This phase II, randomized, double-blind, placebo-controlled study is designed to determine the safety and efficacy of IV epoetin alfa in reducing infarct size among STEMI patients. Because data regarding the safety of IV epoetin alfa are limited in this population, the study comprises two phases: (i) a safety evaluation of escalating doses of epoetin alfa in patients with first STEMI; and (ii) a single-dose efficacy phase comparing effects of the highest acceptable dose (up to 60,000 units) of epoetin alfa versus matching placebo on infarct size among STEMI patients undergoing primary/rescue PCI.
This study is supported by the Intramural Research Program of the National Institute on Aging, the National Institutes of Health, Bethesda, MD, USA. The authors are solely responsible for the design and conduct of this study, all study analyses, the drafting and editing of the paper, and its final contents.
The primary endpoint is infarct size expressed as percentage of LV mass in the territory of the infarct-related artery (IRA), measured by delayed-enhancement cardiac magnetic resonance imaging (DE-CMR) at 2–6 days post study medication administration. Secondary endpoints are (i) change in numbers of circulating EPCs, measured up to 4 h after successful revascularization but before study drug administration and again at 24 and 48 h following study drug administration; and (ii) left ventricular ejection fraction (LVEF), LV volumes, and infarct size as determined by follow-up CMR 12 ± 2 weeks post PCI.
illustrates both phases of REVEAL. The final study population will include 210–250 subjects enrolled at 22 U.S. sites. Enrollment will continue until 110 patients have completed the primary efficacy assessment (CMR at 2–6 days post study drug administration).
Given uncertainty regarding an appropriate dose of epoetin alfa that will confer cardioprotective effects while limiting risk of thrombotic complications in STEMI patients, a dose-escalating design will be employed. During the dose-escalation phase, patients will be randomly allocated in a 2:1 ratio to either 15,000 units of IV epoetin alfa or placebo until 30 patients have received study medication. If the independent data and safety monitoring board (DSMB) concludes that a clinically unacceptable number of events did not occur at this dose, this dose-escalation/evaluation scheme will be repeated for doses of 30,000 and 60,000 units. The DSMB will subsequently assess the safety of the 60,000-unit cohort for enrollment in the efficacy phase (Appendix 1).
In the efficacy phase, participants will be randomly allocated in a 1:1 ratio to receive the highest dose of epoetin alfa deemed acceptable (15,000, 30,000, or 60,000 units) or placebo. Subjects from the dose-escalation phase randomized to the final efficacy dose or placebo will be included in the overall efficacy analysis.
Other treatments will be at the discretion of the treating physician, but concomitant medical therapy consistent with the current standard of STEMI care will be encouraged.1
details REVEAL eligibility criteria. Subjects are eligible if they present with acute STEMI due to total occlusion (TIMI flow grade 0–1) of a major epicardial coronary artery or large branch vessel and undergo successful (<50% residual lesion or TIMI 2–3) primary or rescue angioplasty within 8 h of onset of ischemic symptoms. Subjects with a history of LV dysfunction (LVEF <50%), MI, CABG, or PCI in the IRA will be excluded due to potential confounding of infarct size measurement. Due to EPO’s potential to increase blood viscosity with subsequent risk of thrombosis, patients with hematocrit above the institutional upper limit of normal at time of study drug administration will not be eligible.
The study protocol conforms to International Conference on Harmonization/Good Clinical Practice standards. Participating sites will submit the protocol for approval by the local institutional review board. Subjects will be randomized using the allocation ratio for each dosing cohort and stratified by age (<70, ≥70 y) and infarct location (left anterior descending (LAD) vs. non-LAD), but not by site. The block randomization scheme will use varying block sizes for safety and efficacy phases.
Study drug administration
Epoetin alfa, formulated as a sterile, colorless liquid, will be supplied in single-dose vials identical to placebo. Study medication will be packaged in numbered kits and administered over approximately 10 minutes as a single IV infusion within 4 hours of successful revascularization. Centocor Ortho Biotech provided erythropoietin 20,000 units/mL in 1 mL multidose vials (NDC# 59676-0320-04) and matching placebo free of charge during the initial phase of the study. During the efficacy phase, Centocor Ortho Biotech decided to stop providing study drug and matching placebo. At this point, the NIA (the study sponsor) purchased study medication from Centocor Ortho Biotech through the NIH Clinical Center Pharmacy Department and contracted Florida Biologix (Alachua, FL) to manufacture matching placebo 1 mL sterile saline vials. Neither Centocor Ortho Biotech nor Florida Biologix provided funding for, or had input into the design or the conduct of, the REVEAL trial.
