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Introduction
Although many new drugs, or recently developed drugs were available, the classic antihypertensive drugs, such as
hydrochlorothiazide and nitrendipine, still have their place in the treatment of hypertension. This is because of their
effectiveness and their low cost. Clinically, only approximately 50% of patients achieve optimal blood pressure (BP) control
with monotherapy. Therefore, treatment with two or more antihypertensive drugs of different classes is often
necessary[1_3]. Hydrochlorothiazide, a thiazides diuretic and nitrendipine, a calcium antagonist, are two classic drugs widely used in the
treatment of hypertension. They belong to two different classes of antihypertensives and the mechanisms of action are quite
different. Generally speaking, a combination of two drugs with different action modes may be synergistic. However, there is
no evidence to show whether a combination of hydrochlorothiazide and nitren-dipine is synergistic on BP reduction.
BP is not constant and there exists a spontaneous variation in BP. This spontaneous variation is defined as blood
pressure variability (BPV). It is well known that BP level is an important determinant for the end-organ damage in hypertension.
However, BP level is certainly not the unique determinant for hypertensive organ damage. Recently, it has been proposed
that BPV may be an important factor determining organ damage in
hypertension[4_7]. In other words, antihypertensive
treatment should be aimed not only at reducing BP level but also at reducing
BPV[7_9].
Therefore, the present work was designed to investigate the synergism of hydrochlorothiazide and nitrendipine on both
BP and BPV reduction in spontaneously hypertensive rats (SHR). The possible pharmacological mechanisms of this
synergism are also discussed.
Materials and methods
Animals and chemicals Seventy male SHR, at 16 weeks of age were provided by the animal center of our university. The
animals were divided into seven groups with ten rats in each group. The rats were housed with controlled temperature
(23-25 °C) and lighting (08:00_20:00 light, 20:00_08:00 dark) and with free access to food and tap water. All the animals used in this
work received humane care in compliance with institutional animal care guidelines. Antihypertensive drugs used in this
study are as follows: hydrochlorothiazide (Nanjing Pharmaceutical Co, Nanjing, China) and nitrendipine (Shanghai Sanwei
Pharmaceutical Co, Ltd, Shanghai, China).
Drug administration The two drugs and the combination of these two drugs were dissolved in the 0.8%
carboxymethylcellulose sodium (CMC). After a preliminary study, the dosages were as follow: hydrochlorothiazide 10, 20 mg/kg, nitrendipine
5 mg, 10 mg/kg and the combination of hydrochlorothiazide and nitrendipine 10+5, 20+10 mg/kg. Each group of rats received
one dosage and 0.8% CMC was given to control rats
(n=10 for each group). Drugs were administered by catheter of gastric
fistula implanted 3 d before the experiment.
BP and BPV measurement Systolic BP (SBP), diastolic BP (DBP) and heart period (HP) were continuously recorded
described in previous studies[10,11]. Rats were anesthetized 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 (ig). The catheters
were exteriorized through the interscapular skin. After operation, each animal was treated intramuscularly with a dose of
sodium benzylpenicillin (6×104 IU) and housed individually with controlled temperature (23_25 °C) and with free access to
food and tap water. After a 2-d recovery period, the animals were placed in individual cylindrical cages containing food and
water for BP recording. 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 4-h habituation, at 12:00 the BP signal was digitized by a computerized system
(MPA 2000M, Alcott Biotech, Shanghai, China). Then, at 13:00 the drug was given via the catheter of gastric fistula. SBP,
DBP, and HP values from every heartbeat were recorded for 4 h, up to 17:00, according to our preliminary studies. The mean
values and standard deviation of these parameters for each rat were calculated. The standard deviation of all values obtained
was denoted as the quantitative parameter of variability, ie, SBP variability (SBPV), DBP variability (DBPV).
Probability sum test To determine whether the combination was synergistic, we used the probability sum test
(q test)[10,12]. This test came from classic probability analysis and it was proposed for evaluating the synergism of the combination of
2 drugs. In the present work, we used the following criteria. Compared with the mean values of 1-h baseline (before drug
administration), rats with a decrease of the mean value of 4
h ³20 mmHg (in SBP) and ³15 mmHg (in DBP) were defined as
responders. For SBPV and DBPV, the rats with a decrease or increase
³1.5 mmHg were defined as responders. The formula
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 real percentage of responder and
(PA+PB_P
A×PB) is expected response rate.
