Identification of carboxylesterase-dependent dabigatran etexilate hydrolysis

Article abstract of DOI:10.1124/dmd.113.054353

Dabigatran etexilate (DABE) is an oral prodrug that is rapidly converted to the active thrombin inhibitor, dabigatran (DAB), by serine esterases. The aims of the present study were to investigate the in vitro kinetics and pathway of DABE hydrolysis by human carboxylesterase enzymes, and the effect of alcohol on these transformations. The kinetics of DABE hydrolysis in two human recombinant carboxylesterase enzymes (CES1 and CES2) and in human intestinal microsomes and human liver S9 fractions were determined. The effects of alcohol (a known CES1 inhibitor) on the formation of DABE metabolites in carboxylesterase enzymes and human liver S9 fractions were also examined. The inhibitory effect of bis(4-nitrophenyl) phosphate on the carboxylesterase-mediated metabolism of DABE and the effect of alcohol on the hydrolysis of a classic carboxylesterase substrate (cocaine) were studied to validate the in vitro model. The ethyl ester of DABE was hydrolyzed exclusively by CES1 to M1 (K_m 24.9 6 2.9 μM, V_max 676 6 26 pmol/min per milligram protein) and the carbamate ester of DABE was exclusively hydrolyzed by CES2 to M2 (K_m 5.5 6 0.8 μM; V_max 71.1 6 2.4 pmol/min per milligram protein). Sequential hydrolysis of DABE in human intestinal microsomes followed by hydrolysis in human liver S9 fractions resulted in complete conversion to DAB. These results suggest that after oral administration of DABE to humans, DABE is hydrolyzed by intestinal CES2 to the intermediate M2 metabolite followed by hydrolysis of M2 to DAB in the liver by CES1. Carboxylesterase-mediated hydrolysis of DABE was not inhibited by alcohol. Copyright

Full text of DOI:10.1124/dmd.113.054353

Supplemental Material can be found at:
http://dmd.aspetjournals.org/content/suppl/2013/11/08/dmd.113.054353.DC1.html
1
521-009X/42/2/201–206$25.00
http://dx.doi.org/10.1124/dmd.113.054353
DRUG
M
ETABOLISM AND
DISPOSITION
Drug Metab Dispos 42:201–206, February 2014
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
Identification of Carboxylesterase-Dependent Dabigatran
s
Etexilate Hydrolysis
S. Casey Laizure, Robert B. Parker, Vanessa L. Herring, and Zhe-Yi Hu
Department of Clinical Pharmacy, College of Pharmacy, University of Tennessee Health Science Center, Memphis, Tennessee
Received August 20, 2013; accepted November 8, 2013
ABSTRACT
Dabigatran etexilate (DABE) is an oral prodrug that is rapidly a classic carboxylesterase substrate (cocaine) were studied to
converted to the active thrombin inhibitor, dabigatran (DAB), by
serine esterases. The aims of the present study were to investigate exclusively by CES1 to M1 (K
the in vitro kinetics and pathway of DABE hydrolysis by human per milligram protein) and the carbamate ester of DABE was
carboxylesterase enzymes, and the effect of alcohol on these exclusively hydrolyzed by CES2 to M2 (K 5.5 6 0.8 mM; Vmax 71.1 6
transformations. The kinetics of DABE hydrolysis in two human 2.4 pmol/min per milligram protein). Sequential hydrolysis of DABE
validate the in vitro model. The ethyl ester of DABE was hydrolyzed
m
24.9 6 2.9 mM, Vmax 676 6 26 pmol/min
m
recombinant carboxylesterase enzymes (CES1 and CES2) and in
human intestinal microsomes and human liver S9 fractions were
determined. The effects of alcohol (a known CES1 inhibitor) on the
formation of DABE metabolites in carboxylesterase enzymes and
human liver S9 fractions were also examined. The inhibitory effect
of bis(4-nitrophenyl) phosphate on the carboxylesterase-mediated
metabolism of DABE and the effect of alcohol on the hydrolysis of
in human intestinal microsomes followed by hydrolysis in human
liver S9 fractions resulted in complete conversion to DAB. These
results suggest that after oral administration of DABE to humans,
DABE is hydrolyzed by intestinal CES2 to the intermediate M2
metabolite followed by hydrolysis of M2 to DAB in the liver by CES1.
Carboxylesterase-mediated hydrolysis of DABE was not inhibited
by alcohol.
Introduction
formation of the DAB active metabolite (Satoh et al., 2002; Imai et al.,
2
006). Furthermore, based on the known substrate specificity of these
The direct thrombin inhibitor dabigatran etexilate (DABE) is a new
oral anticoagulant approved in the United States to prevent stroke and
systemic embolism in patients with nonvalvular atrial fibrillation (Connolly
et al., 2009). Dabigatran (DAB), the active moiety, is not orally bioavailable,
so it is administered as a double prodrug (DABE) to improve absorption.
