Metabolic activation of prasugrel: Nature of the two competitive pathways resulting in the opening of its thiophene ring

Article abstract of DOI:10.1021/tx3000279

The mechanism generally admitted for the bioactivation of the antithrombotic prodrug, prasugrel, 1c, is its two-step enzymatic conversion into a biologically active thiol metabolite. The first step is an esterase-catalyzed hydrolysis of its acetate function leading to a thiolactone metabolite 2c. The second step was described as a cytochrome P450 (P450)-dependent oxidative opening of the thiolactone ring of 2c, with intermediate formation of a reactive sulfenic acid metabolite that is eventually reduced to the corresponding active thiol 3c. This article describes a detailed study of the metabolism of 1c by human liver microsomes and human sera, with an analysis by HPLC-MS under conditions allowing a complete separation of the thiol metabolite isomers, after derivatization with 3′-methoxy phenacyl bromide. It shows that there are two competing metabolic pathways for the opening of the 2c thiolactone ring. The major one, which was previously described, results from a P450- and NADPH-dependent redox bioactivation of 2c and leads to 3c, two previously reported thiol diastereomers bearing an exocyclic double bond. It occurs with NADPH-supplemented human liver microsomes but not with human sera. The second one results from a hydrolysis of 2c and leads to an isomer of 3c, 3c endo, in which the double bond has migrated from an exocyclic to an endocyclic position in the piperidine ring. It occurs both with human liver microsomes and human sera, and does not require NADPH. However, it requires Ca²⁺ and is inhibited by paraoxon, which suggests that it is catalyzed by a thioesterase such as PON-1. Chemical experiments have confirmed that hydrolytic opening of thiolactone 2c exclusively leads to derivatives of the endo thiol isomer 3c endo.

Full text of DOI:10.1021/tx3000279

Article
pubs.acs.org/crt
Metabolic Activation of Prasugrel: Nature of the Two Competitive
Pathways Resulting in the Opening of Its Thiophene Ring
Patrick M. Dansette,* Julien Rosi, Justine Debernardi, Gildas Bertho, and Daniel Mansuy
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Universite
Paris Cite, 45 rue des Saints-Peres, 75270 Paris Cedex 06, France
́
Paris Descartes, Sorbonne
́
̀
S
* Supporting Information
ABSTRACT: The mechanism generally admitted for the bioactivation of the antithrombotic prodrug, prasugrel, 1c, is its two-
step enzymatic conversion into a biologically active thiol metabolite. The first step is an esterase-catalyzed hydrolysis of its acetate
function leading to a thiolactone metabolite 2c. The second step was described as a cytochrome P450 (P450)-dependent
oxidative opening of the thiolactone ring of 2c, with intermediate formation of a reactive sulfenic acid metabolite that is
eventually reduced to the corresponding active thiol 3c. This article describes a detailed study of the metabolism of 1c by human
liver microsomes and human sera, with an analysis by HPLC-MS under conditions allowing a complete separation of the thiol
metabolite isomers, after derivatization with 3′-methoxy phenacyl bromide. It shows that there are two competing metabolic
pathways for the opening of the 2c thiolactone ring. The major one, which was previously described, results from a P450- and
NADPH-dependent redox bioactivation of 2c and leads to 3c, two previously reported thiol diastereomers bearing an exocyclic
double bond. It occurs with NADPH-supplemented human liver microsomes but not with human sera. The second one results
from a hydrolysis of 2c and leads to an isomer of 3c, 3c endo, in which the double bond has migrated from an exocyclic to an
endocyclic position in the piperidine ring. It occurs both with human liver microsomes and human sera, and does not require
NADPH. However, it requires Ca2+ and is inhibited by paraoxon, which suggests that it is catalyzed by a thioesterase such as
PON-1. Chemical experiments have confirmed that hydrolytic opening of thiolactone 2c exclusively leads to derivatives of the
endo thiol isomer 3c endo.
