Hepatic microsomal thiol methyltransferase is involved in stereoselective methylation of pharmacologically active metabolite of prasugrel

Article abstract of DOI:10.1124/dmd.114.057661

Prasugrel, a thienopyridine antiplatelet drug, is converted in animals and humans to the pharmacologically active metabolite R-138727 [(2Z)-{1-[(1RS)-2- cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-sulfanylpiperidin-3-ylidene} ethanoic acid], which has two chiral centers, occurring as a mixture of four isomers. The RS and RR isomers are more active than the SS and SR isomers (RS > RR > > SR = SS). The pharmacologically active metabolite is further metabolized to an S-methylated metabolite that is the major identified inactive metabolite in humans. In rat, dog, and human liver microsomes supplemented with S-adenosyl methione, the SS and SR isomers of the active metabolite were extensively S-methylated while the RS and RR isomers were not. Addition of 2,3-dichloromethyl benzylamine (50 μM) completely inhibited the S-methylation reaction, indicating that the microsomal and cytosolic thiol methyltransferase but not the cytosolic thiopurine S-methyltransferase is involved in the methylation. The hepatic intrinsic clearance values for methylation of the RS, RR, SS, and SR isomers (ml/min/kg) were 0, 0, 40.4, and 37.6, respectively, in rat liver microsomes, 0, 0, 11.6, and 2.5, respectively, in dog liver microsomes, and 0, 0, 17.3, and 17.7, respectively, in human liver microsomes, indicating that the RS and RR isomers are not methylated in vitro and that the methylation of SS and SR isomers is high with rat > human > dog. This finding in vitro agreed well with the in vivo observation in rats and dogs, where the S-methylated SS and SR isomers were the major metabolites in the plasma whereas negligible amounts of S-methylated RS and RR isomers were detected after intravenous administration of the pharmacologically active metabolites. Copyright

Full text of DOI:10.1124/dmd.114.057661

Supplemental Material can be found at:
http://dmd.aspetjournals.org/content/suppl/2014/04/14/dmd.114.057661.DC1.html
1521-009X/42/7/1138–1145$25.00
http://dx.doi.org/10.1124/dmd.114.057661
DRUG METABOLISM AND DISPOSITION
Drug Metab Dispos 42:1138–1145, July 2014
Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics
Hepatic Microsomal Thiol Methyltransferase Is Involved in
Stereoselective Methylation of Pharmacologically Active
Metabolite of Prasugrels
Miho Kazui, Katsunobu Hagihara, Takashi Izumi, Toshihiko Ikeda, and Atsushi Kurihara
Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd., Tokyo (M.K., K.H., T.Iz., A.K.); Yokohama
College of Pharmacy, Yokohama (T.Ik.), Japan
Received February 18, 2014; accepted April 14, 2014
ABSTRACT
Prasugrel, a thienopyridine antiplatelet drug, is converted in
cytosolic thiol methyltransferase but not the cytosolic thiopurine
animals and humans to the pharmacologically active metabolite S-methyltransferase is involved in the methylation. The hepatic
R-138727 [(2Z)-{1-[(1RS)-2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]- intrinsic clearance values for methylation of the RS, RR, SS, and
4-sulfanylpiperidin-3-ylidene}ethanoic acid], which has two chiral
centers, occurring as a mixture of four isomers. The RS and RR
isomers are more active than the SS and SR isomers (RS > RR > >
SR = SS). The pharmacologically active metabolite is further
SR isomers (ml/min/kg) were 0, 0, 40.4, and 37.6, respectively, in
rat liver microsomes, 0, 0, 11.6, and 2.5, respectively, in dog liver
microsomes, and 0, 0, 17.3, and 17.7, respectively, in human liver
microsomes, indicating that the RS and RR isomers are not
metabolized to an S-methylated metabolite that is the major methylated in vitro and that the methylation of SS and SR isomers
identified inactive metabolite in humans. In rat, dog, and human
liver microsomes supplemented with S-adenosyl methione, the
SS and SR isomers of the active metabolite were extensively
is high with rat > human > dog. This finding in vitro agreed
well with the in vivo observation in rats and dogs, where the
S-methylated SS and SR isomers were the major metabolites in
S-methylated while the RS and RR isomers were not. Addition of the plasma whereas negligible amounts of S-methylated RS and
2,3-dichloromethyl benzylamine (50 mM) completely inhibited the
S-methylation reaction, indicating that the microsomal and
RR isomers were detected after intravenous administration of the
pharmacologically active metabolites.
Introduction
the second for that at the 19-position of the benzyl group) (Fig. 1). The
rank order of potency of these compounds in inhibiting platelet
aggregation in vitro is: RS isomer . RR isomer .. SS isomer = SR
isomer (Hasegawa et al., 2005). In humans, the pharmacologically
active RS and RR isomers were detected in plasma at about 5-fold
higher levels than the pharmacologically less active SS and SR
isomers after dosing of prasugrel (Wickremsinhe et al., 2007). In rats,
the RS and RR isomers were the major forms detected in plasma, and
the SS and SR isomers were only detected at much lower con-
centrations in plasma (Kazui et al., 2008). In dogs, on the other hand,
the levels of the RS and RR isomers were similar to those of the SS
and SR isomers in plasma (Kazui et al., 2008). The S-methylated
metabolite of the pharmacologically active metabolite was the major
identified metabolite in humans and the second major metabolite in
rats and dogs, indicating that the S-methylation is an important
inactivation pathway of prasugrel (Asai et al., 2006, Farid et al., 2007a,b).
