Effect of Rosuvastatin Versus Atorvastatin Treatment on Paraoxonase- 1 Activity in Men With Established Cardiovascular Disease and a Low HDL-Cholesterol

By Bergheanu, S C Van Tol, A; Dallinga-Thie, G M; Liem, A; Dunselman, P H J; Van Der Bom, J G; Jukema, J W

Key words: Atorvastatin – Cardiovascular disease – Paraoxonase-1 activity – Rosuvastatin ABSTRACT

Objective: Paraoxonase-1 (PON-1) is a high-density lipoprotein (HDL) associated enzyme involved in the protective mechanisms of HDL. Our aim was to compare the effect of treatment with rosuvastatin and atorvastatin on serum PON-1 activity.

Methods: We performed a prespecified prospective study in 68 patients, part of a larger, multicentre randomized study – RADAR (Rosuvastatin and Atorvastatin in different Dosages And Reverse cholesterol transport). Patients aged 40-80 years, all men, with established cardiovascular disease and high-density lipoprotein cholesterol (HDL-C) < 1.0 mmol/L (< 40 mg/dL) entered a 6-week dietary run-in period before receiving treatment with rosuvastatin 10 mg or atorvastatin 20 mg daily for 6 weeks. Doses were increased after 6 weeks to rosuvastatin 20 mg or atorvastatin 40 mg and after 12 weeks to rosuvastatin 40 mg or atorvastatin 80 mg daily. Serum PON-1 activity and lipid profile were determined at baseline, 6 and 18 weeks.

Results: After 18 weeks, the rosuvastatin arm showed a significant increase of PON-1 activity (6.39 U/L, p = 0.02) whereas this was not observed in the atorvastatin arm (1.84 U/L, p = 0.77). The difference between groups did not reach significance (p = 0.11). Both rosuvastatin and atorvastatin resulted in significant (p = 0.0001) and similar increases in HDL-C after 6 weeks [0.06 mmol/L (2.32 mg/dL) vs. 0.05 mmol/L (1.93 mg/dL)] and after 18 weeks [0.10 mmol/L (3.87 mg/dL) vs. 0.10 mmol/L (3.87 mg/dL)].

Conclusions: Rosuvastatin treatment resulted in a significant increment of serum PON-1 activity with increasing dose while this was not observed with atorvastatin.

Introduction

3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors, such as atorvastatin and rosuvastatin, are potent drugs that lower plasma total cholesterol and low-density lipoprotein cholesterol (LDL-C) concentrations1-4. Though LDL-C is the primary target of lipid-modifying therapy for the prevention of cardiovascular disease, the importance of other lipoproteins to the development of cardiovascular disease is becoming increasingly recognized. A 1-mg/dL (0.026 mmol/L) increment in high-density lipoprotein cholesterol (HDL-C) was associated with significant 3.7% (men) and 4.7% (women) decrements in cardiovascular disease mortality rates5. Conversely, low plasma HDL-C levels associate with a high risk for adverse cardiovascular outcome in patients with documented coronary artery disease6.

It has been suggested that high-density lipoprotein (HDL) may have anti-atherogenic potential by enabling reverse cholesterol transport from the arterial wall to the liver for excretion, but also by acting as an anti-oxidative, anti-inflammatory, anti- apoptotic, antithrombotic, anti-infectious and vasodilatatory agent7.

Paraoxonase-1 (PON-1) is an HDL associated enzyme that is potentially contributing to the protective effects of HDL-C8. PON-1 has been demonstrated to protect low-density lipoprotein (LDL) against oxidation, to reverse the biological effects of oxidized LDL and to preserve the function of HDL by blocking its oxidation8-10. Low serum PON-1 activity is an independent risk factor for coronary events in men at high risk because of pre-existing coronary disease or other cardiovascular risk factors11. Paraoxonase activity12 and polymorphisms in the paraoxonase synthase genes associate with premature coronary artery disease and risk of early-onset myocardial infarction respectively13.

Several studies have extensively described the differences between rosuvastatin and atorvastatin in terms of total cholesterol, LDL-C, HDL-C and triglycerides changes14,17. In the present study we aimed to compare the influence of rosuvastatin and atorvastatin treatment on serum PON-1 activity, a protective factor against LDL modification.

Methods

Study population

We used a subset of patients (n = 80) from the RADAR (Rosuvastatin and Atorvastatin in different Dosages And Reverse cholesterol transport study)17 for the characterization of PON-1 activity and lipid profile. As there were only two women in each group we preferred to exclude them from the final analyses. In addition, we decided to exclude patients who did not have complete data for all visits. In total, data from 68 men were analysed. The RADAR study17 was designed and conducted in accordance with the Declaration of Helsinki and in compliance with the ethical principles of Good Clinical Practice. Appropriate ethics committees or institutional review boards approved the study and all patients gave written, informed consent.

