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The Myocardial Ischemia Reduction with Acute Cholesterol Lowering trial: MIRACuLous or not, it's time to change current practice

Abstract

The Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering (MIRACL) study was the first trial to assess whether statins might be of clinical benefit in those with recently unstable coronary disease. MIRACL found that high-dose atorvastatin was safe and reduced the incidence of the composite endpoint, death, non-fatal myocardial infarction, resuscitated sudden cardiac death or emergent rehospitalization for recurrent ischemia at 16 weeks when compared with placebo. Despite a number of important study limitations, MIRACL's findings and the prior observation that inpatient initiation of lipid-lowering therapy is associated with higher rates of subsequent utilization, suggest that it is prudent to begin statin therapy when patients present with an acute coronary syndrome.

The problem

Three published [1–3] and one recently presented [4] randomized placebo-controlled clinical trial have unequivocally demonstrated that 3-Hydroxy-3-methylgluatryl coenzyme A (HMG CoA) reductase inhibitors (statins) reduce the morbidity and mortality associated with coronary disease. These trials found that when compared with placebo, statins significantly reduced the incidence of death, myocardial infarction, unstable angina, percutaneous and surgical coronary revascularization, and stroke in persons with stable coronary disease. Because patients who had experienced an acute coronary syndrome within three to six months of enrollment were excluded, these trials did not assess the effect of lipid-lowering therapy on adverse cardiovascular events in those with recently unstable coronary disease. Whether lipid-lowering therapy would provide incremental benefit if initiated immediately following an acute coronary syndrome is an important issue as the risk of a recurrent adverse cardiac events is much greater in patients with unstable coronary disease than in the stable setting. The Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering (MIRACL) trial set out to answer this question.

The answer?

MIRACL enrolled 3,086 patients within 24–96 hours (mean 63 hours) of admission for unstable angina or a non-Q-wave myocardial infarction and randomized them to 16 weeks of atorvastatin 80 mg or placebo once daily [5]. The major exclusion criteria were: total cholesterol level greater than 270 mg/dL; Q-wave myocardial infarction on admission or during the previous month; and, coronary revascularization in the months before admission, during the index hospitalization or anticipated following hospital discharge. The primary efficacy endpoint was a composite of death, non-fatal myocardial infarction, resuscitated sudden cardiac death or emergent rehospitalization for worsening symptomatic myocardial ischemia. Secondary endpoints included stroke, worsening heart failure, need for coronary revascularization and change in lipid levels throughout the study. On average, patients were 65 years of age, approximately 65% were men, 86% Caucasian and the mean baseline low density lipoprotein (LDL) cholesterol level was 124 mg/dL. Atorvastatin treatment was associated with a 2.6% absolute reduction in the risk of the primary endpoint (14.8% vs. 17.4%; RR relative risk [RR] 0.84, 95% confidence interval [CI] 0.70–1.00, p = 0.048). This reduction was primarily driven by the 2.2% absolute reduction in incidence of emergent rehospitalization for symptomatic myocardial ischemia (6.2% vs. 8.4%; RR 0.74, 95% CI 0.57–0.95, p = 0.02). The risk of death, nonfatal myocardial infarction and resuscitated sudden cardiac death were each no different between the two groups. While there were no significant differences in the incidence of worsening heart failure or need for coronary revascularization, atorvastatin did reduce the incidence of fatal or non-fatal stroke by 0.8% (0.8% vs. 1.6%; RR 0.50, 95% CI 0.26–0.99, p = 0.045). Atorvastatin also significantly reduced total and LDL cholesterol and triglyceride levels but did not significantly change high density lipoprotein (HDL) cholesterol by 16 weeks. By 16 weeks, the adjusted mean LDL cholesterol decreased to 72 mg/dL in atorvastatin-treated patients but increased to 135 mg/dL among placebo-treated patients. No serious adverse events occurred as the result of treatment with atorvastatin, although reversible liver transaminase elevation more than three times the upper limit of normal occurred in 2.5% of atorvastatin-treated versus 0.6% of placebo-treated patients (p < 0.001).