leading to the pharmacologically active thiols 3a and 3b also
requires the involvement of a cytochrome P450-catalyzed
oxidative step with consumption of NADPH and O2 (P450
3A4, P450 2B6, P450 2C9, and P450 2C19 are mainly involved
in oxidation of 2b).8
Prasugrel is the newest member of the class of tetrahy-
drothienopyridine antithrombotic produgs. It is also an
irreversible inhibitor of the platelet receptor P2Y12, after
metabolic conversion in vivo to pharmacologically active 3c3,9
(Figure 1). Because of the presence of two chiral carbons in 3c,
this thiol exists as a mixture of four stereoisomers (two pairs of
enantiomers), and the [R,S] isomer is the most active one.9
Metabolic conversion of prasugrel to 3c involves two enzymatic
reactions (Figure 1): (i) the hydrolysis of its ester function
leading to thiolactone 2c, which seems to be mainly catalyzed
by the hCE2 enzyme in humans,10 and (ii) the oxidative
cleavage of the thioester bond of 2c with the eventual
INTRODUCTION
■
Ticlopidine (Ticlid) 1a, clopidogrel (Plavix, Iscover) 1b, and
prasugrel (Effient) 1c (Figure 1) are antithrombotic prodrugs
of the tetrahydrothienopyridine series that must be metabolized
in vivo into the corresponding pharmacologically active 4-
mercapto-3-piperidinyliden acetic acid derivatives 3a, 3b, and
3c, respectively, to exert their activity as antagonists of the
platelet receptor P2Y12.1−4
Ticlopidine was introduced to the market in 1979 for
prevention of thrombotic stroke. Clopidogrel was first launched
in 1998 in the USA and in 1999 in Europe, for the reduction of
atherosclerotic problems in patients with stroke, myocardial
infarction, or peripheral arterial disease. Their metabolic
activation occurs in two steps that are catalyzed by cytochromes
P450.1,2 The first step is a classical cytochrome P450-
dependent hydroxylation of the thiophene ring by NADPH
and O2 leading to thiolactone metabolites 2a and 2b,
respectively5−7 (Figure 1). It is mainly catalyzed by P450
2C19 and P450 2B6 in the case of 1a and by P450 2C19, P450
1A2, and P450 2B6 in the case of 1b.4,8 The second step
Received: January 18, 2012
Published: April 6, 2012
© 2012 American Chemical Society
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Figure 1. Metabolic activation of tetrahydrothienopyridine antithrombotic drugs into pharmacologically active thiols.
Figure 2. Competing pathways involved in the metabolic opening of the thiophene ring of clopidogrel 1b.
formation of 3c, which is catalyzed by cytochromes P450 3A4,
2B6, 2C9, or 2C19 in humans.4
microsomes17,18 and is present in much lower amounts than 3b
in the sera of 1b-treated patients.19 It has been also shown17,18
that chemical, hydrolytic opening of the thiolactone ring of 2b,
using O-nucleophiles such as CH3O−, only led to 3b endo
derivatives, suggesting that hydrolytic opening of thiolactone
2b, either chemical or enzymatic (PON-1-catalyzed), only led
to endo compounds. It is thus necessary to oxidize 2b (by
P450s) to activate its conjugated CO function to produce the
active cis thiols, 3b (Figure 2). It was recently proposed that
PON-1 was a major determinant of 1b efficacy and that PON-1
Q192R polymorphism might have a key role in the rate of 1b
bioactivation and influence platelet response to 1b and the risk
of stent thrombosis in 1b-treated patients.20 However, since
then, many articles have reported that there is no relationship
between PON-1 polymorphism and 1b efficacy.21−29
Metabolites that should derive from 3c endo, the endo
isomer of 3c (Figure 4), were detected in the plasma, urine, and
feces of patients treated with prasugrel 1c30 and also of mice,
rats, and dogs treated with this drug.31 However, the
mechanism of formation of 3c endo was not determined so
far. Thus, it was interesting to know whether 3c endo could
derive from a thioesterase-catalyzed opening of the thiolactone
ring of metabolite 2c, in competition with the P450-dependent
oxidative opening of 2c leading to 3c, in a manner similar to
what was very recently found in the case of clopidogrel. For
that purpose, we have looked for the formation of such an endo
isomer of 3c during the metabolism of 1c by human liver
microsomes or human serum, by using HPLC methods
Recent data have established a detailed pathway for the
formation of active thiols 3 from thiolactones 2 in the
metabolism of prodrugs 1.11−14 It involves a P450-catalyzed
oxidative opening of thiolactone metabolites 2 leading to the
formation of sulfenic acid intermediates such as 5b and a
reduction of these sulfenic acids into the corresponding thiols 3
by GSH11−13,15 (see for instance Figure 2 for the case of 1b).
