Study design
This randomised, double-blind, controlled study was conducted at the Department of Internal Medicine and Therapeutics, IRCCS Policlinico San Matteo, PAVIA, Italy.
The study protocol was conducted in accordance with the Declaration of Helsinki and its amendments, and the Good Clinical Practice Guidelines. It was approved by the IRCCS Policlinico San Matteo Ethical Committee and all patients provided written informed consent prior to entering the study. TRIAL REGISTRATION: ClinicalTrials.gov NCT02064218.
Patients
We enrolled 151 hypertensive patients with mild to moderate hypertension, type 2 diabetes mellitus, normocholesterolemic [low density lipoprotein cholesterol (LDL-C) < 160 mg/dl], overweight outpatients, aged ≥ 18 of either sex (Table
1).
Table 1
Baseline characteristics of patients in the two treatment groups
N | 75 | 76 |
Age (years) | 60.5 ± 8.9 | 60.7 ± 8.8 |
Sex (male/female) | 37/38 | 36/40 |
BMI (Kg/m2) | 28.5 ± 1.3 | 28.2 ± 1.1 |
Duration of diabetes (months) | 9.3 ± 7.2 | 9.1 ± 7.1 |
Duration of hypertension (months) | 6.4 ± 3.6 | 6.3 ± 3.5 |
eGFR (ml/min/1.73 m2) | 84.1 ± 8.5 | 82.5 ± 7.6 |
Patients were evaluated for eligibility according to the following inclusion criteria:
The exclusion criteria were secondary hypertension, severe hypertension (SBP ≥ 180 mmHg or DBP ≥ 105 mmHg), hypertrophic cardiomyopathies due to etiologies other than hypertension, history of heart failure, history of angina, stroke, transient ischemic cerebral attack, coronary artery bypass surgery or myocardial infarction any time prior to visit 1, concurrent known symptomatic arrhythmia, liver dysfunction (AST or ALT values exceeding 2-fold the upper limit), creatinine > 1.5 mg/dl, known hypersensitivity to the study drugs. Pregnant women as well as women of childbearing potential were excluded.
Suitable subjects, identified from review of case notes and/or computerized clinic registers were contacted personally or by telephone. No changes in anti-diabetic treatment happened during the study.
Treatments
The patients fulfilling the inclusion and exclusion criteria, were randomised to lercanidipine 20 mg/day, or barnidipine, 20 mg/day, in addition to losartan 100 mg/day for 6 months. Both lercanidipine, and barnidipine were supplied as identical, opaque, white capsules in coded bottles to ensure the blind status of the study. Randomisation was done using a drawing of envelopes containing randomisation codes prepared by a statistician. A copy of the code was provided only to the responsible person performing the statistical analysis. The code was only broken after database lock, but could have been broken for individual subjects in cases of an emergency. Medication compliance was assessed by counting the number of pills returned at the time of specified clinic visits. At baseline, we weighed participants and gave them a bottle containing a supply of the study medication for at least 100 days. Throughout the study, we instructed patients to take their first dose of new medication on the day after they were given the study medication. At the same time, all unused medication was retrieved for inventory. All medications were provided free of charge.
Diet and exercise
Patients were already following a controlled-energy diet (near 600 Kcal daily deficit) based on American Heart Association (AHA) recommendations [
16] that included 50 % of calories from carbohydrates, 30 % from fat (6 % saturated), and 20 % from proteins, with a maximum cholesterol content of 300 mg/day and 35 g/day of fibre. Patients were not treated with vitamins or mineral preparations during the study.
Standard diet advice was given by a dietitian and/or specialist doctor. Dietitian and/or specialist doctor periodically provided instruction on dietary intake recording procedures as part of a behaviour modification program and then later used the subject’s food diaries for counselling. Individuals were also encouraged to increase their physical activity by walking briskly for 20 to 30 min, 3 to 5 times per week, or by cycling. The recommended changes in physical activity throughout the study were not assessed.
Assessments
Before starting the study, all patients underwent an initial screening assessment that included a medical history, physical examination, vital signs, and a 12-lead electrocardiogram. We assessed blood pressure (BP) every month, in addition, patients underwent ambulatory blood pressure monitoring (ABPM), at baseline and at the end of the study. We also collected blood sample to assess: fasting plasma glucose (FPG), glycated hemoglobin (HbA1c), some markers indicative of endothelial damage such as high-sensitivity C-reactive protein (Hs-CRP), tumor necrosis factor-α (TNF-α), metalloproteinase-2 (MMP-2) and -9 (MMP-9), soluble vascular adhesion protein-1 (sVCAM-1), soluble intercellular adhesion protein-1 (sICAM-1). We also evaluated some markers of oxidative stress such as isoprostanes and paraoxonase-1 (PON-1).
