Journal of Pharmacological Sciences 133 (2017) 103e109

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Journal of Pharmacological Sciences

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Characterization of microminipigs as an in vivo experimental model for

cardiac safety pharmacology
Suchitra Matsukura a, 1, Yuji Nakamura a, 1, Xin Cao a, Takeshi Wada a,
Hiroko Izumi-Nakaseko a, Kentaro Ando a, Hiroshi Yamazaki b, Atsushi Sugiyama a, *
a
Department of Pharmacology, Faculty of Medicine, Toho University, 5-21-16, Omori-nishi, Ota-ku, Tokyo 143-8540, Japan
b
Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, 3-3165, Higashi-Tamagawa Gakuen, Machida, Tokyo 194-8543,
Japan

a r t i c l e i n f o a b s t r a c t

Article history: We pharmacologically characterized microminipigs as an in vivo experimental model by assessing car-
Received 28 September 2016 diovascular effects of pilsicainide, verapamil and E-4031, which can preferentially inhibit cardiac Naþ,
Received in revised form Ca2þ and Kþ channels, respectively. Intravenous infusion of 1 mg/kg of pilsicainide (n ¼ 4), 0.1 mg/kg of
15 January 2017
verapamil (n ¼ 4) and 0.01 followed by 0.1 mg/kg of E-4031 (n ¼ 5) over 10 min decreased the heart rate,
Accepted 6 February 2017
Available online 11 February 2017
mean blood pressure and ventricular contractility. Moreover, pilsicainide prolonged the PR interval, QRS
width and QTc; verapamil prolonged the PR interval, but shortened the QRS width and QTc; and E-4031
prolonged the QTc, whereas no substantial change was detected in the PR interval or QRS width. Peak
Keywords:
Microminipig
plasma concentrations of pilsicainide, verapamil and E-4031 in microminipigs were 1.7e4.8 times higher
Pilsicainide than those expected in humans and dogs, possibly due to smaller effective volume of drug distribution.
Verapamil The extent of the drug-induced cardiovascular responses was generally greater in microminipigs than in
E-4031 humans and dogs, which could be explained by the following possibilities; namely unique pharmaco-
Cardiovascular effect kinetic profile, less great reflex-mediated increase of sympathetic tone and/or smaller repolarization
reserve in microminipigs. These information may make it feasible to apply this new-type animal to a tool
for assessing cardiac safety profiles of new chemical entities.
© 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological
Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).

1. Introduction In order to overcome such limitation, an extraordinary small

In the field of life science including pharmacological research,
pigs have been expected to become an alternative model to dogs or
monkeys, since the pigs are known to have biologically similar
cardiovascular, respiratory, metabolic and gastrointestinal systems
in addition to the skin to those in humans (1), and experimental use
of dogs and/or monkeys may generate strong concerns among
zoophilism (2). Since miniature pigs weigh between 34 and 68 kg
when they are fully grown (3), and farm pigs typically reach
weights of 136e226 kg (1,2), much larger amount of a test article as
well as breeding space is required to obtain its in vivo proof even
with the use of miniature pigs than dogs or monkeys.
* Corresponding author.
E-mail address: atsushi.sugiyama@med.toho-u.ac.jp (A. Sugiyama).
Peer review under responsibility of Japanese Pharmacological Society.
1 repolarization reserve (9). Indeed, the extent of repolarization
Equally contributed.
size of miniature pigs; namely, microminipigs (Fig. 1), weighing
approximately 7 kg at 6 months of age when they are young
mature, were developed by Fuji Micra Inc. (Shizuoka) (2). However,
background knowledge of microminipigs is still lacking regarding
typical cardiac ion-channel modulators-induced cardiovascular
responses, which will be indispensable to apply this new-type
animal particularly to the field of cardiac safety pharmacology
(4). In the present study, we characterized microminipigs as an
in vivo experimental model by assessing cardiovascular effects of
pilsicainide, verapamil and E-4031, which can preferentially inhibit
cardiac Naþ, Ca2þ and Kþ channels, respectively (5e7).