The S-methylation reaction could be catalyzed by two enzymes:
thiol S-methyltransferase (TMT) and thiopurine S-methyltransferase
(TPMT). These two enzymes differ in their subcellular localization,
Prasugrel is an approved thienopyridine antiplatelet agent for the
reduction of thrombotic cardiovascular events in patients with acute
coronary syndrome who are being managed by percutaneous coronary
intervention (PCI) (Jakubowski et al., 2012). Prasugrel is a prodrug
that requires the metabolic conversion to the pharmacologically active
metabolite R-138727 [(2Z)-{1-[(1RS)-2-cyclopropyl-1-(2-fluorophenyl)-
2-oxoethyl]-4-sulfanylpiperidin-3-ylidene}ethanoic acid] in vivo (Sugidachi
et al., 2000). The pharmacologically active metabolite of prasugrel is a
mixture of four stereoisomers, RS, RR, SS, and SR forms (the first letter
indicates the configuration at the 4-position of the piperidyl group and
Miho Kazui, Katsunobu Hagihara, Takashi Izumi, and Atsushi Kurihara are
employees of Daiichi Sankyo Co., Ltd. Other authors declare no conflict of
interest.
The study was sponsored by Daiichi-Sankyo Co., Ltd, Tokyo, Japan.
dx.doi.org/10.1124/dmd.114.057661.
s This article has supplemental material available at dmd.aspetjournals.org.
ABBREVIATIONS: AUC0–inf, area under the plasma concentration-time curve extrapolated to infinity; CLint, intrinsic clearance; Cmax, maximum
plasma concentration; DCMB, (6)-2,3-dichloro-a-methylbenzylamine hydrochloride; ESI, electrospray ionization; HPLC, high performance liquid
chromatography; LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry; NADP, b-nicotinamide adenine dinucleotide
phosphate sodium salt; ODS, octadecyl silane; R-106583, (2Z)-{1-[2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-(methylsulfanyl)piperidin-3-
ylidene}ethanoic acid; R-121721, (2Z)-(1-{2-[(2,2,3,3-2H4)cyclopropyl]-1-(2-fluorophenyl)-2-oxoethyl}-4-[(2H3)methylsulfanyl]piperidin-3-ylidene)
ethanoic acid; R-135766, (4-{[2-(4-bromophenyl)-2-oxoethyl]sulfanyl}-1-[2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-1,2,5,6-tetrahydropyridin-3-
yl)acetic acid; R-138727, (2Z)-{1-[(1RS)-2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-sulfanylpiperidin-3-ylidene}ethanoic acid; SAM, S-(59-
adenosyl)-L-methionine chloride; TMT, thiol-methyltransferase; TPMT, thiopurine S-methyltransferase; UPLC-UV, ultraperformance liquid
chromatography coupled with ultraviolet detection; Vmax, maximal reaction rate.
1138

Stereoselectivity of Thiol S-Methyltransferase
1139
Fig. 1. Structures of four stereoisomers of the pharma-
cologically active metabolites of prasugrel. The first
letter indicates the configuration at the 4-position of
piperidyl group and the second for that at the 19-
position of benzyl group. *Denotes chiral center.
(beagle) liver microsomes (20 mg protein/ml) were purchased from BD
Biosciences Company (Woburn, MA). Human liver microsomes (pooled from
10 donors) and human liver cytosol (pooled from 10 donors) for identification
of the enzyme involved in the S-methylation of the pharmacologically active
metabolites were purchased from the nonprofit Human & Animal Bridging
Research Organization (Tokyo, Japan).
substrate specificities, inhibitor sensitivities, and regulation. TPMT is
a cytosolic enzyme and inhibited by m-anisic acid whereas TMT is a
microsomal enzyme and inhibited by (6)-2,3-dichloro-a-methylbenzylamine
hydrochloride (DCMB) (Lee and Kim, 1999). TPMT exhibits a genetic
polymorphism (Weinshilboum, 1989), with 89% of whites and African
Americans being extensive metabolizers, 11% intermediate metabolizers,
and 0.33% poor metabolizers (Szumlanski et al., 1996; Hamdan-Khalil
et al., 2003). Our study identified the S-methylating enzyme responsible
for methylating the pharmacologically active metabolite of prasugrel and
determined the stereoselectivity in this metabolic reaction using sub-
cellular preparations of human and animal livers. Based on the assay data
in vitro, we calculated the hepatic intrinsic clearance (CLint) values in
vivo for the stereoselective S-methylation as the major elimination
pathway of the pharmacologically active metabolite of prasugrel, and
examined the relevance of these values to the in vivo profile of the
S-methylated metabolite after intravenous administration of the pharma-
cologically active metabolite of prasugrel R-138727 to rats and dogs.
Animals
Male Sprague-Dawley rats (n = 4) were obtained from Charles River Japan
(Yokohama, Japan) at 8 weeks of age. Four male beagle dogs were originally
obtained from Nihon Nosan Corporation at the age of 6 months and were kept
separately in stainless-steel cages in a controlled animal area. The controlled
animal area was set at a room temperature of 23 6 2°C and 55% 6 10%
relative humidity under a 12-hour cycle of light/dark artificial lighting (7:00
AM–7:00 PM). All experimental procedures were performed in accordance
with the in-house guidelines of the Institutional Animal Care and Use
Committee of Daiichi Sankyo Co., Ltd.
