development of an in vitro screening model for the biosynthesis of ...

0090-9556/02/3004-404–413$7.00
DRUG METABOLISM AND DISPOSITION Vol. 30, No. 4
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 608/969724
DMD 30:404–413, 2002 Printed in U.S.A.
DEVELOPMENT OF AN IN VITRO SCREENING MODEL FOR THE BIOSYNTHESIS OF
ACYL GLUCURONIDE METABOLITES AND THE ASSESSMENT OF THEIR REACTIVITY
TOWARD HUMAN SERUM ALBUMIN
SEBASTIEN BOLZE, NORBERT BROMET, CROISINE GAY-FEUTRY, FREDERIC MASSIERE, ROSELYNE BOULIEU,
AND THIERRY HULOT
Department of Pharmacokinetics and Metabolism, Lipha S.A., Centre de Recherche Lyon-Lacassagne, Lyon, France (S.B., T.H.); Biotec
Centre, Orleans, France (N.B.); Biopredic International, Rennes, France (C.G.F., F.M.); and Département de Pharmacie Clinique, de
Pharmacocinétique et Évaluation des Médicaments, Faculté de Pharmacie, Université Lyon, France (R.B.)
ABSTRACT:
An in vitro screening model was developed to determine the reactivity
of acyl glucuronide metabolites from carboxylic drugs. This
assay is composed of two phases. The first is a phase of biosynthesis
of acyl glucuronides by human liver microsomes (HLM). The
second, during which acyl glucuronides are incubated with human
serum albumin (HSA), consists of assessing the reactivity of acyl
glucuronides toward HSA. Both phases are performed successively
in the same experiment. This model was validated using
eight carboxylic drugs that were well known for their reactivity,
their extent of covalent binding, and their immunological potential.
These products were representative of the scale of reactivity. Each
compound was incubated with HLM at 400 M and metabolized
into acyl glucuronide to different extents, ranging from 5.6% (tol-
Many acidic drugs with carboxylic acid functions are metabolized
to reactive acyl glucuronides. These metabolites are unstable at physiological
pH and can result in free aglycone by hydrolysis and lead to
positional isomers by acyl migration. Acyl migration involves the
transfer of the acyl group from the position 1 to the C-2, C-3, or C-4
position of the glucuronic acid ring, which results in the formation of
isomeric acyl glucuronides (Faed, 1984; Spahn-Langguth and Benet,
1992).
Acyl-migrated glucuronide isomers were shown to bind covalently
to proteins in vitro and in vivo causing potential toxicity (Spahn-
Langguth et al., 1996). The glucuronide-mediated toxicity depends on
the covalent binding of acyl glucuronides to specific target proteins
located in specific tissues. The toxicological mechanisms are still
unknown (Park et al., 1987; Riley and Leeder, 1995; Dansette et al.,
1998).
However, data literature review provides much information regarding
immunologically based and clinically relevant adverse reactions
of several drugs that are probably related to the formation of highly
reactive acyl glucuronides. These drugs include tolmetin, zomepirac,
This work has been presented in part at the 6th International Society for the
Study of Xenobiotics (ISSX) meeting, in Munich on October 8–11, 2001.
Address correspondence to: Sébastien Bolze, MPK department, LIPHA S.A.,
115 Av. Lacassagne, 69003 Lyon, France. E-mail: sebastien.bolze@lipha.fr
(Received October 11, 2001; accepted December 24, 2001)
This article is available online at http://dmd.aspetjournals.org
404
metin) to 89.4% (diclofenac). The first-order aglycone appearance
rate constant and the extent of covalent binding to proteins were
assayed during the incubation of acyl glucuronides formed with
HSA for 24 h. Extensive isomerization phenomenon was observed
for each acyl glucuronide between the two phases. An excellent
correlation was observed (r 2 , 0.94) between the extent of drug
covalent binding to albumin and the aglycone appearance constant
weighted by the percentage of isomerization. This correlation
represents an in vitro reactivity scale, which will be helpful in drug
discovery support programs to predict the covalent binding potential
of new chemical entities. This screening model will also allow
the comparison of acyl glucuronide reactivity for related structure
compounds.
diclofenac, and diflunisal (Hasegawa et al., 1982; Hyneck et al., 1988a,b;
Ojingwa et al., 1988, 1994; Smith et al., 1990, 1992; Kretz-Rommel and
Ding et al., 1994; Boelsterli, 1994; Williams and Dickinson, 1994; Ebner
et al., 1999).