The MIRACuLous

The efficacy and safety findings from MIRACL were unique for a number of reasons. Although lipid-lowering therapy was associated with a significantly lower mortality when initiated early after an acute coronary syndrome in two large observational studies [6, 7], MIRACL was the first randomized trial to suggest that statins confer clinical benefits in this setting. It was also the first trial to identify a short-term (ie, within 16 weeks) clinical benefit from statin therapy; in previous secondary prevention trials, the benefit of statin therapy was not evident for one to two years. And, while clinical trial safety endpoints may be considered less glamorous, MIRACL's most important contribution may have been that high-dose statin therapy was not associated with serious harm, despite its use in the unstable setting. Earlier secondary prevention statin trials had excluded patients with unstable coronary syndromes largely out of theoretical concern that statin-mediated reductions in vascular smooth muscle cell proliferation might destabilize healing plaque. That no harm resulted from this aggressive treatment strategy should allay theoretical fears and by doing so remove a major obstacle to the inpatient initiation of lipid-lowing therapy after coronary events.

The not so MIRACuLous

Despite these unique and important findings, there were a number of inherent study limitations worth noting. First and foremost, the possibility of a null treatment effect cannot be ignored given the wide confidence intervals (and hence marginally significant p value of 0.048) for the effect of atorvastatin on the primary efficacy endpoint. Furthermore, while the number of patients lost to follow up was small, if adverse events had occurred in those treated with atorvastatin (n = 3) but not placebo (n = 8), the overall trial results may have been neutral rather than positive.

The types of events prevented in MIRACL are also worth noting. While rehospitalization for recurrent myocardial ischemia is an important determinant of quality of life and health care costs, other important endpoints were not significantly affected (eg, death, myocardial infarction, resuscitated sudden cardiac death, worsening heart failure, need for coronary revascularization, etc). The question of whether statins can prevent these and other adverse events when initiated soon after an acute coronary syndrome will require further study.

The short duration of follow-up is also particularly troubling. While it is impressive that a clinical benefit was realized after only 16 weeks of statin therapy, the increased risk of adverse clinical events persists throughout the year following an acute coronary syndrome. Without longer clinical follow up, it is not possible to assess the intermediate-term effect (if any) of atorvastatin on hard endpoints such as death or myocardial infarction. To do so would be critical in light of the lack of effect on these important endpoints at 16 weeks. Unfortunately, no late clinical follow up is planned.

There were also a number of limitations that may have hampered the study's generalizability. First, patients who underwent recent revascularization or in whom it was planned were excluded. Specifically, patients who underwent percutaneous transluminal coronary angioplasty (PTCA) or coronary artery bypass graft (CABG) surgery within the previous three or six months respectively were not eligible for inclusion. The investigators reasoned that recurrent ischemic events in this population were likely to result from restenosis or bypass graft closure and that statins would be less likely to affect these processes [8]. Nevertheless, a number of trials have established the benefits of statin therapy early after coronary revascularization [9–11]. Furthermore, a number of recent trials have suggested that higher risk patients with non-ST elevation acute coronary syndromes fair better when an early invasive strategy is applied [12–14] and it is not uncommon for patients to be treated in this fashion. Second, patients with Q-wave myocardial infarction were not eligible for enrollment because it was felt that statins would not influence the development of important prognostic determinants such as left ventricular systolic dysfunction, ventricular arrhythmias or mechanical complications [5]. Nevertheless, patients who develop electrocardiographic Q-waves represent a substantial proportion of all patients with myocardial infarction. While their short-term risk following hospital discharge is lower relative to those with a non-Q-wave myocardial infarction, it is still much greater than in patients with stable coronary disease, and the need for secondary prevention in this population is equally important. Third, despite the high risk nature of enrolled patients (ie, electrocardiogram [ECG] changes and/or other objective evidence of ischemia), the rate of platelet glycoprotein IIb/IIIa inhibitor utilization was quite low (1.1%). Such therapy appears to be cost effective[15, 16], especially among high risk patients and is recommended under current American College of Cardiology/American Heart Association guidelines [17]. Fourth, it may not be possible to ascertain whether these findings apply to all patients with recent acute coronary syndromes regardless of baseline lipid levels. The small difference in number of primary endpoint events between atorvastatin and placebo groups make it difficult to dissect the relationship between baseline lipid levels and treatment effect further. Consequently, it remains uncertain whether one can extrapolate the MIRACL trial results to those who undergo coronary revascularization shortly before or after a coronary event, who present with a Q-wave myocardial infarction, who are treated with platelet glycoprotein IIb/IIIa inhibitors, or who have relatively low admission LDL cholesterol levels.