Materials and Methods
S-Methylation of the Pharmacologically Active Metabolite of Prasugrel in
The pharmacologically active metabolites of prasugrel (R-138727) contain- Human Liver Microsomes and Cytosol
ing four stereoisomers, the S-methylated form of the pharmacologically active
The assay for a TMT reaction was performed using pooled human liver
metabolites containing four stereoisomers (R-106583 [(2Z)-{1-[2-cyclopropyl-
1-(2-fluorophenyl)-2-oxoethyl]-4-(methylsulfanyl)piperidin-3-ylidene}ethanoic
acid]) (Fig. 2), the deuterium-labeled S-methylated metabolite of the
pharmacologically active metabolites of prasugrel containing four stereo-
isomers (R-121721 [(2Z)-(1-{2-[(2,2,3,3-2H4)cyclopropyl]-1-(2-fluorophenyl)-
2-oxoethyl}-4-[(2H3)methylsulfanyl]piperidin-3-ylidene)ethanoic acid]), and
the bromo-phenacyl derivative of the pharmacologically active metabolite
(R-135766 [(4-{[2-(4-bromophenyl)-2-oxoethyl]sulfanyl}-1-[2-cyclopropyl-1-
(2-fluorophenyl)-2-oxoethyl]-1,2,5,6-tetrahydropyridin-3-yl)acetic acid]) were
obtained from Ube Industries, Ltd. (Ube, Japan) (Supplemental Figure 1). Each
stereoisomer of the pharmacologically active metabolites of prasugrel was
synthesized by Daiichi Sankyo Co., Ltd. R-135766 was used as the internal
standard for an in vitro assay of the enantiomers of R-106583 by liquid
chromatography coupled with tandem mass spectrometry (LC-MS/MS).
R-121721 was used as the internal standard for an in vivo assay of R-106583
and its enantiomer (i.e., RR/RS form and SR/SS form of R-106583) by LC-MS/
MS. As the internal standard for the assay of the R-106583 by high-performance
liquid chromatography (HPLC), a-naphthoflavone was obtained from Sigma-
Aldrich Corporation (St. Louis, MO). S-(59-adenosyl)-L-methionine chloride
(SAM), glucose-6-phosphate dehydrogenase from baker’s yeast (G-6-PDH),
D-glucose 6-phosphate disodium salt hydrate (G-6-P), b-nicotinamide adenine
dinucleotide phosphate sodium salt (NADP), L-glutathione reduced (GSH), and
DCMB were also purchased from Sigma-Aldrich Corporation. The derivatizing
reagent for the assay of the pharmacologically active metabolites of prasugrel by
LC-MS/MS (Farid et al., 2007b) m-methoxyphenacyl bromide (MPBr) and
m-anisic acid were obtained from Tokyo Chemical Industry (Tokyo, Japan). All
other reagents were commercially available and of guaranteed reagent grade.
microsomes. The incubation mixture contained 2 mg protein/ml of pooled
human liver microsomes, 0.5 mM of SAM, and 50 mM of the pharmacolog-
ically active metabolite of prasugrel (R-138727 racemic mixture) as the
substrate in a final volume of 400 ml of 100 mM potassium phosphate buffer
(pH 7.9) containing 1 mM EDTA and 0.5% Triton X-100. The mixture without
the substrate was preincubated at 37°C for 5 minutes, and the reaction was
started by the addition of 4 ml of the substrate solution in dimethylsulfoxide
(DMSO). After incubation at 37°C for 0, 5, 10, 20, 30, and 45 minutes, a 50-ml
aliquot of the incubation mixture was collected and added to a mixture of 100
ml of acetonitrile and 50 ml of a solution of a-naphthoflavone as the internal
standard (5 mg/ml in acetonitrile) to terminate the reaction. The mixture was
centrifuged at 20,800g at 4°C for 3 minutes, and 2 ml of the supernatant was
injected into the ultraperformance liquid chromatography coupled with
ultraviolet detection system (UPLC-UV; Waters Corporation to determine the
concentrations of the S-methylated metabolite, R-106583 (details of method to
be explained herein).
The assay for a TPMT reaction was performed using pooled human liver
cytosol. The incubation mixture contained 2.25 mg protein/ml of pooled human
liver cytosol, 0.5 mM of SAM, and 50 mM of R-138727 as the substrate in
a final volume of 300 ml of 400 mM potassium phosphate buffer (pH 6.2). The
sample preparation and assay were performed in the same manner as described
earlier for the assay for a TMT reaction in microsomes. Experiments were
performed in triplicate.
Inhibition of S-Methylation of R-138727, the Pharmacologically Active
Metabolite of Prasugrel, in Human Liver Microsomes and Cytosol
Effects of DCMB as the TMT inhibitor and m-anisic acid as the TPMT
inhibitor on S-methylation were evaluated. DCMB was added at a final
concentration of 5, 50, or 500 mM to the incubation medium consisting of 2 mg
Biologic Samples
Pooled human liver microsomes (20 mg protein/ml), pooled male rat
(Sprague-Dawley) liver microsomes (20 mg protein/ml), and pooled male dog microsomal protein/ml of human liver, 0.5 mM SAM, and 100 mM potassium

1140
Kazui et al.
Fig. 2. Metabolic pathway of prasugrel. Figure does not
illustrate all metabolites of prasugrel that have been
identified.
phosphate buffer (pH 7.9) containing 1 mM EDTA and 0.5% Triton X-100, and
the mixture was preincubated at 37°C for 5 minutes. Similarly, DCMB was
substrate. After incubation at 37°C for 0 and 30 minutes, a 50-ml aliquot of the
incubation mixture was collected and added to a mixture of 100 ml of
added at a final concentration of 5, 50, or 500 mM to the incubation medium acetonitrile and 50 ml of a solution of R-135766 as the internal standard (2 mM
consisting of 2.25 mg protein/ml of human liver cytosol, 0.5 mM SAM, and in acetonitrile) to terminate the reaction. The mixture was centrifuged at
400 mM potassium phosphate buffer (pH 6.2), and the mixture was 20,800g at 4°C for 3 minutes, and 5 ml of the supernatant was injected into an
preincubated at 37°C for 5 minutes. Meta-anisic acid was added at a final
LC-MS/MS system to determine the concentrations of R-106583. This
concentration of 0.1, 1, and 10 mM to the incubation medium consisting of analytical method is referred to as the electrospray ionization (ESI) LC-MS/
2.25 mg protein/ml of human liver cytosol, 0.5 mM SAM, and 400 mM MS single-column method (details of the method explained herein). Experi-
potassium phosphate buffer (pH 6.2), and the mixture was preincubated at
37°C for 5 minutes. Each reaction was initiated by the addition of 50 mM of
the active metabolite in DMSO and terminated after incubation at 37°C for
30 minutes and 45 minutes. A 50-ml aliquot of the incubation mixture was
collected and treated as described in the preceding section on S-methylation
of the pharmacologically active metabolites of prasugrel in human liver
microsomes and cytosol. The assay was conducted by the UPLC-UV
method.
ments were performed in duplicate.