All plasmatic parameters were determined after a 12-h overnight fast. Venous blood samples were taken for all patients between 08.00 and 09.00 A.M. We used plasma obtained by addition of Na2-EDTA, 1 mg/ml, and centrifuged at 3000 g for 15 min at 4 °C. Immediately after centrifugation, the plasma samples were frozen and stored at -80 °C for no more than 3 months. All measurements were performed in a central laboratory.
Blood pressure measurements were obtained from each patient (left arm) in the sitting position by physicians blinded to treatment using a standard mercury sphygmomanometer (Erkameter 3000; ERKA, Bad Tolz, Germany) (Korotkoff I and V) with a cuff of appropriate size. Blood pressure has been always measured in the morning before daily drug intake (i.e, at trough 22-24 h after dosing) and after the subject has rested 10 min in a quiet room. Three successive BP readings were obtained at 1-min intervals and averaged.
Heart rate was measured by pulse palpation for 30 s, just before the BP measurements.
Body weight was measured with light clothes and without shoes and BMI was calculated as the weight in Kg divided by height in m squared.
Glycated hemoglobin level was measured by a high performance liquid chromatography method (DIAMAT, Bio-Rad, USA; normal values 4.2-6.2 %), with intra- and interassay coefficients of variation (CsV) of < 2 % [
17].
Plasma glucose was assayed by glucose-oxidase method (GOD/PAP, Roche Diagnostics, Mannheim, Germany) with intra- and interassay CsV of < 2 % [
18].
High sensitivity C-reactive protein was measured with use of latex-enhanced immunonephelometric assays on a BN II analyser (Dade Behring, Newark, Delaware, USA). The intra- and interassay CsV were 5.7 % and 1.3 %, respectively [
19].
Tumor necrosis factor-α level was assessed using commercially available ELISA kits according to manufacturer’s instructions (Titer-Zyme EIA kit; Assay Designs, Ann Arbor, MI). Intraassay CsV were 4.5 % for low- and 3.6 % for high-concentration samples, whereas the interassay CsV were 6.0 % for low and 11.8 % for high-concentration samples, respectively [
20].
Metalloproteinase-2, and MMP-9 levels were determined by a two-site ELISA methods using commercial reagents (Amersham Biosciences, Uppsala, Sweden). The intra- and interassay CsV for measuring MMP-2 levels were 5.4 %, and 8.3 %, respectively [
21]. The intra- and interassay CsV to evaluate MMP-9 levels were 4.9 %, and 8.6 % [
22].
Soluble intercellular adhesion molecule-1 and sVCAM-1 were assessed using commercially available ELISA kits according to manufacturer instructions (R & D Systems, Minneapolis, MN, USA). The intra- and interassay CsV were < 10 %, respectively [
23,
24].
The level of isoprostanes in serum was determined by commercially available ELISA kit (Cayman Chemicals, Ann Arbor, Mich) [
25].
Paraoxonase 1 activity in serum was measured using paraoxon as a substrate in the presence of 2 mM Ca
+2 in 100 mM Tris-HCL buffer (pH = 8.0) [
26].
Statistical analysis
Data are expressed as mean ± standard deviation (SD). The statistical analysis of the data was performed by the statistical analysis software (SAS) system, version 6.12 (SAS Institute, Inc., Cary, NC, USA). The differences between the two groups in baseline characteristics were analyzed by the two-tailed Student’s
t-test. Comparisons within and between groups were assessed by a mixed ANOVA. Differences between baseline and after 6-months’ treatment in each group in BP and oxidative stress parameters were analyzed with the Wilcoxon signed rank test. Comparisons of changes in BP and oxidative stress parameters between the two groups were performed with the Mann-Whitney
U-test [
28]; we adjusted results for potential confounding factors including SBP. Findings of
p < 0.05 were considered significant. Considering as clinically significant a difference of at least 10 % compared with the baseline and an alpha error of 0.05, the actual sample size was adequate to obtain a power higher than 0.80 for all measured variables.