A Randomized Open-Label Trial to Assess the Effect of Plant Sterols Associated with Ezetimibe in Low-Density Lipoprotein Levels in Patients with Coronary Artery Disease on Statin Therapy

Abstract

Consumption of food products enriched with plant sterols and the use of ezetimibe reduce cholesterol absorption in the intestine and effectively reduce low-density lipoprotein (LDL) plasma levels. We evaluated the therapeutic effect of the ezetimibe+plant sterol association in patients with coronary artery disease still not reaching recommended lipid levels despite the use of statins. We performed a prospective open-label study with 41 patients with stable coronary disease and LDL >70 mg/dL. Patients were randomized into four groups for a 6-week treatment: the control (CT) group remained on the same statin therapy, the ezetimibe (EZ) group received 10 mg/day of ezetimibe, the plant sterol (PS) group received spread enriched with 2 g of plant sterols, and the ezetimibe+PS (EZ+PS) group received 10 mg/day EZ +2 g PS. Initial mean LDL level was 97.4 ± 31.1 mg/dL in control group, 105.1 ± 23.1 mg/dL in EZ group, 95.4 ± 27.7 mg/dL in PS group, and 97.0 ± 8.3 mg/dL in EZ+PS group (P > .05). After 6 weeks of treatment, LDL of patients slightly increased in the control group (+8.9%; P > .05) and dropped in EZ group (−19.1%; P = .06), PS group (−16.6%; P = .01), and EZ+PS group (−27.3%; P < .01). Mean LDL levels after treatment were 70.5 ± 17.9 mg/dL in EZ+PS group, lower than the other groups (control was 106.1 ± 34.9 mg/dL, EZ group was 85.0 ± 35.6 mg/dL, and PS was 79.6 ± 29.7 mg/dL) (P = .05 variance analysis factor [ANOVA]). Body weight, body–mass index, and glucose plasma levels did not change significantly after intervention. The combination of PS+ezetimibe was associated with lower LDL levels and suggests beneficial therapeutic effect against major cardiovascular events.

Introduction

Lowering low-density lipoprotein-cholesterol (LDL) reduces cardiovascular risk in patients with coronary artery disease (CAD).¹ Several studies have shown that LDL levels below 70 mg/dL significantly decrease mortality and cardiovascular events in high-risk populations.^2,3 Statins are considered the first-line therapy for LDL reduction; however, some patients in statin monotherapy cannot achieve LDL target values due to limited efficacy or poor tolerability to a high-intensity treatment. Thus, the association of statins with other drugs or with diet modifications can be helpful in patients unable to achieve recommended targets of LDL values.

Ezetimibe is a selective cholesterol absorption inhibitor that blocks the cholesterol transporter Nieman-Pick C1-like 1 (NPC1L1) protein. Inactivating mutations on the gene that encodes NPC1L1 were found to be associated with lower LDL levels and protection against atherosclerotic disease.⁴ Ezetimibe decreases LDL levels by 15–20% as a monotherapy⁵ and can be more effective in lipid reduction when associated with statins. The combination of ezetimibe with high dose of a potent statin allows up to 70% reduction in LDL serum levels.⁶ Moreover, the LDL reduction obtained by the combination of ezetimibe and a statin translates into clinical benefits in a high-risk population such as patients with chronic kidney disease⁷ and in patients who have had an acute coronary syndrome.⁸

Another effective strategy to reduce cholesterol levels is the association of statins with food enriched with plant sterols (PSs). PSs also inhibit cholesterol absorption by the enterocytes, through a different mechanism from ezetimibe, providing an additional 17% LDL reduction when combined with statins, compared with a statins alone.⁹ It is controversial as to whether the association of ezetimibe and PSs has synergic effects in reducing LDL cholesterol for primary prevention of CAD.^10,11 The outcomes of this association for secondary prevention of CAD have not been tested so far. Therefore, the purpose of this open-label randomized study is to evaluate the therapeutic effects of the association of ezetimibe and PSs in patients with established CAD who could not reach lipid targets despite the use of statins.

