Potent Mechanism-Based Inhibition of Human CYP2B6 by Clopidogrel and Ticlopidine

Article abstract of DOI:10.1124/jpet.103.056127

The thienopyridine derivatives ticlopidine and clopidogrel are inhibitors of ADP-induced platelet aggregation. Pharmacological activity of these prodrugs depends on cytochrome P450 (P450)-dependent oxidation to the active antithrombotic agent. In this study, we investigated the interaction potential of clopidogrel and ticlopidine by using human liver microsomes and recombinantly expressed P450 isoforms. Both clopidogrel and ticlopidine inhibited CYP2B6 with highest potency and CYP2C19 with lower potency, Clopidogrel also inhibited CYP2C9, and ticlopidine also inhibited CYP1A2, with lower potency. Inhibition of CYP2B6 was time- and concentration-dependent, and as shown by dialysis experiments, it was irreversible and dependent on NADPH, suggesting a mechanism-based mode of action. Inactivation was of nonpseudo-first-order type with maximal rates of inactivation (K _inact) for clopidogrel and ticlopidine in microsomes (recombinant CYP2B6) of 0.35 (1.5 min^-1) and 0.5 min^-1 (0.8 min ^-1), respectively, and half-maximal inactivator concentrations (K_I) were 0.5 μM (1.1 μM) for clopidogrel and 0.2 μM (0.8 μM) for ticlopidine. Inhibition was attenuated by the presence of alternative active site ligands but not by nucleophilic trapping agents or reactive oxygen scavengers, further supporting mechanism-based action. A chemical mechanism is discussed based on the known metabolic activation of clopidogrel and on the finding that hemoprotein integrity of recombinant CYP2B6 was not affected by irreversible inhibition. These results suggest the possibility of drug interactions between thienopyridine derivates and drug substrates of CYP2B6 and CYP2C19.

Full text of DOI:10.1124/jpet.103.056127

0022-3565/04/3081-189–197$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics
JPET 308:189–197, 2004
Vol. 308, No. 1
56127/1117480
Printed in U.S.A.
Potent Mechanism-Based Inhibition of Human CYP2B6 by
Clopidogrel and Ticlopidine
Tanja Richter, Thomas E. Mu¨ rdter, Georg Heinkele, Ju¨ rgen Pleiss, Stephan Tatzel,
Matthias Schwab, Michel Eichelbaum, and Ulrich M. Zanger
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (T.R., T.E.M., G.H., M.S., M.E., U.M.Z.);
and Institute of Technical Biochemistry, University of Stuttgart, Stuttgart, Germany (J.P., S.T.)
Received June 27, 2003; accepted September 30, 2003
ABSTRACT
The thienopyridine derivatives ticlopidine and clopidogrel are order type with maximal rates of inactivation (Kinact) for
inhibitors of ADP-induced platelet aggregation. Pharmacologi- clopidogrel and ticlopidine in microsomes (recombinant
cal activity of these prodrugs depends on cytochrome P450 CYP2B6) of 0.35 (1.5 minϪ1) and 0.5 minϪ1 (0.8 minϪ1), respec-
(P450)-dependent oxidation to the active antithrombotic agent. tively, and half-maximal inactivator concentrations (KI) were 0.5
In this study, we investigated the interaction potential of clopi- ␮M (1.1 ␮M) for clopidogrel and 0.2 ␮M (0.8 ␮M) for ticlopidine.
dogrel and ticlopidine by using human liver microsomes and Inhibition was attenuated by the presence of alternative active
recombinantly expressed P450 isoforms. Both clopidogrel and site ligands but not by nucleophilic trapping agents or reactive
ticlopidine inhibited CYP2B6 with highest potency and oxygen scavengers, further supporting mechanism-based ac-
CYP2C19 with lower potency. Clopidogrel also inhibited tion. A chemical mechanism is discussed based on the known
CYP2C9, and ticlopidine also inhibited CYP1A2, with lower metabolic activation of clopidogrel and on the finding that
potency. Inhibition of CYP2B6 was time- and concentration- hemoprotein integrity of recombinant CYP2B6 was not affected
dependent, and as shown by dialysis experiments, it was irre- by irreversible inhibition. These results suggest the possibility of
versible and dependent on NADPH, suggesting a mechanism- drug interactions between thienopyridine derivates and drug
based mode of action. Inactivation was of nonpseudo-first- substrates of CYP2B6 and CYP2C19.
Thrombolytic drugs are widely used in patients with coro- clinical trials showed advantageous effects of clopidogrel in
nary heart disease. These include anticoagulants and anti-
platelet compounds like acetylsalicylic acid, glycoprotein IIb/
IIIa receptor antagonists, and the thienopyridine derivates,
ticlopidine and clopidogrel. The latter substance is being
used more often today because it shows a more rapid onset of
action and a lower incidence of adverse effects, such as neu-
tropenia and thrombotic thrombocytopenic purpura, com-
pared with ticlopidine (Kam and Nethery, 2003). A further
increase in the use of clopidogrel may be expected for the
treatment of patients with acute coronary syndrome, since
combination with acetylsalicylic acid (Mehta et al., 2001).
Both ticlopidine and clopidogrel are not active in vitro and
require hepatic biotransformation for pharmacologic activity,
which is inhibition of ADP-induced platelet aggregation (Savi
et al., 1992). The metabolic activation of clopidogrel has been
investigated in detail. Whereas the majority of clopidogrel is
hydrolyzed by esterases to an inactive carboxylic acid deriv-
ative, microsomal cytochromes P450 (P450) were shown to
catalyze the oxidation of the thiophene ring to 2-oxoclopi-
dogrel (Savi et al., 2000). This intermediate is further acti-
vated by hydrolytic opening of the thiophene ring to the final
active metabolite, which contains a free thiol group that is
able to block the P2Y12 ADP receptor on platelets by forming
a disulfide bond with a cysteine residue, thus preventing the
binding of ADP to the receptor (Savi et al., 2000). The P450
isoenzyme responsible for clopidogrel activation was initially
suggested to be CYP1A2 (Savi et al., 1994), but subsequent
studies indicated that CYP3A4 is the most active isozyme in
human liver with lower but still significant amounts metab-
olized by CYP1A2 and CYP2B6 (Clarke and Waskell, 2002).
This work was supported by Grant ZA 245/2-1 of the Deutsche Forschungs-
gemeinschaft and by the Robert Bosch Foundation, Stuttgart, Germany.
Previous meeting abstracts: Richter T, Klein K, Mu¨rdter TE, Eichelbaum
M, Schwab M, and Zanger UM (2002) ThioTEPA and clopidogrel are specific
mechanism-based inhibitors of human CYP2B6. Proceedings of the joint an-
nual fall meeting, German Society for Biochemistry and Molecular Biology
(GBM) and German Society for Experimental and Clinical Pharmacology and
Toxicology (DGPT), Halle (Saale), Germany, 2002 Sep 7–10 (www.gbm-on-
line.de; DOI:10.1240/sav_gbm_2002_h_000185)
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
DOI: 10.1124/jpet.103.056127.
ABBREVIATIONS: P450, cytochrome(s) P450; ESI, electrospray ionization; HPLC, high-performance liquid chromatography; DMSO, dimethyl
sulfoxide; OR, NADPH-P450 oxidoreductase.
189

190
Richter et al.