In summary, RADAR was an open-label, randomized, parallel-group, multicentre, phase IIIb study conducted in 29 centres in The Netherlands. Patients (male = 418/female = 43) were aged between 40 and 80 years and had established cardiovascular disease as described earlier17. Inclusion criteria: at enrolment fasting plasma HDL-C concentrations of < 1.0 mmol/L (40 mg/dL) and fasting plasma triglycerides

Measurements and statistical analyses

Blood was collected at the start of the run-in, at baseline and after 6, 12 and 18 weeks during treatment. Patients were fasted for 12 h and abstained from alcohol for the same period prior to blood sampling. Plasma and serum were isolated after centrifugation at 40[degrees]C, 800xg (3000 rpm) for 15min and stored at – 80[degrees]C for further analyses. Plasma cholesterol and triglyceride levels were analyzed using the COVANCE virtual central laboratory (VCL) concept. Plasma LDL-C was calculated using the Friedewald formula. Plasma HDL-C was measured by a direct enzymatic method based on polyethylene glycol (PEG)-modified enzymes using a Hitachi 911 autoanalyser (Roche Diagnostics, Mannheim, Germany). Serum PON-1 activity was analysed spectrophotometrically with paraoxon as substrate. In short, 10 and 20 [mu]L of serum were added to 1 mL incubation medium containing 50mM Tris-HCL buffer (pH 8.0), with 1 mM CaCl^sub 2^ and 6mM paraoxon. The reaction was monitored for up to 30min at 25[degrees]C by measuring the appearance of p- nitrophenol at 412 nm in a CARY 1E UV visible spectrophotometer. PON- 1 activity is expressed in U/L. One U is equal to 1 [mu]mol of p- nitrophenol formed per minute.

Figure 1. RADAR study design

Baseline values and absolute variations during the treatment presented a skewed distribution and, therefore, median values and interquartile ranges were computed instead of mean values and confidence intervals. The differences between the visits, in each group, are presented as the median of the differences, in absolute value. For comparison between visits, in the same group, we used Wilcoxon test for two related samples. The between-group comparisons at baseline and at the two time points were performed using the Mann- Whitney U test for two independent samples. The proportion of diabetes patients and the use of concomitant medication were compared between the two groups using the chi-square test. SPSS for Windows version 12.0.1 was used for all analyses.

Results

The baseline characteristics were similar for the two arms of the study group (Table 1). The median age was 66 years for both groups. The baseline values of HDL-C ranged between 0.25mmol/L (9.67mg/dL) and 0.87mmol/L (33.64mg/dL) with a median of 0.64mmol/L (24.75mg/ dL) for the rosuvastatin group and 0.62mmol/L (23.98mg/dL) for the atorvastatin group because HDL-C < 1 mmol/L (40mg/dL) was among the inclusion criteria.

The use of concomitant medications (number of patients) in the rosuvastatin (n = 34) and atorvastatin (n = 34) groups throughout the study period did not significantly differ: angiotensin- converting enzyme inhibitors 13 versus 12, beta-blocking agents 21 versus 26, calcium-channel blockers 9 versus 11, aspirin 26 versus 31, clopidogrel 3 versus 3, vitamin K antagonists 8 versus 3 and thiazides 5 versus 5 [p> 0.05 for all comparisons).

PON-1 activity, HDL-C, LDL-C, total cholesterol and triglycerides were measured after 6 weeks of 10 mg daily rosuvastatin or 20 mg daily atorvastatin and after the last 6 weeks period with the highest dose of 40 mg daily of rosuvastatin or 80 mg daily of atorvastatin. The absolute changes are presented in Table 2. During treatment, rosuvastatin resulted in a uniform increment of serum PON- 1 activity with increasing dose (Figure 2). At the end of 18 weeks treatment, the rosuvastatin arm showed a significant increase of PON- 1 activity from baseline (6.39 U/L, p = 0.02) whereas this was not observed in the atorvastatin arm (1.84 LVL, p = 0.77). The difference between groups did not reach statistical significance (p = 0.11). Both rosuvastatin and atorvastatin resulted in significant (p = 0.0001) and similar increases in HDL-C after 6 weeks [0.06mmol/ L (2.32mg/dL) vs. 0.05mmol/L (1.93mg/dL)] and after 18 weeks [0.10mmol/L (3.87mg/dL) vs. 0.10mmol/L (3.87mg/dL)].

