Introduction

Acute myocardial infarction remains a public health problem and a main cause of death, often premature, in many developing countries [1]. Despite improved clinical outcomes by early thrombolysis and/or angioplasty, in-hospital mortality in ST-elevation myocardial infarction (STEMI) is still high, approximating 5% in the Global Registry of Acute Coronary Events [2]. Beyond the acute phase, adverse ventricular remodelling, heart failure, and mortality are directly related to infarct size and to left ventricular (LV) dysfunction [3, 4]. Thus, cardiomyocyte salvage remains a primary aim of effective treatment.

Experimental studies indicate that cardiomyocytes threatened by necrotic or apoptotic damage can be rescued by growth factors, such as erythropoietin (Epo) [5] and insulin-like growth factor-1 (IGF-1) [6, 7]. Indeed, Epo and its receptor (EpoR) exert biological functions that go beyond erythropoiesis [8, 9] and that include broad antiapoptotic actions, mediated by Bcl-2 and Bcl-XL, proangiogenetic, and antinflammatory effects [8, 9]. Moreover, the Epo–EpoR system enhances the synthesis and bioavailability of constitutive nitric oxide (NO) [10], through endothelial NO synthase transcription and activation, increases coronary flow-reserve [11], recruits bone-marrow and/or cardiac pluripotential precursor cells [12, 13], and has membrane-stabilizing and insulin-sensitizing actions [5], in synergy with IGF-1 [14]. Epo is thus emerging as a potentially important heart and vascular protective hormone [15].

EPAMINONDAS: design and end points

EPAMINONDAS (Exogenous erythroPoietin in Acute Myocardial Infarction: New Outlook aNd Dose Association Study) is a multicenter, prospective, randomized, double-bind, placebo-controlled, dose-finding, phase II trial testing the effects on infarct size of two intravenous (i.v.) doses of epoietin-α, administered on the first 3 days of hospitalization to a homogeneous group of 102 patients with a first STEMI. The first dose of drug or placebo is given within 12 hours (h) of primary percutaneous coronary intervention (PCI).

The primary end point is to assess the dose-dependent effects of Epo compared to placebo on infarct size. The latter is estimated by serum CK-MB 24 h time–concentration curves in the acute phase, and by LV wall motion score index, LV ejection fraction, and extent of “delayed enhancement” by contrast-enhanced magnetic resonance (MR), assessed at discharge and at 12 months’ follow-up.

The secondary endpoints are to assess the effects of two doses of Epo versus placebo on the incidence of major adverse events and on LV remodelling at 12 months’ follow-up. The incidence of major adverse events [defined as cardiovascular death, death from other cause, nonfatal myocardial infarction, nonfatal ischemic stroke, cardiogenic shock, rehospitalization for recurrent ischemia, heart failure, pulmonary embolism or other life-threatening conditions, sustained ventricular arrhythmias, atrial fibrillation, major bleeds (including intracranial), deep vein thrombosis, new onset hemodialysis or ultrafiltration, mechanical ventilation, uncontrolled hypertension] is recorded by 3-monthly interviews, hospital records, and clinical visits with 12-lead ECGs at follow-up. LV remodelling is assessed by echocardiography and cardiac MR, based on global and regional changes in geometry and function from discharge to follow-up.

Methods

Patients

Consecutive patients with diagnostic signs and symptoms of STEMI, satisfying the study criteria (Fig. 1), and with a Thrombolysis In Myocardial Infarction (TIMI) flow grade 0-1 in the infarct-related artery on initial angiography are eligible. Immediately after successful PCI, patients are asked for written informed consent and, if they agree, are assigned—according to a pre-defined randomization scheme—to receive Epo or placebo on top of optimal standard medical care. Neither the patient, nor the attending physician, nor the staff performing echocardiography, MR, or the clinical follow-up are aware of the assigned treatment. A total of 102 patients are scheduled.