A major contribution of CYP3A to metabolic clopidogrel ac-
tivation was confirmed by a clinical study that demonstrated
attenuation of the antiplatelet activity of clopidogrel by the
CYP3A4 substrate, atorvastatin, and modulation of the in-
hibitory effect by inhibitors and inducers of CYP3A4, sug-
gesting competitive inhibition of clopidogrel activation by the
alternative substrate (Lau et al., 2003).
Cytochromes P450 and Human Liver Microsomes. Recombi-
nant cytochromes P450 coexpressed with NADPH-P450 oxidoreduc-
tase (OR) in insect cells (supersomes) were purchased from BD
Gentest (Woburn, MA). Human liver microsomes were prepared
from surgically removed liver tissue as described previously (Lang et
al., 2001). The study was approved by the ethics committees of the
Medical Faculties of the Charite´, Humboldt-University Berlin, and of
the University of Tu¨bingen, and written informed consent was ob-
tained from each volunteer as well as from each patient.
Chemical Syntheses of Bupropion Hydrochloride, Hy-
droxybupropion Hydrochloride, and [2H3]Hydroxybupropion
Hydrochloride. Bupropion hydrochloride, hydroxybupropion hy-
drochloride, and the internal standard [2H3]hydroxybupropion hy-
drochloride were synthesized using a modification of the method
described by Mehta and Raleigh (1974). In brief, 3-chloropropiophe-
none or [3Ј,3Ј,3Ј-2H3]3-chloropropiophenone was brominated, and
the product was used for amination with an excess of the correspond-
ing amine. The resulting raw products were purified by preparing
the hydrochlorides and recrystallization from 2- propanol/isooctane.
Details will be published elsewhere.
Chemical Syntheses of 4ꢀ-Hydroxymephenytoin (3-Methyl-
5-ethyl-5-(4-hydroxyphenyl)-hydantoin) and [2H3]4ꢀ-Hydroxy-
mephenytoin (3-Methyl-5-ethyl-5-(4-hydroxyphenyl)-hydan-
toin). 5-Ethyl-5-(4-hydroxyphenyl)-hydantoin was prepared from
4-hydroxypropiophenone, potassium cyanide, and ammonium car-
bonate in 50% ethanol under pressure at 110°C for 24 h. Methylation
of 5-ethyl-5-(4-hydroxyphenyl)-hydantoin with dimethyl sulfate or
[2H6]dimethyl sulfate and sodium hydroxide in ethanol resulted in
4Ј-hydroxymephenytoin and [2H3]4Ј-hydroxymephenytoin, respec-
tively.
Several sulfur-containing substances with structural sim-
ilarities to clopidogrel were shown to be irreversible inhibi-
tors of cytochromes P450. The thiophene derivative, tic-
rynafen (tienilic acid), was intensively investigated because
it induced immunoallergic hepatitis in a subset of patients
who developed so-called anti-liver-kidney microsome anti-
body type 2 autoantibodies. It was shown that these inhibi-
tory autoantibodies were directed against CYP2C9, the pri-
mary P450 isozyme involved in the liver metabolism of the
drug (Beaune et al., 1987). Covalent modification of CYP2C9
protein by ticrynafen was found to be the cause for irrevers-
ible enzyme inhibition and presumably for initiation of im-
munoallergic reactions against changed autoepitopes (Lopez-
Garcia et al., 1994). Another thiophene derivative, the
thienopyridine ticlopidine, which differs structurally from
clopidogrel only by the absence of a carboxymethyl group,
caused drug interactions with substrates of CYP2C19 like
phenytoin (Donahue et al., 1997) and omeprazole (Tateishi et
al., 1999). The drug was subsequently shown to be a selective
mechanism-based inhibitor of CYP2C19 (Ha-Duong et al.,
2001). Metabolic activation of ticlopidine, however, has not
been studied in as great detail as clopidogrel.
Because of the structural similarities between clopidogrel
and ticlopidine on the one side and ticrynafen on the other
side, we hypothesized that irreversible and possibly rather
selective interactions with P450 enzymes may also occur
with clopidogrel. Until now, neither in vitro investigations
nor in vivo clinical studies with the aim to evaluate the drug
interaction profile of this substance have been performed.
The aim of this study, therefore, was to investigate the in-
hibitory potential of clopidogrel toward human drug-metab-
olizing cytochromes P450. We also applied structural homol-
ogy modeling of the involved cytochromes P450 (Bathelt et
al., 2002) to study interactions with clopidogrel on a molec-
ular level.
Chemical Synthesis of [2H5]5-Ethyl-5-phenyl-hydantoin
([2H5]Nirvanol). [2H6]Benzene was acylated with propionyl chlo-
ride and aluminum chloride in 1,2-dichloroethane to obtain
[2H5]propiophenone, which was used to synthesize [2H5]5-ethyl-5-
phenyl-hydantoin with potassium cyanide and ammonium carbon-
ate.
Determination of Catalytic P450 Activities. Details for each
assay are described below. All assays were performed with recombi-
nant cytochromes P450 (2.5–5 pmol) or human liver microsomes
(50–100 ␮g of protein) in a final volume of 250 ␮l with inhibitors as
indicated. After equilibrating the reaction mixture at 37°C for 3 min,
preincubation with inhibitors was started by adding 25 ␮l of 10-fold
concentrated NADPH-regenerating system (final concentrations, 5
mM MgCl2, 4 mM glucose 6-phosphate, 0.5 mM NADPϩ, and 4 U/ml
glucose 6-phosphate dehydrogenase) and performed for the indicated
times at 37°C. Subsequently, enzyme reactions were started by the
addition of substrate. Reactions were terminated and processed as
described below.
Materials and Methods
Bupropion hydroxylation was performed with CYP2B6 or human
liver microsomes at the concentrations indicated in 0.1 M sodium
phosphate buffer, pH 7.4, and with triethylenethiophosphoramide
(10 ␮M) as a CYP2B6 control inhibitor (Rae et al., 2002). Enzyme
reactions were carried out using 500 ␮M bupropion with different
incubation times for different assays. The reactions were stopped by
adding 50 ␮l of 1 N HCl. After addition of the internal standard
d3-OH-bupropion (100 pmol), the samples were centrifuged at 16,000
g for 5 min. The supernatant was directly injected into the HPLC
system. The metabolite hydroxy-bupropion was separated and de-
tected by HPLC-ESI-mass spectrometry using an HPLC system (HP
1100; Agilent Technologies, Waldbronn, Germany) equipped with a
Prontosil-C18 AQ column (150 ϫ 3 mm, 3 ␮M particle size; Bischoff,
Leonberg, Germany) and a mass spectrometer (Agilent Technolo-
gies). Elution was performed with a gradient of 16 (1% acetic acid/
water) and 84% (1% acetic acid/acetonitrile) to 45/55% from 0 to 16
min. The dynamic range for detection of hydroxybupropion was 1 to
500 pmol per incubation, and assay accuracy over the calibration
range was Ͻ8%. Formation of hydroxybupropion was linear with
Chemicals. Clopidogrel was kindly provided by SANOFI Re-
search Center (Montpellier, France). Ticlopidine, NADPϩ, NADPH,
diethyldithiocarbamate, sulfaphenazole, superoxide dismutase,
N-acetylcysteine, DMSO, glutathione, 7-ethoxycoumarin, coumarin,
umbelliferone, quinidine, and sodium hydrosulfite were purchased
from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany).
Furafylline was a kind gift from U. Fuhr (University of Cologne,
Germany). Glycerine was purchased from Roth (Karlsruhe, Ger-
many), and Emulgen 911 was provided by Kao-Atlas (Tokyo, Japan).
