Trial Information

Synergistic Effect of Simvastatin and Ezetimibe on Lipid and Pro-inflammatory Profiles in Pre-diabetic Subjects

Long-term benefits of statins on prevention of cardiovascular events have been consistently
shown in several populations. It has been demonstrated that, with particular regard to
subjects at high risk, the lower LDL-cholesterol levels, the lower the incidence of
cardiovascular outcomes. Beneficial effects of statins can be attributed to their
lipid-lowering ability as well as to additional benefits. The so-called pleiotropic effects
on low grade inflammation status have been described in subsets of subjects with different
cardiovascular profiles. The most common inflammatory marker used in clinical practice is
the high-sensitivity C-reactive protein (CRP) level, but a number of others has also been
investigated.

Disturbances of glucose metabolism accompanied by insulin resistance are pro-inflammatory
conditions which may accelerate atherosclerosis. Diabetic populations are at high
cardiovascular risk and strict control of lipoprotein concentrations is recommended. Several
recent studies showed the efficacy of statins on primary and secondary prevention of
cardiovascular events in diabetic populations. The goal of 100 mg/dL for LDL-cholesterol may
be too elevated for subjects at very high risk for whom a target of 70 mg/dl has been
suggested. High statin doses may be necessary, which increases its adverse effects. Given
that statin monotherapy may be insufficient for the desirable reduction in LDL levels, a
combination of lipid-lowering agents has become frequent in clinical practice. In
particular, statin and ezetimibe combination has been shown to be very effective in reducing
total and LDL cholesterol levels.

Ezetimibe is a specific cholesterol absorption inhibitor that acts at the brush border of
the small intestine, blocking the absorption of dietary and biliary cholesterol and plant
sterols, resulting in intracellular cholesterol depletion via the Niemann-Pick C1-like
transporter. Adding ezetimibe to statin therapy induces a 15% reduction in LDL levels
compared with only 6% achieved by doubling the dose of statins. Data concerning the
pleiotropic effects of this combination are controversial. One study, in which CRP level was
used as the inflammatory marker, found that a combination of simvastatin and ezetimibe
produced an incremental effect in lowering CRP, independently of the improvement in
lipoprotein concentrations. Although few studies have confirmed this finding, as far as we
know, data regarding simvastatin-ezetimibe combination induced-changes in serum
interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-a) levels are lacking. We tested
the hypothesis that this combination would induce improvement in inflammatory status, as
reflected by leukocyte count and CRP, IL-6 and TNF-α levels.

Therefore, this study evaluates whether the combination of lipid-lowering effects of
low-to-moderate dose of simvastatin and ezetimibe also results in a synergistic effect that
reduces the pro-inflammatory status of pre-diabetic subjects with mild-to-moderate
hypercholesterolemia.

Subjects and Methods:

Participants were selected from the Federal University of São Paulo outpatient clinics. The
study was approved by the institutional ethical committee and all participants were provided
with written informed consent.

Eligible subjects were men and women, aged from 18 to 75 years, with a body mass index (BMI)
ranging from 25 to 40 kg/m2 and pre-diabetes (impaired glucose tolerance or impaired fasting
glucose). Entry criteria required triglyceride levels ≤ 350 mg/dl and LDL cholesterol ≤ 200
mg/dl, stable blood pressure and no evidence of cardiovascular, hepatic or renal diseases.
Subjects were not taking anti-inflammatory agents or others interfering with lipid or
glucose metabolism. Eligible participants were recruited from June 2005 to May 2006.
Two-hundred-ninety subjects with weight excess, with or without family history of diabetes,
were screened for the interventional protocol and 50 with impaired glucose tolerance (IGT)
or impaired fasting glucose (IFG) were randomly assigned to 2 groups that would receive
ezetimibe 10 mg/d (n = 25) or simvastatin 20 mg/d (n = 25), preceded by a 2-week run-in
period. Monotherapies were maintained for 12 weeks; thereafter the drugs were combined in
each group for an additional 12-week period. All participants received individual counseling
for a healthy lifestyle and had monthly visits. Treatment was to be discontinued prematurely
if transaminases exceeded 3 times the upper limit of reference or creatine phosphokinase
(CPK) 10 times, but no subject met these criteria. One of the ezetimibe group dropped out
due to non-compliance.

Baseline, 12-week and 24-week blood samples were drawn in the morning, after a 12-hour fast,
for glucose, lipid profile, including apolipoprotein A-I and B, leukocyte count and
inflammatory markers were made. A LDL-cholesterol goal of 100 mg/dl was used in the present
study (8,22).

Laboratory analysis: Plasma glucose, transaminases, CPK and creatinine were determined by
routine methods. Serum lipid levels (total cholesterol, HDL-cholesterol, and triglycerides)
were analyzed by commercially available tests (Roche Diagnostics GmbH, Mannheim, Germany).
Blood samples were stored at -20ºC until determinations of apolipoproteins and inflammatory
markers. Apolipoprotein A-I and B were measured by immunoturbidimetry (Olympus Life and
Material Science Europa GmbH, Lismeeham, Ireland), with an intra-assay coefficient of
variability (CV) of 1.26-1.30% and 0.93-1.17% respectively, and an inter-assay CV of
1.43-1.55% and 1.10-1.46%, respectively. High-sensitivity CRP (Immulite - DPC, Los Angeles,
CA, USA), TNF-α and IL-6 (Immulite - Euro/DPC, Llanberis, Gwynedd, UK) were determined by
chemiluminescent immunometric assay. The sensitivity of CRP assay was 0.01 mg/dL
(intra-assay CV 4.2-6.4% and inter-assay CV 4.8-10%), of TNF-α assay 1.7 pg/mL (intra-assay
CV 2.6-3.6%, inter-assay CV 4.0-6.5%) and of IL-6 assay was 2.0 pg/mL (intra-assay CV
3.5-6.2%, inter-assay CV 5.1-7.5%).

Statistical analysis: Data were expressed as mean values and standard errors or deviations.
Unpaired Student's t test was used to compare groups at baseline and chi-square to assess
differences between qualitative data. One-way ANOVA for repeated measures was used to
evaluate the effect of drugs over time and to compare data between groups of subjects
according to the type of therapy. Pairwise contrasts were made by comparing least-square
mean estimates, and P values adjusted for multiple comparisons using the Bonferroni Holm
method. The level of significance was set at P < 0.05. Correlation between variables was
tested by the Pearson coefficient, also employed to assess whether changes in variables over
time were correlated. Data analysis was performed using Statistical Analysis System
software, version 8.2 (SAS Institute, Cary, NC).