The DABE prodrug is quickly converted to the active compound because
DAB plasma concentrations peak rapidly after an oral dose and the two
primary intermediate metabolites, as well as the DABE plasma concen-
trations, are very low (Stangier et al., 2007, 2008). This finding indicates
that DABE undergoes a high first-pass metabolism prior to reaching the
systemic circulation.
enzymes, it would be predicted that CES1 hydrolyzes the DABE ethyl
ester, whereas CES2 would metabolize the carbamate ester (Imai, 2006;
Hu et al., 2013).
However, the specific hydrolytic pathways and the relative contribu-
tions of CES1 and CES2 to the formation of the DAB active metabolite
have not been studied. Identification of the specific in vivo metabolic
pathway is essential for understanding potential factors affecting the
safety and efficacy of DABE, given the concentration-dependent
anticoagulant effect of the DAB active metabolite and the correlation
between DAB plasma concentrations and risk of stroke and bleeding
(Stangier et al., 2007, 2008; Harper et al., 2012, Reilly et al., 2013). A
The conversion of DABE to DAB is a two-step process involving
hydrolysis of an ethyl ester and a carbamate ester producing the active
moiety (Blech et al., 2008). Experiments conducted in vitro using human
growing body of evidence suggests that a number of factors can affect
the catalytic activity of CES1 and CES2 and result in changes in drug
liver microsomes confirm that enzymatic hydrolysis by serine hydro- disposition (Laizure et al., 2013). One such factor is drug interactions
lases is the primary pathway for the formation of DAB from DABE and that inhibit carboxylesterase function (Parker and Laizure, 2010; Zhu
that the cytochrome P450 system plays no significant role in forming et al., 2010; Rhoades et al., 2012). It is well established that alcohol is an
the active metabolite (Blech et al., 2008). Although the specific inhibitor of carboxylesterase-mediated cocaine hydrolysis (Farre et al.,
esterases involved in DABE metabolism have not been identified, the 1997; Cami et al., 1998; Song et al., 1999; Laizure et al., 2003; Parker
structure of DABE would suggest that human carboxylesterase-1 (CES1) and Laizure, 2010). Whether this effect of alcohol is specific for cocaine
and human carboxylesterase-2 (CES2) are likely to play a role in the or is more broadly applicable to other CES1 substrates is uncertain.
However, recent work showing that alcohol inhibits the hydrolysis of
the CES1 substrate drug methylphenidate in humans suggests that the
This research was supported by the National Institutes of Health National
hydrolysis of other CES1 substrate drugs might also be inhibited (Patrick
et al., 2007; Bell et al., 2011). Given the large number of individuals
who consume alcohol and its known effects on CES1 hydrolysis,
Institute of General Medical Sciences [Grant R15-GM096074].
dx.doi.org/10.1124/dmd.113.054353.
s This article has supplemental material available at dmd.aspetjournals.org.
ABBREVIATIONS: BE, benzoylecgonine; BNPP, bis(4-nitrophenyl) phosphate; CE, cocaethylene; CES1, human carboxylesterase 1; CES2, human
carboxylesterase 2; CLint, in vitro intrinsic clearance; COC, cocaine; DAB, dabigatran; DAB-d3, dabigatran-d3; DABE, dabigatran etexilate; EME,
ecgonine methyl ester; HIM, human intestinal microsome; HLS9, human liver S9; HPLC, high-performance liquid chromatography; LC, liquid
chromatography; LC-MS/MS, liquid chromatography/triple quadrupole mass spectrometry.
201

2
02
Laizure et al.
understanding the effect of alcohol on carboxylesterase-mediated DABE similar to that of the metabolic stability studies except the final substrate
concentrations were 0.1, 0.2, 0.5, 1, 5, 10, 50, and 100 mM in the incubation
system. The final acetonitrile concentration was 1% for all assays. The optimal
incubation time was 5 minutes. The final protein concentration was 0.25 mg/ml
for recombinant CES1, CES2, HLS9, and HIM. All reactions were run in
triplicate.
In Vitro Inhibition. Procedures to determine the effect of alcohol on the
hydrolysis could have important implications for the safety and efficacy
of this agent.
Therefore, the objectives of this study are to characterize the human
carboxylesterase-mediated DABE metabolic pathway and to determine
the effect of alcohol on carboxylesterase-mediated hydrolysis of DABE.
carboxylesterase-mediated hydrolysis of DABE were similar to the enzyme
kinetics study. However, DABE was first mixed with alcohol at increasing
concentrations (0, 12.5, 25, 50, and 100 mM) in the incubation tubes and then
recombinant enzyme or HLS9 was added. The alcohol concentration was selected
based on the reported human exposure levels (Umulis et al., 2005). Inhibition was
Materials and Methods
DABE was purchased from TLC PharmaChem Inc. (Mississauga, ON,
Canada). DAB and dabigatran-d3 (DAB-d3) were the products of Toronto tested under two conditions to determine the effect of alcohol on the formation of
Research Chemicals Inc. (North York, ON, Canada). Bis(4-nitrophenyl)phosphate the intermediate M1 and M2 metabolites (condition A) and DAB (condition B).