Glucose 6-phosphate was obtained from Roche Diagnostics (Mann-
heim, Germany). Glucose 6-phosphate-dehydrogenase was pur-
chased from Calbiochem (Schwalbach, Germany). Ketoconazole was
obtained from Ultrafine Chemicals (Manchester, England). Trieth-
ylenethiophosphoramide was obtained from Wyeth Pharma GmbH
(Muenster, Germany). (S)-Mephenytoin was a kind gift from U. A.
Meyer (Biozentrum, Basel, Switzerland). Propafenone, verapamil,
and metabolites were obtained from Knoll (Ludwigshafen, Ger-
many). Paroxetine was obtained from GlaxoSmithKline (Welwyn
Garden City, Hertfordshire, UK).

Suicide Inhibition of CYP2B6 by Clopidogrel
191
time up to 30 min and linear with protein between 5 and 200 ␮g of hydroxybupropion was 5 to 500 pmol per incubation, and assay
microsomal protein.
accuracy over the calibration range was Ͻ14%.
Verapamil O-demethylation and verapamil N-demethylation were
Coumarin-7-hydroxylation reactions were performed according to
assayed as described by von Richter et al. (2000). The reaction buffer the protocol of BD Gentest with minor modifications (http://www.
consisted of 50 mM potassium phosphate buffer, pH 7.4, and 30 mM gentest.com/products/tissue_frac/prod_inserts/hlm_meth.shtm).
MgCl2. Incubations were carried out as described above using 100 Briefly, the incubation mixture consisted of 200 ␮M coumarin in 100
␮M verapamil and a 30-min incubation time with recombinant mM Tris, pH 7.5, and diethyldithiocarbamate (100 ␮M) was used as
CYP2C8 for verapamil O-demethylation and 10-min incubation time control inhibitor. The reactions were stopped after 15 min with 100
with CYP3A4 for verapamil N-demethylation. Ketoconazole (100 ␮M ␮l of aqueous 20% (w/v) trichloroacetic acid. The samples were vor-
for CYP2C8, 10 ␮M for CYP3A4) was used as control inhibitor (in 1 texed, centrifuged at 16,000 rpm for 5 min, and 100 ␮l of supernatant
or 0.1% DMSO, respectively, as vehicle control). The reactions were was diluted in 1.9 ml of 100 mM Tris buffer pH 9.0. The formation of
stopped by the addition of 1.7 ml of cold ethanol. After addition of the umbelliferone was determined fluorometrically as described above.
internal standard [2H3]norverapamil (50 pmol), samples were vor- The dynamic range for detection of hydroxybupropion was 1 to 500
texed, centrifuged at 16,000g for 5 min, and the supernatant was pmol per incubation, and assay accuracy over the calibration range
dried under nitrogen and finally dissolved in 150 ␮l of mobile phase. was Ͻ15%.
Analysis of the metabolites [2-(4-hydroxy-3-methoxyphenyl)-8-(3,4-
dimethoxy-phenyl)-6-methyl-2-isopropyl-6-azaoctanitrile], [2-(3,4-di- liver microsomes (50–100 ␮g), bupropion (500 ␮M), and either clo-
methoxy-phenyl)-8-(4-hydroxy-3-methoxyphenyl)-6-methyl-2-isopro- pidogrel or ticlopidine (0.1–10 ␮M) were equilibrated in 0.1 N sodium
Inhibition Studies in Human Liver Microsomes. Human
pyl-6-azaoctanitrile], and norverapamil was performed by HPLC- phosphate buffer, pH 7.4, at 37°C for 3 min. After addition of 25 ␮l of
ESI-mass spectrometry as described for bupropion-hydroxylation NADPH-regenerating system, reactions were allowed to proceed for
assay using a LUNA C8 column (150 ϫ 3 mm i.d., 5-␮m particle size; 15 min and analyzed as described for the bupropion hydroxylase
Penomenex, Aschaffenburg, Germany). The metabolites were sepa- assay. The effect of nucleophilic trapping agents (10 mM glutathione
rated with 5 mM ammonium acetate, pH 4.2/acetonitrile as the or N-acetylcysteine) or scavengers of reactive oxygen species (0.1%
mobile phase run with a gradient from 29:71 to 50:50 within a DMSO or 1000 units of superoxide dismutase) was tested by adding
runtime of 15 min. The dynamic range for detection of verapamil
metabolites was 1 to 500 pmol per incubation, and assay accuracy with inhibitors. Substrate protection was analyzed accordingly with
over the calibration range was Ͻ14%. 7-ethoxycoumarin (1 mM) and 0.5 ␮M clopidogrel or ticlopidine (con-
these compounds at the indicated concentrations prior to incubation
Propafenone-5-hydroxylation was analyzed according to Hofmann trols without inhibitors) in the incubation mixture and determina-
et al. (2000) in 0.1 M sodium phosphate buffer, pH 7.4, using 5 ␮M tion of residual bupropion hydroxylase activity as described above.
quinidine as control inhibitor. Propafenone concentration was 2 ␮M, Substrate protection with the inhibitor paroxetine (50 ␮M) and 0.5
and incubations were performed for 20 min. The reactions were ␮M clopidogrel or ticlopidine (controls without inhibitors) was ana-
stopped by adding 1.7 ml of cold ethanol. After addition of 200 pmol lyzed accordingly after removal of paroxetine as CYP2B6 inhibitor by
of [2H7]5-hydroxy-propafenone as internal standard, samples were extensive dialysis as described below.
vortexed, centrifuged at 16,000 g for 5 min, and the supernatant was
Dialysis Experiments. Human liver microsomes (100 ␮g, con-
dried under nitrogen and finally dissolved in 150 ␮l of mobile phase taining 4.4 pmol of CYP2B6, as determined by Western blot; Lang et
(70% 12 mM ammonium acetate/30% acetonitrile). The metabolite al., 2001) were incubated with or without 10 ␮M of clopidogrel or
5-hydroxy-propafenone was separated and detected by HPLC-ESI- ticlopidine and with or without NADPH-regenerating system for 15
mass spectrometry, essentially as described for the verapamil as- min as described for the inactivation assay. The samples were then
says, using an endcapped Lichrospher RP-18 column (150 ϫ 3 mm immediately dialyzed against 0.1 M sodium phosphate buffer, pH 7.4
i.d., 5-␮m particle size; Merck, Darmstadt, Germany). The dynamic (3 ϫ 2 l, 2 h each) at 4°C in QuixSep Micro Dialyzer capsules (Orange
range for detection of hydroxybupropion was 5 to 500 pmol per Scientific, Braine-l’Alleud, Belgium) and a regenerated cellulose tu-
incubation, and assay accuracy over the calibration range was Ͻ10%. bular membrane with molecular mass cutoff of 12 kDa (Roth). Bu-
(S)-Mephenytoin-N-demethylation and (S)-mephenytoin-4Ј-hy- propion hydroxylase activity was then determined with 500 ␮M
droxylation were carried out in 0.1 M sodium phosphate buffer, pH
bupropion and 15-min incubation time as described.
