The CardiAMP Heart Failure trial: A randomized controlled pivotal trial of high-dose autologous bone marrow mononuclear cells using the CardiAMP cell therapy system in patients with post–myocardial infarction heart failure: Trial rationale and study design

Accepted Manuscript The CardiAMP Heart Failure trial: A Randomized Controlled Pivotal Trial of High Dose Autologous Bone Marrow Mononuclear Cells Using the CardiAMP Cell Therapy System in Patients with Post Myocardial Infarction Heart Failure: Trial Rationale and Study Design

Amish N. Raval MD FSCAI FACC, Thomas D. Cook, Henricus J. Duckers, Peter V. Johnston, Jay H. Traverse, William T. Abraham, Peter A. Altman, Carl J. Pepine PII: DOI: Reference:

S0002-8703(18)30100-5 doi:10.1016/j.ahj.2018.03.016 YMHJ 5655

To appear in: Received date: Accepted date:

13 December 2017 24 March 2018

Please cite this article as: Amish N. Raval MD FSCAI FACC, Thomas D. Cook, Henricus J. Duckers, Peter V. Johnston, Jay H. Traverse, William T. Abraham, Peter A. Altman, Carl J. Pepine , The CardiAMP Heart Failure trial: A Randomized Controlled Pivotal Trial of High Dose Autologous Bone Marrow Mononuclear Cells Using the CardiAMP Cell Therapy System in Patients with Post Myocardial Infarction Heart Failure: Trial Rationale and Study Design. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ymhj(2018), doi:10.1016/j.ahj.2018.03.016

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The CardiAMP Heart Failure trial: A Randomized Controlled Pivotal Trial of High Dose Autologous Bone Marrow Mononuclear Cells Using the CardiAMP Cell Therapy System in Patients with Post Myocardial Infarction Heart Failure: Trial Rationale and Study

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Design

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RCT# NCT02438306

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Amish N. Ravala, Thomas D. Cookb, Henricus J. Duckersc, Peter V. Johnstond, Jay H. Traversee, William T. Abrahamf, Peter A. Altmanc, Carl J. Pepineg a

Division of Cardiovascular Medicine, Department of Medicine and Biomedical Engineering, University of Wisconsin School of Medicine and Public Health, Madison, WI Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison

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Biocardia Inc. San Carlos, CA.

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Minneapolis Heart Institute Foundation at Abbott Northwestern Hospital, Minneapolis, USA

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Division of Cardiovascular Medicine, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD

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Departments of Medicine, Physiology, and Cell Biology, Division of Cardiovascular Medicine, and the Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio. g

Division of Cardiovascular Medicine, Department of Medicine, University of Florida,Gainesville, FL

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Short Title: Raval. The CardiAMP Heart Failure Trial Rationale and Study Design

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Corresponding Author:

Amish N. Raval MD, FSCAI, FACC, FAHA Associate Professor of Medicine Division of Cardiovascular Medicine Department of Medicine and Biomedical Engineering University of Wisconsin School of Medicine and Public Health H4/568 Clinical Sciences Center 600 Highland Ave, Madison, WI 53792-3248, USA Office:(608) 263-0836 Cell: (608) 347-8074 Fax: (608) 263-0405 Email: [email protected]

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Journal Subject Terms: Cell Therapy, Stem cells, Heart Failure, Chronic Ischemic Heart Disease, Treatment. Abstract Background: Heart failure following myocardial infarction is a common, disabling and deadly

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condition. Direct injection of autologous bone marrow mononuclear cells (BM MNCs) into the myocardium may result in improved functional recovery, relieve symptoms and improve other

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cardiovascular outcomes.

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Methods: CardiAMP-HF is a randomized, double-blind, sham-controlled, pivotal trial designed to investigate the safety and efficacy of autologous BM MNC treatment for patients

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with medically refractory and symptomatic ischemic cardiomyopathy. The primary endpoint is

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change in six-minute walk distance adjusted for major adverse cardiovascular events at 12 months following treatment. Particularly novel aspects of this trial include a cell potency assay

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to screen subjects who have bone marrow cell characteristics that suggest a favorable response to treatment, a point of care treatment method, a high target dose of 200 million

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high cell retention.

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cells and an efficient transcatheter intramyocardial delivery method that is associated with

Conclusions: This novel approach may lead to a new treatment for those with ischemic

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heart disease suffering from medically refractory heart failure. Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT02438306 Key words: Cell, Heart Failure, Treatment, Trial, Catheter

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Introduction Heart failure following myocardial infarction affects ~5 million Americans and is a common reason for hospitalization, which impairs quality of life and consumes enormous health care

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resources1. The mortality rate of these patients is high and similar to that of advanced cancer.

