Ezetimibe is effective in providing additional low-density lipoprotein (LDL) cholesterol lowering when coadministered with statins, but its effect beyond LDL cholesterol lowering is unknown. Oxidized LDL (ox-LDL) is a better predictor of adverse cardiovascular events than standard lipid parameters. The objective of this study was to investigate the effect of ezetimibe on ox-LDL. A total of 100 patients with coronary artery disease or coronary artery disease equivalent were randomized to atorvastatin 40 mg/day and ezetimibe 10 mg/day or to atorvastatin 40 mg/day and placebo. LDL cholesterol, LDL cholesterol subfractions, and ox-LDL were measured at baseline and after 8 weeks of therapy. The ezetimibe group had a larger reduction in total LDL cholesterol compared to placebo. This was due mainly to a larger reduction in large buoyant LDL (24% vs 10%, p = 0.008). Ox-LDL level did not change in the placebo group (50 ± 13 vs 51 ± 13 U/L), while it decreased in the ezetimibe group, from 51 ± 13 to 46 ± 10 U/L (p = 0.01 vs baseline and p = 0.02 vs final level in placebo). The change in ox-LDL correlated significantly with those in total LDL and in large buoyant LDL (r = 0.6 and r = 0.5, respectively, p <0.01 for both), but not with that of small dense LDL, high-density lipoprotein, or very low density lipoprotein. In conclusion, this study demonstrates that ezetimibe decreases ox-LDL cholesterol through reductions in total LDL cholesterol and in large buoyant LDL cholesterol.
Oxidized low-density lipoprotein cholesterol (ox-LDL) is a new marker that was shown to better predict cardiovascular risk than standard lipid parameters in apparently “healthy” patients as well as in those with coronary artery disease (CAD). Ox-LDL is caused by the oxidation of LDL cholesterol when it enters the arterial wall. It is involved in the very early steps of atherogenesis, such as endothelial injury, expression of adhesion molecules, and leukocyte recruitment, as well as foam cell and thrombus formation. Although most clinical trials using lipid-lowering therapy have measured the effect of the treatment on standard lipid parameters, very few have tested the efficacy against ox-LDL cholesterol. Statins were shown in few studies to reduce atherogenic ox-LDL cholesterol beyond LDL cholesterol lowering. This may explain at least part of their clinical benefit. Ezetimibe, in contrast, is a new cholesterol absorption inhibitor that is frequently added to statins in patients who have less than optimal levels of LDL cholesterol. Although ezetimibe is very effective in providing additional LDL cholesterol lowering when coadministered with statins, the effect of ezetimibe on ox-LDL cholesterol remains unknown. Recent trials testing the efficacy of ezetimibe in combination with statins on surrogate end points of CAD, such as endothelial function and carotid atherosclerosis, yielded conflicting results. Given the controversy surrounding its use, the effect of ezetimibe on more atherogenic lipid parameters (beyond LDL lowering) is important to understand. Our study was designed to test the effect of ezetimibe on ox-LDL cholesterol.
Methods
This was a prospective, randomized, double-blind, placebo-controlled trial. The objective was to test the effect of ezetimibe 10 mg/day coadministered with atorvastatin 40 mg/day on levels of ox-LDL cholesterol in patients with stable CAD or CAD equivalent. Patients were recruited from outpatient clinics of Hôtel-Dieu de France University Hospital. The protocol was approved by the hospital’s ethics committee, and written informed consent was obtained from all patients. The study began in October 2008 and ended in July 2009.
Patients with stable CAD or CAD equivalent were included in the study. CAD was defined as ≥1 of the following: presence of ≥1 lesion causing > 50% diameter stenosis on coronary angiography, history of myocardial infarction, and history of coronary revascularization, either percutaneously or by coronary artery bypass grafting. CAD equivalent was defined as the presence of diabetes mellitus requiring medications, history of ischemic stroke, or history of peripheral vascular disease. Lipid levels were not entry criteria. Patients who were receiving statin therapy were allowed to enter the study as long as the potency of the statin used was ≤20 mg/day of atorvastatin. This meant that patients who were statin naive or patients who were taking atorvastatin 10 to 20 mg/day, simvastatin 10 to 40 mg/day, pravastatin 10 to 40 mg/day, or fluvastatin 80 mg/day could be enrolled in the study. All patients receiving atorvastatin ≥40 mg/day and those receiving any dose of rosuvastatin were excluded. Other exclusion criteria were age >80 years; treatment with ezetimibe or other cholesterol absorption inhibitor, niacin, or fibrate within the past 3 months; history of acute coronary syndromes or coronary artery revascularization (percutaneous or surgical) within the past 3 months; an ejection fraction <35% or a history of moderate to severe heart failure (New York Heart Association class >II); creatinine clearance <30 ml/min; and creatine phosphokinase or aspartate amino transferase >2 times the upper normal limit.
