Background: preliminary data have shown that erythrocyte-bound apolipoprotein B (ery-apoB) may be protective against atherosclerosis. However, statins may have an effect on ery-apoB.

Objective: to investigate the effect of statins on ery-apoB levels.

Study design: a non-randomized intervention study. Ery-apoB will be measured twice in volunteers who are on statin therapy for medical reasons. After a baseline measurement of ery-apoB volunteers will discontinue their statin use for a period of six weeks followed by a second measurement of ery-apoB. Consecutively subjects will start with their original statin therapy again.

Study population: patients on statin therapy aged 18 years or older.

Intervention: temporary discontinuation of statin therapy for a period of six weeks.

Main study endpoints: changes in ery-apoB levels.

Risks, burden and benefits on participation: volunteers will visit the outpatient clinic twice, the second visit will be exactly six weeks after the first visit. The volunteers' general practitioner and medical specialist (internist or cardiologist) will be informed about their participation. Subjects have to fast for 10 hours before every visit and venous blood samples will be drawn on both visits (a total of 36ml of blood). Subjects will discontinue their usual statin therapy for a period of six weeks. No major risks are involved with temporary discontinuation of statin therapy in stable chronic cardiovascular disease. Volunteers will receive 25 euro's in total for participation. Participation serves to further investigate the relation of statins and potentially beneficial binding of apoB on erythrocytes.

INTRODUCTION:

Previously it has been shown that atherogenic apolipoprotein (apo) B containing lipoproteins bind to erythocytes.1-3 Studies from our laboratory have shown that the binding of apoB on erythrocytes (ery-apoB) may reflect a situation of protection against cardiovascular disease (CVD).4 Mean ery-apoB is significantly lower in subjects with known CVD compared to healthy controls. However, the use of statins could be an important confounding factor, because of its known LDL-C and serum apoB lowering effects.5, 6 In our on-going study population 91% of the patients with CVD were on statin therapy compared to only 19% of the subjects without CVD. Preliminary data showed at least some effect of statin therapy on ery-apoB. A modest, but significant negative correlation was found between ery-apoB and serum apoB and LDL-cholesterol in subjects without statins (Pearson's r -0.172 and -0.185, P < 0.05) but these correlations were lost in subjects using statins. Statins are known for their inhibition of HMG-CoA reductase, which is necessary for the liver to synthesize cholesterol, and increase the expression of hepatic LDL-receptors resulting in lowering of LDL-cholesterol and its related apoB.7, 8 In theory ery-apoB may be lowered by statins as well, which could be an explanation for the differences in ery-apoB between subjects with or without CVD. This interventional study is to explore the effects of statin therapy on ery-apoB.

AIM:

To investigate the effects of statin therapy on plasma levels of erythrocyte-bound apolipoprotein B.

HYPOTHESIS:

Ery-apoB will increase after statin withdrawal due to higher total plasma apoB.

METHODS AND MATERIALS:

Study population Subjects will be males and females aged 18 years or older who are on statin therapy for medical purposes like primary or secondary hypercholesterolemia or the presence of CVD. Exclusion criteria are unstable angina pectoris, an acute cardiovascular event (i.e. myocardial infarction, percutaneous coronary intervention, stroke or bypass surgery) in the last 6 months or the use of any other lipid lowering drug like ezetimibe or fibrates besides statin therapy in the last 6 months.

Study design

A total of 53 subjects will visit the outpatient clinic twice. The following procedures will be performed during the first visit:

• Questionnaire to obtain information about patient's medical history, medication profile and the general practitioners contact information.
• Measurement of anthropometric variables (blood pressure, length, weight and waist).
• Venipuncture (1 Clot tube, 1 Heparin tube, 1 EDTA tube and 1 Glucose tube, a total of 18ml of blood).
• Temporary discontinuation of statin therapy for 6 weeks. Six weeks after the first visit participating subjects will attend the outpatient clinic again for another venipuncture (1 Clot tube, 1 Heparin tube, 1 EDTA tube and 1 Glucose tube, a total of 18ml of blood). Subjects will start again with their original statin therapy afterwards (same type of drug and dosage). However, statin therapy will be initiated earlier if the subject suffers from an acute cardiovascular event during the study period. Study subjects will be monitored by phone for adverse events 8 weeks after finishing the study.

Intervention Statin therapy will be discontinued for a period of six weeks.