7.4. Control inhibitors were 10 ␮M sulfaphenazole for (S)-mepheny-
P450 Reduced CO-Difference Spectroscopy. Recombinant
toin-N-demethylation (CYP2C9) and 100 ␮M ketoconazole (1% CYP2B6 (0.6 nM, obtained by expressing wild-type CYP2B6 cDNA in
DMSO as vehicle) for (S)-mephenytoin-4Ј-hydroxylation (CYP2C19). insect cells; details will be published elsewhere) and OR (0.6 nM;
(S)-Mephenytoin concentrations were 1 mM and 200 ␮M, respec- purified from rat liver) were incubated in 0.1 N sodium phosphate
tively. The reactions were stopped with 1.7 ml of cold ethanol after
buffer, pH 7.4, in the presence or absence of clopidogrel (10 ␮M) or
30 and 15 min, respectively. [2H3]4Ј-OH-(S)-mephenytoin and ticlopidine (10 ␮M), respectively. Inhibition was started by adding
[2H5]nirvanol (100 pmol/each) were added as internal standards. The NADPH-regenerating system. Controls were incubated without
samples were vortexed and centrifuged at 16,000 rpm for 5 min. The NADPH-regenerating system. The reactions were allowed to proceed
supernatant was dried under nitrogen and finally dissolved in 150 ␮l for 15 min and were then stopped with 1.75 ml of quenching buffer
of mobile phase consisting of 30% acetonitrile/70% water. The me- (0.1 M sodium phosphate buffer, pH 7.4, 10% glycerol, and 0.5%
tabolites were analyzed by HPLC-ESI-mass spectrometry using a Emulgen 911). Dithionite was added, the samples were gently bub-
column as described for the propafenone-5-hydroxylation assay. The bled with CO for 15 s, and the reduced carbonyl spectrum was
dynamic range for detection of hydroxybupropion was 1 to 500 pmol
recorded between 400 and 500 nm on a Unicam UV/VIS spectropho-
per incubation, and assay accuracy over the calibration range was tometer (Thermo Nicolet, Cambridge, UK). Before termination with
Ͻ12%.
quenching buffer, 25-␮l samples were taken to determine bupropion
7-Ethoxycoumarin O-deethylation was assayed as described by hydroxylase activity as described above.
Yamazaki et al. (1999b). Determination of CYP1A2 activity was
Kinetic Inhibition Studies with Clopidogrel and Ticlopi-
carried out with 10 ␮M ethoxycoumarin, and CYP2E1 activity was dine. All incubations were carried out at 37°C with either recombi-
determined using 200 ␮M ethoxycoumarin. Diethyldithiocarbamate nant CYP2B6 ϩ OR supersomes (BD Gentest) or 100 ␮g of human
(10 ␮M) and furafylline (10 ␮M) were used as control inhibitors for liver microsomes. The samples were equilibrated with different con-
CYP2E1 and CYP1A2, respectively. The formation of umbelliferone centrations of clopidogrel and ticlopidine (ranging from 0.05–1 ␮M)
was determined fluorometrically with a 1420 Victor spectrophotom- for 3 min at 37°C, and after the addition of NADPH-regenerating
eter (PerkinElmer Wallac, Turku, Finland) set at 460 nm using an system, the samples were incubated for 0 to 15 min as indicated.
excitation wavelength of 355 nm. The dynamic range for detection of Subsequently, 25 ␮l of the preincubation mixture was transferred to

192
Richter et al.
225 ␮l of enzyme activity assay mixture, consisting of 0.1 M sodium
phosphate buffer, pH 7.4, 500 ␮M bupropion, and NADPH-regener-
ating system, prewarmed to 37°C. After 6 min of incubation, the
reactions were stopped with 50 ␮l of 1 N HCl. After the addition of
100 pmol of the internal standard, d3-hydroxy-bupropion, the sam-
ples were vortexed and centrifuged for 5 min (16,000g). The super-
natant was directly injected into the HPLC system (HP 1100; Agilent
Technologies) and analyzed as described for bupropion hydroxylation
assay.
Homology Modeling and Docking. For modeling and docking
experiments of CYP2B6 and CYP2C19, previously developed homol-
ogy models were used (Bathelt et al., 2002). The docking of clopi-
dogrel into CYP2B6 was done by using AutoDock 3.05 (Morris et al.,
1998). Charges were assigned by Assisted Model Building with En-
ergy Refinement (University of California, San Francisco). The struc-
ture of the ligand was calculated using Gaussian98 (Gaussian Inc.,
Pittsburgh, PA) with a Hartree-Fock method and the 6-31G* basis
set. For docking, mass-centered grid maps for the active site were
generated at 0.18-Å spacing and 126 ϫ 126 ϫ 126 grid points. The
Lamarckian genetic algorithm and the pseudo-Solis and Wets meth-
ods were applied for minimization. The number of generations was
set to 27,000. Random starting positions, orientations, and torsions
were used for the ligand. One thousand runs were performed with a
maximum number of 1.5 ꢁ 106 energy evaluations. A two-step proce-
dure was used for classification of the results of each job. First, the
calculated free energy was used to rank the docked conformations.
Then, all docked conformations with the lowest distance from the
heme oxygen to the hydrogen at position 2 of the thiophene ring were
extracted.
Data Analysis. Enzyme kinetic data were analyzed according to
the method of Silverman (1995). The half-time of enzyme inactiva-
tion (t1/2) was calculated from the initial slopes of the remaining
enzyme activity, plotted semilogarithmically against the preincuba-
tion time. The half-time of enzyme inactivation thus obtained was
plotted against the reciprocal of the respective clopidogrel and ticlo-
pidine concentrations (Kitz-Wolson plot). The concentration required
for half-maximal inactivation (KI) and the maximum inactivation
rate constant (kinact) were determined from the intercepts on the
abscissa and ordinate, respectively.
Fig. 1. Selectivity of inhibition by clopidogrel and ticlopidine in recombi-
nant P450 enzymes. Cytochrome P450 enzymes recombinantly coex-
pressed with OR in insect cells (supersomes) were incubated with 1 ␮M
(hatched bars) or 10 ␮M (filled bars) clopidogrel (A) or ticlopidine (B).
P450 activities were determined using appropriate marker drug assays
as detailed in Table 1. For each P450, control activity measured in the
absence of inhibitor was set at 100%. Data represent the means of two
independent experiments.
Results
Effect of Clopidogrel and Ticlopidine on P450 Mono-
oxygenase Activities. Initial experiments were performed
to confirm the inhibitory effect of ticlopidine on CYP2C19 shown). At 10 ␮M, both bupropion hydroxylation and (S)-
(Ha-Duong et al., 2001) and to investigate whether the struc- mephenytoin-4Ј-hydroxylation were almost completely inhib-
ture-related clopidogrel had a similar effect. Indeed, incuba- ited by both substances. A difference in selectivity between
tions of recombinant CYP2C19 with 1 ␮M of clopidogrel or clopidogrel and ticlopidine (10 ␮M) was observed in that the
ticlopidine reduced (S)-mephenytoin-4Ј-hydroxylation by 60 former also inhibited CYP2C9 by 53%, whereas the latter
and 69%, respectively (Fig. 1). To determine the selectivity of inhibited CYP1A2 by 51% (Fig. 1). Other cytochromes P450
this inhibition toward catalytic activities representing other were still not markedly affected at this concentration.
