In vitro drug metabolism by human carboxylesterase 1: Focus on angiotensin-converting enzyme inhibitors

Article abstract of DOI:10.1124/dmd.113.053512

Carboxylesterase 1 (CES1) is the major hydrolase in human liver. The enzyme is involved in the metabolism of several important therapeutic agents, drugs of abuse, and endogenous compounds. However, no studies have described the role of human CES1 in the activation of two commonly prescribed angiotensinconverting enzyme inhibitors: enalapril and ramipril. Here, we studied recombinant human CES1-and CES2-mediated hydrolytic activation of the prodrug esters enalapril and ramipril, compared with the activation of the known substrate trandolapril. Enalapril, ramipril, and trandolapril were readily hydrolyzed by CES1, but not by CES2. Ramipril and trandolapril exhibited Michaelis-Menten kinetics, while enalapril demonstrated substrate inhibition kinetics. Intrinsic clearances were 1.061, 0.360, and 0.02 ml/min/mg protein for ramipril, trandolapril, and enalapril, respectively. Additionally, we screened a panel of therapeutic drugs and drugs of abuse to assess their inhibition of the hydrolysis of p-nitrophenyl acetate by recombinant CES1 and human liver microsomes. The screening assay confirmed several known inhibitors of CES1 and identified two previously unreported inhibitors: the dihydropyridine calcium antagonist, isradipine, and the immunosuppressive agent, tacrolimus. CES1 plays a role in the metabolism of several drugs used in the treatment of common conditions, including hypertension, congestive heart failure, and diabetes mellitus; thus, there is a potential for clinically relevant drug-drug interactions. The findings in the present study may contribute to the prediction of such interactions in humans, thus opening up possibilities for safer drug treatments. Copyright

Full text of DOI:10.1124/dmd.113.053512

1521-009X/42/1/126–133$25.00
http://dx.doi.org/10.1124/dmd.113.053512
DRUG METABOLISM AND DISPOSITION
Drug Metab Dispos 42:126–133, January 2014
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
In Vitro Drug Metabolism by Human Carboxylesterase 1: Focus on
Angiotensin-Converting Enzyme Inhibitors
Ragnar Thomsen, Henrik B. Rasmussen, Kristian Linnet, and The INDICES Consortium
Section of Forensic Chemistry, Department of Forensic Medicine, Faculty of Health Sciences, University of Copenhagen,
Copenhagen, Denmark (R.T., K.L.); and Institute of Biological Psychiatry, Mental Health Centre Sct. Hans, Copenhagen University
Hospital, Roskilde, Denmark (H.B.R.)
Received June 27, 2013; accepted October 18, 2013
ABSTRACT
Carboxylesterase 1 (CES1) is the major hydrolase in human liver.
The enzyme is involved in the metabolism of several important
therapeutic agents, drugs of abuse, and endogenous compounds.
However, no studies have described the role of human CES1
in the activation of two commonly prescribed angiotensin-
respectively. Additionally, we screened a panel of therapeutic
drugs and drugs of abuse to assess their inhibition of the
hydrolysis of p-nitrophenyl acetate by recombinant CES1 and
human liver microsomes. The screening assay confirmed
several known inhibitors of CES1 and identified two previously
converting enzyme inhibitors: enalapril and ramipril. Here, we unreported inhibitors: the dihydropyridine calcium antagonist,
studied recombinant human CES1- and CES2-mediated hydro- isradipine, and the immunosuppressive agent, tacrolimus. CES1
lytic activation of the prodrug esters enalapril and ramipril, plays a role in the metabolism of several drugs used in the
compared with the activation of the known substrate trandolapril.
treatment of common conditions, including hypertension, con-
Enalapril, ramipril, and trandolapril were readily hydrolyzed by gestive heart failure, and diabetes mellitus; thus, there is
a
CES1, but not by CES2. Ramipril and trandolapril exhibited
Michaelis-Menten kinetics, while enalapril demonstrated sub-
strate inhibition kinetics. Intrinsic clearances were 1.061, 0.360,
and 0.02 ml/min/mg protein for ramipril, trandolapril, and enalapril,
potential for clinically relevant drug-drug interactions. The findings
in the present study may contribute to the prediction of such
interactions in humans, thus opening up possibilities for safer drug
treatments.
Introduction
Several of the drugs prescribed for these diseases are substrates of
CES1. Likewise, drug addicts may abuse several drugs that can be
metabolized by CES1 concomitantly. These groups are particularly
prone to the potential for clinically relevant drug interactions that
involve CES1.