Conscious animals usually have more repolarization reserve
than healthy human subjects, making the animals less sensitive for
detecting drug-induced QT-interval prolongation (8). Halothane
anesthesia is known to suppress cardiac IKs and IKr together with
autonomic tone to some extent, resulting in a decrease of the

http://dx.doi.org/10.1016/j.jphs.2017.02.002
1347-8613/© 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
104 S. Matsukura et al. / Journal of Pharmacological Sciences 133 (2017) 103e109
Fig. 1. An extraordinary small size miniature pig; microminipig, weighing approxi-
mately 7 kg at 6 months of age when it was young mature.
reserve has been considered to be similar between the healthy
conscious human subjects and halothane-anesthetized dogs (8);
thus, the drug-induced QT-interval prolongation in the halothane-
anesthetized dogs well mimics that in healthy human subjects (5, 7,
9). In this study, microminipigs were anesthetized with halothane
inhalation in order to directly compare their drug-induced car-
diovascular responses with previous reports in dogs obtained un-
der the same anesthetic conditions (5e7). We suggest this study
can provide important information indicating advantage and
disadvantage of microminipigs when they are used as an alternative
in vivo model to dogs or monkeys for cardiac safety pharmacology
studies.
2. Materials and methods
Microminipigs of 5 ± 1 months old, weighing 8.1 ± 0.6 kg (n ¼ 13)
were obtained from Fuji Micra Inc. All experiments were approved
by the Toho University Animal Care and User Committee (No. 14-
51-275) and performed in accordance with the Guidelines for the
Care and Use of Laboratory Animals of Toho University.
2.1. Cardiovascular variables
Microminipigs of 8 ± 2 months old, weighing 8.6 ± 1.1 kg
(n ¼ 13), were pre-anesthetized by an intramuscular injection of
ketamine (16 mg/kg)/xylazine (1.6 mg/kg). A 24G cannula was
introduced into a superficial auricular vein, and 20 mg/body of
propofol was intravenously injected for inducing anesthetic con-
dition. After intubation with a 5 mm cuffed endotracheal tube, 1.0%
halothane vaporized with 100% oxygen was inhaled with a
volume-limited ventilator (SN-480-3; Shinano Manufacturing Co.,
Ltd., Tokyo). Tidal volume and respiratory rate were set at 10 mL/kg
and 15 strokes/min, respectively. Two sets of clinically available
6F-size catheter sheath sets (FAST-CATH™ 406108, St. Jude Medi-
cal Daig Division, Inc., Minnetonka, MN, USA) were inserted into
the right femoral artery and vein, respectively. Heparin calcium in
a dose of 100 IU/kg was intravenously administered through the
catheter sheath placed at the right femoral vein to prevent blood
clotting. A pig-tail catheter was introduced into the left ventricle
through the catheter sheath placed at the right femoral artery for
measuring the left ventricular pressure. Left ventricular pressure
at a time point of peak of R wave on electrocardiogram was defined
as left ventricular end-diastolic pressure (10). The maximum up-
stroke velocity of the left ventricular pressure (LVdP/dtmax) and
left ventricular end-diastolic pressure were obtained during sinus
rhythm to estimate the contractility and the preload to the left
ventricle, respectively. The electrocardiogram was obtained from
the A-B lead. The QT interval was corrected with Van de Water's
formula: QTc ¼ QT  0.087  (RR  1000) (11), since the formula
was originally developed for dogs and the heart rate of micro-
minipigs was similar to that of dogs under the halothane anes-
thesia in our preliminary experiments.
2.2. Experimental protocol
The aortic and left ventricular pressures together with the
electrocardiogram were monitored by using a polygraph system
(RM-6000, Nihon Kohden, Co., Tokyo), and analyzed with a real-
time fully automatic data analysis system (Win VAS 3 for Win-
dows ver. 1.1R24v; Physio-Tech, Co., Ltd., Tokyo). Three recordings
of consecutive complexes were used to calculate the mean for the
cardiovascular variables. After the basal assessment, pilsicainide in
a dose of 1 mg/kg was intravenously infused over 10 min, and each
variable was assessed at 5, 10, 15, 20 and 30 min after the start of
administration (n ¼ 4). The effect of verapamil at a dose of 0.1 mg/
kg was assessed in the same manner to that of pilsicainide (n ¼ 4).