Time to change current practice

Although MIRACL and the two aforementioned cohort studies suggest that lipid-lowering agents exert short-term clinical benefits when initiated soon after an acute coronary syndrome, this remains an open question. Even if these findings are not confirmed after further study, one could still make a compelling argument that lipid-lowering therapy (barring contraindications) should be initiated early and universally in patients who present with an acute coronary syndrome: First, the long-term safety and effectiveness of statins for the secondary prevention of stable coronary disease is well-established [1–3]; Second, as evidenced by MIRACL, these agents are safe when initiated at the time of hospitalization for an acute coronary syndrome; Third, the in-hospital initiation of lipid-lowering therapy appears to promote greater long-term utilization of these agents [18–21]. Finally, although lipid levels may be unreliable in the setting of an acute coronary syndrome (excepting total :HDL and LDL:HDL cholesterol ratios [22]) the overwhelming majority of patients with coronary disease will ultimately require both pharmacologic and non-pharmacologic lipid-lowering interventions to attain recommended cholesterol targets [23–25]; newer guidelines are even more stringent [26]. Furthermore, data from the recently presented Heart Protection Study suggest that clinical benefits may accrue independent of baseline cholesterol level [4]. Thus, to withhold lipid-lowering therapy from patients who present with an acute coronary syndrome would be to accept the status quo, and to date our efforts at cholesterol lowering in the secondary prevention setting have been dismal [27, 28].

More MIRACLes ahead?

The ascertainment and quantification of any incremental benefit conferred by statin therapy initiated early after an acute coronary syndrome will require confirmation. There is currently only one ongoing randomized placebo-controlled trial of early versus delayed statin therapy in this setting, A-2-Z (Aggrastat to Zocor, Merck) [29]. The A-2-Z study is evaluating the efficacy of early treatment with simvastatin in 4,500 patients following an episode of unstable angina or a non-Q wave myocardial infarction. In the first four months, patients will be randomized to simvastatin 40 mg daily or placebo. Thereafter, those patients treated with simvastatin in the first phase will receive 80 mg of simvastatin daily and those treated with placebo, 40 mg of simvastatin daily. The primary composite endpoint is the occurrence of cardiovascular death, non-fatal myocardial infarction, or rehospitalization for an acute coronary syndrome (ACS) at one year. If A-2-Z demonstrates significant reductions in the incidence of adverse events during the first four months, it would suggest an incremental clinical benefit from initiating these agents early after an acute coronary syndrome. If benefits accrue, but do so later during follow up, it would be difficult to discriminate between the effects of more aggressive vs. earlier lipid lowering therapy.

The Pravastatin or Atorvastatin Evaluation and Infection Therapy (PROVE IT) trial is looking at 4,000 patients within 10 days of an acute coronary syndrome and randomizing them to either pravastatin 40 mg or atorvastatin 80 mg daily [29]. Patients will be observed over at least 1.5 years for the occurrence of myocardial infarction or other cardiovascular events. Unlike, MIRACL and the A-2-Z trials, this study will not assess the efficacy of early statin therapy after an acute coronary syndrome; rather, it will examine the role of more vs. less aggressive lipid-lowering in this setting.

In 2002, many would consider it unethical to withhold statins from patients with established coronary disease. This makes it unlikely that additional placebo-controlled trials will be carried out in this area. Future secondary prevention studies should look at patients with stable or unstable disease and will need to address the comparative efficacy of different statins (or newer agents), assess the incremental benefit of combination therapy [30] and determine whether there is a serum cholesterol 'floor' below which reductions are unlikely to provide further clinical benefit.

