Characterization of species differences in tissue diltiazem deacetylation identifies Ces2a as a rat-specific diltiazem deacetylase

Article abstract of DOI:10.1124/dmd.115.064089

Diltiazem, a calcium channel blocker, is mainly metabolized via demethylation or deacetylation in humans. Diltiazem demethylation is catalyzed by cytochrome P450 2D6 and 3A4. Although it was previously reported that the area under the curve ratio of deacetyldiltiazem to diltiazem after oral dosing with diltiazem in rats was sevenfold higher than in humans, the molecular mechanisms underlying this species difference remain to be clarified. In the present study, we compared the diltiazem deacetylase activity in liver, intestinal, renal, and pulmonary microsome preparations of human and experimental animal tissues to identify the specific deacetylase enzyme(s) involved in deacetylation. Diltiazem deacetylase activity was detected in rat liver and small intestine microsome preparations, but not in those from human, monkey, dog, and mouse tissues. Further purification of rat liver microsome (RLM) proteins identified four carboxylesterase (Ces) enzymes (Ces1d, Ces1e, Ces1f, and Ces2a) as potential candidate deacetylases. On the basis of their tissue distribution, the Ces2a enzyme was considered to be the enzyme that was responsible for diltiazem deacetylation. Furthermore, recombinant rat Ces2a expressed in Sf21 cells displayed efficient diltiazem deacetylase activity with similar Km values as RLM. In addition, the inhibitory characteristics of various chemical inhibitors were similar between recombinant rat Ces2a and RLM. In conclusion, we determined that only rat tissues were able to catalyze diltiazem deacetylation. The characterization of Ces enzymes in animal species, as undertaken in this study, will prove useful to predict the species-specific pharmacokinetics differences between the in vivo models used for drug development.

Full text of DOI:10.1124/dmd.115.064089

Supplemental material to this article can be found at:
http://dmd.aspetjournals.org/content/suppl/2015/05/15/dmd.115.064089.DC1.html
1
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521-009X/43/8/1218–1225$25.00
RUG METABOLISM AND DISPOSITION
http://dx.doi.org/10.1124/dmd.115.064089
Drug Metab Dispos 43:1218–1225, August 2015
Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics
Characterization of Species Differences in Tissue Diltiazem
Deacetylation Identifies Ces2a as a Rat-Specific
s
Diltiazem Deacetylase
Takaya Kurokawa, Tatsuki Fukami, and Miki Nakajima
Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan
Received February 26, 2015; accepted May 15, 2015
ABSTRACT
Diltiazem, a calcium channel blocker, is mainly metabolized via
demethylation or deacetylation in humans. Diltiazem demethylation (Ces1d, Ces1e, Ces1f, and Ces2a) as potential candidate deacetylases.
is catalyzed by cytochrome P450 2D6 and 3A4. Although it was On the basis of their tissue distribution, the Ces2a enzyme was
previously reported that the area under the curve ratio of considered to be the enzyme that was responsible for diltiazem
deacetyldiltiazem to diltiazem after oral dosing with diltiazem in deacetylation. Furthermore, recombinant rat Ces2a expressed in
rats was sevenfold higher than in humans, the molecular mecha- Sf21 cells displayed efficient diltiazem deacetylase activity with
nisms underlying this species difference remain to be clarified. In similar Km values as RLM. In addition, the inhibitory characteristics
the present study, we compared the diltiazem deacetylase activity of various chemical inhibitors were similar between recombinant
in liver, intestinal, renal, and pulmonary microsome preparations of rat Ces2a and RLM. In conclusion, we determined that only rat
(RLM) proteins identified four carboxylesterase (Ces) enzymes
human and experimental animal tissues to identify the specific tissues were able to catalyze diltiazem deacetylation. The charac-
deacetylase enzyme(s) involved in deacetylation. Diltiazem deace- terization of Ces enzymes in animal species, as undertaken in this
tylase activity was detected in rat liver and small intestine
study, will prove useful to predict the species-specific pharmaco-
microsome preparations, but not in those from human, monkey, kinetics differences between the in vivo models used for drug
dog, and mouse tissues. Further purification of rat liver microsome development.
Introduction
(Laizure et al., 2013) and lipid metabolism (Zhao et al., 2005). In
mammals, CES enzymes are divided into five families. The CES1 and
CES2 families are primarily involved in drug hydrolysis. In humans and
dogs, both the CES1 and CES2 families consist of a single isoform. In
the cynomolgus monkey, although the CES1 family consists of a single
isoform, the CES2 family appears to consist of two isoforms (Williams
et al., 2010, 2011). In contrast, a number of CES isoforms exist in
rodents. Eight Ces1 isoforms (Ces1a–Ces1h) and eight Ces2 isoforms
The calcium channel blocker diltiazem, which is widely used
for treating hypertension and angina pectoris, is metabolized via
N-demethylation, deacetylation, and O-demethylation (Fig. 1) (Yeung
et al., 1990; Molden et al., 2002). N-demethylation and deacetylation
are the major metabolic pathways, and the area under the curve (AUC)
ratio of O-demethyldiltiazem, deacetyldiltiazem, and N-demethyldiltiazem
reported in humans was 1:3.4:28, respectively (Molden et al., 2002).