Carboxylesterase 1 (CES1) is the predominant hydrolase in the
human liver (Imai, 2006). The enzyme is involved in the metabolism
of several therapeutic agents, drugs of abuse, and endogenous com-
pounds. CES1 activates several ester-containing prodrugs, including
oseltamivir, dabigatran etexilate, mycophenolate mofetil, and a num-
ber of angiotensin-converting enzyme (ACE) inhibitors, including
trandolapril, imidapril, benazepril, and quinapril (Takai et al., 1997;
Shi et al., 2006; Zhu et al., 2009; Fujiyama et al., 2010; Hu et al.,
2013). The enzyme also inactivates a multitude of drugs, including
methylphenidate, pethidine, clopidogrel, rufinamide, and oxybutynin
(Zhang et al., 1999; Sun et al., 2004; Tang et al., 2006; Williams et al.,
2011; Sato et al., 2012).
ACE-inhibitors are commonly prescribed antihypertensive agents.
Of importance, clinical studies have failed to show consistent
antihypertensive action with these agents in individuals that received
kidney transplants (Hiremath et al., 2007; Cross et al., 2009). Because
these agents are activated by CES1, a possible mechanism for this lack
of effect could be the simultaneous administration of immunosup-
pressive drugs that inhibit CES1. A previous study demonstrated that
the immunosuppressive agent, mycophenolate mofetil, was a substrate
for CES1 (Fujiyama et al., 2010). Other immunosuppressive agents
have not been tested for their activity as esterase inhibitors. Although
several ACE inhibitors have been shown to be substrates of CES1, no
study has investigated the human enzyme responsible for the
activation of two of the most commonly prescribed compounds in
this family: enalapril (EPL) and ramipril (RPL). Thus, in the present
study, we explored the metabolism of these compounds by CES1 and
CES2, in comparison with the known CES1 substrate trandolapril
(TPL). Additionally, we screened a panel of cardiovascular, anti-
platelet, and anticoagulant drugs; drugs of abuse; and immunosup-
pressive agents for their inhibitory potential against CES1. We further
examined the inhibition kinetics of some identified inhibitors.
Polypharmacy is common in the treatment of hypertension, diabetes
mellitus, congestive heart failure, and other multifactor diseases.
This work is part of the INDICES research collaboration (Individualized drug
therapy based on pharmacogenomics: Focus on carboxylesterase 1, CES1) and
financed by the Danish Council for Strategic Research, Programme Commission
on Individuals, Disease, and Society [Grant 10-092792/DSF].
Primary laboratory of origin: Section of Forensic Chemistry, Department of
Forensic Medicine, Faculty of Health Sciences, University of Copenhagen,
Frederik V’s vej 11, DK-2100 Copenhagen, Denmark
See Appendix for a list of all partners in the INDICES Consortium.
dx.doi.org/10.1124/dmd.113.053512.
ABBREVIATIONS: ACE, angiotensin-converting enzyme; CES1, carboxylesterase 1; CES2, carboxylesterase 2; DMSO, dimethyl sulfoxide; EPL,
enalapril; EPLA, enalaprilat; HLM, human liver microsomes; PNPA, p-nitrophenyl acetate; RPL, ramipril; RPLA, ramiprilat; TPL, trandolapril; TPLA,
trandolaprilat.
126

In Vitro Metabolism of ACE-Inhibitors by Human CES1
127
Materials and Methods
a standard Michaelis-Menten equation or a substrate inhibition equation, where
a second substrate molecule was treated as an uncompetitive inhibitor (Cornish-
Bowden, 2012). The two fits were compared with an extra-sum-of-squares
F-test. The test compares the improvement of sum-of-squares with the more
complicated model (the substrate inhibition model) versus the loss of degrees of
freedom. The null-hypothesis was that the data were adequately described by
the standard Michaelis-Menten equation. P values , 0.05 were considered
significant. Nonlinear regression fitting and statistical analysis was performed
with Prism, version 6.02 (GraphPad Software, Inc., San Diego, CA). Intrinsic
clearance (CLint) was calculated by dividing Vmax by Km.
Screening Assay. We screened the inhibitory potential of a variety of
therapeutic drugs and drugs of abuse with the well-known esterase substrate,
p-nitrophenyl acetate (PNPA). The incubations were performed in 96-well,
pureGrade BRANDplates (BRAND, Wertheim, Germany) in 100 mM
phosphate buffer at 37°C in a final volume of 200 ml. Human CES1 and
HLM were used for the enzyme source. Dimethyl sulfoxide (DMSO) was used
for the dissolution of all compounds. The final concentration of DMSO was 2%
v/v in all incubations, including the controls. An initial study was performed to
analyze the effect of this concentration of DMSO on enzyme activity. The
substrate and inhibitor were premixed to allow simultaneous addition of both.