Abbreviations

HMG CoA:

3-Hydroxy-3-methylgluatryl coenzyme A

MIRACL:

Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering

LDL:

low density lipoprotein

RR:

relative risk

CI:

confidence interval

HDL:

high density lipoprotein

PTCA:

percutaneous transluminal coronary angioplasty

CABG:

coronary artery bypass graft

ECG:

electrocardiogram

A-2-Z:

Aggrastat to Zocor

ACS:

acute coronary syndrome

PROVE IT:

Pravastatin or Atorvastatin Evaluation and Infection Therapy.

References

  1. The 4S Investigators: Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet. 1994, 344 (8934): 1383-1389. 10.1016/S0140-6736(94)90566-5.

    Google Scholar 

  2. The LIPID Investigators: Prevention of cardiovascular events and death with pravastatin in patients with coronary heart disease and a broad range of initial cholesterol levels. The Long-Term Intervention with Pravastatin in Ischaemic Disease (LIPID) Study Group. N Engl J Med. 1998, 339 (19): 1349-1357. 10.1056/NEJM199811053391902.

    Article  Google Scholar 

  3. Sacks FM, Pfeffer MA, Moye LA, Rouleau JL, Rutherford JD, Cole TG, Brown L, Warnica JW, Arnold JM, Wun CC, Davis BR, Braunwald E: The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. N Engl J Med. 1996, 335 (14): 1001-1009. 10.1056/NEJM199610033351401.

    Article  CAS  PubMed  Google Scholar 

  4. HPS Investigators: MRC/BHF Heart Protection Study of cholesterol-lowering therapy and of antioxidant vitamin supplementation in a wide range of patients at increased risk of coronary heart disease death: early safety and efficacy experience. Eur Heart J. 1999, 20 (10): 725-741. 10.1053/euhj.1998.1350.

    Article  Google Scholar 

  5. Schwartz GG, Olsson AG, Ezekowitz MD, Ganz P, Oliver MF, Waters D, Zeiher A, Chaitman BR, Leslie S, Stern T: Effects of atorvastatin on early recurrent ischemic events in acute coronary syndromes: the MIRACL study: a randomized controlled trial. JAMA. 2001, 285 (13): 1711-1718. 10.1001/jama.285.13.1711.

    Article  CAS  PubMed  Google Scholar 

  6. Aronow HD, Topol EJ, Roe MT, Houghtaling PL, Wolski KE, Lincoff AM, Harrington RA, Califf RM, Ohman EM, Kleiman NS, Keltai M, Wilcox RG, Vahanian A, Armstrong PW, Lauer MS: Effect of lipid-lowering therapy on early mortality after acute coronary syndromes: an observational study. Lancet. 2001, 357 (9262): 1063-1068. 10.1016/S0140-6736(00)04257-4.

    Article  CAS  PubMed  Google Scholar 

  7. Stenestrand U, Wallentin L: Early statin treatment following acute myocardial infarction and 1-year survival. JAMA. 2001, 285: 430-436. 10.1001/jama.285.4.430.

    Article  CAS  PubMed  Google Scholar 

  8. Schwartz GG, Oliver MF, Ezekowitz MD, Ganz P, Waters D, Kane JP, Texter M, Pressler ML, Black D, Chaitman BR, Olsson AG: Rationale and design of the Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering (MIRACL) study that evaluates atorvastatin in unstable angina pectoris and in non-Q-wave acute myocardial infarction. Am J Cardiol. 1998, 81 (5): 578-581. 10.1016/S0002-9149(97)00963-6.

    Article  CAS  PubMed  Google Scholar 

  9. Flaker GC, Warnica JW, Sacks FM, Moye LA, Davis BR, Rouleau JL, Webel RR, Pfeffer MA, Braunwald E: Pravastatin prevents clinical events in revascularized patients with average cholesterol concentrations. J Am Coll Cardiol. 1999, 34 (1): 106-112. 10.1016/S0735-1097(99)00145-X.

