The Synergism Of Amlodipine And Telmisartan On The Treatment Of Hypertension In Rats

INTRODUCTIONHypertensive complications such as stroke, heart failure, renal failure and myocardial infarction are often lethal. End-organ damage (EOD) is the early phase of these complications in hypertension. It is well accepted that reduction of blood pressure (BP) can effectively reduce or reverse EOD and consequently prevent the possible complications in hypertension.Randomized controlled trials have shown that single drug treatment is usually not adequate to achieve BP goal in most hypertensive patients. Initiating therapy with more than one agent offers the potential advantages of achieving blood pressure control more rapidly and avoiding dose-related adverse effects of individual drugs by producing greater BP reduction at lower doses of the component agents.Amlodipine, a calcium channel blocker (CCB), and telmisartan, an AT(1) receptor blocker (ARB), are two widely used drugs in the treatment of hypertension and the prevention of hypertensive complications. They belong to two different classes of antihypertensives and the mechanisms of action are quite different for these two drugs. So they might have synergistic effect on BP reduction. In fact, the beneficial effects of the combination of amlodipine and ARB on endothelial function have been found to be greater than the effect of either drug alone. However, it remains to be established whether the combination of amlodipine and telmisartan would have a synergistic effect on blood pressure reduction, and to be founded the optimal combining proportion of these 2 drugs and their effects on hypertensive organ damages. Therefore, the present work was designed to investigate the optimal combining proportion and the preventive effect of the combination on the myocardial hypertrophy and renal injury in hypertensive rats.METHODSAnimals and chemicalsAmlodipine was purchased from Zhejiang Pharmaceutical Co. Ltd (Zhejiang, China) and telmisartan was purchased from Jiangsu Pharmaceutical Co. Ltd (Jiangsu, China). Male Sprague–DawleyN (SD) rats (used for preparation of hypertensive models) were purchased from the Sino-British SIPPR/BK Lab Animal Ltd (Shanghai, China). Male spontaneously hypertensive rats (SHR) were provided by the animal center of our university. The rats were housed with controlled temperature (23–25℃) and lighting (08:00–20:00 h light, 20:00–08:00 h 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.BP measurements Systolic BP (SBP), diastolic BP (DBP) and heart period (HP) were continuously recorded described in previous studies. 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. In experiment 1 and 2, the other catheter was placed into the stomach through a midabdominal incision for the drugs administration. The catheters were exteriorized through the interscapular skin. After operation, each animal was treated intramuscularly with a dose of penicillin (8×104 IU) and housed individually with controlled temperature (23-25°C) and with free access to food and tap water. After one day recovery, 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 about 4-hr habituation, the BP signal was digitized by a microcomputer. SBP, DBP and HP values from every heartbeat were determined on line. The mean values of these parameters during a period of time for each rat were calculated. The standard deviation of the mean was calculated and defined as the quantitative parameter of variability; that is systolic blood pressure variability (SBPV), diastolic blood pressure variability (DBPV) and heart period variability (HPV).Preparation of two-kidney, one-clip (2K1C) hypertensive ratsMale SD rats weighing 180–200 g were anaesthetized with a combination of ketamine (40 mg/kg) and diazepam (6 mg/kg). The left renal artery of each animal was isolated through a flank incision, and a silver clip (0.25-mm internal gap) was placed on the left renal artery. All animals were fed standard rat chow and tap water ad libitum. On day 30 after operation, BP was measured using the previously mentioned methods. The rats with SBP>140 mmHg were used for this study, and received the treatment with different drugs.Morphological examination.In experiment 3, after determination of blood pressure, the animals were killed and the right kidney, thoracic aorta, and heart were dissected quickly. Morphological examination was performed using the previously mentioned methods. Ratios of heart weight to body weight (HW/BW), left ventricular weight to body weight (LVW/BW) and aortic weight to the length of aorta (AW/length) were calculated. Histopathological observation was also carried out with our conventional method.Glomerulosclerosis scoreFor the semi-quantitative evaluation of glomerulular damage, the glomerulosclerosis score (GSS) was defined as previously described. On the light microscopic specimens, approximately 50 glomeruli from the outer cortex and the same number of glomeruli from the inner cortex