Safety and effectiveness of granulocyte-colony stimulating factor in advanced chronic heart failure Jacob Joseph, MD Veterans Affairs Healthcare System- Boston and Boston University Am J Cardiol 2006; 97:

Stem cell mobilization in AMI vs CHF Activated homing signals Activated homing signals Better mobilization Better mobilization Poor vascularity Poor vascularity Less electrical heterogeneity if successful myocardial regeneration Less electrical heterogeneity if successful myocardial regeneration Safety (Collagenolysis due to neutrophil proteases) Safety (Collagenolysis due to neutrophil proteases) Weak homing signals Less mobilization Better myocardial milieu Significant electrical heterogeneity Safety (electrical heterogeneity, neutrophil mobilization)

Rationale for a phase I study of G-CSF in advanced heart failure Stem cells may not be adequately mobilized in advanced heart failure Stem cells may not be adequately mobilized in advanced heart failure Bone Marrow Hypoperfusion Bone Marrow Hypoperfusion Cytokines Cytokines Microscopic myocardial injury (ongoing myocyte necrosis with replacement fibrosis due to neurohormonal stimulation and ischemia) may produce homing of stem cells Microscopic myocardial injury (ongoing myocyte necrosis with replacement fibrosis due to neurohormonal stimulation and ischemia) may produce homing of stem cells Safety issues unknown Safety issues unknown

Escalating Dose Protocol

Inclusion and Exclusion Criteria INCLUSION Age ≥ 18 years; NYHA III or IV; LVEF < 35% Age ≥ 18 years; NYHA III or IV; LVEF < 35% Background treatment with standard therapy for heart failure ≥ 3 months Background treatment with standard therapy for heart failure ≥ 3 months ICD in situ ICD in situEXCLUSION Unstable ischemic syndrome Unstable ischemic syndrome Stroke or transient ischemic attacks within last 3 months Stroke or transient ischemic attacks within last 3 months Severe organ dysfunction/illnesses limiting survival ≤ 6 months Severe organ dysfunction/illnesses limiting survival ≤ 6 months

Study Protocol Measurement Base line TreatmentFollow-up Day/Week/ Mo D1D2D3D4D5D6D8D10 W6W6 M3M3 M9M9 G-CSFXXXXX ChemistryXXXXXXXXXXX CBCXXXXXXXXXXX CD 34 + XXXXXXXXXXX CytokinesXXXX Echo XXX Optison Echo XXX

Endpoints Primary endpoint CD 34+ cells above 10 cells/microliter CD 34+ cells above 10 cells/microliter Secondary endpoints Lack of significant changes (twice baseline measurements) in laboratory parameters during the entire study (safety endpoint) Lack of significant changes (twice baseline measurements) in laboratory parameters during the entire study (safety endpoint) Increase in LVEF at 9 months Increase in LVEF at 9 months Changes in cytokine levels Changes in cytokine levels

Patient Age (years) LVEF(%) EtiologyIschemic Non Ischemic Blood Pressure (mmHg) 131/77122/69139/98113/6491/64140/83 Heart Rate (Beats per minute) Serum creatinine (mg/dL) White Blood Cell Count (x10 3 /dL) New York Heart Association Class III IVIII Dose (mg/kg) Mean age (±SEM) was 63 ±4.2 years; mean LVEF(±SEM) was 18.5±1.8% Baseline Characteristics

BaselinePeak Patient #WBC count (X10 3 /  l  CD34 +ve cell count (cells/  l) WBC count (X10 3 /  l  CD34 +ve cell count (cells/  l) Mean±SEM6.9±13.6± ±8*38.7±13* * p value < 0.05 compared to corresponding baseline group Hematologic parameters

CD 34+ cell mobilization

CD34+ cell mobilization vs. WBC increase

Side Effects Pt 1 Increased AlkP 2 Increased AlkP, Calf cramping 3 Increased AlkP, Pain (calf, legs, back) 4 Increased AlkP 5 Increased AlkP, decreased UOP, increased Cr, shoulder & back pain 6 Increased AlkP, Pain at the injection site

ParameterBaseline9 monthp value (paired t- test) Mean Left Ventricular Ejection Fraction (all patients)18.5± ± Mean Left Ventricular Ejection Fraction (ischemic cardiomyopathy patients) 20.3± ± Left Ventricular End Diastolic Dimension (all patients)6.8±0.46.6± Left Ventricular End Diastolic Dimension (ischemic cardiomyopathy patients) 6.5±0.56.1± Left Ventricular End Systolic Dimension (all patients)6.3±0.35.9± Left Ventricular End Systolic Dimension (ischemic cardiomyopathy patients) 5.9±0.45.5± Effects on LV structure and function

Cytokines in Heart failure Proinflammatory cytokine levels are increased in heart failure IL-6 and TNF alpha Anti-inflammatory cytokines are decreased in advanced chronic heart failure IL-10 and IL-10/TNF ratio IL-10/TNF alpha ratio correlates with response to treatment of CHF Clin Sci 2003; 105: 45-50; Eur Heart J 2003; 24:

CytokineBaseline level (pg/ml) Peak level (pg/ml) P value Tumor Necrosis Factor-   2.8+/ /-1.0NS Interferon-   14.4+/ /-10.3NS IL-21.4+/ /-0.7NS IL-44.8+/ /-1.1NS IL-52.7+/ /-1.0NS IL / / Mean cytokine levels in study patients

Conclusions A low dose of GCSF (5  g/kg/day) for 5 days can safely mobilize stem cells in advanced systolic heart failure A low dose of GCSF (5  g/kg/day) for 5 days can safely mobilize stem cells in advanced systolic heart failure WBC counts should be monitored for safety WBC counts should be monitored for safety GCSF-induced stem cell mobilization may result in favorable long term effects on left ventricular function, especially in ischemic heart failure GCSF-induced stem cell mobilization may result in favorable long term effects on left ventricular function, especially in ischemic heart failure GCSF has a favorable effect on cytokine profile GCSF has a favorable effect on cytokine profile

Future directions Could there be differences in response of ischemic vs. non-ischemic cardiomyopathy? Could there be differences in response of ischemic vs. non-ischemic cardiomyopathy? What is the optimal frequency of repeated cycles of GCSF? What is the optimal frequency of repeated cycles of GCSF? What is the role of cytokine modulation by GCSF mobilized cells? What is the role of cytokine modulation by GCSF mobilized cells? What is the mechanism of and clinical predictors of improvement in LV function? What is the mechanism of and clinical predictors of improvement in LV function?

Acknowledgements Asem Rimawi, MD Asem Rimawi, MD Jawahar L.Mehta, MD, PhD Jawahar L.Mehta, MD, PhD Paulette Mehta, MD Paulette Mehta, MD Michele Cottler-Fox, MD Michele Cottler-Fox, MD