K. Muta et al. / Biochemical Pharmacology 98 (2015) 659–670
661
ꢁ
2.2. Animals
at 37 C. After the 30-min (for HLC) or 2-min (for recombinant
ꢁ
enzymes) incubation at 37 C, the reaction was terminated by
C57BL/6J mice (8-weeks old females, 16–21 g) were obtained
the addition of 200 mL of ice-cold methanol. After the removal of
from SLC Japan (Hamamatsu, Japan). Mice were housed in an
institutional animal facility in a controlled environment (temper-
the protein by centrifugation at 9500 ꢂ g for 5 min, a 20-
mL
portion of the supernatant was subjected to HPLC as described
above.
ꢁ
ature 25 ꢀ1 C and 12-h light/dark cycle) with access to food and
water ad libitum. Mice were acclimated for one week prior to use in
experiments. Animal maintenance and treatment were conducted
in accordance with the National Institutes of Health Guide for
Animal Welfare of Japan, and the protocols were approved by the
Institutional Animal Care and Use Committee of Kanazawa
University, Japan.
2.5. Inhibitory effects of esterase inhibitors on acebutolol hydrolase
activities in HLM and HIM
To clarify the involvement of esterases in human tissues, an
inhibition analysis for acebutolol hydrolysis was performed by using
representative esterase inhibitors. Vinblastine is a CES2 and AADAC
inhibitor, telmisartan and loperamide are CES2 specific inhibitors,
and digitonin is a CES1 specific inhibitor [21]. PMSF is a serine
hydrolase inhibitor [22]. Organophosphates, such as BNPP and DFP,
are known to be general CES inhibitors [23,24]. Eserine is a CES2 and
AADAC inhibitor as well as a cholinesterase inhibitor [9,25]. The
concentrations of vinblastine, telmisartan, loperamide, digitonin,
PMSF, BNPP, DFP, and eserine were 50, 20, 5, 200, 100, 10, 100, and
2.3. Preparation of acetolol, a hydrolytic metabolite of acebutolol, by
the forced hydrolysis of acebutolol
To 50
m
L of 100 mM acebutolol, 50
m
L of 1 M hydrochloric acid
ꢁ
was added and incubated at 100 C for 5 h and then neutralized by
the addition of 50 mL of 1 M sodium hydrate. Complete hydrolysis
of acebutolol was confirmed by HPLC, and the hydrolyzed product
was confirmed to be acetolol by the LC–MS/MS as described below.
100 mM, respectively. Vinblastine, telmisartan, loperamide, digito-
nin, and PMSF were dissolved in DMSO such that the final
concentration of DMSO in the incubation mixture was 1.0%. The
other inhibitors were dissolved in distilled water. The experimental
procedure and conditions were the same as above except that the
concentration of acebutolol was 10 mM. We confirmed that 1.0%
DMSO did not inhibit the acebutolol hydrolase activity.
2
.4. Acebutolol hydrolase and acetolol N-acetyltransferase activities
Acebutolol hydrolase activity was determined as follows: a
typical incubation mixture (final volume of 0.2 mL) contained
00 mM potassium phosphate buffer (pH 7.4) and various enzyme
1
sources (HLM, HIM, Sf21 cell homogenates expressing human
recombinant CES1, CES2, and AADAC, which were prepared for our
previous studies [19,20]: 0.4 mg/mL, 0.2 mg/mL, 0.25 mg/mL,
2.6. Detection of a putative reactive metabolite as a NAC conjugate
0
.63 mg/mL, and 0.25 mg/mL, respectively). We confirmed that
Procainamide is metabolized to N-hydroxyprocainamide by
CYP2D6, and then autooxidized to nitroso-procainamide [17],
which causes DILE [16]. The hydrolyzed metabolite of acebutolol is
hydroxyarylamine, which has a structure that is similar to
procainamide. Because nitroso metabolites are often trapped with
NAC, a study to investigate whether a NAC conjugate could be
formed from acetolol was performed. A typical incubation mixture
(final volume of 0.2 mL) contained 100 mM potassium phosphate
the rate of acetolol formation was linear with respect to the protein
concentration (<1.0 mg/mL human microsomal protein and
<
0.8 mg/mL Sf21 cell homogenates expressing esterases) and
incubation time (<30 min). Acebutolol hydrochloride was dis-
solved in distilled water. The reaction was initiated by the addition
of 0.5–50 mM acebutolol after a 2-min preincubation at 37 C. After
a 20-min incubation at 37 C, the reaction was terminated by the
ꢁ
ꢁ
+
addition of 200
m
L of ice-cold methanol. After removal of the
L portion of
buffer (pH 7.4), an NADPH-generating system (50 mM NADP ,
protein by centrifugation at 9500 ꢂ g for 5 min, a 20-
m
2
50 mM MgCl and 10 unit/mL G6PDH), 50 mM NAC, and HLM
the supernatant was subjected to HPLC. The HPLC analysis was
performed using an L-7100 pump (Hitachi, Tokyo, Japan), an
L-7200 autosampler (Hitachi), an L-7405 UV detector (Hitachi), and
a D-2500 chromato-integrator (Hitachi) equipped with a Wakopak
(0.8 mg/mL) or human recombinant CYP enzymes (25 pmol/mL).
