1. Cardiovascular Cell Therapy: What’ s New? On Clinical Scenarios and Clinical Trials Stefan P. Janssens, MD, PhD Department of Cardiology University of Leuven, Belgium Madrid, April 24 th, 2008 No disclosures

2. Preclinical In vitro In vivo Phase II RCT Surrogate endpoint Clinical mechanism Phase III RCT Clinical need Clinical outcome In vitro In vivo Pre- clinical Surrogate endpoint Clinical Mechanism Phase II RCT Clinical need Clinical outcome Phase III RCT

3. 30 years later: VALIANT study (14,703 post-MI pts EF<35%, clin CHF) 1 y mortality 1 y death, re- MI, CHF rehosp Pfeffer et al. NEJM 2003 Cardiac Regeneration in 2008: the stem cell approach Premise: myocyte deficit contributes to dysfunctional phenotype? Caulfield et al. Circ 1976 Infarct size (% LV mass) Shock, DeathCHF 48% 28% 13% 26%

4. Stem Cells: from Bench to Bedside Clinical Scenarios 1.Acute Myocardial Infarction with significantly impaired LV function: - is safety & efficacy sufficiently established to start a Phase III randomized, controlled outcome trial? or - are additional innovative, mechanistic Phase II studies required?

5. 5 Stem Cell Therapy in Post-MI Patients with Depressed LV Function is Safe BOOSTLEUVENASTAMIREPAIR-AMI Follow-up (months)1812 Control/Placebon=30 Cn=34 Pn=50 Cn=103 P Mortality1 (3%)006 (6%) Reinfarction0106 (6%) Revascularisation4(13%)2(6%)11(22%)37(36%) BMC groupn=30n=33n=50n=101 Mortality02(6%) # 02(2%) Reinfarction1(3%) 1(2%)0 Revascularisation5(17%)2(6%)13(26%)22(22%) (Adapted from Arnesen et al. Lancet 2007;369,2142) # unrelated to BMC

6. RC Trials using Intracoronary BMC post MI LEUVEN-AMI MI size -28% (P=0.03) ND P=n.s. ND + 2.8% (P=n.s.)* * 18-months follow-up

7. Stem Cells: from Bench to Bedside Clinical Scenarios What does an increase in global LVEF of a few % points mean?

8. Infarct Size Reduction in Reperfused STEMI Infarct Size Reduction (%) Small Infarcts (<17% LV mass) Large Infarcts (>17% LV mass) Small Infarcts (n=29) Large Infarcts (n=29) Age58±1161±11 BP adm140/82128/80* LV-EDP18±526±8 ** IRA LAD1115 RCA1712 Cx12 Time to PCI284±180292±173 Max Trop60±28115±68 ** MedsACE- /BB/Stat/ Asp

9. Infarct Size Determines Global and Regional Functional Recovery in Reperfused STEMI Change in LV-EF (%) Small Infarcts (<17% LV mass) n=29 Large Infarcts (>17% LV mass) n=29 Δ+ 3% P=0.003 P=NS SWT (mm) Small Infarcts (<17% LV mass) n=29 Large Infarcts (>17% LV mass) n=29 BorderInfarctBorderInfarct P=0.01 P=0.003 P=NS

10. Bone Marrow Cell Transfer Post-AMI Infarct size and Coronary Flow Reserve (Doppler) (Schachinger et al., NEJM 2006; 355:1210-21) BMCPlac 20 -20 10 -10 0 <48.9% >48.9% P=0.002 P=0.81 (52)(41)(40)(54) Baseline EF (%) Δ EF (%) (Erbs et al., Circulation 2007) (n=30)(n=28)

11. Infarct Size Determines Global Functional Recovery in Reperfused STEMI Change in LV-EF (%) Large Infarcts (>20% LV mass, n=20) Change in LV-ESVI (mL) CON BMSC Baseline 1 year P=0.06 for interaction P=0.07 for interaction 44 48 43 45

12. Wall Motion Score Index and Ejection Fraction for Risk Stratification after AMI Predictors of mortality (forward Cox PHA) HRP Age (per 10y) 1.65 <.0001 Kilip Class 1.44 <.0001 (per 1 increase) WMSI 1.15 <.0001 (per 0.2 increase) (Moller et al. Am Heart J 2006;151:419-25)

13. Powerful MRI and TDI Analysis of Biological Signals: Infarct Transmurality & Segmental Contraction Coronary occlusion 20 min60 min3h>6h LV apex mid base Time (ms) -20 -10 0 02004006008001000 Strain Septum 4 months (%) -30 AVCAVO -20 -10 0 200400600800 -30 AVCAVO Baseline Strain Septum (%)