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Guideline-based therapy, using medications that block maladaptive neurohormonal pathways,

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is often only partially effective and not universally tolerated. Left ventricular assist devices and heart transplant may be offered in advanced heart failure, but device failure, stroke, infection,

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and organ shortages limit these approaches. Cell based therapy represents an innovative approach to recover contractile heart function and improve clinical outcomes. However,

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despite two decades of investigation, no Food and Drug Administration (FDA) cell treatments

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for cardiovascular disease have been approved.

Adult bone marrow contains a large reservoir of stem and progenitor cells capable of

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differentiating into hematopoietic, endothelial and mesenchymal lineages. In addition, numerous pre-clinical cardiac studies have shown that paracrine signaling by bone marrow-

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derived stem cells promotes microcirculatory adaptation, immune modulation and

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cytoprotection through anti-apoptotic effects, facilitating post-infarct cardiac recovery, in part via recruitment of endogenous cardiac stem/progenitor cells2-12. Improvements in left

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ventricular myocardial perfusion and contractile function have been demonstrated in small and large animal models following intramyocardial injection of bone marrow-derived stem cells13-19. These encouraging pre-clinical findings paved the way for several Phase I and II clinical trials of autologous bone marrow cell therapy for chronic ischemic heart disease and postmyocardial infarct recovery20-35. For example, the Transendocardial Autologous Bone Marrow in Myocardial Infarction (TABMMI) heart failure trial treated 20 chronic ischemic heart failure patients with autologous bone marrow derived mononuclear cells (BM MNC) using a 3

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transendocardial helical injection catheter24, 35. Left ventricular ejection fraction and exercise tolerance were improved at up to 5 years follow-up, with no treatment emergent major adverse events. Subsequently, the Transendocardial Autologous Cells in Ischemic Heart Failure Trial (TAC-HFT) compared autologous BM MNC treatment and autologous bone

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marrow derived mesenchymal stem cell treatment with two corresponding placebo arms in

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patients with ischemic heart failure32. Transendocardial delivery of BM MNC was shown to be

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safe at 30 days and 1 year. When the BM MNC treated group (n=19) was compared to a pooled placebo group (n=20) in the study, quality of life as measured by the Minnesota Living

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with Heart Failure Questionnaire (MLWHFQ) and six minute walk distance (6MWD) were

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improved at 12 months, although the latter was not statistically significant (p = 0.005 and p = 0.11, respectively). In a separate analysis34 in which the BM MNC treated group (n=19) was

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compared to the matched placebo group (n=10), there was significant improvement in 6MWD

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at 12 months [BM MNC group (+14.3 meters, CI -15.4 to 43.9) versus the placebo group (42.0 meters, CI -102.1 to 18.1), P=0.049]. In this analysis, the MLWHFQ score also improved

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in the BM MNC group compared to a deterioration in the corresponding placebo group at 12 months [BM MNC group -7.7, SD 24.8 versus Placebo group +9.7, SD 24.8 (P=0.038)].

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Likewise, all other pre-specified endpoints favored the BM MNC group whereas the placebo

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treated patients deteriorated over 12 month follow-up. Finally, multiple imputation and rank adjustment for missing 6MWD results due to intervening major adverse cardiovascular events (MACE) was applied to the data, revealing greater statistical confidence of the between group differences in 6MWD (refer to SUPPLEMENT 1 for more detail). The safety of the treatment approach combined with the improvements in 6MWD and quality of life observed in the Phase I (TABMMI) and Phase II (TAC-HFT) trials provide compelling evidence to support further study of BM MNC treatment of patients with chronic heart failure due to ischemic heart disease (IHD). 4

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A 2016 Cochrane meta-analysis of 38 randomized-controlled trials assessing effects of BM MNCs in 1907 heart failure patients with IHD found that BM MNC treatment significantly reduced all-cause mortality, non-fatal myocardial infarction, and arrhythmias at 12 month follow-up, although heterogeneity among the trials limited the robustness of the analysis36, 37.

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The totality of clinical trial evidence to date supports that intramyocardial delivery of

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controlled trials are required to establish clinical efficacy.