Plasma Concentrations of the S-Methylated Metabolites after Intravenous
Administration of the Pharmacologically Active Metabolites of Prasugrel
to Rats and Dogs
Administration and Plasma Sample Preparations. R-138727, a mix-
ture of the four stereoisomers of the pharmacologically active metabolite of
prasugrel, was dissolved in saline to prepare a 1 mg/ml solution and used
for intravenous administration. Rats (n = 4) and dogs (n = 4) were fasted
overnight and administered R-138727 intravenously at a dose of 1 mg/kg.
At 0.033, 0.083, 0.25, 0.5, and 1 hour after the dose, approximately 0.4 ml
of blood was collected from the jugular vein (rat) or the cephalic vein (dog)
with a heparinized syringe. Each blood sample was transferred into
a sampling tube containing 10 ml of 500 mM m-methoxyphenacyl bromide
(MPBr) in acetonitrile to derivatize the sulfhydryl-containing substances to
chemically stable substances. The mixture was vortexed and allowed to
stand at room temperature for about 10 minutes for the derivatization
reaction. The blood samples were centrifuged at 21,600g for 3 minutes at
4°C to separate the plasma for the assay of each stereoisomer of the
S-methylated metabolites.
S-Methylation of Each Isomer of the Pharmacologically Active Metabolite
of Prasugrel in Rat, Dog, and Human Liver Microsomes
The assays were performed using rat, dog, and human liver microsomes. The
incubation mixture contained 2 mg liver microsomal protein/ml of each species,
an NADPH-generating system (2.5 mM b-NADP, 22.5 mM D-glucose-6-
phosphate, 10 mM magnesium chloride, and 0.5 units/ml of glucose-6-
phosphate dehydrogenase), 0.5 mM of SAM, and 100 mM of either of four
stereoisomers of the pharmacologically active metabolite of prasugrel, SS, SR,
RS, or RR form in DMSO solution, as the substrate in a final volume of 500 or
800 ml of 100 mM potassium phosphate buffer (pH 7.9) containing 1 mM
EDTA and 0.5% Triton X-100.
A mixture without the substrate was
preincubated at 37°C for 5 minutes, and the reaction was started by the
addition of 5 or 8 ml of a solution of the substrate. After incubation at 37°C for
0, 5, 15, 30, 60, 90, and 120 minutes, a 50-ml aliquot of the incubation mixture
was collected and added to a mixture of 100 ml of acetonitrile and 50 ml of
a solution of a-naphthoflavone as the internal standard (5 mg/ml in acetonitrile)
to terminate the reaction. The mixture was centrifuged at 20,800g at 4°C for
3 minutes, and 2 ml of the supernatant was analyzed by the UPLC-UV method
to determine the concentrations of the S-methylated metabolites. Experiments
were performed in duplicate.
Assay of S-Methylated Metabolites of the Pharmacologically Active
Metabolite of Prasugrel (UPLC-UV Method, Nonstereoselective)
The S-methylated metabolite enantiomers produced from the pharmacolog-
ically active metabolite of prasugrel, in the rat, dog, and human liver
microsomes were measured using a UPLC system (Acquity Ultra Performance
system; Waters Corporation). After injection of the sample (2 ml) to an
ACQITY UPLC BEH C18 column (2.1 ꢀ 50 mm, particle size of 1.7 mm;
Waters Corporation), the S-methylated metabolite was separated as a single
peak via a gradient system with a flow rate of 0.5 ml/min. R-106583 (racemic
mixture) was used as standard the S-methylated metabolites. The mobile phase
Enzyme Kinetic Parameters for S-Methylation of Each Stereoisomer of the
Pharmacologically Active Metabolites of Prasugrel
The incubation mixture contained 2 mg protein/ml of the liver microsomes consisted of a mixture of distilled water and trifluoroacetic acid (1000:0.05, v/v,
from rat, dog, or human liver, an NADPH-generating system, 0.5 mM SAM, solvent A) and a mixture of acetonitrile, distilled water, and trifluoroacetic acid
5 mM glutathione and 0.5, 1, 2, 5, 10, 50, 100, 200, or 400 mM of one of four
isomers of the pharmacologically active metabolite of prasugrel (RS, RR, SS,
(400:600:0.05, v/v/v, solvent B). The elution was started with 90% solvent A
and 10% solvent B, and the proportion of solvent B was increased from 10% to
or SR form) in DMSO as the substrate in a final volume of 250 ml of 100 mM 55% in 10 minutes, from 55% to 100% in 14 minutes, and returned to the initial
potassium phosphate buffer (pH 7.9) containing 1 mM EDTA and 0.5% Triton condition in 19 minutes. Detection was performed at 220 nm for R-106583 and
X-100. A mixture without the substrate was preincubated at 37°C for 5 minutes, at 280 nm for a-naphthoflavone, the internal standard, using a photodiode array
and the reaction was started by the addition of 2.5 ml of a solution of the detector (Waters Corporation).

