by HPLC.

768 Chem. Res. Toxicol., Vol. 15, No. 6, 2002
Akira et al.
F igu r e 4. HPLC chromatogram of PRG after incubation in 100
mM phosphate buffer (pH 7.4) at 37 °C for 1 h. 1, 4-acyl; 2, 1R-
acyl; 3, PRG; 4, 3-acyl; 5, 2-acyl; 6, PR.
Ta ble 1. 1H NMR Ch em ica l Sh ifts of th e Glu cu r on id e
Rin g of Isom er ic Glu cu r on id es in 2H2Oa
isomer
H1
H2
H3
H4
H5
2R-acyl
2â-acyl
3R-acyl
3â-acyl
4R-acyl
4â-acyl
5.54 d
∼5.0b
5.35 d
4.84 d
5.34 d
c
5.03 dd
∼5.0b
4.12 t
3.91 m
5.46 t
5.28 m
4.11 t
3.93 t
3.70 t
3.70 t
3.89 t
∼3.9b
5.15 t
5.17 t
4.20 d
3.85 d
4.26 d
∼3.9b
4.38 d
4.03 d
F igu r e 5. Profiles for acyl migration and hydrolysis of PRG
in 100 mM phosphate buffer (pH 7.4) at 37 °C. Each point
represents the mean of values obtained from triplicate incuba-
tions.
3.96 dd
3.66 dd
3.77 dd
3.47 dd
2H4 deuterioxide was added to the deuterium oxide
solution of PRG contained in an NMR tube followed by
1H NMR analysis, the compound degraded to form an
equilibrated mixture of the R- and â-anomers (R/â ) ca.
1) of 2-acyl, 3-acyl, and 4-acyl isomers. This experiment
directly demonstrated the occurrence of acyl migration
followed by rapid mutarotation.
a
The signals are assigned based on chemical shifts, splitting
patterns, spin-spin coupling constants, and integrals and on the
connectivity information from the COSY experiments (42). The
splitting patterns are indicated as follows: d, doublet; dd, double
of doublets; t, triplet; m, multiplet. Coupling constants fall into
characteristic ranges of 3.6-3.8 Hz for J (H1-H2) in R isomers,
7.7-8.0 Hz for J (H1-H2) in â isomers, and 9.2-10.2 Hz for
couplings between H2-H3, H3-H4, and H4-H5. For all isomers,
the protons of the probenecid moiety have similar chemical shifts:
δ 8.25-8.30 (d, aromatic protons), δ 7.99-8.00 (d, aromatic
protons), δ 3.18-3.20 (t, CH2 of side chain), δ 1.52-1.55 (m, CH2
A minor peak observed at tR 5.2 min in Figure 4 was
identified to be the 1R-acyl isomer by co-chromatography
with the authentic compound synthesized here. When the
b
of side chain), δ 0.81-0.83 (t, CH3 of side chain). The specific
1
peak was analyzed by H NMR following the collection
assignment of the H1 and H2 resonances of 2â-acyl and the H4
and H5 resonances of 3â-acyl cannot be made because of their
closeness and overlap. c The signal is presumed to be concealed
behind the H2HO signal.
by preparative HPLC, the 2-acyl, 3-acyl, and 4-acyl
isomers were observed, and the spectral pattern was
similar to that of the equilibrated mixture formed from
PRG in the phosphate buffer. On the other hand, the
1
and sonication, and incubated at 65 °C for 12 h to hydrolyze
and release covalently bound PR. To the incubation mixture
were added 100 µL of 10% hydrochloric acid to acidify (pH ca.
1) and 50 µL of a solution of ketoprofen in acetonitrile (4 µM)
as an internal standard. The mixture was extracted with 500
µL of cyclohexane/diethyl ether (1/1, v/v) followed by centrifuga-
tion (3000g × 10 min). The organic layer was separated and
evaporated to dryness by a stream of dry nitrogen. The residue
was redissolved in 50 µL of the mobile phase, and 10 µL portions
of the solution were subjected to HPLC. A linear calibration
curve with a coefficient of variation of 0.999 was obtained using
blank plasma containing known amounts of PR (36-730 pmol)
and ketoprofen (197 pmol).
direct H NMR spectroscopic analysis of the equilibrated
mixture from PRG showed a doublet at δ 6.45 corre-
sponding to the H1 proton chemical shift of the PR 1R-
acyl glucuronide, which demonstrated the formation of
the 1R-acyl isomer from PRG. From these experimental
results, it was found that the compound was extremely
unstable and decomposed during the collection proce-
dures. The degradation of the 1R-acyl isomer in phos-
phate buffer (pH 7.4) at 37 °C was followed by HPLC.
With time, the peak due to the 1R-acyl isomer very
rapidly decreased with concurrent and sequential ap-
pearance of the 2-acyl, 3-acyl, and then 4-acyl isomers.