Patients who met the inclusion criteria were initially seen in the fasting state for measurement of total cholesterol, LDL cholesterol, very low density lipoprotein (VLDL) cholesterol, high-density lipoprotein (HDL) cholesterol, LDL cholesterol subclasses (LDL 1 to LDL 7 ), mean LDL cholesterol particle size, ox-LDL cholesterol, as well as creatine phosphokinase and aspartate amino transferase. Patients were then equally randomized to receive either atorvastatin 40 mg/day plus ezetimibe 10 mg/day (ezetimibe group) or atorvastatin 40 mg/day plus placebo (placebo group). Patients who were receiving statin therapy before randomization had their initial statins stopped and replaced by atorvastatin 40 mg/day, as mentioned previously. Consequently, all patients were shifted to a more potent dose of statin, and all of them shared the same dose and type of statin. The duration of treatment was 8 weeks, at the end of which patients had the same blood tests performed. Compliance with therapy was assessed by follow-up phone calls and by pill count.
Routine blood chemistry (creatine phosphokinase and aspartate amino transferase) and total cholesterol were analyzed using fresh blood samples according to established enzymatic methods in our laboratory. HDL, VLDL, and LDL cholesterol as well as LDL cholesterol subfractions and mean LDL particle size were measured on fresh serum using the Lipoprint system (Quantimetrix Corporation, Redondo Beach, California). This method uses polyacrylamide gel electrophoresis to divide LDL into 7 fractions from near VLDL (LDL 1 ) to near HDL (LDL 7 ). LDL 1 and LDL 2 are classified as large buoyant LDL, and LDL 3 to LDL 7 are classified as small dense LDL. This technique also measures the concentration of VLDL and HDL cholesterol, as well as mean LDL particle size.
Ox-LDL was measured in a core laboratory in Sweden, in plasma samples that were stored at −80°C and that were batch analyzed at the end of the study. A sandwich enzyme-linked immunosorbent assay was used (Mercodia, Uppsala, Sweden). This assay uses the monoclonal antibody 4E6 directed against an oxidation epitope in the apolipoprotein B100 moiety of LDL that is generated because of the substitution of ≥60 lysine residues of apolipoprotein B100 with aldehydes. It was used in most recent studies that investigated the relation of ox-LDL with CAD. All tests were done in duplicate by laboratory technicians who were not aware of treatment allocation. The intra- and interassay coefficients of variation were <3.9% and 9.8%, respectively.
The primary efficacy end point was the reduction in ox-LDL from baseline to final assessment at the end of 8 weeks of treatment. The secondary efficacy end points were the reduction in subfractions of LDL cholesterol and the improvement in LDL particle size.
Categorical variables are expressed as absolute values and percentages and were compared using chi-square tests. Continuous variable are represented as mean ± SD. Baseline and post-treatment values in each group were compared using the paired Student’s t test. Mean values between the 2 groups (ezetimibe and placebo) were compared using independent Student’s t test. Correlations between the changes in marker levels were performed using Pearson’s correlation test. All tests were 2 tailed, and p values <0.05 were considered statistically significant. SPSS version 15.0 was used for statistical analysis.
Results
A total of 100 patients were included in the trial, 50 in each group. The inclusion criteria are listed in Table 1 and were similar between the 2 groups. Of note, 90% of patients had CAD, and only 10% had CAD disease equivalent. Similarly, 90% of patients were receiving statin therapy at the time of randomization. Simvastatin was most commonly used (53 patients), followed by atorvastatin (30 patients). The types and doses of the statins used were similar between the 2 groups. Baseline demographics, clinical characteristics and concomitant medical therapy are listed in Table 2 . All patients took their investigational treatment according to the protocol for a period of 8 consecutive weeks. There were no side effects that required the cessation of therapy, and there were no clinical events. None of the patients had elevations of creatine phosphokinase or of aspartate amino transferase >2 times the upper limit of normal.
