Abstract

Aims

The predominance of small dense low-density lipoproteins (sdLDLs) has been associated with increased cardiovascular risk. The effect of ezetimibe on LDL subfraction distribution has not been fully elucidated. This study assessed by gradient gel electrophoresis the effects of ezetimibe alone, simvastatin alone, and their combination on sdLDL subfraction distribution.

Methods and results

A single-centre, randomized, parallel three-group open-label study was performed in 72 healthy men with a baseline LDL-cholesterol (LDL-C) concentration of 111 ± 30 mg/dL (2.9 ± 0.8 mmol/L). They were treated with ezetimibe (10 mg/day, n = 24), simvastatin (40 mg/day, n = 24), or their combination (n = 24) for 14 days. Blood was drawn before and after the treatment period. Generalized estimating equations were used to assess the influence of drug therapy on LDL subfraction distribution, controlling for within-subject patterns (clustering). We adjusted for age, body mass index, and baseline concentrations of LDL-C and triglycerides. Ezetimibe alone changed LDL subfraction distribution towards a more atherogenic profile by significantly increasing sdLDL subfractions (LDL-IVA +14.2%, P = 0.0216 and LDL-IVB +16.7%, P = 0.039; fully adjusted Wald χ2 test). In contrast, simvastatin alone significantly decreased the LDL-IVB subfraction (−16.7%, P = 0.002). This effect was offset when simvastatin was combined with ezetimibe (LDL-IVB +14.3%, P = 0.44). All three treatments decreased the large, more buoyant LDL-I subfraction, the effects of ezetimibe being the most pronounced (ezetimibe –13.9%, P < 0.0001; combination therapy −7.3%, P = 0.0743; simvastatin −4.6%, P < 0.0001).

Conclusion

In healthy men, treatment with ezetimibe alone is associated with the development of a pro-atherogenic LDL subfraction profile. Potentially atheroprotective effects of simvastatin are offset by ezetimibe.

This study is registered with ClinicalTrials.gov, identifier no. NCT00317993.

Introduction

Ezetimibe represents the first of a new class of lipid-lowering agents, the cholesterol absorption inhibitors. It is able to reduce low-density lipoprotein cholesterol (LDL-C) by 15–25% when given as monotherapy or added on an ongoing statin treatment.1,2 Owing to the complementary mechanism of action of ezetimibe and statins (inhibition of cholesterol absorption and synthesis, respectively) and to their additive effects on LDL-C lowering, their combination is widely used to achieve reductions in LDL-C of up to 60%.3,4

However, a substantial body of evidence suggests that the ‘quality’, and not only the ‘quantity’, of LDL exerts a direct influence on cardiovascular risk (reviewed in Superko and Gadesam5 and Rizzo and Berneis6). Low-density lipoprotein consists of a set of discrete subfractions with distinct molecular properties, among them size and density. In normal subjects, seven major LDL subfractions can be identified [I (large), IIA and IIB (medium), IIIA and IIIB (small), and IVA and IVB (very small)]. Low-density lipoprotein-I is the largest and least dense and LDL-IVB is the smallest, most dense particle.7 The predominance of small dense LDL (sdLDL) and of small LDL particle size (diameter <258 Å) has been associated with increased cardiovascular risk.5,8–11 In this context, the LDL-IVB subfraction has been found to be the single best lipoprotein predictor for atherosclerotic disease progression.12

Statins have been shown to have either no or only a moderate effect on LDL subclass distribution or particle size.13 Few studies have so far assessed the effects of ezetimibe on LDL particle size and/or subfraction distribution (as reviewed in Rizzo et al.14), with conflicting results. Furthermore, most of these trials included subjects with concomitant metabolic disorders such as obesity, hypercholesterolaemia, diabetes, and the metabolic syndrome, and with a variety of co-medications with unknown effects on lipoproteins.

The recent ENHANCE trial found that although the addition of ezetimibe (10 mg/day) to simvastatin (80 mg/day) in patients with heterozygous familial hypercholesterolaemia caused an additional 16.5% reduction in LDL-C, it did not significantly affect the primary endpoint, i.e. the mean change in intima–media thickness (IMT), compared with simvastatin monotherapy.15 Subsequently, the value of ezetimibe in the arena of cardiovascular prevention was questioned, and although various theories have been proposed to explain these surprising results, the actual reasons remain unclear.16,17

The purpose of the present study was to test the hypothesis that ezetimibe may alter LDL subfraction distribution and particle size towards a pro-atherogenic profile in healthy subjects.

Methods

Study design

Low-density lipoprotein particle size and subfractions were analysed from frozen samples of a single-centre, randomized, parallel three-group open-label study that investigated the effects of ezetimibe and simvastatin, alone or in combination, on lipid metabolism. The primary results of this randomized trial have been reported previously.4,18 A total of 72 subjects were randomized to receive ezetimibe (10 mg/day), simvastatin (40 mg/day), or ezetimibe (10 mg/day) plus simvastatin (40 mg/day) for 2 weeks (n = 24 for each group). Ezetimibe and simvastatin were taken once a day in the evening. Blood was drawn before the initiation of treatment and at the end of the treatment period, and the samples were analysed in a blinded manner.

Subjects

Inclusion criteria were age between 18 and 60 years, body mass index (BMI) between 18.5 and 30 kg/m2, fasting LDL-C concentrations <190 mg/dL, triglyceride concentrations <250 mg/dL, and normal blood pressure (<140/90 mmHg). Subjects who had received lipid-lowering drugs within 12 weeks prior to study entry, those with a history of excessive alcohol intake, liver disease, renal dysfunction (glomerular filtration rate <60 mL/min), coronary heart disease, diabetes mellitus or other endocrine disorders, eating disorders, a history of recent substantial (>10%) weight change, a history of obesity (BMI > 35 kg/m2), or taking medications known to affect lipoprotein metabolism were excluded from the study.

The protocol was approved by the Ethics Committee of the University of Cologne, and all subjects gave written informed consent. The study conformed to the Declaration of Helsinki. All subjects completed the study. Body weight did not change in any treatment group. The subjects did not use any extra medications, had no illnesses, and did not deviate from the study protocol. No serious side effects were reported.

Biochemical analyses

Blood was drawn by venipuncture in the morning after a 12 h fast to obtain serum for analysis of lipids. Total cholesterol, LDL-C, and high-density lipoprotein cholesterol (HDL-C) as well as triglycerides were determined by enzymatic methods (CHOD-PAP and GPO-PAP; Roche Diagnostics, Mannheim, Germany) on the day of blood collection in the laboratories of the Cologne University Medical Center (inter-assay coefficient of variation for total cholesterol, LDL-C, HDL-C, and triglycerides was 1.09, 2.79, 0.81, and 1.72%, respectively). Serum was obtained by centrifugation at 1600 g for 30 min at 4°C within 15 min after venipuncture and aliquots were stored immediately at −80°C for future analysis.