drug-metabolizing cytochromes P450, recombinant super-
Mechanism-Based Inactivation of CYP2B6 by Clopi-
somes were analyzed with appropriate assays using P450 dogrel and Ticlopidine. Kinetic experiments in human
isoform-specific inhibitors as positive controls (Table 1). Sur- liver microsomes revealed unusual inhibition kinetics not
prisingly, recombinant CYP2B6 was even more potently in- explained by reversible inhibition mechanisms (data not
hibited than CYP2C19. Both clopidogrel and ticlopidine in- shown). In particular, we observed that inactivation of bu-
hibited bupropion hydroxylation, a specific marker reaction propion hydroxylation was concentration- and time-depen-
for CYP2B6, by more than 90% at 1 ␮M concentrations. dent when microsomes were preincubated with clopidogrel
Incubations of human liver microsomes with 1 ␮M clopi- and ticlopidine. To investigate whether the inactivation was
dogrel or ticlopidine and NADPH-regenerating system also the result of an irreversible mechanism, we performed dial-
reduced bupropion hydroxylase activity by up to 98% (data ysis experiments in which liver microsomes were incubated
not shown). Control incubations with NADPH-regenerating with both compounds in the presence or absence of NADPH-
system in the absence of inhibitor showed an approximately regenerating system. As shown in Fig. 2, extensive dialysis of
20% loss of enzyme activity during a 30-min incubation time, samples incubated with inhibitor (10 ␮M) and NADPH-re-
which was not prevented by the addition of catalase (data not generating system did not lead to recovery of bupropion hy-

Suicide Inhibition of CYP2B6 by Clopidogrel
193
TABLE 1
Summary of incubation conditions for cytochrome P450 marker assays
Assay conditions were as described under Materials and Methods. Results are means of two to four independent experiments. Control activity without inhibitor was set at
100%.
Substrate
Concentration
Inhibitor
Concentration
P450
Reaction
Control Inhibitor
Residual Activity
␮M
␮M
%
1A2
2A6
2B6
2C8
2C9
2C19
2D6
2E1
3A4
7-Ethoxycoumarin O-deethylation
Coumarin 7-hydroxylation
10
200
50
100
10
200
2
200
100
Furafylline
10
100
10
100
10
100
5
8.4
30.7
11.8
N.D.
34.7
41.7
40.7
1.2
Diethyldithiocarbamate
Triethylenethiophosphoramide
Ketoconazole
Bupropion hydroxylation
Verapamil O-demethylation
(S)-Mephenytoin N-demethylation
(S)-Mephenytoin 4’-hydroxylation
Propafenone 5-hydroxylation
7-Ethoxycoumarin O-deethylation
Verapamil N-demethylation
Sulfaphenazole
Ketoconazole
Quinidine
Diethyldithiocarbamate
Ketoconazole
1
100
18.2
N.D., not detectable.
Fig. 3. Substrate protection by alternative active site ligands. Human
liver microsomes (100 ␮g) were incubated with either clopidogrel (C,
hatched bars; 0.5 ␮M) or ticlopidine (T, open bars; 0.5 ␮M) in 0.1 N
sodium phosphate buffer, pH 7.4, at 37°C for 3 min in the presence or
absence of either paroxetine (P; 50 ␮M) or 7-ethoxycoumarine (E; 1 mM).
After addition of 25 ␮l of NADPH-regenerating system, reactions were
allowed to proceed for 15 min. Residual bupropion hydroxylase activity
was determined as described under Materials and Methods. Results
represent the means of two independent experiments.
Fig. 2. Mechanism-based irreversible inhibition of microsomal bupropion
hydroxylation. Human liver microsomes (100 ␮g) were incubated for 15
min with NADPH-regenerating system and 10 ␮M clopidogrel or ticlopi-
dine, respectively. Controls were incubated without inhibitor and with or
without NADPH-regenerating system, as indicated. After extensive dial-
ysis, residual bupropion hydroxylase activity was determined. The means
of two independent experiments are shown.
surable effect on CYP2B6 inhibition caused by clopidogrel
and ticlopidine (data not shown). To analyze whether inhibi-
droxylase activity, whereas samples incubated with inhibitor tion of bupropion hydroxylase activity was due to destruction
in the absence of NADPH-regenerating system regained full of the heme moiety, we recorded reduced CO-difference spec-
activity compared with controls without inhibitor. Further- tra of recombinant CYP2B6 incubated with clopidogrel and
more, Fig. 3 shows that inhibition of CYP2B6 by the thien- ticlopidine in the presence or absence of NADPH-regenerat-
opyridines was attenuated by the presence of alternative ing system. There was no visible destruction of the heme
active site ligands. Addition of the competitive inhibitor, component compared with controls without inhibitors under
paroxetine (50 ␮M; Hesse et al., 2000), to the primary incu- conditions where enzymatic activity was completely blocked
bation mixture together with clopidogrel (0.5 ␮M) completely (data not shown).
protected the enzyme from being inhibited, whereas less
Kinetic Analysis of Inactivation of Bupropion Hy-
effective protection was observed toward ticlopidine as the droxylation. Detailed kinetic investigation of mechanism-
inhibitor. The alternative substrate, 7-ethoxycoumarin, based inhibition reactions requires separation of the inacti-
showed the opposite selectivity, namely higher protective vation step from observation of substrate metabolism, which
effect toward ticlopidine (Fig. 3). Taken together, these re- is usually achieved by dialysis or by diluting the incubation
sults demonstrated unequivocally the irreversible and mech- mixtures. We therefore determined residual bupropion hy-
anism-based inhibition type by both thienopyridine deri- droxylase activity in 10-fold diluted incubation mixtures af-
vates. To further characterize the inactivation of CYP2B6, ter preincubation with inhibitor (see Materials and Methods).
the effect of the nucleophilic trapping agents glutathione (10 Inactivation was time- and concentration-dependent in both
mM) and N-acetylcysteine (10 mM) and the effect of the cases, proceeded very rapidly, and showed saturation. Figure
reactive oxygen scavengers DMSO (0.1%) and superoxide 4 shows the microsomal inactivation kinetics of clopidogrel
dismutase (1000 units) was tested by incubating human liver and ticlopidine, respectively, at various concentrations of
microsomes (50 ␮g) and inhibitors (10 ␮M) as described un- inhibitor. Inactivator concentrations for half-maximal inac-
der Materials and Methods. None of the agents had a mea- tivation (KI) due to mechanism-based enzyme inhibition were

194
Richter et al.
Fig. 5. Kinetics of inactivation of recombinant CYP2B6 by different
concentrations of clopidogrel (A) and ticlopidine (B), respectively. For
details on incubation and determination of activity, see Materials and
Methods. Supersomes (5 pmol) from insect cells expressing CYP2B6 were
incubated for indicated periods in the presence of NADPH-generating
system and clopidogrel or ticlopidine as indicated: ꢂ, 0 ␮M; f, 0.05 ␮M;
Œ, 0.1 ␮M; F, 0.2 ␮M; ▫, 0.3 ␮M; ‚, 0.4 ␮M; E, 0.5 ␮M; and ꢃ, 1 ␮M. The
insets show the corresponding Kitz-Wilson plots.
Fig. 4. Kinetics of inactivation of microsomal bupropion hydroxylation by
different concentrations of clopidogrel (A) and ticlopidine (B). For details
on incubation and determination of activity, see Materials and Methods.