Fig. 1
figure 1

Inclusion and exclusion criteria for enrollment in EPAMINONDAS

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Treatment

Prior to or at the time of primary PCI, standard antithrombotic treatment for STEMI is administered, consisting of aspirin, heparin, clopidogrel, and abciximab [1]. On arrival into the coronary care unit and within 12 h of PCI, enrolled patients are randomly assigned to placebo or one of two Epo doses (100 or 200 IU/kg/day). Active drug or placebo are diluted in 100 ml of saline and administered i.v. over 30 min for the first 3 days, under regular blood pressure and continuous ECG monitoring. The double-blind administration is ensured by a treatment code unknown to physicians, nurses and patients. Drug or placebo is prepared, under medical supervision, by nurses not involved in the study, according to instructions contained in predefined packages provided by the Pharmacy. Standard treatment, including β-blockade, lipid-lowering therapy, and angiotensin-converting enzyme inhibition or angiotensin-II receptor blockade, is additionally prescribed [1].

Clinical and laboratory measures

Blood pressure, heart rate, and ECG are monitored in the coronary care unit and then at regular time points until discharge. Major adverse events (as defined above) are recorded during hospitalization and up to 12 months’ thereafter. Before discharge and at 12 months, the following are also performed and recorded: physical exam, 12-lead ECG, 2-D echocardiogram, and cardiac MR.

LV remodelling is defined as a >20% change in end-diastolic volume at follow-up compared to pre-discharge values. Absolute changes in LV ejection fraction, contractility by wall motion score index, and MR contrast-enhanced first-pass and delayed enhancement perfusion imaging at follow-up versus pre-discharge are also recorded. During follow-up, patients are assessed every 3 months, either as out-patients or by telephone call. An independent data safety monitoring board receives real-time clinical information, performs interim safety analyses at 25, 50, 75 and 100% recruitment, and provides regular feedback to the investigators.

Biochemical measurements

Six-hourly serial blood samples are drawn from the time of enrolment up to 24 h, for the serum determination of creatine-kinase (CK)-MB. Full blood cell and reticulocyte count, AST and ALT are measured daily during the first 3 days, and before discharge. A venous blood sample for future determinations is also taken, processed, and stored, at baseline, discharge and follow-up.

Statistical analysis

The sample size of 102 patients has been selected to detect differences in the primary endpoint variables (expressed as means and standard deviations and estimated according to current literature) with a power of 0.8 and an alfa value of 0.01. Bonferroni’s correction is applied to repeated comparisons. Comparisons are made between Epo and placebo-treated patients, and among the three different treatment arms. Statistical analyses are performed using the SPSS program (version 11).

Centers and financial support

EPAMINONDAS involves three clinical centers located in Rome (Italy), each enrolling 34 patients and performing/recording clinical and laboratory exams, as per protocol. The scientific coordinator of the investigation and head of the research unit within the Department of Cardiovascular Medicine of the “Gemelli” University Hospital is Dr. F. Andreotti. This center acts as core laboratory for randomization and for the centralized biochemical analyses. Head of the research unit within the Department of Cardiology of the Sant’Andrea Hospital of the 2nd Faculty of the University “La Sapienza” is Dr. C. Autore. This center acts as core laboratory for cardiac MR, in collaboration with the “Gemelli” Radiology department. Head of the research unit within the Department of Cardiology of the Umberto I Hospital of the 1st Faculty of the University “La Sapienza” is Prof. L. Agati. This center acts as echocardiographic core laboratory. EPAMINONDAS is funded by the Italian “Ministero dell’Istruzione, dell’Università e della Ricerca” (PRIN 2006_6063891).