Non-denaturing polyacrylamide gradient gel electrophoresis (GGE) of serum was performed in the laboratory of K.B. at the University Hospital Zurich, Switzerland, in a blinded manner. Samples were shipped from Germany in dry ice and immediately analysed by GGE without re-freezing. Previous studies have shown that freezing and thawing has no effect on the measurement of LDL subfractions.19 Gradient gel electrophoresis was performed at 10–14°C in 2–16% polyacrylamide gradient gels. Gels were subjected to electrophoresis for 24 h at 125 V in tris borate buffer (pH 8.3) as described previously.7,20 Gels were fixed and stained for lipids in a solution containing oil red O in 60% ethanol at 55°C. Gels were placed on a light source and photographed using a Luminescent Image Analyzer, LAS-3000 of Fujifilm. Migration distance for each absorbance peak was determined and the molecular diameter corresponding to each peak was calculated from a calibration curve generated from the migration distance of size standards of known diameter, which includes carboxylated latex beads (Duke Scientific, Palo Alto, CA, USA), thyroglobulin, and apoferritin (HMW Std, Pharmacia, Piscataway, NJ, USA) having molecular diameters of 380, 170, and 122 Å, respectively, and lipoprotein calibrators of previously determined particle size. The coefficient of variation in repeated measurements was 1.3%. Low-density lipoprotein subfraction distribution (LDL-I, -IIA, -IIB, -IIIA, -IIIB, -IVA, and -IVB) as percentage of total LDL was calculated as described previously.7

Statistical analysis

Descriptive data are presented as mean values (SD) unless otherwise stated. We performed multivariate analyses using generalized estimating equations to assess the influence of therapy on LDL subclass distribution, controlling for within-subject patterns (clustering). We adjusted for age, BMI, and baseline concentrations of LDL-C and triglycerides. Statistical analyses were conducted using Stata version 9 (StataCorp, College Station, TX, USA). We used Stata's xtgee command to model panel data. All reported P-values were calculated two-sided. Statistical significance was assumed at P-values <0.05.

Results

Baseline subject characteristics are shown in Table 1 and were not different among the three treatment groups. The flow of participants through the trial is shown in Figure 1. All subjects completed the study and their adherence was excellent, as based on pill counts [mean (SD) adherence, 99.1 (3.7)%]. As shown in Table 2, total cholesterol and LDL-C levels significantly decreased in all treatment groups (P < 0.001 for all), whereas triglycerides decreased only in the groups receiving simvastatin. High-density lipoprotein-cholesterol concentrations remained unchanged in all groups.

Table 1

Demographic data and biochemical baseline characteristics of the study participants (total n = 72)

Parameter Total cohort (n = 72) Ezetimibe only (n = 24) Ezetimibe plus simvastatin (n = 24) Simvastatin only (n = 24) 
Age (years) 32 (9) 29 (7) 34 (11) 32 (9) 
Height (cm) 181 (7) 181 (7) 181 (7) 182 (6) 
Weight (kg) 85 (12) 82 (11) 84 (12) 87 (12) 
BMI (kg/m225.7 (3.2) 25.0 (3.3) 25.8 (3.1) 26.4 (3.2) 
Fasting plasma glucose (mg/dL) 88 (8) 87 (6) 89 (2) 86 (7) 

 
Smoking status 
 Current smoker [n (%)] 21 (29) 7 (29) 6 (25) 8 (33) 
 Ex-smoker [n (%)] 9 (12.5) 4 (17) 3 (13) 2 (8) 
 Never smoker [n (%)] 42 (58.3) 13 (54) 15 (63) 14 (58) 

 
Serum lipoproteins 
 Total cholesterol (mg/dL) 189 (35) 180 (28) 194 (41) 194 (34) 
 LDL cholesterol (mg/dL) 111 (30) 105 (23) 116 (35) 113 (30) 
 HDL cholesterol (mg/dL) 64 (15) 64 (13) 61 (14) 65 (18) 
 Triglycerides (mg/dL) 95 (43) 78 (32) 106 (48) 101 (45) 
LDL particle size (Å) 276 (9) 279 (7) 273 (9) 277 (11) 

 
LDL subclasses 
 LDL-I (%) 37.2 (6.7) 39.6 (5.5) 35.4 (7.3) 36.8 (6.7) 
 LDL-IIA (%) 17.4 (3.6) 16.3 (2.9) 19.1 (4.6) 16.8 (2.3) 
 LDL-IIB (%) 16.0 (4.3) 14.2 (1.8) 18.1 (5.3) 15.7 (4.1) 
 LDL-IIIA (%) 10.7 (2.6) 10.3 (1.6) 10.6 (2.4) 11.3 (3.5) 
 LDL-IIIB (%) 4.7 (1.0) 5.1 (1.0) 4.3 (1.0) 4.9 (0.9) 
 LDL-IVA (%) 6.8 (1.6) 7.2 (1.4) 6.2 (1.8) 7.0 (1.6) 
 LDL-IVB (%) 7.3 (2.1) 7.4 (1.7) 6.5 (2.2) 8.0 (2.2) 
Parameter Total cohort (n = 72) Ezetimibe only (n = 24) Ezetimibe plus simvastatin (n = 24) Simvastatin only (n = 24) 
Age (years) 32 (9) 29 (7) 34 (11) 32 (9) 
Height (cm) 181 (7) 181 (7) 181 (7) 182 (6) 
Weight (kg) 85 (12) 82 (11) 84 (12) 87 (12) 
BMI (kg/m225.7 (3.2) 25.0 (3.3) 25.8 (3.1) 26.4 (3.2) 
Fasting plasma glucose (mg/dL) 88 (8) 87 (6) 89 (2) 86 (7) 

 
Smoking status 
 Current smoker [n (%)] 21 (29) 7 (29) 6 (25) 8 (33) 
 Ex-smoker [n (%)] 9 (12.5) 4 (17) 3 (13) 2 (8) 
 Never smoker [n (%)] 42 (58.3) 13 (54) 15 (63) 14 (58) 

 
Serum lipoproteins 
 Total cholesterol (mg/dL) 189 (35) 180 (28) 194 (41) 194 (34) 
 LDL cholesterol (mg/dL) 111 (30) 105 (23) 116 (35) 113 (30) 
 HDL cholesterol (mg/dL) 64 (15) 64 (13) 61 (14) 65 (18) 
 Triglycerides (mg/dL) 95 (43) 78 (32) 106 (48) 101 (45) 
LDL particle size (Å) 276 (9) 279 (7) 273 (9) 277 (11) 

 
LDL subclasses 
 LDL-I (%) 37.2 (6.7) 39.6 (5.5) 35.4 (7.3) 36.8 (6.7) 
 LDL-IIA (%) 17.4 (3.6) 16.3 (2.9) 19.1 (4.6) 16.8 (2.3) 
 LDL-IIB (%) 16.0 (4.3) 14.2 (1.8) 18.1 (5.3) 15.7 (4.1) 
 LDL-IIIA (%) 10.7 (2.6) 10.3 (1.6) 10.6 (2.4) 11.3 (3.5) 
 LDL-IIIB (%) 4.7 (1.0) 5.1 (1.0) 4.3 (1.0) 4.9 (0.9) 
 LDL-IVA (%) 6.8 (1.6) 7.2 (1.4) 6.2 (1.8) 7.0 (1.6) 
 LDL-IVB (%) 7.3 (2.1) 7.4 (1.7) 6.5 (2.2) 8.0 (2.2) 

BMI, body mass index; LDL, low-density lipoprotein; HDL, high-density lipoprotein. Data are presented as mean (SD) or counts (percentages). There were no significant differences between the three treatment groups.

