Therapeutic	Autologous CD34+ Cells (Auto-CD34+ cells)
Study Phase	Phase II
Protocol Title	A double blind, prospective, randomized, placebo-controlled study to determine the tolerability, efficacy, safety and dose range of intramyocardial injections of G-CSF mobilized Auto-CD34+ cells for reduction of angina episodes in subjects with refractory chronic myocardial ischemia. (ACT34-CMI)
Objective	Primary Objective: The primary objective of the Phase 2 clinical trial is to demonstrate reduction in angina episodes, evaluate the efficacy, tolerability and safety of two doses of Auto-CD34+ cells administered via intramyocardial injection to subjects with refractory chronic myocardial ischemia (CMI).
Study Design	This is a prospective, randomized, double blind, placebo-controlled study to evaluate the efficacy, tolerability, and safety of two doses of Auto-CD34+ cells when delivered by intramyocardial injection. A single administration of Auto-CD34+ cells will be dosed at either 1 × 105 or 5 × 105 (±10%) cells/kg body weight, up to a maximum of 100 kg, and compared to subjects receiving placebo. There will be 50 subjects per group.
	All subjects will receive subcutaneous injections of Granulocyte Colony Stimulating Factor (G-CSF) at a dose of 5 μg/kg/day for 5 days to mobilize CD34+ cells from the bone marrow to the peripheral blood. Prior to undergoing apheresis, complete blood counts will be performed on days 1, 2, 3, 4 and 5. Fluorescent-activated cell sorter (FACS) analysis will be performed on days 4 and 5, to determine the number of CD34+ cells in the circulation. On day 5, apheresis will be performed using the Amicus Blood Cell Separator (Fenwal) or an alternative approved apheresis system (e.g. Cobe Spectra), according to the manufacturer’s instructions for mononuclear cell collection. The endpoint of each collection will be the processing of up to 2-5 total blood volumes (TBV) and will be based on the circulating CD34+ cell count on the day of apheresis and subject tolerance for the apheresis procedure. <15 CD34+ cells/μL whole blood → 5 TBV 15-25 CD34+ cells/μL whole blood → 4 TBV 26-50 CD34+ cells/μL whole blood → 3 TBV >50 CD34+ cells/μL whole blood → 2 TBV
	On the day of cell injection, the apheresis product will be enriched for CD34+ cells using the Isolex 300i Magnetic Cell Selection System (Baxter Healthcare). Quality control testing will be performed on the apheresis product and on the final selected product. After the final selected product is tested and determined to meet release specifications, subjects will be randomly assigned to 1 × 105, 5 × 105 (± 10%) CD34+ cells/kg body weight (up to 100 kg) or to placebo (0.9% NaCl (saline) plus 5% autologous plasma) at this point in the trial. The subject will undergo cardiac catheterization with the NOGA™ electromechanical mapping system. This system is used to identify ischemic but viable regions of the myocardium as targets for cell delivery. CD34+ cells will be delivered in 10 intramyocardial injections of 0.2 mLs each into the target areas of myocardial ischemia using the MyoStar injection catheter (Biosense Webster, Inc., a Johnson & Johnson company).
Number of subjects	150
Study population	Male or female subjects who are 21-80 years of age with refractory chronic myocardial ischemia on maximal therapy who are not suitable candidates for conventional revascularization
Inclusion Criteria	Male or female subjects who are 21-80 years of age, inclusive. Subjects with CCS functional class III or IV chronic refractory angina as evaluated by the Core Lab independent interview. Subjects without control of their angina symptoms, in spite of maximal tolerated doses of anti-angina drugs, must be on optimal therapy for their angina (1,2,3a), and on a stable anti-angina medication regimen for at least 1 month prior to entering the screening period of the study (3b). This means that their current treatment must include (all 3 below): Aspirin (81 mg) or clopidogrel or ticlopidine Statins at maximum tolerated dose At least 2 of the following anti-angina medications – Beta Adrenergic Blocking Agents, Calcium Channel Blocker, ACE Inhibitors, long-acting nitrates – at maximum tolerated dose, unless not tolerated because of significant side effects. Stable doses of the anti-angina medications for 1 month prior to entering the screening of the study. Subjects must be identified as unsuitable for conventional revascularization. A local independent interventional cardiologist and cardio- thoracic surgeon will review the subject’s angiogram to determine if the subject is eligible for revascularization. Additional factors which will be considered in determining the candidacy of a subject for revascularization: 1) Technical factors, such as the availability of conduits for bypass (veins, internal mammary arteries, radial arteries), the suitability of native arteries for accepting bypass grafts, calcification of the aorta (making cross-clamping difficult and higher risk); or for percutaneous revascularization, left main disease, restenosis, small vessels, diffuse disease, severe tortuosity or other anatomic considerations. 2) Comorbidities, such as chronic obstructive lung disease, asthma, morbid obesity, diabetes, active infection, arthritis, etc. All subjects must have a recent coronary angiogram (within the last 12 months) to document the coronary anatomy and to verify the revascularization procedures. Candidates for this study must meet at least one of the following criteria: 1) Total occlusion of an epicardial coronary artery. 