    Article  CAS  PubMed  Google Scholar 

  10. Serruys PW, Foley DP, Jackson G, Bonnier H, Macaya C, Vrolix M, Branzi A, Shepherd J, Suryapranata H, de Feyter PJ, Melkert R, van Es GA, Pfister PJ: A randomized placebo-controlled trial of fluvastatin for prevention of restenosis after successful coronary balloon angioplasty; final results of the fluvastatin angiographic restenosis (FLARE) trial. Eur Heart J. 1999, 20 (1): 58-69. 10.1053/euhj.1998.1150.

    Article  CAS  PubMed  Google Scholar 

  11. Christenson JT: Preoperative lipid control with simvastatin protects coronary artery bypass grafts from obstructive graft disease. Am J Cardiol. 2001, 88 (8): 896-869. 10.1016/S0002-9149(01)01901-4.

    Article  CAS  PubMed  Google Scholar 

  12. The FRISC II Investigators: Invasive compared with non invasive treatment in unstable coronary-artery disease: FRISC II prospective randomised multicentre study. FRagmin and Fast Revascularisation during Instability in Coronary artery disease Investigators. Lancet. 1999, 354 (9180): 708-715. 10.1016/S0140-6736(99)07349-3.

    Article  Google Scholar 

  13. Cannon CP, Weintraub WS, Demopoulos LA, Vicari R, Frey MJ, Lakkis N, Neumann FJ, Robertson DH, DeLucca PT, DiBattiste PM, Gibson CM, Braunwald E: Comparison of early invasive and conservative strategies in patients with unstable coronary syndromes treated with the glycoprotein IIb/IIIa inhibitor tirofiban. N Engl J Med. 2001, 344 (25): 1879-1887. 10.1056/NEJM200106213442501.

    Article  CAS  PubMed  Google Scholar 

  14. Solomon DH, Stone PH, Glynn RJ, Ganz DA, Gibson CM, Tracy R, Avorn J: Use of risk stratification to identify patients with unstable angina likeliest to benefit from an invasive versus conservative management strategy. J Am Coll Cardiol. 2001, 38 (4): 969-976. 10.1016/S0735-1097(01)01503-0.

    Article  CAS  PubMed  Google Scholar 

  15. Szucs TD, Meyer BJ, Kiowski W: Economic assessment of tirofiban in the management of acute coronary syndromes in the hospital setting: an analysis based on the PRISM PLUS trial. Eur Heart J. 1999, 20 (17): 1253-1260. 10.1053/euhj.1999.1526.

    Article  CAS  PubMed  Google Scholar 

  16. Mark DB, Harrington RA, Lincoff AM, Califf RM, Nelson CL, Tsiatis AA, Buell H, Mahaffey KW, Davidson-Ray L, Topol EJ: Cost-effectiveness of platelet glycoprotein IIb/IIIa inhibition with eptifibatide in patients with non-ST-elevation acute coronary syndromes. Circulation. 2000, 101 (4): 366-371.

    Article  CAS  PubMed  Google Scholar 

  17. Braunwald E, Antman EM, Beasley JW, Califf RM, Cheitlin MD, Hochman JS, Jones RH, Kereiakes D, Kupersmith J, Levin TN, Pepine CJ, Schaeffer JW, Smith EE, Steward DE, Theroux P, Alpert JS, Eagle KA, Faxon DP, Fuster V, Gardner TJ, Gregoratos G, Russell RO, Smith SC: ACC/AHA guidelines for the management of patients with unstable angina and non-ST-segment elevation myocardial infarction. A report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee on the Management of Patients With Unstable Angina). J Am Coll Cardiol. 2000, 36 (3): 970-1062. 10.1016/S0735-1097(00)00889-5.

    Article  CAS  PubMed  Google Scholar 

  18. Cambou JP, Grenier O, Ferrieres J, Danchin N: Secondary prevention of patients with acute coronary syndrome in France. The Lancet Conference. 1999, 75- , Copenhagen. Abstracts book

    Google Scholar 

  19. Muhlestein JB, Home BD, Bair TL, Li Q, Madsen TE, Pearson RR, Anderson JL: Usefulness of in-hospital prescription of statin agents after angiographic diagnosis of coronary artery disease in improving continued compliance and reduced mortality. Am J Cardiol. 2001, 87 (3): 257-261. 10.1016/S0002-9149(00)01354-0.