Thus, models for predicting the glucuronide-mediated toxicity of
new chemical entities are desirable particularly for all drug discovery
support programs. Benet et al. (1993) found on six drugs a correlation
between the extent of covalent binding to protein and the global
degradation rate constant of -1-O-acyl glucuronide. But, this correlation
seems to be less evident when literature data are implemented
in the model (Spahn-Langguth et al., 1996). The global degradation
rate constant of -1-O-acyl glucuronide may not be used as a direct
measure of potential protein reactivity. As covalent binding to proteins
primarily occurs via acyl-migrated isomers, a distinction between
the degradation due to hydrolysis and the degradation due to
acyl migration is certainly necessary.
On the other hand, studies assessing the reactivity of acyl glucuronides
need a preliminary step for the chemical synthesis or the
isolation from urine or microsomal incubation of the -1-O-acyl
glucuronide standard. This step is often the limiting factor for an early
detection of highly reactive acyl glucuronides for new chemical
entities.
The objective of this study was to develop a screening model for the
reactivity of acyl glucuronides. This reactivity was assessed by the
instability (hydrolysis isomerization) of these acyl glucuronides
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and by their extent of covalent binding to human serum albumin. The
model developed allowed in the same experiment, i) the production of
acyl glucuronide by human liver microsomes, ii) the determination of
its hydrolysis and isomerization rate constant, and iii) the determination
of the extent of covalent binding with HSA. 1 The model was
evaluated with eight acidic drugs metabolized into acyl glucuronide
(tolmetin, zomepirac, diclofenac, fenoprofen, ketoprofen, ibuprofen,
suprofen, and furosemide). These drugs have been extensively studied
and represent a large scale of reactive products (Smith et al., 1986;
Hyneck et al., 1988a; Castillo and Smith, 1991; Volland et al., 1991;
Hayball et al., 1992; Dubois et al., 1993, 1994; Smith and Liu, 1993;
Castillo et al., 1995; Ebner et al., 1999; Mizuma et al., 1999). Tolmetin
and zomepirac were withdrawn from the market because of hypersensitivity
reactions; ibuprofen is considered to be the safest nonsteroidal
anti-inflammatory drug, and furosemide shows very low levels of covalent
binding.
Materials and Methods
The experiment was divided into two steps. The first phase (“biosynthesis”
phase) was elaborated to synthesize acyl glucuronide by human liver microsomes.
The second phase (“reactivity” phase) was dedicated to the determination
of the hydrolysis and isomerization rate constant of 1-O-acyl glucuronide
and the extent of covalent binding to HSA.
Chemicals. Tolmetin, zomepirac, diclofenac, suprofen, fenoprofen, ketoprofen,
ibuprofen, furosemide, HSA (fraction V), UDP-N-acetylglucosamine,
UDP-glucuronic acid, and bovine -glucuronidase were purchased from Sigma
(l’Isle d’Abeau Chesnes, France). The pool of human liver microsomes (29
donors) used in this study was prepared by Biopredic International (Rennes,
France). All other reagents and solvents were of analytical grade and obtained
from Sigma or Merck (Darmstadt, Germany).
In Vitro Biosynthesis of Acyl Glucuronides. Test compounds (tolmetin,
zomepirac, fenoprofen, ketoprofen, ibuprofen, suprofen, diclofenac, and furosemide;
Fig. 1) were incubated at 400 M in triplicate for 4hat37°C with
human liver microsomes (3 mg/ml) in 100 mM Tris buffer, pH 7.4, containing
1% dimethyl sulfoxide, 5 mM MgCl2, 5 mM UDP-glucuronic acid, and 1 mM
UDP-N-acetylglucosamine in a final volume of 4.8 ml. Two 400-l aliquots
were withdrawn at time points 0 and 4 h. The reaction was stopped by protein
precipitation with the addition of 1 ml of 4% trifluoroacetic acid in acetonitrile
(pH lowered to 3–4) and then centrifuged at 1500 rpm for 10 min. Supernatants
were collected and stored at 80°C until analysis for the determination
of the amount of acyl glucuronide synthesized.