The final concentrations of substrate and inhibitor were 50 mM and 100 mM,
respectively; the final protein concentrations were 10 mg/ml. The hydrolytic
product, p-nitrophenol, was determined by measuring the absorbance at 405 nm
after 3 minutes with a Sunrise microplate reader (Tecan, Grödig, Austria). The
sampling time was tested to be in the linear range of the reaction. The assay was
performed in triplicate. Substrate in buffer without enzyme was included as
a negative control. The results were corrected for spontaneous hydrolysis by
subtracting the absorbance of the negative control and comparing with a
positive control containing enzyme, but no inhibitor.
Inhibition Kinetics and Pattern. The inhibition constant Ki was determined
for selected inhibitors of CES1, with TPL as substrate. The analytical method
for TPLA is described in the analytical methods section below. Substrate
concentrations were 0.1, 0.2, 0.5, and 1 mM. Five concentrations of inhibitors
were used, including zero. The final protein concentration was 50 mg/ml. Ki
constants were determined by fitting the data with a competitive, non-
competitive, uncompetitive, or mixed-mode inhibition model with Prism
software.
Analytical Methods. Formations of TPLA, RPLA, and EPLA were deter-
mined with liquid chromatography–tandem mass spectrometry. Chromatographic
separation was performed with an Agilent 1100 series high-performance liquid
chromatography system from Agilent Technologies (Santa Clara, CA). The
system consisted of a binary pump, a degasser, a column compartment with
thermostat, and a 1200 series autosampler. We used a Zorbax SB-C18 column
(3.5 mm, 2.1 Â 50 mm) for TPLA and ramiprilat (RPLA), and a Chromsep
SS100 Pursuit3 column (3 mm, 3 Â 100 mm) for EPLA (both from Agilent
Technologies). Isocratic chromatographic methods were developed to separate
the prodrugs from their respective metabolites. The mobile phase was 0.1%
formic acid and isopropanol (65:35, v/v) for TPLA, 0.1% formic acid and
isopropanol (72:25, v/v) for EPLA, and 10 mM ammonium formate and
methanol (40:60, v/v) for RPLA. Flow rates were 0.3 ml/min for TPLA,
0.5 ml/min for EPLA, and 0.2 ml/min for RPLA. A column temperature of
50°C was used for all methods. Injection volume was 5 ml for TPLA, 3 ml for
EPLA, and 10 ml for RPLA. Run times were 2 minutes for TPLA and
3 minutes for EPLA and RPLA.
Chemicals, Reagents, and Reference Compounds. p-Nitrophenyl acetate,
morphine, and fentanyl were from Sigma-Aldrich (St. Louis, MO); p-nitrophenol
was from Fluka (Buchs, Switzerland). Amphetamine, cocaine, methamphet-
amine, 3,4-methylenedioxy-N-methylamphetamine, methadone, 6-monoacetyl-
morphine, and heroin were from Lipomed AG (Arlesheim, Switzerland).
Carvedilol was from GlaxoSmithKline (London, United Kingdom). Dabiga-
tran, dabigatran etexilate, and trandolaprilat-d5 were from Toronto Research
Chemicals (Toronto, Canada). Metoprolol, pethidine, diazepam, and nitrazepam
were from Nycomed Denmark (Roskilde, Denmark). Spironolactone was from
Ercopharm A/S (Kvistgard, Denmark). Eplerenone, amlodipine, and sirolimus
were from Pfizer (New York City, NY). Verapamil was from GEA (Copenhagen,
Denmark). Diltiazem was from Norpharma (Hørsholm, Denmark). Furosemide
was from Hoechst AG (Frankfurt, Germany). Bendroflumethiazide was from
LEO Pharma (Ballerup, Denmark). Ketamine was from Parke-Davis (Ponty-
pool, United Kingdom). Clopidogrel, ramipril, ramiprilat, and irbesartan were
from Sanofi-Aventis (Paris, France). Zopiclone was from Actavis (Zug,
Switzerland). Simvastatin, bisoprolol, and enalapril were from Merck & Co.