The acyl migration was predominant over hydrolysis. It
was ambiguous whether the degradation follows first-
order kinetics because the degradation was too rapid to
follow the initial decrease. The compound decreased to
half its initial concentration after 1 min of incubation.
The time-depencence of degradation of PRG in buffer
(pH 7.4) at 37 °C is shown in Figure 5. PRG decreased
according to apparent first-order kinetics to form the
2-acyl, 3-acyl, and 4-acyl isomers in that order, and the
acyl migration predominated over hydrolysis, as reported
for other drug acyl glucuronides (1). At 75 min, 96% of
PRG had disappeared, and only 4% hydrolyzed to PR.
The degradation of PRG did not follow the apparent first-
order kinetics after 75 min, and a small amount of PRG
remained for many hours. The degradation half-life was
calculated to be 0.27 ( 0.003 h using the first five points.
Resu lts
Rea ctivity of P RG in Bu ffer Solu tion s. The reac-
tivity of PRG in phosphate buffer (pH 7.4) at 37 °C was
examined using HPLC. Four major peaks due to degra-
dation products including the aglycon emerged after 1 h
of incubation as shown in Figure 4. The eluates corre-
sponding to individual peaks were collected by prepara-
tive HPLC and analyzed by 1H NMR and COSY in order
to identify the degradation products. The detected NMR
resonances and assignments are summarized in Table
1. The NMR spectra showed that the products were
anomeric pairs of 2-acyl (tR 7.9 min), 3-acyl (tR 6.9 min),
and 4-acyl isomers (tR 3.9 min). The anomeric ratios (R/
â) were aproximately 1/1 in all cases. When ammonium-

Acyl Migration of Probenecid Glucuronide
Chem. Res. Toxicol., Vol. 15, No. 6, 2002 769
F igu r e 6. HPLC chromatograms of the 3-acyl isomer after
incubation in 100 mM phosphate buffer (pH 7.4) at 37 °C.
Incubation periods are indicated by the chromatograms.
F igu r e 8. Profiles for acyl migration and hydrolysis of PRG
in plasma at 37 °C. Each point represents mean of values
obtained from triplicate incubations.
F igu r e 7. HPLC chromatograms of (A) blank plasma and (B)
plasma spiked with PRG after incubation at 37 °C for 1 h. I.S.,
internal standard (sulfaphenazole).
F igu r e 9. HPLC chromatograms of hydrolyzed pellets obtained
from (A) blank plasma and (B) plasma spiked with PRG after
incubation at 37 °C for 1 h. I.S., internal standard (ketoprofen).
The 1R-acyl isomer appeared as rapidly as the 2-acyl
isomer, and the concentration closely followed that of the
2-acyl isomer, which means the rapid equilibrium be-
tween the 1R-acyl isomer and the 2R/â-acyl isomer. The
2-acyl, 3-acyl, and 4-acyl isomers were relatively stable
to hydrolysis under the present conditions.
PRG was stable in phosphate buffer (pH 3.0) at 37 °C
during 24 h incubation. However, it underwent hydrolysis
to PR (2%) and rearrangement to 2-acyl isomer (13%) in
phosphate buffer (pH 5.0) at 37 °C during 24 h incuba-
tion. When the 3-acyl isomer (R/â ) ca. 1), that was not
contaminated with the 1R- and 1â-acyl isomers at all, was
incubated in phosphate buffer (pH 7.4) at 37 °C, the
compound degraded with the concurrent appearance of
the 2-acyl and 4-acyl isomers, and after 30 min, small
amounts of PRG and 1R-acyl isomer appeared, as shown
in Figure 6.
Rea ctivity of P RG in P la sm a . The reactivity of PRG
in intact plasma (unbuffered) at 37 °C was investigated.
Aliquots of the reaction mixture were analyzed by HPLC
after deproteinization. As shown in Figure 7, the 2-acyl,
3-acyl, and 4-acyl isomers and PR were well resolved
although no 1R-acyl isomer was observed because of the
hindrance by endogenous peaks. To quantitate PRG, the
isomeric glucuronides, and PR, the suitability of various
sulfa drugs as the internal standard was examined, as
Selen et al. have successfully determined PR in plasma
by HPLC using sulfamethazine as the internal standard
(43). Consequently, sulfaphenazole was found to be most
suitable. The peak due to PRG decreased with time
according to apparent first-order kinetics with concurrent
and sequential appearance of the 2-acyl, 3-acyl, and
4-acyl isomers as in the buffer, as shown in Figure 8. The
half-life of PRG, 0.17 ( 0.007 h, was lower than that
found in the buffer solution (pH 7.4) at 37 °C. A small
amount of PRG remained for many hours after most of
the PRG degraded, as in the buffer. Although the acyl
migration predominated over hydrolysis, the rate of
hydrolysis was much larger than that in the buffer at
pH 7.4. The accelerated hydrolysis appeared to be
responsible for the decrease in half-life. At 45 min, more
than 90% of PRG had disapperaed, and 20% hydrolyzed
to PR. PRG and the isomeric glucuronides almost com-
pletely hydrolyzed to PR after 8 h of incubation. This
instability to hydrolysis in plasma is probably due not
only to the higher pH value but also to enzymic hydroly-
sis by esterase and albumin (44, 45).