(
BNPP), fluorescein diacetate, cocaine (COC), benzoylecgonine (BE), and ecgonine For condition A (low DABE depletion), the incubation time was 5 minutes and
methyl ester (EME) were purchased from Sigma-Aldrich (St. Louis, MO). Absolute the protein concentrations in the incubations were 0.01, 0.025, and 0.025 mg/ml
alcohol (200 proof) was from Decon Laboratories (King of Prussia, PA). High- for CES1, CES2, and HLS9, respectively. For condition B (high DABE
performance liquid chromatography (HPLC)–grade acetonitrile and methanol were
depletion), the incubation time was 10 and 5 minutes for the carboxylesterase
purchased from Fisher Scientific (Pittsburgh, PA). Liquid chromatography (LC)- enzymes and HLS9 fractions, respectively. The protein concentrations were 0.025
mass spectrometry–grade formic acid was purchased from Sigma-Aldrich (St. mg/ml for the carboxylesterase enzymes and 1.0 mg/ml for HLS9 fractions. The
Louis, MO). HPLC-grade water was prepared with an in-house Milli-Q Advantage inhibitory effect of BNPP on the carboxylesterase-mediated metabolism of
A10 Ultrapure water purification system (Bedford, MA). Recombinant human DABE was tested as a positive control. The concentrations of BNPP were 0, 1, 5,
carboxylesterase 1b (named as CES1 hereafter) and 2 (CES2) are from baculovirus 25, and 125 mM.
transfected insect cells (BD Supersomes); human liver S9 (HLS9) fractions (150
The effects of alcohol on cocaine hydrolysis were determined to validate the
pooled donors of mixed sex) and human intestinal microsomes (HIMs, 7 pooled in vitro model for alcohol-mediated inhibition of carboxylesterase activity.
donors of mixed sex) were obtained from BD Gentest (San Jose, CA). Human liver Cocaine metabolic stability when incubated with recombinant human CES1
cytosol has significant CES1 and CES2 activity; therefore, HLS9, which contains and CES2 was evaluated for determination of linear metabolite formation
both cytosolic and microsomal enzymes, was used for in vitro metabolism studies conditions with respect to the incubation time. Recombinant CES1 and CES2
instead of microsomes (Takahashi et al., 2009).
protein and cocaine concentrations in the incubation were 0.25 mg/ml and
10 mM, respectively. Incubation times were 0, 5, 15, 30, and 60 minutes. On
the basis of the metabolic stability results, a 60-minute incubation time was
used in the alcohol inhibition study. Final alcohol concentrations were 0, 12.5,
25, 50, 100, and 200 mM.
In Vitro Metabolic Stability. The metabolic stability of DABE in
incubations containing recombinant human CES1, CES2, CES1/CES2 mixture,
and HLS9 were performed. Assays were conducted in duplicate (triplicate for
HLS9) in 96-well cluster tubes with a total assay volume of 100 ml in each well
(
at 37°C). The assay buffer was 0.1 M potassium phosphate, pH 7.4. Incubation
LC-MS/MS Analyses. LC-MS/MS–based assays were used to measure the
substrates and their metabolites. The LC-MS/MS system consisted of a Shimadzu
times were 0, 5, 15, 30, and 60 minutes. Final protein concentrations were
0
.025, 0.025, 0.025/0.025, and 0.25 mg/ml for CES1, CES2, CES1/CES2 HPLC separation module (Milford, MA) and an AB Sciex 3000 triple quadrupole
mixture, and HLS9, respectively. Substrate concentrations in the incubations mass spectrometer (Toronto, Canada) with a turbo ion spray (electrospray
were 200 nM, which were similar to the human plasma concentrations of DAB
ionization) source. The LC separation for all of the analytes was achieved on
(
Stangier, 2008). The final acetonitrile concentration was not greater than 1% a 5.0-mm Agilent Eclipse Plus C18 column (50 mm  2.1 mm inside diameter;
for all assays. Assays were initiated by adding the substrate/buffer mix to the
Santa Clara, CA) at 24°C. Mobile phases were as follows: methanol/water, 1:99
enzyme/buffer mix. The metabolic depletion of fluorescein diacetate by each (v/v), containing 2.5 mM formic acid, for phase A; and methanol/water, 99:1
carboxylesterase enzyme was tested as a positive control to validate the enzyme (v/v), modified with the same electrolyte, for phase B. A fast gradient
activity (data not shown). Chemical stability of substrate in assay buffer was
examined as the negative control. The reactions were terminated by the addition
chromatographic method was used, which we have employed successfully for
the sensitive analysis of other drugs (Hu et al., 2011). This method is as
of an equal volume of ice-cold acetonitrile containing 200 nM internal standard follows: B 5% from 0.00 to 0.50 minutes, B 100% from 0.51 to 2.00 minutes,
DAB-d3). After centrifugation at 16,000g for 5 minutes, 10 ml supernatant was and B 5% from 2.01 to 4.50 minutes.
(
injected into the liquid chromatography/triple quadrupole mass spectrometry
The precursor-product ion pairs (singly protonated species) used for multiple
reaction monitoring of DABE, DAB, DAB-d3, COC, BE, EME, and CE were
(
LC-MS/MS) instrument.