14. Improved contraction (%) 0 20 40 60 80 CONTROL BMC (29 of 53) (33 of 63) (14 of 32) (25 of 83) (6 of 13) (9 of 18) (9 of 25) (10 of 87) Improved Regional Contraction in Dysfunctional Segments indicates BMC Functional Repair Lancet 2006; 367:113-121 -20 -15 -10 -5 0 Baseline5 d2 mo4 mo 1 yr (n=232) Infarcted segments *** ES Strain (%) BMSC Control 0-25% 26-50% 51-75% 76-100% 0-25% 26-50% 51-75% 76-100% P<0.05 for interaction * *

15. Schachinger, V. et al. Eur Heart J 2006 27:2775-2783 Kaplan-Meier event-free survival analysis

16. Cardiac Regeneration in 2008: Clinical Scenarios VALIANT study (14,703 post-MI pts EF<35%, clin CHF) 1 y mortality 1 y death, re- MI, CHF rehosp Pfeffer et al. NEJM 2003 13% 26% Power calculations for Outcome study: Significant Reduction in combined Clinical EP (death, recurrent MI, CHF hospitalizations) requires ± 1,200 pts

17. Bone Marrow Cell Therapy Anno 2008: Limitations for Cinical Benefit Modest improvement in cardiac function in 4 RCTs of BMC transfer is attributable to: –limited homing, engraftment, and survival of BMCs lack of cardiac muscle regeneration IC injection 18 F-FDG labeled BMSC: 1.3 - 2.6% homing infarct region (Hofmann et al. Circ 2005) limited progenitor cell functionality in sick patients

18. Stem Cells: from Bench to Bedside Clinical Scenarios 2.Acute myocardial infarction with significantly impaired LV function: - Focus on cell enhancement strategies - Labeling and in vivo tracking of different progenitor cell populations 1.luciferase bioluminescence 2.Genetic or histochemical marker (GFP, Endorem, DiI,..) 3.PET/CT: compare timing of delivery and route of administration 1.Acute Myocardial Infarction with significantly impaired LV function: - clinical outcome trial (phase III, confirmed safety & efficacy) versus - innovative, mechanistic studies (phase II)

19. Stem Cells: from Bench to Bedside Cell Enhancement Strategies Priming of Progenitor Cells Priming of Target Tissue Impaired PC phenotype & non-responders: ~ CV Risk factors ~ progenitor cell modification Hostile target milieu: ~ oxidant stress ~ microvascular obstruction ~ transmigration - residency

20. BMC and EPC cell transfer 3 hours after I/R I/R injury L2G85 FVB WT FVB Hypothesis Imaging of Bone Marrow Mononuclear Cell Homing in Ischemic Myocardium

21. Day 1Day 2Day 6Day 4Day 8Day 10Day 14Day 21 I/R Sham

22. Reperfusion Therapy frequently Associated with Microvascular Obstruction (MVO) Cx occlusion Successful PCI Incidence postPCI: >60% Persistent MVO J. Bogaert & S. Janssens, Eur J Rad 2007 47 (9) 4 mo 46 (8) LV-EF (%) P=NS 3-4 d LV-EDV (mL) P=0.014 162 (33)175 (43) 3-4 d4 mo

23. BMSC (n=17) CON (n=19) MVO Global LV Function Recovery in AMI Patients with and without Microvascular Obstruction 44 46 48 50 52 54 56 58 60 62 1 week4 months P = 0.63 P = 0.60 LV-EF (%) 12 months BMSC (n=11) CON (n=9) No MVO P = 0.05 P = 0.36 +3.5% +5.5%

24. Direct Labeling of Stem Cells Using Positron Emission Tomography Radionuclides T1/2 = 109 min Transport via GLUT Phosphorylated by hexokinase (= metabolic trapping) Good labeling efficiency Poor retention Substrate for cardiomyocytes 2-[18F]fluorodeoxyglucose (FDG) (Ma et al. 2005) 90 min post injection 60 min post injection 30 min post injection Liver Heart Bladder

25. Direct Labeling of Stem Cells Using Positron Emission Tomography Radionuclides T1/2 = 109 min No transport, no enzymatic reaction Incorporation in cell membrane Good labeling efficiency Good retention No substrate for cardiomyocytes Hexadecyl-4-[18F]fluorobenzoate (HFB) (Ma et al. 2005) Liver Heart Bladder 18 F-HFB 90 min post injection 60 min post injection 30 min post injection

26. 18 F-HFB BMCs vs Free label Injection post MI

27. Stem Cells: from Bench to Bedside Cell Enhancement Strategies Priming & Labeling of Progenitor cells statins p38 inhibitors PPAR  eNOS enhancers Integrin activators Cardiac specification…. gene transduction: Akt, eNOS,… PET- MRI tracers Mechanical activation Cytokines / Growth factors: –IGF-1, HGF, SDF-1, PDGF,…. NO Priming of Target Tissue Impaired EPC phenotype & non-responders ~ CV Risk factors ~ post MI cell modification Hostile target milieu ~ oxidant stress ~ microvascular obstruction ~ transmigration - residency