(Whitehouse Station, NJ). Tacrolimus was from Fujisawa Pharmaceutical
(Osaka, Japan). Azathioprine and labetalol were from Glaxo Wellcome
(London, United Kingdom). Propranolol was from Zeneca (London, United
Kingdom). Atorvastatin was from Bie & Berntsen (Herlev, Denmark). Sotalol
was from Bristol-Myers Squibb (New York City, NY). Enalaprilat-d5 was from
CDN isotopes (Quebec, Canada). Trandolapril and trandolaprilat were from
Roussel (Paris, France). Pindolol was from DuraScan (Odense, Denmark).
Enalaprilat was from USP (Rockville, MD). Nimodipine was from Bayer
Denmark (Frederiksberg, Denmark). Isradipine was from Sandoz (Basel,
Switzerland). Lercanidipine was from Recordati (Milano, Italy). Valsartan was
from Novartis (Basel, Switzerland). Pooled human liver microsomes (HLMs),
recombinant human CES1 (CES1b/CES1A1), and CES2 (prepared from
baculovirus-infected High-Five insect cells) were obtained from BD Gentest
(Woburn, MA). Phosphate buffer (100 mM, pH 7.4) was produced from
potassium phosphate and dipotassium phosphate (both from Merck, Darmstadt,
Germany). Other solvents and chemicals were commercially available and of
liquid chromatography–mass spectrometry grade.
Detection of CES1 and CES2 Metabolism. The hydrolysis of TPL, RPL,
and EPL by recombinant human CES1 and CES2 was investigated. Substrate
(100 mM) was incubated for 20 minutes at 37°C with a final protein con-
centration of 100 mg/ml. Negative controls were included in which substrate
was incubated in buffer without enzyme. The hydrolytic products were
quantified as described in the analytical methods section below. Hydrolysis
rates were corrected for spontaneous hydrolysis (i.e., nonenzymatic hydrolysis)
by subtracting any product formed in the negative controls.
Kinetic Studies. Michaelis-Menten kinetic parameters were determined for
TPL, RPL, and EPL in recombinant human CES1. An initial study was
performed to establish time and protein linearity of the reactions by measuring
product formed at several time points (5, 10, 15, 20, and 30 minutes) using
three protein concentrations (50, 100, and 150 mg/ml). Final protein and
substrate concentrations, as well as incubation times, were selected such that
a quantifiable amount of hydrolysis product was formed and so that the
sampling was done in the linear range of the reactions. No more than 20% of
substrate was hydrolyzed at the time of sampling. Substrate concentrations
were 0.2–4 mM for TPL, 0.1–4 mM for RPL, and 0.05–4 mM for EPL. Final
recombinant CES1 protein concentrations were 50 mg/ml for TPL and RPL,
and 100 mg/ml for EPL. No organic solvent was present in the incubations. The
drug and enzyme combinations were incubated in 96-well, twin.tec polymerase
chain reaction plates (Eppendorf, Hamburg, Germany) in 100 mM phosphate
buffer at 37°C, in a final reaction volume of 50 ml. Control incubations with no
enzyme were performed for each compound. Reactions (8 minutes for RPL,
10 minutes for TPL, and 20 minutes for EPL) were terminated by transferring
20 ml to an equal volume of cold acetonitrile with 0.5% formic acid and the
corresponding internal standard—500 nM trandolaprilat (TPLA)-d5 for TPL or
RPL, and 1,000 nM enalaprilat (EPLA)-d5 for EPL. Next, 110 ml of mobile
phase was added, and the samples were centrifuged at 2,000g, 5°C, for
A Quattromicro triple quadrupole tandem mass spectrometer (Waters
Corporation, Milford, MA) was coupled to the high-performance liquid
chromatography system. Data were acquired in positive electrospray ionization
mode with Masslynx 4.1 software (Waters). Mass spectrometry conditions were
optimized by infusing a standard solution through a T-piece and engaging the
autotune function in the software. Multiple reaction monitoring was used for
data collection. Monitored mass transitions were m/z 403.2 . 170.1, m/z 389.3
. 206.2, and m/z 349.1 . 206.2 for TPLA, RPLA, and EPLA, respectively,
and m/z 408.2 . 170.1 and m/z 354.3 . 211.2 for TPLA-d5 and EPLA-d5,
respectively. Cone voltage and collision energy of 30 V and 20 eV,
10 minutes. The supernatant was analyzed by liquid chromatography–tandem respectively, were applied for all analytes except for EPLA-d5, where a cone
mass spectrometry as described in the analytical methods section below. The voltage of 25 V was applied. Other mass spectrometry parameters were
hydrolysis rate versus the substrate concentration was plotted and fit with
capillary voltage 1 kV, source temperature 120°C, desolvation temperature

128
Thomsen et al.
