Common use of Study Objectives Clause in Contracts

Study Objectives. The primary endpoint of the study was the change in FMD between 24 months and baseline. Secondary endpoints were the change in absolute diameter (Dmax-D), the time to peak (Tmax), the change in Nitroglycerin Mediated Dilation (NMD) and the FMD-to-NMD ratio(FMD/NMD). Comparisons between standard measurements for FMD at 1 minute after cuff deflation and for NMD at 3,4 or 5 minutes after Nitroglycerin administration and real maximum values obtained by beat-to-beat analysis were analyzed as an exploratory endpoint. Patients returned to the study site after a 12 hours fast at 3, 6, 12, 18 and 24 months when blinded lipid and safety measurements (creatinin kinase, ALT) were performed. Ultrasound measurements were performed at baseline and 24 months. Two years follow-up for clinical events was performed for all 250 patients. Ultrasound imaging was performed with an Acuson Aspen scanner with a linear array 7.5 MHz probe. All images were recorded digitally for off-line, blinded, analysis by an independent core laboratory, Heart Core, Leiden, the Netherlands. During the study, all measurements were performed by the same two, certified, ultrasonographers. Fasting subjects were examined in the supine position. Heart rate was continuously moni- tored by three-lead ECG. Mean common carotid artery Intima-Media Thickness (CCA IMT) was measured as reported earlier 27. Briefly, the left and right distal 1.0 cm of the common carotid arteries, near and far walls, were examined longitudinally in the angle resulting in an optimal and maximal IMT (while avoiding plaques). For each segment, three R-wave trig- gered images were stored. Mean IMT was measured, when possible, over the entire 1 cm of the vessel segment. CCA IMT was obtained by averaging the mean IMT’s of far and near wall, left and right. For FMD the right arm was placed in extension in the elbow, hand in supination, wrist and elbow supported by foam cushions. An optimal longitudinal image of the brachial artery at, or just above the elbow, was established and kept stable using a specially designed fixative. To obtain clearer images, a water bag was placed between the transducer and the skin. At baseline, 15 consecutive R-wave triggered beats were stored. A cuff placed just distally from the elbow was inflated to 50 mm Hg above systolic blood pressure (up to a maximum of 230 mm Hg) for four minutes. After deflation, R-wave frozen images were recorded for every beat, during 5 minutes. After 10 minutes rest again 15 R-wave triggered beats were stored. Subsequently two puffs of nitroglycerin (0.8 mg) spray were given sublingually, upon which again R-wave frozen images were recorded for every beat during 5 minutes. Lumen diameter (D) was defined as the distance between the media-adventitia interfaces of far and near wall. Using an automated contour detection system, D was measured semi- automatically by placing a cursor on the media-adventitia interfaces. FMD was defined as the percentage increase in brachial artery diameter within 30 to 120 seconds after ischemia ((Dmax-D)/ D). NMD is defined as the percentage increase within five minutes after nitroglyc- erin. Earlier studies in our institute reported reliability coefficients of 99%, 99% and 67% for baseline diameter, peak diameter and FMD respectively 28. In a recent report on variability of FMD (using a continuous method like we did) in DM2, CVs for baseline diameter, peak diameter and FMD were 2.7, 2.5 and 29.7%, respectively29. All laboratory measurements were performed at the Department of Clinical Chemistry and Hematology of the Leyenburg Hospital, according to ISO 15189 standard procedures. Blood samples were collected from the subjects after a 12 hour fast. EDTA tubes were used for the determination of HbA1c. Liver enzymes and lipids were measured in serum. A urine sample was collected for the determination of the albumin-to-creatinin ratio. Serum or plasma was isolated by centrifugation at 1700 g (2900 rpm) for 5 minutes. Serum levels of total cholesterol and triglycerides were measured by enzymatic methods on a Synchron LX20-analyzer (Beckman Coulter, Brea, USA). LDL cholesterol was calculated according to the Friedewald formula. If triglycerides were > 4.5 mmol/l, LDL cholesterol was measured directly with the use of a reagent kit (Genzyme Diagnostics). HDL cholesterol levels were determined after dextran sulfate-magnesium precipitation of apolipoprotein B-containing lipoproteins. Creatinin kinase and ALT were measured by an enzymatic rate method on a Synchron LX20 multichannel chemistry analyzer, according to IFCC-methods. HbA1c was measured by HPLC on a Variant II (BioRad, USA). For the urine sample, a Jaffé rate method was used for the measurement of creatinine on a Synchron LX20-analyzer, while albumin was measured by rate nephelometry. The number of patients needed to detect a difference in FMD of 2% after 2 years (expected SD 4%) with a power of 80 % (α = 0.05) was 63 patients in each group. The primary treatment comparison was between placebo and statin therapy in patients completing the study, as on-treatment analysis. Changes from baseline within each treatment group were analyzed using Student’s paired t-test. Comparisons of the effects between the treatment groups were performed using Student’s independent samples t-test. Stepwise regression techniques were used to investigate the effect on baseline FMD and on changes in FMD of baseline character- istics, carotid IMT and duration of cerivastatin versus simvastatin use. To test the equivalence of cerivastatin 0.4 mg and simvastatin 20 mg, LDL levels before and after the switch to sim- vastatin were compared using Student’s paired t-test. Correlation between changes in FMD and changes in lipid levels were evaluated by calculating Pearson’s correlation coefficients. Comparison between beat-to-beat analysis and standard methods was performed using the Student’s paired t-test and Bland Altman analysis30. Analyses were performed using SPSS 11.0 for Windows software. All analyses were 2-sided, with a level of significance of α = 0.05.