N-Demethyldiltiazem and deacetyldiltiazem have coronary vasodila-
tion activity, with approximately one-fifth and one-half the potency
of diltiazem, respectively (Yabana et al., 1985). In humans, diltiazem
N- and O-demethylation are catalyzed by cytochrome P450 (CYP)
(
Ces2a–Ces2h) are found in mice. In rats, five Ces1 isoforms (Ces1a–
Ces1f) and seven Ces2 isoforms (Ces2a–Ces2j) exist (Holmes et al.,
010). According to a previous report (Imai, 2006), in primates, CES1
2
is mainly expressed in the liver and CES2 is highly expressed in the
small intestine, kidneys, and liver. In dogs, both CES1 and CES2 are
expressed in the liver, but neither is expressed in the small intestine. In
mice, most Ces1 isoforms are expressed in the liver, and only
a portion, such as Ces1e, Ces1f, and Ces1g, is expressed in the small
intestine, whereas Ces2 isoforms are mostly expressed in the small
intestine (Jones et al., 2013). The expression profile of Ces isoforms in
rats remains to be comprehensively determined.
3
A4 and 2D6, respectively, whereas the esterase(s) responsible for
diltiazem deacetylation has not been identified.
Esterases are expressed in various organs, including the liver, small
intestine, kidneys, and lungs, and contribute to the hydrolysis of
a number of clinically used drugs containing ester, amide, or thioester
bonds (Fukami and Yokoi, 2012). Carboxylesterases (CES) are well-
known to catalyze the hydrolysis of various drugs and xenobiotics
In humans, the substrate specificity of the CES1 and CES2 enzymes
can partly be explained by the relative size of the acyl and alcohol
groups found within the compound (Fukami and Yokoi, 2012). Indeed,
CES1 prefers compounds with a large acyl group and a small alcohol
group, whereas CES2 prefers compounds with a small acyl group
and a large alcohol group. Recently, Ozaki et al. (2013) measured
This work was supported in part by a Grant-in-Aid for Scientific Research (C)
from the Japan Society for the Promotion of Science [23590174].
dx.doi.org/10.1124/dmd.115.064089.
s This article has supplemental material available at dmd.aspetjournals.org.
ABBREVIATIONS: AADAC, arylacetamide deacetylase; AUC, area under the curve; BNPP, bis(4-nitrophenyl) phosphate; CES, carboxylesterase;
CYP, cytochrome P450; DFP, diisopropyl fluorophosphate; DMSO, dimethylsulfoxide; HPLC, high-performance liquid chromatography; NaF,
sodium fluoride; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl fluoride; RLM, rat liver microsome; RT-PCR, reverse transcription-
polymerase chain reaction; sRLM, solubilized RLM.
1218

Species Differences in Diltiazem Deacetylation
1219
Human Plasma. Human plasma samples (from five healthy Japanese
volunteers: 22- to 30-year-old males) were obtained according to our previous
report (Hioki et al., 2011). The use of human plasma samples was approved
by the Ethics Committees of Kanazawa University (Kanazawa, Japan; No.
2
16).
Diltiazem Deacetylase Activity. Diltiazem deacetylase activities were
determined as follows: a typical incubation mixture (final volume of 0.2 ml)
contained 100 mM potassium phosphate buffer (pH 7.4) and enzyme sources
(
0
tissue microsomes and rat liver cytosol, 0.2 mg/ml; solubilized RLM [sRLM],
.5 mg/mL; 50–70% ammonium sulfate precipitated fraction, 20 mL; DEAE
Sephacel fraction, 20 mL; Superdex 200 fraction, 10 mL; hydroxyapatite
chromatography fraction, 20 mL; Sf21 cell homogenates expressing esterases,
0
.1 mg/ml; plasma, 2.5 mL). In a preliminary study, we confirmed that the rates
of deacetyldiltiazem formation were linear with respect to the protein con-
centration (,1.4 mg/ml) and incubation time (,50 minutes). Diltiazem was
dissolved in distilled water. The reactions were initiated by the addition of
diltiazem (at a final concentration of 200 mM) after a 2-minute preincubation
period at 37ꢀC. After the 30-minute incubation, the reactions were terminated
by the addition of 200 ml ice-cold methanol. The supernatant obtained by
centrifugation at 12,000g for 5 minutes was diluted 10-fold with mobile
phase, and a 40-mL aliquot of the diluted supernatant was subjected to high-
performance liquid chromatography (HPLC). The HPLC analysis was per-
formed using an L-2130 pump (Hitachi, Tokyo, Japan), an L-2200 autosampler
Fig. 1. Diltiazem metabolic pathways in humans.
the hydrolase activity of several rat Ces enzymes toward paraben
derivatives and found that the substrate specificity of the Ces1 and
Ces2 families does not necessarily correspond with that in humans.
(
(
Hitachi), an L-2400 UV detector (Hitachi), and a D-2500 Chromato-Integrator
Hitachi) equipped with a Wakopak eco-ODS column (5 mm particle size,
4
.6 mm i.d. Â 150 mm; Wako Pure Chemical Industries). The eluent was
In addition, it has been shown that most substrates were hydrolyzed monitored at 237 nm with a noise-base clean Uni-3 (Union, Gunma, Japan),
by multiple isoforms in rats (Robbi and Beaufay, 1994; Sanghani which can reduce the noise by integrating the output and increasing the signal
et al., 2002). Because the substrate specificity of each Ces isoforms by threefold after differentiating the output and by an additional fivefold after
amplification with an internal amplifier, resulting in the maximal 15-fold am-
in rats remains to be characterized, it is very difficult to predict
plification of the signal. The mobile phase was composed of a 35% methanol/
the rat isoforms that are involved on the basis of their chemical
2
7% acetonitrile solution containing 5 mM ammonium acetate and 0.02%
structures.
triethylamine. The flow rate was set at 1.0 ml/min. The column temperature
was set at 35ꢀC. The quantification of deacetyldiltiazem was obtained by
comparing the HPLC peak height with that of an authentic standard. Because
diltiazem is partially deacetylated in a nonenzymatic manner, the total amount
of deacetyldiltiazem in the control mixture, incubated without the enzyme
Yeung et al. (1990) reported that the deacetyldiltiazem to diltiazem
AUC ratio after a single oral dose of diltiazem was higher in rats
0.82) than in humans (0.12), dogs (0.58), and rabbits (0.15), implying
(
the existence of species differences in diltiazem deacetylase activity.