Table 2

Plasma lipids, low-density lipoprotein size, and low-density lipoprotein subfraction distribution before and after treatment

Parameter Ezetimibe only
 
Ezetimibe plus simvastatin
 
Simvastatin only
 
 Before therapy After therapy Mean per cent change P-valuea Before therapy After therapy Mean per cent change P-valuea Before therapy After therapy Mean per cent change P-valuea 
Lipoprotein concentrations 
 Total cholesterol, mg/dL 180 (28) 159 (23) −11.2 (9.7) 0.0002 194 (41) 121 (25) −36.9 (8.1) <0.0001 194 (34) 145 (24) −24.7 (7.9) <0.0001 
 LDL cholesterol (mg/dL) 105 (23) 80 (16) −22.1 (10.2) <0.0001 116 (35) 47 (19) −59.6 (9.7) <0.0001 113 (30) 67 (22) −40.7 (11.5) <0.0001 
 HDL cholesterol (mg/dL) 64 (13) 65 (16) +1.7 (11) 0.35 61 (14) 60 (14) −1.5 (8.5) 0.23 65 (18) 65 (16) +.7 (11.1) 0.88 
 Triglycerides (mg/dL) 78 (32) 88 (49) +27 (79) 0.57 106 (48) 90 (36) −8.9 (29.7) 0.0288 101 (45) 82 (39) −11.8 (39.9) 0.0386 
LDL particle size (Å) 279 (7) 279 (10) +.2 (3.3) 0.22 273 (9) 276 (8) +1.1 (2.6) 0.0975 277 (11) 276 (8) −.4 (1.9) 0.22 

 
LDL subclass composition 
 LDL-I (%) 39.6 (5.5) 33.9 (5.9) −13.9 (11.8) <0.0001 35.4 (7.3) 31.8 (4.7) −7.3 (19.1) 0.0743 36.8 (6.7) 34.5 (5.7) −4.6 (16.7) <0.0001 
 LDL-IIA (%) 16.3 (2.9) 16.1 (2.2) −.5 (9.8) 0.89 19.1 (4.6) 18.7 (4.3) +.6 (20.1) 0.73 16.8 (2.3) 17.3 (2.0) +4.0 (10.1) 0.59 
 LDL-IIB (%) 14.2 (1.8) 15.8 (3.1) +11.2 (15.8) 0.0003 18.1 (5.3) 17.6 (3.2) +3.4 (27.2) 0.37 15.7 (4.1) 17.1 (3.1) +11.3 (16.3) 0.0002 
 LDL-IIIA (%) 10.3 (1.6) 12.1 (1.8) +19.5 (21.0) <0.0001 10.6 (2.4) 11.5 (1.8) +13.6 (28.5) 0.0609 11.3 (3.5) 12.5 (2.6) +15.4 (24.3) <0.0001 
 LDL-IIIB (%) 5.1 (1.0) 5.6 (1.0) +11.9 (19.2) 0.0021 4.3 (1.0) 5.2 (1.2) +27.0 (30.0) 0.0017 4.9 (.9) 5.6 (1.0) +17.3 (24.1) 0.0011 
 LDL-IVA (%) 7.2 (1.4) 8.2 (2.0) +14.2 (25.9) 0.0216 6.2 (1.8) 7.8 (2.2) +28.5 (32.1) 0.0002 7.0 (1.6) 6.9 (1.6) +2.5 (30.7) 0.0179 
 LDL-IVB (%) 7.4 (1.7) 8.3 (2.2) +16.7 (40.4) 0.0392 6.5 (2.2) 7.3 (2.9) +14.3 (30.8) 0.44 8.0 (2.2) 6.1 (1.8) −16.7 (37.4) 0.002 
Parameter Ezetimibe only
 
Ezetimibe plus simvastatin
 
Simvastatin only
 
 Before therapy After therapy Mean per cent change P-valuea Before therapy After therapy Mean per cent change P-valuea Before therapy After therapy Mean per cent change P-valuea 
Lipoprotein concentrations 
 Total cholesterol, mg/dL 180 (28) 159 (23) −11.2 (9.7) 0.0002 194 (41) 121 (25) −36.9 (8.1) <0.0001 194 (34) 145 (24) −24.7 (7.9) <0.0001 
 LDL cholesterol (mg/dL) 105 (23) 80 (16) −22.1 (10.2) <0.0001 116 (35) 47 (19) −59.6 (9.7) <0.0001 113 (30) 67 (22) −40.7 (11.5) <0.0001 
 HDL cholesterol (mg/dL) 64 (13) 65 (16) +1.7 (11) 0.35 61 (14) 60 (14) −1.5 (8.5) 0.23 65 (18) 65 (16) +.7 (11.1) 0.88 
 Triglycerides (mg/dL) 78 (32) 88 (49) +27 (79) 0.57 106 (48) 90 (36) −8.9 (29.7) 0.0288 101 (45) 82 (39) −11.8 (39.9) 0.0386 
LDL particle size (Å) 279 (7) 279 (10) +.2 (3.3) 0.22 273 (9) 276 (8) +1.1 (2.6) 0.0975 277 (11) 276 (8) −.4 (1.9) 0.22 

 
LDL subclass composition 
 LDL-I (%) 39.6 (5.5) 33.9 (5.9) −13.9 (11.8) <0.0001 35.4 (7.3) 31.8 (4.7) −7.3 (19.1) 0.0743 36.8 (6.7) 34.5 (5.7) −4.6 (16.7) <0.0001 
 LDL-IIA (%) 16.3 (2.9) 16.1 (2.2) −.5 (9.8) 0.89 19.1 (4.6) 18.7 (4.3) +.6 (20.1) 0.73 16.8 (2.3) 17.3 (2.0) +4.0 (10.1) 0.59 
 LDL-IIB (%) 14.2 (1.8) 15.8 (3.1) +11.2 (15.8) 0.0003 18.1 (5.3) 17.6 (3.2) +3.4 (27.2) 0.37 15.7 (4.1) 17.1 (3.1) +11.3 (16.3) 0.0002 
 LDL-IIIA (%) 10.3 (1.6) 12.1 (1.8) +19.5 (21.0) <0.0001 10.6 (2.4) 11.5 (1.8) +13.6 (28.5) 0.0609 11.3 (3.5) 12.5 (2.6) +15.4 (24.3) <0.0001 
 LDL-IIIB (%) 5.1 (1.0) 5.6 (1.0) +11.9 (19.2) 0.0021 4.3 (1.0) 5.2 (1.2) +27.0 (30.0) 0.0017 4.9 (.9) 5.6 (1.0) +17.3 (24.1) 0.0011 
 LDL-IVA (%) 7.2 (1.4) 8.2 (2.0) +14.2 (25.9) 0.0216 6.2 (1.8) 7.8 (2.2) +28.5 (32.1) 0.0002 7.0 (1.6) 6.9 (1.6) +2.5 (30.7) 0.0179 
 LDL-IVB (%) 7.4 (1.7) 8.3 (2.2) +16.7 (40.4) 0.0392 6.5 (2.2) 7.3 (2.9) +14.3 (30.8) 0.44 8.0 (2.2) 6.1 (1.8) −16.7 (37.4) 0.002 

Data are presented as mean (SD). Each group comprised n = 24 subjects.

aWald χ2 test after adjusting for age, body mass index, baseline LDL cholesterol, and triglycerides.

Figure 1

CONSORT flow diagram: flow of participants through the trial.

Figure 1

CONSORT flow diagram: flow of participants through the trial.