Human liver microsomes (100 ␮g) were incubated for the indicated times
in the presence of NADPH-generating system and clopidogrel or ticlopi-
dine as indicated: ꢂ, 0 ␮M; f, 0.05 ␮M; Œ, 0.1 ␮M; F, 0.2 ␮M; ▫, 0.3 ␮M;
‚, 0.4 ␮M; E, 0.5 ␮M; and ꢃ, 1 ␮M. The insets show the corresponding
Kitz-Wilson plots.
ticlopidine were 1.5 and 0.8 minϪ1, and t1/2 were 0.5 and 0.9
min, respectively.
calculated to be 0.5 ␮M for clopidogrel and 0.2 ␮M for ticlo-
pidine by transferring the data into a Kitz-Wilson plot (insets
in Figs. 4 and 5). The inactivation process was of a non-
pseudo-first-order type. Kinact, the maximal rate of inactiva-
tion, at saturating concentrations of clopidogrel and ticlopi-
Discussion
The present in vitro study provides the first data on the
dine was 0.35 and 0.5 minϪ1, respectively. Accordingly, the cytochrome P450 interaction profile of the platelet aggrega-
time required for half of the enzyme molecules to be inactived tion inhibitor, clopidogrel, and demonstrates that it acts as a
(t1/2) was 2 and 1.4 min, respectively. Figure 5 shows the potent mechanism-based inhibitor of human CYP2B6. In ad-
inactivation of recombinant CYP2B6 by clopidogrel and ticlo- dition, the structure-related derivative ticlopidine was shown
pidine, respectively, which was very similar to that in human to inhibit CYP2B6 by a similar mechanism and potency.
liver microsomes and was analyzed as described above. KI These conclusions are based on experiments with human
were 1.1 and 0.8 ␮M, respectively. Kinact for clopidogrel and liver microsomes and with recombinant cytochromes P450 in

Suicide Inhibition of CYP2B6 by Clopidogrel
195
which bupropion hydroxylation was measured as CYP2B6- pion hydroxylase activity in human liver microsomes and in
selective marker enzyme activity (Faucette et al., 2000; recombinant CYP2B6-expressing insect cell membranes pro-
Hesse et al., 2000). The specific findings were that inhibition ceeded with almost identical characteristics. Inactivation
of bupropion hydroxylation by clopidogrel and by ticlopidine proceeded very rapidly within the first few min and slowed
was 1) time- and concentration-dependent, 2) NADPH-de- down thereafter, probably due to consumption of the inhibi-
pendent, 3) irreversible, 4) not affected by scavengers of tor by metabolism or by hydrolysis. The irreversible and
reactive oxygen species or by nucleophilic trapping agents, 5) NADPH-dependent nature of inhibition was demonstrated
reduced by the presence of alternative active site ligands, and by dialysis experiments. Inhibition of bupropion hydroxyla-
6) not associated by a decrease in spectrally detected P450. tion occurred only in the presence of NADPH during incuba-
Investigation of the inhibition profiles of the two drugs in- tion with the inhibitor, strongly suggesting that catalytic
cluded the nine cytochromes P450 1A2, 2A6, 2B6, 2C8, 2C9, turnover was required for inhibition (Fig. 2). Although these
2C19, 2D6, 2E1, and 3A4, representing the major drug-me- experiments were performed with microsomal protein, it is
tabolizing cytochromes P450 in human liver. In addition to unlikely that a P450 other than 2B6 is involved in the acti-
CYP2B6, CYP2C19 was also inhibited by both substances, vation of the inhibitor, because recombinant 2B6 was inhib-
albeit at lower potency. Whereas mechanism-based inhibi- ited with practically the same potency as the microsomal
tion of CYP2C19 by ticlopidine has been reported before, the enzyme (Figs. 4 and 5) and because the microsomal inhibition
more potent inhibition of CYP2B6 by this substance had been was not affected by scavengers of reactive oxygen species or
overlooked in that study because CYP2B6 was not included by nucleophilic trapping agents. Further evidence for the
(Ha-Duong et al., 2001). Differences between clopidogrel and involvement of the CYP2B6 active site in the activation of the
ticlopidine with respect to the P450-inhibition profile were thienopyridine inhibitors is provided by the observation that
only observed at higher concentrations in that the former alternative active site ligands effectively attenuated their
also inhibited CYP2C9 whereas the latter inhibited CYP1A2, inhibitory potency. An interesting observation was made
but these inhibitions did not exceed about 50% at 10 ␮M that the competitive CYP2B6 inhibitor, paroxetine, was a
inhibitor concentrations compared with over 90% inhibition more potent attenuator of clopidogrel-mediated inhibition,
of CYP2B6 at 1 ␮M concentrations (Fig. 1).
whereas the alternative substrate, 7-ethoxycoumarin, had a
Time- and concentration-dependent inactivation of bupro- more potent effect on ticlopidine-mediated inhibition. It is
Fig. 6. Possible mechanism for irreversible inhibition of CYP2B6 by clopidogrel. The hypothetical mechanism is based on 1) the known metabolic
activation and pharmacological action of clopidogrel (left) and 2) the observation that inhibition is not accompanied by heme destruction. Alternative
pathways leading to the inactivated P450 may proceed via hydrolysis or via an additional oxidative step catalyzed by the P450.

196
Richter et al.
conceivable that these differences reflect subtle differences
between these substances regarding their steric occupation of
the active site of the enzyme.
Spectral analysis of recombinant CYP2B6 did not indicate
any destruction of the heme moiety during inhibition (data
not shown), suggesting that alkylation of the apoprotein
should be the major event leading to inhibition. Although the
precise mechanism remains to be investigated, reasonable
speculations are possible on the basis of hemoprotein stabil-
ity and in analogy to the known mechanism of irreversible
inhibition of the P2Y12-ADP receptor. The initial step in the
microsomal activation of clopidogrel was shown to be cyto-
chrome P450-dependent oxidation to 2-oxo-clopidogrel (Savi
et al., 2000). The major contribution to this step appears to be
catalyzed by CYP3A4, although only indirect evidence was
provided in a recent study (Clarke and Waskell, 2002). De-
spite not being active in vitro, antiaggregating activity of
2-oxo-clopidogrel was demonstrated ex vivo, suggesting that
it represents an intermediate that can be converted to the
active metabolite. The structure of the active metabolite was
finally determined to be a hydrolyzed derivative with an
opened thiophene ring and a highly reactive thiol function
(Fig. 6; Pereillo et al., 2002), which blocks the receptor by
forming disulfide bonds with extracellular cysteine residues
(Ding et al., 2003). By analogy, a similar mechanism may
explain cytochrome P450 inhibition by thienopyridines. The
first step could be conversion to the 2-oxo-derivative by the
P450. This intermediate may then form a disulfide bond with
an available cysteine, either after hydrolysis of the 2-oxo-
derivative as in the case of ADP-receptor alkylation or, alter-
natively, after another cycle of P450-dependent oxidation
(Fig. 6). This mechanism was shown to be consistent with
Fig. 7. Clopidogrel docked to the active site of CYP2B6 in an orientation
favorable for oxidation of the 2 position of the thiophene ring. Stick model
of the substrate (yellow), heme, and residues that form the substrate-
binding pocket is shown.
protein homology modeling results. Docking of clopidogrel of this finding. Clinically relevant interactions between sub-
into the binding site of CYP2B6 resulted in a number of strates of CYP2C19 and ticlopidine have already been re-
complexes with similar energy between Ϫ8 and Ϫ9 kcal/mol. ported, although CYP2C19 is inhibited with lower potency
In 1% of the complexes, clopidogrel was oriented with the (Donahue et al., 1997; Tateishi et al., 1999). The pharmaco-
hydrogen in the 2 position of the thiophene ring at a distance logical significance of CYP2B6 has long remained unrecog-
between 3.0 and 3.5 Å to the reactive oxygen of the heme (Fig. nized in part due to the lack of suitable probes (Ekins and
7). Because this position is chemically highly reactive, a Wrighton, 1999). Furthermore, the content of CYP2B6 in
small percentage of correctly oriented species would be suf- human liver was recently shown to be much higher than
ficient for a highly regioselective oxidation. The product previously estimated (Gervot et al., 1999; Lang et al., 2001).
