RIGENERA 2.0 (Recupero Dall'Infarto Miocardico Con G-CSF E Nuovi Esempi di Rigenerazione Avanzata) Project: "The Combined Effect of Subcutaneous Granulocyte - Colony Stimulating Factor and Myocardial Contrast Echocardiography With Intravenous Infusion of Sulphur Hexafluoride on Post-infarction Left Ventricular Function"

1. INTRODUCTION 1.1. BACKGROUND AND RATIONALE OF THE STUDY The long term prognosis of patients suffering from acute myocardial infarction (AMI) has progressively improved since the introduction of reperfusion therapies and in particular primary angioplasty [1]. In the setting of ST-elevation myocardial infarction (STEMI), the immediate reopening of acutely occluded coronary arteries via primary angioplasty is the treatment of choice to salvage ischemic myocardium. However, the sudden re-initiation of blood flow can lead to a local acute inflammatory response with further endothelial and myocardial damage. This phenomenon, described as 'reperfusion injury', may explain why, despite optimum myocardial reperfusion, the short-term mortality after AMI approaches 7% [1] and the incidence of heart failure approaches 15-20% [2][3]. Despite the use of full conventional treatment, including ACE inhibitors, beta-blockers, aldosterone inhibitors and diuretics, in the context of randomised controlled trials yearly mortality rates of patients with post-infarction heart failure are still in the range of 10-13% and rehospitalisation for worsening of heart failure occurs at a yearly rate of 6-8 % [4]. Registry data indicate a more dismal outcome in real world clinical experience. A major reason for the high morbidity and mortality is that the heart has an inadequate regenerative response to the myocardial necrosis sustained following AMI; cell death from the ischemic damage can lead to progressive ventricular dilation and dysfunction through the processes of adverse left ventricular remodelling. However, the discovery of tissue resident cardiac stem cells in the mammalian heart [5] has challenged the long held belief that the heart is a terminally-differentiated organ and opens up the possibility of using bone marrow derived stem cells to repair the heart. Indeed, recent experimental studies documented that bone marrow-derived cell (BMC) injection into the infarcted heart stimulates the formation of newly formed cardiac myocytes, although the origin of these myocytes is still a matter of debate[6]. Numerous pre-clinical studies provided high degree of evidence that bone marrow derived cells do contribute to cardiac repair after acute myocardial injury, limit infarct expansion and improve cardiac function most likely via a paracrine mechanism of action. Pre-clinical evidence is corroborated by several small to intermediate size clinical trials demonstrating beneficial effects of bone marrow derived cells on top of the state-of-the-art reperfusion treatment [7]. For this reason, European Community has recently funded within FP-7 "The effect of intracoronary reinfusion of bone marrow derived mononuclear cells (BM-MNC) on all cause-mortality in acute myocardial infarction (BAMI)" project aimed to definitively demonstrate the efficacy of BM-MNC in patients with myocardial infarction.

Considering that recovery of post-infarction LV function is favourably correlated to bone marrow derived stem cell mobilization induced by endogenous Granulocyte-Colony Stimulating Factor (G-CSF) [8-9], therapeutic stem cell mobilization was proposed as an alternative method to ameliorate post-infarction LV function. Several basic studies have supported this notion suggesting that stem cell mobilization might improve LV function by increasing stem cell homing in the damaged myocardium in addition to a favourable direct effect of G-CSF on LV myocardium [10-12].

On these bases several studies have evaluated the effect of G-CSF on LV function in patients with myocardial result with mixed results [13]. In general, G-CSF was proven safe but the effect appeared modest. However, in patients with large myocardial infarction the effect was significant and clinically relevant approaching a 5% increase in LVEF. In particular in the Rigenera Trial conducted at the Catholic University of the Sacred Heart of Rome 41 patients with large anterior wall AMI at high risk of unfavorable remodeling were randomized 1:2 to Lenograstim (rhu G-CSF, Myelostim 34, Italfarmaco) (10 μg/kg/day for 5 days) or to conventional therapy. After a median follow-up of 5 months patients treated with Lenograstim exhibited improvement of 5% in LVEF, in the absence of LV dilation. In contrast, patients treated conventionally exhibited significant LV dilation in the absence of an improvement in LVEF [14]. In the present study, a subgroup of 8 patients randomized to Lenograstim received also the administration of a second-generation ultrasound contrast agent containing sulphur hexafluoride (SonoVue®, Bracco, Milan, Italy) administered intravenously (5 ml at 1 ml/min) for myocardial opacification to collect diagnostic images by Myocardial Contrast Echocardiography. Interestingly, a post hoc analysis, revealed that only patients receiving Sonovue® showed a significant benefit on LV function [unpublished data]. This could be due to an increased homing effect of mobilized bone marrow derived stem cells due to the myocardial destruction of contrast medium-microbubbles by 3.5 MHz ultrasounds produced by the Echo machine. As this regard, two basic studies demonstrated that the highly focused ultrasound-mediated stimulation of microbubbles increased the migration of stem cells across the myocardial endothelium into the post-ischemic myocardium in vivo [15-16]. Moreover, long term (10 years) follow up data of the RIGENERA trial have been recently collected, confirming the safety of the procedure and a significant improvement of the quality of life assessed by NYHA functional class, Seattle Heart Failure Model, Minnesota Living with Heart Failure Questionnaire (p<0.005) when compairing patients treated with G-CSF versus standard therapy. Data regarding efficacy, in terms of LVEF, end-diastolic volume and end-systolic volume are currently under analysis.

On the basis of the previous results, The Recupero dall'Infarto miocardico con G-CSF E Nuovi Esempi di Rigenerazione Avanzata (RIGENERA 2.0) trial will specifically test the efficacy of the combined effect of subcutaneous Granulocyte - Colony Stimulating Factor and sulphur hexafluoride on post-infarction left ventricular function.

1.2 BENEFIT-RISK ASSESSMENT In every clinical trial, anticipated benefits need to be weighed against the study associated risks. In previous investigations where G-CSF has been used, no G-CSF associated adverse reactions were reported. Only the MAGIC trial was prematurely stopped due to possible safety concerns in treated patients in terms of an unexpectedly high restenosis rate in stented vessels [17]. However, only three patients in the G-CSF group received angiographic follow-up, thus no clear conclusion about the incidence of restenosis should have reasonably be drawn from this study. In all other studies using G-CSF adverse events were comparable between patients and controls [13]. Nevertheless G-CSF is largely used in oncology for treatment of febrile neutropenia and in hematology for this indication and for mobilization of stem cells for transplantation. As this regard G-CSF is considered reasonably safe without any association with a documented increase in malignancies [18].