Analytical determinations Basic parameters for renal function as well as glucose, CRP, total cholesterol, HDL-C and TG will be determined using Synchrom LX analyzer (Beckman Coulter, Anaheim CA, USA). LDL-C values will be calculated using the Friedewald formula. ApoAI and ApoB will be determined by rate nephelometry using IMAGE by commercially available kits provided by Beckman (Beckman Coulter Anaheim CA, USA). Blood cell counts will be determined automatically using LH analyzer (Beckman Coulter, CA, USA). The leukocyte differentiation will be determined as a five-part differentiation on the same instruments. ApoB on blood cells will be detected by indirect immunofluorescence evaluated by flow cytometry, previously validated and published.9

Required materials

• Flow cytometer (Beckman Coulter, USA)
• Validated measurement protocol for ery-apoB on flow cytometry
• Venous blood drawing equipment and blood tubes
• Funding: Stichting Onderzoek & Ontwikkeling Sint Franciscus Gasthuis and Klinisch Chemisch Laboratorium Sint Franciscus Gasthuis cover the costs

Investigational product The medication group of statins is the product of investigation. The most frequently prescribed statins are simvastatin, atorvastatin, rosuvastatin, pravastatin and fluvastatin with dosages ranging from 5mg to 80mg administered once daily.10 Statins inhibit HMG-CoA reductase, which is necessary for the liver to synthesize cholesterol, and subsequently lowers LDL-cholesterol and its related apo B.7, 8 Atorvastatin and rosuvastatin show the longest terminal half-life (11-14 hours) compared to the other statins (1-3 hours).11 Bioavailability of statins is low due to an extensive first-pass effect intestinally and at the liver and all but pravastatin show active metabolites.11 All active metabolites of statins will be eliminated after discontinuation of statins for six weeks.

Statistics Subjects will serve as their own control group. Therefore, the paired T test will be used to test for significant differences in ery-apoB before and after withdrawal of statin therapy. The differences in ery-apoB before and after withdrawal of statin therapy are checked for normality using the Shapiro-Wilk test. A P-value below 0.05 will be regarded as statistical significant.

Ongoing observational studies have shown a mean difference 0.25 between subjects with and without statin therapy. A recently completed pilot has shown a SD of the difference of 0.64 instead of previously estimated SD of 0.50. To demonstrate a significant difference of ery-apoB with a mean difference of 0.25 a.u. and a SD of the difference of 0.64, at least 53 subjects should be included in the analysis (power is 80%, alpha is 0.05 two-sided).

Safety reporting The investigator will inform the subjects and the reviewing accredited METC if anything occurs, on the basis of which it appears that the disadvantages of participation may be significantly greater than was foreseen in the research proposal. The study will be suspended pending further review by the accredited METC, except insofar as suspicion jeopardize the subjects' health. The investigator will take care that all subjects are kept informed.

Adverse events (AE) are defined as any undesirable experience occurring to a subject during the study, whether or not considered related to the investigated treatment. All adverse events reported spontaneously by the subject or observed by the investigator or his staff will be recorded. A serious adverse event (SAE) is any untoward medical occurrence or effect that at any dose results in death, is life threatening, requires hospitalization or prolongation or existing in patients' hospitalization, results in persistent disability of incapacity or affects the safety of the subjects in any way. These serious adverse events will be reported through the web portal "Toetsing Online" to the accredited METC that approved the protocol.

Adverse events that may be expected regarding the withdrawal of statins for a period of six weeks are the occurrence of an acute cardiovascular event or "statin withdrawal syndrome". Studies have shown that discontinuation of statin therapy is associated with worse outcomes after an acute coronary syndrome, stroke or vascular surgery.12 However, no increase in adverse outcomes was observed in patients with stable chronic coronary artery disease following the temporary discontinuation of statin therapy.12 The prevalence of statin related muscle complaints is 1-10%.13 However, the risk of statin related muscle complaints after restarting the statin after withdrawal of four weeks will be very small since study subjects were already on the same statin before participation of the study. There is no clear evidence of any significant harm or effects on quality of life caused by statins in a group of 34.272 study subjects.14

Inclusion Criteria:

• 18 years or older
• use of a statin

Exclusion Criteria:

• use of other lipid lowering drugs, other than statins
• a cardiovascular event in the past 6 months