    Article  CAS  PubMed  Google Scholar 

  20. Fonarow GC, Gawlinski A, Moughrabi S, Tillisch JH: Improved treatment of coronary heart disease by implementation of a cardiac hospitalization atherosclerosis management program (CHAMP). Am J Cardiol. 2001, 87: 819-822. 10.1016/S0002-9149(00)01519-8.

    Article  CAS  PubMed  Google Scholar 

  21. Aronow HD, Novaro GM, Prosper DM, Lincoff AM, Topol EJ, Nissen SE: Initiation of lipid-lowering therapy during hospitalization for coronary intervention is a powerful predictor of subsequent compliance (abstract). Eur Heart J. 2001, 22: 271-10.1053/euhj.2000.2434.

    Article  Google Scholar 

  22. Wattanasuwan N, Khan IA, Gowda RM, Vasavada BC, Sacchi TJ: Effect of acute myocardial infarction on cholesterol ratios. Chest. 2001, 120 (4): 1196-1199. 10.1378/chest.120.4.1196.

    Article  CAS  PubMed  Google Scholar 

  23. DeBusk RF, Miller NH, Superko HR, Dennis CA, Thomas RJ, Lew HT, Berger WE, Heller RS, Rompf J, Gee D, et al: A case-management system for coronary risk factor modification after acute myocardial infarction. Ann Intern Med. 1994, 120 (9): 721-729.

    Article  CAS  PubMed  Google Scholar 

  24. Schrott HG, Bittner V, Vittinghoff E, Herrington DM, Hulley S: Adherence to National Cholesterol Education Program Treatment goals in postmenopausal women with heart disease. The Heart and Estrogen/Progestin Replacement Study (HERS). The HERS Research Group. JAMA. 1997, 277 (16): 1281-1286. 10.1001/jama.277.16.1281.

    Article  CAS  PubMed  Google Scholar 

  25. Pearson TA, Laurora I, Chu H, Kafonek S: The lipid treatment assessment project (L-TAP): a multicenter survey to evaluate the percentages of dyslipidemic patients receiving lipid-lowering therapy and achieving low-density lipoprotein cholesterol goals. Arch Intern Med. 2000, 160 (4): 459-467. 10.1001/archinte.160.4.459.

    Article  CAS  PubMed  Google Scholar 

  26. Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, And Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III). JAMA. 2001, 285 (19): 2486-2497.

  27. EUROASPIRE I and II Group: Clinical reality of coronary prevention guidelines: a comparison of EUROASPIRE I and II in nine countries. Lancet. 2001, 357: 995-1001. 10.1016/S0140-6736(00)04235-5.

    Article  Google Scholar 

  28. Fonarow GC, French WJ, Parsons LS, Sun H, Malmgren JA: of lipid-lowering medications at discharge in patients with acute myocardial infarction: data from the National Registry of Myocardial Infarction. Circulation. 2001, 103 (1): 38-44.

    Article  CAS  PubMed  Google Scholar 

  29. Gotto AM: Ongoing clinical trials of statins. Am J Cardiol. 2001, 88 (4 Suppl): 36F-40F. 10.1016/S0002-9149(01)01876-8.

    Article  CAS  PubMed  Google Scholar 

  30. Brown BG, Zhao XQ, Chait A, Fisher LD, Cheung MC, Morse JS, Dowdy AA, Marino EK, Bolson EL, Alaupovic P, Frohlich J, Albers J: Simvastatin and niacin, antioxidant vitamins, or the combination for the prevention of coronary disease. N Engl J Med. 2001, 345: 1583-1592. 10.1056/NEJMoa011090.

    Article  CAS  PubMed  Google Scholar 

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Correspondence to Herbert D Aronow.

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Dr Aronow has received honoraria as a speaker and advisory board member for Pfizer and as a speaker for Merck.

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Aronow, H.D. The Myocardial Ischemia Reduction with Acute Cholesterol Lowering trial: MIRACuLous or not, it's time to change current practice. Trials 3, 3 (2002). https://doi.org/10.1186/1468-6708-3-3

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