These previous observations prompted us to clarify the molecular sources, was subtracted from the amount of deacetyldiltiazem obtained in the
mechanisms behind this particular species difference between humans presence of the enzyme sources. The activities were measured in triplicate, and
and experimental animals.
the mean value was calculated.
The kinetic analyses of diltiazem deacetylation were performed between 50
and 1000 mM. The parameters were estimated from fitted curves using software
Materials and Methods
Chemicals and Reagents. Diltiazem hydrochloride, deacetyldiltiazem, regression analyses.
(
KaleidaGraph, Synergy Software, Reading, PA) designed for nonlinear
phenylmethylsulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DFP),
Purification of the Diltiazem Deacetylating Enzymes. All of the
and eserine sulfate were purchased from Wako Pure Chemical Industries procedures were carried out at 4ꢀC. The RLM were solubilized in 100 mM
(
Osaka, Japan). Bis(4-nitrophenyl) phosphate (BNPP) sodium salt, sodium potassium phosphate buffer (pH 7.4) containing 20% glycerol, 1 mM EDTA,
fluoride (NaF), and ethopropazine hydrochloride were obtained from Sigma- 0.1% tergitol type-NP9, and 0.3% sodium cholate for 8 hours and were then
Aldrich (St. Louis, MO). All primers were commercially synthesized at
Hokkaido System Sciences (Sapporo, Japan). Other chemicals were of the
highest commercially available grade.
centrifuged at 105,000g for 1 hour. The collected supernatant represented the
sRLM. Proteins in the sRLM precipitated between 50 and 70% saturated
ammonium sulfate were dissolved in 20 mM Tris-HCl buffer (pH 7.4) con-
Tissue Microsomes. Human liver (pooled, n = 50) and intestinal micro- taining 20% glycerol, 1 mM EDTA, and 0.1% tergitol type-NP9 and then were
somes (pooled, n = 7) were purchased from Corning (Corning, NY). Human dialyzed against the same buffer to remove the ammonium sulfate. The di-
renal (pooled, n = 6) and pulmonary microsomes (individual) were purchased alyzed fraction was applied to a DEAE Sephacel anion exchange column (2.5
from KAC (Kyoto, Japan). Monkey liver (pooled, n = 10, males) and intestinal  4.0 cm; GE Healthcare, Buckinghamshire, UK) equilibrated with 20 mM
microsomes (pooled, n = 10, males) and dog liver (pooled, n = 8, male) and
Tris-HCl buffer (pH 7.4) containing 20% glycerol, 1 mM EDTA, and 0.1%
intestinal microsomes (pooled, n = 3, males) were obtained from Xenotech tergitol type-NP9. The proteins were eluted using the gradient method in
Lenexa, KS). Protease inhibitors, such as phenylsulfonyl fluoride, leupeptine, addition to the equilibration buffer containing 1 M KCl at a rate of 0.5 mL/min.
aprotinin, and bestatin, were not included in the suspension buffer.
The eluate fractions were collected continuously as 5-mL aliquots, and their
(
Pooled liver, jejunum, renal, and pulmonary microsomes were prepared from protein concentrations were determined according to the Bradford method
three rats (7-week-old Sprague-Dawley male rats, 210–230 g) and three mice (Bradford, 1976). The diltiazem deacetylation activity of the fractions was
(
(
6-week-old C57BL/6J male mice, 20–25 g) purchased from SLC Japan evaluated, as described above. Fractions showing activity (fractions 20–23)
Hamamatsu, Japan), according to the method described in our previous study were pooled and centrifuged at 4000–5000g using a Centricon YM-30
(Kobayashi et al., 2012). For the purification of diltiazem deacetylating enzyme(s), (Millipore, Billerica, MA) to concentrate the proteins and exchange the buffer.
rat liver microsomes (RLM) were prepared from five pooled samples from
-week-old Sprague-Dawley female rats (140–160 g). The protein concentrations
Concentrated proteins were diluted in 50 mM potassium phosphate buffer
(pH 7.4) containing 20% glycerol, 150 mM NaCl, and 0.1% tergitol type-NP9.
7
were determined according to the Bradford method (1976) using g-globulin as The diluted proteins were then applied to a Superdex 200 10/300 GL gel
a standard.
filtration column (GE Healthcare) equilibrated with 50 mM potassium phosphate

1
220
Kurokawa et al.
The sequences and position of the primers are shown in Supplemental Table 1.
A 1-ml portion of the reverse-transcribed mixture was added to a PCR mixture
containing 0.4 mM each primer and 12.5 ml SYBR Premix Ex Taq solution in
a final volume of 25 ml. After an initial denaturation step at 95ꢀC for 30
seconds, amplification was performed with 40 cycles of denaturation, annealing,
and extension. The PCR conditions for each Ces enzyme are shown in
Supplemental Table 2. The amplified products were monitored by measuring
the increase in the intensity of the SYBR Green (Takara Bio, Shiga, Japan)
dye that binds to the amplified double-stranded DNA during the PCR. The
target mRNA copy number in the samples was defined based on a standard
curve using the PCR products generated using real-time RT-PCR primer
pairs. The specificity of all of the primer pairs was confirmed by PCR product
digestion with the appropriate restriction enzymes and sequence analysis. The
Ces mRNA expression levels were normalized with glyceraldehyde-3-
phosphate dehydrogenase mRNA levels.