The sequential metabolism of DABE in HIMs (step 1) and HLS9 fractions m/z 628.3→289.1, 472.2→289.1, 475.3→292.2, 304.3→182.1, 290.3→168.1,
step 2) was also conducted. The incubation volume was 100 ml for each well in 200.3→182.1, and 318.3→196.1, respectively. The LC eluent was introduced
(
the first step. Three independent incubations were prepared for 0-, 5-, 15-, 30-, to the electrospray ionization source at a flow rate of 0.40 ml/min over the
and 60-minute incubations (step 1). For the 60-minute incubations (step 1), 15
period of 0.3–2.2 minutes. One internal standard, DAB-d3, was used for
independent incubation samples were performed. The incubations in three of quantification of all of the analytes. Matrix-matched standard curves of the
these samples were terminated after 60 minutes. For the remaining 12 samples analyte/internal standard peak area ratio of a given analyte versus the nominal
(
5
step 2), an equal volume of HLS9 (100 ml) was added and further incubated for concentration in nanomoles were linear with correlation coefficients .0.99.
, 15, 30, or 60 minutes (three samples for each time point). The final protein The lower limit of quantification was 1.37 nM for all of the analytes except for
concentration was 0.25 mg/ml for HIMs in the first step, whereas the concentration EME, which was 12.3 nM. The within-run and between-run assay accuracies
was 0.50 mg/ml for HLS9 fractions in the second step. All of the reactions were ranged from 93% to 109% and from 95% to 108%, respectively, whereas the
terminated by the addition of an equal volume of ice-cold acetonitrile and then ranges of precision values for the assays were from 1.8% to 12.5% and from
analyzed by LC-MS/MS.
The stability of DABE (1000 nM) in human plasma was tested with and without
1.5% to 14.4%, respectively. The two intermediate metabolites (M1 and M2) in
the study samples were quantified by our recently developed assay (Hu et al.,
BNPP (400 mM), a known specific carboxylesterase inhibitor. The experimental 2013).
procedure was similar to that of the stability studies with human carboxylesterases
Data Analysis. Michaelis constant (K
except the final acetonitrile concentration was 1% for all assays. The reaction was values were determined by nonlinear regression analysis of rates of metabolite
m
) and maximum velocity (Vmax)
terminated by the addition of a 3-fold volume of ice-cold acetonitrile.
In Vitro Enzyme Kinetics. To determine enzyme kinetics (K
formation as a function of substrate concentration using GraphPad Prism software
(version 5.0; GraphPad Software Inc., San Diego, CA). In vitro intrinsic clearance
m
, Vmax, and
CLint), DABE was incubated with recombinant CES1, CES2, HLS9, and HIM
under linear metabolite formation conditions. The experimental procedure was
(CLint) was calculated from the ratio of Vmax to K
figures are the mean 6 standard deviation.
m
. All data presented in the

Dabigatran Etexilate Hydrolysis
203
Results
In Vitro Metabolic Stability. To identify the specific enzymes
responsible for DABE hydrolysis, separate incubations using recombi-
nant CES1 and CES2 were conducted. Incubations using a mixture of
recombinant CES1 and CES2 were also performed to assess the combined
effect of these enzymes. The results of these experiments are summarized
in Fig. 1 and show that CES1 converts DABE to the intermediate
metabolite M1, whereas CES2 mediates the formation of intermediate
metabolite M2. Furthermore, only a small quantity of the DAB active
metabolite is formed in individual CES1 or CES2 incubations (Fig. 1).
In contrast, the formation of DAB in incubations containing both CES1
and CES2 was approximately 4- and 12-fold higher compared with
CES1 or CES2 alone, respectively. The metabolic profile of DABE in
HLS9 fractions is shown in Fig. 2. Both M1 (major form) and M2
(minor form) were formed in HLS9 fractions. A moderate amount of
DAB was also formed (Fig. 2).
The sequential hydrolysis of DABE in HIMs and HLS9 fractions is
shown in Fig. 3. The metabolic depletion of DABE in HIMs showed
that M2 was the major metabolite and only a small quantity of DAB
was formed (Fig. 3A, step 1). After addition of HLS9 fractions, M2
was rapidly and completely hydrolyzed to DAB (Fig. 3B, step 2).
Fig. 2. In vitro hydrolysis of DABE in HLS9.
In Vitro Alcohol Inhibition. The effects of alcohol and BNPP on
The stability study of DABE in human plasma showed that less than the hydrolysis of DABE by recombinant CES1 and CES2 are shown
5% of DABE was converted to M1 after a 60-minute incubation (the in Fig. 4. Alcohol showed no significant inhibitory effect on the
2
amounts of M2 and DAB formed were very low; data shown in hydrolysis of DABE in CES1 or CES2. However, carboxylesterase-
Supplemental Figure 1). The addition of the carboxylesterase inhibitor mediated hydrolysis of DABE was almost completely inhibited by
BNPP did not affect this process, suggesting that the slow hydrolysis BNPP (Fig. 4). The effects of alcohol and BNPP on the hydrolysis of
of DABE in human plasma was spontaneous or mediated by other DABE in HLS9 fractions are shown in Fig. 5. The results are similar
enzymes.
