Effect of Ezetimibe on Platelet Aggregation and LDL Tendency to Peroxidation

Condition(s) targeted: Hypercholesterolemia

Intervention: Simvastatin (Drug); Ezetimibe (Drug)

Phase: Phase 4

Status: Completed

Sponsored by: Ziv Hospital

Official(s) and/or principal investigator(s):
Osamah Hussein, MD, Principal Investigator, Affiliation: Ziv Hospital

The aim of our study is to Estimate the reduction of LDL by ezetimibe in hypercholesterolemic patients on simvastatin. Investigate the effect of LDL lowering by ezetimibe on platelet activity and LDL tendency to peroxidation in hypercholesterolemic patients on simvastatin therapy

The hypothesis is that:

1. LDL lowering by ezetimibe on-top of simvastatin in patients on fixed dose of simvastatin can reduce platelet aggregation, due to the potential decreasing of cholesterol content in the platelet membranes.

2. LDL lowering by ezetimibe can lower LDL tendency to peroxidation.

Official title: Phase 4 Study on Effect of Ezetimibe on Platelet Aggregation and LDL Tendency to Peroxidation In Hypercholesterolemic Patients on Simvastatin

Study design: Treatment, Non-Randomized, Open Label, Active Control, Single Group Assignment

Detailed description: The hypothesis is that:

1. LDL lowering by ezetimibe on-top of simvastatin in patients on fixed dose of simvastatin can reduce platelet aggregation, due to the potential decreasing of cholesterol content in the platelet membranes.

2. LDL lowering by ezetimibe can lower LDL tendency to peroxidation.

The aim of our study is:

to Estimate the reduction of LDL by ezetimibe in hypercholesterolemic patients on simvastatin. Investigate the effect of LDL lowering by ezetimibe on platelet activity and LDL tendency to peroxidation in hypercholesterolemic patients on simvastatin therapy.

The importance of the study:

To investigate if ezetimibe has pleiotrophic additive effect on top of statin, which can justify its use in hypercholesterolemic patients, while waiting studies with clinical outcomes.

The question of what is more important, LDL reduction or the pleiotrophic effect of statins is intensified after the release of the results of PROVE-IT study (11).

Patients and methods:

Twenty hypercholesterolemic patients on fixed dose of simvastatin with LDL>130 or >100 mg/dL (Re. inclusion criteria) and triglycerides<300 mg/dL without active coronary heart disease, will be enrolled.

Five healthy volunteers will be enrolled for validation purposes.

Criteria for inclusion:

1. Hypercholesterolemic patients on stable simvastatin dose for at least one month.

2. Age ≥18 years on stable AHA step 1 diet.

3. Patients without CHD or with one risk factors ; LDL > 130 mg/dL and for Patients with CHD or CHD risk equivalent(clinical manifestations of non-coronary forms of atherosclerotic disease) or with 2 risk f actors (cigarette smoking, hypertension (BP ≥140/90 mm Hg or on antihypertensive medication), low HDL cholesterol (<40 mg/dL), family history of premature CHD;LDL>100 mg/dL

4. Patients `on at least simvastatin treatment of 20 mg per day.

5. CPK, ALT and AST < 1. 5 X upper limit of normal at baseline.

Criteria for exclusion:

1. Women currently receiving cyclical hormones.

2. Treatment with psyllium, other fiber based laxatives, phytosterol margarines, or other OTCs that affect serum lipids, unless treated with a stable regimen for > 6 weeks and the patient agrees to continue this regimen for the duration of the trial.

3. Oral corticosteroids unless used as replacement therapy for pituitary/adrenal disease and a stable regimen for at least 6 weeks.

4. Lipid lowering agents including fish oils and QUESTRAN taken within 6 weeks.

5. Active coronary heart disease: unstable angina, acute myocardial infarction, CABG or PTCA within the last 3 months.

6. Women with childbearing potential unless on safe contraception.

7. Psychiatric disease with defect in judgement.

8. Severe renal or hepatic disease.

9. Uncontrolled hypo- or hyperthyroidism.

10. Contraindication for ezetimibe or simvastatin treatment. The patients will continue on their treatment with simvastatin for 6 weeks, and then the patients will be treated by the same dose of simvastatin combined with ezetimibe 10 mg/day for other 6 weeks.

Blood tests for lipids, liver (ALT,AST,GGT,Alkaline phosphatase and bilirubin) and renal function tests (urea and creatinine), complete blood count, general urine test, LDL tendency to peroxidation, platelet aggregation, cholesterol content in platelet membranes and electrocardiogram will be done at base line, after 6 weeks and after 12 weeks.

