Cilnidipine as an agent to lower blood pressure without sympathetic nervous activation as demonstrated by iodine-123 metaiodobenzylguanidine imaging in rat hearts.

Abstract

Administration of short-acting antihypertensive agents to patients with ischemic heart disease results in increased sympathetic nervous activity and is associated with worsened outcomes. Cilnidipine is an agent which blocks not only L-type calcium channels at the smooth muscle in the artery, but also N-type calcium channels at the presynaptic terminal. The goal of the present study was to determine the effect of cilnidipine on sympathetic nervous activity as on agent which blocks both L-type and N-type calcium channels at the presynaptic terminal, on sympathetic nervous activity in an experimental rat model using iodine-123 metaiodobenzylguanidine (MIBG) myocardial imaging.
Fourteen-week-old Wistar-Kyoto rats were divided into 3 separate groups: CTR group (control: distilled water administered), Nif group (nifedipine administered), or Cil group (cilnidipine administered). Agents were administered via a stomach tube, followed by injection of MIBG via the femoral vein. Systolic blood pressure (SBP) and heart rate (HR) were measured by tail-cuff plethysmography just prior to administration of antihypertensive drugs and 150 minutes later. Initial imaging (Ce) and delayed imaging (Cd) were defined as the sum of density counts in the region of interest created by adjusting to myocardial edge, and were corrected for both physical decay and weight. The myocardial washout rate (WR) was defined as the percent change in the count density from the initial to delayed images.
Significant decreases in SBP were seen in the Nif group (from 132 +/- 3 mmHg to 85 +/- 5 mmHg, p < 0.0001) and the Cil group (from 128 +/- 4 mmHg to 92 +/- 7 mmHg, p = 0.0008), whereas no significant change in SBP was noted in the CTR group (from 123 +/- 5 mmHg to 127 +/- 3 mmHg). HR significantly increased in the Nif group (from 290 +/- 12/min to 378 +/- 14/min, p < 0.0001) but not in the CTR (from 278 +/- 3/min to 300 +/- 6/min) or Cil (from 291 +/- 6/min to 303 +/- 5/min) groups. WR was significantly greater in the Nif group (64.7 +/- 0.5%) when compared to the CTR (56.4 +/- 1.2%, p = 0.0031) or the Cil (55.4 +/- 2.2%, p = 0.0016) groups.
In contrast to nifedipine, administration of cilnidipine did not result in increased myocardial sympathetic nervous activation.

Full text

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Original Article
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Vol. 17, No. 4, 2003
Annals of Nuclear Medicine Vol. 17, No. 4, 321–326, 2003
ORIGINAL ARTICLE
Received October 29, 2002, revision accepted April 4, 2003.
For reprint contact: Takatoshi Sakaki, M.D., Department of
Internal Medicine, Cardiovascular Division, Hyogo College
of Medicine, 1–1, Mukogawa-cho, Nishinomiya 663–8501,
JAPAN.
E-mail: takatosi@hyo-med.ac.jp
INTRODUCTION
INCREASED sympathetic nervous activity following ad-
ministration of short-acting antihypertensive drugs to
patients with ischemic heart disease is associated with a
poor prognosis,1–3 whereas use of long-acting calcium
antagonists have beneficial effects on cardiovascular
mortality and/or morbidity.4 Studies have demonstrated
Cilnidipine as an agent to lower blood pressure without sympathetic
nervous activation as demonstrated by iodine-123
metaiodobenzylguanidine imaging in rat hearts
Takatoshi SAKAKI,* Hitoshi NARUSE,* Miho MASAI,* Keiko TAKAHASHI,*
Mitsumasa OHYANAGI,* Tadaaki IWASAKI* and Minoru FUKUCHI**
*Department of Internal Medicine, Cardiovascular Division, Hyogo College of Medicine
**Department of Internal Medicine, Hyogo College of Medicine
Background: Administration of short-acting antihypertensive agents to patients with ischemic
heart disease results in increased sympathetic nervous activity and is associated with worsened
outcomes. Cilnidipine is an agent which blocks not only L-type calcium channels at the smooth
muscle in the artery, but also N-type calcium channels at the presynaptic terminal. The goal of the
present study was to determine the effect of cilnidipine on sympathetic nervous activity as on agent
which blocks both L-type and N-type calcium channels at the presynaptic terminal, on sympathetic
nervous activity in an experimental rat model using iodine-123 metaiodobenzylguanidine (MIBG)
myocardial imaging. Methods: Fourteen-week-old Wistar-Kyoto rats were divided into 3 separate
groups: CTR group (control: distilled water administered), Nif group (nifedipine administered), or
Cil group (cilnidipine administered). Agents were administered via a stomach tube, followed by
injection of MIBG via the femoral vein. Systolic blood pressure (SBP) and heart rate (HR) were
measured by tail-cuff plethysmography just prior to administration of antihypertensive drugs and
150 minutes later. Initial imaging (Ce) and delayed imaging (Cd) were defined as the sum of density
counts in the region of interest created by adjusting to myocardial edge, and were corrected for both
physical decay and weight. The myocardial washout rate (WR) was defined as the percent change
in the count density from the initial to delayed images. Results: Significant decreases in SBP were
seen in the Nif group (from 132 ± 3 mmHg to 85 ± 5 mmHg, p < 0.0001) and the Cil group (from
128 ± 4 mmHg to 92 ± 7 mmHg, p = 0.0008), whereas no significant change in SBP was noted in
the CTR group (from 123 ± 5 mmHg to 127 ± 3 mmHg). HR significantly increased in the Nif group
(from 290 ± 12/min to 378 ± 14/min, p < 0.0001) but not in the CTR (from 278 ± 3/min to 300 ±
6/min) or Cil (from 291 ± 6/min to 303 ± 5/min) groups. WR was significantly greater in the Nif
group (64.7 ± 0.5%) when compared to the CTR (56.4 ± 1.2%, p = 0.0031) or the Cil (55.4 ± 2.2%,
p = 0.0016) groups. Conclusion: In contrast to nifedipine, administration of cilnidipine did not
result in increased myocardial sympathetic nervous activation.
Key words: MIBG, washout rate, N-type Ca channel blocker, myocardial sympathetic nervous
activation