2-oxo-clopidogrel would leave the active site through the The growing list of clinically relevant substrates of CYP2B6
substrate access channel. Interestingly, comparative model- includes the antidepressant and antismoking agent bupro-
ing showed that in both CYP2B6 and CYP2C19, the metab- pion, which is almost exclusively metabolized by this isozyme
olite would get into tight contact to a cysteine located near (Hesse et al., 2000), and the antineoplastic agents cyclophos-
the substrate channel (data not shown). Of course, other phamide and ifosfamide, the former of which is metabolically
reaction mechanisms, which may involve thiophene S-oxida- activated mainly by CYP2B6 with some contributions of cy-
tion or -epoxidation, for example, cannot be excluded at this tochromes P450 3A4 and 2C9 (Roy et al., 1999), whereas the
time.
latter is being deactivated (Granvil et al., 1999). CYP2B6 has
Other potent mechanism-based inhibitors of CYP2B6 in- also been shown to catalyze the major route of metabolism for
clude a series of xanthates, which are, however, not in clin- the anesthetics propofol (Court et al., 2001) and ketamine
ical use (Yanev et al., 1999), 17-␣-ethynylestradiol (Kent et (Yanagihara et al., 2001), the MAO-B inhibitor selegiline
al., 2001), certain methyladamantane derivatives (Stiborova (Hidestrand et al., 2001), and the antiretroviral agent efa-
et al., 2002), and phencyclidine (Jushchyshyn et al., 2002). virenz (Ward et al., 2003). CYP2B6 also contributes to the
Potent competitive inhibitors are the antiretroviral drugs metabolism of environmental toxicants and substances of
ritonavir, efavirenz, and nelfinavir (Hesse et al., 2001) and abuse like nicotine and others (Yamazaki et al., 1999a). Since
triethylenethiophosphoramide (Rae et al., 2002). Compared these drugs are widely used, drug interactions with clopi-
with these inhibitors, the results of this study revealed clo- dogrel or ticlopidine may not be uncommon, although none
pidogrel and ticlopidine as the most potent inhibitors of have been reported to date to our knowledge. At least one
CYP2B6 known to date, with KI values for the microsomal example of a clinically relevant drug interaction involving
inactivation of about 0.5 and 0.2 ␮M, respectively. It is there- CYP2B6 has, however, been described. The anticancer drug
fore important to consider the pharmacological consequences triethylenethiophosphoramide was shown to cause a signifi-

Suicide Inhibition of CYP2B6 by Clopidogrel
197
tivation of human cytochrome P450 2B6 by phencyclidine. Drug Metab Dispos
31:46–52.
Kam PC and Nethery CM (2003) The thienopyridine derivates (platelet adenosine
diphosphate receptor antagonists), pharmacology and clinical development. An-
aesthesia 58:28–35.
Kent UM, Mills DE, Rajnarayanan RV, Alworth WL, and Hollenberg PF (2001)
Effect of 17-alpha-ethynylestradiol on activities of cytochrome P450 2B (P450 2B)
enzymes: characterization of inactivation of P450s 2B1 and 2B6 and identification
of metabolites. J Pharmacol Exp Ther 300:549–558.
Lang T, Klein K, Fischer J, Nu¨ssler AK, Neuhaus P, Hofmann U, Eichelbaum M,
Schwab M, and Zanger UM (2001) Extensive genetic polymorphism in the human
CYP2B6 gene with impact on expression and function in human liver. Pharmaco-
genetics 11:399–415.
Lau WC, Waskell LA, Watkins PB, Neer CJ, Horowitz K, Hopp AS, Tait AR, Carville
DG, Guyer KE, and Bates ER (2003) Atorvastatin reduces the ability of clopidogrel
to inhibit platelet aggregation: a new drug-drug interaction. Circulation 107:32–
37.
Lopez-Garcia MP, Dansette PM, and Mansuy D (1994) Thiophene derivatives as new
mechanism-based inhibitors of cytochromes P450: inactivation of yeast-expressed
human liver cytochrome P-450 2C9 by tienilic acid. Biochemistry 33:166–175.
Mehta NB and Raleigh NC (1974), inventors; Burroughs Wellcome (now Glasxo-
SmithKline), assignee. Meta chloro substituted-␣-butylaminopropiophenones.
U.S. patent 3,819,706. 1974 June 25.
Mehta SR, Yusuf S, Peters RJ, Bertrand ME, Lewis BS, Natarajan MK, Malmberg
K, Rupprecht H, Zhao F, Chrolavicius S, et al. Clopidogrel in Unstable angina to
prevent Recurrent Events trial (CURE) Investigators (2001) Effects of pretreat-
ment with clopidogrel and aspirin followed by long-term therapy in patients
undergoing percutaneous coronary intervention: the PCI-CURE study. Lancet
358:527–533.
Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, and Olson AJ
(1998) Automated docking using a Lamarckian genetic algorithm and an empirical
binding free energy function. J Comput Chem 19:1639–1662.
cant reduction in the plasma levels of the active metabolite of
cyclophosphamide when administered first (Huitema et al.,
2000). As mentioned above, triethylenethiophosphoramide
was found to be a potent and selective inhibitor of CYP2B6
(Rae et al., 2002).
In conclusion, we found that the two thienopyridines, clo-
pidogrel and ticlopidine, are highly potent, irreversible inhib-
itors of CYP2B6. We provided strong evidence that inhibition
involves a mechanism-based process, and we suggested a
chemical mechanism, which has to be investigated in further
detail in future experiments. Since clopidogrel and ticlopi-
dine are among the most potent CYP2B6 inhibitors known
today, these findings may be of clinical relevance. In addi-
tion, these substances may be useful as in vitro or in vivo
probes to estimate the relative contribution of CYP2B6 to
drug metabolism.
Acknowledgments
We gratefully acknowledge Kathrin Klein, Stuttgart, for helpful
discussions and Andreas Nu¨ssler, Berlin, for providing human liver
samples.
Pereillo JM, Maftouh M, Andrieu A, Uzabiaga MF, Fedeli O, Savi P, Pascal M,
Herbert JM, Maffrand JP, and Picard C (2002) Structure and stereochemistry of
the active metabolite of clopidogrel. Drug Metab Dispos 30:1288–1295.
Rae JM, Soukhova NV, Flockhart DA, and Desta Z (2002) Triethylenethiophosphor-
amide is a specific inhibitor of cytochrome P450 2B6: implications for cyclophos-
phamide metabolism. Drug Metab Dispos 30:525–530.
Roy P, Yu LJ, Crespi CL, and Waxman DJ (1999) Development of a substrate-
activity based approach to identify the major human liver P-450 catalysts of
cyclophosphamide and ifosfamide activation based on cDNA-expressed activities
and liver microsomal P-450 profiles. Drug Metab Dispos 27:654–666.
Savi P, Combalbert J, Gaich C, Rouchon MC, Maffrand JP, Berger Y, and Herbert
JM (1994) The antiaggregating activity of clopidogrel is due to a metabolic acti-
vation by the hepatic cytochrome P450–1A. Thromb Haemostasis 72:313–317.
Savi P, Herbert JM, Pflieger AM, Dol F, Delebassee D, Combalbert J, Defreyn G,
Maffrand JP (1992) Importance of hepatic metabolism in the antiaggregating
activity of the thienopyridine clopidogrel. Biochem Pharmacol 44:527–532.