28. Stem Cells: from Bench to Bedside Informative Cell Delivery Studies REGENT Poland ( NCT 00313339 ) - recruitment complete R, open label, safety/efficacy: BMNC vs CD34 + /CxCR4 + vs CON post AMI (EF<40%) Prim EP: LVEF and volumes (Echo and angio) ---> 2008? SWISS AMI ( NCT 00355186) (EF evaluate after 60 MYSTAR Austria ( NCT 00384982) (4 arm 360 pts, LVEF<45%, comp 21-42 d vs 3 mo post-AMI and IC vs IM delivery vs combination) Prim EP:  perfusion defect and LVEF by gated SPECT, NOGA ---> 2008 - 2009? AMORCYTE REPAIR US ( NCT 00313339) (Ph I, CD34 + cells post AMI, n=40) (www.ClinicalTrials.gov)

29. Stem Cells: from Bench to Bedside Informative Cell Delivery Studies REVEAL US ( NCT 00378352 ) Ph I/II: dose finding -> RCT Db blind, safety/efficacy: EPCs vs CON post AMI Prim EP: Infarct size (MRI), n=210 NEURONYX US ( NCT 00361855) & PROVACEL Osiris, US (NCT 00114452) Ph I, RCT, safety escalating doses allogeneic hBM-derived SCs-MSC n=18 and 48 MAGIC-Cell-5-Combicytokine (Korea, ± 120 pts, 1:1:2) Ph II, RCT: safety/efficacy CON vs G-CSF+MN Cell apheresis vs G-CSF+MN Cells + EPO CHF or Chronic refractory ischemia - Surgery: –BMNC during CABG Berlin ( NCT 00462774) 60 pts, CD133 + cells in infarct border zone LVEF<35%), MRI EP:  LVEF -Refractory ischemia Kobe ( NCT 00221182) 10 pts, CD34 + cells IC, sestamibi SPECT scans -BMNC and CABG for CHF Helsinki (NCT 00418418) RCT, dble blind, efficacy; Incl if CABG and EF 15-45%; prim EP:  LVEF by MRI at 6 mo -TABMMI Biocardia endocardial delivery chronic infarcts Argentina (NCT 00507468) Ph I, safety, prior AMI with LVEF<40%, n=20 pts (www.ClinicalTrials.gov)

30. Sca-1+ cells c-Kit + cells SP cells Endothelial Progenitor Cells Hematopoietic SCs Mesenchymal SCs Hemangioblasts SP cells MAPC Sca-1+ cells Myoblasts SP cells Mesenchymal SCs SPcells PLURIPOTENT Acute MI Chronic Ischemia Cardiac Stem Cells Cardiac Stem Cells Revascularisation using CSC US Kentucky (NCT 00474461) - Ph I: n=40; - RAA resection during CABG - Reinfusion of CSC after 4 mo if LVEF < 40% Anversa P.et al. Circulation 2007

31. Acknowledgments - X. Liu, MD, PhD - P. Pokreisz, PhD - T. Vandendriessche - M. Chua - K. Sipido, MD, PhD - C. Dubois, MD - G. Marsboom, PhD - O. Gheysens, MD - S. Vandenwyngaert, MSc - H. Gillijns, BSc - M. Pellens, BSc Cardiology lab and CTG SCIL - C. Verfaillie, MD, PhD - M. Boogaerts, MD, PhD - L. Mortelmans, MD, PhD KUL & Univ Hospital - F. Van de Werf, MD, PhD - G. Marchal, MD, PhD - J. Bogaert, MD, PhD - G. Bormans, PhD - A. Verbruggen, MD, PhD Stanford University - S. Gambhir, MD, PhD Harvard University - K. Bloch, MD

33. Conclusions Modest effects of BM-derived progenitor cell transfer in AMI is likely attributable to limited homing and engraftment and lack of cardiomyogenesis. Comprehensive 3D-MRI analysis suggests early infarct imaging correlates with diverging patterns of functional and structural recovery post-MI. Progenitor cell transfer is best reserved for patients with large MI, at risk for developing maladaptive remodeling and heart failure. Potential confounding factors including MVO and cell functionality, warrant focused trials.

34. Stem Cells: from Bench to Bedside Cell Enhancement Strategies 1.Labeling and (in vivo) tracking of different progenitor cell populations In vivo tracking: - luciferase bioluminescence - PET/CT Genetic marker (Lentiviral infection GFP, RFP,..) Histochemical labeling (Endorem, DiI, …) 2. Factors limiting Functionality/Survival of progenitor cell populations (hypoxia and tissue ischemia) (microvascular obstruction)