The in vitro irreversible binding of PRG to plasma
proteins was examined. The protein pellets obtained from
the incubated plasma were exhaustively washed followed
by hydrolysis of the adducts and extraction of the
liberated PR. A typical chromatogram of the extract is
shown in Figure 9. Ketoprofen was useful as the internal
standard for the determination of PR (46). The covalent
binding rapidly proceeded and reached a plateau after 2
h of incubation as shown in Figure 10, where the
maximal binding, 146 ( 4.8 pmol/mg of protein, was
achieved. The protein binding was found to be a signifi-

770 Chem. Res. Toxicol., Vol. 15, No. 6, 2002
Akira et al.
tween the neighboring hydroxyl groups occurs via o-acid
ester intermediates, and the initial acyl migration step
from the 1â-acyl glucuronide to the 2â-acyl isomer is
considered to be irreversible whereas the rearrangement
between the 2R/â-, 3R/â-, and 4R/â-acyl isomers is revers-
ible (1). The sequential appearance of 2-acyl, 3-acyl, and
4-acyl isomers with disappearance of PRG (Figures 5 and
8) is in accordance with this mechanism. However, the
concentration of PRG did not follow the first-order
degradation kinetics after most of the PRG degraded. As
the concentration of PRG was higher than predicted at
the late time points, it was possible that the compound
exists in an equilibrium favoring the corresponding 2R/
â-acyl isomer. The equilibrium was demonstrated to exist
by the experiments in Figure 6, where a low level of the
1â-acyl isomer was rapidly formed from the 3â-acyl
isomer via the 2â-acyl isomer. Similar deviation from
first-order kinetics at the late time points was also
observed in (S)-naproxen 1â-acyl glucuronide (54). Hansen-
Moller et al. (3) presented evidence for minor regenera-
tion of diflunisal 1â-acyl glucuronide from the corre-
sponding 4â-acyl isomer at pH 8.0 or a mixture of the
acyl-migrated isomers at pH 8.5. However, all other
studies on rearrangement of acyl glucuronides have
concluded that this particular migration seems not to
occur.
The PR 1R-acyl isomer was found to exist in an
equilibrium favoring the corresponding 2R/â-acyl isomer
in buffer at pH 7.4 and 37 °C. The isomer was much less
stable than PRG in terms of acyl migration as well as
overall degradation. The formation and rapid degradation
are of great interest in connection with the mechanism
and kinetics (10) of acyl migration and the structure-
acyl migration relationships (8, 47). The isomer may have
special reactivity such as the direct reaction with impor-
tant proteins via the transacylation mechanism because
of the characteristic conformation (1,2-cis) different from
PRG (1,2-trans). Thus, the 1R-acyl isomer may be a
significant species in terms of bioreactivity and toxicity,
even though the concentration is low compared with
other acyl-migrated isomers. There was no evidence for
the formation of 1R-acyl isomer until very recently, which
would be because no authentic sample was obtained, and
the compound was very unstable and existed in a low
concentration. Corcoran et al. (56) have first reported that
a low level of 1R-acyl isomer exists in the incubation
mixture (pH 7.4, 37 °C) of (S)-naproxen 1â-acyl glucu-
ronide purified from human urine. This paper presented
another example of the formation of 1R-acyl isomer.
The half-life (0.17 h) of PRG in plasma at pH 7.4 and
37 °C was much lower than that (∼0.75 h) reported in
the literature (36), where urine from a PR-treated human
subject was used as the source of PRG to incubate with
human plasma. The previous half-life is thought to be
overestimated because the HPLC method used did not
discriminate between PRG and its isomeric glucuronides.
It is needless to say that the higher pH value in our study
is at least partly responsible for the difference. PRG was
found to be very unstable and mainly decompose to the
acyl-migrated products also in plasma. The present
results mean that when the concentration of PRG in
blood is measured in the pharmacokinetic studies, PRG
may decompose during treatments of blood samples even
though appreciable amounts of PRG originally exist. On
the other hand, PRG is relatively stable in urine, the pH
of which is generally acidic (35). The high reactivity in
F igu r e 10. Time course of covalent binding of PRG to proteins
in human plasma at 37 °C. initial concentration of PRG, 109
mM; bars indicate (SD (n ) 3).
cant pathway in the reaction of PRG in plasma since the
maximum amount corresponded to approximately 10%
of the initial concentration of PRG. The adduct was at
least stable for more than 8 h. The protein binding was
considered to proceed mainly via the Schiff’s base mech-
anism because the adducts were largely formed after
most of the PRG degraded to the isomeric glucuronides.