LDL tendency to peroxidation:

Blood samples will be collected into sodium ethylene diamine-tetraacetic acid (Na2EDTA) (1mM) from patients and from controls at baseline and after three months, will be centrifuged at 1500 xg for 10 minutes at room temperature. LDL will be separated from the plasma by discontinuous gradient ultracentrifugation (13) at 4ºC and will be dialyzed against saline sodium ethylene diamine-tetraacetic acid (1mM). LDL protein concentration will be determined by the method of Lowry (14).

LDL oxidation studies will be performed each time on fresh LDL samples. Before oxidation, LDL will be dialyzed against saline 1. 006 with Na2EDTA 1mM, pH=7. 4, and then will be dialyzed against phosphate buffered saline at 4°C to remove Na2EDTA. It will be then diluted with PBS to 0. 2 mg of protein per ml. The lipoprotein will be incubated in the presence of 5μM CuSO4 at 37ºC. Measuring conjugated dienes formation at 234nm continuously monitored the kinetics of LDL oxidation (15). For each LDL sample we will received a curve consisting of three consecutive phases: lag phase (lag time in minutes required for the initiation of CuSO4-induced LDL oxidation), propagation phase and a final plateau phase.

Platelet separation:

For platelet studies, venous blood (30 ml) will be collected through siliconized syringes into acid citrate dextrose solution(1. 4% citric acid, 2. 5% sodium citrate, and 2% dextrose) at a ratio of 9: 1 (v: v) for washed platelets (WP)preparation. WP will be prepared by centrifugation at 240g for 20 min. The platelet bellet will be washed twice in 5 mmol Hepes buffer, pH 7. 4 (140 mmol NaCl, 2 mmol KCL, 1 mmol MgCl2, 5 mmol Hepes, 12 mmol NaHCO3 and 5. 5 mmol of glucose). For the preparation of WP suspension, 15 L of acetic acid (1mmol) will be added to 1 ml of platelet suspension throughout WP preparation in order to ensure acidic conditions which are required for platelet resuspension. This procedure will reduce the medium pH to 6. 5 and it does not influence the aggregation response of the WP.

Platelet aggregation:

Collagen (Nycomed, germany) will be used as the aggregating agent at a concentration of 4 g/ml (this concentration can cause up to 60% aggregation amplitude in WP). Platelet aggregation will be performed at 37ºC in aggregometer using hepes as a reference system. Results will be expressed as the extent of maximal aggregation (% of maximal amplitude) and also as the slope of the aggregation curve (cm/min).

Cholesterol content in platelet membranes:

Platelets will be washed three times with Hepes buffer, and then sonicated twice for 20 seconds at 80 watt. Platelet lipids will be extracted with hexane: isopropanol (3: 2, v: v). The cholesterol content will be measured in the dried hexane phase by the method of Chiamori et al (16). Platelet protein will be determined using the method of Lowry (14).

Statistical analysis:

Student's t-test will be used for comparing the two means, whereas analysis of variance (ANOVA) will be used when the results of the study group will be compared with the control group. Data are presented as mean ± standard deviation.

Spearsman's correlation coefficients will be calculated to determine the relation between changes in lipid parameters and changes in LDL tendency to oxidation and platelet aggregation.

By assumption that treatment will reduce the aggregation percent and slope by 30% and LDL tendency to peroxidation by 25% in 20 patients, p will be < 0. 001.

Minimum age: 18 Years. Maximum age: N/A. Gender(s): Both.

Criteria:

Inclusion Criteria:

1. Hypercholesterolemic patients on stable simvastatin dose for at least one month.

2. Age ≥18 years on stable AHA step 1 diet.

3. Patients without CHD or with one risk factors ; LDL > 130 mg/dL and for Patients with CHD or CHD risk equivalent(clinical manifestations of non-coronary forms of atherosclerotic disease) or with 2 risk f actors (cigarette smoking, hypertension (BP ≥140/90 mm Hg or on antihypertensive medication), low HDL cholesterol (<40 mg/dL), family history of premature CHD;LDL>100 mg/dL

4. Patients `on at least simvastatin treatment of 20 mg per day.

5. CPK, ALT and AST < 1. 5 X upper limit of normal at baseline.

Exclusion Criteria:

1. Women currently receiving cyclical hormones.

2. Treatment with psyllium, other fiber based laxatives, phytosterol margarines, or other OTCs that affect serum lipids, unless treated with a stable regimen for > 6 weeks and the patient agrees to continue this regimen for the duration of the trial.