Material and Methods

Subjects

After approval by the independent ethics committee of the involved institution, we selected patients over 18 years old undergoing statin treatment for secondary prevention of CAD, with a fixed dosage for at least 3 months previous to the beginning of this study, still not able to reach LDL <70 mg/dL. All patients were required to have a history of acute coronary event in the past or coronary angiography demonstrating significant coronary disease. We did not include in this study patients who were already taking ezetimibe and/or PSs. Initial recruitment was performed based on the availability of LDL level, and a confirmatory lipid profile was performed before the randomization. Those who required statin dosage modification at physician's discretion during the intervention were excluded. All participants provided written informed consent.

Study protocol

Eligible patients were randomly assigned to four groups: the control (CT) group, where no additional treatment was provided besides statin already in use; the ezetimibe (EZ) group, where patients received a fixed dose of 10 mg/day; the PS group, where patients received 30 g/day of PS-enriched vegetable spread (equals 2 g PS); the ezetimibe+PS (EZ+PS) group, where patients received both ezetimibe and the vegetable spread on the top of statin. Ezetimibe was donated by Merck Sharp–Dohme (Zetia^®); vegetable spread was provided by Unilever (Becel Pro-Activ^®—composition is presented in Table 1). Both companies had no other role in this study but the donation of the respective products.

Table 1.

Nutritional Composition of the Vegetable Spread (Becel Pro-Activ) as Provided by Manufacturer

Nutritional facts per 10 g (1 tablespoon)
Content per serving
Energy	31 kcal = 129 kJ
Carbohydrate	0 g
Sugars	0 g
Protein	0 g
Total fat	3.4 g
Saturated fatty acids	0.8 g
Trans fat	0 g
Fiber	0 g
Sodium	32 mg
Vitamin A	80 μg
Vitamin D	0.75 μg
Vitamin E	2.0 mg

Patients were treated during a 6-week period and were advised to keep the same diet pattern reported in their nutritional evaluation throughout the study. It was strongly recommended to the physicians to maintain statin dosage/treatment unchanged during the intervention period. Randomization was performed in a 1:1 ratio and the outcome assessors were unaware of patient's treatment allocation.

Procedures

Anthropometric evaluations and laboratory exams (blood glucose test, total cholesterol, LDL, high-density lipoprotein [HDL], triglycerides, and C-reactive protein [CRP]) were performed in all participants at baseline and after the 6-week intervention. LDL level was determined by direct enzymatic colorimetric method.

Nutritional evaluation

All patients were required to provide a full 3-day food report, describing all food and liquid intake. The nutritionist then analyzed the reports, and calculation of macro- and micronutrients of each patient's diet was performed using an open-access software, validated in the Brazilian population (Nutwin).¹² Analyses were performed regarding the food composition, recommended daily allowances, and proportions of polyunsaturated fatty acids, monounsaturated, and saturated fatty acids. Since this analysis was performed before the study intervention, the vegetable spread was not included in the calculation. Baseline daily consumption of phytosterols was not assessed, but according to literature, nonvegetarian individuals consume from 0.2 to 0.4 g of phytosterols/phytostanols daily.¹³

Primary and secondary endpoints

The primary endpoint was to evaluate LDL level variations during a 6-week treatment after the addition of PS and/or ezetimibe to statin treatment.

Secondary endpoints included any change in non-LDL cholesterol, total cholesterol, HDL, very low-density lipoprotein (VLDL), triglyceride levels, and high-sensitivity CRP levels.

Statistical analysis

The results obtained in the study are expressed as mean and standard deviation or as frequencies and percentages. Variance analysis factor (ANOVA), Student's t-test, or nonparametric Kruskal–Wallis test was used to compare independent groups. Normality condition of the variables was evaluated by the Kolmogorov–Smirnov test and homogeneity of variances by the Levène test. To analyze dichotomic variation between the groups, the chi-square test and Fisher exact test were used. P values <.05 were considered statistically significant.