Reduction of these sulfenic acids by GSH would occur via the
intermediate formation of GSH conjugate disulfides that are
reduced to thiols 3 either by GSH itself11,12 or by the
glutaredoxin−thioredoxin system.16 These sulfenic acid inter-
mediates have been trapped by dimedone during the oxidative
metabolism of thiolactones 2 by rat and human liver
microsomes or by several recombinant human liver cyto-
chromes P450.11,12
A new metabolic pathway resulting in the opening of the
thiolactone ring of clopidogrel metabolite 2b has been
discovered quite recently.17,18 It leads to an isomer of 3b, 3b
endo, in which the double bond has migrated into the
piperidine ring (Figure 2). This isomer is formed upon
treatment of 2b with human liver microsomes and human
serum17,18 and is detected in the serum of 1b-treated patients.19
Its formation from 2b seems to be mainly catalyzed by
paraoxonase-1 (PON-1) (Figure 2).17,18 It is noteworthy that
3b endo is formed in much lower amounts than 3b upon
metabolism of 2b by NADPH-supplemented human liver
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Figure 3. HPLC profiles of incubations of 1c with human liver microsomes with (B) or without (A) NADPH, or with human serum (C), using MS
detection after derivatization with 3′-methoxy-phenacyl bromide and MS2 spectra (parent ion at m/z = 498) of the three derivatized thiol
metabolites, 3c′ (2 diastereomers, E and F) and 3c′endo (D and G correspond to HPLC profiles A and C, respectively).
Incubations of 1c with Human Sera. Human sera were obtained
allowing a good separation of the isomers of 3c and trapping of
these thiols by 4′-methoxy phenacyl bromide. This article shows
that metabolite 3c endo is formed upon thioesterase-dependent
opening of thiolactone 2c during the metabolism of 1c by
human liver microsomes and human serum. Hydrolytic
formation of 3c endo from 2c is in competition with the
P450-dependent oxidative opening of the 2c thiolactone ring
leading to the cis diastereomers 3c, as previously found in the
metabolism of 2b (Figure 2).17,18 This article also shows that
chemical opening of thiolactone 2c by sodium methylate only
leads to an endo thiol isomer derivative, 3c endo methyl ester.
according to a previously published protocol and were given by J. F.
Chasse.
́
32 Typical incubations were performed in potassium phosphate
buffer (0.1 M, pH 7.4) containing 2 mM CaCl2, human serum (10 mg
protein/mL), 1c (100 μM), and a reducing agent (20 mM ascorbic
acid or 5 mM GSH), at 37 °C for 30 min. Reactions were stopped and
analyzed as described above for microsomal incubations.
HPLC-MS Studies. Studies were performed on a Surveyor HPLC
instrument coupled to a LCQ Advantage ion trap mass spectrometer
(Thermo, Les Ulis, France), using a Gemini C18 column (100 × 2
mm, 3 μm; Phenomenex) and a gradient starting at 40% B for 1 min
then increasing linearly to 100% B in 15 min (A = 10 mM ammonium
acetate buffer, pH 4.6, and B = CH3CN/CH3OH/H2O (7:2:1)) at 200
μL/min. Mass spectra were obtained by electrospray ionization (ESI)
in positive ionization mode detection under the following conditions:
source parameters, sheeth gas, 20; auxiliary gas, 5; spray voltage, 4.5
kV; capillary temperature, 200 °C; capillary voltage, 15 V; and m/z
range for MS recorded generally between 300 and 700 (except for
exploratory experiments with a wider range 300−800). MS2 energy
was tested between 20 and 40 eV and was generally 35 eV. For all
products, the indicated parent ions corresponded to M + H+.
EXPERIMENTAL PROCEDURES
■
Chemicals and Biochemicals. Prasugrel base (racemate), 1c, was
obtained from Bepharm (Shangai, China). Oxo-prasugrel base
(racemate), 2c, was obtained from Twisun Pharma (Shangai,
China). All other products including enzymes were from Sigma-
Aldrich (St. Quentin Fallavier, France).