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autologous bone marrow mononuclear cells is safe; however, more robust randomized-

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The pivotal CardiAMP-HF trial is a novel approach to the problem of post myocardial infarction heart failure. It will assess patients with New York Heart Association (NYHA) functional class

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II-III heart failure, who are selected based on their bone marrow potency, whether intramyocardial injection of high dose autologous bone marrow mononuclear cells will improve

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ambulatory function and quality of life, and prevent MACE. Prior to entry, patients will be

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screened using a cell potency assay that measures the stem cell characteristics of their bone marrow. Those subjects meeting the selection criteria will have their BM MNCs harvested and

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immediately isolated using a point-of-care device in the cardiac catheterization laboratory.

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The CardiAMP-HF cell product will be then be delivered into the myocardium using a novel

Methods:

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and efficient helical needle tipped transendocardial delivery catheter.

CardiAMP-HF is a pivotal, randomized controlled trial to investigate the safety and efficacy of the CardiAMP cell therapy system (BioCardia, San Carlos, CA) in bone marrow potency selected patients with post-myocardial infarction NYHA functional class II-III heart failure (NCT02438306) enrolled in up to 40 clinical centers in the United States. The trial has two cohorts: 1) a roll-in phase with a prospective, open-label, uncontrolled, patient cohort to evaluate on-site physician and coordinator training in the various procedures/tests and ensure 5

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timely transfer of materials and information between sites and core laboratories to create best practices before opening additional enrolling sites (n=10), and 2) the prospective, doubleblinded, randomized (3:2 treatment vs. sham) cohort to establish clinical efficacy (n=250) (Figure 1). The trial is being conducted under FDA Investigational Device Exemption (IDE)

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number 16057. Enrollment is anticipated to be completed by June 2019, and study

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completion by June 2021. Study Population

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Subjects between 21 and 90 years of age, with chronic, symptomatic ischemic left ventricular dysfunction and NYHA functional class II-III are eligible for enrollment. Written informed

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consent is obtained by members of the investigational site with institutional review board (IRB)

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approval. Chronic ischemic left ventricular dysfunction is defined as a left ventricular ejection fraction (EF) of 20-40% after a remote (>6 months) myocardial infarction (MI). All subjects are

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required to be receiving an “optimized” regimen of guideline-directed-medical therapy for at least 3 months prior to treatment. All subjects must be ambulatory and capable of performing

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a 6MWD test with a distance >100 meters but < 450 meters. Key inclusion and exclusion

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criteria for the CardiAMP HF trial are summarized in Table 1.

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Cell Potency Assay

All subjects enrolled in the trial first undergo a 5mL bone marrow aspirate from the iliac crest during the screening period, which is 4 to 45 days prior to randomization. The aspirate is shipped overnight to an independent Cell Analysis Core Laboratory (Center for Cell and Gene Therapy, Baylor University, Houston TX). The cellular aspirate is tested for pre-defined markers that have pro-angiogenic and cardio-reparative potential. The results are expressed as a Cell Potency Assay (CPA) score. All investigators are masked to the details and specific

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results of this assay in any given patient, other than whether or not the patient’s bone marrow qualifies. This proprietary CPA is detailed in the approved FDA IDE. The CardiAMP HF bone marrow CPA is derived from observations in prior ischemic heart

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disease cell therapy trials. For example, a lower limit of the effective dose threshold for CD34+

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cells is one component of the assay. CD34+ cells are associated with favorable angiogenic and regenerative properties in a number of pre-clinical and clinical cardiovascular disease

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trials27, 28, 38-41. In the FOCUS trial31, which tested intramyocardial injections of unselected BM

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MNCs in ischemic heart failure patients, it was observed that those patients who received higher doses of CD34+ cells were correlated with further improvements in left ventricular

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ejection fraction (LVEF). Every 3% higher level of CD34+ cells was associated with, on average, a 3.0% greater absolute unit increase in LVEF. Furthermore, transendocardial

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administration of CD34+ cells were associated with improvements in exercise tolerance in the

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ACT34+ chronic myocardial ischemia trial28, the RENEW trial42, and in recently published patient level pooled analysis of a combination of chronic myocardial ischemia trials43. Based

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on calculations performed on these prior data, it is estimated that ~70% of subjects will meet

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the pre-specified CPA criteria, and therefore, qualify for enrollment. Enrolled subjects will undergo a second, larger volume, bone marrow aspirate to obtain the treatment product as

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described below in Treatment Procedure. Subjects who fail to meet the CPA criteria will not be enrolled in the randomized phase of the trial. Treatment Procedure All subjects who meet screening criteria will undergo aspiration of 60 mL of bone marrow from the iliac crest under local anesthesia and mild sedation if required. The bone marrow aspirate is immediately processed using a point-of-care system (CardiAMP™ Cell Separator, Biocardia, San Carlos, CA) in the cardiac catheterization laboratory (Figure 2). The process is a density7

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tuned dual buoy separation to isolate the BM MNC fraction that typically takes ~25 minutes. Patients will receive a dose targeted to yield ~200 million autologous bone marrow mononuclear cells, identical to the phase II TAC-HFT trial32.