In Vitro Enzyme Kinetics. The enzyme kinetics results are shown DAB can be quantified in both conditions A and B using HLS9.
in Table 1 and Supplemental Figure 2. The CLint values for the
Alcohol-mediated inhibition of cocaine metabolism was used to
to those observed with the recombinant carboxylesterase enzymes.
formation of M1 in CES1 and M2 in CES2 were 27.2 and 12.9 ml/min validate the in vitro carboxylesterase substrate–alcohol interaction model.
per milligram protein, respectively. In contrast, CLint values were Only CES1 converted cocaine to BE (Supplemental Figure 3A) and
#
0.3 ml/min per milligram protein for formation of M2 in CES1 and the addition of alcohol resulted in the formation of the transesterification
M1 in CES2. Although the Vmax for the formation of M1 by CES1 was product cocaethylene (data not shown). In contrast, both CES1 and
.5-fold higher than the formation of M2 by CES2, the K
for the CES2 enzymes catalyzed the hydrolysis of COC to EME (Supplemental
latter conversion was much lower (5.5 mM) than that of M1 formation Figure 3A). Alcohol significantly inhibited the hydrolysis of COC to BE
24.9 mM). The Km value for M1 formation in HLS9 fractions was and EME (Supplemental Figure 3B).
9
m
(
comparable with that in the recombinant CES1 preparation (33.5 mM
versus 24.9 mM). In addition, the kinetics for the formation of M2 was
Discussion
m
comparable between HIMs and recombinant CES2 (K 8.6 mM versus
The primary findings of this study are that CES1 catalyzes the
hydrolysis of DABE to M1 and CES2 catalyzes the hydrolysis of
DABE to M2. There was little cross-reactivity of carboxylesterase
hydrolysis, suggesting that both enzymes are required for the formation
of the active metabolite, DAB. We also found that alcohol did not
inhibit carboxylesterase-mediated DABE hydrolysis by CES1 or CES2.
The formation of DABE hydrolysis products shown in Table 1 and
Fig. 1 is consistent with the reported substrate specificities of CES1
and CES2 with CES1 hydrolyzing DABE to the M1 metabolite (ester
hydrolysis) and CES2 hydrolyzing DABE to the M2 metabolite (carbamate
hydrolysis) (Imai, 2006). Carboxylesterases are considered relatively
substrate nonspecific, so it is common for a hydrolysis site to be susceptible
to both CES1 and CES2 (although usually one predominates). However,
as Table 1 shows, recombinant CES1 and CES2 only produce M1 and
M2, respectively, and fail to produce the alternate metabolite. It is also
apparent from Table 1 that the inability of CES1 and CES2 to hydrolyze
the carbamate and ester groups, respectively, also applies to the M2 and
M1 metabolites because the individual incubations conducted with
recombinant CES1 and CES2 produce only small quantities of the DAB
active metabolite.
5
.5 mM). The CLint for M2 formation in HLS9 fractions was much
less than that for the formation of M1 in HLS9 fractions (2.0 ml/min
per milligram protein versus 35.0 ml/min per milligram protein).
Both M1 and M2 metabolites were formed in HLS9 fractions (Fig. 2).
The metabolite concentration pattern was as expected since CES1
Fig. 1. DABE (200 nM) metabolite formation in recombinant CES1, CES2, and
CES1/CES2 mixture (60-minute incubation).

2
04
Laizure et al.
Fig. 3. Sequential hydrolysis of DABE (200 nM) in
HIMs (A) (step 1) and HLS9 fractions (B) (step 2).
Because the incubations for step 2 (B) were diluted
after the addition of HLS9, the resulting concentration
of DABE and its metabolites in (B) are normalized to
2
00 nM.
accounts for 80%–95% of hydrolase activity in the liver (Imai et al., to M2 by intestinal CES2. The M2 metabolite is then subject to further
006). Thus, the high M1 concentrations are consistent with the high first-pass hydrolysis by hepatic CES1, resulting in formation of the DAB
2
levels of hepatic CES1 activity. Similarly, since M2 formation is active metabolite. This proposed metabolic pathway is supported by the
dependent on CES2 hydrolysis, the low M2 concentrations reflect the sequential hydrolysis of DABE by HIMs and HLS9 fractions shown in
low level of hepatic CES2 expression. The incubation of DABE in Fig. 3. In addition, this proposed metabolic pathway is consistent with the
HLS9 fractions also shows a low amount of DAB active metabolite anatomic locations of CES1 (liver) and CES2 (intestine) expression
formation. Compared with oral administration of DABE to humans, in (Satoh et al., 2002; Imai et al., 2006), the specific susceptibility of the
vitro incubations in HLS9 fractions cannot account for the significant ethyl and carbamate esters to hydrolysis by CES1 and CES2 (Table 1),
prehepatic formation of M2 from DABE by CES2 in the intestine and the disposition of DABE demonstrated in human studies (Blech et al.,
(Imai et al., 2006; Imai and Ohura, 2010). This intestinal M2 formation
is supported by sequential hydrolysis experiments showing formation of
the M2 intermediate metabolite in HIMs followed by the subsequent
complete conversion to DAB in HLS9 fractions (Fig. 3).