Stereoselectivity of Thiol S-Methyltransferase
1141
Assay of S-methylated Metabolite of the Pharmacologically Active Metabolite Acquity UPLC System (Waters Corporation) with an ODS column (Acquity
of Prasugrel (ESI-LC-MS/MS Single-Column Method, Nonstereoselective))
UPLC BEH C18, 2.1 mm ꢀ 100 mm, 1.7 mm, Waters Corp.) and a chiral
column (Chiralcel OJ-RH, 2ꢀ150 mm, 5 mm; Daicel Chemical Industries, Ltd.,
Osaka, Japan) at a flow rate of 0.25 ml/min with a mobile phase consisting of
acetonitrile, distilled water, 1 M ammonium acetate, and trifluoroacetic acid
(TFA) [275:715:10:0.5 (v/v/v)]. The two diastereomeric pairs (RR/RS and SS/
SR) of R-106583 were separated chromatographically by the ESI-LC-MS/MS
double-column method. Therefore, the ratios of the two diastereomeric pairs
(RR/RS and SS/SR) of the S-methylated metabolite in plasma were determined
using this assay.
The total concentration of the S-methylated metabolite in the plasma was
determined using a slight modification of the ESI-LC-MS/MS single-column
method: API5000 (Applied Biosystems/MDS SCIEX) in the positive-ion
detection mode with the ESI interface and LC-20A UFLC system (Shimadzu
Corpororation, Kyoto, Japan) consisting of L-column ODS (2.1 ꢀ 150 mm,
particle size of 5 mm; Chemicals Evaluation and Research Institute, Fukuoka,
Japan) and a gradient system. Elution was started with 50% solvent A (distilled
water and formic acid, 990:10 v/v) and 50% solvent B (acetonitrile), and the
proportion of solvent B was increased linearly to 75% in 2 minutes, maintained
at 75% for 6 minutes, and returned to the initial condition in 6.1 minutes.
For determination of the enzyme kinetic parameters of the S-methylation of
the pharmacologically active metabolite of prasugrel, the S-methylated
metabolite was measured by the ESI-LC-MS/MS single-column method, using
R-106583 as the standard. The Quattro-LC MS/MS system (Micromass UK.,
Ltd., Manchester, United Kingdom) was used in the positive-ion detection
mode with the ESI interface. The peak area of the m/z 364→206 for R-106583
was measured against the peak areas of the m/z 548→206 for the internal
standard (R-135766). Separation by HPLC was conducted using an Alliance
2695 Separations Module (Waters Corporation) with an octadecyl silane
column (Inertsil ODS-3, 2.1 mm ꢀ 150 mm, 5 mm; GL Sciences, Torrance,
CA) at a flow rate of 0.2 ml/min with a mobile phase consisting of methanol,
distilled water, and trifluoroacetic acid (TFA) [520:480:0.5 (v/v/v)].
Assay of the Stereoisomers of the S-Methylated Metabolite in Plasma (ESI-
LC-MS/MS Double-Column Method)
For determination of the plasma concentrations of the stereoisomers of the S-
methylated metabolite after intravenous administration of the pharmacologi-
cally active metabolite of prasugrel (R-138727) to rats and dogs, an ESI-LC-
MS/MS system equipped with two columns was used. API5000 (Applied
Biosystems/MDS SCIEX, Foster City, CA) was used in the positive-ion
detection mode with the ESI interface. The peak area of the m/z 364→206 for
Data Handling
The activity for producing the S-methylated metabolite (V, pmol/min/mg
R-106583 was measured against the peak areas of the m/z 371→153 for the protein), V/S (ml/min/mg protein), and the inhibition ratio (%) were calculated
internal standard (R-121721). Separation by UPLC was conducted using an according to the following equations.
Theꢀ concentrationðmMÞofꢀ theꢀ product ꢀ 1000
Vðpmol=min=mgꢀ proteinÞ ¼
ð1Þ
ð2Þ
ð3Þ
Incubationꢀ timeðminÞ ꢀ proteinꢀ concentrationðmg=mLÞ
V
Theꢀ meanꢀ Vðpmol=min=mgꢀ proteinÞ
ðmL=min=mgꢀ proteinÞ ¼
S
Theꢀ nominalꢀ concentrationꢀ ofꢀ eachꢀ substrateðmMÞ
ðTheꢀ meanꢀ Vꢀ withoutꢀ inhibitor 2 theꢀ meanꢀ Vꢀ withꢀ inhibitorÞ ꢀ 100
Theꢀ meanꢀ Vꢀ withoutꢀ inhibitor
Inhibitionꢀ ratioð%Þ ¼
intrinsic clearance (CLint in ml/min/kg b.wt.) for the S-methylation in the
animals was calculated according to eq. 4 and eq. 5. The microsomal protein
(mg/g liver) of rats (44.8), humans (48.8), and dogs (77.9) and the weight of the
liver (g) per body weight (kg) in rats (10 g/0.25 kg), humans (1800 g/70 kg),
and dogs (320 g/10 kg) were from the literature reports (Davies and Morris,
1993; Iwatsubo et al., 1997).
The values for V, V/S, and the inhibition ratio were expressed to one decimal
place.
The Michaelis-Menten constant (Km in mM) and maximal reaction rate (Vmax
in pmol/min/mg protein) were calculated from the Eadie-Hofstee plots of the
reaction rate (V) against V/S using Microsoft Office Excel 2003 (version SP2;
Microsoft Corporation, Redmond, WA). The y-intercept and the slope obtained
from the Eadie-Hofstee plots indicate Vmax and Km values, respectively. The
Vmaxðpmol=min=mgꢀ proteinÞ
CLint;ꢀ inꢀ vitroðmL=min=mgꢀ proteinÞ ¼
ð4Þ
KmðmMÞ
CLint;ꢀ inꢀ vitro ꢀ theꢀ microsomalꢀ proteinðmg=gꢀ liverÞ ꢀ theꢀ weightsꢀ ofꢀ theꢀ liverꢀ wereꢀ g=B:W:ðkgÞ
CLintðmL=min=kgÞ ¼
ð5Þ
1000

1142
Kazui et al.