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Immediately following bone marrow aspiration, patients are randomized 3:2 to either the BM

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MNC therapy group or a sham procedure group. Randomization will be stratified for patients

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who have cardiac resynchronization therapy (CRT) at entry using a computerized block randomization method.

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All subjects undergo percutaneous femoral artery access, left heart catheterization and ventriculography in two orthogonal projections. Subjects randomized to active treatment then

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undergo 10 transendocardial injections of 0.5 ml high dose BM MNC in the peri-infarct

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myocardial segments. Target segments are selected based on a 17 segment bulls’ eye maps depicting myocardial wall thickness and local wall motion, determined by baseline contrast–

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enhanced echocardiography. Injections are performed using the Helix™ dual lumen,

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intramyocardial injection catheter and the Morph® deflectable guide catheter (BioCardia, San Carlos, CA; Figure 3). Diluted contrast solution is infused through a separate contrast lumen in

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the Helix catheter to verify engagement of the target myocardium. Subjects randomized to sham treatment will likewise undergo bone marrow aspiration, placement of an arterial sheath,

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and left heart catheterization with ventriculography. Thereafter, the interventionalist and staff will follow a scripted sham protocol to mimic the activities of an injection procedure to maintain subject blinding. After the treatment, a separate team of physicians and study personnel who are blinded to the treatment assignment will perform the follow-up visits with the subject. Outcomes The primary outcome is a composite that combines change in 6MWD with missing values ranked according to the severity of heart failure defined as first occurrence of a major 8

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cardiovascular event (MACE) among all-cause mortality, hospitalization for worsening heart failure, non-fatal recurrent myocardial infarction, placement of a left ventricular assist device, or heart transplantation. The 6MWD will be measured at baseline, and at intervals of 3 months over the first 12 months, and then again at 24 months. For each time point, two 6MWD

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measurements will be performed under identical conditions, with at least a 1-hour recovery

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between measurements. The longest 6MWD distance at each time point will be used for the

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analysis. If the difference between the two 6MWD tests is >10%, a third test will be performed

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and the longest 6MWD will be used for analysis.

Secondary outcomes will be adjudicated following a hierarchical scheme and gatekeeper

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approach. These include: i) overall 12-month survival (non-inferiority), ii) freedom from heart failure MACE at 12-months; non-inferiority), iii) change in quality of life as measured by the

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Minnesota Living with Heart Failure Questionnaire44 at 12-months (superiority), iv) Time to first

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MACE at 12-months (superiority), and v) overall 12 month survival (superiority). Additional secondary outcomes will include treatment-emergent serious adverse events at 30-

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days, days alive out of hospital, New York Heart Association (NYHA) heart failure Functional

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Class, echocardiographic measures of change in ejection fraction and left ventricular endsystolic and end-diastolic dimensions. Echocardiographic analysis is performed by a blinded

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independent imaging core laboratory (Yale Cardiovascular Research Group, New Haven, CT). All clinical events are adjudicated by an independent clinical events committee (CEC). An independent data safety monitoring committee monitors the study for safety outcomes and futility in achieving the primary efficacy outcomes. Importantly, prior transendocardial injection studies have shown modestly elevated cardiac biomarkers up to 48-hours after the procedure, without any clinical evidence of an acute coronary syndrome, including signs or symptoms, electrocardiographic changes or novel wall motion abnormalities on echocardiography. 9

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Therefore, the clinical relevance of these modestly elevated biomarkers following transendocardial injection is unclear45. The independent CEC has chosen to define “peri procedural infarction” as an adverse event occurring within the first 48-hours after the study procedure, in association with at least a 5 x the upper limit of normal (ULN) increase in CK-MB

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and/or 35 x ULN increase in Troponin T, in combination with evidence of prolonged ischemia,

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as demonstrated with prolonged chest pain, electrocardiographic (ST changes, new Q

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waves), or imaging evidence of new regional wall motion abnormalities according to the Joint ESC/ACCF/AHA/WHF Taskforce for the Universal Definition of Myocardial Infarction46. All

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peri-procedural biomarker changes will be captured, but only those that meet these criteria will

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be considered adverse events and will be evaluated on a case-to-case basis by the CEC.

