Forty-two hypercholesterolaemic subjects, who were statin intolerant and at high risk for CVD events, were screened for participation; 34 were randomized. High risk was defined as meeting National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III) criteria.1
Heterozygous FH subjects (≥30 years for men and ≥45 years for women) were also classified as high risk.12–14
Familial hypercholesterolaemia was diagnosed by genotyping or by fulfilling the criteria as outlined by the World Health Organization (FH: report of the WHO, 1998).
In the present study, patients were considered to be ‘statin intolerant’ if they were unable to tolerate at least two different statins due to side effects of any kind. Participants did not use other lipid-lowering drugs unless the dose had been stable for >8 weeks prior to screening. At screening, fasting LDL-c was ≥3.4 mmol/L and plasma triglyceride levels <2.3 mmol/L. Haemoglobin A1c (HbA1c) was ≤8.0, alanine aminotransferase (ALT) ≤1.5× the upper limit of normal (ULN), and serum creatine phosphokinase <3× ULN. Alcohol consumption had to be ≤3 U (30 g) per day and ≤12 U (120 g) per week for male subjects, and ≤2 U (20 g) per day and ≤8 U (80 g) per week for female subjects. All study participants were enrolled at one site in the Netherlands. The study protocol was approved by the local institutional review board. All subjects gave written informed consent. The study was performed in compliance with the standards of Good Clinical Practice (CPMP/ICH/135/95) and the declaration of Helsinki (Washington 2002). During the study, the protocol was amended to allow the inclusion of subjects with FH as well as subjects with controlled type II diabetes mellitus.
Participants were randomized at a 2:1 ratio, active to placebo. Participants, investigators, and study staff were blinded to the treatment assignment with the exception of the personnel who prepared the study drug. The study drug was administered subcutaneously at a dose of 200 mg/week from Week 1 until Week 26.11
Pre-specified efficacy endpoints included per cent change in LDL-c from the baseline to 2 weeks after the last dose. The other endpoints included per cent change in total cholesterol, apoB, HDL-c, triglycerides, non-HDL-c, VLDL, LDL/HDL ratio, ApoA1 and lipoprotein a [Lp(a)] concentrations as well as change in the particle size and number from the baseline to 2 weeks after the last dose. Safety was determined using the incidence of treatment-emergent AEs, clinical laboratory evaluations, vital signs, electrocardiograms, and physical examination findings. Due to the long half-life of mipomersen, the treatment period was followed by a 6-month evaluation period with visits at Weeks 28, 32, 40, and 50.
Lipid and lipoprotein analysis
Fasting blood and urine samples were taken after at least 10 h of fasting at visit during Weeks 1, 3, 5, 7, 9, 11, 13, 17, 21, 25, 28, 32, 40, and 50. Fasting blood samples were analysed for lipids and lipoproteins by MedPace (Cincinnati, OH, USA). apoB, apoA1, and Lp(a) concentrations were determined by rate nephelometry; and total cholesterol and triglycerides were measured by standard enzyme-based colorimetric assays. High-density lipoprotein cholesterol (HDL-c) was determined by an enzyme-based colorimetric assay after dextran sulfate precipitation. Low-density lipoprotein cholesterol and non-HDL-c were calculated. Lipoprotein particles were analysed by nuclear magnetic resonance spectroscopy (MRS) as described previously.15
The safety and tolerability of mipomersen was assessed by determining the incidence, severity, and possible relationship to the study drug of AEs and laboratory parameters, including blood chemistry, routine haematology, coagulation, and urinalysis. Vital signs were recorded at Weeks 1, 2, 3, 5, 7, 9, 11, 13, 15, 17, 21, 25, 28, 32, 40, and 50. Full physical examination was performed at screening and at Weeks 13, 28, and 50. A 12-lead electrocardiogram was recorded at screening and at Week 28.
Three-Tesla proton MRS was used to quantify IHTG concentration.16,17
The IHTG concentration of >5.6% was defined as reflecting hepatic steatosis.18
Intrahepatic triglyceride values were quantified by one assessor who was masked to treatment assignment. Initially, MRS was recommended for subjects with persistent transaminase levels ≥3× ULN or for medical reasons. Following the observation of moderate hepatic steatosis in one patient, MRS was performed if ALT levels ≥2× ULN at any time during treatment. If IHTG content was ≥10%, MRS measurements were repeated around Weeks 28 and 50. In case hepatic steatosis persisted, MRS was repeated until IHTG was <10% or stabilized. Subjects with persistent transaminase increases ≥2× ULN and IHTG ≥ 20% were referred to a hepatologist. In patients requiring liver biopsy, the hepatic macrovesicular steatosis and steatohepatitis score was determined according to Kleiner et al
A sample size of 30 patients (20 mipomersen and 10 placebo) was planned for this study assuming a standard deviation of per cent change in LDL-c ≤ 20%. A two-sided t-test with an α level of 0.05 was expected to provide ≥90% power to detect a 30% difference in LDL-c per cent reduction between the two groups.
The study database was housed by an electronic data collection vendor (Almac, Souderton, PA, USA). Investigators had full access to the data. Data analysis as defined in the protocol was performed by a clinical research organization MedPace. Post hoc analysis was performed by the investigators. The sponsor had no influence on the interpretation of the results. Baseline characteristics were summarized using descriptive statistics. For the efficacy parameters, baseline was defined as the mean of the value at screening and the last value prior to the first dose. For the safety parameters, baseline was defined as the last value prior to the first dose. The primary efficacy time point was defined as the visit closest to 2 weeks after the last dose of study treatment.
Percentage change from the baseline for lipid parameters was compared between treatment groups using the t-test or the Wilcoxon rank-sum test for data with a skewed distribution. The difference between the highest and lowest IHTG content during follow-up was used to estimate the increase in IHTG content attributable to mipomersen. In a post hoc analysis, a comparison of each patient's highest and lowest IHTG content was tested using the Wilcoxon signed-rank test. Spearman's rank correlation coefficients were calculated to assess the relationship between ALT increases, IHTG content, and apoB levels. Software utilized for the analyses was SAS version 9.2 (SAS Institute, Cary, NC, USA). All statistical tests were two-sided with a significance level of 0.05. Data were expressed as mean ± standard deviation, unless specified otherwise.