The differences between rosuvastatin and atorvastatin, in respect to total cholesterol, LDL-C and triglycerides failed to reach significance, both at 6 weeks and at 18 weeks (Table 2).

Discussion

Our findings show during 18 weeks of treatment with 3 incremental doses, PON-1 activity was only significantly increased from baseline in the rosuvastatin group. However, when compared directly with atorvastatin, the difference failed to reach significance. In this subgroup of RADAR, rosuvastatin and atorvastatin, in general, resulted in similar changes in lipid profile with those already described in the RADAR study17. Although in the present study the differences between the statin groups did not reach statistical significance, LDL-C and total cholesterol changes were numerically greater in the rosuvastatin arm. These results are supported by other studies18-21. In the present sub-study the increase in HDL-C was very similar after treatment with rosuvastatin or atorvastatin (Table 2).

PON-1 was initially described as an organophosphate hydrolyzing enzyme, one of the three entities of the paraoxonase family. PON-1 is synthesized by the liver and secreted in the blood where it is found associated with HDL. Although the precise role of PON-1 in humans remains unclear, PON-1 may be responsible for inhibiting LDL oxidation8 and for preserving the function of HDL9,10, which further ensures many anti-atherogenic properties among which reverse cholesterol transport, anti-oxidative role and inhibition of inflammatory processes7. PON-1 may also have a protective role against endothelial dysfunction by its capability to hydrolyze homocysteine metabolites such as thiolactones22,23. A number of in vivo studies in mice show a potential PON-1 therapeutic role to prevent the development of atherosclerotic lesions. When fed a high- cholesterol diet, PON-1 deficient mice developed significantly larger aortic lesions than their wild-type and heterozygous counterparts24. In contrast, transgenic mice with expression of human PON-1 and elevated serum PON-1 activity developed significantly smaller atherosclerotic lesions relative to their controls25. In humans, serum PON-1 activity rather than genetic variation in the PON-1 gene, predicts vascular disease26 and a recent study shows a strong association of PON-1 activity with angiographie severity and extent of coronary artery disease, independent of age, hypertension, smoking, abnormal glucose regulation and HDL-cholesterol27.

Table 1. Baseline characteristics of the study population (n = 68)

Table 2. Difference from baseline after 6 weeks and 18 weeks of treatment in the study population (n = 68)

To our knowledge this is the first study to investigate the effect of rosuvastatin on serum PON-1 activity. Atorvastatin significantly increased serum PON-1 activity in a number of studies28-32. One study29 demonstrated that, when patients were divided into two groups according to baseline HDL-C levels (cut-off value ~ 1 mmol/L (40 mg/dL)), atorvastatin therapy increased PON-1 activity especially inpatientswithbaselineHDL-C> 1 mmol/L (40 mg/ dL). The study group included men and women with various types of dyslipidaemia. However, the present study differs substantially because only male patients with low baseline plasma HDL-C [< 1.0 mmol/L (40 mg/dL)] were included. Furthermore we investigated changes in serum PON-1 activity during 3 different statin dosages across an 18-week period. For these reasons it is difficult to make a comparison between our results and those already published.

Figure 2. Paraoxonase-1 (PON-1) activity (%) change at Week 6 and Week 18. Box and whisker plot shelving median values, interquartile ranges (box) and extreme values (whiskers) among the rosuvastatin group (n = 34) and the atorvastatin group (n = 34). ‘o’ represents outliers

Recently it was shown that aspirin may compete for the hydrolysis of paraoxon and, therefore, influence the PON-1 activity33. We did not find any significant difference between aspirin users within the two groups and since aspirin use is associated with higher concentrations and activities of PON-1(34) we may speculate that the numerically higher number of patients taking aspirin within the atorvastatin group may only strengthen our present results.

The limitations of the present study include the open-label design and the selection of men with established cardiovascular disease and low levels of HDL-C only. Another possible limitation is the lack of determination of major PON-1 genotypes and their effect on statin-induced PON-1 activity increase. A recent study showed, however, that Q192R and L55M PON-1 polymorphisms did not affect the PON-1 response to statin treatment with fluvastatin35. Further and larger studies are necessary to clarify the differences in effects on PON-1 activity between various statins in a wider category of patients and to compare possible benefits of one statin over another in terms of cardiovascular protection.

Conclusion

High-dose rosuvastatin may result in a consistent and higher increase of PON-1 activity when compared with atorvastatin and, therefore, rosuvastatin might offer a superior cardiovascular protective effect at various therapeutic regimens.

Acknowledgements

We wish to thank Professor Dr. Jan P. Vandenbroucke, Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, The Netherlands for his contribution to the design of this study. L. M. Scheek performed the PON-I activity assays. The RADAR study was supported by Astra-Zeneca.