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Statistical Analysis Plan

The primary endpoint is a comparison of a composite of the change in distance walked in 6-

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minutes and heart failure MACE between the treatment group and the sham-control group at 12 months (superiority outcome). If the primary endpoint is significantly improved (p<0.05),

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then a series of pre-specified secondary endpoints will be analyzed in a hierarchical fashion

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using a gatekeeper approach (described in the “Statistical Analysis Plan” in SUPPLEMENT 2). These secondary endpoints are ranked as follows: 1) overall survival at 12-months as a non-

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inferiority outcome, 2) freedom from MACE defined as the composite of all-cause death, hospitalization for worsening heart failure, non-fatal recurrent myocardial infarction, placement of a left ventricular assist device or heart transplant at 12-months as a non-inferiority outcome, 3) change in quality of life as measured by Minnesota Living with Heart Failure Questionnaire at 12 months as a superiority outcome, 4) time to first MACE at 12 months as a superiority outcome and 5) overall survival at 12 months as a superiority outcome. Hierarchical

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secondary endpoints will be evaluated sequentially, conditional on all treatment comparisons for previous endpoints reaching statistical significance. It is expected that a proportion of subjects will either die or experience a non-lethal MACE

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event that will preclude the assessment of the 6MWD. Therefore, the statistical analysis plan

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is designed to account for these missing variables. The MACE component of the primary

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endpoint will be ranked according to the severity of clinical outcome, with the inability to perform the 6MWD due to death of the subject awarded as lowest rank. Progressive ranks are

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assigned for intervening non-lethal MACE events that prevent the performance of the 6MWD test. The highest rank would be for those individuals who achieve a successful 6MWD without

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intervening death or MACE. The analysis will use a semi-parametric proportional odds model with terms for treatment and baseline 6MWD. This approach has greater power than using

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change in 6MWD and the adjustment for baseline implicitly accounts for baseline in an optimal

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way. Missing responses not attributable to either death or an intervening non-lethal MACE will be handled using multiple imputation. Sensitivity analyses that conduct the imputations under

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a range of models that capture plausible deviations from the assumption, that data are

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missing at random, will be used to ensure a robust result. A separate risk benefit/analysis will be performed to study the treatment effect on intervening MACE events that do not prevent

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the successful performance of the 6MWD test. All randomized subjects will be followed and analyzed for both safety and efficacy endpoints on an intention to treat basis. Continuous variables will be summarized using descriptive statistics, and categorical variables will be summarized by frequencies and percentages. Sample Size Considerations: Based on results of the TAC-HFT study, a sample size of 250 in the randomized phase is expected to provide adequate power for the primary outcome, whereas the sample size was also based on the first hierarchical secondary outcome of non-

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inferiority analysis for all-cause mortality. The non-inferiority margin of 10% can be excluded from a 95% confidence interval with 90% power if the true survival rate in the cell treatment arm is 95% and the true survival rate in the sham arm is 96%. Details of the sample size

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justification are provided in SUPPLEMENT 2.

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The trial sponsor is Biocardia Inc. (San Carlos, CA). The authors are solely responsible for the

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design and conduct of this study, all study analyses, the drafting and editing of the paper and its final contents.

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Discussion

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The CardiAMP-HF trial is intended to expand and extend the results of the Phase II TAC-HFT study. The current study includes, to our knowledge, the first human use of a Cell Potency

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Assay (CPA) to select patients who are likely to yield therapeutic cells and a high dose of BM

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MNC cells. It will test the hypothesis that intramyocardial delivery of high dose BM MNCs isolated with a point-of-care device will improve a clinical composite of 6MWD and MACE in

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NYHA functional class II-III ischemic heart failure patients with an LVEF of 20-40%. The use of a Cell Potency Assay (CPA) to select patients who are more likely to yield therapeutic cells,

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the high effective dose of BM MNCs that will be injected, and the point of care treatment approach are intended to maximize therapeutic efficacy, limit dose variability and improve

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treatment efficiency.

The CPA was developed to both enrich the enrollment of subjects that will have a greater yield of therapeutic cells and to reduce heterogeneity across subjects. The rationale for this approach is based on previous clinical trials where selected MNC subpopulations were administered in chronic myocardial ischemia patients28, 42 and were associated with improved outcomes. Reduced angina frequency was observed in the ACT-34 and RENEW trials that tested transendocardial injection of CD34+ cells in coronary artery disease patients with 12

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refractory chronic angina28, 37. Furthermore, in the FOCUS31, TIME47, and Late Time48 trials, certain cell sub-populations were associated with improved outcome following BM MNC treatment in patients with heart failure and recent or remote MI49. Age, and comorbidities were

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also implicated as factors for responsiveness to cell therapy.