The calculated enzymatic kinetic parameters were expressed to two decimal
places.
For calculating the plasma concentrations of each stereoisomer of the
plasma concentrations were expressed to three significant figures. The mean
values and S.D. of the concentration and peak area ratio are calculated and
expressed to three significant figures using Microsoft Office Excel 2003 (SP2;
S-methylated metabolite produced after intravenous administration of the Microsoft Corporation). After dosing of R-138727, the enantiomer ratio of RS/
pharmacologically active metabolites of prasugrel to rats and dogs, we used
Analyst 1.4.1 and Analyst 1.4.2 (Applied Biosystems/MDS SCIEX). The
RR or SS/SR isomers of R-106583 was calculated using the peak area ratios of
RS/RR and SS/SR isomers by eq. 6.
Peakꢀ areaꢀ ratioꢀ ofꢀ RS=RRꢀ orꢀ SS=SRꢀ isomers
Enantiomerꢀ ratioꢀ ofꢀ RS=RRꢀ orꢀ SS=SRꢀ isomersð%Þ ¼
ꢀ 100
ð6Þ
Peakꢀ areaꢀ ratioꢀ ofðRS=RRꢀ isomers þ SS=SRꢀ isomersÞ
The plasma concentrations of R-106583 enantiomers (SS/SR isomers and
RS/RR isomers) were calculated by eq. 7.
Plasmaꢀ concentrationsꢀ ofꢀ R-106583ꢀ enantiomers
Plasmaꢀ concentrationsꢀ ofꢀ R-10683ðng=mLÞ ꢀ enantiomerꢀ ratioꢀ RS=RRꢀ orꢀ SS=SRꢀ isomersð%Þ
ðRS=RRꢀ orꢀ SS=SRꢀ isomersÞðng=nLÞ ¼
100
ð7Þ
The pharmacokinetic parameters of R-106583 enantiomers were calculated pharmacologically active metabolite of prasugrel in the liver micro-
using the computer program WinNonlin Professional (version 4.0.1; Pharsight
Corporation, Mountain View, CA) based on the noncompartmental model.
somes and cytosol.
Stereoselectivity and Species Difference in S-Methylation of the
Pharmacologically Active Metabolite of Prasugrel In Vitro. The
activity for S-methylation of SS, SR, RR, and RS isomers of the
pharmacologically active metabolite of prasugrel was determined
Results
S-Methylation of the Pharmacologically Active Metabolites of separately using rat, dog, and human liver microsomes. The SS and
Prasugrel, R-138727, in Human Liver Microsomes and Cytosol. SR forms were S-methylated in rat, dog, and human liver microsomes
The S-methylation of the pharmacologically active metabolite of at varying activities, with the rat liver microsomes showing the highest
prasugrel was measured in human liver microsomes and cytosol. activity, followed by the human and dog liver microsomes in that
R-138727, the mixture of four stereoisomers of the pharmacologically order (Fig. 5). Interestingly, neither the RS form nor the RR form were
active metabolites, was used as the substrate, and the production of the S-methylated in the liver microsomes of any of the three species.
total amount of S-methylated isomers (R-106583) was determined by
Enzyme Kinetics in S-Methylation of Each Enantiomer of the
UPLC. As shown in Fig. 3, the S-methylating activity was 56.7 6 5.0 Pharmacologically Active Metabolite of Prasugrel. We determined
pmol/min/mg protein (mean 6 S.D., n = 3) in the microsomes and the enzyme kinetic parameters for the S-methylation of the SS and SR
11.2 6 1.2 pmol/min/mg protein (mean 6 S.D., n = 3) in the cytosol, forms of the pharmacologically active metabolite of prasugrel in rat,
indicating that the activity in human liver microsomes was higher than dog, and human liver microsomes as shown in Table 1. In the case of
the activity in human liver cytosol.
the SS form, the Km values for rat, dog, and human liver microsomes
Inhibition of S-Methylation of the Pharmacologically Active were 32.58, 155.24, and 26.29 mM, respectively, and the Vmax values
Metabolite of Prasugrel. Two S-methylating enzymes are known were 734.80, 722.79, and 362.01 pmol/min/mg protein, respectively.
TMT in microsomes and TPMT in cytosols (Lee and Kim, 1999). In the case of the SR form, the Km values for rat, dog, and human liver
Regarding TMT, low activity was observed in the cytosolic fraction microsomes were 79.34, 208.04, and 17.26 mM, respectively, and the
also, which was likely due to a soluble isoform of TMT as reported Vmax values were 1665.74, 205.30, and 243.50 pmol/min/mg protein,
previously because DCMB inhibited the cytosolic S-methylating respectively.
activity (Glauser et al., 1992). The inhibitory effects of DCMB, the
The CLint values (ml/min/kg) for S-methylation of the SS and SR
TMT inhibitor, on the S-methylating activity for the pharmacologi- forms of the pharmacologically active metabolites of prasugrel in rat,
cally active metabolite of prasugrel in the human liver microsomes dog, and human in vivo were calculated based on the in vitro data as
were determined as shown in Fig. 4. The addition of DCMB at shown in Table 1, and were 40.41, 11.62, and 17.28 ml/min/kg,
a concentration of 50 mM or 500 mM completely inhibited the respectively, for the SS form, and 37.61, 2.47, and 17.71 ml/min/kg,
production of the S-methyl metabolite in the liver microsomes. respectively, for the SR form. Therefore, the data indicate that rats
Additionally, DCMB at a concentration of 5, 50, or 500 mM show the highest CLint in S-methylation of both enantiomers, followed
completely inhibited the production of the S-methyl metabolite in the by humans and dogs in that order.