Discussion

Non-hematopoietic effects of Epo

Epo’s predominant role is red cell production through prevention of erythroid precursor cell apoptosis and by promoting proliferation and maturation of erythroid progenitor cells [14]. Non-hematopoietic effects of Epo, however, have been recently described [15, 16], consistent with the discovery of Epo receptors on the same cells that synthesize Epo, as well as on cardiomyocytes, cardiac fibroblasts, and endothelial, retinal, gastric, prostate and vascular smooth muscle cells [15]. Epo production occurs mainly in peritubular kidney cells under the control of hypoxia inducible factor-1 [17], but Epo mRNA has recently been found also in extrarenal tissues, including liver, spleen, brain, lung, bone marrow, and reproductive organs [15].

Potential cardioprotective effects of Epo

Reasons to believe that Epo may improve cardiac function in patients with acute myocardial infarction are based on a large body of evidence [16]. Initial experiments in rats showed consistent cardioprotection by Epo following ischemia-reperfusion or permanent coronary artery ligation [18–20], through multiple antiapoptotic pathways, independent of increases in hematocrit. High doses given intraperitoneally (i.p., 5,000 IU/kg/day) for seven consecutive days, starting before or after transient (30 min) coronary artery occlusion, compared with saline, reduced cardiomyocyte loss by 50% [19]. Even a single i.p. dose (3,000 IU/kg), compared with saline, decreased apoptosis and reduced infarct size to approximately 1/3 when given immediately after permanent coronary artery occlusion [20]. In contrast, subcutaneous injections (5,000 IU/kg/day) starting 24 h before, at the time of, and for 5 days after permanent left anterior descending coronary artery ligation, did not improve long-term cardiac function in the same animal species [21]. In a similar model, a single i.v. dose (3,000 IU/kg), compared with saline, decreased apoptosis and reduced infarct size when given immediately or 4, 8, or 12 h after infarction, but not after 24 h [22]. On balance, these data suggest that Epo administration in rats is effective when given i.p. or i.v. (but not subcutaneously) and when started within a day after infarction. For a broad review of animal and clinical studies investigating the effects of Epo on myocardial necrosis and/or apoptosis, see Riksen et al. [16]. Benefits of Epo on ischemic myocardium have been reported to occur in rats, mice, rabbits and dogs, but not in pigs or sheep [16].

To date, studies investigating the potential protective effects of Epo in human cardiac ischemia are extremely limited. Accordingly, several trials are being performed in the attempt to translate the cardioprotection found in experimental models to patients with acute myocardial infarction.

Safety of Epo in cardiac patients

Epo has been administered to heart failure patients with or without diabetes, anemia or renal failure [23, 24]. In these trials, subcutaneous doses, ranging from 15,000–30,000 IU/week for 3 months to 4,000 IU/week for 1 year, showed benefits, in terms of reduced length and rate of hospitalizations, reduced progression of renal impairment, increased LV ejection fraction, and improved exercise tolerance, in the absence of Epo-related complications [23, 24].

The administration of Epo to patients with acute cardiovascular diseases may raise concerns stemming from potential prothrombotic effects, increased hematocrit, and hypertension [25]. Initial data reporting an increased frequency of thrombotic events came from retrospective non-randomized studies [26, 27] or from prospective randomized studies conducted in chronically diseased populations receiving prolonged Epo dosings (>400 IU/kg/week for months or years) [28]. Increased hematocrits have been observed particularly with simultaneous iron administration. Among 40 acute ischemic stroke patients, i.v. epoietin (33,000 IU/day) for the first 3 days compared to saline resulted in significantly improved neurological outcomes and no safety concerns at 30 days [29]. Another investigation assessed the effects of relatively high doses of epoietin α (40,000 IU/week for up to 3 weeks compared to placebo) in 1,460 critically ill medical, surgical or trauma patients [30]. Among those receiving Epo, there was no effect on transfusion rates, with an increased rate of thrombotic vascular events at 140 days (16.5 vs. 11.5%, P = 0.008) [30]. However, Epo was associated with a striking and significant reduction of mortality at 29 and 140 days among the prespecified group of trauma patients who, on average, were 20 years younger than the other patient groups [30]. Thus, Epo may exert beneficial effects in specific groups, but high total doses may cause thrombotic complications, especially in older patients not receiving antithrombotic therapy.