Discu ssion
The half-life of PRG (0.27 h) in phosphate buffer (pH
7.4) at 37 °C was found to be much smaller than that
(ca. 0.4 h) reported in the literature (35). In the previous
report, relatively impure PRG was used, and the HPLC
separation of PRG from its isomeric glucuronides was not
considered. Thus, it is considered that the measured
amounts of PRG included the amounts of acyl-migrated
products and the half-life was overestimated. Rates of
degradation of 1â-acyl glucuronides depend on the struc-
ture of the aglycon as well as pH and temperature (8,
47). The published first-order degradation half-lives in
buffer at pH 7.4 and 37 °C vary from one compound to
another in the range of 0.27-79 h (48). The half-lives
(h) of relatively unstable acyl glucuronides have been
reported as follows: tolmetin (49) 0.27; zenarestat (50)
0.42; zomepirac (51) 0.45; diclofenac (52) 0.51; (R)-keto-
profen (10) 0.66; diflunisal (53) 0.67; (R)-naproxen (6)
0.92. The lability of PRG was comparable to that of
tolmetin that is the most labile acyl glucuronide reported
to date. Tolmetin was withdrawn from the market
because of severe adverse events that were most likely
due to the formation of new antigens followed by auto-
immune responses. Thus, PRG is least stable of all the
drug 1â-acyl glucuronides clinically used. The extent of
formation of the potentially immunotoxic protein adducts
has been postulated to be ralated to the extent of acyl
migration (54). As PRG largely degrades by acyl migra-
tion, it may be most reactive to proteins of all the drug
1â-acyl glucuronides although in vivo the extent of
systemic exposure toward the acyl glucuronide and the
participation of some enzymes must be also considered
(55).
The scheme of acyl migration shown in Figure 1 is
generally accepted, where succesive acyl migration be-

Acyl Migration of Probenecid Glucuronide
Chem. Res. Toxicol., Vol. 15, No. 6, 2002 771
naproxen glucuronide diastereomers to proteins. J . Pharmaco-
kinet. Biopharm. 23, 379-395.
plasma may be thus one convincing reason PRG was not
detected in human plasma after dosing of PR despite the
urinary excretion of large amounts of PRG, although the
high clearance and biosynthesis of the glucuronide in the
kidney tubule are also other possible reasons (36).
(7) Sidelmann, U. G., Lenz, E. M., Spraul, M., Hofmann, M., Troke,
J ., Sanderson, P. N., Lindon, J . C., Wilson, I. D., and Nicholson,
J . K. (1996) 750 MHz HPLC NMR spectroscopic studies on the
separation and characterization of the positional isomers of the
glucuronides of 6,11-dihydro-11-oxodibenz[b,e]oxepin-2-acetic acid.
Anal. Chem. 68, 106-110.
There has been no report on the irreversible protein
binding of PRG in plasma whereas the binding to human
serum albumin in buffer has been examined by Hansen-
Moller et al. (35). They reported that a large amount of
adducts, 3.9 nmol/mg of human serum albumin, was
formed in 6 h of incubation, that was more than 20 times
larger than the maximum binding in plasma obtained
here. Although there have been many papers on the
reactivity of drug 1â-acyl glucuronides in human plasma,
it is difficult to compare the results because the pH values
of plasma samples as well as the treatments (buffered
or unbuffered) after blood sampling are not frequently
described despite the lability of plasma pH. The increase
in pH will accelerate the chemical hydrolysis and rela-
tively decrease the acyl migration and the subsequent
covalent binding. The reaction rate of binding will also
be influenced by the molar ratio between the acyl
glucuronide and the plasma proteins. The maximum
amount (pmol/mg of protein) of protein binding, the
period (h) required to reach the maximum, and the initial
concentration (µM) of acyl glucuronide are reported as
follows: clofibric acid (57) 111, 4-8, 76; diflunisal (58)
171, 4, 198; (S)-ibuprofen (59) 150, 12, 131; ibufenac (59)
185, 6, 136. Although the maximum amount of protein
adducts of PRG is comparable to those of other acyl
glucuronides, the period to reach the maximum is rela-
tively short. Thus, PRG appears to be among acyl
glucuronides that are most reactive to plasma pro-
teins.
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In summary, PRG was found very unstable in phos-
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significant amounts of protein adducts in plasma prob-
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proteins could provide a possible explanation for the
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