Significant changes in LDL subfraction distribution were observed in all treatment groups after adjusting for age, BMI and baseline concentrations of LDL-C and triglycerides (Wald χ2P < 0.05). The results are depicted in Figure 2. Adjusted comparisons within individual subclasses showed that ezetimibe treatment significantly increased LDL-IIB (+11.2%), LDL-IIIA (+19.5%), LDL-IIIB (+11.9%), LDL-IVA (+14.2%), and LDL-IVB (+16.7%) (Table 2 and Figure 2A). Combination treatment (ezetimibe plus simvastatin) significantly increased LDL-IIIB (+27%) and LDL-IVA (+28.5%) (Table 2 and Figure 2B). Treatment with simvastatin alone significantly increased LDL-IIB (+11.3%), LDL-IIIA (+15.4%), LDL-IIIB (+17.3%), and LDL-IVA (+2.5%), but significantly decreased LDL-IVB, the most atherogenic LDL subfraction (−16.7%, P = 0.002) (Figure 2C). This effect was offset when ezetimibe was added to simvastatin (Table 2, and Figures 2B and 3).

Figure 2

Low-density lipoprotein subclass distribution (in %) and changes from baseline. Low-density lipoprotein subclass distribution in the ezetimibe monotherapy group (A), combination treatment group (ezetimibe plus simvastatin) (B), and simvastatin monotherapy group (C). Significant changes, as determined by generalized estimating equations (Wald χ2P-values), adjusting for age, body mass index, baseline low-density lipoprotein cholesterol, and triglycerides, are indicated by asterisks [*P < 0.05, (*)P < 0.1]. Data shown are mean values (SEM).

Figure 2

Low-density lipoprotein subclass distribution (in %) and changes from baseline. Low-density lipoprotein subclass distribution in the ezetimibe monotherapy group (A), combination treatment group (ezetimibe plus simvastatin) (B), and simvastatin monotherapy group (C). Significant changes, as determined by generalized estimating equations (Wald χ2P-values), adjusting for age, body mass index, baseline low-density lipoprotein cholesterol, and triglycerides, are indicated by asterisks [*P < 0.05, (*)P < 0.1]. Data shown are mean values (SEM).

Figure 3

Per cent change from baseline in low-density lipoprotein subfractions in the three treatment groups. Data shown are mean values (SEM).

Figure 3

Per cent change from baseline in low-density lipoprotein subfractions in the three treatment groups. Data shown are mean values (SEM).

All treatments decreased the larger, more buoyant LDL-I (Table 2 and Figures 2AC). The decrease was most pronounced in the ezetimibe alone group (−13.9%), followed by the combination group (−7.3%) and smallest in the simvastatin alone group (−4.6%; Figure 3). The reported within-group changes did not reach statistical significance in between-group comparisons due to the overall small sample size.

In multivariate analyses there was a significant influence of baseline LDL-C concentrations on changes in LDL subfraction distribution in the ezetimibe alone group. The increase in atherogenic sdLDL was more pronounced when baseline LDL-C was higher and vice versa (data not shown).

None of the treatments had an effect on overall LDL particle size (Table 2).

Discussion

One essential finding of our study is that treatment with ezetimibe alone or in combination with a statin increases sdLDL proportions, thus resulting in a more pro-atherogenic LDL subfraction profile. Small dense LDL has been accepted as an emerging cardiovascular risk factor by the National Cholesterol Education Program Adult Treatment Panel III.21 Moreover, a consensus statement endorsed by the American Diabetes Association and the American College of Cardiology advocated measuring LDL particle concentration in subjects at high risk for cardiometabolic disorders and pointed out the pro-atherosclerotic effects of sdLDL.22 The mechanisms through which sdLDL may promote atherosclerosis include increased endothelial permeability, impaired clearance from the circulation, easier oxidation and glycation, and increased ability to bind to proteoglycans in the vessel wall.23,24

Although it cannot be fully excluded that the increased risk associated with smaller LDL phenotype may also be a consequence of the broader pathophysiology of which sdLDL are a part (e.g. high triglycerides, low HDL-C, increased LDL particle number, obesity, insulin resistance, diabetes, and metabolic syndrome),23,25–27 some studies have shown that sdLDLs are a strong and independent predictor of coronary artery disease (CAD).28 Other studies have investigated whether the therapeutic modification of LDL subfractions reduces cardiovascular risk. Such investigations used angiographic changes as outcome variables and have reported benefit in patients with a predominance of sdLDL who received treatment such as statins and bile acid-binding resins that tend to reduce the amount of such particles.8,29,30

In fact, various lipid-lowering drugs are able to favourably alter sdLDL, and fibrates and nicotinic acid seem to be the most effective in this respect (reviewed in Gazi et al.31 and Rizzo et al.32). As we were also able to show in the present study, simvastatin has been found to have either no or only a marginal net effect on LDL subfraction distribution.13 This is true for the majority of the statins.33 Interestingly, rosuvastatin, the latest statin to be introduced in the market, seems to be more efficient in modulating plasma lipids and LDL subfractions (reviewed in Rizzo et al.13,34). In contrast, the effects of the cholesterol absorption inhibitor ezetimibe on LDL size and subfraction distribution have been contradictory. Ezetimibe monotherapy was found to be associated with a small but significant decrease in sdLDL concentrations and increase in LDL particle size in patients with primary dyslipidaemia,35 mixed hyperlipidaemia,36 and in obese and overweight patients with hypercholesterolaemia.37 On the other hand, Ose et al.38 in a 12-week trial found no effects of ezetimibe monotherapy on sdLDL concentrations and LDL particle size in patients with hypercholesterolaemia. Moreover, in patients with mixed hyperlipidaemia Tribble et al.39 found that ezetimibe caused reductions in both the large and sdLDL subfractions and had no effects on LDL particle size. Furthermore, Geiss et al.40 found no effect of ezetimibe on LDL subfraction distribution in patients concomitantly treated with LDL apheresis and statins. Three recent studies support our findings. Winkler et al.41 showed that in patients with the metabolic syndrome, the combination of simvastatin with ezetimibe reduces LDL radius of the sdLDL subfractions even further. Moreover, Stojakovic et al.42 found that in patients at high risk for coronary events, the addition of ezetimibe to fluvastatin does not result in any further reduction of dense LDL compared with fluvastatin alone. Tomassini et al.43 in a 6-week trial compared the effects of ezetimibe/simvastatin with atorvastatin on lipoprotein subfractions in patients with type 2 diabetes and hypercholesterolaemia. Ezetimibe monotherapy was not evaluated. They found that neither the combination of ezetimibe/simvastatin nor atorvastatin alone significantly affected the LDL-IV cholesterol subfraction. However, when subjects with triglyceride levels <200 mg/dL were examined, a population similar to that of our study, they also observed an increase, albeit not significant, of the LDL-IV subfraction in the ezetimibe/simvastatin-treated patients. To our knowledge, the present study is the first one to examine whether ezetimibe modulates LDL size and subfraction distribution in healthy individuals, a model which in a sense reflects ezetimibe's ‘true’ effects on a normal metabolic background.