Microsomal Incubations. Human liver microsomes (pool, 10 mg
protein/mL) were obtained from BD-Gentest (Le Pont de Claix,
France). Typical incubations were performed in 200 μL of potassium
phosphate buffer (0.1 M, pH 7.4) containing 2 mM CaCl2,
microsomes (1 mg protein/mL), 1c (100 μM), and a reducing
agent (20 mM ascorbic acid or 5 mM GSH) with or without the
NADPH generating system (1 mM NADP, 15 mM glucose-6-
phosphate, and 2 unit/mL of glucose-6-phosphate dehydrogenase) at
37 °C for 30 min. Reactions were stopped by adding one-half volume
of CH3CN and 3′-methoxy phenacyl bromide (MPBr) (final
concentration 4 mM) for thiol derivatization. After 10 min of
incubation, 25 μL/mL CH3COOH was added, and proteins were
removed by centrifugation at 13,000g.
Chemical Experiments on 1c. Reaction of 1c with CH3ONa in
CH3OH. A solution of 2 mM 1c in CH3OH was treated with 120
mM CH3ONa in CH3OH at 20 °C for 45 min. After dilution
with 10 vol 100 mM phosphate buffer at pH 7.4, the final
product was analyzed by HPLC-MS, as such or after
derivatization with MPBr, as described above, or after oxidation
to 6, the disulfide of 3c endo methyl ester (Figure 5), with 5
mM iodine. Compound 6 was purified by loading on a SepPak
C18 column (Waters), washing with H2O, eluting by CH3OH,
1
and analyzing by H NMR (in CD2Cl2) after evaporation of
CH3OH.
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Figure 4. Two competing pathways involved in the metabolic opening of the thiophene ring of prasugrel 1c. Because of the presence of 2 chiral
carbons (benzylic carbon and carbon bearing the SH function) in metabolite 3c, this metabolite can exist as a mixture of 2 diastereomers (4 optically
active isomers [R,S] and [S,R] and [R,R] and [S,S]).
1
NMR Experiments. H NMR spectra of 6 were done on a Bruker
Avance 400 spectrometer (500 MHz) at 27 °C. Chemical shifts are
given in ppm relative to (CH3)4Si.
Table 1. Formation of 3c and 3c endo upon Metabolism of
1c by Human Liver Microsomes
b
(nmol/mg protein/30 min)
conditions
3c′
3c′ endo
RESULTS
■
a
complete system
22
4
5.7
6
1
1
Metabolism of 1c by Human Liver Microsomes.
Incubation of 1c with human liver microsomes was done for
30 min at 37 °C in the presence of NADPH, which is a
required cofactor for P450-dependent activities,33 and of a
reducing agent (20 mM ascorbic acid or 5 mM GSH) to reduce
the sulfenic acid intermediate 5c into the corresponding thiol
3c (Figure 4).11,12 An HPLC-MS study of the incubate, after
derivatization of the thiol metabolites by treatment with MPBr,
showed the formation of the phenacyl derivatives of three thiol
isomers (Figure 3) exhibiting a parent ion (ESI+) correspond-
ing to M + H+ and characterized by a peak at m/z = 498. Two
of these thiol isomers exhibited identical MS2 spectra, with
major fragments of the parent ion (m/z = 498) at m/z = 348,
318, 206, 177, and 149, as previously described30,34 for the 3′-
methoxy phenacyl derivatives (3c′) of the two cis-thiol
diastereomers 3c (Figure 4).
The third derivatized thiol metabolite exhibited a different
MS2 spectrum characterized by a major fragment of the parent
ion (m/z = 498) at m/z = 206 (Figure 3), which was identical
to that previously described for the 3′-methoxy phenacyl
derivative of the endo thiol isomer, 3c′endo.30 This major ion
at m/z = 206 should correspond to a retro Diels−Alder
fragmentation of the dihydropyridine ring of 3c′endo. Such a
fragmentation generally occurs in the case of the derivatives of
3b endo and 3c endo thiols, and the appearance of the
corresponding very intense fragments seems to be a character-
istic of these endo isomers.18,30 Analyses of the reaction
mixtures were also done on the thiol metabolites themselves
without derivatization. Three thiol metabolites exhibiting the
expected parent ion at m/z = 350 could be detected with a ratio
almost identical to that found for their phenacyl derivatives.
