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Introduction
Blood pressure (BP) is not constant, and can vary
spontaneously. This variation is defined as blood pressure
variability (BPV). BPV is increased in hypertensive humans
and animals[1-3]. Furthermore, BPV is positively related to
the severity of organ damage in hypertensive humans and
rats[4-6]. In other words, increased BPV can produce organ
damage. Therefore, it has been proposed that
antihypertensive drugs with a BP-stabilizing effect would act to protect
organs in the treatment of hypertension. We have
demonstrated that long-term treatment with ketanserin, candesartan,
nitrendipine , a combination of nitrendipine and atenolol,
and a hydrochlorothiazide mixture not only decrease BP, but
also decrease BPV, and have obvious effects on organ
protec-tion in spontaneously hypertensive rats as well. Importantly,
organ protection was attributed to the decrease in
BPV[7-10].
Clinically, combination therapy against hypertension
using 2 or more drugs from different classes can produce
better drug efficacy[11]. Furthermore, the use of such
synergistic therapy is also recommended for the initial treatment
of hypertension[12]. Both b-blockers and dihydropyridine
calcium antagonists are widely used in the treatment of
hypertension. Atenolol and amlodipine are examples of
long-acting drugs from these 2 classes. We hypothesized that
these 2 drugs combined would produce a synergistic effect
in the treatment of hypertension. Therefore, this study was
designed to investigate the synergistic effects of atenolol
and amlodipine on both lowing and stabilizing BP in 2K1C
renovascular hypertensive rats (RVHR).
Materials and methods
Drugs and drug administration Amlodipine (Nanjing
Pharmaceutical Co, Nanjing, China), atenolol (Shanghai
Second Pharmaceutical Co, Shanghai, China) and a
combination of these 2 drugs were dissolved in 0.8%
carboxymethylcellulose sodium (CMC). The doses used were as
follows: atenolol 10.0 mg/kg , amlodipine 1.0 mg/kg and 5.0+0.5 mg/kg, 10.0+1.0 mg/kg, and 20.0+2.0 mg/kg combinations
of the 2 drugs. Five groups of rats received
antihypertensive drugs and one group of rats received 0.8% CMC as
control. Drugs were administered by a catheter in a gastric
fistula implanted 3 days before treatment.
Animals and RVHR preparation Male Sprague-Dawley
rats (160-180 g), purchased from the Sino-British SIPPR/BK
Lab Animal Ltd (Shanghai, China), were anesthetized with a
combination of ketamine (40 mg/kg) and diazepam (6 mg/kg).
The right renal artery of each animal was isolated through a
flank incision as described
previously[13], and a silver clip
(0.2 mm internal gap) was placed on the renal artery. Five
weeks after placement of the clip, the systolic blood
pressure (SBP) of rats was measured by using the tail-cuff method
(CB10; Alcott Biotech Co, Shanghai, China). In total, 48
RVHR whose SBP was greater than 160 mmHg were used in
this study. Rats were kept in a controlled temperature (23
°C-25 °C) and lighting (light 08:00-20:00, dark
20:00-08:00) environment, and had free access to food and tap water. All
the animals used in this work received humane care in
compliance with institutional animal care guidelines.
BP and BPV measurement SBP, diastolic blood
pressure (DBP) and heart period (HP) were continuously recorded
using a previously described
technique[10]. Briefly, rats were anesthetized by injection (ip) with a combination of ketamine
(40 mg/kg) and diazepam (6 mg/kg). A floating polyethylene
catheter was inserted into the lower abdominal aorta via the
left femoral artery for BP measurement, and another catheter
was placed into the stomach via a mid-abdominal incision
for drug administration. The catheters were exteriorized
through the interscapular skin. After a 2-d recovery period,
the animals were placed for BP recording in individual
cylindrical cages containing food and water. The aortic catheter
was connected to a BP transducer via a rotating swivel that
allowed the animals to move freely in the cage. After
approximately 14-h habituation, at 9:00 o¡¯clock the BP signal
was begun to be digitized by a microcomputer. One hour
later, at 10:00 o¡¯clock the drug was given via the catheter in
the gastric fistula. SBP, DBP, and HP values were recorded
beat-to-beat for 25 h, up to 10:00 o¡¯clock on the second day.
The mean values of these parameters during a designated
period were calculated and served as SBP, DBP and HP
values. The standard deviation of all values obtained over
24 h was denoted as the quantitative parameter of variability;
that is, SBP variability (SBPV), DBP variability (DBPV), and
HP variability (HPV) for each rat.