Meanwhile, E-4031 was intravenously infused at a low dose of
0.01 mg/kg over 10 min, and each parameter was assessed at 5, 10,
15, 20 and 30 min after the start of drug administration (n ¼ 5).
Then, additional E-4031 was infused at a high dose of 0.1 mg/kg
over 10 min, and each variable was assessed at 5, 10, 15, 20, 30, 45
and 60 min after the start of administration. The doses of pilsicai-
nide, verapamil and E-4031 in this study were determined based on
the previous reports (5e7,12).
2.3. Plasma drug concentration
A volume of 2 mL of blood was drawn from the femoral artery
with heparinized syringe to measure the plasma drug concentra-
tion at 10, 15 and 30 min after the start of pilsicainide and verap-
amil; at 5, 10, 15 and 30 min after the start of the low-dose of
E-4031; and at 5, 10, 15, 30 and 60 min after the start of the high-
dose of E-4031. The blood samples were centrifuged at 1,500g for
30 min at 4  C to prepare their plasma, which were then stored
at 80  C until the drug concentration was measured. The plasma
concentrations of pilsicainide, verapamil and E-4031 were deter-
mined with HPLC (13e15).
2.4. Drugs
Ketamine (Ketalar®, Daiichi Sankyo Co., Ltd., Tokyo), xylazine
(Celactal®, Bayer Healthcare Co., Tokyo), propofol (Frensenius Kabi
Co., Ltd., Tokyo), halothane (Fluothane®, Takeda Pharmaceutical Co.,
Ltd., Osaka), heparin calcium (Caprocin®, Sawai Pharmaceutical Co.,
Ltd., Osaka), pilsicainide hydrochloride (Sunrhythm Inj®, Daiichi
Sankyo Co., Ltd., Tokyo), verapamil hydrochloride (Vasolan Inj®,
Eisai Co., Ltd., Tokyo) and E-4031 (SigmaeAldrich Co., LLC, St. Louis,
MO, USA) were purchased. Pilsicainide hydrochloride and verap-
amil hydrochloride were diluted with normal saline in a concen-
tration of 1 and 0.1 mg/mL, respectively, whereas E-4031 was
dissolved with normal saline in concentrations of 0.01 and 0.1 mg/
mL.
2.5. Statistical analysis
Data are presented as the mean ± S.E.M. The statistical signifi-
cances within a parameter were evaluated by one-way, repeated-
measures analysis of variance (ANOVA) followed by Contrasts as a
post-hoc test for mean values comparison. A p-value <0.05 was
considered to be statistically significant.
 S. Matsukura et al. / Journal of Pharmacological Sciences 133 (2017) 103e109
3. Results
Representative tracings showing the effects of pilsicainide,
verapamil and E-4031 on the electrocardiogram, aortic pressure
and left ventricular pressure are depicted in Fig. 2. Time courses of
changes in the plasma concentration; the heart rate, mean blood
pressure, left ventricular end-diastolic pressure and LVdP/dtmax;
and the PR interval, QRS width, QT interval and QTc are summarized
in Fig. 3 (n ¼ 4 for pilsicainide and verapamil; n ¼ 5 for E-4031).
3.1. Pharmacokinetic and pharmacodynamic profile of pilsicainide
The peak plasma concentration of pilsicainide was observed at
10 min after the start of the infusion, which was 2.37 ± 0.22 mg/mL.
The pre-drug control values (C) of the heart rate, mean blood
pressure, left ventricular end-diastolic pressure and LVdP/dtmax
were 83 ± 6 bpm, 58 ± 3 mmHg, 11 ± 2 mmHg and 612 ± 43 mmHg/
s, respectively. Pilsicainide significantly decreased the heart rate,
mean blood pressure and LVdP/dtmax for 10e30 min after the start
of administration, whereas no significant change was observed in
the left ventricular end-diastolic pressure. The pre-drug control
values (C) of the PR interval, QRS width, QT interval and QTc were
128 ± 9 ms, 88 ± 9 ms, 418 ± 37 ms and 441 ± 39, respectively.
Pilsicainide significantly prolonged the PR interval, QRS width, QT
interval and QTc for 10e30 min.