In the present study, we investigated a group of healthy men to assess the effects of ezetimibe, simvastatin, and their combination on LDL particle size and subfraction distribution. We were able to show that treatment with ezetimibe alone or in combination with a statin did not alter LDL particle size but altered the LDL subfraction distribution towards increased concentrations of atherogenic small dense particles. Although simvastatin alone also increased LDL-III subfractions, it significantly decreased the smallest, most dense LDL fraction (LDL-IVB), which has been found to be the best lipoprotein predictor of atherosclerotic disease progression, even if it represents only a minor fraction of total LDL.12 This potentially atheroprotective effect of simvastatin was offset when ezetimibe was co-administered. These findings may, at least partially, explain the lack of additional benefit of ezetimibe added to simvastatin on atherosclerosis progression, measured as changes in IMT, despite a significant additional reduction in LDL-C levels, observed in the ENHANCE study.15 Although there is still no consensus on the clinical significance of surrogate markers of cardiovascular risk, such as IMT,44 it should be pointed out that data from a subsequent study with ezetimibe were also disappointing; the Simvastatin and Ezetimibe in Aortic Stenosis (SEAS) study showed that treatment with ezetimibe (10 mg/day) plus simvastatin (40 mg/day) significantly reduced LDL-C concentrations in patients with aortic stenosis, compared with placebo, but did not affect the composite primary endpoint of aortic-valve events and ischaemic events.45 Furthermore, the recently published trial ARBITER6-HALTS casts some more doubts on the clinical benefits of ezetimibe.46 In specific, the trial showed that while extended-release niacin added to a statin causes a smaller LDL-C reduction compared with ezetimibe added to a statin, niacin had greater efficacy than ezetimibe regarding beneficial changes in IMT. The findings of our trial may at least partially explain the lack of antiatherosclerotic effects of ezetimibe, despite its stronger LDL-C lowering than the comparators.

Interestingly, we found that under treatment with ezetimibe alone, a significant association existed between baseline LDL-C concentrations and the pro-atherogenic changes of the LDL subfractions. Considering that the population of the current study was normocholesterolaemic and that ezetimibe is prescribed to patients with much higher LDL-C levels, it can be postulated that the pro-atherogenic effects of ezetimibe would be even more pronounced in the latter population. Although it could be argued that ezetimibe is usually prescribed along with a statin, a group in which such an association was not observed, it should be pointed out that ezetimibe monotherapy is a widely used alternative for the treatment of hypercholesterolaemia in patients with statin intolerance.47

Limitation of the study is the fact that the clinical relevance of our findings remains to be established. Another limitation is the fact that no a priori power calculations were made for changes in LDL subfractions because the primary outcome parameter of the study was change in LDL-C. Therefore, statistical between-group changes did not reach significance due to the overall small sample size. Strengths of the study include its randomized design and robust statistical methodology, the blinded measurements of LDL subclasses, and the use of a ‘drug-naïve’ population, devoid of co-medications and co-morbidities, which could potentially alter lipid metabolism, and excellent treatment adherence. Treatment duration was relatively short, which does not exclude that the observed effects could be even more pronounced during long-term treatment, especially considering the different plasma residence times of light LDL (1.7–2 days) and dense LDL (2.4–5 days).48,49

A 2-week treatment duration was chosen for this study since the lipid-lowering effects of simvastatin reach maximum at day 14 and remain stable thereafter.50 This was first shown in 2001 by the group of Michael Davidson and was later confirmed for simvastatin and other statins.50–53 Regarding ezetimibe, Bays et al.54 first showed that the maximum LDL-C-lowering effect is present after 2 weeks of treatment, after which it remains stable. This finding was confirmed also by others.

In conclusion, our findings suggest that treatment with ezetimibe alone is associated with the development of a pro-atherogenic LDL subfraction profile. Moreover, potentially atheroprotective effects of simvastatin are offset by ezetimibe when co-administered. Cardiovascular event outcome trials, which are underway, will hopefully provide additional insights into the effects of ezetimibe on cardiovascular events.

Funding

This study was funded in part by MSD Sharp and Dohme, Germany, and the Wilhelm-Doerenkamp Foundation, Cologne (Germany).

Conflict of interest: the sponsors had no role in the design of the study and had no influence on the interpretation of the data and writing of the manuscript. K.B. has received research grants from Astra-Zeneca and the Swiss National Science Foundation and is a consultant for Takeda. M.R. has received honoraria from Astra-Zeneca. G.A.S. has received grant support from the Swiss National Research Foundation and Astra-Zeneca, and honoraria and consulting fees from MSD Sharp and Dohme and Astra-Zeneca. W.K. has received honoraria from and has an advisory board relationship with MSD Sharp and Dohme and a research grant from Bayer Healthcare. I.G.-B. has received honoraria from MSD Sharp and Dohme.

Acknowledgements

We would like to thank Cornelia Zwimpfer for her excellent technical assistance and Dr Jan Bremer for clinical assistance in performing the study.

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RH
Lipka
LJ
Lebeaut
AP
Yang
B
Mellars
LE
Cuffie-Jackson
C
Veltri
EP
Effectiveness and tolerability of ezetimibe in patients with primary hypercholesterolemia: pooled analysis of two phase II studies
Clin Ther
 , 
2001
, vol. 
23
 (pg. 
1209
-
1230
)

Author notes

These authors contributed equally to this work.

Supplementary data

Comments

4 Comments
LDL-subclass changes with ezetimibe and combination ezetimibe plus simvastatin
12 July 2010
Andrew M Tershakovec (with Thomas A Musliner MD and Joanne E Tomassini PhD)

Ezetimibe alone or in combination with simvastatin increases small dense low-density lipoproteins in healthy men: a randomized trial. Berneis et al, doi:10.1093/eurheartj/ehq181

The study of LDL-subclass changes with lipid-lowering therapy by Berneis et al is inconsistent with the large body of data on the effects of ezetimibe on LDL subclasses. The results from lipoprotein subclass assessment in >5,000 hyperlipidemic patients in ezetimibe-related trials of 6-64 week durations overall have shown that ezetimibe alone or combined with statins reduces LDL-C across the size spectrum, including sdLDL-C (1-6). These therapies also reduce sdLDL-C substantially more in patients with high baseline sdLDL-C levels, such as those with elevated triglycerides, versus those with low sdLDL-C levels (1,4,6,7). While lowering LDL-C across the LDL-subclass size range, ezetimibe, ezetimibe statins and statin monotherapy generally have little effect on particle size/distribution pattern (1-7).

In addition, the study by Berneis et al has several shortcomings which limit the ability to form appropriate conclusions. The study was conducted in normocholesterolemic subjects, the treatment period was only two weeks (since the original study was designed to assess changes in gene expression, not primarily lipoprotein changes), the treatment groups were small (n=24) and between-group differences in subclass changes were not significant. The title of the paper, "Ezetimibe alone or in combination with simvastatin increases small dense low-density lipoproteins..." is misleading given that the actual levels of all of the LDL subspecies were decreased, not increased. Finally, even if one was to accept that the small differences in proportions of LDL subspecies seen were real, the clinical significance of such differences in LDL particle-size distribution in the context of therapeutic interventions that decrease particle number across the size-range is unknown and entirely speculative.

The extension of the conclusions drawn from this short, small study, with all its limitations, to broad clinical pronouncements is inappropriate. Such assertions exclude the consideration of wider information regarding the ENHANCE, SEAS and ARBITER 6 trials and other clinical studies, suggesting that the anti-atherosclerotic effect of ezetimibe is consistent with what would be expected from the degree of LDL -C lowering it provides (8-15). The well-demonstrated improvements in LDL -C, apoB, and non-HDL-C with ezetimibe in numerous studies, the well- documented reductions in LDL-subspecies across the spectrum of particle size, and the available clinical outcomes data (15) support the expectation of clinical benefit. Definitive confirmation awaits the results of the IMPROVE-IT trial (16).