350°C, cone gas flow 30 L/h, and desolvation gas flow 800 L/h. Argon was
used as the collision gas.
The measuring range was 1–400 mM for all analytes. The methods were
validated for precision and accuracy by analyzing blank samples spiked at four
concentration levels; quadruplicate determinations were performed on three
different days. The precision (CV%) was ,7%, and the accuracy was 91% to
111% for all methods.
Results
Hydrolytic Activities of CES1 and CES2 for TPL, RPL, and
EPL. The hydrolytic activities of recombinant human CES1 and
CES2 were measured for TPL, RPL, and EPL. CES2 showed no
hydrolytic activity above the spontaneous hydrolysis toward any of the
three ACE inhibitors. In contrast, all three compounds were readily
hydrolyzed by CES1 (Fig. 1). The hydrolytic activity of CES1 was
roughly 10 times higher for RPL and TPL than for EPL.
Kinetics of CES1-Mediated Hydrolysis of EPL, TPL, and RPL.
We investigated the kinetics of recombinant CES1-mediated hydro-
lysis of EPL, TPL, and RPL. All three reactions were linear up to at
least 30 minutes at the protein concentrations used in the final assay.
Hydrolysis rates were plotted versus substrate concentrations (Fig. 2).
EPL displayed obvious substrate inhibition kinetics, illustrated by
a lack of linearity at low v/[S] on the Eadie-Hofstee plot (inset in Fig.
2C). For this compound, an extra-sum-of-squares F-test was used to
compare fitting to the standard Michaelis-Menten equation and the
substrate inhibition equation. The F-test revealed the inclusion of an
extra parameter (Ksi, the inhibition constant for a second substrate
molecule) to be justified (P value , 0.0001). For EPL, the standard
Michaelis-Menten equation was therefore rejected, and the substrate
inhibition equation applied. The kinetic parameters are summarized in
Table 1. Intrinsic clearance (CLint) was approximately 3-fold higher
for RPL than for TPL and roughly 18-fold higher for TPL than for
EPL.
Inhibitory Effects of Therapeutic Drugs and Drugs of Abuse on
the Hydrolase Activity of Recombinant Human CES1 and HLM.
We investigated a panel of therapeutic drugs and drugs of abuse by
measuring their inhibition of human CES1 and HLM hydrolysis of
Fig. 2. Determination of kinetic constants of the hydrolysis of three ACE inhibitors
by CES1. The hydrolysis of TPL (A), RPL (B), and EPL (C) were determined at
substrate concentrations of 50–4,000 mM. Km and Vmax constants were estimated by
nonlinear regression using Prism. Eadie-Hofstee plots are provided in the insets.
Data represent mean 6 S.E.M. (n = 4).
Fig. 1. Enzymatic activity of CES1 and CES2 on the hydrolysis of three ACE
inhibitors. Substrates EPL, TPL, and RPL (100 mM) were incubated with
recombinant human CES1 and CES2 at 37°C for 20 minutes. PNPA (100 mM)
was included as a positive control. The corresponding hydrolytic products were
determined and normalized by time and protein contents. Values represent mean
6 S.E.M. (n = 4).

In Vitro Metabolism of ACE-Inhibitors by Human CES1
129
TABLE 1
inhibition character. Higher substrate concentrations would likely
reveal a more pronounced substrate inhibition character for this com-
pound, which, unfortunately, was not possible due to limitations in
solubility. Of the three tested compounds, RPL was most efficiently
hydrolyzed by CES1, with a CLint of 1.061 ml/min/mg protein. The
catalytic efficiency of CES1 for EPL was much lower (18-fold and 53-
fold lower, respectively) than for TPL and RPL. Both RPL and TPL
are more lipophilic compounds than EPL, due to the additional cy-
clopentane and cyclohexane rings, respectively, fused to the
pyrrolidine ring. The increased lipophilic character of RPL and TPL
relative to EPL is likely to improve hydrophobic interactions with
CES1, which would lead to increased CES1 catalytic efficiency. The
fact that no CES2-mediated hydrolysis was detected for any of the
three compounds suggested that the primary site for activation of these
prodrugs is in the liver, rather than the intestinal wall, where CES2 is
most highly expressed (Schwer et al., 1997). This finding was
consistent with those of Pang et al., who found no significant EPLA
in portal venous plasma after delivery of EPL into the superior
mesenteric artery with an in situ perfused rat intestine-liver preparation
(Pang et al., 1985). Zhu and coworkers previously determined the
kinetic parameters of TPL hydrolase activity by recombinant CES1
and found a Km of 639.9 mM and a Vmax of 103.6 nmole/min/mg
protein (Zhu et al., 2009). These results are somewhat different from
the values determined in the present study. However, Zhu et al. did not
use the commercially available enzyme, but instead constructed and
purified their own recombinant version. Differences in construction
methods could lead to variations in post-translational modifications
and hence enzyme activity and affinity. Additionally, they used
a maximum substrate concentration of 2 mM, while a maximum of
4 mM was used in the present study. The higher maximal concen-
tration leads to more accurate determination of Vmax and hence Km.