Savi P, Pereillo JM, Uzabiaga MF, Combalbert J, Picard C, Maffrand JP, Pascal M,
and Herbert JM (2000) Identification and biological activity of the active metab-
olite of clopidogrel. Thromb Haemostasis 84:891–896.
Silverman RB (1995) Mechanism-based enzyme inactivators. Methods Enzymol 249:
240–283.
Stiborova M, Borek-Dohalska L, Hodek P, Mraz J, and Frei E (2002) New selective
inhibitors of cytochromes P450 2B and their application to antimutagenesis of
tamoxifen. Arch Biochem Biophys 403:41–49.
Tateishi T, Kumai T, Watanabe M, Nakura H, Tanaka M, and Kobayashi S (1999)
Ticlopidine decreased the in vivo activity of CYP2C19 as measured by omeprazole
metabolism. Br J Clin Pharmacol 47: 454–457.
von Richter O, Eichelbaum M, Schonberger F, and Hofmann U (2000) Rapid and
highly sensitive method for the determination of verapamil, [2H7]verapamil and
metabolites in biological fluids by liquid chromatography-mass spectrometry.
J Chromatogr B Biomed Sci Appl 738:137–147.
References
Bathelt C, Schmid RD, and Pleiss J (2002) Regioselectivity of CYP2B6: homology
modeling, molecular dynamics simulation, docking. J Mol Model 8:327–335.
Beaune P, Dansette PM, Mansuy D, Kiffel L, Finck M, Amar C, Leroux JP, and
Homberg JC (1987) Human anti-endoplasmic reticulum autoantibodies appearing
in a drug-induced hepatitis are directed against a human liver cytochrome P-450
that hydroxylates the drug. Proc Natl Acad Sci USA 84:551–555.
Clarke TA and Waskell LA (2002) The metabolism of clopidogrel is catalyzed by
human cytochrome P450 3A and is inhibited by atorvastatin. Drug Metab Dispos
31:53–59.
Court MH, Duan SX, Hesse LM, Venkatakrishnan K, and Greenblatt DJ (2001)
Cytochrome P-450 2B6 is responsible for interindividual variability of propofol
hydroxylation by human liver microsomes. Anesthesiology (Hagerst) 94:110–119.
Ding Z, Kim S, Dorsam RT, Jin J, and Kunapuli SP (2003) Inactivation of the human
P2Y12 receptor by thiol reagents requires interaction with both extracellular
cysteine residues, Cys17 and Cys270. Blood 101:3908–3914.
Donahue SR, Flockhart DA, Abernethy DR, and Ko JW (1997) Ticlopidine inhibition
of phenytoin metabolism mediated by potent inhibition of CYP2C19. Clin Phar-
macol Ther 62:572–577.
Ekins S and Wrighton SA (1999) The role of CYP2B6 in human xenobiotic metabo-
lism. Drug Metab Rev 31:719–754.
Faucette SR, Hawke RL, Lecluyse EL, Shord SS, Yan B, Laethem RM, and Lindley
CM (2000) Validation of bupropion hydroxylation as a selective marker of human
cytochrome P450 2B6 catalytic activity. Drug Metab Dispos 28:1222–1230.
Gervot L, Rochat B, Gautier JC, Bohnenstengel F, Kroemer H, de Berardinis V,
Martin H, Beaune P, and de Waziers I (1999) Human CYP2B6: expression, induc-
ibility and catalytic activities. Pharmacogenetics 9:295–306.
Granvil CP, Madan A, Sharkawi M, Parkinson A, and Wainer IW (1999) Role of
CYP2B6 and CYP3A4 in the in vitro N-dechloroethylation of (R)- and (S)-
ifosfamide in human liver microsomes. Drug Metab Dispos 27:533–541.
Ha-Duong NT, Dijols S, Macherey AC, Goldstein JA, Dansette PM, and Mansuy D
Ward BA, Gorski JC, Jones DR, Hall SD, Flockhart DA, and Desta Z (2003) The
cytochrome P450 2B6 (CYP2B6) is the main catalyst of efavirenz primary and
secondary metabolism: implication for HIV/AIDS therapy and utility of efavirenz
as a substrate marker of CYP2B6 catalytic activity. J Pharmacol Exp Ther 306:
287–300.
Yamazaki H, Inoue K, Hashimoto M, and Shimada T (1999a) Roles of CYP2A6 and
CYP2B6 in nicotine C-oxidation by human liver microsomes. Arch Toxicol 73:65–
70.
(2001) Ticlopidine as
P4502C19. Biochemistry 40:12112–12122.
a selective mechanism-based inhibitor of cytochrome
Hesse L, Venkatakrishnan K, Court M, von Moltke L, Duan X, Shader RI, and
Greenblatt DJ (2000) CYP2B6 mediates the in vitro hydroxylation of bupropion:
potential drug interactions with other antidepressants. Drug Metab Dispos 28:
1176–1183.
Hesse LM, von Moltke LL, Shader RI, and Greenblatt DJ (2001) Ritonavir, efavirenz
and nelfinavir inhibit CYP2B6 activity in vitro: potential drug interactions with
bupropion. Drug Metab Dispos 29:100–102.
Hidestrand M, Osxarson M, Salamen JS, Nyman L, Pelkonen O, Turpeinen M, and
Ingelman-Sundberg M (2001) CYP2B6 and CYP2C19 as the major enzymes re-
sponsible for the metabolism of selegiline, a drug used in the treatment of Par-
kinson’s disease, as revealed from experiments with recombinant enzymes. Drug
Metab Dispos 29:1480–1484.
Hofmann U, Pecia M, Heinkele G, Dilger K, Kroemer HK, and Eichelbaum M (2000)
Determination of propafenone and its phase I and phase II metabolites in plasma
and urine by high-performance liquid chromatography-electrospray ionization
mass spectrometry. J Chromatogr B Biomed Sci Appl 748:113–123.
Huitema AD, Kerbusch T, Tibben MM, Rodenhuis S, and Beijnen JH (2000) Reduc-
tion of cyclophosphamide bioactivation by thioTEPA: critical sequence-dependency
in high-dose chemotherapy regimens. Cancer Chemother Pharmacol 46:119–127.
Jushchyshyn MI, Kent UM, and Hollenberg PF (2002) The mechanism-based inac-
Yamazaki H, Tanaka M, and Shimada T (1999b) Highly sensitive high-performance
liquid chromatographic assay for coumarin 7-hydroxylation and 7-ethoxycoumarin
O-deethylation by human liver cytochrome P450 enzymes.
Biomed Sci Appl 721:13–19.
J Chromatogr B
Yanagihara Y, Kariya S, Ohtani M, Uchino K, Aoyama T, Yamamura Y, and Iga T
(2001) Involvement of CYP2B6 in n-demethylation of ketamine in human liver
microsomes. Drug Metab Dispos 29:887–890.
Yanev S, Kent UM, Pandova B, and Hollenberg PF (1999) Selective mechanism-
based inactivation of cytochromes P-450 2B1 and P-450 2B6 by a series of xan-
thates. Drug Metab Dispos 27:600–604.
Address correspondence to: Dr. Ulrich M. Zanger, Dr. Margarete Fischer-
Bosch Institute of Clinical Pharmacology, Auerbachstr. 112, D-70376 Stutt-
gart, Germany. E-mail: uli.zanger@ikp-stuttgart.de