References

1. Bays HE, Conard SE, Leiter L, Bird S, Hanson ME, Jensen E, Tershakovec AM. Are post-treatment low density lipoprotein subclass pattern analyses potentially misleading? J Am Coll Cardiol 2010;55(10 (Supp 1)):A53.

2. Farnier M, Perevozskaya I, Taggart W, Kush D, Mitchel Y. Effects of the co-administration of ezetimibe/simvastatin and fenofibrate on the lipoproteins sub-classes and on the LDL size profile in patients with mixed hyperlipidemia. Diabetes & Metabolism 2007;33:S59-S60.

3. Ose L, Reyes R, Johnson-Levonas AO, Sapre A, Tribble DL, Musliner T. Effects of ezetimibe/simvastatin on lipoprotein subfractions in patients with primary hypercholesterolemia: an exploratory analysis of archived samples using two commercially available techniques. Clin Ther 2007;29:2419-2432.

4. Tomassini JE, Mazzone T, Goldberg RB, Guyton JR, Weinstock RS, Polis A, Jensen E, Tershakovec AM. Effect of ezetimibe/simvastatin compared with atorvastatin on lipoprotein subclasses in patients with type 2 diabetes and hypercholesterolaemia. Diabetes Obesity & Metabolism 2009;11:855-864.

5. Tribble DL, Farnier M, Macdonell G, Perevozskaya I, Davies MJ, Gumbiner B, Musliner TA. Effects of fenofibrate and ezetimibe, both as monotherapy and in coadministration, on cholesterol mass within lipoprotein subfractions and low-density lipoprotein peak particle size in patients with mixed hyperlipidemia. Metabolism-Clinical and Experimental 2008;57:796-801.

6. Fazio S, Guyton JR, Polis AB, Adewale AJ, Tomassini JE, Tershakovec AM. Long-term Effect of triple combination ezetimibe/simvastatin extended-release niacin on cholesterol content of lipoprotein subclasses in patients with hyperlipidemia. J Clin Lipid 2009;3: 228-229.

7. Kalogirou M, Tsimihodimos V, Gazi I, Filippatos T, Saougos V, Tselepis AD, Mikhailidis DP, Elisaf M. Effect of ezetimibe monotherapy on the concentration of lipoprotein subfractions in patients with primary dyslipidaemia. Curr Med Res Opin 2007;23:1169-1176.

8. Toth PP, Maki K. A Commentary on the implications of the ENHANCE (Ezetimibe and Simvastatin in Hypercholesterolemia Enhances Atherosclerosis Regression) Trial: Should ezetimibe move to the "Back of the Line" as a therapy for dyslipidemia? J Clin Lipid 2008;2:313-317.

9. Stein EA. Additional lipid-lowering trials using surrogate measurements of atherosclerosis by carotid intima-media thickness: more clarity or confusion? J Am Coll Cardiol 2008;52:2206-2209.

10. Duivenvoorden R, Vergeer M, Kastelein JJP. Niacin compared with ezetimibe. N Engl J Med 2010;362:1046-1047.

11. Blumenthal RS, Michos ED: The HALTS trial--halting atherosclerosis or halted too early? N Engl J.Med. 2009;361:2178-2180.

12. Kastelein JJ , Bots ML. Statin therapy with ezetimibe or niacin in high-risk patients. N Engl J .Med 2009;361:2180-2183.

13. Sibley CT, Gottlieb I, Cox C, Godoy G, Spooner AE, Bluemke DA, Lima JA. Comparative effect of statins vs niacin on MRI measured regression of carotid atherosclerosis in a randomized clinical trial: the NIA plaque study. Circulation 2009;120:S376.

14. Fleg JL, Mete M, Howard BV, Umans JG, Roman MJ, Ratner RE, Silverman A, Galloway JM, Henderson JA, Weir MR, Wilson C, Stylianou M, Howard WJ. Effect of statins alone versus statins plus ezetimibe on carotid atherosclerosis in type 2 diabetes: the SANDS (Stop Atherosclerosis in Native Diabetics Study) trial. J Am Coll Cardiol 2008;52:2198-2205.

15. Holme I, Boman K, Brudi P, Egstrup K, Gohlke-Baerwolf C, Kesaniemi YA, Malbecq W, Rossebo AB, Wachtell K, Willenheimer R, Pedersen TR. Observed and predicted reduction of ischemic cardiovascular events in the Simvastatin and Ezetimibe in Aortic Stenosis trial. Am J Cardiol 2010;105:1802-1808.

16. Califf RM, Lokhnygina Y, Cannon CP, Stepanavage ME, McCabe CH, Musliner TA, Pasternak RC, Blazing MA, Giugliano RP, Harrington RA, Braunwald E. An update on the IMProved reduction of outcomes: Vytorin Efficacy International Trial (IMPROVE-IT) design. Am Heart J 2010;159:705- 709.

Conflict of Interest:

Drs. Musliner, Tershakovec and Tomassini are employees of Merck and own stock/stock options in the company.

Submitted on 12/07/2010 8:00 PM GMT
Small dense LDL after ezetimibe treatment (study Berneis et al.)
12 July 2010
Ioanna Gouni-Berthold (with Kaspar Berneis, Manfredi Rizzo, Heiner K. Berthold, Giatgen A. Spinas, Wilhelm Krone)

We read with interest the letter of Drs. Musliner, Tershakovec and Tomassini, all employees and stockholders of Merck & Co., Ltd.

The following is a point-by-point response to their comments:

"1. The results from lipoprotein subclass assessment in >5,000 hyperlipidemic patients in ezetimibe-related trials of 6-64 week durations overall have shown that ezetimibe alone or combined with statins reduces LDL-C across the size spectrum, including sdLDL-C (1-6). These therapies also reduce sdLDL-C substantially more in patients with high baseline sdLDL-C levels, such as those with elevated triglycerides, versus those with low sdLDL-C levels (1,4,6,7). While lowering LDL-C across the LDL- subclass size range, ezetimibe, ezetimibe statins and statin monotherapy generally have little effect on particle size/distribution pattern (1-7)."

The interpretation of the literature quoted by Musliner et al. to support these statements is misleading and in part incorrect.

Their references 1, 2 and 6 are abstracts, not peer-reviewed publications, and can therefore not be cited as high-level evidence.

The study of their reference 3 (Ose et al.) found no significant reduction of sdLDL-IV by ezetimibe, simvastatin or the combination. As discussed in our publication (1), LDL-IV is the most proatherogenic fraction of sdLDL.

Tomassini et al. (their reference 4) did not examine the effects of ezetimibe monotherapy on sdLDL, but found that the combination of ezetimibe with simvastatin increased sdLDL-IV in subjects with triglyceride levels of < 200 mg/dl, a finding which is in complete agreement with the results of our study.

Similarly, Tribble et al. (their reference 5) also found no reduction of sdLDL-IV by ezetimibe and, finally, the study of their reference 7 is a non-randomized trial which does not report data on the effects of ezetimibe specifically on sdLDL-IV.

Thus, the conclusions of Musliner et al. that ezetimibe alone or in combination with statins reduces sdLDL-C are erroneous. It should also be stated that 6 of the 7 studies cited by them were (co-) authored by employees of the manufacturer of the drug, thus limiting their independent nature.