Several known inhibitors were confirmed in the screening assay.
These included clopidogrel, diltiazem, and verapamil. Clopidogrel
is a prodrug that is oxidized to a 2-oxo intermediate, followed
by conversion to the pharmacologically active thiol metabolite.
Clopidogrel is a known substrate for CES1; in fact, CES1 is the
primary esterase that inactivates clopidogrel and its 2-oxo- and thiol-
metabolites (Bouman et al., 2011). This is consistent with our finding
that clopidogrel was also a potent inhibitor of CES1. Diltiazem and
verapamil were recently identified as inhibitors of human CES1 and
CES2 (Yanjiao et al., 2013). In addition, both drugs are known
inhibitors of CYP3A4 (Sutton et al., 1997; Wang et al., 2004). Thus,
the inhibition of CES1 and CES2 provides an additional mechanism
for drug-drug interactions caused by diltiazem and verapamil, and
it widens the range of potentially affected drugs. Carvedilol was
previously determined as an inhibitor of imidapril hydrolase activity in
human liver microsomes and cytosol (Takahashi et al., 2009). The
present study provides evidence that the effect on imidapril hydrolase
in these preparations is most likely caused by inhibition of CES1.
Carvedilol was the only b-blocking agent out of the seven tested that
demonstrated inhibition of CES1. Thus, inhibition of CES1 did not
Kinetic parameters for hydrolysis of three ACE inhibitors by recombinant
human CES1
Substrate
Km
Vmax
Ksi
CLint
mM
nmol/min/mg protein
mM
ml/min/mg protein
Trandolapril
Ramipril
Enalapril
1734
901
1721
624
956
34
—
—
0.360
1.061
0.020
2956
PNPA. The concentration of DMSO at 2% v/v was found to have only
a minor impact on PNPA hydrolysis in both recombinant CES1 (92 6
0.2% residual activity compared with a control without DMSO, mean
6 S.E.M., n = 3) and HLM (98 6 1.6%, mean 6 S.E.M., n = 3).
The results for 26 therapeutic drugs and dabigatran (the active
metabolite of the prodrug dabigatran etexilate) are shown in Fig. 3. In
general, the tested drugs showed comparable inhibition patterns for the
recombinant enzyme and HLM, although some compounds showed
less inhibition of HLM than of CES1.
No inhibition was detected for dabigatran etexilate and its active
metabolite. The two tested statin drugs, simvastatin and atorvastatin,
both inhibited the enzyme with 0.2% and 44.1% residual activity,
respectively, of the recombinant enzyme. The known substrate and
inhibitor of CES1, clopidogrel, showed potent inhibition of recombi-
nant CES1, with only 12.0% residual activity. The b-blocking agent,
carvedilol, suppressed all but 13.9% of recombinant CES1 activity;
the other b-blocking agents tested showed no major inhibition of
CES1. The two nondihydropyridine calcium channel blockers,
diltiazem and verapamil, demonstrated potent inhibition of recombi-
nant CES1, with 20.6% and 30.0% residual activity, respectively. All
four dihydropyridine calcium channel blockers tested inhibited the
enzyme with variable potency. Of these, isradipine and amlodipine
showed the most potent inhibition of recombinant CES1, with 17.6%
and 40.6% residual activity, respectively. The diuretic agents and
angiotensin II receptor antagonists tested did not exhibit any
noteworthy inhibition. Tacrolimus showed moderate inhibition with
28.4% remaining activity of recombinant CES1, while no major
inhibition was observed for sirolimus or azathioprine.
The results for 10 common drugs of abuse, 6-monoacetylmorphine,
two benzodiazepine drugs, and zopiclone are shown in Fig. 4. Of these
compounds, only cocaine showed potent inhibition, with 8.8% and
67.8% residual activity in the recombinant enzyme and HLM,
respectively.