| Variable | Ezetimibe + Atorvastatin (n = 50) | Placebo + Atorvastatin (n = 50) |
|---|---|---|
| Coronary stenosis >50% | 19 (38%) | 23 (46%) |
| Previous myocardial infarction | 18 (36%) | 12 (24%) |
| Percutaneous coronary intervention | 23 (46%) | 15 (30%) |
| Coronary artery bypass graft surgery | 26 (52%) | 21 (42%) |
| Diabetes mellitus | 18 (36%) | 21 (42%) |
| Stroke | 1 (2%) | 1 (2%) |
| Peripheral vascular disease | 6 (12%) | 5 (10%) |
| Variable | Ezetimibe + Atorvastatin (n = 50) | Placebo + Atorvastatin (n = 50) |
|---|---|---|
| Age (years) | 64 ± 8 | 65 ± 11 |
| Men | 44 (88%) | 41 (82%) |
| Smoking | 14 (28%) | 12 (24%) |
| Hyperlipidemia ‡ | 47 (94%) | 43 (86%) |
| Hypertension † | 35 (70%) | 38 (76%) |
| Family history of CAD | 21 (42%) | 14 (28%) |
| Body mass index (kg/m 2 ) | 27 ± 3 | 28 ± 4 |
| Aspirin | 45 (90%) | 43 (86%) |
| Clopidogrel | 11 (22%) | 11 (22%) |
| ACE inhibitors or ARBs | 41 (82%) | 34 (68%) |
| β blockers | 37 (74%) | 35 (70%) |
| Calcium channel blockers | 8 (16%) | 18 (36%) ⁎ |
| Nitrates | 10 (20%) | 11 (22%) |
| Diuretics | 6 (12%) | 6 (12%) |
| Oral antidiabetic drugs | 15 (30%) | 17 (34%) |
| Insulin | 6 (12%) | 6 (12%) |
⁎ p = 0.023; for all other comparisons between the 2 groups, p = NS.
† Blood pressure ≥140/90 mm Hg before the initiation of antihypertensive therapy.
‡ LDL cholesterol ≥130 mg/dl before the initiation of lipid-lowering drugs.
The values of different lipid parameters are listed in Table 3 . Patients were well treated with statins before inclusion in the study, as attested by mean baseline LDL cholesterol levels of 102 mg/dl in the ezetimibe group and 99 mg/dl in the placebo group (p = NS). The coadministration of ezetimibe with atorvastatin resulted in a more important reduction of total LDL cholesterol compared to placebo (average reduction 20% vs 10%, p = 0.01). This was due mainly to a larger reduction in the subfraction of large buoyant LDL cholesterol by ezetimibe (average reduction 24% vs 10% with placebo, p = 0.008). The reduction in small dense LDL cholesterol was similar between the 2 groups (average reduction 32% and 36%, p = NS). As a result, the mean level of large buoyant LDL cholesterol at the end of therapy was statistically lower in the ezetimibe group, while the mean levels of small dense LDL cholesterol were equal ( Table 3 ). The mean increase in LDL particle size was also similar between the 2 groups ( Table 3 ). HDL cholesterol level was not affected by either therapy, while that of VLDL cholesterol decreased, but the reduction reached statistical significance only in ezetimibe-treated patients.
| Variable | Ezetimibe + Atorvastatin (n = 50) | Placebo + Atorvastatin (n = 50) | p Value |
|---|---|---|---|
| Total cholesterol (mg/dl) | |||
| Baseline | 176 ± 35 | 168 ± 26 | NS |
| Final | 145 ± 19 | 154 ± 20 | 0.025 |
| p value | <0.001 | <0.001 | |
| LDL cholesterol (mg/dl) | |||
| Baseline | 102 ± 29 | 99 ± 21 | NS |
| Final | 77 ± 10 | 86 ± 14 | <0.001 |
| p value | <0.001 | <0.001 | |
| Large buoyant LDL (mg/dl) | |||
| Baseline | 56 ± 18 | 52 ± 13 | NS |
| Final | 40 ± 8 | 45 ± 10 | 0.007 |
| p value | <0.001 | <0.001 | |
| Small dense LDL (mg/dl) | |||
| Baseline | 4 ± 4 | 5 ± 7 | NS |
| Final | 2 ± 2 | 2 ± 4 | NS |
| p value | 0.001 | 0.001 | |
| Mean LDL size (Å) | |||
| Baseline | 268 ± 3 | 268 ± 4 | NS |
| Final | 270 ± 3 | 270 ± 4 | NS |
| p value | 0.006 | 0.002 | |
| HDL cholesterol (mg/dl) | |||
| Baseline | 37 ± 8 | 37 ± 8 | NS |
| Final | 38 ± 7 | 37 ± 9 | NS |
| p value | NS | NS | |
| VLDL cholesterol (mg/dl) | |||
| Baseline | 36 ± 8 | 33 ± 10 | NS |
| Final | 29 ± 11 | 30 ± 9 | NS |
| p value | 0.01 | NS | |
| Ox-LDL (U/L) | |||
| Baseline | 51 ± 13 | 50 ± 13 | NS |
| Final | 46 ± 10 | 51 ± 13 | 0.02 |
| p value | 0.01 | NS |
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