The reaction was initiated by the addition of 100
a 2-min preincubation at 37 C. After a 40-min incubation at 37 C,
m
M acetolol after
ꢁ
ꢁ
the reaction was terminated by the addition of 200
methanol. After removal of the protein by centrifugation at
L portion of the supernatant was
mL of ice-cold
eco-ODS column (5-
m
m particle size, 4.6 mm i.d. ꢂ150 mm; Wako
Pure Chemical Industries, Osaka, Japan). The eluent was monitored
at 232 nm with a noise-base clean Uni-3 (Union, Gunma, Japan).
The mobile phase was (A) 10% acetonitrile containing 25 mM
potassium dihydrogen phosphate (pH 4.0) and (B) 15% acetonitrile
containing 25 mM potassium dihydrogen phosphate (pH 4.0). The
conditions for elution were as follows: 100% A (0–7 min), 100–0% A
9500 ꢂ g for 5 min, a 20-
m
subjected to LC–MS/MS. The LC equipment comprised an
HP1100 system including a binary pump, an automatic sampler,
and a column oven (Agilent Technologies, Santa Clara, CA), which
was equipped with a ZORBAX SB-C18 column (2.1 ꢂ50 mm,
3.5 mm; Agilent Technologies). The column temperature was set
ꢁ
(
(
7–8 min), 0% A (8–16 min), 0–100% A (16–17 min), and 100% A
17–19 min). The flow rate was 1.0 mL/min. The column tempera-
at 25 C, and the flow rate was 0.2 mL/min. The mobile phase was
0.1% formic acid (A) and acetonitrile including 0.1% formic acid (B).
The conditions for elution were as follows: 10% B (0–3.5 min),
10–20% B (3.5–4.5 min), 20% B (4.5–6 min), 20–10% B (6–7 min),
and 10% B (7–10 min). The LC was connected to a PE Sciex
API2000 tandem mass spectrometer (AB Sciex, Framingham, MA)
operated in the positive electrospray ionization mode. The turbo
ꢁ
ture was 35 C. Acetolol was quantitated by comparing the HPLC
peak height with that of a standard produced as described above.
The kinetic parameters were estimated from the fitted curves using
a computer program (KaleidaGraph; Synergy Software, Reading,
PA) designed for nonlinear regression analysis.
Acetolol N-acetyltransferase activity was determined as
follows: a typical incubation mixture (final volume of 0.2 mL)
contained 100 mM potassium phosphate buffer (pH 7.4), 1 mM
acetyl-CoA, 0.1 mM dithiothreitol (DTT), 0.1 mM EDTA, and
enzyme sources (HLC: 1.0 mg/mL, baculovirus-infected insect
cells (BTI-TN-5B1-4) homogenates expressing human recombi-
nant NAT1 and NAT2: 0.25 ng/mL). The reaction was initiated by
ꢁ
gas was maintained at 550 C. Nitrogen was used as the nebulizing,
turbo, and curtain gas at 60, 85, and 30 psi, respectively. Parent
and/or fragment ions were filtered in the first quadrupole and
dissociated in the collision cell using nitrogen as the collision gas.
In the multiple reaction monitoring (MRM) mode, m/z ion
transitions 444.2 and 116.0 were monitored. The analytical data
were processed using Analyst software (version 1.5; Applied
Biosystems, Foster City, CA).
the addition of 0.5–200 mM acetolol after a 2-min preincubation