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Identifying biomarkers that are likely to predict therapeutic response to bone marrow-derived

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mononuclear cell therapy has been proposed in heart failure, recent acute myocardial infarction and critical limb ischemia. For example, the FOCUS trial demonstrated that

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transendocardial injection of unselected BM MNCs in patients with ischemic cardiomyopathy was safe, but did not improve the co-primary outcomes of maximal oxygen consumption and

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perfusion31. However, left ventricular ejection fraction (LVEF) did improve in the BMMNC treated patients and those with increased LVEF had were younger than median age (<62

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years) and had higher numbers of CD34+ and CD133+ cells in their BM31. Additionally,

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subjects who responded favorably had higher frequencies of B cells, CXCR4+ BM MNCs, fewer endothelial colony- forming cells and monocytes/macrophages in the bone marrow49.

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Demonstrating cardioregenerative responsiveness based on constituent bone marrow

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elements will be tested prospectively in the CardiAMP-HF trial. In an analysis of 63 biomarkers and cytokines in baseline plasma samples from myocardial infarction patients

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treated with BM MNCs or with cell free placebo, several markers were associated with favorable responses to cell therapy50. These bone marrow cell and plasma cytokines markers were also linked to endothelial progenitor cell function51. Similarly, expression of senescence genes was associated with increased mobilization efficiency and functional responsiveness of autologous CD133+ cells in no-revascularization option critical limb ischemia patients, in the Stem Cell Revascularization in Patients with Critical Limb Ischemia (SCRIPT-CLI) randomized trial52. The CPA approach used in the CardiAMP-HF trial also acts as a quality control on patient and dose variability, one of the great difficulties in autologous cell therapy trials to date. 13

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In addition to selection by CPA, CardiAMP-HF patients will be treated with a high cell dose. The target dose of 200 million MNCs is higher than most prior trials and will be delivered via transendocardial injection using a helical catheter previously shown to yield high acute cell retention within the myocardium compared to other methods53. Transendocardial and direct

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intramyocardial cell delivery have been used extensively in pre-clinical models and human

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ischemic heart disease trials with an excellent safety profile54-56. This minimally invasive route

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offers the ability to deliver cells to all potential myocardial target segments as compared to intracoronary cell infusion, which relies on patent coronary arteries for delivery and can result

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in microvascular obstruction due to cell plugging. Indeed, a meta-analysis of pre-clinical

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studies demonstrated that transendocardial delivery of mesenchymal stem cells in ischemia models improved cardiac outcomes to a greater extent than other delivery methods57.

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The target cell dose in the CardiAMP Heart Failure Trial of 200 million BM MNCs is based on

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the dose used in the Phase II TAC-HFT trial32, and is a higher dose than previously has been used in randomized controlled trials of intramyocardial delivery of BM MNC. A BM MNC dose

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response was suggested by Silva et al. in a pre-clinical chronic ischemia model, where the

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~200 million cell dose showed greater efficacy than lower doses. Clinical trials using higher doses of BM MNCs also suggest a dose effect. Tse et al. reported in the PROTECT-CAD trial

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that intramyocardial injection of 42.0 ± 28.0 million MNCs improved exercise time, LVEF, and NYHA class25. Van Ramshorst et al. delivered 98 ± 6 million MNC showing a modest, but significant improvement in myocardial perfusion compared to placebo26. Perin et al. delivered 99.0 ± 5.6 million MNC and showed an improvement in LVEF, but failed to show an effect on other pre-specified primary endpoints, including left ventricular end systolic volume, maximal oxygen consumption, or reversibility on SPECT perfusion31. Most recently, in the REGENERATE-IHD trial, ischemic cardiomyopathy patients treated with 124.7 ± 89.2 million MNC experienced improvements in cardiac contractile function and heart failure symptoms58. 14

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To achieve a high “effective” cell dose, the CardiAMP study utilizes a helical injection catheter (“Helix” - see Figure 3) designed to maximize myocardial cell retention. Mitsutake et al. demonstrated acute retention of radiolabeled BM MNC was three-fold higher using the Helix catheter compared to straight needle injections53. In addition, transendocardial cell injection

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using straight-needle catheters are associated with variable cell retention compared to

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intracoronary infusion59-61. The higher absolute BM MNC dose generated by the CardiAMP

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Cell Separation device, coupled with three fold higher estimated cell retention efficiency is expected to lead to an effective BM MNC dose up to five-fold higher than that delivered by

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Mathur et al.58, which is the next highest cell dose delivered by intramuscular injection in a

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clinical trial to date.