Reactivity of Acyl Glucuronides. At the end of the 4-h incubation duration,
the remaining mixture was centrifuged at 1500 rpm for 20 min (withdrawal
of microsomes). Supernatants (3.0 ml) were then transferred into
capped new tubes and incubated for various times with 0.5 mM HSA. At the
sample times of 15 and 30 min and 1, 2, 4, 6, and 24 h, a 300-l aliquot was
withdrawn and transferred into a tube containing 1 ml of 4% trifluoroacetic
acid in acetonitrile and then centrifuged at 1500 rpm for 10 min. Supernatants
were collected and stored at 80°C. Pellets were washed with 1 ml of 5%
aqueous trifluoroacetic acid (gentle shaking for 10 min and then centrifugation
at 1500 rpm for 10 min) and then three times successively with 1 ml of
methanol (gentle shaking for 10 min and then centrifugation at 1500 rpm for
10 min). Washed pellets and dry residues from supernatants from the last
washing step only were stored at 80°C until analysis.
Controls were performed in parallel and treated identically except that no
cofactor was added during the incubation with microsomes. One 400-l
aliquot was withdrawn after 0 and 4hofincubation in the microsome mixture
and treated like the aliquots of the biosynthesis phase. The residual mixture
was centrifuged at 1500 rpm for 20 min; then supernatants (1.0 ml) were
transferred into capped new tubes and underwent the reactivity phase (i.e.,
incubation for various times with 0.5 mM HSA). After an incubation period of
6 and 24 h, a 300-l aliquot was withdrawn and treated like the other aliquots
of the reactivity phase.
Analytical Method. Incubation samples generated for each drug were
IN VITRO ASSESSMENT OF ACYL GLUCURONIDE REACTIVITY
1 Abbreviations used are: HSA, human serum albumin; QCs, quality controls. .
405
FIG. 1.Structures of the eight drugs for which in vitro potential of metabolism
by human microsomes and the extent of covalent binding with HSA were
investigated.
analyzed by a generic liquid chromatography-tandem mass spectrometry
method. The analytical column was a Hypersil BDS (125 4-mm i.d.;
Thermoquest; Thermo Finnigan MAT; San Jose, CA). Separation of the
different acyl glucuronide isomers from their aglycone was achieved using a
gradient elution mode. The mobile phase was a mixture of acetonitrile/10 mM
acetate ammonium buffer (70:30, v/v) 0.5% acid acetic for solvent A and
acetonitrile/10 mM acetate ammonium buffer (4:96, v/v) for solvent B. The
gradient profile was adjusted for each compound with a flow rate of 1 ml/min
and run time around 15 min. Detection and quantification were performed by
tandem mass spectrometry using a turbo ion spray ion source (API 365;
Applied Biosytems, Toronto, ON, Canada).
Concentrations of aglycone, 1-O--acyl glucuronide, and acyl glucuronide
isomers were determined in supernatants of the biosynthesis and of reactivity
phase. The principle of the assay is summed up below. Each sample was
divided into three aliquots. In the first aliquot, free aglycone concentration (i)
was determined. The second aliquot was incubated with 1000 units of bovine
-glucuronidase at 37°C for2htocleave 1-conjugates and liberate the
corresponding aglycone part. A positive control with phenolphthalein-1-Oglucuronide
was examined to verify the -glucuronidase enzyme activity. The
aglycone concentration found (ii) minus the free aglycone concentration (i)
determined earlier corresponded to the 1-O--acyl glucuronide concentration.
In the same way, the third aliquot was submitted to alkaline hydrolysis (1 N
KOH at 80°C for 3 h) to hydrolyze all acyl glucuronides present into their
corresponding aglycone. The concentration of acyl glucuronide isomers was
estimated as the difference between total aglycone concentration (iii) and
aglycone concentration coming from the cleavage of 1-conjugates (ii). As
mentioned earlier, two 400-l aliquots were withdrawn at time points 0 and 4 h
of the biosynthesis phase, and one of these aliquots was used to check the total
hydrolysis of the 1-O--acyl glucuronide and the chemical stability of the acyl
glucuronides isomers during the 2-h incubation period with -glucuronidase.