Kinetics of Inhibition by Diltiazem and Verapamil on CES1
Hydrolase Activity. Both diltiazem and verapamil inhibited the
recombinant human CES1 hydrolysis of TPL (Fig. 5). The kinetics of
the inhibition was determined in two independent experiments and the
results summarized in Table 2.
Discussion
In this study, we identified two novel substrates of human CES1, appear to be a class effect for this family of drugs. Cocaine was
EPL, and RPL. These drugs are among the most commonly prescribed previously shown to inhibit heroin and 4-methylumbelliferyl acetate
drugs in the ACE-inhibitor family. Thus, they are included in the hydrolysis in enzyme purified from human liver (Kamendulis et al.,
group of drugs metabolized by CES1 that are typically prescribed for 1996; Brzezinski et al., 1997). Studies have shown conflicting data on
cardiovascular diseases, such as congestive heart failure and hyper- whether the drug was a substrate for CES1. Early studies, using CES1
tension, but also for diabetes mellitus. The kinetics of CES1 hydrolysis enzyme purified from human liver, determined that cocaine was
of RPL and TPL were fitted with the standard Michaelis-Menten a substrate (Dean et al., 1991; Brzezinski et al., 1994), but a later
equation, while EPL displayed obvious substrate inhibition kinetics, study, using recombinant CES1 enzyme, failed to detect any CES1-
and a modified equation that took into account the uncompetitive mediated hydrolysis of cocaine (Hatfield et al., 2010). The present
inhibition by a second substrate molecule was therefore employed study showed cocaine to be a potent inhibitor of recombinant CES1
for this compound. The kinetics of TPL also hinted at a substrate (8.8% residual activity); however, there was only a minor inhibition of

130
Thomsen et al.
Fig. 3. Inhibition of CES1 by therapeutic drugs. Inhibitory effects
of 26 cardiovascular, antiplatelet, anticoagulant, and immunosup-
pressant drugs and dabigatran on the hydrolysis of 50 mM PNPA in
recombinant human CES1 or HLM. Concentrations of tested
compounds were 100 mM. Values represent mean 6 S.E.M. (n = 3).
Control activity was 382 nmol/min/mg protein for CES1 and 838
nmol/min/mg protein for HLM.
HLM (67.8% residual activity). This discrepancy could be a result of a weaker inhibitor of CES1 (Yanjiao et al., 2013). Thus, it appears that
cocaine being metabolized by other esterases present in HLM. most calcium channel blockers of the dihydropyridine class inhibit
The four tested dihydropyridine calcium channel blockers all CES1 to varying degrees. Dihydropyridine calcium antagonists possess
inhibited CES1 to varying degrees, with isradipine and amlodipine two ester groups at positions 3 and 5 of the 1,4-dihydropyridine ring.
having the greatest inhibition potential. Isradipine is previously Most of these drugs are transformed in vivo, where the dihydropyridine
unreported as an inhibitor of human CES1. Yanjiao and coworkers ring is oxidized to pyridine, and one or both of the ester groups may be
identified nitrendipine and felodipine as strong, and amlodipine as cleaved (Weidolf et al., 1984; Stopher et al., 1988; Böcker et al., 1990).
Fig. 4. Inhibition of CES1 by common drugs of abuse and related
compounds. Inhibitory effects of common drugs of abuse, including
two benzodiazepines, zopiclone, pethidine, fentanyl, and 6-mono-
acetylmorphine (6MAM) on the hydrolysis of 50 mM PNPA in
recombinant human CES1 or HLM. Concentrations of tested
compounds were 100 mM. Values represent mean 6 S.E.M. (n = 3).
Control activity was 382 nmol/min/mg protein for CES1 and 838
nmol/min/mg protein for HLM. MDMA, 3,4-methylenedioxy-N-
methylamphetamine.

In Vitro Metabolism of ACE-Inhibitors by Human CES1
131
However, this finding needs to be further examined in clinical
studies.
Simvastatin was confirmed as a potent inhibitor of CES1. Addi-
tionally, atorvastatin moderately inhibited the enzyme. In the study by
Fukami et al., several statin drugs were tested as inhibitors of CES1
(Fukami et al., 2010). Their study found the lactone-containing statins,
simvastatin and lovastatin, to be more potent inhibitors of CES1 than
the statins existing as hydroxy acids. The results in the present study
confirm this observation, with simvastatin being a more potent inhib-
itor than atorvastatin; the latter compound not containing a lactone.
For some compounds there were discrepancies between the degree
of inhibition in recombinant CES1 and HLM, with some compounds
having a greater inhibition potential of the recombinant enzyme.
Cocaine, clopidogrel, diltiazem, and isradipine were among the com-
pounds that inhibited recombinant CES1 more potently than HLM.