Identical results were obtained in incubations using 2c
instead of 1c as a starting product.
+ 20 μM N-benzyl-imidazole
1.7 0.5 (7%)
<0.1 (<0.5%)
<0.1 (<0.5%)
<0.1 (<0.5%)
<0.1 (<0.5%)
− ascorbate
− NADPH
5.8 +1
5.5
1
− NADPH − CaCl2
− NADPH + 500 μM paraoxon
− NADPH − CaCl2 + 5 mM EDTA <0.1 (<0.5%)
3.6 1 (63%)
2.8 0.8 (49%)
1.9 0.9 (33%)
a
Complete system: incubations of 100 μM 1c with human liver
microsomes (1 mg protein/ml) in 0.1 M phosphate buffer at pH 7.4
containing 2 mM CaCl2, 20 mM ascorbic acid, and a NADPH
generating system (1 mM NADP, 15 mM glucose-6-phosphate, 2
unit/mL of glucose-6-phosphate dehydrogenase), at 37 °C for 30 min;
analysis by HPLC-MS after derivatization with MPBr, as described in
Experimental Procedures. Mean values
independent experiments. Values in parentheses are % activities
relative to those of the complete system. Under identical conditions
but with a 1c concentration of 12 μM, closer to that found in
prasugrel-treated patients,4 the 3c/3c endo ratio observed with the
complete system increased to 10.
b
SD from at least 3
incubation of 1c with NADPH-supplemented microsomes
almost completely inhibited the formation of the cis
diastereomers 3c′, whereas it did not significantly modify the
formation of 3c′ endo (Table 1). These data indicated that the
formation of the cis-thiol isomers 3c is P450-dependent.
Since the formation of the endo thiol isomer 3b endo upon
the metabolism of 1b by human liver microsomes or human
sera appeared to be mainly catalyzed by PON-1, identical
microsomal incubations of 1c without NADPH were performed
in the presence of 500 μM paraoxon, an usual substrate of
PON-1.36,37 Table 1 shows that this led to a 50% inhibition of
3c endo formation. Moreover, incubations in the absence of
CaCl2 led to a marked decrease of 3c endo formation, and
addition of 5 mM ethylenediamine tetraacetate (EDTA) led to
an even greater decrease of this formation (Table 1). PON-1
activity is dependent on the presence of Ca2+ ions.36 Thus, the
above results show that the formation of the cis diastereomers
3c is dependent on NADPH and is catalyzed by P450s, whereas
that of their isomer 3c endo is not dependent on NADPH and
When identical microsomal incubations of 1c were done in
the absence of NADPH, there was no formation of the cis
isomers 3c′ and the only isomer that could be detected was 3c′
endo (Table 1). Moreover, the addition of N-benzylimidazole
(20 μM), a well-known inhibitor of microsomal P450s,35 to the
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Figure 5. Formation of 3c endo methyl ester and its dithioether 6 upon reaction of 1c with CH3ONa in CH3OH.
1
Figure 6. H NMR spectrum of compound 6.
is catalyzed by an esterase such as PON-1. This conclusion was
completely confirmed by the results of incubations of 1c under
conditions identical to those of the complete system of Table 1
except for the absence of a reducing agent (ascorbate or GSH).
Under those conditions, one only observed the formation of 3c
endo. This is in agreement with the fact that formation of 3c
endo from a simple hydrolysis of 2c does not require the
presence of a reducing agent contrary to that of 3c, which
involves the reduction of sulfenic acid 5c (Figure 4).
Metabolism of 1c by Human Sera. Incubation of 100 μM
1c with human serum (10 mg protein mL−1), which contains
PON-1 but not P450s, for 30 min at 37 °C, and a study of the
reaction mixture by HPLC-MS after derivatization using MPBr
under the above-described conditions showed the formation of
only one thiol isomer, 3c endo (Figure 3C). In this case,
incubations with or without a reducing agent such as ascorbate
led to almost identical results.
Chemical Opening of the Thiolactone Ring of 2c.