liver cytosol. On the other hand, m-anisic acid at a concentration of
Plasma Concentrations of the S-Methylated Metabolites after
0.1, 1, or 10 mM did not show any inhibitory effects on the Intravenous Administration of the Pharmacologically Active
production of the S-methyl metabolite in the liver cytosol (Fig. 4). Metabolite to Rats and Dogs. R-138727, the mixture of four
These data indicate that TMT catalyzes the S-methylation of the isomers of the pharmacologically active metabolite of prasugrel was

Stereoselectivity of Thiol S-Methyltransferase
1143
Fig. 5. S-methylation of the single enantiomers of the pharmacologically active
metabolite of prasugrel in rat, dog, and human liver microsomes. The rat, dog, and
human liver microsomes were incubated with 0.5 mM SAM and 100 mM the single
enantiomer of the pharmacologically active metabolites of prasugrel at 37°C for 30
minutes. Data are expressed as the mean of duplicate experiments.
Fig. 3. S-methylation of the pharmacologically active metabolite of prasugrel in
human liver microsomes and cytosol. The human liver microsomes or cytosol was
incubated with 0.5 mM SAM and 50 mM of the pharmacologically active metabolite
of prasugrel at 37°C for 30 minutes or 45 minutes. Data are expressed as mean 6
S.D. of three experiments.
difficult to separate from each other using an ODS-column and
a chiral column in tandem. The in vitro data demonstrated that the
steric configuration at the 4-position of the piperidyl group was
the crucial position for the S-methylation reaction (Fig. 5); therefore,
the measurement of the RS and RR isomers together or the SS and SR
isomers together was appropriate. Based on the plasma concentrations
of the RS and RR isomers and the SS and SR isomers of S-methylated
metabolite in rats and dogs, the pharmacokinetic parameters of S-methylated
metabolites were calculated (Table 2).
administered intravenously at a dose of 1 mg/kg to rats and dogs.
The plasma concentrations of the S-methylated metabolites were
measured as a mixture of the RS and RR isomers or as a mixture of
SS and SR isomers by LC-MS/MS as shown in Fig. 6 (rat) and Fig. 7
(dog). We measured the combined concentration of the RS and RR
isomers and the SS and SR isomers because the pair of the RS and
RR isomers and the pair of SR and SS isomers were technically
As shown in Fig. 6 and Fig. 7, the amount of the SS and SR isomers
of the S-methylated metabolite was greater than that of the RS and RR
isomers of the S-methylated metabolite in both animal species. The
maximum plasma concentration (Cmax) and area under the plasma
concentration-time curve extrapolated to infinity (AUC0-inf) for the SS
and SR isomers of S-methylated metabolites in rats were 614 6 124
ng/ml and 421 6 95.4 ng·h/ml, and the AUC0–inf for the SS and SR
isomers of S-methylated metabolites was about 8.5 times higher than
that for the RS and RR isomers of S-methylated metabolite (Table 2).
The Cmax and AUC0–inf for the SS and SR isomers of S-methylated
metabolites in dogs were 156 6 21.2 ng/ml and 134 6 42.6 ng·h/ml,
and the AUC0–inf for the SS and SR isomers of S-methylated
metabolite was about 76.6 times higher than that for the RS and RR
isomers of S-methylated metabolite (Table 2). In comparison with the
AUC0–inf for the SS and SR isomers in rats and dogs, the AUC0–inf of
rats was about 3 times greater than that of dogs (Table 2).
Discussion
Previous clinical studies demonstrated that the S-methylated me-
tabolite of the pharmacologically active metabolite of prasugrel is the
main metabolite identified in human plasma after oral administration
of prasugrel (Asai et al., 2006; Farid et al., 2007a,b), indicating that
the S-methylation is a major elimination pathway for the pharmaco-
logically active metabolite. TMT and TPMT have been reported as the
enzymes responsible for the S-methylation of xenobiotics (Glauser
et al., 1992). In the present study, we identified the enzyme involved
in the S-methylation of the pharmacologically active metabolites of
prasugrel as TMT based on the localization of the activity being
predominantly in the microsomal fraction and sensitive to the TMT
inhibitor DCMB. Although the liver cytosol showed about one-fifth of
the S-methylating activity in the microsomes, the cytosolic activity
Fig. 4. Inhibitory effects of DCMB and m-anisic acid on S-methylation of the
pharmacologically active metabolite of prasugrel in human liver microsomes and
cytosol. (A) S-methyl metabolite formation activity from the pharmacologically
active metabolite with or without DCMB in pooled human liver microsomes. (B)
S-methyl metabolite formation activity from the pharmacologically active metabolite
with or without DCMB and m-anisic acid in pooled human liver cytosol. Data are
expressed as mean 6 S.D. of three experiments.

1144
Kazui et al.
TABLE 1
Apparent enzyme kinetic parameters and estimated CLint values in S-methylation of SS and SR forms of the
pharmacologically active metabolite of prasugrel
The assays were performed by using rat, dog, and human liver microsomes. Data are expressed as mean of duplicate experiments.
CLint
and CLint values were scaled to eq. 4 and eq. 5.
in vitro
Pharmacologically Active
Metabolite Substrate
Km
Vmax
CLint
CLint
in vitro
mM
pmol/min/mg protein
ml/min/mg protein
ml/min/kg b.wt.