Collectively, these experiments demonstrate that hydrolysis of the
DABE double prodrug by both CES1 (ethyl ester) and CES2 (carbamate
ester) is required for formation of the DAB active moiety. When this
drug is given to humans, DAB is rapidly formed with peak concentrations
occurring within 2 hours after an oral dose, with negligible plasma
concentrations of M1, M2, and DABE (Blech et al., 2008; Stangier et al.,
2008). This indicates that the hydrolysis of both the ethyl and carbamate
ester sites undergo high first-pass metabolism prior to reaching the
systemic circulation. On the basis of these findings, a proposed metabolic
pathway for DABE after oral administration to humans is shown in Fig. 6.
Our data suggest that DABE undergoes extensive presystemic conversion
TABLE 1
Enzyme kinetic parameters for DABE hydrolysis in recombinant CES1, CES2,
pooled HLS9 fractions, and pooled HIMs
All reactions were run in triplicate and results are presented as mean 6 S.D.
Metabolic Reaction
K
m
V
max
CLint
mM
pmol/min per mg protein
ml/min per mg protein
CES1
DABE → M1 24.9 6 2.9
676 6 26
27.2
DABE → M2
CES2
N.D.
N.D.
N.D.
DABE → M1
DABE → M2
HLS9
DABE → M1 33.5 6 4.1
DABE → M2 15.4 6 1.9
HIM
DABE → M1
DABE → M2
N.D
5.5 6 0.8
N.D.
71.1 6 2.4
N.D.
12.9
Fig. 4. The effect of alcohol (left) and BNPP (right) on the carboxylesterase-
mediated metabolism of DABE in recombinant CES1 or CES2 enzymes. The
concentrations of the resulting metabolites without inhibitors were set as 100%.
Condition A (5-minute incubation, low DABE depletion) and condition B
1174 6 54
30.8 6 1.1
35.0
2.0
(
10-minute incubation, high DABE depletion) were used to test the effect of
N.D
8.6 6 0.9
N.D.
207.1 6 5.7
N.D.
24.1
alcohol on the formation of intermediate metabolites (M1 and M2) and the final
metabolite (DAB), respectively. The concentration of DAB in the incubation with
CES2 was too low to be detected under condition B.
N.D., kinetic parameters not determined due to the low concentration of metabolites detected.

Dabigatran Etexilate Hydrolysis
205
2008; Stangier et al., 2008). If there were large first-pass formation of the
M1 metabolite via CES1-mediated ester hydrolysis, then high plasma
concentrations of M1 would be expected, because this metabolite would
not be subsequently exposed to the high CES2 activity in the intestines. In
this case, DAB formation would depend on the relatively low levels of
hepatic CES2. Thus, as shown in Fig. 6, we propose that the conversion
of DABE to M1 to DAB represents a minor metabolic pathway with the
primary formation of the DAB active metabolite occurring via CES2-
mediated hydrolysis of DABE to M2 that is subsequently hydrolyzed by
hepatic CES1 to DAB.
Alcohol has been shown to inhibit cocaine hydrolysis catalyzed by
CES1 and CES2 (Roberts et al., 1993; Song et al., 1999) and the
hydrolysis of methylphenidate (Bourland et al., 1997) and clopidogrel
(Tang et al., 2006) (all by CES1) in microsomes or HLS9 fractions. In
human studies, cocaine and methylphenidate hydrolysis are signifi-
cantly inhibited by the consumption of alcohol and the transesterifica-
tion products cocaethylene and ethylphenidate are produced (Farre
et al., 1997; Cami et al., 1998; Patrick et al., 2007). These studies show
that the inhibition of carboxylesterase hydrolysis and the formation of
transesterified metabolites are not unique to the cocaine-alcohol in-
teraction and may occur with other CES1 substrates. However, unlike
cocaine, dabigatran hydrolysis was not affected by alcohol and no
transesterified product was formed (see Figs. 4 and 5). Although BNPP
significantly inhibited the hydrolysis of DABE by CES1 and CES2, even
alcohol concentrations up to 100 mM did not significantly affect the
hydrolysis of DABE by CES1 or CES2. Thus, although past studies
Fig. 5. The effect of alcohol (left) and BNPP (right) on the carboxylesterase- clearly demonstrate that the inhibition of CES1 by alcohol occurs with
mediated metabolism of DABE in HLS9. The concentrations of the resulting some CES1 substrates, the lack of effect on DABE hydrolysis indicates
metabolites without inhibitors were set as 100%. Conditions A (5-minute incubation,
that the inhibitory effects of alcohol on CES1 cannot be generalized to all
low DABE depletion) and condition B (5-minute incubation, high DABE depletion)
CES1 substrates. The potential mechanism(s) for the inability of alcohol
to inhibit DABE hydrolysis are unclear. Although speculative, the lack of
were used to test the effect of alcohol on the formation of intermediate metabolites
M1 and M2) and the final metabolite (DAB), respectively.