In summary, the CardiAMP treatment “strategy” has several advantages. First, the autologous

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cell therapy approach avoids the potential for immune rejection. While an off-the-shelf

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allogeneic approach has conceptual appeal, the inherent need for cell manipulation, cleanroom facilities for large scale cell production and storage, the shipment considerations, and

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contamination risk all present challenges. Second, the point-of-care approach to BM-MNC

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harvest, preparation and delivery utilized in this trial can easily be implemented within an existing interventional lab workflow. Relative accessibility, low cost, ease of procurement and

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simple cell preparation procedure, absence of immune response make autologous cell therapy an appealing adjunct to advanced heart failure therapy. CardiAMP-HF’s primary clinical composite endpoint uses 6MWD and MACE. The 6MWD has been shown to predict survival in patients with moderate to severe heart failure62-65. This simple to perform, patient-oriented, functional test was used as a primary efficacy endpoint in the Multicenter InSync Randomized Clinical Evaluation (MIRACLE) trial of cardiac resynchronization therapy (CRT), which enrolled a similar heart failure population to that

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proposed in CardiAMP-HF. In MIRACLE, six month 6MWD improved +39 meters versus +10 meters (CRT vs. control, p=0.001, n= 453 subjects randomized). This trial result contributed to the FDA decision to approve the Medtronic InSync CRT device66. In this study, MLWHFQ was also improved in the treatment group compared to control (–18.0 vs. –9.0 points, p= 0.001).

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Notably, the relative differences in the point estimates between CRT and control for 6MWD

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and quality of life scores were smaller than the observed treatment benefit of BM MNCs

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observed in the aforementioned analysis of the TAC-HFT (CardiAMP Phase II) trial34. The

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CardiAMP Phase III randomized trial is designed to confirm these treatment benefits. Conclusion: The CardiAMP pivotal trial will test whether transendocardial injection of a high

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effective dose autologous BM MNCs obtained from NYHA class II-III ischemic heart failure patients, who are selected by a novel bone marrow potency assay designed to assess the

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likelihood of a therapeutic effect, improves ambulatory function, quality of life and survival.

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Success in this treatment approach may lead to a novel treatment option for those suffering with medically refractory ischemic heart failure.

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Acknowledgements: : The authors would like to acknowledge the contributions of Adrian

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Gee and April Durett of the Center for Cell & Gene Therapy, Baylor College of Medicine, Texas Children’s Hospital in Houston; Maria Cabreira of the Stem Cell Center, Texas Heart

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Institute, Houston for their support of CardiAMP HF stem cell studies. In addition, Mary Jo Rizzo, Dr.Lavanya Bellumkonda, Dr.Lissa Sugeng Dr. Alexandra Lansky of Yale University are acknowledged for their contributions for the echocardiographic image analysis in the CardiAMP HF study. Finally, Michael Kolber, Cheryl Wong Po Foo, Andrew Mackenzie, and of Biocardia and Dr. Jerome Fleg of NHLBI are acknowledged for contributions toward developing the CardiAMP-HF trial protocol.

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Disclosures: The CardiAMP-HF trial is sponsored, in part, by Biocardia Inc. (San Carlos, CA). Authors ED and PA are employees of Biocardia Inc. The remaining authors are

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members of the executive steering committee for the CardiAMP-HF trial.

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the comparison of medical therapy, pacing, and defibrillation in heart failure (COMPANION) trial. Circulation. 2006;114:2766-2772 Yusuf S, Pfeffer MA, Swedberg K, Granger CB, Held P, McMurray JJ, Michelson EL, Olofsson B, Ostergren J. Effects of candesartan in patients with chronic heart failure and preserved leftventricular ejection fraction: The CHARM-PRESERVED trial. Lancet. 2003;362:777-781 Zannad F, McMurray JJ, Krum H, van Veldhuisen DJ, Swedberg K, Shi H, Vincent J, Pocock SJ, Pitt B. Eplerenone in patients with systolic heart failure and mild symptoms. The New England journal of medicine. 2011;364:11-21 Varela-Roman A, Grigorian L, Barge E, Bassante P, de la Pena MG, Gonzalez-Juanatey JR. Heart failure in patients with preserved and deteriorated left ventricular ejection fraction. Heart. 2005;91:489-494