2) Are at high risk for percutaneous coronary angioplasty of treatment zone(s) based upon clinical or anatomic considerations including but not limited to the following: diabetes, left main disease, pulmonary hypertension, severe proximal vessel tortuosity, severe bendpoint obstructions, diffuse disease (>2 cm in length), small vessel (<2 mm reference diameter), stenoses which are either diffuse (>2 cm in length) or distal, incessant restenotic lesions, unfavorable bifurcation stenosis, and degenerated or thrombosed saphenous vein grafts. Subjects must have objective evidence of inducible ischemia or viable myocardium in the potential target injection zone, as manifested by any one of the following criteria: under-perfused myocardium as shown on their initial nuclear scan SPECT done during screening. The presence of myocardial perfusion abnormalities in ≥2 segments of the standard 17-segment model are required for entry into this clinical study. There must be partial or complete reversibility present on the SPECT study, or primarily fixed perfusion abnormalities that are associated with wall motion and thickening on the gated SPECT study. Eligibility will be determined by the SPECT Core lab analysis of the screening SPECT image. Or, ST-segment shifts ≥0.5 mm documented on the screening exercise treadmill test, as determined by the exercise testing core laboratory. A left ventricular ejection fraction ≥25% by ECHO or SPECT (as determined by the core lab) at screening. Subjects must experience at minimum an average of 7 angina/or anginal equivalent episodes per week, as recorded in the 28-Day Angina and Angina Medication Use Diary. Subjects must be able to complete a minimum of 3 minutes but no more than 10 minutes on a treadmill following the Modified Bruce Protocol. Subjects must experience angina or anginal equivalent episodes during the screening exercise treadmill test. Female subjects must either be no longer capable of reproduction or using medically valid contraception to prevent pregnancy during the study. Subject must understand the nature of the procedure and provide written informed consent prior to the procedure. Subject must be willing and able to comply with specified follow-up evaluations.
Exclusion Criteria	Predominant congestive heart failure symptoms (CHF). a). Subjects who have been hospitalized with a primary diagnosis of CHF in the prior 6 months; b). Subjects with evidence of pulmonary edema that requires acute intervention such as the administration of intravenous therapy e.g. diuretics, inotropic agents, or vasodilator therapy, c). Subjects with physical findings consistent with cardiogenic shock (See Note 3). Myocardial infarction (Q wave or non-Q wave) within 60 days of treatment. Myocardial infarction is defined according to standard practice on the basis of symptoms evocative of acute coronary ischemia accompanied by diagnostic change in the ECG and/or cardiac enzymes (troponin, CK/CKMB ratio). (See Note 3). Successful or partially successful coronary revascularization procedures (any vessel) within 6 months of study enrollment (See Note 3). Placement of a bi-ventricular pacemaker for cardiac re-synchronization therapy (CRT) for heart failure in the past 90 days. (See Note 3). Documented stroke or transient ischemic attack (TIA) within 60 days of study enrollment (See Note 3). History of moderate to severe aortic stenosis (aortic valve area <1.5 cm2) or severe aortic insufficiency; severe mitral stenosis (mitral valve area <1.5 cm2); or severe mitral insufficiency. The area of the aortic and mitral valve must be confirmed at the pre-treatment echocardiogram. Subjects with any prosthetic aortic valve replacement. Evidence of any life threatening arrhythmia that requires intervention (e.g. third degree AV block, sustained ventricular tachycardia, sustained ventricular fibrillation, prolonged sinus arrest etc.) on the 24-hour Holter monitor (See Note 3). Life threatening arrhythmia that is successfully treated with an ICD is not exclusionary. Splenomegaly and/or severe co-morbidity associated with a reduction in life expectancy of less than 1 year, such as chronic medical illness (i.e., severe chronic obstructive pulmonary disease, renal failure or cancer [exceptions – in-situ skin cancer or fully removed skin cancer other than melanoma, in-situ cervical cancer, or cancer free for 5 years with no history of a stem cell transplant]). Subjects with sickle cell disease or sickle cell trait. Subjects with platelet counts greater than 10% above the upper limit of normal or a platelet count below 100,000 if on Clopidogrel or 50,000 without Clopidogrel. (See Note 3) Subjects with a hematocrit <30%. (See Note 3). Serum Creatinine > 2.5 mg/dl. (See Note 3) Any clinically significant laboratory abnormality on screening laboratories. Currently enrolled in another investigational device or drug trial (IDE or IND) that has not completed the protocol required primary follow-up period (excludes 15 year follow-up of gene therapy trials) History of alcohol or drug abuse within 3 months of screening. (See Note 3) Joint, peripheral vascular disease or neurologic disease that severely limits treadmill walking. Chronic obstructive pulmonary disease that severely limits walking or forced expiratory volume in one second (FEV1) <30% predicted. Females who are pregnant or lactating. Female subjects who are capable of reproduction and will not use medically valid contraception to prevent pregnancy during the study. Subjects who test positive for HIV, hepatitis B or hepatitis C, or are on chronic immunosuppressive medications or have had a prior stem cell transplant. Subjects with a known hypersensitivity to E. coli-derived proteins, or to any component of Neupogen (Filgrastim) or G-CSF. Subjects who have a significant psychiatric disorder or mental disability that could interfere with the subject’s ability to provide informed consent and/or comply with protocol procedures.