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that third-generation dihydropyridine-based calcium an-
tagonists possess less sympathetic nervous activation
effects than short-acting dihydropyridines.5 Cilnidipine,
an agent developed as a new class of calcium channel
blockers, which blocks not only L-type calcium channels
at the smooth muscle in the artery, but also N-type calcium
channels at the presynaptic terminal of the sympathetic
nerve.6,7
Measurement of the ratio of high-frequency compo-
nent (HF) to low-frequency component (LF) in heart rate
variability spectral analysis and iodine-123 metaiodo-
benzylguanidine (MIBG) imaging have been employed
to assess cardiac sympathetic nervous activity and the
severity of congestive heart failure.8–11 However, only a
few has been reported to assess changes in sympathetic
nervous activity following administration of various anti-
hypertensive agents.12,13 The goal of the present study
was to determine the effect of cilnidipine on sympathetic
nervous activity as an agent which blocks both L-type and
N-type calcium channels at the presynaptic terminal, on
sympathetic nervous activity in an experimental rat model
using iodine-123 metaiodobenzylguanidine (MIBG) myo-
cardial imaging.
METHODS
Anesthetized 14-week-old Wistar-Kyoto rats (Japan SLC,
Inc.) were divided into 3 groups: CTR group (control:
distilled water, n = 7), Nif group (nifedipine, 9 mg/kg,
Bayer Yakuhin, Ltd., n = 6), and Cil group (cilnidipine, 9
mg/kg, Ajinomoto Co., Inc., Mochida Pharmaceutical
Co., Ltd., n = 6). Anesthesia was performed first with
ether (Wako Pure Chemical Industries, Ltd.) inhalation
prior to drug administration and then with ether inhalation
anesthesia and pentobarbital (30 mg/kg I.P., Dainabot
Co., Ltd.) just prior to MIBG intravenous infusion.
Nifedipine and cilnidipine were dissolved in a 5%
solution of Arabic Gum (Wako Pure Chemical Industries,
Ltd.). Drugs and distilled water were administered via a
stomach tube. One hour later, MIBG [133.2 MBq (3.6
mCi)/kg, specific activity 74 GBq (2000 mCi)/µg, Daiichi
Radioisotope Laboratories, Ltd., Japan] was injected via
the femoral vein. A 10-minute static acquisition was made
every 30 minutes (initial) and 4 hours (delayed) after
injection of MIBG in the anterior view. Cardiac images
were acquired after each static acquisition, using a gamma
camera (PRISM2000XP) (Picker International, Inc.)
equipped with a pinhole collimator (Fig. 1). This pinhole
collimator was used with an aperature 2 mm for high-
resolution. Rats were restrained (limbs, upper teeth) in the
Fig. 1 Experimental schema. Nif: nifedipine, Cil: cilnidipine,
NA: plasma noradrenaline.
Fig. 2 MIBG imaging and calculation of WR. Initial imaging
(Ce) and delayed imaging (Cd) were defined as the sum of
density counts in the region of interest created adjust to myocar-
dial edge.
Fig. 3 Changes in SBP before and after administration of
antihypertensive drugs. SBP: systolic blood pressure. Open
bars: before administration of antihypertensive drugs, filled
bars: after administration of antihypertensive drugs. CTR group
(no antihypertensive drug), Nif group (nifedipine), Cil group
(cilnidipine).