JWJ is an established clinical investigator of the Netherlands Heart Foundation (2001D032).

References

1. Malhotra HS, Goa KL. Atorvastatin: an updated review of its pharmacological properties and use in dyslipidaemia. Drugs 2001;61:1835-81

2. Schaefer EJ, McNamara JR, Tayler T, et al. Effects of atorvastatin on fasting and postprandial lipoprotein subclasses in coronary heart disease patients versus control subjects. Am J Cardiol 2002;90:689-96

3. Olsson AG, McTaggart F, Raza A. Rosuvastatin: a highly effective new HMG-CoA reductase inhibitor. Cardiovasc Drug Rev 2002;20:303-28

4. Schuster H. Rosuvastatin – a highly effective new 3-hydroxy-3- methylglutaryl coenzyme A reductase inhibitor: review of clinical trial data at 10-40mg doses in dyslipidemic patients. Cardiology 2003;99:126-39

5. Gordon DJ, Probstfield JL, Garrison RJ, et al. High-density lipoprotein cholesterol and cardiovascular disease. Four prospective American studies. Circulation 1989;79:8-15

6. Genest JJ, McNamara JR, Salem DN, Schaefer EJ. Prevalence of risk factors in men with premature coronary artery disease. Am J Cardiol 1991;67:1185-9

7. Assmann G, Nofer JR. Atheroprotective effects of high-density lipoproteins. Annu Rev Med 2003;54:321-41

8. Mackness MI, Arrol S, Durrington PN. Paraoxonase prevents accumulation of lipoperoxides in low-density lipoprotein. FEBS Lett 1991;286:152-4

9. Oda MN, Bielicki JK, Ho TT, et al. Paraoxonase 1 overexpression in mice and its effect on high-density lipoproteins. Biochem Biophys Res Commun 2002;290:921-7

10. Aviram M, Rosenblat M, Bisgaier CL, et al. Paraoxonase inhibits high-density lipoprotein oxidation and preserves its functions. A possible peroxidative role for paraoxonase. J Clin Invest 1998;101:1581-90

11. Mackness B, Durrington P, McElduff P, et al. Low paraoxonase activity predicts coronary events in the Caerphilly Prospective Study. Circulation 2003;107:2775-9

12. Sarkar PD, TMS, Madhusudhan B. Association between paraoxonase activity and lipid levels in patients with premature coronary artery disease. Clin Chim Acta 2006;373:77-81

13. Morray B, Goldenberg I, Moss AJ, et al. Polymorphisms in the paraoxonase and endothelial nitric oxide synthase genes and the risk of early-onset myocardial infarction. Am J Cardiol 2007;99:1100-5

14. Ballantyne CM, Bertolami M, Hernandez Garcia HR, et al. Achieving LDL cholesterol, non-HDL cholesterol, and apolipoprotein B target levels in high-risk patients: Measuring Effective Reductions in Cholesterol Using Rosuvastatin therapY (MERCURY) II. Am Heart J 2006; 151:975-9

15. Schuster H, Barter PJ, Stender S, et al. Effects of switching statins on achievement of lipid goals: Measuring Effective Reductions in Cholesterol Using Rosuvastatin Therapy (MERCURY I) study. Am Heart J 2004;147:705-13

16. Leiter LA, Rosenson RS, Stein E, et al. Efficacy and safety of rosuvastatin 40 mg versus atorvastatin 80 mg in high-risk patients with hypercholesterolemia: results of the POLARIS study. Atherosclerosis 2007; Jan 15 [Epub ahead of print].

17. Jukema JW, liem AH, Dunselman PH, et al. LDL-C/HDL-C ratio in subjects with cardiovascular disease and a low HDL-C: results of the RADAR (Rosuvastatin and Atorvastatin in different Dosages And Reverse cholesterol transport) study. Curr Med Res Opin 2005;21:1865- 74

18. Clearfield MB, Amerena J, Bassand JP, et al. Comparison of the efficacy and safety of rosuvastatin lOmg and atorvastatin 20 mg in high-risk patients with hypercholesterolemia – Prospective study to evaluate the Use of Low doses of the Statins Atorvastatin and Rosuvastatin (PULSAR). Trials 2006;7:35

19. Davidson M, Ma P, Stein EA, et al. Comparison of effects on low-density lipoprotein cholesterol and high-density lipoprotein cholesterol with rosuvastatin versus atorvastatin in patients with type IIa or IIb hypercholesterolemia. Am J Cardiol 2002;89:268-75