Treatment Groups	Test: 1 × 105 (±10%) or 5 × 105 (±10 %) CD34+ cells / kg body weight (up to a maximum of 100 kg)
	Control: placebo (0.9 % NaCl (saline) plus 5% autologous plasma) Mode of Administration: Intramyocardial injection
Duration of Treatment	Mobilization with G-CSF is 5 days.
	Apheresis is performed in one day on day 5 of mobilization CD34+ selection is performed on day 6
	Mapping and injection of stem cells or placebo is a 2-3 hour procedure performed on day 6.
	Subjects will be hospitalized for 24-hour observation after cell injection.
	The subject will then be followed for 12 months.
	In a separate protocol, subjects will be followed for 12 months after ending participation in this protocol.
Efficacy Variable	Primary Efficacy variable is frequency of angina episodes per week, when comparing subjects receiving injection of CD34+ cells to placebo.
	Secondary Efficacy variables are divided into two categories, symptom relief and myocardial perfusion, and function measurement endpoints.
	Symptom Relief: ETT, anti-anginal medication, pedometer measurements, CCS functional class and QOL [SAQ, SF-36, Dyspnea Questionnaire, Euro 5 Questionnaire], and the combined rate of MACE events.
	Myocardial perfusion and function measurements: SPECT and cardiac MRI.
Safety Variable	Adverse event reporting, MACE, physical examination, vital signs, ECHO, laboratory parameters, revascularization procedures (as defined by new diseased vessel or progression of disease in a vessel not believed to be the cause of baseline angina), hospitalization rates for cardiac related admissions and Emergency Department/Acute Care Service visits for cardiac related admissions will assess safety.
Pharmacoeconomics	Pharmacoeconomics will be evaluated by assessment of hospitalization rates, emergency room visits, revascularization procedures (as defined by a new diseased vessel or progression of disease in a vessel not believed to be the cause of baseline angina), and changes in medication.
Statistical Methods	A log linear model (Poisson regression) will be performed on the frequency of angina at baseline and six months. The independent parameters in the model will be treatment group (as randomized) and visit (baseline, 6 months), and the interaction between treatment group and visit. The baseline value will be used as a covariate. Contrasts will be constructed on the difference between 6 months and baseline. Since this analysis is done on the log scale, these contrasts will take the form of relative risks. Missing data will be imputed using last value carried forward. The primary analysis will be with the intent-to-treat (ITT) population.
	A secondary analysis will be done using the actual dose of cells the subject received. The independent parameters in the model will be dose of cells the subject actually received, visit (baseline, 6 months) and their interaction. The baseline value will be used as a covariate.
	Similar analyses will be performed using all available visits. Repeated measures analyses will be used including all visits. Data will be summarized by visit for each treatment group. The difference between the treated subjects and control subjects (as randomized) will also be summarized at each visit. Missing data will not be imputed for this analysis.
	One interim analysis is planned. This is for administrative purposes only and will not affect the conduct of the study. An independent committee will conduct the analysis. All study personnel will remain blinded until the end of the study.
	For the secondary efficacy parameters, analysis of variance with repeated measures will be performed on continuous data. Generalized linear models will be used to analyze ordinal and categorical data. The independent parameters in the model will be treatment group and visit (baseline, 6 months) and the interaction between treatment group and visit. The baseline value will be used as a covariate.
	Similar analysis will be performed using all available visits.
	Adverse events will be listed by subject and summarized in tabular format by and within standard of care (SOC) and treatment group. Listings and tables will be provided for treatment-emergent adverse events (defined for an individual subject as an event not present prior to beginning study medication, or, if present prior to beginning study medication, an event that increases in intensity, is considered related to the study medication, or becomes serious during the treatment or follow-up phases of the study). Separate listings and/or tables will also be provided for adverse events by maximum intensity, drug-related adverse events, serious adverse events, adverse events resulting in discontinuation of study medication, and deaths.
	Vital Signs assessment of the significance of the mean changes from baseline to each follow-up evaluation point will be made within treatment groups, using paired t-tests. Comparisons between treatment groups with respect to mean changes from baseline will be made using ANOVA/Categorical – Chi-square, or Fisher’s Exact as appropriate.
	Laboratory results will be summarized (summary statistics for quantitative assays, contingency tables for qualitative assays) by treatment group in tabular format. Assessment of the significance of the mean changes from baseline to each follow-up visit will be made within treatment groups for quantitative assays, using paired t-tests. Comparisons between treatment groups with respect to mean changes from baseline in selected quantitative assays will be made using analysis of variance (ANOVA). Individual subject values identified as abnormal (outside the lab normal ranges) and substantially abnormal will be listed. Frequency tables will be produced for selected assays summarizing shifts from pretreatment to the last assessment while on treatment and the final visit.