3. Oral corticosteroids unless used as replacement therapy for pituitary/adrenal disease and a stable regimen for at least 6 weeks.

4. Lipid lowering agents including fish oils and QUESTRAN taken within 6 weeks.

5. Active coronary heart disease: unstable angina, acute myocardial infarction, CABG or PTCA within the last 3 months.

6. Women with childbearing potential unless on safe contraception.

7. Psychiatric disease with defect in judgement.

8. Severe renal or hepatic disease.

9. Uncontrolled hypo- or hyperthyroidism.

10. Contraindication for ezetimibe or simvastatin treatment. The patients will continue on their treatment with simvastatin for 6 weeks, and then the patients will be treated by the same dose of simvastatin combined with ezetimibe 10 mg/day for other 6 weeks.

Internal Medicine Department A ,Ziv Goverment Hospital, Safed 13100, Israel

Related publications:

Bays HE, Moore PB, Drehobl MA, Rosenblatt S, Toth PD, Dujovne CA, Knopp RH, Lipka LJ, Lebeaut AP, Yang B, Mellars LE, Cuffie-Jackson C, Veltri EP; Ezetimibe Study Group. Effectiveness and tolerability of ezetimibe in patients with primary hypercholesterolemia: pooled analysis of two phase II studies. Clin Ther. 2001 Aug;23(8):1209-30. Erratum in: Clin Ther 2001 Sep;23(9):1601.

Davidson MH, McGarry T, Bettis R, Melani L, Lipka LJ, LeBeaut AP, Suresh R, Sun S, Veltri EP. Ezetimibe coadministered with simvastatin in patients with primary hypercholesterolemia. J Am Coll Cardiol. 2002 Dec 18;40(12):2125-34.

van Heek M, Austin TM, Farley C, Cook JA, Tetzloff GG, Davis HR. Ezetimibe, a potent cholesterol absorption inhibitor, normalizes combined dyslipidemia in obese hyperinsulinemic hamsters. Diabetes. 2001 Jun;50(6):1330-5.

Sager PT, Melani L, Lipka L, Strony J, Yang B, Suresh R, Veltri E; Ezetimibe Study Group. Effect of coadministration of ezetimibe and simvastatin on high-sensitivity C-reactive protein. Am J Cardiol. 2003 Dec 15;92(12):1414-8.

Davis HR Jr, Compton DS, Hoos L, Tetzloff G. Ezetimibe, a potent cholesterol absorption inhibitor, inhibits the development of atherosclerosis in ApoE knockout mice. Arterioscler Thromb Vasc Biol. 2001 Dec;21(12):2032-8.

Lavy A, Brook GJ, Dankner G, Ben Amotz A, Aviram M. Enhanced in vitro oxidation of plasma lipoproteins derived from hypercholesterolemic patients. Metabolism. 1991 Aug;40(8):794-9.

Hussein O, Frydman G, Frim H, Aviram M. Reduced susceptibility of low density lipoprotein to lipid peroxidation after cholestyramine treatment in heterozygous familial hypercholesterolemic children. Pathophysiology. 2001 Aug;8(1):21-28.

Surya II, Akkerman JW. The influence of lipoproteins on blood platelets. Am Heart J. 1993 Jan;125(1):272-5. Review. No abstract available.

Ardlie NG, Selley ML, Simons LA. Platelet activation by oxidatively modified low density lipoproteins. Atherosclerosis. 1989 Apr;76(2-3):117-24.

Osamah H, Mira R, Sorina S, Shlomo K, Michael A. Reduced platelet aggregation after fluvastatin therapy is associated with altered platelet lipid composition and drug binding to the platelets. Br J Clin Pharmacol. 1997 Jul;44(1):77-83.

Aviram M. Plasma lipoprotein separation by discontinuous density gradient ultracentrifugation in hyperlipoproteinemic patients. Biochem Med. 1983 Aug;30(1):111-8.

Knopp RH, Gitter H, Truitt T, Bays H, Manion CV, Lipka LJ, LeBeaut AP, Suresh R, Yang B, Veltri EP; Ezetimibe Study Group. Effects of ezetimibe, a new cholesterol absorption inhibitor, on plasma lipids in patients with primary hypercholesterolemia. Eur Heart J. 2003 Apr;24(8):729-41.

Starting date: February 2005
Ending date: August 2007
Last updated: August 13, 2007