for each kidney were graded according to the degree of sclerosis: 0, no mesangial expansion; 1, mild mesangial expansion (<30% of a glomerular area); 2, moderate mesangial expansion (30-60% of a glomerular area); 3, marked mesangial expansion (>60% of a glomerular area); and 4, sclerosis was global. This was performed by one observer in a blind fashion using coded slides. A weighted composite sclerosis score was then calculated for each kidney according to the following formula: glomerulosclerosis score [1×(number of grade 1 glomeruli)+2×(number of grade 2 glomeruli)+3×(number of grade 3 glomeruli)+4×(number of grade 4 glomeruli)]×100 /(number of glomeruli observed).Experimental protocolsExperiment 1: effect of a single dose of different combinations of amlodipine and telmisartan on the BP in SHRTwo hundred and fifty SHR aged 40 weeks were used in this study. They were divided into 23 groups (n=10-11 per group), and treated by the following drugs: amlodipine (1mg, 2mg, 4 mg/kg), telmisartan (4, 8, 12, 16, 20 mg/kg) and the combination of amlodipine and telmisartan (4+4, 2+4, 4+8, 4+12, 1+4, 2+8, 4+16, 2+12, 1+8, 2+16, 2+20, 1+12, 1+16, 1+20 mg/kg). The drugs were dissolved in the 0.8% carboxymethylcellulose sodium (CMC) and were given via a catheter inserted into the stomach at the same time as aortic catheter implantation. After about 4-hr habituation, the BP and HP before drug administration and during 6-hr after drug administration were determined in conscious freely moving rats. Before this experiment, six SHR were given a dose of vehicle (0.8% CMC). No significant changes in BP and HP were observed in these control rats.Experiment 2: effect of the single dose of the combination of amlodipine and telmisartan (1:6) on the BP in 2K1C rats The rats with SBP>140 mmHg after 2K1C operation were used for this study. Animals were divided into six groups (n=10-11 per group). Amlodipine (1mg/kg), telmisartan (6 mg/kg) and their combinations (1:6: 0.5+3, 1+6, 2+12 mg/kg) were dissolved in the 0.8% carboxymethylcellulose sodium (CMC). Drugs were administered by catheter of gastric fistula implanted 1d before the experiment. After about 4-hr habituation, the BP and HP before drug administration and during 6-hr after drug administration were determined in conscious freely moving rats.Experiment 3: effect of long-term treatment of amlodipine and telmisartan in optimal combining proportion on the BP, EOD in SHRSeventy five SHR were divided into six groups (n=12-13 per group). Amlodipine and telmisartan were mixed in the rat chow. The consumption of rat chow containing drugs was determined previously. The content of drugs in the rat chow was calculated according to the chow consumption. The daily ingested dose of amlodipine, telmisartan and their combinations (1:6) were about 1, 6 3.5, 7 and 14 mg/kg per day, respectively. The control group received the same diet without any drugs. After 4 months of drug administration, the BP was recorded during 6-hr, then the BPV was calculated in conscious freely moving rats. Histopathological examinations were performed after BP recording with mentioned methods.Probability sum testTo determine if the drugs were acting synergistically, we used the probability sum test (q test). Compared with the mean values in the control group of rats, treated rats with a decrease in blood pressure >15 mmHg were defined as responders. The formula used is as follows: q=PA+B/(PA+PB?PA×PB). Here, A and B indicate drug A and drug B; P is the percentage of responders in each group. PA+B is the real percentage of responders and (PA+PB–PA×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 was <0.85, the combination was thought to be antagonistic, when q > 1.15, the combination was thought to be synergistic, and when q was between 0.85 and 1.15, the combination was thought to be additive.Statistical analysisData are expressed as mean±SD. The comparisons between pre- and postdrug were made by paired t tests and between 2 groups by unpaired t tests. The comparisons among different groups were made by one-way analysis of variance. P<0.05 was considered statistically significant.RESULTSThere is a synergistic interaction between amlodipine and telmisartan on BP reduction. The optimal proportion was found at 1:6. The synergistic effect of this proportion (1:6) was also seen in 2K1C rats. Long-term treatment with this combination presents a beneficial effect on the reduction of BP and BPV. The EOD, including myocardial hypertrophy, glomerular atrophy and fibrosis were significantly attenuated by this combination.CONCLUSSIONIn conclusion, there is a synergistic interaction between amlodipine and telmisartan on BP reduction in different hypertensive animals. The optimal proportion of the two drugs is 1:6.We found that long-term treatment with the combinations of the two drugs in fixed appropriate (1:6) presents not only with regard to reduction in BP, but also with regard to reduction in BPV and prevention of EOD.