The total hydrolysis of the 1-O--acyl glucuronide and the chemical stability
of the acyl glucuronides isomers were evaluated by direct graphic assessment
of the peak’s area, as shown in Fig. 2. This preliminary step was performed
before the analysis of all the samples.
The extent of covalent binding yield to human serum albumin was evaluated
for each drug. Extensively washed protein pellets obtained during the second
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406 BOLZE ET AL.
FIG. 2.Principle of assay.
①, liquid chromatography/tandem mass spectrometry chromatogram obtained after drug incubation with microsomes or HSA; 1-O-acyl glucuronide, 2-, 3-, and 4-O-acyl
glucuronide were separated and free aglycone drug was assayed. ②, samples were hydrolyzed with -glucuronidase to estimate 1-O-acyl glucuronide (obtained by the
difference of ② ①). ③, the acyl glucuronide isomer concentration was obtained after alkaline hydrolysis (③ ②).
incubation period were submitted to alkaline hydrolysis (1 N KOH at 80°C for
3 h). Moles of aglycone released by this procedure were considered as
irreversibly bound to HSA. Protein pellets from control samples, performed
without cofactors, were also submitted to alkaline hydrolysis. Although no
aglycone release was expected, the detected levels were considered as background
noise and were subtracted from the results obtained.
Samples for the determination of free aglycone and the 1-O--acyl glucuronide
concentration were just diluted in mobile phase from 1/10 to 1/40,
depending on the sensitivity of the compound in mass spectrometry before
injection into the analytical system. Calibration curves, from 50 to 10,000
ng/ml, were prepared by spiking the adequate amount of standard (ketoprofen,
diclofenac, suprofen, tolmetin, zomepirac, furosemide, fenoprofen, and ibuprofen)
in the blank matrix. Samples for the determination of acyl glucuronide
isomers and the covalent binding concentration were extracted by a solid-phase
extraction method using Oasis HLB cartridges (Waters, Saint Quentin en
Yvelines, France). Briefly, the cartridges were conditioned by 1 ml of methanol
followed by 1 ml of water. Then, the samples were loaded onto the
cartridges. The cartridges were washed with 1 ml of water. Elution was based
on 1 ml of the mixture acetonitrile/10 mM ammonium acetate buffer (75:25,
v/v) 0.05% acetic acid. Calibration curves, from 5 to 1000 ng/ml, were
prepared in the chromatographic mobile phase. Dry residues from the last
washing fraction of protein pellets were redissolved in 1 ml of chromato-
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TABLE 1
Percentages of drugs metabolized into acyl glucuronide and of acyl glucuronide
isomerized between the biosynthesis phase and the reactivity phase
Mean S.D. (n 3); The percentage of acyl glucuronides formed is determined by
parent drug depletion. The isomerization percentage was defined as the amount of the 1-Oacyl
glucuronide transformed into isomers during the process between the two phases.
Compound Name Acyl Glucuronide Formed Isomerisation
%
Tolmetin 5.6 4.8 92.8 18.1
Zomepirac 22.5 10.5 42.4 20.4
Suprofen 68.6 2.1 32.0 8.3
Diclofenac 89.4 0.5 72.2 3.4
Fenoprofen 55.8 0.4 51.0 12.2
Ibuprofen 53.3 6.0 30.6 8.5
Ketoprofen 62.8 5.4 32.4 4.9
Furosemide 19.2 2.9 0.0 0.0
graphic mobile phase and analyzed to ensure the exhaustiveness of the washing
procedure. Only traces of free aglycone or free acyl glucuronide should still
remain.
Analyte peak areas (determined by mass spectrometry) were used for
quantification together with the different calibration curve (external calibration).
Quality control samples were performed for each phase of the model at
three concentrations (15, 150, and 300 M). Cofactors were added at the end
of the incubation period for QCs of the biosynthesis phase. QCs of the
reactivity phase were incubated with HSA for the selected duration and then
treated as an experimental incubation. QCs were used to ensure accuracy and
precision of the method. All QCs showed accuracy within 80 to 120%.