Probability sum test To determine whether the drugs
were acting synergistically, we used the probability sum test.
This test comes from classic probability analysis and is
useful for evaluating the synergistic interactions between 2 drugs
(q test)[10,14,15].
In the present work, we used the following criteria.
Compared with the mean values from control rats, treated rats
with a decrease in BP (SBP or DBP) >20 mmHg were defined
as responders and rats with a decrease in BP£20 mmHg were
defined as non-responders. For BPV (SBPV or DBPV), the
criterion was 2 mmHg. The formula used is as follows:
q=PA+B/(PA
+PB-PA×
PB). Here, A and B indicate drug A and
drug B; P (probability) is the percentage of responders in
each group. PA+B is the real percentage of responders and
(PA+PB-P
A×PB) is the expected response rate.
PA+PB is the sum of the probabilities when drug A and drug B are used
alone.
PA×PB is the probability of rats responding to both
drugs when they were used alone. When q<0.85, the
combination is antagonistic, when q>1.15, the combination is
synergistic, and when q is between 0.85 and 1.15, the
combination is additive.
Statistical analysis Data were expressed as mean±SEM.
Comparisons between values obtained in the same group
before and after drug administration were made using the
paired t-test. Comparisons among groups were carried out
using ANOVA followed by Duncan¡¯s test. P<0.05 was
considered statistically significant.
Results
Effects of atenolol and amlodipine on BP and HP at 24 h
after treatment The effects of atenolol and amlodipine alone
and in combination on BP and HP at 24 h after administration
in 2K1C RVHR are shown in Table 1. The mean values of
these variables 1 h before administration serve as the
control values and are defined as "before"; the mean values at
24 h after administration are defined as "after". We found
that SBP was significantly decreased in all 3 groups treated
with both atenolol and amlodipine when compared with
"before". A significantly lower DBP was also found in 2
groups of rats treated with both atenolol and amlodipine
(10.0+1.0 mg/kg and 20.0+2.0 mg/kg) when compared with "before".
Relative to "before" , the mean HP value at 24 h after
administration was higher only in rats treated with atenolol (10.0
mg/kg) alone. Of the 3 groups treated with both atenolol and
amlodipine, we found that both the intermediate and the high
doses reduced SBP at 24 h after administration more
effectively than the low dose.
Effects of atenolol and amlodipine on BP and HP for the
24-h period after administration We found that the average
SBP in control rats during the 24 h after administration was
171.0±3.5 mmHg. The average SBP over this time was
significantly lower in all 5 groups treated with either atenolol, or
amlodipine, or both when compared with the control group
(Figure 1). The minimal decrease (-13.0 mmHg) was found in
rats treated with amlodipine alone and the maximal decrease
(-39.0 mmHg) was found in rats treated with both atenolol
and amlodipine at a high dose (20.0+2.0 mg/kg). Compared
with control, the decrease in average DBP in the
experimental groups during the 24 h after administration was also
significant but not so profound. The mean value of HP during
this time was only increased in the group treated with atenolol
(10.0 mg/kg) alone. Of the 3 groups treated with both atenolol
and amlodipine, both the intermediate and the high doses
reduced SBP more obviously than the low dose (Figure 1).
Effects of atenolol and amlodipine on BPV and HPV for
the 24-h period after administration Compared with the
control group (13.00±0.53 mmHg for SBPV and 9.40±0.64
mmHg for DBPV), a significant decrease in BPV was found in
groups treated with both atenolol and amlodipine at an
intermediate (8.80±0.85 mmHg for SBPV and 7.60±0.53 mmHg for
DBPV) and high dose (8.50±0.71 mmHg for SBPV and 7.30±
0.53 mmHg for DBPV) (Figure 2).
Synergistic interaction of atenolol and amlodipine on
BP and BPV for the 24-h period after
administration Based on the results presented in Figure 1, the effectiveness of the
decrease in BP was calculated for rats individually. Rats
with a decrease in BP >20 mmHg (relative to controls) were
defined as responders and those with a decrease in BP
£20 mmHg as non-responders. The results of probability testing
are presented in Table 2. We arrived at q values of 2.29 for
SBP and 1.45 for DBP for the combination of both atenolol
and amlodipine (10.0+1.0 mg/kg). Compared with the mean
values for control rats, rats with a decrease in SBPV or DBPV
>2 mmHg were defined as responders. According to this
criterion, the q values were 1.41 for SBPV and 1.60 for DBPV
for the combination of both atenolol and amlodipine (10.0+1.0 mg/kg).