Phase II studies of Epo in STEMI patients

Currently, at least four randomized controlled trials, each of ≥100 patients with STEMI, are testing the effects of i.v. Epo administration on infarct size. An initial single-center, open-label pilot investigation evaluated the safety and tolerability in 22 non-anemic patients with acute STEMI randomized to a single bolus of 300 μg of darbepoetin alfa (~60,000 IU of epoietin α) or to no additional medication before primary PCI [31]. Adverse events were not observed during a 30-day follow-up. LV ejection fraction did not differ significantly at 4 months (52 ± 3% for darbepoetin vs. 48 ± 5% for controls) [31]. An ongoing multicenter, randomized, open-label trial is enrolling 466 patients with STEMI within 3 h after successful primary PCI to receive a single 60,000 IU bolus of epoietin (unspecified) on top of standard medical care vs. only standard medical care. The effect of Epo on LV ejection fraction is assessed by planar radionuclide ventriculography after 6 weeks from the acute event [32].

REVEAL (Reduction of infarct Expansion and Ventricular remodelling with Erythropoietin After Large myocardial infarction) is a double-blind, randomized, placebo-controlled, dose-finding, parallel-phase II study coordinated by the Duke University Medical Center, enrolling 210 patients with first STEMI, within 8 h from the onset of symptoms and treated by successful primary or rescue PCI. The primary aim is to evaluate the effects of a single administration of epoietin α (15,000, 30,000 or 60,000 IU in a dose-escalation safety phase followed by an efficacy phase) on infarct size and LV remodelling. Infarct size is assessed by cardiac MR within 2–6 days of the study medication, and again approximately 3 months later [33]. REVIVAL-3 (Regeneration of Vital Myocardium in ST-Segment Elevation Myocardial Infarction by Erythropoietin) is a German double-blind, placebo-controlled, randomized trial that has recently reported results on 138 patients with a first STEMI undergoing primary PCI. Immediately after balloon inflation and after 24 and 48 h, 68 patients received epoietin β (33,000 IU/dose) and 70 received placebo. The primary end point was LV ejection fraction at 6 months’ follow-up measured by MR. LV function and infarct size were not significantly different at discharge in the two groups. There was a trend towards a higher rate of adverse events at 6 months’ follow-up in the Epo treated group [34]. A recent pilot study administered a single i.v. dose of 40,000 IU of Epo to patients presenting with non-ST segment elevation acute coronary syndromes: plasma levels of troponin I and creatine kinase were not reduced by Epo [35]. The main characteristics of all these studies are shown in Table 1.

Table 1 Overview of randomized controlled studies investigating the effects of Epo in patients with acute myocardial infarction
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EPAMINONDAS is the 4th large phase II study of Epo in STEMI patients, contributing to the search of novel treatments aimed at reducing early mortality and late heart failure. It has the following distinctive features: it is double-bind and dose-finding; the first administration occurs after successful PCI, with three consecutive dosings; the total Epo dose (for body weights of 70 kg) is moderate (<50,000 IU), rather than high (>60,000 IU); the dose of active drug is weight-adjusted; finally, the extent of follow-up is 12 months. EPAMINONDAS will contribute to define Epo’s safety and potential efficacy (or lack thereof) in reducing infarct size in STEMI patients. To date, among the 68 enrolled patients, the rate of adverse events at follow-up has been low with no significant differences among the three treatment arms [36].

In conclusion, Epo is a hematopoietic hormone found to have potential cardioprotective effects against prolonged ischemia through antiapoptotic properties, promotion of neovascularization, and stem cell-mediated repair. Although animal studies have shown protection against infarcted myocardium, initial clinical testing has failed to show striking benefits. The conclusion of current phase II, randomized, placebo-controlled trials is awaited to more fully assess the cardiac effects of Epo administered at different doses and times, and to carefully monitor its safety profile in patients with STEMI.