Reaction of 1c with CH3ONa in CH3OH at 20 °C for 45 min
led to the hydrolysis of the acetate function of 1c and the
opening of the 2c thiolactone ring with the formation of only
one thiol isomer, the methyl ester of 3c endo, with an 80%
yield. Its mass spectrum exhibited the expected parent ion at
m/z = 364 with a major fragment at m/z = 206.30 Because of
the relative unstability of this thiol, its further characterization
was done at the level of the dithioether 6 obtained by the
oxidation of 3c endo methyl ester with iodine (Figure 5). The
structure of this product was established from its mass
spectrum (parent ion with a peak at m/z = 725 and MS2
main fragments at m/z = 520 and 206; see Figure S1 of
Supporting Information for the UV and MS spectrum of 6) and
1
its H NMR spectrum that exhibited characteristic signals at
3.33 and 2.61 ppm for the allylic hydrogens α to the COOCH3
1
and amine groups (Figure 6). A detailed analysis of the H
NMR spectrum of 6 in CD2Cl2, using 2D NMR methods
(correlation spectroscopy (COSY), heteronuclear multiple
bond correlation (HMBC), and heteronuclear single quantum
coherence spectroscopy (HSQC)) allowed us to assign all the
signals to the protons of the molecule (Figure 6).
These data confirmed in the case of thiolactone 2c what was
already observed with thiolactone 2b derived from clopidogrel
1b.18 Reaction of nucleophiles such as CH3ONa on 2b (or 2c)
preferentially occurs on the thioester carbon of 2b′ (or 2c′), the
tautomer of 2b (or 2c′), which is more electrophilic than the
thioester carbon of 2b (or 2c), which is conjugated to a double
bond (Figure 5).
DISCUSSION
■
The above results on the metabolism of 1c by human liver
microsomes showed that, after microsomal esterase-catalyzed
hydrolysis of 1c to 2c,10 two pathways resulting in the opening
of the 2c thiolactone ring are occurring. The major one results
from a P450- and NADPH-dependent oxidation leading to the
already described3,4,12,13,38 cis-thiol diastereomers 3c. The
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second one does not require any oxidative step and leads to the
endo thiol isomer 3c endo (Figure 4). It occurs both in liver
microsomes and in human sera, does not depend on the
presence of NADPH, depends on the presence of Ca2+, and is
inhibited by paraoxon (Table 1). The enzyme(s) involved in
the hydrolysis of 2c to 3c endo remain(s) to be determined;
however, because of the observed effects of Ca2+ and paraoxon
on the formation of 3c endo (Table 1) and of a quite recent
report showing that microsomal transformation of 2b into 3b
endo was catalyzed by recombinant PON-1,39 it is likely that
the formation of 3c endo from 2c by human liver microsomes
could be catalyzed by PON-1. Accordingly, quite recent,
preliminary data showed us that recombinant, purified human
PON-1 (from Chesapeake-Perl, USA)40 did catalyze the
hydrolysis of 2c into 3c endo, with a Km value very similar
to that found for the formation of 3c endo from 2c by human
liver microsomes (Dansette, P., Leven, D., and Mansuy, D.,
unpublished work). In relation with this role of PON-1 in the
metabolism of clopidogrel and prasugrel, it is noteworthy that
homocysteine thiolactone has been proposed as a physiological
substrate of human PON-1.41
Our data showing that 3c endo methyl ester is the only thiol
isomer formed upon reaction of CH3ONa with 1c suggest that
O-nucleophiles preferentially react on the thioester carbon of
the more reactive tautomer 2c′ that is in equilibrium with 2c
(Figure 5). This should be true for the enzymatic hydrolysis of
the thioester function of 2c by thioesterases such as PON-1 in
liver microsomes and human sera. Accordingly, literature data
reported that PON-1 readily catalyzed the hydrolysis of
lactones, whereas it was inactive on unsaturated lactones
involving a less reactive carbonyl moiety conjugated to a double
bond.37 Thus, the only way to open the thiolactone ring of 2c
with formation of the cis-thiol isomers, 3c, is to oxidize its S
atom with the intermediate formation of thioester sulfoxide 4c
(Figure 4).12 The thioester carbon of 4c is much more reactive
than that of 2c and should rapidly react with water to give
sulfenic acid 5c, as previously reported.12 Reduction of 5c with
GSH or ascorbic acid12 eventually leads to 3c.
hydrolysis in humans is the formation of thiol 3c.30 This is in
agreement with our in vitro data on the metabolism of prasugrel
(100 μM) by human liver microsomes showing a 3c/3c endo
ratio of 3.8 (Table 1). Under identical conditions but with a
prasugrel concentration, 12 μM, closer to that found in
prasugrel treated patients,4 the 3c/3c endo ratio increases to 10
(see footnotes in Table 1).