"2. In addition, the study by Berneis et al. has several shortcomings which limit the ability to form appropriate conclusions. The study was conducted in normocholesterolemic subjects, the treatment period was only two weeks (since the original study was designed to assess changes in gene expression, not primarily lipoprotein changes), the treatment groups were small (n=24) and between-group differences in subclass changes were not significant. The title of the paper, "Ezetimibe alone or in combination with simvastatin increases small dense low-density lipoproteins..." is misleading given that the actual levels of all of the LDL subspecies were decreased, not increased. Finally, even if one was to accept that the small differences in proportions of LDL subspecies seen were real, the clinical significance of such differences in LDL particle-size distribution in the context of therapeutic interventions that decrease particle number across the size-range is unknown and entirely speculative."

All these points have been thoroughly addressed and discussed in our paper (1) and we are glad to reiterate them here.

As we state in our publication (1) we studied a "drug-naive" population to avoid co-medications and co-morbidities, which could potentially alter lipid metabolism. Interestingly, we found that under treatment with ezetimibe alone, there was a significant association between baseline LDL-C concentrations and a shift towards a more pro- atherogenic pattern in the LDL subfractions. Therefore it can be postulated that, when ezetimibe is administered to hypercholesterolemic patients, the pro-atherogenic effects described would be even more pronounced.

The short treatment period, if anything, suggests that the observed effects could be even more pronounced during long-term treatment, considering that the large LDL has a shorter plasma residence time (1.7 to 2 days) compared to sdLDL (2.4 to 5 days).

We regret that the authors find the title misleading. However, it is not possible to conclude from calculation of percent distribution of subclasses and LDL cholesterol concentrations that there would have been a significant quantitative reduction of sdLDL, as demonstrated before (2). The cholesterol content of very large LDL particles is generally higher as compared with sdLDL particles (cholesterol content in large LDL-I and LDL- IIA is 52 and 55%, respectively, cholesterol content in small LDL-IVA and LDL-IVB is 49 and 47%, respectively). In addition, LDL-I and -IIA represent 54.6% of all LDL particles, while LDL-IVA together with LDL-IVB represent only 14.1% of all LDL particles (refer to table 1 of Berneis & Krauss 2002 (2)). Therefore, a decrease in LDL-C is very well compatible with a reduction of large LDL particles, while sdLDL increased during treatment with ezetimibe. Interestingly, although the proportion of sdLDL is small, it has been shown repeatedly that the increase in CAD risk can be predicted by these small particles independently from total or LDL cholesterol. As discussed in our study, these small particles probably have intrinsic properties which transcend mechanistic thinking that cholesterol lowering must be followed by clinical benefit in any circumstance. We would like to point out again that we explicitly described changes of percent distribution of LDL subclasses and did not speculate about cholesterol quantities. Cholesterol content in the subfractions was not measured.

Regarding the clinical significance of our study, we already pointed out in our publication that "the clinical relevance of our findings remains to be established".

"3. The extension of the conclusions drawn from this short, small study, with all its limitations, to broad clinical pronouncements is inappropriate. Such assertions exclude the consideration of wider information regarding the ENHANCE, SEAS and ARBITER 6 trials and other clinical studies, suggesting that the anti-atherosclerotic effect of ezetimibe is consistent with what would be expected from the degree of LDL -C lowering it provides (8-15). The well-demonstrated improvements in LDL- C, apoB, and non-HDL-C with ezetimibe in numerous studies, the well- documented reductions in LDL-subspecies across the spectrum of particle size, and the available clinical outcomes data (15) support the expectation of clinical benefit. Definitive confirmation awaits the results of the IMPROVE-IT trial (16)."

These statements are incorrect and distort the facts. The ENHANCE study (3) demonstrated that the additional LDL-C lowering by ezetimibe did not result in a significant difference in intima-media thickness (IMT) as compared with simvastatin alone. In the SEAS study (4) it was found that simvastatin and ezetimibe did not reduce the composite primary outcome of combined aortic-valve events and ischemic events in patients with aortic stenosis. And finally, the ARBITER 6 study (5) revealed that niacin had greater efficacy regarding the change in mean carotid IMT than ezetimibe, even though the latter caused a significantly greater decrease in LDL-C. In that study greater reductions of LDL-C concentrations in the ezetimibe group were associated with an increase in the carotid IMT.

The statement by Musliner et al. that there are "available clinical outcomes data" supporting ezetimibe's effects by referring to a post hoc analysis of the SEAS trial requires no further comment.

As stated in our paper (1) we are all eagerly awaiting the results of the IMPROVE-IT trial, a cardiovascular event outcome trial. However, final data collection for primary outcome measure for this trial will not occur before June 2013 (Clinical trials.gov, accessed July 7, 2010).

In closing, the fact remains that although ezetimibe significantly decreases LDL-C, there is no evidence showing a clinical benefit on patient-related hard endpoints by ezetimibe, either as monotherapy or co- administered with a statin. We believe that our study may contribute to explaining this lack of benefit.

References

1. Berneis K, Rizzo M, Berthold HK, Spinas GA, Krone W, Gouni- Berthold I. Ezetimibe alone or in combination with simvastatin increases small dense low-density lipoproteins in healthy men: a randomized trial. Eur Heart J 2010;31:1633-1639.

2. Berneis KK, Krauss RM. Metabolic origins and clinical significance of LDL heterogeneity. J Lipid Res 2002;43:1363-1379.

3. Kastelein JJ, Akdim F, Stroes ES, Zwinderman AH, Bots ML, Stalenhoef AF, Visseren FL, Sijbrands EJ, Trip MD, Stein EA, Gaudet D, Duivenvoorden R, Veltri EP, Marais AD, de Groot E, ENHANCE investigators. Simvastatin with or without ezetimibe in familial hypercholesterolemia. N Engl J Med 2008;358:1431-1443.

4. Rossebo AB, Pedersen TR, Boman K, Brudi P, Chambers JB, Egstrup K, Gerdts E, Gohlke-Barwolf C, Holme I, Kesaniemi YA, Malbecq W, Nienaber CA, Ray S, Skjaerpe T, Wachtell K, Willenheimer R. Intensive lipid lowering with simvastatin and ezetimibe in aortic stenosis. N Engl J Med 2008;359:1343-1356.

5. Taylor AJ, Villines TC, Stanek EJ, Devine PJ, Griffen L, Miller M, Weissman NJ, Turco M. Extended-release niacin or ezetimibe and carotid intima-media thickness. N Engl J Med 2009;361:2113-2122.

Kaspar Berneis, Giatgen Spinas University Hospital, Zurich, Switzerland

Manfredi Rizzo University of Palermo, Palermo, Italy

Heiner K. Berthold Charite University Medicine, Berlin, Germany

Ioanna Gouni-Berthold, Wilhelm Krone University of Cologne, Cologne, Germany

ioanna.berthold@uni-koeln.de

Conflict of Interest:

None declared

Submitted on 12/07/2010 8:00 PM GMT
Dear Editor
26 July 2010
Michel Farnier

I read with great interest the article by Berneis et al. Ezetimibe alone or in combination with simvastatin increases small dense low-density lipoproteins in healthy men: a randomized trial (1). This study has evaluated the evolution of LDL sub-classes during ezetimibe, simvastatin and ezetimibe/simvastatin treatments in a population of healthy men. The interpretation of the observed data is really surprising and it seems hard to accept the authors' conclusion. In summary, as expected, at baseline in healthy volunteers with normal levels of LDL cholesterol and triglycerides, the proportion of small dense (sd) LDL particles was low (LDL-IVA 6.8% and LDL-IVB 7.3%). The effects of ezetimibe alone, simvastatin alone or combination therapy on LDL- cholesterol is mainly the reflect of the increase on the fractional catabolic rate of LDL apoB-100 (2) and it's well known that large LDL particles are preferentially uptaken by the liver. This decrease in large LDL particles induces of course a change in the proportion of LDL subclasses remaining in the plasma, but not an increase in the number of sd LDL particles. Such a conclusion needs a determination of either the number of the particles or the apoB content in each LDL subclasses. In other words, the small increase (about 1%) in the proportion of sd LDL is only due to the preferential decrease in large LDL particles. Moreover, we (3-5) and others (6-8) have consistently reported that ezetimibe alone or in combination with simvastatin can reduce cholesterol mass within all LDL subclasses, with a greater effect on large LDL subfraction. In the specific situation of patients with mixed dyslipidemia for which sd LDL particles were preponderant, ezetimibe alone induced a trend to a shift for more dense to more buoyant LDL particles (3). Globally, at the opposite of the effect observed during for example a fenofibrate treatment (3,9), ezetimibe alone, simvastatin alone and ezetimibe plus simvastatin combination therapies have little effect on LDL distribution pattern.