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Inclusion Criteria 1. Age >21 and <90 years 2. New York Heart Association Class II or III 3. Chronic ischemic left ventricular dysfunction secondary to MI as defined by: - Previous MI (>6 months) documented by history that includes elevated cardiac biomarkers or ECG changes consistent with MI - Prior thrombolytic therapy, percutaneous coronary intervention or coronary artery bypass surgery 4. Left ventricular ejection fraction ≥20% and ≤40% assessed by echo core laboratory 5. All patients should receive stable, evidence based, medical and device therapy for heart failure or post 67 infarction left ventricular dysfunction (2013 ACC/AHA Heart Failure Guideline ) for at least 3 months prior to randomization. Pharmacotherapy: Stable pharmacologic therapy is defined as changes not exceeding halving or doubling the medications listed below. - Angiotensin converting enzyme inhibitors or angiotensin II receptor blockers or nitrate/hydralazine combination with investigator discretion after factoring in patient intolerances - Beta blockers approved for heart failure with investigator discretion after factoring in patient intolerances - Diuretics and aldosterone inhibitors Device therapy: cardiac resynchronization therapy (CRT) or CRT-Defibrillator therapy - CRT or CRT-D implant must be >3 months before randomization - Patients anticipated to be eligible for CRT or CRT-D within 6 months are excluded 6. Acceptable Cell Potency Assay Score as determined by the Cell Analysis Core Lab 7. Able to provide informed consent

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Exclusion Criteria 1. Severe lung disease (asthma or chronic obstructive pulmonary disease), orthopedic, muscular, or neurologic conditions that could limit the ability to perform the 6 Minute Walk Distance Test. 2. 6 Minute Walk Distance <100 meters or >450 meters 3. Ideal candidate for coronary revascularization 4. Patients anticipated to be eligible for CRT or CRT-D within 6 months are excluded 5. Severe mitral or tricuspid regurgitation as previously documented or measured by the echocardiographic core lab 6. Presence of a mechanical aortic valve or heart constrictive device 7. Presence of moderate or severe aortic valve stenosis or regurgitation 8. Acute coronary syndrome within 3 months 9. Life threatening arrhythmias or QTc interval >55ms on screening ECG 10. Baseline glomerular filtration rate <50 ml/min / 1.73m2 11. Hematological abnormality as evidenced by hematocrit <30%, white blood cell <4,500/µl or platelet values, <100,000/µl 12. Liver dysfunction, as evidenced by enzymes (AST and ALT) greater than three times the ULN) 13. Coagulopathy (INR ≥1.3) not due to a reversible cause (i.e., Coumadin). Patients on Coumadin will be withdrawn before the procedure and confirmed to have an INR <1.3. 14. Organ transplant recipient 15. Malignancy within 5 years (i.e., patients with prior malignancy must be disease free for 5 years), except curatively-treated basal cell carcinoma, squamous cell carcinoma of skin, or cervical carcinoma 16. Serum positive for hepatitis B/C and/or HIV. 17. History of serious radiologic contrast allergy 18. Pregnant, nursing female or female of childbearing potential while not practicing effective contraceptive methods for at least 30 days prior to randomization

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Figure 1. CardiAMP-HF trial workflow. ECG=electrocardiogram, 6MWD=six meter walk distance, HF=Heart Failure, BMMNC’s= Bone marrow mononuclear cells, PPM=permanent pacemaker, ICD=implanted cardiac defibrillator, MLWHFQ = Minnesota Living with Heart Failure Questionnaire, MACE=major adverse cardiac events

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Figure 2. CardiAMP-HF “Point of Care” Cell Isolation Process. From left to right, bone marrow aspirate is obtained from the posterior iliac crest and transferred into the cell separation chamber. The chamber is centrifuged and removed. The cell poor plasma layer is removed and the remaining nucleated product is drawn into a 10 mL syringe. This cell product is immediately distributed into 1 mL syringes for therapeutic transendocardial injections.

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Figure 3. Helix Transendocardial Delivery System. The Helix catheter has a helicalshaped needle with a distal lumen to administer the cell therapy and a more proximal lumen for injection of dilute contrast, which is used to verify transendocardial catheter engagement (left image). The Helix catheter is telescoped within the steerable Morph guide (right image). Both devices are positioned retrograde across the aortic valve into the left ventricle for transendocardial therapeutic injections.

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