Results

Baseline characteristics

The baseline clinical characteristics and laboratory data of all 41 randomized patients are described in Table 2. There was no significant difference in clinical characteristics between the four groups. Regarding statin therapy, the great majority of patients were under treatment with simvastatin, with only two patients on atorvastatin and one on rosuvastatin.

Table 2.

Baseline Clinical and Laboratory Characteristics

	Control (n = 10)	Ezetimibe (n = 10)	Plant sterols (n = 11)	Ezetimibe+plant sterols (n = 10)	P
Age (years)	60.2 ± 7.8	61.5 ± 14.0	65.9 ± 7.8	60.6 ± 12.0	ns^a
Male, n (%)	7 (70)	6 (60)	7 (64)	4 (40)	ns
Weight (kg)	78.8 ± 17.9	82.3 ± 12.8	82.0 ± 20.0	78.8 ± 17.5	ns
Height (cm)	167.4 ± 8.8	162.8 ± 10.8	164.5 ± 6.8	161.4 ± 11.3	ns
BMI (kg/m²)	27.8 ± 4.0	31.2 ± 5.5	30.1 ± 5.8	30.0 ± 5.1	ns
Systolic BP (mmHg)	115.3 ± 14.9	146.6 ± 21.9	136.8 ± 19.1	141.8 ± 26.6	ns
Glucose (mg/dL)	96.7 ± 14.8	101.4 ± 8.18	127.1 ± 53.23	119.5 ± 53.0	ns
TC (mg/dL)	172 ± 40.9	185.4 ± 22.8	175.5 ± 32.32	169.6 ± 22.5	ns
HDL (mg/dL)	47.3 ± 11.0	48.9 ± 13.26	44.3 ± 8.1	45.9 ± 8.1	ns
LDL (mg/dL)	97.4 ± 31.1	105.1 ± 23.1	95.4 ± 25.7	97.0 ± 8.27	ns
TG (mg/dL)	135.5 ± 60.3	156.8 ± 72.92	184.4 ± 70.9	143.5 ± 67.7	ns
VLDL (mg/dL)	27.3 ± 11.9	31.4 ± 14.6	36.7 ± 14.1	28.7 ± 13.57	ns
Non-HDL (mg/dL)	124.7 ± 36.0	136.5 ± 29.2	132.1 ± 35.2	125.7 ± 17.9	ns
hs-CRP (mg/dL)	2.59 ± 5.9	1.49 ± 1.4	0.86 ± 2.0	1.21 ± 1.1	ns

Data are mean ± SD or n (%).

ns = not statistically significant for P < .05.

BMI, body–mass index; BP, blood pressure; HDL, high-density lipoprotein; hs-CRP, high-sensitivity C-reactive protein; LDL, low-density lipoprotein; TC, total cholesterol; TG, triglycerides; VLDL, very low-density lipoprotein.

Analysis of the food record showed an elevated caloric intake of proteins, lipids, saturated fatty acids, cholesterol, sodium, and potassium, as well as a deficit in intake of fibers, polyunsaturated, and monounsaturated fatty acids at baseline. All diet characteristics at baseline were similar between the four groups (Table 3).

Table 3.

Dietary Daily Intake

	Control (n = 10)	Ezetimibe (n = 10)	PS (n = 11)	Ezetimibe+PS (n = 10)	P
Calories (kcal)	2411.5 ± 669.6	2481.9 ± 386.4	2581.6 ± 497.7	2575.0 ± 411.3	ns
Carbohydrates (g)	281.3 ± 87.7	300.4 ± 68.8	297.7 ± 75.5	288.7 ± 60.0	ns
Protein (g)	115.8 ± 22.7	107.6 ± 20.4	126.3 ± 37.2	114.0 ± 35.5	ns
Lipids (g)	92.7 ± 28.2	102.1 ± 12.9	108.5 ± 34.7	104.0 ± 23.3	ns
Fibers (g)	5.8 ± 1.9	5.0 ± 2.4	7.0 ± 3.2	6.2 ± 2.2	ns
Saturated FA (g)	32.8 ± 11.2	35.2 ± 7.0	37.9 ± 13.7	34.1 ± 11.0	ns
PUFA (g)	11.8 ± 3.3	14.5 ± 5.9	12.3 ± 4.6	16.6 ± 3.7	ns
MUFA (g)	15.7 ± 5.0	18.5 ± 7.3	21.3 ± 7.4	24.8 ± 8.3	ns
Cholesterol (mg)	302.5 ± 78.7	353.4 ± 110.1	379.3 ± 188.8	340.0 ± 121.6	ns
Sodium (mg)	5633.5 ± 1541.2	5383.8 ± 1318.4	5499.3 ± 2710.3	4985.6 ± 724.1	ns
Potassium (mg)	3500.5 ± 1159.1	3107.2 ± 751.6	3695.6 ± 1369.0	3561.7 ± 1141.9	ns