Construction of an Expression System for Rat Ces2a in Sf21 Cells. An
expression system for rat Ces2a was constructed using the Bac-to-Bac
Baculovirus Expression System (Invitrogen, Carlsbad, CA), according to the
manufacturer’s protocol. The rat Ces2a cDNA was obtained by RT-PCR from
a rat liver RNA sample using the Ces2a SalI and the Ces2a XhoI primers
Fig. 2. Diltiazem deacetylase activity in liver, small intestine, kidney, and lung
microsomes from human, monkey, dog, rat, and mouse. Tissue microsomes (0.2 mg/mL)
were incubated with diltiazem (200 mM) for 30 minutes. Kidney and lung
microsomes from monkey and dog were not available commercially. Each column
represents the mean 6 S.D. of triplicate determinations. †, Not detected; N, not
available.
(
Supplemental Table 1). The nucleotide sequence (referred to as accession no.
NM_144743) was confirmed by DNA sequence analysis performed at
FASMAC (Kanagawa, Japan). The PCR product was transferred into the
pFastBac1 vector using the appropriate restriction enzymes. The pFastBac1
vector containing the Ces2a cDNA was transformed into DH10Bac-competent
cells, followed by transposition of the inserts into bacmid DNA. Nonrecom-
binant bacmid DNA (mock) was also prepared using the same procedure. The
steps following the infection of Spodoptera frugiperda Sf21 cells (Invitrogen) with
bacmid DNA were performed according to the method described previously, with
slight modifications (Iwamura et al., 2012).
Inhibition Studies on Diltiazem Deacetylase Activity. To confirm the
deacetylase activity of rat Ces2a for diltiazem in rat livers, we performed
inhibition studies using recombinant rat Ces2a and RLM with typical esterase
inhibitors. Organophosphates, such as BNPP and DFP, are general Ces
inhibtors (Heymann and Krisch, 1967; Yamaori et al., 2006). PMSF is a
general serine esterase inhibitor (Johnson and Moore, 2000). Eserine, NaF, and
ethopropazine are cholinesterase inhibitors (Preuss and Svensson, 1996;
Yamaori et al., 2006; Takahashi et al., 2009). Eserine is also an inhibitor of
human CES2 (Takahashi et al., 2009). The final inhibitor concentration was
buffer (pH 7.4) containing 20% glycerol, 150 mM NaCl, and 0.1% tergitol
type-NP9. The proteins were eluted using the same buffer at a rate of 0.25
mL/min, and the diltiazem deacetylase activity of each 0.5-mL fraction was
evaluated. Fractions with enzyme activity (fractions 30–32) were pooled and
applied to a hydroxyapatite column (1.5 Â 3.0 cm; Bio-Rad, Hercules, CA)
equilibrated with 100 mM potassium phosphate buffer (pH 7.4) containing
2
0% glycerol, 1 mM EDTA, and 0.1% tergitol type-NP9. The proteins were
then eluted using the gradient method with the addition of an equilibration
buffer at 300 mM. The eluted fractions (1 mL) were then subjected to SDS-
PAGE, followed by silver staining. The diltiazem deacetylase activity of the
fractions containing proteins weighing approximately 60 and 70 kDa on
silver staining was measured, as described above. The final fractions showing
enzyme activity (fraction 56–67) were pooled and concentrated using a
Centricon YM-30.
1
00 mM for BNPP, DFP, and PMSF; 1 mM for eserine and NaF; and 10 mM
for ethopropazine. DFP, PMSF, and ethopropazine were dissolved in dimethylsulf-
oxide (DMSO). The final concentration of DMSO in the incubation mixture
was 1%. The other inhibitors were dissolved in distilled water. The ex-
perimental procedure and conditions were the same as described above. It
was confirmed that a 1% DMSO concentration did not inhibit diltiazem
deacetylase activity, and the activity in the control samples was also
determined in the presence of 1% DMSO.
Gel Electrophoresis and Protein Identification. SDS-PAGE was
performed according to the method described previously, with slight modifi-
cations (Tabata et al., 2004). Briefly, proteins were separated on a 7.5%
polyacrylamide gel. After electrophoresis, the gels were stained with a 0.05%
Coomassie Brilliant Blue solution. The protein band was excised using
a clean scalpel, and the amino acid sequence was analyzed at Wako Pure
Chemical Industries.
RNA Preparation from Rat Tissues and Reverse Transcription-
Polymerase Chain Reaction Analyses. The abdominal cavities of rats were
opened, and several tissues were excised. Total RNA samples from the rat liver,
jejunum (enterocytes), kidneys, and lungs were freshly isolated using RNAiso.