SS form
Rat
Dog
Human
SR form
Rat
32.58
155.24
26.29
734.80
722.79
362.01
22.55
4.66
13.77
40.41
11.62
17.28
79.34
208.04
17.26
1665.74
205.30
243.50
20.99
0.99
14.11
37.61
2.47
17.71
Dog
Human
seems due to the cytosolic isoform of TMT (Glauser et al., 1992) as important than the configuration of the 19-position of the benzyl group.
DCMB inhibited both the microsomal and cytosolic S-methylating The configuration of the chiral carbon adjacent to the thiol group seems to
activities. Additionally, m-anisic acid had little effect on the cytosolic be the key for substrate recognition, but both of the two chiral carbon
S-methylating activities. Taken together, the S-methylation of the configurations appear to be important for platelet inhibition activity.
pharmacologically active metabolite was not catalyzed by TPMT, which
was the enzyme showing a genetic polymorphism, but by TMT which metabolites in rats and dogs after intravenous administration of the
was the enzyme showing no genetic polymorphism. pharmacologically active metabolite, and the results demonstrated that
We also measured the plasma concentrations of the S-methylated
The RS and RR isomers of the pharmacologically active metabolite are the S-methylated metabolite in vivo was almost exclusively the SS and
more active in inhibiting platelet aggregation than the SS and SR isomers SR isomers. Additionally, as shown in Table 1, the combined CLint
of the active metabolite (Hasegawa et al., 2005). Interestingly, the SR and value obtained in vitro for the SS and SR isomers in rats (78.0 ml/min/
SS isomers of the active metabolite were found to be extensively kg) was about 5.5 times greater than that in dogs (14.1 ml/min/kg),
methylated in vitro whereas the RS and RR isomers of the active and this difference approximated the 3.1-fold greater AUC0-inf in rats
metabolite were not, demonstrating that TMT catalyzes the S-methylation over that in dogs (Table 2). Although in vitro the RS and RR isomers
reaction in a stereoselective manner (Fig. 5). It was reported that were not S-methylated, the S-methylated metabolite was detected in
2-mercaptopyrazine, diethyldithiocarbamate, and dihydroziprasidone, which both rat and dog plasma at much lower amounts. At this time, it is
is a metabolite of ziprasidone (an antipsychotic drug), were metabolized by unclear why the RS and RR isomers of the S-methylated metabolites
TMT; however, stereoselectivity of TMT in liver microsomes (Glauser were detected in both rat and dog plasma after intravenous
et al., 1993; Lee and Kim, 1999; Obach et al., 2012) was not reported.
administration of the pharmacologically active metabolite. Collec-
This study is the first report describing stereoselectivity in S-methylation tively, findings in vitro of stereoselective S-methylation were reflected
catalyzed by TMT. Specifically, only the SS form and SR form were in the observations in vivo in both rats and dogs.
S-methylated in the liver microsomes of all three species, demonstrating
Recently, the pharmacokinetics of enantiomers of clopidogrel active
that the configuration of the 4-position of the piperidyl group is far more metabolites in patients with cardiovascular diseases were reported
Fig. 6. Plasma concentrations of the S-methylated metabolite (R-106583)
enantiomers after intravenous administration of the racemic mixture of the
pharmacologically active metabolite of prasugrel to rats at a dose of 1 mg/kg: RS/
RR isomers (d) and SS/SR isomers (s). Data are expressed as mean 6 S.D. of four
animals.
Fig. 7. Plasma concentrations of the S-methylated metabolite (R-106583)
enantiomers after intravenous administration of the racemic mixture of the
pharmacologically active metabolite of prasugrel to dogs at a dose of 1 mg/kg: RS/
RR isomers (d) and SS/SR isomers (s). Data are expressed as mean 6 S.D. of four
animals.

Stereoselectivity of Thiol S-Methyltransferase
1145
TABLE 2
Pharmacokinetic parameters of R-106583 enantiomers after intravenous administration of R-138727 to rats and dogs at
a dose of 1 mg/kg
The pharmacokinetic parameters of R-106583 enantiomers were calculated using the computer program WinNonlin Professional
(version 4.0.1, Pharsight Corporation) based on the noncompartmental model. Data are expressed as mean 6 S.D. of four animals.
Rat
Dog
Pharmacokinetic Parameters
RS+RR Isomers
SS+SR Isomers
RS+RR Isomers
SS+SR Isomers
t1/2 (h)
Cmax (ng/ml)
AUC0–1 h (ng·h/ml)
AUC0–inf (ng·h/ml)
0.446 6 0.239
59.7 6 2.15
36.0 6 3.81
49.3 6 18.1
0.167 6 0.0964
614 6 124
351 6 62.6
421 6 95.4
0.270 6 0.112
2.40 6 1.52
1.50 6 1.04
1.75 6 1.41
0.424 6 0.106
156 6 21.2
103 6 21.9
134 6 42.6
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In conclusion, our results indicate that TMT is involved in the
stereoselective S-methylation of the pharmacologically active
metabolite of prasugrel, and this stereoselectivity in S-methylation
will contribute to the higher plasma concentrations of RS and RR
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animals and humans.
Acknowledgments
The authors thank Dr. Mary Pat Knadler (Eli Lilly and Company) for her
helpful comments and discussion on this manuscript.
Authorship Contributions
Participated in research design: Kazui, Kurihara.
Conducted experiments: Kazui, Hagihara.
Performed data analysis: Kazui, Hagihara, Kurihara.
Wrote or contributed to the writing of the manuscript: Kazui, Izumi, Ikeda,
Kurihara.
Address correspondence to: Miho Kazui, Drug Metabolism & Pharmacokinetics
Research Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-
Ku, Tokyo, 140-8710, Japan. E-mail: kazui.miho.yk@daiichisankyo.co.jp