Data Analysis. The degradation rate was defined as the initial loss of the
1-O--acyl glucuronide component. Hydrolysis was defined as the initial
formation of the aglycone, and acyl migration (isomerization) was defined as
the formation of positional isomers. According to Sidelmann et al. (1996), the
hydrolysis rate was calculated as the degradation rate corrected for the formation
of positional isomers, and the acyl migration rate was calculated as the
degradation rate corrected for hydrolysis. Kinetic data of degradation of acyl
glucuronides were calculated by nonlinear regression analysis of the measured
data using the equation for first-order reaction kinetics, C C(0)e kt .Inthe
same way, aglycone release kinetic data were analyzed by nonlinear regression
analysis using the equation for first-order reaction kinetics, C C(0)e kt .
Results
Biosynthesis of Acyl Glucuronides. The first step consisted in acyl
glucuronide synthesis by human liver microsomes in straight conditions
(400 M substrate; 3 mg/ml microsomal proteins; 4 h incubation).
The conditions retained were able to produce acyl glucuronide
for the eight compounds tested. The percentage of metabolism or the
percentage of acyl glucuronides formed was determined by the quantification
of parent drug depletion during the 4-h incubation periods.
This percentage of metabolism ranged from 5.6% (tolmetin) to 89.4%
(diclofenac) (Table 1). Thus, this first step allowed an estimation of
the capability of each drug to be metabolized into acyl glucuronide.
Reactivity Assessment with Human Serum Albumin. Instability
assessment. During the second step, the supernatant of the first step
was incubated with 0.15 mM phosphate buffer containing 0.5 mM
HSA for 24 h. The time-dependent degradation of 1-O-acyl glucuronide
and the appearance of its isomers and hydrolyzed aglycone were
monitored for each drug. An example of the time course observed for
each species derived from diclofenac and ibuprofen acyl glucuronide
is shown in Fig. 3. The sum of the initial concentrations observed is
lower than expected (400 M). A binding (reversible or irreversible)
on microsomes and a drug loss during centrifugation could explain
this decrease on concentrations. This phenomenon should not effect
the data interpretation because we always referred to the initial acyl
glucuronide concentration of the second phase. 1-O-Acyl glucuronides
were mostly expected to be detected at the beginning of the
IN VITRO ASSESSMENT OF ACYL GLUCURONIDE REACTIVITY
407
incubation with HSA. However, extensive acyl migration occurred
during the process between the two steps. A majority of acyl glucuronide
isomers were detected from early kinetic points. The analytical
method developed allowed good separation between 1-O-acyl
glucuronide and its isomers but not totally between the isomers
themselves. This isomer resolution was time consuming and not
compatible with a screening purpose. Therefore, high isomerization
could not be seen since the levels of isomers remained constant. Only
the time-dependent degradation of acyl glucuronide isomers by hydrolysis
could be followed. 1-O-Acyl glucuronide levels remained
low over the incubation period. This phenomenon observed for diclofenac
and ibuprofen was also observed for the other compounds
studied. The percentage of isomerization between the two steps is
presented in Table 1 for each compound. The determination of the
aglycone appearance and acyl glucuronide degradation rate was
shown in Fig. 4. Apparent first-order degradation and the appearance
constants for all compounds are listed in Table 2.
Irreversible binding to HSA. The time dependence for irreversible
binding to HSA of each acyl glucuronide studied was also investigated
during this second step. The extent of covalent binding was
expressed in millimoles of aglycone covalently bound per mole of
protein. As shown in Fig. 5, all acyl glucuronides rapidly produced a
covalent adduct with HSA. The maximum yield was achieved after 4
to 6 h of incubation, except for fenoprofen for which maximum
covalent binding was achieved after 24 h of incubation. The maximum
extent of covalent binding varied from 0.43 to 8.20 mmol irreversibly
bound/mol of protein (Fig. 6A). However, the amount of drug irreversibly
bound was obviously related to the amount of acyl glucuronide
present at the beginning of the reactivity phase. Thus, the extent
of covalent binding was normalized to protein content and expressed
as the percentage of total acyl glucuronide present at the beginning of
the reactivity phase. Percentages of covalent binding ranged from 5.7
to 0.34% (Fig. 6B). This expression of the extent of covalent binding
changed the compound ranking. Tolmetin, which is known to be the
most reactive acyl glucuronide, changed from a low intrinsic value of
covalent binding (Fig. 6A) to the highest percentage of covalent
binding related to a low amount of acyl glucuronide (Fig. 6B). On the
other hand, furosemide remained in the same position no matter what
expression system was chosen, thus showing the low covalent binding
capacity of furosemide acyl glucuronide (Benet et al., 1993; Mizuma
et al., 1999). The ranking according to the amount of drug irreversibly
bound observed for the eight compounds studied was consistent with
the literature data. Indeed, tolmetin, zomepirac, and diclofenac (Smith
et al., 1986; Hyneck et al., 1988a; Munafo et al., 1989) are considered
as reactive products, whereas ibuprofen, ketoprofen, and furosemide
(Dubois et al., 1993; Castillo and Smith, 1995; Presle et al., 1996;
Mizuma et al., 1999) are mentioned as safer products.