20. Schwartz GG, Bolognese MA, Tremblay BP, et al. Efficacy and safety of rosuvastatin and atorvastatin in patients with hypercholesterolemia and a high risk of coronary heart disease: a randomized, controlled trial. Am Heart J 2004;148:e4

21. Stender S, Schuster H, Barter P, et al. Comparison of rosuvastatin with atorvastatin, simvastatin and pravastatin in achieving cholesterol goals and improving plasma lipids in hypercholesterolaemic patients with or without the metabolic syndrome in the MERCURY I trial. Diabetes Obes Metab 2005;7:430-8 22. van Himbergen TM, van Tits LJ, Roest M, Stalenhoef AF. The story of PON1 : how an organophosphate-hydrolysing enzyme is becoming a player in cardiovascular medicine. Neth J Med 2006;64:34-8

23. Jakubowski H. Calcium-dependent human serum homocysteine thiolactone hydrolase. A protective mechanism against protein N- homocysteinylation. J Biol Chem 2000;275:3957-62

24. Shih DM, Gu L, Xia YR, et al. Mice lacking serum paraoxonase are susceptible to organophosphate toxicity and atherosclerosis. Nature 1998;394:284-7

25. Tward A, Xia YR, Wang XP, et al. Decreased atherosclerotic lesion formation in human serum paraoxonase transgenic mice. Circulation 2002; 106:484-90

26. Jarvik GP, Hatsukami TS, Carlson C, et al. Paraoxonase activity, but not haplotype utilizing the linkage disequilibrium structure, predicts vascular disease. Arterioscler Thromb Vasc Biol 2003;23:1465-71

27. Graner M, James RW, Kahri J, et al. Association of paraoxonase-1 activity and concentration with angiographie severity and extent of coronary artery disease. J Am Coll Cardiol 2006;47:2429-35

28. Kassai A, Illyes L, Mirdamadi HZ, et al. The effect of atorvastatin therapy on lecithin:cholesterol acyltransferase, cholesteryl ester transfer protein and the antioxidant paraoxonase. Clin Biochem 2007;40:1-5

29. Kural BV, Orem C, Uydu HA, et al. The effects of lipid- lowering therapy on paraoxonase activities and their relationships with the oxidant-antioxidant system in patients with dyslipidemia. Coron Artery Dis 2004; 15:277-83

30. Sardo MA, Campo S, Bonaiuto M, et al. Antioxidant effect of atorvastatin is independent of PONl gene T(-107)C, Q192R and L55M polymorphisms in hypercholesterolaemic patients. Curr Med Res Opin 2005;21:777-84

31. Paragh G, Torocsik D, Seres I, et al. Effect of short term treatment with simvastatin and atorvastatin on lipids and paraoxonase activity in patients with hyperlipoproteinaemia. Curr Med Res Opin 2004;20:1321-7

32. Harangi M, Seres I, Varga Z, et al. Atorvastatin effect on high-density lipoprotein-associated paraoxonase activity and oxidative DNA damage. Eur J Clin Pharmacol 2004;60:685-91

33. Santanam N, Parthasarathy S. Aspirin is a substrate for paraoxonase-like activity: implications in atherosclerosis. Atherosclerosis 2007;191:272-5

34. Blatter-Garin MC, Kalix B, De Free S, James RW. Aspirin use is associated with higher serum concentrations of the anti-oxidant enzyme, paraoxonase-1. Diabetologia 2003;46:593-4

35. Christidis DS, Liberopoulos EN, Kakafika AI, et al. Effect of paraoxonase 1 polymorphisms on the response of lipids and lipoprotein-associated enzymes to treatment with fluvastatin. Arch Med Res 2007;38:403-10

CrossRef links are available in the online published version of this paper: http://www.cmrojournal.com

Paper CMRO-4024_3, Accepted for publication: 29 June 2007

Published Online: 09 August 2007

doi: 10.1185/030079907X226104

S. C. Bergheanu(a), A. Van Tol(b), G. M. Dallinga-Thie(b), A. Liem(c), P. H. J. Dunselman(d), J. G. Van Der Bom(a) and J. W. Jukema(a)

a Leiden University Medical Center, Leiden, The Netherlands

b Erasmus University Medical Center, Rotterdam, The Netherlands

c Oosterschelde Hospital, Goes, The Netherlands

d Amphia Hospital, Breda, The Netherlands

Address for correspondence: J. Wouter Jukema, MD, PhD, Professor of Cardiology, Department of Cardiology C5-P, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. Tel.: +31 715266695; Fax.: +31 715266885: e-mail: j.w.jukema@lumc.nl

Copyright Librapharm Sep 2007

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