3.2. Pharmacokinetic and pharmacodynamic profile of verapamil
The peak plasma concentration of verapamil was observed at
10 min after the start of the infusion, which was 627 ± 77 ng/mL.
The pre-drug control values (C) of the heart rate, mean blood
pressure, left ventricular end-diastolic pressure and LVdP/dtmax
were 72 ± 4 bpm, 48 ± 6 mmHg, 12 ± 1 mmHg and 456 ± 39 mmHg/
s, respectively. Verapamil significantly decreased the heart rate and
mean blood pressure for 5e30 min, and the LVdP/dtmax for
15e30 min after the start of administration, whereas no significant
change was observed in the left ventricular end-diastolic pressure.
The pre-drug control values (C) of the PR interval, QRS width, QT
interval and QTc were 132 ± 7 ms, 80 ± 5 ms, 444 ± 19 ms and
458 ± 16, respectively. Verapamil significantly prolonged the PR
interval for 10e30 min; but shortened the QRS width for
10e20 min and the QTc for 10e30 min, whereas no significant
change was observed in the QT interval.
3.3. Pharmacokinetic and pharmacodynamic profile of E-4031
The peak plasma concentrations of the low and high doses of E-
4031 were observed at 10 min after the start of the infusion, which
were 29 ± 12 and 594 ± 309 ng/mL, respectively. The pre-drug
control values (C) of the heart rate, mean blood pressure, left
ventricular end-diastolic pressure and LVdP/dtmax were 82 ± 19
bpm, 66 ± 7 mmHg, 14 ± 1 mmHg and 734 ± 56 mmHg/s,
respectively. The low dose decreased the heart rate for 15e20 min,
whereas no significant change was detected in the other car-
diohemodynamic variables. The high dose decreased the heart rate
for 5e30 min, the mean blood pressure for 5e60 min and the LVdP/
dtmax for 10e30 min, whereas no significant change was observed
in the left ventricular end-diastolic pressure. The pre-drug control
values (C) of the PR interval, QRS width, QT interval and QTc were
132 ± 14 ms, 73 ± 5 ms, 416 ± 47 ms and 426 ± 31, respectively. The
low dose prolonged the QT interval and QTc for 5e30 min, and high
dose prolonged them for 5e60 min, whereas no significant change
was observed in the PR interval or QRS width.
105
4. Discussion
In the present study, we pharmacologically characterized
microminipig as an in vivo experimental animal by assessing typical
cardiac ion-channel modulators-induced cardiovascular responses;
and found that most of the results were directionally similar to those
reported in humans and dogs, but each extent of their changes was
greater in microminipigs at equivalent doses (5e8,16e20).
4.1. Pharmacokinetic profile
Pilsicainide in the dose of 1 mg/kg over 10 min provided peak
plasma concentration of 2.37 mg/mL in microminipigs. Meanwhile in
a previous clinical phase I study, the maximum plasma concentra-
tions of pilsicainide after intravenous infusions of 0.25, 0.50 and
0.75 mg/kg over 10 min have been reported to be 0.34, 0.54 and
1.05 mg/mL (n ¼ 10 for each dose), respectively (16). Supposing that
1 mg/kg of pilsicainide was infused over 10 min in these subjects,
estimated peak plasma concentration would be approximately
1.4 mg/mL, which suggests that the plasma concentration could be
1.7 times higher in microminipigs than in humans. Meanwhile in
dogs anesthetized with sodium pentobarbital, when 1 mg/kg of
pilsicainide was injected, plasma concentration reached 1.4 ± 0.1 mg/
mL (n ¼ 6) after 2 min (17), indicating that the plasma concentration
was also 1.7 times higher in microminipigs.
Verapamil in the dose of 0.1 mg/kg over 10 min provided peak
plasma concentration of 627 ng/mL in microminipigs. Meanwhile in
patients with chronic stable angina who were taking at least 40 mg/
day of oral propranolol, the plasma concentration was 214 ± 108 ng/
mL (n ¼ 10) at the cessation of intravenous 2 min infusion of 0.1 mg/
kg of verapamil (18). As well regarding a clinical phase I study in
healthy males, the maximum plasma concentration of verapamil
after intravenous infusion of 5 mg/body over 5 min was 168 ± 11 ng/
mL (n ¼ 3) according to interview form from the manufacturer (Eisai
Co., Ltd.). These previous information suggest that the plasma con-
centration might be at least 2.9 times higher in microminipigs than
in humans. Similarly in the halothane-anesthetized dogs, the
maximum plasma concentration was 132 ± 67 ng/mL (n ¼ 6) after
one-shot injection of 0.1 mg/kg of verapamil (19), indicating that the
plasma concentration was at least 4.8 times higher in microminipigs.