Analysis of the last washing fraction of protein pellets revealed that
the washing procedure was sufficiently exhaustive for most of the
compounds. The mean values of aglycone found were below the limit
of quantification (5 ng/ml) for ketoprofen, diclofenac, suprofen,
tolmetin, and zomepirac and were equal to 77 ng/ml for furosemide,
63 ng/ml for fenoprofen, and 29 ng/ml for ibuprofen. The same results
were obtained for the analysis of covalent binding in controls. Background
noise levels found for furosemide, fenoprofen, and ibuprofen
were subtracted from the results obtained.
Discussion
The objective of this work was to develop a screening model to
assess the reactivity of acyl glucuronides. This screening model al-
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408 BOLZE ET AL.
FIG. 3.Time courses of rearrangement and hydrolysis of diclofenac (A) and ibuprofen (B) acyl glucuronide in 0.15 mM phosphate buffer containing 0.5 mM HSA, pH
7.4, at 37°C.
Each point and vertical bar represent the mean S.D. of three independent series.
lowed us to assess the instability of the biosynthesized acyl glucuronides
and to rank compounds according to their maximum covalent
binding to HSA.
The first biosynthesis phase was designed with straightforward
experimental conditions for standardization purpose. Being easy to
work on, human liver microsomes were used for the production of
acyl glucuronides. High protein concentration (3 mg/ml), use of
glucuronosyl transferase activities activator (UDP-N-acetylglucosamine),
high drug concentration (400 M) of incubation, and long
incubation duration (4 h) were chosen as standard conditions to
maximize the biosynthesis of acyl glucuronide but were not specifically
optimized for each case. Consequently, the linearity of acyl
glucuronide formation could not been studied for each compound.
However, the selected conditions should allow comparison of acyl
glucuronidation potential between compounds from the same chemical
family. In the same way, human serum albumin was chosen as a
reference target protein to assess the extent of covalent binding during
the reactivity phase. The extent of covalent binding was shown to vary
greatly depending on the nature of albumin preparation used (Williams
and Dickinson, 1994; Ebner et al., 1999). Thus, in vitro assays
on covalent binding to proteins can be expected to be highly variable.
Moreover, plasma proteins are not the only targets of covalent binding.
Acyl glucuronides can irreversibly bind to several tissues or organ
macromolecules. Unfortunately, irreversible binding on all proteins
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FIG. 4.Nonlinear regression analysis of the degradation of the whole acyl glucuronide (top) and appearance of aglycone (bottom) during incubation at 37°C with
HSA, pH 7.4.
, tolmetin; f, zomepirac; Œ, ketoprofen; ‚, fenoprofen; F, suprofen; E, furosemide; , diclofenac; ✳, ibuprofen.
cannot be assessed in a screening process. Furthermore, HSA remains
the protein most extensively studied. It is widely distributed in the
plasma compartment and easily related to the immune system. The
knowledge gathered on covalent binding of acyl glucuronide and HSA
allowed the comparison and validation of the results achieved with
this model. A product showing a significant covalent binding to HSA
in our model will require specific attention during the development
process. The constant values calculated for the disappearance of acyl
glucuronides were lower than those previously published, especially
for zomepirac and tolmetin (Hasegawa et al., 1982; Ojingwa et al.,
1994). In fact, those reported values represented the global degradation
of 1-O-acyl glucuronide (hydrolysis isomerization), whereas
the values presented in this study only represent the degradation of
isomeric forms. In the conditions of analysis described here, the exact
IN VITRO ASSESSMENT OF ACYL GLUCURONIDE REACTIVITY
TABLE 2
Apparent first-order degradation of various acyl glucuronides and aglycone
appearance constants measured during incubation phases with 0.5 mM HSA, pH
7.4, at 37°C
Mean S.D. (n 3).