CMR scans will be performed at 2–6 days and 12 ± 2 weeks post study medication administration. Each examination will use cine-CMR for ventricular volumes and function, and DE-CMR for infarct size assessment and evaluation of microvascular obstruction.23,24
Images will be analyzed in blinded fashion in a central core laboratory (Duke Cardiovascular Magnetic Resonance Center, Durham, NC) (Appendix 2). outlines parameters to be measured on CMR techniques, provides an analysis schematic, and summarizes CMR endpoints.
EPC collection and analysis
Blood specimens will be collected using 8 mL cell preparation tubes (Becton-Dickinson) at baseline immediately before and at 24 (± 12 h) and 48 h (± 12 h) after study drug administration and processed by local site personnel.25
The mononuclear cell fraction will be isolated using density gradient centrifugation according to manufacturer’s instructions. Plasma will be removed and stored at −80° C and the cell layer transferred to a 50 mL conical tube, where cells will be washed with phosphate-buffered saline. Mononuclear cells will be suspended in a 10% DMSO supplied solution, cryopreserved at −80° C, and shipped on dry ice to the Duke EPC core lab. Samples will be thawed, cells washed and recovered, and EPCs enumerated based on expression of cell surface markers (CD34, CD133, and vascular endothelial growth factor receptor-2) as well as on levels of aldehyde dehydrogenase, a marker of multiple progenitor cell subtypes.26
Plasma samples will form a biorepository for subsequent analysis. Because interpatient EPC levels vary considerably, primary EPC analysis will focus on changes in EPC counts over the course of the study.
Concomitant medications, clinical events, procedures, and blood samples will be collected at initial hospitalization, at 7, 14, and 30 days, and at 12 weeks ().
Adverse events and additional safety assessments
For subjects receiving study medication, serious adverse events (SAEs) will be collected from randomization to 12 ± 2 weeks post randomization. For randomized subjects who do not receive study medication, SAEs will be recorded from randomization through 7 days after randomization. Nonserious adverse events (AEs) will be collected from randomization to 7 days after study medication administration.
Safety assessments will also include changes in hemoglobin levels during the first week and at 14 ± 4 days following study medication administration, and there will be careful evaluation for possible complications of IV epoetin alfa with regard to death or thrombotic events (Appendix 3).
Sample size calculation
The primary outcome of REVEAL is CMR-delineated infarct size (expressed as percentage of LV mass) in the territory of the IRA as measured at 2–6 days after study medication administration in subjects randomized to the highest acceptable dose of epoetin alfa or placebo.
The sample size for the first three cohorts in the dose-escalation phase is fixed, with 30 treated subjects at each dose level. Statistical power for the primary comparison is based on subjects in the highest acceptable dose group among these three cohorts, in a stratified analysis with subsequent enrollment into that group. The stratification will adjust for implicit differences that may exist between patients recruited in the dose-escalation and single-dose efficacy phases. Data regarding infarct size from a number of unpublished sources reveals consistently non-normal distribution; therefore, conventional methods of sample-size estimation were rejected in favor of an empirical approach based on available data carefully matched to the conditions of the present study.27
We determined that a sample size of 55 subjects per arm plus the safety cohort subjects (20 active, 10 placebo) will provide adequate power to detect a difference of ≥20% in infarct size between patients receiving placebo and those receiving the highest acceptable dose of epoetin alfa.
Data will be analyzed on a modified intent-to-treat basis: all subjects who receive study medication will be included, regardless of treatment assignment or protocol compliance. If problems occur with incorrect treatment assignments, an as-treated analysis may also be performed. Baseline differences for continuous variables will be evaluated using the t test or Wilcoxon rank-sum test; the Fisher exact mid-p test will be used for categorical variables. Treatment effects will be evaluated based on a two-sided significance level of 0.05, unless otherwise stated.
The primary efficacy comparison will be tested by means of a log-rank test. Secondary analyses investigating the hypotheses that (i) IV epoetin alfa alters the change in number of circulating EPCs from baseline and (ii) IV epoetin alfa will reduce infarct size and improve measures of LV function and remodeling at 12 ± 2 weeks after study medication administration will be performed. Infarct size at follow-up MRI (12 ± 2 weeks after study medication administration) will be evaluated using ANCOVA, with infarct size at 2–6 days as a covariate. Similarly, secondary efficacy parameters of systolic and diastolic LV volumes and LVEF (%), measured at 12 ± 2 weeks after study medication administration, will be evaluated using ANCOVA, with baseline measures as covariates.
Primary and secondary analyses will also be analyzed according to IRA (LAD vs. non-LAD) and patient age (<70 or ≥70 years).
There will be one formal interim test of efficacy after 20 subjects have been randomly assigned to each treatment group in the single-dose efficacy phase and have completed the first CMR. A Haybittle-Peto rule will be used for the interim test, which will be made using an alpha level of 0.001. Due to rounding, this will result in no penalty for the final comparison alpha at 0.05 and an overall alpha level of 0.05 will be maintained.