(PA+PB) indicates the sum of the probabilities when drug A and drug B is used alone.
(PA×PB) is the probability of rats responding to
both drugs when they were used alone, that is assuming the two drugs act independently. When
q<0.85 the combination is antagonistic; when
q>1.15 it is synergistic. And when
q is between 0.85 and 1.15, it is additive.
Statistical analysis Data were expressed as mean±SEM. Comparisons among groups were made by using ANOVA and
comparisons between values obtained in the same group before and after drug administration were made by using the
paired t-test. P<0.05 was considered statistically significant.
Results
Effects of nitrendipine and hydrochlorothiazide alone and in combination on BP in
SHR The baseline SBP in control SHR was 175±12 mmHg and DBP was 110±14 mmHg. BP remained unchanged after administration of vehicle (Table 1). The
baseline SBP and DBP in all 6 treated groups of SHR were similar to those in the control group. SBP and DBP (means of a
period of 4 h) were significantly decreased in rats treated with the combination and with the high dose of hydrochlorothiazide
or nitrendipine alone. The minimal decrease in SBP (-6 mmHg) was observed in rats treated with a small dose (10 mg/kg) of
hydrochlorothiazide and the maximal decrease in SBP (-41 mmHg) in rats treated with the combination of hydrochlorothiazide
and nitrendipine at high doses (20+10 mg/kg). A significant dose-effect relationship was found in the combination group.
The decrease in 4-h DBP was not as profound as the decrease in SBP. Only the combination of hydrochlorothiazide and
nitrendipine at high doses (20+10 mg/kg) decreased the DBP of rats more than 20 mmHg. In terms of HP, only a slight
tachycardia (shortened HP) was seen in rats treated with large-dose nitrendipine and the combination at a large dose (Table
1).
Effects of nitrendipine and hydrochlorothiazide alone, and in combination on SBPV and DBPV after administration in
SHR The mean values of BPV over 1 h just before drug administration served as the baseline. The mean values of BPV over
4 h after drug administration were compared with baseline values. The baseline SBPV and DBPV in control SHR were
9.67±0.16 mmHg and 6.51±0.19 mmHg, respectively. BPV remained unchanged after administration of the vehicle. The baseline BPV
in all 6 treated groups of SHR was similar to those in the control group. It was found that SBPV significantly decreased in rats
treated with the combination and with the high dose of nitrendipine alone and DBPV significantly decreased only in rats
treated with the combination (Figure 1).
Synergism of hydrochlorothiazide and nitrendipine on SBP and SBPV in
SHR Based on the results presented in Table 2, the effectiveness of the decrease in SBP was calculated individually. Rats with a decrease in SBP
(³20 mmHg) were defined as responder and with a decrease in SBP (<20 mmHg) as non-responder. According to this criterion, the probability was
defined as the ratio of responder by the total number rats studied in a group. The result of the probability is presented in
Table 2. Using the formula described in the method section, we have
q-values of 1.79 for the low-dose group (10+5 mg/kg)
and 1.23 for the high-dose group (20+10 mg/kg) with the combination of hydrochlorothiazide and nitrendipine.
These q values were >1.15, and the combination was synergistic. However,
q values for DBP were not the same as for SBP. The
synergism was only seen in the large-dose group
(q=1.30). BPV was calculated using the data obtained during a period of 4
h after the drug administration. Compared with the baseline, a decrease of SBPV
(³1.5 mmHg) was defined as responder. According to this criterion and using the formula described in the Method section, we have the
q-values 1.79 for the low dose group and 1.39 for the high-dose group with the combination of hydrochlorothiazide and nitrendipine. In terms of the effect
on DBPV, we have q values of 1.50 for the low-dose group and 2.22 for the large-dose group. As all
q values were >1.15, the combination was judged as synergistic.