Compound Name Acyl Glucuronide Degradation Rate Aglycone Appearance Rate
Tolmetin 0.055 0.020 0.106 0.049
Zomepirac 0.025 0.008 0.103 0.027
Suprofen 0.046 0.021 0.035 0.018
Diclofenac 0.050 0.016 0.081 0.023
Fenoprofen 0.021 0.014 0.044 0.011
Ibuprofen 0.028 0.003 0.037 0.013
Ketoprofen 0.037 0.010 0.016 0.005
Furosemide 0.006 0.011 0.000 0.001
h 1
409
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410 BOLZE ET AL.
FIG. 5.Time-dependent irreversible binding of several aglycone after incubation of their respective acyl glucuronide (produced by human liver microsomes;
incubation of 400 M aglycone) in human serum albumin solution 0.5 mM at 37°C, pH 7.4.
Data are the average of triplicate measurements. , tolmetin; f, zomepirac; Œ, ketoprofen; ‚, fenoprofen; F, suprofen; E, furosemide; , diclofenac; ✳, ibuprofen.
FIG. 6.The ranking of compounds according to their extent of covalent binding expressed in millimoles irreversibly bound per mole of protein and normalized by
protein content (A) and expressed as the percentage of total acyl glucuronide present at the beginning of the reactivity phase (B).
Data are the average of triplicate measurements; bars indicate S.D.
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FIG. 7.Correlation between the extent of covalent binding (protein content normalized and expressed as the percentage of total acyl glucuronide present at the
beginning of the reactivity phase) versus the degradation rate constant (h1 ) (A) and the aglycone appearance rate constant (h 1 ) (B) for the in vitro incubation of
various acyl glucuronide with HSA (0.5 mM).
The solid line represents correlation with eight drugs; the dotted line represents correlation with six drugs.
distribution of isomers could not be determined. Only the hydrolysis
of acyl glucuronide isomers could be examined. Therefore, the greater
the isomerization process was compared with hydrolysis, the lower
the degradation constant was. Thus, the values observed in this study
were obviously lower than the published values. The results achieved
showed that the acyl glucuronide isomer degradation constant seemed
to be less interesting because we could not distinguish whether the
acyl glucuronide isomers were strongly isomerized or were very
stable. This could be illustrated by the low constant value observed for
furosemide. The constant determined for the rate of aglycone appearance
could then be an alternative parameter for the assessment of acyl
glucuronide instability. Indeed, the appearance of aglycone in the
second incubation medium came from the hydrolysis of 1-O-acyl
glucuronide and its isomers. The rate of aglycone release could be an
indicator of the instability and therefore of the reactivity of acyl
glucuronide.
IN VITRO ASSESSMENT OF ACYL GLUCURONIDE REACTIVITY
411
The in vitro extent of covalent binding to HSA allowed the rank of
acyl carboxylic drugs according to their reactivity potential. However,
as the relation between covalent binding and toxicological effect is
still unclear, we suggest extending the predictability of our model by
integrating a second parameter, which is the instability of the acyl
glucuronides. For this purpose, correlation between the amount of
covalent binding observed and the instability of each acyl glucuronide
was attempted.
First, we tried to reproduce the correlation described by Benet
(Benet et al., 1993) between the moles of drug maximally bound
irreversibly per mole of protein versus the degradation rate constant
for the -1-O-acyl glucuronide conjugates. The maximum covalent
binding (normalized to protein content and expressed as the percentage
of total acyl glucuronide present) observed was not correlated
with the global degradation rate constant for acyl glucuronide isomers
(Fig. 7A). Indeed, the global degradation rate constant of acyl gluc-
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412 BOLZE ET AL.