(
Fig. 6. Proposed in vivo metabolic pathway of orally administered DABE in humans. The thickness of each arrow indicates the relative contribution of each transformation
to the hydrolysis of DABE to DAB. Circles indicate the hydrolysis sites by CES1 (blue) and CES2 (green).

2
06
Laizure et al.
Farré M, de la Torre R, González ML, Terán MT, Roset PN, Menoyo E, and Camí J (1997)
Cocaine and alcohol interactions in humans: neuroendocrine effects and cocaethylene metab-
olism. J Pharmacol Exp Ther 283:164–176.
Harper P, Young L, and Merriman E (2012) Bleeding risk with dabigatran in the frail elderly.
N Engl J Med 366:864–866.
Hu Z, Sun Y, Du F, Niu W, Xu F, Huang Y, and Li C (2011) Accurate determination of the
anticancer prodrug simmitecan and its active metabolite chimmitecan in various plasma sam-
ples based on immediate deactivation of blood carboxylesterases. J Chromatogr A 1218:
formation of transesterified metabolites and the absence of alcohol-mediated
inhibition of DABE hydrolysis may be linked. Other CES1 substrate
drugs that are inhibited by alcohol also undergo transesterification,
including cocaine, methylphenidate, clopidogrel, and meperidine (Bourland
et al., 1997; Farre et al., 1997; Song et al., 1999; Tang et al., 2006; Patrick
et al., 2007). In addition, the molecular weight of DABE is 2- to 3-fold
higher than these other CES1 substrate drugs that are susceptible to
inhibition/transesterification with alcohol. This could potentially affect the
conformational orientation or access to the CES1 active site pocket (Imai
et al., 2006).
6
646–6653.
Hu ZY, Parker RB, Herring VL, and Laizure SC (2013) Conventional liquid chromatography/
triple quadrupole mass spectrometry based metabolite identification and semi-quantitative es-
timation approach in the investigation of in vitro dabigatran etexilate metabolism. Anal Bioanal
Chem 405:1695–1704.
Imai T (2006) Human carboxylesterase isozymes: catalytic properties and rational drug design.
Drug Metab Pharmacokinet 21:173–185.
DABE is a unique prodrug requiring hydrolysis at two sites to form
the active direct thrombin inhibitor. A recent analysis demonstrating
that both stroke and bleeding risk in patients with atrial fibrillation are
directly linked to DAB plasma concentrations suggests that understanding
DABE’s metabolic pathway and factors affecting it are crucial for
assessing benefits and risks of therapy (Reilly et al., 2013). We
attempt to address this issue in this report with our results showing that
both CES1 and CES2 are essential for DAB active metabolite
formation. Characterizing this drug’s metabolic pathway is a crucial
first step in identifying how factors affecting the activity of CES1 and
CES2 may have important effects on this drug’s disposition, and in
turn, efficacy and safety. The common assumption applied to DABE
and other carboxylesterase substrate drugs is that these agents are not
subject to significant variation in disposition. However, a growing
body of evidence indicates that these enzymes may be affected by
numerous factors including drug–drug interactions and genetic variability
in activity (Laizure et al., 2013). Further investigation is warranted to
understand the relationship between factors affecting DAB formation
and therapeutic response and toxicity.
Imai T, Taketani M, Shii M, Hosokawa M, and Chiba K (2006) Substrate specificity of car-
boxylesterase isozymes and their contribution to hydrolase activity in human liver and small
intestine. Drug Metab Dispos 34:1734–1741.
Imai T and Ohura K (2010) The role of intestinal carboxylesterase in the oral absorption of
prodrugs. Curr Drug Metab 11:793–805.
Laizure SC, Mandrell T, Gades NM, and Parker RB (2003) Cocaethylene metabolism and in-
teraction with cocaine and ethanol: role of carboxylesterases. Drug Metab Dispos 31:16–20.
Laizure SC, Herring V, Hu Z, Witbrodt K, and Parker RB (2013) The role of human carbox-
ylesterases in drug metabolism: have we overlooked their importance? Pharmacotherapy 33:
2
10–222.
Parker RB and Laizure SC (2010) The effect of ethanol on oral cocaine pharmacokinetics reveals
an unrecognized class of ethanol-mediated drug interactions. Drug Metab Dispos 38:317–322.
Patrick KS, Straughn AB, Minhinnett RR, Yeatts SD, Herrin AE, DeVane CL, Malcolm R, Janis
GC, and Markowitz JS (2007) Influence of ethanol and gender on methylphenidate pharma-
cokinetics and pharmacodynamics. Clin Pharmacol Ther 81:346–353.
Reilly PA, Lehr T, Haertter S, Connolly SJ, Yusuf S, Eikelboom JW, Ezekowitz MD, Nehmiz G,
Wang S, and Wallentin L (2013) The effect of dabigatran plasma concentrations and patient
characteristics on the frequency of ischemic stroke and major bleeding in atrial fibrillation
patients in the RE-LY Trial [published online ahead of print September 26, 2013]. J Am Coll
Cardiol doi: 10.1016/j.jacc.2013.07.104.