There are several possible explanations for this finding, including
other PNPA-hydrolyzing enzymes found in the microsomal prepara-
tion, inhibitors being metabolized by other enzymes in HLM,
differences in protein binding and thus of free drug concentrations,
and possible differences in the structure of the recombinant form of the
enzyme and the natural enzyme found in HLM preparations, caused
by, for example, post-translational modifications.
Most of the identified inhibitors of CES1 are compounds containing
an ester or amide bond, and are thus possible substrates of the enzyme.
However, two of the identified inhibitors, carvedilol and verapamil, do
not contain any such bonds. There are no particularly reactive groups,
such as aldehydes, alkenes, or phenols, in the structure of these com-
pounds, so an irreversible inhibition mode is unlikely. Also, verapamil
was found to inhibit the TPL hydrolase activity of recombinant CES1
in a noncompetitive manner (Table 2). This means that the compound
binds equally well to the free enzyme and the enzyme-substrate
complex (Cornish-Bowden, 2012). It is thus likely that verapamil
binds to an allosteric site on the enzyme. However, discerning the
binding mechanism for these compounds is difficult without crystal
structures of CES1 cocrystallized with these compounds, but molecular
docking studies may help elucidate the binding region.
Fig. 5. Determination of inhibition constants for inhibition by diltiazem (A) and
verapamil (B) on the trandolapril hydrolase activities of recombinant CES1. Plotted
values represent mean 6 S.E.M. (n = 2).
Fukami and coworkers identified simvastatin as an inhibitor of
recombinant human CES1 hydrolysis of imidapril; they determined
a Ki of 0.11 mM (Fukami et al., 2010). The present study confirmed
the strong inhibition of CES1 by simvastatin. We also found that
diltiazem and verapamil (Ki 9.0 6 0.5 mM and 16.0 6 1.1 mM,
respectively) were weaker than simvastatin at inhibiting CES1 hydro-
lysis of TPL. Little or no clinical interactions have been demonstrated
The oxidation seems to be mostly catalyzed by the 3A4 isoform of the
cytochrome P450 enzymes (Katoh et al., 2000). It was also demon-
strated that CYP3A4 was capable of oxidative cleavage of the ester
bond in nifedipine (Funaki et al., 1989). To our knowledge, no study
has investigated a potential role of esterases in hydrolytic cleavage of
ester bonds at the 3 or 5 position in dihydropyridine calcium antag- between EPL/RPL and simvastatin (Shionoiri, 1993; Meyer et al.,
onists. Considering that several of the dihydropyridine calcium channel 1994); therefore, these drugs might also possess a low potential for
blockers are inhibitors of CES1, carboxylesterases may at least con-
tribute to the metabolism of these drugs.
One of the immunosuppressive agents, tacrolimus, moderately
inhibited CES1. Thus, this inhibition could play a role in the lack of
effect of ACE inhibitors seen in renal-transplant recipients who are
receiving concomitant immunosuppressive therapy with this agent.
affecting the pharmacokinetics of ACE inhibitors. However, with
the lengthening list of CES1 substrates and inhibitors, the possibility
of clinically significant interactions has increased for scenarios in
which several of these drugs are administered concomitantly. Addi-
tionally, genetic variations that cause reduced expression and/or
activity of CES1 may further increase this possibility. Polymor-
phisms in the gene or promoter region for CES1 have been shown
to cause significant changes in the pharmacokinetics of and/or the
clinical response to methylphenidate, oseltamivir, clopidogrel, and
imidapril (Geshi et al., 2005; Zhu et al., 2008; Tarkiainen et al.,
2012; Lewis et al., 2013). Individuals with CES1 polymorphisms
that produce a poor CES1 phenotype in relation to metabolism of
substrates, may be at increased risk of experiencing drug interactions
that involve CES1 substrates and inhibitors. Considering the wide
usage of many of these drugs, the potential for clinically relevant
drug interactions should not be ignored.
TABLE 2
Inhibition constants for inhibition of trandolapril hydrolase activity in recombinant
CES1 by diltiazem and verapamil
Inhibitor
Kia
Inhibition type
Diltiazem
Verapamil
9.0 6 0.5
16.0 6 1.1
Competitive
Noncompetitive
a
Mean 6 S.E.M. for two independent determinations.

132
Thomsen et al.
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Conducted experiments: Thomsen.
Performed data analysis: Thomsen.
Wrote or contributed to writing of the manuscript: Thomsen, Rasmussen,
Linnet.

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