Real-time reverse transcription-polymerase chain reaction (RT-PCR) was
Results
Diltiazem Deacetylase Activity in the Microsomes from Human,
Monkey, Dog, Rat, and Mouse Tissues. Diltiazem deacetylase
performed for the quantitative determination of Ces mRNA using a MX3000P activity in the liver, small intestine, kidney, and lung microsomes
real-time polymerase chain reaction (PCR) system (Stratagene, La Jolla, CA). isolated from human, monkey, dog, rat, and mouse tissues was
TABLE 1
Purification of the enzyme(s) involved in diltiazem deacetylation in RLM
Total Protein
mg
Total Activity
nmol/min
Specific Activity
nmol/min/mg
Purification
Fold
Recovered Activity
%
Step
RLM
sRLM
969.2
937.9
212.2
175.8
1.4
538.3
381.3
478.2
629.8
475.7
150.0
0.6
0.4
2.3
3.6
338.6
125.0
1.0
0.7
4.1
6.5
609.6
225.0
100.0
70.8
88.8
117.0
88.4
27.9
5
0–70% (NH
4
2
) SO
4
DEAE Sephacel
Superdex 200
Hydroxyapatite
0.1

Species Differences in Diltiazem Deacetylation
1221
measured at 200 mM substrate concentration (Fig. 2). These organs ammonium sulfate precipitation, the 50%–70% ammonium sulfate-
were selected because they are the main organs involved in drug saturated fraction exhibited the highest deacetylase activity and was
metabolism and express several esterases. Diltiazem deacetylase purified on a DEAE Sephacel anion exchange column. By monitoring
activity was only detected in rats. The activity in the male rat liver the protein concentration and diltiazem deacetylase activity of the
and intestinal microsomes was 3.18 6 0.05 and 0.29 6 0.01 eluate, we observed that the candidate protein was mainly found in
nmol/min/mg protein, respectively. The activity in the female rat liver fractions 20–23. The pooled proteins (fractions 20–23) were
was 4.23 6 0.13 nmol/min/mg protein, which is 1.3-fold greater than concentrated using a Centricon YM-30 and subsequently loaded on
the activity measured in male rats (data not shown). In contrast to the a Superdex 200 gel filtration column. Again, the active fractions
microsomes, the rat liver cytosol samples showed no activity (data not (fractions 30–32) were pooled and were subsequently subjected to
shown).
hydroxyapatite chromatography purification. Fractions 53–71 dis-
Purification and Identification of the Enzyme(s) Responsible for played some activity, but the highest activity was found in fractions
Diltiazem Deacetylation. To identify the enzyme responsible for 62–64 (10.5–10.8 nmol/min/mL fraction) (Fig. 3B). In these fractions,
diltiazem deacetylation, we sought to purify the enzyme(s) from two bands of approximately 60 and 70 kDa were detected by SDS-
female rat liver microsomes. The efficiency of protein recovery for PAGE and silver staining (Fig. 3A). The intensity of the 60-kDa
each step of the purification procedure is shown in Table 1. The protein band was correlated with the diltiazem deacetylase activity
protein staining profile and diltiazem deacetylase activity are shown in found in fractions 58–68. The intensity of the 70-kDa protein band
Fig. 3. Rat liver microsomes were solubilized in a solution containing was highest in fraction 50, which showed lower diltiazem deacetylase
tergitol type-NP9 and sodium cholate, and we confirmed that the activity (0.6 nmol/min/mL fraction) than fractions 62–64 (data not
diltiazem deacetylase activity of sRLM was retained (70.8%). Upon shown). Overall, compared with the initial RLM fraction, we achieved
a 225-fold purification of enzymatically active proteins with a 0.12%
yield. However, the specific activity in the final step was lower than in
the Superdex 200-purified fractions (Table 1). This decrease may have
been caused by the deterioration of the hydroxyapatite after multiple
uses. Finally, based on the amino acid sequence analysis of the excised
6
0-kDa protein band, Ces1d (accession no. NP_579829), Ces1e
(NP_113753), Ces1f (NP_001096829), and Ces2a (NP_653344)
emerged as candidate proteins (Table 2).
mRNA Expression Levels of Rat Ces Enzymes. To determine
which of the Ces isoforms are responsible for diltiazem deacetylation
among the four candidates, their tissue distribution at the mRNA
expression level was evaluated in comparison with the activities of
the rat tissue microsomes, as shown in Fig. 2. In addition, the mRNA
levels of the other Ces isoforms were also evaluated. All of the mRNA
levels were determined as the copy numbers normalized to glyceraldehyde-
3
-phosphate dehydrogenase levels (Fig. 4). In the Ces1 family, Ces1a
was not detected in the liver, small intestine, kidneys, or lungs. The
expression of Ces1c was limited to the liver, whereas Ces1d, Ces1e,
and Ces1f were expressed in various organs. In the Ces2 family, Ces2a
was expressed in the liver and at a lower level in the small intestine.
Ces2c, Ces2g, and Ces2h were expressed in the liver, small intestine,
and kidneys. These Ces2 isoforms were also detected in the lungs, but
only in female rats. Ces2e was expressed in the liver and barely
expressed in the small intestine of female rats. The expression of Ces2i
and Ces2j was limited to the liver and small intestine, respectively.
The only isoform for which the expression profile corresponded to the
diltiazem deacetylase activity profile was Ces2a.
Kinetic Analyses of the Dilitiazem Deacetylase Activity of
Recombinant Rat Ces2a and RLM. To examine whether rat Ces2a
might be responsible for diltiazem deacetylation, we performed a
kinetic analysis of the diltiazem deacetylase activity of a recombinant
Ces2a protein (Fig. 5; Table 3). The activities of both recombinant
Ces2a and RLM followed the Michaelis-Menten kinetics model. The
Km, Vmax, and CLint values of recombinant Ces2a were 116.8 6 5.3 mM,
Fig. 3. Purification of the enzyme(s) responsible for diltiazem deacetylation from
RLM. (A) Protein profiles of sRLM and RLM fractionated by ammonium sulfate
and column chromatography. The proteins were separated by SDS-PAGE and were
visualized by silver staining: lane 1, molecular weight standard; lane 2, 30 mg
proteins; lane 3, 10 mg proteins; lane 4, 6 mg proteins; lane 5, 8 mg proteins; lane 6,
TABLE 2
Candidate proteins involved in diltiazem deacetylation in rat
Protein
Gene
Molecular Weight
pI
Accession No.