Hourly analysis of the effects of nitrendipine and hydro-chlorothiazide alone and in combination on
BP Figure 2 shows hourly analysis of the effects of hydrochlorothiazide and nitrendipine alone and in combination on BP. In the control group,
SBP remained unchanged in the 1st to 4th hour after vehicle administration (data not shown). It was found that SBP was
significantly decreased in the 2nd hour after the treatment with a small dose of hydro-chlorothiazide, in the 2nd and 3rd hour
after the treatment with a small dose of nitrendipine, and in the 2nd to 4th hour after the treatment with a small dose of the
combination (upper panel of Figure 2). In large dose groups, SBP was significantly decreased in the 1st to 3rd hour after the
treatment with hydrochloro-thiazide, in the 2nd and 3rd hours after the treatment with nitrendipine, and in all 4 hour studied
after the treatment with combination (bottom panel of Figure 2). From the hourly analysis, it was found that the combination
of hydrochlorothiazide and nitrendipine not only potentiated but also prolonged BP reduction.
Discussion
The main findings of the present work may be summarized as follows: (i) there existed an important synergism of
hydrochlorothiazide and nitrendipine on SBP reduction. This synergism was greater when treated with small doses; (ii) there
existed a synergism of hydrochlorothiazide and nitrendipine on BPV reduction; (iii) the combination of these two drugs
prolonged the antihypertensive effect in SHR.
Theoretically, using two antihypertensive agents with different modes of action can increase blood pressure control.
Hydrochlorothiazide and nitrendipine are two drugs widely used and often used in
combination[12,13]. It was reported that the
combination was found to be effective in lowering BP to desirable levels with minimal side
effects[13]. Practically, however, it is not easy to judge whether a combination of two drugs is synergic or not. The probability sum test was first proposed by
Jin in 1980[10] and adapted for evaluating the synergism of two antihypertensive drugs recently. With this method, we
have studied the synergic effects on BP lowering and BPV reduction of two combinations: atenolol and nitrendipine, atenolol
and amlodipine[12,14,15]. This method was also used in the present work to evaluate the possible synergism of
hydrochlorothiazide and nitrendipine.
Usually, antihypertensive drugs are used orally in hypertensive patients. To mimic this clinical administration, drugs are
given by gavage through the mouth in animal studies. However, stress is unavoidable when the gavage is used. In the
present study, drugs were given ig by using a gastric catheter implanted 3 d before the
experiments[10,15]. This method allows
us to record the observing variables during or immediately after drug administration.
The present work clearly demonstrated the synergism of hydrochlorothiazide and nitrendipine on SBP reduction in
SHR. This synergism is greater when the treatment is with small doses
(q=1.79) than with large doses (q=1.23). Inter-estingly,
the combination of these drugs could prolong their antihypertensive effect. Even in large doses, there was no effect in the
4th hour after the administration of hydro-chlorotiazide or nitrendipine. However, the antihypertensive effect of the
combination of these drugs lasted more than 4 h even in the small-dose group. In terms of DBP, the synergism was not obvious as
SBP, and it was only seen in the large-dose group. It is well known that hydrochlorothiazide reduces the blood volume in
acute treatment and nitrendipine dilate the resistant arteries. The great difference in their action modes might be the basis of
synergism when they were used in combination.
BPV is associated with hypertensive organ damage in
SHR[7,16]. Furthermore it was found that high BPV alone, without
hypertension, could also induce organ damage in sinoaortic denervated normotensive
rats[15,16]. Recently, it was reported
that BPV reduction contributed importantly to the organ protective effects of some antihypertensive
drugs[7_9]. Therefore, we propose BPV reduction as a new strategy for the treatment of
hypertension[7,8]. In the present work, it was found that the
monotherapies with hydrochlorotiazide or nitrendipine had no or minor effects on BPV reduction. However, the combination
of hydrochlorotiazide and nitrendi-pine possessed a significant effect on BPV reduction. This effect will be beneficial in the
treatment of hypertension.
In conclusion, the combination of hydrochlorothiazide and nitrendipine possessed an obvious synergism on both BP
lowering and BPV reduction in conscious free-moving SHR.
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