E-4031 in doses of 10 mg/kg over 10 min and 100 mg/kg over
10 min attained peak plasma concentrations of 29 and 594 ng/mL in
microminipigs, respectively. Meanwhile in patients with apparently
normal electrophysiological study results, E-4031 in doses of 1.5, 3
and 6 mg/kg over 5 min provided peak plasma concentrations of
4.43 ± 5.78 (n ¼ 5), 9.07 ± 1.31 (n ¼ 4) and 13.30 ± 5.94 (n ¼ 7) ng/
mL (20). Supposing that 10 mg/kg of E-4031 was infused over 10 min
in these populations, estimated peak plasma concentration would
be 16 ng/mL, which suggests that the plasma concentration could
be 1.8 times higher in microminipigs than in humans. Similarly in
the halothane-anesthetized dogs, the maximum plasma concen-
tration of E-4031 was 182 ± 48 ng/mL (n ¼ 4) after intravenous
infusion of 0.1 mg/kg over 10 min, indicating that the plasma
concentration was 3.3 times higher in microminipigs under the
same experimental protocol (7).
Circulating blood volume has been reported to be 77 and
79e90 mL/kg in humans and dogs, respectively, whereas that in
miniature pigs was shown to be 65 mL/kg (21). Moreover, the
whole body fat content of miniature pigs has been reported to be
9.3e24.3% (22), which could be lower than that of humans. Since
microminipig was newly developed from miniature pigs, we assume
that both circulating blood volume and whole body fat content
would be similar between microminipigs and miniature pigs. Thus,
these previous knowledge; namely, smaller effective volume of
drug distribution in microminipigs, may partly explain why the
106 S. Matsukura et al. / Journal of Pharmacological Sciences 133 (2017) 103e109

Fig. 2. Typical tracings showing the AeB lead electrocardiogram (ECG), aortic pressure (AoP) and left ventricular pressure (LVP) at pre-drug control (Control) (left panels); and
15 min after the start of 1 mg/kg of intravenous infusion of pilsicainide (15 min after 1 mg/kg), 15 min after the start of 0.1 mg/kg of intravenous infusion of verapamil (15 min after
0.1 mg/kg) and 10 min after the start of 0.1 mg/kg of intravenous infusion of E-4031 (10 min after 0.1 mg/kg) (right panels).
 S. Matsukura et al. / Journal of Pharmacological Sciences 133 (2017) 103e109 107

Fig. 3. Time courses changes in the plasma drug concentration (Conc.), heart rate (HR), mean blood pressure (MBP), left ventricular end-diastolic pressure (LVEDP), maximum
upstroke velocity of the left ventricular pressure (LVdP/dtmax), PR interval (PR), QRS width (QRS), QT interval (QT) and corrected QT by Van de Water's formula (QTc) after 1 mg/kg,
i.v. of pilsicainide (n ¼ 4); 0.1 mg/kg, i.v. of verapamil (n ¼ 4); and 0.01 followed by 0.1 mg/kg, i.v. of E-4031 (n ¼ 5 except for plasma drug concentration which was n ¼ 4). Data are
presented as the mean ± S.E.M. Closed symbols represent significant differences from each control value (C) by p < 0.05.