The conclusion of the authors is inappropriate: it has not been observed more sd LDL particles during treatment with ezetimibe alone or in combination with simvastatin.

References

1. Berneis K, Rizzo M, Berthold H, et al. Ezetimibe alone or in combination with simvastatin increases small dense low-density lipoproteins in healthy men: a randomized trial. Eur Hear J 2010; 31: 1633 -1639.

2. Tremblay AJ, Lamarche B, Hogue J-C, Couture P. Effects of ezetimibe and simvastatin on apolipoprotein B metabolism in males with mixed hyperlipidemia. J Lipid Res 2009; 50: 1463-1471.

3. Farnier M, Freeman MW, Macdonell G, et al. Efficacy and safety of the coadministration of ezetimibe with fenofibrate in patients with mixed hyperlipidemia. Eur Heart J 2005; 26: 897-905.

4. Tribble DL, Farnier M, Macdonell G, et al. Effects of fenofibrate and ezetimibe, both as monotherapy and in coadministration on cholesterol mass within lipoprotein subfractions and low-density lipoprotein peak particle size in patients with mixed hyperlipidemia. Metabolism 2008; 57: 796-801.

5. Farnier M, Perevozskaya I, Taggart WV, Kush D, Mitchel YB. VAP II analysis of lipoprotein subclasses in mixed hyperlipidemic patients on treatment with ezetimibe/simvastatin and fenofibrate. J Lipid Res 2008; 49: 2641-2647.

6. Kologirou M, Tsimihodimos V, Gazi I, et al. Effect of ezetimibe monotherapy on the concentration of lipoprotein subfractions in patients with primary dyslipidaemia. Curr Med Res Opin 2007; 23: 1169-1176.

7. Tomassini JE, Mazzone T, Golberg RB, et al. Effect of ezetimibe/simvastatin compared with atorvastatin on lipoprotein subclasses in patients with type 2 diabetes and hypercholesterolaemia. Diab Obesity Metab 2009 ; 11 : 855-864.

8. Ansquer J-C, Bekaert I, Guy M, Hanefeld M, Simon A and the Study Investigators. Efficacy and safety of coadministration of fenofibrate and ezetimibe compared with each as monotherapy in patients with type IIb dyslipidemia and features of the metabolic syndrome. Am J Cardiovasc Drugs 2009; 9: 91-101.

9. Farnier M, Roth E, Gil-Extremera, et al. Efficacy and safety of the coadministration of ezetimibe/simvastatin with fenofibrate in patients with mixed hyperlipidemia. Am Heart J 2007; 153: 335e1-335e8.

Conflict of Interest:

Dr Farnier has received grant/research support and speaker's honoraria from and served as a consultant and advisor for Astra-Zeneca, Genzyme, Kowa, Merck and Co, Merck-Schering-Plough, Novartis, Pfizer, Recordati, Sanofi-Aventis, SMB, Solvay and Takeda.

Submitted on 26/07/2010 8:00 PM GMT
Effects of ezetimibe on small dense LDL
26 July 2010
Ioanna Gouni-Berthold

We read with interest the letter of Dr. Farnier. Here is our response:

1) The author suggests that our conclusion states that ezetimibe causes an increase in the number of sdLDL particles. This is incorrect since nowhere in the manuscript such a statement is made.

2) Dr. Farnier's statements regarding the increase of the fractional catabolic rate (FCR) of apoB-100-containing lipoproteins with statins, ezetimibe and their combination are

a) irrelevant to the findings of our study and do not alter them in the least

b) controversial, since statins can lower apoB-100 levels by decreasing production rates (PR) and increasing fractional catabolic rates (FCR), with somewhat variable effects that may depend on the patient population and the specific statin regimen (Tremblay AJ et al. JLR 2009). For example lovastatin in patients with familial combined hyperlipidemia had no effect on the FCR of LDL apoB-100, but it decreased apoB-100 PR (Cuchel M et al. ATVB 1997).

3) Now let's examine the references Dr. Farnier is quoting:

Reference 3 states that, "in the specific situation of patients with mixed dyslipidemia for which sdLDL particles were preponderant, ezetimibe alone induced a trend to a shift for more dense (note: we assume he means "from" more dense) to more buoyant LDL particles". In this study a) the changes he is referring to were not significant b) the various sdLDL subfractions were not measured.

Reference 4 In this study Tribble et al. found no reduction of sdLDL-IV by ezetimibe.

Reference 5 In this study, sponsored by the company that makes ezetimibe and where all authors except for Dr. Farnier were employees of that same company, there is no ezetimibe monotherapy arm, therefore the effects of ezetimibe on the various sdLDL subfractions are not addressed.

Reference 6 is a non-randomized trial which does not report data on the effects of ezetimibe on sdLDL-IV.

Reference 7 In this study the effects of ezetimibe monotherapy on sdLDL were not examined. Furthermore, the authors found that the combination of ezetimibe with simvastatin increased sdLDL-IV in subjects with triglyceride levels of < 200 mg/dl, a finding which is in complete agreement with the results of our study.

In Reference 8 and 9 the various sdLDL subfractions were not even measured.

In closing, based on the results of our study and the existing evidence we find the statement of Dr. Farnier that "Globally, ... ezetimibe alone, simvastatin alone and ezetimibe plus simvastatin combination therapies have little effect on LDL distribution pattern" unfounded and misleading.

Kaspar Berneis, Giatgen Spinas

University Hospital, Zurich, Switzerland

Manfredi Rizzo

University of Palermo, Palermo, Italy

Heiner K. Berthold

Charite University Medicine, Berlin, Germany

Ioanna Gouni-Berthold, Wilhelm Krone

University of Cologne, Cologne, Germany

ioanna.berthold@uni-koeln.de

Conflict of Interest:

Dr. Berneis has received research grants from Astra-Zeneca and the Swiss National Science Foundation and is a consultant for Takeda. Dr. Rizzo has received honoraria from Astra-Zeneca. Dr. Berthold has no conflict of interest. Dr. Spinas has received grant support from the Swiss National Research Foundation and Astra-Zeneca, and honoraria and consulting fees from MSD Sharp & Dohme and Astra-Zeneca. Dr. Krone has received honoraria from and has an advisory board relationship with MSD Sharp & Dohme and a research grant from Bayer Healthcare. Dr. Gouni-Berthold received honoraria from MSD Sharp & Dohme.

Submitted on 26/07/2010 8:00 PM GMT