0
.6 mg proteins; lane 7, 0.1 mg proteins. The open and closed arrowheads indicate
the 70- and 60-kDa protein bands, respectively. (B) Diltiazem deacetylase activity in
the fractions eluted from hydroxyapatite chromatography and approximately 60- and
Ces1d
Ces1e
Ces1f
Ces2a
Ces1d
Ces1e
Ces1f
Ces2a
62,147
61,669
62,308
61,802
9.41
5.71
6.29
6.19
NP_579829
NP_113753
NP_001096829
NP_653344
7
0-kDa protein bands of each fraction. The 60- and 70-kDa protein bands were
visualized by silver staining. For the diltiazem deacetylase activity, 20 mL fractions
were incubated with 200 mM diltiazem for 30 minutes.

1
222
Kurokawa et al.
Fig. 4. The mRNA expression profiles of rat Ces isoforms in liver, small intestine, kidney, and lung from male and female rats. Total RNA samples were freshly isolated
from three rats, and pooled samples were used. The copy numbers of Ces mRNA were determined by real-time RT-PCR analyses and were normalized with glyceraldehyde-
3
-phosphate dehydrogenase levels. A standard curve for the determination of copy number was constructed using PCR products with each primer pair used for real-time RT-
PCR. Each column represents the mean 6 S.D. of triplicate determinations. †, Not detected.
1
8.3 6 0.5 nmol/min/mg protein, and 156.8 6 3.1 mL/min/mg protein, protein, respectively. Thus, recombinant Ces2a showed a similar Km
respectively (Table 3). Those values for the RLM were 359.4 6 value as the RLM, suggesting that rat Ces2a is the main enzyme
.7 mM, 11.6 6 0.4 nmol/min/mg protein, and 32.2 6 0.3 mL/min/mg responsible for diltiazem deacetylation in the rat liver.
9

Species Differences in Diltiazem Deacetylation
1223
activity (data not shown). Our results support a previous report by
LeBoeuf and Grech-Belanger (1987) showing that diltiazem was
deacetylated in the rat liver and small intestine, but not in the kidneys
or lungs. Although deacetyldiltiazem was detected in human plasma
after an oral dose of diltiazem (Yeung et al., 1990), no diltiazem
deacetylase activity was detected in the microsomes of human livers,
the small intestine, the kidneys, and the lungs (Fig. 2). In addition, we
confirmed that human plasma did not show diltiazem deacetylase
activity in an examination of plasma samples from five healthy
Japanese volunteers (data not shown). This apparent contradiction
may be partly accounted for by dilitiazem deacetylation that occurs in
other organs that were not examined. In addition, during a 30-minute
incubation of 200 mM diltiazem at 37ꢀC, 7.8% and 0.25% of the
added diltiazem were enzymatically (with 0.2 mg/mL RLM) and
nonenzymatically deacetylated, respectively. Therefore, nonenzymatic
deacetylation may be another reason for the detection of deacetyldil-
tiazem in human plasma. Initially, it was suggested that the enzyme(s)
catalyzing diltiazem deacetylation in the main drug metabolism
Fig. 5. Kinetic analyses of diltiazem deacetylation by recombinant rat Ces2a and organs, such as the liver and small intestine, was expressed only in
RLM. Recombinant rat Ces2a (0.1 mg/mL) or RLM (0.2 mg/mL) were incubated
with diltiazem for 30 minutes. The diltiazem deacetylase activity was measured by
rats.
To identify the enzyme(s) responsible for diltiazem deacetylation in
HPLC. Each point represents the mean 6 S.D. of triplicate determinations.
rats, we purified enzymatically active proteins from rat liver micro-
somes. Through several purification steps (Table 1; Fig. 3A), we
The Effects of Chemical Inhibitors on Diltiazem Deacetylation finally restricted the candidate dilitiazem deacetylating enzymes to
by Recombinant Ces2a and RLM. To investigate whether Ces2a is proteins weighing approximately 60 kDa (Fig. 3). The amino acid
the principal enzyme for diltiazem deacetylation in the rat liver, we sequences of these 60-kDa proteins were analyzed, and four Ces
compared the effects of various esterase inhibitors on the diltiazem enzymes, Ces1d, Ces1e, Ces1f, and Ces2a (Table 2), were identified
deacetylase activity of recombinant Ces2a and RLM (Fig. 6). The as candidates. Using purified Ces enzymes, Hosokawa et al. (1990)
activity of both recombinant Ces2a and RLM was efficiently inhib- reported that Ces1f preferentially hydrolyzes malathion and palmitoyl-
ited by BNPP, DFP, PMSF, and eserine, but not by NaF and CoA, that Ces1d selectively hydrolyzes butanilicaine, and that Ces1d
ethopropazine. Such similarity in the inhibition profiles between and Ces1e hydrolyze isocarboxazid. Additionally, Robbi and Beaufay
recombinant Ces2a and RLM supported the hypothesis that Ces2a is (1994) have reported that recombinant Ces1e hydrolyzes acetanilide.
the enzyme that is responsible for diltiazem deacetylation in rats.
Among those chemicals, by acyl group size, the descending order is
palmitoyl-CoA, isocarboxazid, butanilicaine, malathion, and acetan-
ilide. In addition, Ozaki et al. (2013) investigated the substrate
specificity of rat Ces isoforms using parabens with various alkyl chain
lengths, although the substrate specificities of Ces1d, Ces1e, and
Ces1f could not be clearly determined from the size of the alcohol or
acyl moieties. In the same report, although information regarding the
substrate specificity of rat Ces2 was very limited, it was revealed that
rat Ces2a appears to prefer compounds with a large alcohol moiety,
which is a characteristic of diltiazem. Because we considered that it
would be difficult to determine which Ces isoform is responsible for
diltiazem deacetylation on the basis of the chemical structure of their
substrates, we observed the expression levels of Ces isoforms in the rat
liver, small intestine, kidneys, and lungs (Fig. 4). Because antibodies
specific for each Ces isoform are not available, we determined the
mRNA expression levels in this study.