108 S. Matsukura et al. / Journal of Pharmacological Sciences 133 (2017) 103e109

plasma drug concentrations in microminipigs were 1.7e4.8 times approximately 1, 5, 2 and 2 times greater in microminipigs than in
higher than those in humans and/or dogs, while variability in fat- dogs under the same protocol, respectively (5).
solubility of each drug as well as difference of drug-metabolizing Verapamil prolonged the PR interval by 38%, suggesting that
enzyme activities among the species might have also affected verapamil may inhibit Ca2þ channel of microminipigs' hearts (8),
their plasma concentrations (23), which needs to be further whereas it shortened the QRS width by 7% and QTc by 7%. In
elucidated. humans, verapamil in intravenous infusion of 2.5, 5 and 10 mg/
body over 4e5 min hardly alter the PR interval, QRS width and QTc
according to the interview form from the manufacturer (Eisai Co.,
4.2. Cardiohemodynamic effects
Ltd.). Meanwhile in our previous study with the halothane-
anesthetized canine model, intravenous infusion of 0.3 mg/kg
Pilsicainide decreased the heart rate by 14%, ventricular con-
over 30 min of verapamil prolonged the PR interval by 41%, whereas
tractile force by 44% and mean blood pressure by 35%. Directionally
no significant change was observed in the QRS width or QTc (6).
similar but trivial changes in the heart rate and mean blood pressure
Abbreviation of QTc by verapamil confirmed in microminipigs has
to those in microminipigs were described in healthy subjects who
not been demonstrated in humans or dogs, indirectly suggesting
received a single intravenous infusion 0.25, 0.50 and 0.75 mg/kg of
that Kþ channel density in the heart; namely, repolarization
pilsicainide over 10 min (16). Also, the decreases in the heart rate,
reserve, was smaller in microminipigs than in humans and dogs.
ventricular contractile force and mean blood pressure in micro-
Moreover, the abbreviation of repolarization period might have
minipigs were approximately 2, 2.5 and 12 times more potent than
enhanced the recovery of Naþ channel function, which might
those in the pentobarbital-anesthetized, open chest mongrel dogs,
explain verapamil-induced shortening of the QRS width in
respectively which were observed after intravenous bolus injection
microminipigs.
of 1 mg/kg of pilsicainide (17). Meanwhile, the results were different
The low and high doses of E-4031 prolonged the QT interval by
from those obtained with the halothane-anesthetized closed-chest
24% and 39%, and QTc by 21% and 34%, respectively; whereas no
beagle dogs (5), in which slight but significant positive chronotropic
significant change was detected in the PR interval or QRS width,
and inotropic effects were observed under the same protocol, sug-
indicating that E-4031 can selectively inhibit the Kþ channel of
gesting that the extent of hypotension-induced, reflex-mediated
microminipigs' hearts. In humans, E-4031 in doses of 1.5, 3 and 6 mg/
increase of sympathetic tone may be greater in the halothane-
kg over 5 min followed by 0.1, 0.2 and 0.4 mg/kg per min for 60 min
anesthetized dogs than in microminipigs.
prolonged the QT interval by 5, 9 and 13%; and QTc by 7, 11 and 15%,
Verapamil decreased the heart rate by 33%, the ventricular
respectively, whereas it did not alter the atrioventricular nodal or
contractile force by 59% and the mean blood pressure by 47% in
intraventricular conductions (20). In our previous study using the
microminipigs. In humans, verapamil did not alter the heart rate or
halothane-anesthetized canine model, the low and high doses of E-
blood pressure after the intravenous infusion of 2.5, 5 and 10 mg/
4031 prolonged the QT interval by 17% and 34%, and QTc by 15% and
body over 4e5 min according to the interview form from the
30%, respectively, under the same protocol (7). The longer QT in-
manufacturer (Eisai Co., Ltd.). While the heart rate and ventricular
terval and QTc at pre-drug basal condition in microminipigs
contractile force in our previous canine study were hardly altered
(416 ± 47 ms and 426 ± 31) compared with those in humans
after the intravenous infusion of 0.3 mg/kg over 30 min, it
(350 ± 19 ms and 379 ± 24) and dogs (280 ± 5 ms and 313 ± 8,
decreased the mean blood pressure (6), of which extent of change
respectively) may have underestimated the percent changes in the
was approximately 3 times smaller than that in microminipigs.
prolongation of microminipigs (7, 20), suggesting that the use of
The low and high doses of E-4031 decreased the heart rate in a
absolute value and/or absolute change in QT interval and QTc may
dose-related manner by 14% and 25%, respectively; moreover, the
be more reliable analysis than the percent change for
high dose decreased the ventricular contractile force by 12% and the
microminipigs.
mean blood pressure by 10%. There has been no report describing
Thus, microminipigs are considered to be more sensitive to
clinically significant cardiohemodynamic effects of E-4031 in
detect the drug-induced electrophysiological effects than humans
humans (20). Meanwhile, these results in microminipigs were
and dogs, possibly due to the unique pharmacokinetic profile and
inconsistent with our previous ones in the halothane-anesthetized
smaller repolarization reserve.
dog, in which the ventricular contractile force increased, but no
significant change was detected in the heart rate or mean blood
4.4. Study limitations
pressure (7).