Rhoades JA, Peterson YK, Zhu HJ, Appel DI, Peloquin CA, and Markowitz JS (2012) Prediction
and in vitro evaluation of selected protease inhibitor antiviral drugs as inhibitors of carbox-
ylesterase 1: a potential source of drug-drug interactions. Pharm Res 29:972–982.
Roberts SM, Harbison RD, and James RC (1993) Inhibition by ethanol of the metabolism of
cocaine to benzoylecgonine and ecgonine methyl ester in mouse and human liver. Drug Metab
Dispos 21:537–541.
Satoh T, Taylor P, Bosron WF, Sanghani SP, Hosokawa M, and La Du BN (2002) Current
progress on esterases: from molecular structure to function. Drug Metab Dispos 30:488–493.
Song N, Parker RB, and Laizure SC (1999) Cocaethylene formation in rat, dog, and human
hepatic microsomes. Life Sci 64:2101–2108.
Authorship Contributions
Participated in research design: Laizure, Parker, Herring, Hu.
Conducted experiments: Hu.
Performed data analysis: Hu.
Wrote or contributed to the writing of the manuscript: Laizure, Parker,
Herring, Hu.
Stangier J (2008) Clinical pharmacokinetics and pharmacodynamics of the oral direct thrombin
inhibitor dabigatran etexilate. Clin Pharmacokinet 47:285–295.
Stangier J, Rathgen K, Stähle H, Gansser D, and Roth W (2007) The pharmacokinetics, phar-
macodynamics and tolerability of dabigatran etexilate, a new oral direct thrombin inhibitor, in
healthy male subjects. Br J Clin Pharmacol 64:292–303.
Stangier J, Stähle H, Rathgen K, Roth W, and Shakeri-Nejad K (2008) Pharmacokinetics and
pharmacodynamics of dabigatran etexilate, an oral direct thrombin inhibitor, are not affected by
moderate hepatic impairment. J Clin Pharmacol 48:1411–1419.
Takahashi S, Katoh M, Saitoh T, Nakajima M, and Yokoi T (2009) Different inhibitory effects in
rat and human carboxylesterases. Drug Metab Dispos 37:956–961.
References
Tang M, Mukundan M, Yang J, Charpentier N, LeCluyse EL, Black C, Yang D, Shi D, and Yan
B (2006) Antiplatelet agents aspirin and clopidogrel are hydrolyzed by distinct carbox-
ylesterases, and clopidogrel is transesterificated in the presence of ethyl alcohol. J Pharmacol
Exp Ther 319:1467–1476.
Umulis DM, Gürmen NM, Singh P, and Fogler HS (2005) A physiologically based model for
ethanol and acetaldehyde metabolism in human beings. Alcohol 35:3–12.
Zhu HJ, Appel DI, Peterson YK, Wang Z, and Markowitz JS (2010) Identification of selected
therapeutic agents as inhibitors of carboxylesterase 1: potential sources of metabolic drug
interactions. Toxicology 270:59–65.
Bell GH, Novak AJ, Griffin WC, 3rd, and Patrick KS (2011) Transdermal and oral dl-
methylphenidate-ethanol interactions in C57BL/6J mice: transesterification to ethylphenidate
and elevation of d-methylphenidate concentrations. J Pharm Sci 100:2966–2978.
Blech S, Ebner T, Ludwig-Schwellinger E, Stangier J, and Roth W (2008) The metabolism and
disposition of the oral direct thrombin inhibitor, dabigatran, in humans. Drug Metab Dispos 36:
3
86–399.
Bourland JA, Martin DK, and Mayersohn M (1997) Carboxylesterase-mediated transesterification
of meperidine (Demerol) and methylphenidate (Ritalin) in the presence of [2H6]ethanol: pre-
liminary in vitro findings using a rat liver preparation. J Pharm Sci 86:1494–1496.
Brzezinski MR, Spink BJ, Dean RA, Berkman CE, Cashman JR, and Bosron WF (1997) Human
liver carboxylesterase hCE-1: binding specificity for cocaine, heroin, and their metabolites and
analogs. Drug Metab Dispos 25:1089–1096.
Cami J, Farré M, González ML, Segura J, and de la Torre R (1998) Cocaine metabolism in humans
after use of alcohol. Clinical and research implications. Recent Dev Alcohol 14:437–455.
Connolly SJ, Ezekowitz MD, Yusuf S, Eikelboom J, Oldgren J, Parekh A, Pogue J, Reilly PA,
Themeles E, and Varrone J, et al.; RE-LY Steering Committee and Investigators (2009)
Dabigatran versus warfarin in patients with atrial fibrillation. N Engl J Med 361:1139–1151.
Address correspondence to: Dr. S. Casey Laizure, Department of Clinical
Pharmacy, College of Pharmacy, University of Tennessee, Room 358, 881
Madison Ave., Memphis, TN 38163. E-mail: claizure@uthsc.edu