None of the variables differed significantly between the groups (P > .05 for all comparisons).

Data are mean ± SD.

MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids.

Laboratory assessment

Serum level of LDL remained unchanged in the CT group. In the PS group, LDL level decreased to 79.6 ± 22.7 mg/dL (16.6% reduction; P = .008). In patients who received ezetimibe 10 mg/day, LDL levels decreased to 85.0 ± 35.6 mg/dL (19.1% reduction; P = .06). The largest reduction was observed in the EZ+PS group, where mean LDL level decreased to 70.5 ± 17.9 mg/dL (27.3% reduction; P = .003) (Fig. 1).

FIG. 1.

Individual variation in LDL levels with different interventions. Group as follows: (A) control group; (B) ezetimibe group; (C) PS group; (D) ezetimibe+PS group. LDL, low-density lipoprotein.

Lower total cholesterol levels were detected in those treated with PS and ezetimibe (EZ) alone (153.2 ± 26.2, P = .004, and 162.6 ± 34.3, P = .0057, respectively). Non-HDL cholesterol levels were lower in the PS and ezetimibe (EZ) alone treatment and also in the ezetimibe-PS (EZ+PS) group (149.4 ± 23.8, P = .077). There were no differences among the groups concerning HDL, VLDL, triglycerides, and CRP levels. Biochemical parameters before and after intervention are shown in Table 4.

Table 4.

Biochemical Parameters Before and After Intervention

	Groups
	Control (n = 10)	Ezetimibe (n = 10)	Plant Sterols (n = 11)	Ezetimibe+plant sterols (n = 10)	P^a
Glucose (mg/dL)
Pre	96.7 ± 14.8	101.4 ± 8.18	127.1 ± 53.23	119.5 ± 53.0	ns
Post	95.2 ± 10.5	100.4 ± 10.8	123.36 ± 37.35	120.5 ± 41.0
P value^b	ns	ns	ns	ns
TC (mg/dL)
Pre	172 ± 40.9	185.4 ± 22.8	175.5 ± 32.32	169.6 ± 22.5	.020
Post	179.9 ± 46.7	162.6 ± 34.3	153.2 ± 26.2	149.4 ± 23.87
P value^b	ns	.0057	.004	.077
HDL (mg/dL)
Pre	47.3 ± 11.0	48.9 ± 13.26	44.3 ± 8.1	45.9 ± 8.1	ns
Post	45.5 ± 12.3	51.0 ± 16.8	43.4 ± 6.8	48.7 ± 10.6
P value^b	ns	ns	ns	ns
LDL (mg/dL)
Pre	97.4 ± 31.1	105.1 ± 23.1	95.4 ± 25.7	97.0 ± 8.27	.004
Post	106.1 ± 34.9	85.0 ± 35.6	79.6 ± 22.7	70.5 ± 17.9
P value^b	ns	.06	.008	.003
TG (mg/dL)
Pre	135.5 ± 60.3	156.8 ± 72.92	184.4 ± 70.9	143.5 ± 67.7	ns
Post	141.8 ± 85.1	133.0 ± 52.0	151.2 ± 47.5	150.9 ± 88.4
P value^b	ns	ns	ns	ns
VLDL (mg/dL)
Pre	27.3 ± 11.9	31.4 ± 14.6	36.7 ± 14.1	28.7 ± 13.57	ns
Post	28.3 ± 16.9	26.6 ± 10.4	30.2 ± 9.4	30.2 ± 17.6
P value^b	ns	ns	ns	ns
Non-HDL (mg/dL)
Pre	124.7 ± 36.0	136.5 ± 29.2	132.1 ± 35.2	125.7 ± 17.9	.006
Post	134.4 ± 46.1	111.6 ± 39.6	109.9 ± 27.7	100.7 ± 27.8
P value^b	ns	.007	.005	.038
hs-CRP (mg/dL)
Pre	2.59 ± 5.9	1.49 ± 1.4	0.86 ± 2.0	1.21 ± 1.1	ns
Post	0.98 ± 1.8	1.37 ± 2.9	0.34 ± 0.41	0.76 ± 1.6
P value^c	ns	ns	ns	ns