Discussion
The purpose of using fixed-dose combinations of 2
different kinds of antihypertensive drugs in the treatment of
hypertension is to obtain increased BP control and to
enhance compliance by using a single tablet that is taken once
or twice daily[16]. Furthermore, by combining 2 different
agents at lower doses, the clinical and metabolic side effects
that would be produced by either drug at higher doses can
be minimized[17]. Therefore, fixed-dose combinations of
antihypertensive drugs could potentially increase BP control,
simplify dosage regimens, improve compliance, decrease
dose-dependent side effects, and reduce costs as the
first-line treatment for
hypertension[18]. These advantages make
combination antihypertensive therapy the recommended
initial treatment, particularly in patients with end-organ
damage (EOD) or more severe initial
hypertension[19,20]. However, high BP is not a unique factor determining hypertensive EOD.
Parati et al found that for patients with similar mean
hypertension levels for the 24 h after treatment, those whose BPV
levels were lower had less severe organ damage than those
with higher BPV levels[4]. Our previous study found that in
60-week-old spontaneously hypertensive rats , the severity
of organ damage was positively related to BP
(r=0.31-0.32,
n=50, P<0.05) and BPV levels
(r=0.63-0.65, n=50, P<0.01),
and BPV was positively related to the extent of organ damage,
so BPV might be a new determinant of
EOD[6,21,22].
Therefore, it seems very important to emphasize the role
of BPV in antihypertensive therapy. However, it is not clear
how BPV can be controlled in the treatment of hypertension.
We have previously proposed 2 ways to reduce BPV in
antihypertensive therapy[6]: (1) find antihypertensive drugs that
decrease BPV even at a dose that does not affect BP, for
example ketanserin; and (2) treat patients with long-acting
antihypertensive drugs, for example candesartan or
amlo-dipine. In the present work, we investigated a third way to
control BPV in the treatment of hypertension, that is,
combination therapy.
Both b-blockers and dihydropyridine calcium antagonists
are widely used in antihypertensive therapy. The
combination of a b-blocker and a dihydropyridine calcium antagonist
is a logical choice[23], and is effective for treating
hypertensive patients with chronic renal insufficiency and left
ventricular hypertrophy[24]. Theoretically, calcium
antagonists are vasodilators and tend to increase plasma renin, therefore
combining them with b-blockers is a good
idea[25]. A combination of these compounds can also neutralize the side
effects of both, for example the initial heart rate increases
induced by dihydropyridine calcium antagonists, and the rise
in peripheral resistance elicited by some
b-blockers[26].
In terms of the synergistic effects of atenolol (10.0
mg/kg) and amlodipine (1.0 mg/kg) on BP reduction, the
findings of the present work are: (1) the combination of atenolol
and amlodipine significantly decreases BP 24 h after
administration, whereas neither drug alone had an obvious
influence; (2) both atenolol or amlodipine alone reduced the
average BP for the 24 h period after administration by less
than 15 mmHg, but in combination they decreased SBP by
up to 35 mmHg and DBP by approximately 25 mmHg. The
q values for SBP and DBP for the 24 h period after
administration were 2.29 and 1.45, respectively, which are higher than
1.15, the threshold value for synergistic effects. Concerning
the synergism of combined atenolol and amlodipine on the
reduction in BPV for the 24 h period after administration, we
found that BPV was not influenced by treatment with atenolol
or amlodipine alone, but was markedly reduced when they
were used in combination. The q values for SBPV and DBPV
for the 24 h period after administration were 1.41 and 1.60,
respectively. These findings demonstrate that the
combination of atenolol and amlodipine has a significant synergistic
effect on lowing BP and stabilizing BPV in RVHR.
Our previous studies have demonstrated that long-term
(4 months) treatment with a hydrochlorothiazide mixture,
which consisted of 5 drugs at low doses, markedly reduced
BP and BPV in spontaneously hypertensive
rats[9]. Long-term (3 months) treatment with atenolol and nitrendipine had
a significant protective effect on organs in spontaneously
hypertensive rats[8].
We conclude that atenolol and amlodipine in
combination have a synergistic effect in lowering and stabilizing BP
in RVHR. Combination therapy is likely to be the optimal
way to control BP and reduce BPV in the treatment of
hypertension.
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