We found that the Ces1 isoforms were highly expressed in the liver,
which is similar to humans (Satoh et al., 2002). It is noteworthy that
Ces1e and Ces-like1, which are probably isoforms in the Ces1 family,
were expressed in the rat small intestine. It has been reported that
Ces1e was highly expressed in the rat intestine (Mentlein et al., 1987),
although CES1 was not expressed in the human small intestine at
protein levels (Taketani et al., 2007). The Ces2 isoforms, except for
Ces2j, were commonly expressed in the liver, whereas some isoforms
were moderately expressed in the small intestine. Our results are
supported by previous papers (Mello et al., 2008; Ohura et al., 2014)
that reported Ces isoform mRNA expression in the rat liver, jejunum,
or ileum. Compared with these previous studies, an advantage of the
present study was the determination of the expression profiles of all of
the Ces isoforms in rats. Among the candidate isoforms (Ces1d,
Discussion
In humans, diltiazem is primarily metabolized to N-demethyldiltia-
zem, O-demethyldiltiazem, and deacetyldiltiazem by CYP3A4,
CYP2D6, and esterase(s), respectively (Molden et al., 2002; Williams
et al., 2002); however, the esterase(s) responsible for the deacetylation
remained to be identified. Yeung et al. (1990) reported that the AUC
ratio of deacetyldiltiazem to diltiazem after an oral dose of diltiazem
was much higher in rats (0.82) than in humans (0.12), suggesting the
existence of species differences in the enzyme activity and/or substrate
specificity of the esterase(s) involved in diltiazem deacetylation. In
this study, we sought to clarify the molecular underpinnings of this
species difference in diltiazem deacetylase activity between humans
and experimental animals via in vitro experiments.
First, we measured the diltiazem deacetylase activity of tissue
microsomes isolated from humans, monkeys, dogs, rats, and mice
(
Fig. 2). Among the tested samples, diltiazem deacetylase activity was
detected only in the liver and small intestine microsomes of rats. In
addition, we confirmed that rat plasma had no diltiazem deacetylase
TABLE 3
Kinetic parameters of diltiazem deacetylation in RLM and recombinant Ces2a
K
m
V
max
CLint
Enzyme Source
mM
nmol/min/mg
mL/min/mg
RLM
Recombinant Ces2a
359.4 6 9.7
116.8 6 5.3
11.6 6 0.4
18.3 6 0.5
32.2 6 0.3
156.8 6 3.1

1
224
Kurokawa et al.
addition, it has been reported that Ces1f protein is expressed in the
rat kidney (Yan et al., 1994). Because the rat kidney and lung
microsomes did not show any activity (Fig. 2), the contribution of
Ces1f to diltiazem deacetylation would be quite low. There are no
reports regarding diltiazem deacetylation by recombinant Ces1e. It
was demonstrated that the rat Ces1e protein is highly expressed in
the liver and intestine and is expressed in the kidneys at relatively
low levels (Mentlein et al., 1987), which is almost consistent with the
mRNA expression results of this study. Diltiazem deacetylation was
not detected in rat kidney microsomes. These observations reminded
us of the major contribution of Ces2a to diltiazem deacetylation in
rats.
To confirm the role of rat Ces2a in diltiazem deacetylation, we
measured the diltiazem deacetylase activity of recombinant Ces2a and
compared its Km value with that in RLM (Table 3; Fig. 5). The
similarity of Km values between them suggested that Ces2a is re-
sponsible for diltiazem deacetylation in the rat liver. Furthermore, this
idea was further supported by the similarity of the inhibition profiles
of RLM and recombinant Ces2a, as determined with various inhibitors
(
Fig. 6). Because information regarding potent inhibitors against rat
Fig. 6. Inhibitory profile of various chemical inhibitors against diltiazem Ces2a is lacking, we used general inhibitors that are used for human
deacetylase activity. Recombinant rat Ces2a (0.1 mg/mL) or RLM (0.2 mg/mL)
were incubated with 200 mM diltiazem for 30 minutes. The controlled activity values
inhibitor (Takahashi et al., 2009), also inhibited rat Ces2a activity.
by recombinant rat Ces2a and RLM were 9.55 6 0.14 and 4.23 6 0.13 nmol/min/mg
CES enzymes. Eserine, which is known to be a human CES2
Although it is known that eserine potently inhibits human arylacetamide
deacetylase (AADAC) activity (Watanabe et al., 2010), we con-
firmed that recombinant rat AADAC did not show any diltiazem
deacetylation (data not shown). In addition, we evaluated the effects
protein, respectively. Each column represents the mean 6 S.D. of triplicate
determinations.
Ces1e, Ces1f, and Ces2a), only Ces2a showed an expression profile of calcium chloride (1 mM) on diltiazem deacetylation in RLM,
that corresponded to the profile of diltiazem deacetylase activity. because paraoxonase is known to be activated by calcium chloride
Furthermore, the sex difference in the diltiazem hydrolase activity (Hioki et al., 2011). However, the activation was not observed (94.7 6
(
higher in females) was consistent with the difference in the Ces2a 0.7% of control, data not shown). Based on these results, we
expression level in the liver (Fig. 4). Consequently, Ces2a was suggested concluded that rat Ces2a is the enzyme responsible for diltiazem
to be the enzyme responsible for diltiazem deacetylation.
deacetylation.