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Vol. 17, No. 4, 2003
supine position in order to fix the imaging point and
prevent body motion artifact. Systolic blood pressure
(SBP) and heart rate (HR) were measured by tail-cuff
plethysmography (Softron Co. Ltd., Japan) just prior to
administration of antihypertensive drugs and 150 minutes
later. The room temperature was maintained at 24°C
throughout the experiment.
After the delayed imaging, the rats were sacrified and
blood sample was collected immediately. Blood samples
were store at −80°C. The plasma noradrenaline (NA)
concentraition was determined by a high-performance
liquid chromatography.
Initial imaging (Ce) and delayed imaging (Cd) were
defined as the sum of density counts in the region of
interest created by adjusting to myocardial edge, and were
corrected for both physical decay and weight. The myo-
cardial washout rate (WR) was defined as the percent
change in the count density between the initial and de-
layed images, and was calculated by following equation:
WR (%) = [(Ce − Cd)/Ce] × 100 (Fig. 2).
Statistical analysis
Data are expressed as mean ± S.E. Probability value
<0.05 was considered significant. SBP and HR in the 3
groups (CTR, Nif, Cil) were compared using 2-way
ANOVA before and after administration of antihyper-
tensive drugs. Ce, Cd, WR and NA were compared among
the 3 groups (CTR, Nif, Cil) using 1-way ANOVA. The
difference in the SBP and HR, before and after adminis-
tration of antihypertensive drugs (∆SBP and ∆HR, re-
spectively) were compared among the 3 groups (CTR,
Nif, Cil) using 1-way ANOVA. Post-hoc test (Scheffe)
was performed if significant differences were detected by
ANOVA.
RESULTS
Significant decreases in SBP were seen in the Nif group
(from 132 ± 3 mmHg to 85 ± 5 mmHg, p < 0.0001) and the
Cil group (from 128 ± 4 mmHg to 92 ± 7 mmHg, p =
0.0008), whereas no significant change in SBP was noted
Fig. 4 Changes in HR before and after administration of
antihypertensive drugs. HR: heart rate. Open bars: before ad-
ministration of antihypertensive drugs, filled bars: after admin-
istration of antihypertensive drugs. CTR group (no antihyper-
tensive drug), Nif group (nifedipine), Cil group (cilnidipine).
Fig. 5 Mean WR of each group. WR: washout rate. CTR group
(no antihypertensive drug), Nif group (nifedipine), Cil group
(cilnidipine).
Fig. 6 Mean Ce of each group. Ce: initial imaging. CTR group
(no antihypertensive drug), Nif group (nifedipine), Cil group
(cilnidipine).
Fig. 7 Mean Cd of each group. Cd: delayed imaging. CTR group
(no antihypertensive drug), Nif group (nifedipine), Cil group
(cilnidipine).

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Takatoshi Sakaki, Hitoshi Naruse, Miho Masai, et al
in the CTR group (from 123 ± 5 mmHg to 127 ± 3 mmHg).
There was no significant difference in ∆SBP between the
Nif (47 ± 6 mmHg) and the Cil (36 ± 8 mmHg) groups
(Fig. 3). HR significantly increased in the Nif group (from
290 ± 12/min to 378 ± 14/min, p < 0.0001) but not in the
CTR (from 278 ± 3/min to 300 ± 6/min) or Cil (from 291
± 6/min to 303 ± 5/min) groups. ∆HR was significantly
larger in the Nif group (89 ± 21/min) than in the CTR
group (22 ± 6/min, p = 0.0077) or Cil group (12 ± 9/min,
p = 0.0035) (Fig. 4).
WR was significantly greater in the Nif group (64.7 ±
0.5%) than in the CTR (56.4 ± 1.2%, p = 0.0031) or the Cil
(55.4 ± 2.2%, p = 0.0016) groups (Fig. 5). Ce was
significantly larger in the Nif group (65960 ± 5215) than
in the Cil group (43330 ± 1882) (p = 0.0084) (Fig. 6). Cd
did not differ among the groups (24628 ± 1737, 23235 ±
1696, and 19457 ± 1759 in the CTR, Nif, and Cil groups,
respectively) (Fig. 7).
NA was significantly greater in the Nif group (442 ±
104 pg/ml) than in the CTR group (170 ± 42 pg/ml) (p =
0.0336), and there were no significant difference between
the CTR group and the Cil group (281 ± 42 pg/ml) (Fig.
8).
Fig. 8 Mean NA of each group. NA: plasma noradrenaline.
CTR group (no antihypertensive drug), Nif group (nifedipine),
Cil group (cilnidipine).
Fig. 9 Blood pressure regulation and sympathetic nervous activation. Baroreceptors distributed in the
carotid and aorta perceive decreases in blood pressure and stimulate increases in blood pressure via the
nucleus tractus solitarius and vasomotor centers in the medulla oblongata. Peripheral sympathetic
outflow increases in response to this process with concomitant activation of N-type calcium channels
and secretion of NA resulting in an increase in heart rate. Increases in HR, WR and NA were not seen
after cilnidipine administration. This is likely secondary to antagonism of N- and L-type Ca channel in
the cardiac sympathetic nervous terminal rather than a centrally mediated phenomenon, as cilnidipine
does not pass through the blood brain barrier.