Thus, microminipigs are considered to have higher sensitivity in
There are some limitations in this study. First, further analysis
detecting the drug-induced direct cardiohemodynamic effects than
using various drugs except for pilsicainide, verapamil and E-4031
humans and dogs, possibly due to smaller effective volume of drug
may be necessary to support the usefulness of microminipigs.
distribution and less great reflex-mediated increase of sympathetic
Second, there has been no established correction method for QT
tone.
interval of the halothane-anesthetized microminipigs. We
compared the drug-induced changes in the QTc values corrected
4.3. Electrophysiological effects by Van de Water's formula with those by Bazett's and Fridericia's
formuli which have been used for humans. The results were
Pilsicainide prolonged the PR interval by 36%, QRS width by essentially the same except that statistical significance was not
150%, QT interval by 31% and QTc by 28%, suggesting that pilsicai- detected by Bazett's or Fridericia's formula at 10 min after pilsi-
nide may more preferentially inhibit the Naþ channel than Ca2þ cainide; but the significance was observed by the formuli at 5 min
and Kþ channels of microminipigs' hearts (8). Meanwhile in healthy after verapamil. Further studies need to be performed to establish
human subjects, a single intravenous infusion of pilsicainide in optimal correction method for QT interval of microminipigs. Third,
doses of 0.25, 0.50 and 0.75 mg/kg over 10 min prolonged the PR we compared the drug-induced responses in microminipigs with
interval by 8, 18 and 16%, whereas no significant change was those of dogs, which was not performed with other experimental
observed in the QRS width or QTc (16). In the halothane- animals like guinea pigs, since there are previous information of
anesthetized canine model, directionally similar changes were dogs assessed under the same anesthetic conditions (5e7).
observed, but the extent of prolongation in each variable was Recently, we started to analyze the effects of pilsicainide and
 S. Matsukura et al. / Journal of Pharmacological Sciences 133 (2017) 103e109
verapamil on the electrocardiogram of the halothane-anesthetized
guinea-pig model (n ¼ 5 for each drug; unpublished observation).
Meanwhile, the effects of E-4031 on the halothane-anesthetized
guinea-pig model have been previously reported (n ¼ 5) (24).
According to the results by us and others, the effects of pilsicainide,
verapamil and E-4031 on the PR interval, QRS width, QT interval
and QTc in guinea pigs were qualitatively similar to those in
microminipigs, but the extents of changes in each electrocardio-
gram variable were less great in guinea pigs than in microminipigs.
5. Conclusions
The present study characterized microminipigs as an in vivo
experimental model by assessing the cardiovascular effects of pil-
sicainide, verapamil and E-4031 comparing those of humans and
dogs for the first time. The peak plasma concentrations of pilsi-
cainide, verapamil and E-4031 in microminipigs are 1.7e4.8 times
higher than those expected in dogs possibly due to smaller effective
volume of distribution of the drugs and less whole body fat
content in microminipigs than in dogs. The extent of the drug-
induced cardiovascular responses was generally greater in micro-
minipigs than in dogs, which could be explained by the following
possibilities; namely unique pharmacokinetic profile, less great
reflex-mediated increase of sympathetic tone and/or smaller
repolarization reserve in microminipigs. These information may
make it feasible to apply this new-type animal to a tool for
assessing cardiac safety profiles of new chemical entities like other
well-established animal models (8,12,24e26).
Conflict of interest statement
The authors have no conflicts of interest.
Acknowledgments
This study was supported in part by AMED Grant (#AS2116907E)
and JSPS KAKENHI (#16K08559). The authors thank Dr. Norie
Murayama, Ms. Asami Mori, Ms. Misako Nakatani and Mrs. Yuri
Ichikawa for their technical assistance.
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