Previously, it was reported that the rat Ces1f isoform, the trivial
Why isn’t diltiazem deacetylated in the human liver and small
name of which is carboxylesterase pI 6.2, catalyzes diltiazem deacetylation intestine? When we measured the diltiazem deacetylase activity using
Vmax: 3.0 nmol/min/mg protein of purified Ces1f) (Luan et al., recombinant human CES1, CES2, and AADAC, which are represen-
997). However, it was unclear whether Ces1f was the sole enzyme tative esterases involved in tissue drug hydrolysis, none of them
(
1
exhibiting diltiazem hydrolysis because the activity was evaluated displayed any activity (data not shown). The amino acid sequence
only by recombinant rat Ces1d and Ces1f. In the present study, we homology between human CES2 and rat Ces2a is approximately 67%
found that Ces1f was expressed in rat kidneys and lungs (Fig. 4). In (Fig. 7). The amino acids within the catalytic triad Ser, Glu, and His
Fig. 7. Alignment of amino acid sequences of human
CES2 (NP_003860), rat Ces2a (NP_653344), and mouse
Ces2a (NP_598721). Amino acids identical to rat Ces2a are
shown in gray boxes. Bold letters in boxes indicate the
conserved amino acids, which are part of an oxyanion hole
(
(
GG), catalytic triad (S, E, and H), and retention sequence
HXEL).

Species Differences in Diltiazem Deacetylation
1225
LeBoeuf E and Grech-Bélanger O (1987) Deacetylation of diltiazem by rat liver. Drug Metab
Dispos 15:122–126.
Luan L, Sugiyama T, Takai S, Usami Y, Adachi T, Katagiri Y, and Hirano K (1997) Purification
and characterization of pranlukast hydrolase from rat liver microsomes: the hydrolase is
identical to carboxylesterase pI 6.2. Biol Pharm Bull 20:71–75.
Molden E, Johansen PW, Bøe GH, Bergan S, Christensen H, Rugstad HE, Rootwelt H, Reubsaet
L, and Lehne G (2002) Pharmacokinetics of diltiazem and its metabolites in relation to
CYP2D6 genotype. Clin Pharmacol Ther 72:333–342.
Mello T, Nakatsuka A, Fears S, Davis W, Tsukamoto H, Bosron WF, and Sanghani SP (2008)
Expression of carboxylesterase and lipase genes in rat liver cell-types. Biochem Biophys Res
Commun 374:460–464.
Mentlein R, Ronai A, Robbi M, Heymann E, and von Deimling O (1987) Genetic identification of
rat liver carboxylesterases isolated in different laboratories. Biochim Biophys Acta 913:27–38.
Ohura K, Tasaka K, Hashimoto M, and Imai T (2014) Distinct patterns of aging effects on the
expression and activity of carboxylesterases in rat liver and intestine. Drug Metab Dispos 42:
264–273.
Ozaki H, Sugihara K, Watanabe Y, Fujino C, Uramaru N, Sone T, Ohta S, and Kitamura S (2013)
Comparative study of the hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and
dodecylparaben by microsomes of various rat and human tissues. Xenobiotica 43:1064–1072.
Preuss CV and Svensson CK (1996) Arylacetamide deacetylase activity towards mono-
acetyldapsone: species comparison, factors that influence activity, and comparison with
are conserved, and the sequences around the serine residue in the
active site are also highly similar between species. Although the
mouse Ces2a, which is highly expressed in the duodenum, jejunum,
and ileum (Jones et al., 2013), shows a high level of homology with
the rat Ces2a at the amino acid level (88%), no diltiazem deacetylase
activity was detected in mouse tissue microsomes (Fig. 2). Crystal
structure analysis would be useful to obtain the answer to these
questions, but one can imagine that subtle differences in amino acid
residues in Ces2 between the rat and other species would determine
the specificity of substrate recognition.
In conclusion, we found that diltiazem deacetylase activity was
detected only in the rat liver and small intestine and that Ces2a is the
enzyme responsible for this activity. In addition, our study clarified the
expression profile of all of the rat Ces isoforms in several tissues. The
present study provides useful information regarding rat Ces enzymes
and will help us to understand the species-specific differences in drug
pharmacokinetics between humans and rats.
2
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Robbi M and Beaufay H (1994) Cloning and sequencing of rat liver carboxylesterase ES-3
egasyn). Biochem Biophys Res Commun 203:1404–1411.
(
Sanghani SP, Davis WI, Dumaual NG, Mahrenholz A, and Bosron WF (2002) Identification of
microsomal rat liver carboxylesterases and their activity with retinyl palmitate. Eur J Biochem
2
69:4387–4398.
Authorship Contributions
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.
Tabata T, Katoh M, Tokudome S, Nakajima M, and Yokoi T (2004) Identification of the cytosolic
carboxylesterase catalyzing the 59-deoxy-5-fluorocytidine formation from capecitabine in hu-
man liver. Drug Metab Dispos 32:1103–1110.
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.
Taketani M, Shii M, Ohura K, Ninomiya S, and Imai T (2007) Carboxylesterase in the liver and
small intestine of experimental animals and human. Life Sci 81:924–932.
Participated in research design: Kurokawa, Fukami, Nakajima.
Conducted experiments: Kurokawa, Fukami.
Contributed new reagents or analytic tools: Kurokawa, Fukami.
Performed data analysis: Kurokawa, Fukami.
Wrote or contributed to the writing of the manuscript: Kurokawa, Fukami,
Nakajima.
Watanabe A, Fukami T, Takahashi S, Kobayashi Y, Nakagawa N, Nakajima M, and Yokoi T
(
2010) Arylacetamide deacetylase is a determinant enzyme for the difference in hydrolase
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