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DISCUSSION
The use of short-acting calcium channel blockers in
hypertensive patients with ischemic heart disease is asso-
ciated with poor outcomes, and thus, the use of slow-
onset, long-acting antihypertensive drugs is preferred. It
is reported that in comparison with the placebo group, the
blood pressure is lower in resting and exercise test on
cilnidipine, but the heart rate does not change.14
Various methods have been employed to measure car-
diac sympathetic nervous activation, including HF/LF
ratio in heart rate variability spectral analysis and WR in
MIBG imaging. However, HF/LF ratio is an imperfect
measure of sympathetic nervous activation as LF is af-
fected by both sympathetic and parasympathetic nerve
activity.15 In contrast, MIBG is an excellent measure of
sympathetic nervous activity16 and can yield data in the
form of visual imaging or quantitative indices (heart-to-
mediastinum ratio and WR).
While MIBG has been employed in patients with heart
failure, there are few studies that describe its use in
assessing changes in myocardial sympathetic nervous
activity with various antihypertensive agents. The reports
using MIBG in clinical hypertensive patients compared
changes in sympathetic nervous activity after admin-
istration of enalapril, nitrendipine, amlodipine and
cilnidipine12,13 and demonstrated that WR decreased with
enalapril, amlodipine and cilnidipine in comparison to
nitrendipine. However, chronic effect of antihypertensive
drugs was assessed in these reports, and there has been no
report to assess acute effect with a similarly protocol.
In the present study, both HR and WR were increased
in the Nif group, and SBP was reduced in the Nif and Cil
groups compared to control (anesthesia only). While WR
was higher in the Nif group when compared to the Cil
group, ∆SBP did not differ between the two groups,
suggesting that SBP decreased without sympathetic ner-
vous activation following administration of cilnidipine.
We speculate that the effect of cilnidipine is mediated
through antagonism of the N-type Ca channel.
A key regulator of blood pressure is the carotid and
aortic baroreflex. Baroreceptors distributed in the carotid
and aorta perceive decreases in blood pressure and stimu-
late increases in blood pressure via the nucleus tractus
solitarius and vasomotor centers in the medulla oblongata
(Fig. 9). Peripheral sympathetic outflow increases in
response to this process with concomitant activation of N-
type calcium channels and secretion of NA resulting in an
increase in heart rate. In the present study, increases in
HR, WR and NA were not seen after cilnidipine adminis-
tration. This is likely secondary to antagonism of N- and
L-type Ca channel in the cardiac sympathetic nervous
terminal rather than a centrally mediated phenomenon, as
cilnidipine does not pass through the blood brain barrier.17
Further, T-type calcium channels that are distributed
in the sinus node were also unlikely to be affected by
cilnidipine and nifedipine.
In the present study, NA levels were greater in the Nif
group than in the control groups, while there was no
significant difference between the Cil group and the
control group. These data are consistent with previous
studies performed in spontaneously hypertensive rats
(SHR),18 and other studies have demonstrated that there
was no significant difference in NA levels when compar-
ing 14-week-old WKY and SHR.19,20
Increased WR was associated with high Ce in the Nif
group. We speculate that this may be due to: 1) increased
reuptake of norepinephrine accompanied by increased
exocytosis, and 2) a different influence on reuptake of
norepinephrine by Ca channel blockers.
Study limitation
The action time of Nif was shorter than Cil, thus this
difference might cause more sympathetic nervous activa-
tion in Nif group. We could not resolve the problem in the
present study. Controversy would continue as to whether
acute effect of the antihypertensive drugs in the present
study brought chronic effect in the previous reports by
Sakata, et al.
CONCLUSION
These data demonstrated that cilnidipine lowers blood
pressure without the increase in sympathetic nervous
activity that is seen with nifedipine. These observations
may contribute to improvements in the clinical selection
of antihypertensive agents in different patient popula-
tions.
ACKNOWLEDGMENTS
We thank Mr. Minoru Inoue, Mr. Hiroyuki Kurosawa, Mr.
Syuhsaku Tazawa, Mr. Satoshi Ishida and the staff at Daiichi
Radioisotope Laboratories, Ltd., Japan.
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