FIG. 8.Correlation between the extent of covalent binding (protein content normalized and expressed as the percentage of total acyl glucuronide present at the
beginning of the reactivity phase) versus the aglycone appearance rate constant weighted by the percentage of isomerization (between biosynthesis and reactivity
phase) (h 1 ) during in vitro incubation of various acyl glucuronide with HSA (0.5 mM).
uronide isomers was not a better parameter for reactivity prediction
than the global acyl glucuronide degradation rate constant described
by Benet. A correlation was also searched for between covalent
binding and the hydrolysis rate. The aglycone appearance rate constant
corresponded to the global acyl glucuronide hydrolysis rate. A
satisfactory correlation was obtained for six of eight drugs (r 2 , 0.89).
When zomepirac and tolmetin were taken into account, the correlation
was less satisfactory (r 2 , 0.62) (Fig. 7B). The hydrolysis rate constant
was the same for both compounds, whereas the extent of covalent
binding was higher for tolmetin. The rate of hydrolysis (i.e., the rate
of aglycone appearance) does not discriminate sufficiently to explain
the reactivity of all compounds. For most of the acyl glucuronides
under study, isomerization was found to occur between the first and
the second incubation. The extent of this phenomenon seemed to be
more or less important for each acyl glucuronide (Table 1). The extent
of this isomerization was certainly related to the instability of the
1-O-acyl glucuronide formed and its covalent binding capacity. Indeed,
the formation of isomeric forms via acyl migration is a prerequisite
for covalent binding to proteins by “imine” mechanism. Therefore,
the aglycone appearance rate constant was weighted by the
percentage of isomerization between the two incubation steps. An
excellent correlation was then obtained between the maximal amount
of bound drug, expressed as percentage of acyl glucuronide present in
the incubation medium, and the aglycone appearance rate constant
weighted by the percentage of isomerization (r 2 , 0.94) (Fig. 8). The
results presented here showed that the extent of covalent binding
could be predicted on the basis of acyl glucuronide hydrolysis rate
combined with acyl migration propensity. The combination of these
two parameters seemed to be more accurate for covalent binding
prediction than the 1-O-acyl glucuronide degradation rate used by
Benet. The correlation was still confirmed even when data from
products like ibuprofen, suprofen, and diclofenac were added to the
correlation. Thus, an in vitro reactivity scale was drawn up. The rank
of the drugs tested was consistent with the literature. Tolmetin appeared
as the most reactive, furosemide as the least. Ibuprofen and
ketoprofen showed a similar low reactivity, whereas diclofenac,
which has not been previously evaluated, showed a level of reactivity
higher than zomepirac. In these conditions, suprofen appeared as a
low reactive product. Smith showed that 0.75% of suprofen acyl
glucuronide became covalently bound to HSA after 6hofincubation
(Smith and Liu, 1993). The 0.62% value found in this study was found
in fairly good accordance with Smith’s data.
The structural relationship between acyl glucuronide degradation
and covalent binding put forward by Benet (Benet et al., 1993) was
also observed in this study. Acyl glucuronides of -unsubstituted
acetic acid derivatives such as tolmetin and zomepirac (Fig. 1) exhibited
the highest covalent binding. Mono--substituted acetic acids
(fenoprofen) showed intermediate levels of covalent binding. At last,
fully substituted -acetic acids, such as furosemide, led to the lowest
irreversible binding. Additional compounds not tested by Benet complied
with this structural relationship. Indeed, diclofenac, an -unsubstituted
acetic acid, demonstrated high levels of covalent binding,
whereas ketoprofen, ibuprofen, and suprofen (mono--substituted
acetic acids) showed intermediate levels of irreversible binding. This
observation must be confirmed with more compounds and could be
taken into account in the drug design process.
For the first time, a screening model allowing, in one experiment,
the formation of acyl glucuronide metabolite by human microsomes
and the assessment of its reactivity was presented. An excellent
correlation (r 2 , 0.94) was found between the maximal amount of
covalently bound drug (normalized to protein amount and expressed
as percentage of total acyl glucuronide synthesized in the incubation
medium) and the aglycone appearance rate constant weighted by the
percentage of isomerization. This correlation represents an in vitro
reactivity scale, which will help predict the covalent binding potential
of new chemical entities.
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