One-factor ANOVA.

Student^'s t-Test.

Kruskal–Wallis nonparametric test.

ANOVA, variance analysis factor.

Discussion

Lowering LDL levels in patients with CAD are the cornerstone for cardiovascular risk reduction. In the present study, we demonstrated that combining two cholesterol absorption inhibitors with statins may be an efficacious strategy to patients still not achieving their lipid goals.

Cholesterol plasma concentration depends on several inter-related processes, such as absorption, synthesis, transport, storage, catabolism, and excretion. Statins are considered the first-line agents for lipid lowering, exerting this effect by inhibiting HMG-CoA reductase.¹⁴ The reduction in cholesterol synthesis leads to an upregulation of LDL receptors and, consequently, to a greater clearance of LDL particles from plasma. As a result, this lipid-lowering effect clearly translates into cardiovascular risk reduction, as demonstrated in several clinical trials.¹⁵ However, many high-risk patients are still unable to meet their lipid goals despite the use of statins.^16,17

The rationale to combine cholesterol absorption inhibitors with statins relies on the fact that decreased synthesis of cholesterol in the hepatocytes promotes a compensatory increase in intestinal cholesterol absorption, as demonstrated in animal models¹⁸ and humans.¹⁹ Therefore, the association of a cholesterol absorption inhibitor with statin leads to a more aggressive reduction in LDL levels. Mikhailidis et al. ²⁰ have demonstrated in a meta-analysis involving 5039 patients that ezetimibe coadministered with ongoing statin therapy provides additional 23.6% reduction in LDL. A meta-analysis combining the results of eight randomized controlled trials has also shown benefits of the association of PS or stanol-enriched foods with a statin treatment, with an additional 13.6 mg/dL reduction in LDL.²¹ Currently, the association of both ezetimibe and PS has been marginally exploited since their different mechanisms of action make it reasonable to assume the possibility of synergic effects. Ezetimibe selectively inhibits the transport of cholesterol across the intestine mucosa by preventing it from binding to NPC1L1, the cholesterol transporter located in the brush border membrane of the enterocyte.²² On the other hand, PS seems to compete with dietary and biliary cholesterol for inclusion into the micelles, thus decreasing the amount of cholesterol available for uptake by the enterocyte. Recent studies have also shown that PSs and plant stanols may also influence intestinal cholesterol metabolism by increasing transintestinal cholesterol excretion, which is the hepatobiliary cholesterol secretion mediated by hepatic ABCG5/ABCG8.²³ The efficacy of PS may decrease in its oxidized forms, less absorbed than cholesterol. However, different from oxidized cholesterol, a potent proatherogenic molecule, there is no clear role for oxyphytosterols in atherosclerosis development so far.²⁴

Plant sterols and stanols have similar effects on the inhibition of cholesterol absorption, reducing LDL-C levels by 6% to 12%. There is a dose–response effect between PS consumption and reduction of cholesterol concentrations with daily intakes of PSs up to ∼3 g/d.²⁵ Since most studies evaluated the 2 g/day dose, this is the recommended intake for a broad range of hypercholesterolemic patients, from those who do not qualify for pharmacological treatment to those at high risk, but not reaching their lipid goals despite adequate pharmacological tretament.²⁶ In our study, we evaluated the supplementation on a vegetable spread, but other forms of supplementation are available and are equally effective.²⁷

