Synergism Of Amlodipine And Candesartan On Lowering, Stabilizing Blood Pressure And Organ Protection

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. Preventing or reversing EOD is therefore essential in the treatment of hypertension. The blood pressure (BP) level is a well known determinant for hypertensive EOD, and BP reduction is very important for organ protectioni. However, BP is certainly not the unique determinant for EOD. Blood pressure variability (BPV) is found as another important factor determining the end organ damage (EOD) in hypertension. In consideration of these, it has been proposed that an antihypertensive drug with a BP-stabilizing effect would benefit the hypertensives additionally. Randomized controlled trials have shown that single drug treatment usually is not adequate to achieve blood pressure goal in most hypertensive patients.Therefore, treatment with two or more antihypertensive drugs of different classes is often necessary. It is even suggested that the use of combination therapy will be appropriate as initial treatment. Generally speaking, a combination of two drugs with different action modes may be synergistic. Our hypothesis was that the appropriate combination therapy might make better use of the synergism in the treatment of hypertension. Besides, initiating therapy with appropriate proportion offers the potential advantages of achieving blood pressure control more rapidly and avoiding dose-related adverse effects of individual drugs by producing greater blood pressure reduction at lower doses of the component agents. The present work was designed to test this hypothesis. Both angiotensin II type 1 receptor blocker candesartan and calcium channel blocker amlodipine were used in the studies.MEHTOD AnimalsTwo hundred female SHR, at 12 weeks of age and seventh five male SHR, at 6 weeks of age were provided by the Animal Center of Second Military Medical University. One hundred male Spargue-Dawley rats (used for preparation of hypertensive models) were purchased from the Sino-British SIPPR/BK Lab Animal Ltd. (Shanghai, China). The rats were housed with controlled temperature (23?25℃) and lighting (08:00–20:00 light, 20:00–08:00 dark) and with free access to chow and tap water. All the animals used in this work received humane care in compliance with institutional animal care guidelines.Preparation of 2K1C hypertensive ratsMale Sprague-Dawley 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, as described previously, and a silver clip (0.2-mm internal gap) was placed on the right renal artery. All animals were fed standard rat chow and tap water ad libitum. Four weeks after placement of the clip, these rats received the treatment with different drugs and the rats with systolic blood pressure >130 mmHg were used for this study.Drugs and drug administrationAntihypertensive drugs used in this study are as follows: candesartan (Yongning Pharmaceutical Co. Zhejiang, China) and amlodipine (Shuanghe Pharmaceutical Co. Ltd. Beijing, China).In acute SHR studies, two hundred female SHR were divided into twenty groups with ten rats in each group. 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: amlodipine 0.5mg, 1mg, 2mg, 3mg/kg, candesartan 1mg, 2mg, 3mg, 4mg, 6mg, 7mg/kg and the combination of amlodipine and candesartan 0.5+1.0,0.5+2.0,0.5+4.0,1.0+1.0,1.0+2.0,1.0+3.0,1.0+4.0,1.0+6.0,1.0+7.0,2.0+1.0,2.0+2.0,2.0+4.0mg/kg. Each group of rats received one dosage. Drugs were administered by catheter of gastric fistula implanted 1d before the experiment.In acute 2K1C studies, fifty five male Sprague-Dawley rats with systolic blood pressure >130 mmHg were divided into six groups with ten or nine rats in each group. The two drugs and the combination of these two drugs were dissolved in the 0.8% carboxymethylcellulose sodium (CMC). The dosages were as follow: amlodipine 1mg/kg, candesartan 2mg/kg and the combination of amlodipine and candesartan 0.5+1, 1+2, 2+4 mg/kg. Each group of rats received one dosage. Drugs were administered by catheter of gastric fistula implanted 1d before the experiment.In chronic SHR studies, seventh five hundred male SHR were divided into six groups with twelve or thirteen rats in each group. Amlodipine and candesartan 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, such that the ingested dose of amlodipine and candesartan were about 1 and 2 mg·kg-1·d-1 and 0.5+1, 1+2, 2+4 mg·kg-1·d-1 for the combination of the two drugs. The control group received the same diet without the drugs. After 5 months of drug administration, BP was recorded during 6h, and then BPV was calculated. And histopathological examinations were performed after BP, BPV.Intra-arterial blood pressure measurementsSystolic 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, and another catheter was placed into the stomach via a mid-abdominal incision for drug administration. In chronic studies, the catheter of gastric fistula was not necessary. The catheters were exteriorized through the interscapular skin. After operation, each animal was housed individually with controlled temperature (23–25℃) and with free access to food and tap water.After a 1d 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 3h 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 6 h, up to 20:00, 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, i.e., SBP variability (SBPV), DBP variability (DBPV).Morphological examinationIn chronic studies, after determination of blood pressure, rats were sacrificed for end-organ damage evaluation. The rat was weighed and killed by decapitation. The thoracic and peritoneal cavities were immediately opened. The right kidney, aorta and heart were excised and rinsed in cold physiological saline. The right kidney was blotted, and weighed. The left ventricle was isolated, blotted, and weighed. At the same time, the aorta was cleaned of adhering fat and connective tissue. Just below the branch of the left subclavicular artery, a 30-mm-long segment of thoracic aorta was harvested, blotted, and weighed. Ratios of heart weight to body weight (VW/BW),left ventricular weight to body weight (LVW/BW), right ventricular weight to body weight (RVW/BW), and aortic weight to the length of aorta (AW/length) were calculated.Probability sum testTo determine whether the combination was synergistic and the best proportion of the two drugs was in combination, probability sum test was used. This is a classic statistical analysis and it was proposed for evaluating the synergism of the combination of two drugs (q-test). BP mean values during two periods (1h before and 6h after drug administration) were calculated. The differences of BP between these two periods were taken as the effectiveness of drug on BP. Rat with a decrease in BP (≥15 mmHg) was defined as responder and with a decrease in BP (<15 mmHg) as non-responder. The formula used to calculate the synergism (q) of the drug combination was 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?PA×PB) is the expected response rate. (PA+PB) indicates the sum of the probabilities when drug A and drug B were used alone. (PA×PB) is the probability of rats responding to both drugs when they were used alone, i.e., 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 analysisData are expressed as mean±S.E. The differences among groups were evaluated using analysis of variance followed by a two-tailed Student's unpaired t-test 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 significantCONCLUSSIONThe main findings of the present work are that: (1) there is a synergistic interaction between amlodipine and candesartan on blood pressure reduction. The synergism interaction is not constant but proportion dependent. The synergism is highest when the dose appropriate of the two drugs is 1:2. (2) The long term treatment with the combination of the two drugs in fixed appropriate presents a synergistic interaction with regard as not only blood pressure reduction, but also blood pressure variability reduction, and end-organ protection. (3) The synergism of the two drugs can also be found in 2K1C hypertensive rats.