It is thus very likely that the pharmacological effects of
prasugrel are mainly due to the P450 dependent transformation
of its thiolactone metabolite to thiol 3c.3,4,12,13 The cis-thiol
metabolites 3 derived from ticlopidine, clopidogrel, and
prasugrel are generally considered as the pharmacologically
active species that are responsible for the inhibition of the
P2Y12 platelet receptor after the formation of a covalent
disulfide bond with a cysteine residue of this protein.4,45,46
Another possible mechanism for this inactivation of the P2Y12
receptor could be the formation of such a covalent bond from
reaction of their sulfenic acid metabolites 5 or of the
glutathionyl adducts deriving from their reaction with GSH,
with a cysteine SH residue of the P2Y12 platelet receptor.12,15
These sulfenic acid metabolites are electrophilic species that
could covalently bind to other proteins and could be at the
origin15 of some of the secondary toxic effects reported for
these tetrahydrothienopyridine antithrombotic prodrugs.47 In
that regard, one must note that more strongly electrophilic
intermediates, such as thiophene sulfoxides,48 which are formed
in the first step of bioactivation of ticlopidine and clopidogrel,
could also be at the origin of the secondary toxic effects of these
two drugs. Thus, ticlopidine and clopidogrel are mechanism
based inactivators of P450 2B649−51 and 2C19,6,52 and
activation of their thiophene ring to reactive metabolites
which bind covalently to the enzyme has been demonstra-
ted.6,51 By comparison, 2-oxoclopidogrel and prasugrel
thiolactone are much less inhibitory.50−53 Anyway, direct
roles of sulfenic acid metabolites 5 in the pharmacological or
secondary toxic effects of ticlopidine, clopidogrel, and prasugrel
remain to be established.
The three thiol isomers that we have detected upon the
metabolism of 1c by human liver microsomes in the presence
of NADPH and/or several products deriving from their further
metabolism have been found in the plasma, urine, and feces of
prasugrel treated patients and in the plasma, urine, and feces of
mice, rats, and dogs treated with this drug.30,31 From these data,
it seems that further metabolism of 3c in vivo would involve a
methylation or a cysteinylation of its thiol function. The
resulting S-methyl metabolite may be further oxidized to the
corresponding sulfoxide and/or conjugated with the formation
of the corresponding acyl-glucuronide. Further metabolism of
3c endo in the same species30,31 appears to be even more
complex. S-Cysteinylation and S-methylation of the thiol
function of 3c endo seem to occur, and further transformations
of S-methylated 3c endo identical to those found for S-methyl-
3c also appear to occur. Another pathway seems to be involved
in the case of 3c endo. It would result in the loss of the sulfur
atom of its thioenol function with the formation of a ketone
metabolite that would be eventually reduced to the
corresponding alcohol.30,31
ASSOCIATED CONTENT
■
S
* Supporting Information
Mass spectra and UV spectrum of the endo-thiol dimer methyl
ester 6. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +33 142862191. Fax: +33 142868387. E-mail: patrick.
dansette@parisdescartes.fr.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank J. F. Chasse
Descartes) for a gift of human sera.
́
(INSERM UMR-S 747, Universite
́
Paris
ABBREVIATIONS
Previous literature data showed that one of the cis-thiol
isomers 3c is a very good inhibitor of platelet aggregation via its
irreversible binding to the P2Y12 receptor,3,9,42,43 whereas thiol
3c endo does not have antiplatelet activity in vitro.44 They also
showed that the major metabolic pathway following prasugrel
■
EDTA, ethylene diamine tetraacetic acid; HPLC-MS, high
performance liquid chromatography−mass spectrometry; GSH,
glutathione; MPBr, 3′-methoxy phenacyl bromide; PON-1,
paraoxonase-1; P450, cytochrome P450
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Hurbin, F., and Sultan, E. (2011) An improved method for specific and
quantitative determination of the clopidogrel active metabolite isomers
in human plasma. Thromb. Haemost. 105, 696−705.
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