Jakulj et al. ¹¹ have performed the first study to evaluate the association of ezetimibe and PS in moderately hypercholesterolemic individuals on primary prevention for CAD and not taking statins. After 4 weeks of intervention, ezetimibe as a monotherapy, PSs, and the association of both reduced LDL plasma levels by 22.2%, 5%, and 25.2%, respectively. There was no significant superiority in the association of ezetimibe and PSs compared with ezetimibe alone. Lin et al. ¹⁰ have demonstrated discordant findings while evaluating 21 statin naive patients with mild hypercholesterolemia. In this study, in which nutritional aspects and fecal cholesterol excretion were strictly controlled, the association of PSs with ezetimibe provided greater LDL reduction in comparison with ezetimibe as a monotherapy. Malina et al. ²⁸ have demonstrated in a prospective study (including 86 patients) that addition of PSs to different lipid-lowering therapies improves lipid profile, with no effects on markers of cholesterol synthesis and absorption. In this study, a significant additive effect of PS supplementation on total cholesterol and LDL by 7.7% and 6.5%, respectively, was observed. However, PSs added to ezetimibe nonsignificantly reduced LDL cholesterol in their study, in contrast with our finding, in which the largest reduction in LDL was seen in the EZ+PS group, with a 27.3% reduction of LDL.

Our study assessed the association of ezetimibe and PSs in patients already taking statins for hypercholesterolemia, but still not meeting the desirable lipid target values. We believe that absorption might be playing an important role on persistent high-LDL levels.²⁹ Similarly, Farnier et al. ³⁰ have evaluated 618 patients classified as high risk for cardiovascular disease that was already on statin monotherapy, but unable to meet LDL target levels. Patients were randomized in two different groups; patients from the first group were switched to a more potent statin therapy with rosuvastatin and patients from the other group started an association of ezetimibe with simvastatin. The largest LDL reduction and the greatest percentage of patients meeting the target LDL <70 mg/dL were observed in the group that received double therapy with ezetimibe and simvastatin. Authors assumed that patients with poor therapeutic response to statins tend to have a better response to ezetimibe, suggesting that these patients could be greater cholesterol absorbers.

Since the proposed intervention combines two distinct mechanisms of action upon intestinal absorption of cholesterol and could be dependent upon diet, all patients were evaluated through a 3-day food record. Interestingly, we observed a homogeneously inappropriate dietary pattern in all four groups, with high-fat diet in addition to an excess of saturated fat, cholesterol, and sodium intake. There was a deficiency in dietary fiber as well as poly- and monounsaturated fats. These results are similar to those found in a study in which hypercholesterolemic subjects were submitted to dietary intervention, including low-fat stanol ester-containing margarines.³¹

Our sample size is a limitation of this study. Sample calculation was performed to detect differences between active interventions and control group, but it does not allow definite conclusions between different treatments. Another limitation is the fact that we do not know if the lipid reduction obtained with the combination of PSs and ezetimibe on top of statin may have clinical relevance on cardiovascular events. To this date, no study has demonstrated any association between consumption of PSs and reduction in cardiovascular events. Studies that evaluated surrogate markers were unable to demonstrate any improvement with PS intake as a single intervention.^32
–34 On the other hand, the combination of ezetimibe with statin was proven to be effective in reducing cardiovascular events in a high-risk population. In the IMPROVE-IT trial,⁸ cardiovascular risk reduction obtained through this combination was similar to the predicted LDL reduction with statin therapy alone from CTT data published in 2010.³⁵ These evidences allow us to hypothesize that further reduction in LDL levels, obtained through a more aggressive cholesterol absorption inhibition, could provide greater cardiovascular protection.

In conclusion, patients with CAD already taking statins and not reaching target lipid levels benefit from ezetimibe+PS association for LDL reduction. It still remains unclear whether this new approach will translate into clinical benefits, reducing major cardiovascular events, as already demonstrated with high-dose statin therapy.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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