1. Cardiac Cell Repair Therapy A Clinical Perspective CP1212505-6 Madrid 2007

2. CP1184400-3 Cell Repair Therapy – Current Status Underlying factors A compelling clinical need An opportunity for a therapeutic breakthrough in CV disease Supportive preclinical data Promising early clinical experience Intuitively appealing

3. Irrespective Cell number ‘Universality’ of Benefit in Animals and Clinical Studies Benefits noted within 72 hr Effective early & late post MI Allogenic stem cells Autologous stem cells ExtracardiacIntracardiac  LVEF  LVEF  Infarct size  Infarct size Improved diastolic function Improved diastolic function  LV volumes  LV volumes  Perfusion  Perfusion 6,700-fold range Murry: Circ, 2005 Method of delivery CP1256603-9 Site of delivery

4. Protection Repair Regeneration Inotropism Cellular Transdifferentiation into cardiac myocytes Fusion Mechanical support/ scaffolding Potentially Beneficial Mechanisms Cytokine/Paracrine Activation of resident cardiac stem cells Cytokine-induced proliferation of residual myocytes Angiogenesis Inflammation Metabolic modulation  Apoptosis CP1236917-7

5. Cell Repair Therapy – Safety Issues CP1252958-1 Potential concerns Aberrant cell differentiation Documented in animal models Cell numbers – ? Multiorgan seeding Accelerated atherosclerosis/ angiogenesis Prothrombotic risk Cell dependent Adjuvant therapy, eg, GCSF ? ? Oncogenesis Cell Time Arrhythmogenesis Dependent ?

6. Cell Repair Therapy Lessons Learned – Clinical Studies CP1224326-1 Safe and feasible Can modestly improve LV function Improved perfusion and remodeling after MI Greatest improvement in areas with greatest damage Can modestly improve LV function Improved perfusion and remodeling after MI Greatest improvement in areas with greatest damage Majority of implanted cells disappear within 1 wk No evidence of myocyte regeneration using existing therapies Majority of implanted cells disappear within 1 wk No evidence of myocyte regeneration using existing therapies Discordant results of double-blind RCT generate new questions.

7. CP1264589-7 Meta-Analysis of BMC Therapy in IHD Adel-Lafif (in press) 18 studies 18 studies 999 pt 999 pt Outcome: Mean Change in LVEF Study or subcategoryNo.No. RCTs Subtotal (95% CI)412395 Test for heterogeneity: Chi 2 = 59.81, df = 13 (P<0.00001), I 2 = 78.3% Test for overall effect: Z = 3.42 (P=0.0006) Study or subcategoryNo.No. RCTs Subtotal (95% CI)412395 Test for heterogeneity: Chi 2 = 59.81, df = 13 (P<0.00001), I 2 = 78.3% Test for overall effect: Z = 3.42 (P=0.0006) WMD (random) 95% CI WMD (random) 95% CI -10-505 10 Favors BMC treated Favors cntl Cohort studies Subtotal (95% CI)8782 Test for heterogeneity: Chi 2 = 4.32, df = 5 (P=0.51), I 2 = 0% Test for overall effect: Z = 2.83 (P=0.005) Cohort studies Subtotal (95% CI)8782 Test for heterogeneity: Chi 2 = 4.32, df = 5 (P=0.51), I 2 = 0% Test for overall effect: Z = 2.83 (P=0.005) Total (95% CI)499477 Test for heterogeneity: Chi 2 = 64.73, df = 19 (P<0.00001), I 2 = 70.6% Test for overall effect: Z = 4.14 (P<0.0001) Total (95% CI)499477 Test for heterogeneity: Chi 2 = 64.73, df = 19 (P<0.00001), I 2 = 70.6% Test for overall effect: Z = 4.14 (P<0.0001) 3.66%

8. Schächinger: NEJM, 2006 REPAIR MI – 1-Year Outcomes 204 Patients CP1250698-2 Placebo BMSC Placebo BMSC Pt (%) Pt (%) Death/MI P=0.02 Death/MI revasc Death/MI revasc P=0.01 Death/MI IRA revasc Death/MI IRA revasc P=0.08 Death/MI CHF hospitalization Death/MI CHF hospitalization P=0.006

9. The Rigorous Scrutiny of Large Trials Pexilizumab Post PPCI COMMA Trial 814 Patients 90-Day Mortality Infarct size – no difference Placebo Pexilizumab Placebo Pexilizumab Granger: Circ, 2003 Pt (%) Pt (%) P=0.014 JAMA 2007 All- cause mortality APEX AMI 5,745 Patients 30-Day Mortality CP1250698-1

10. Mesenchymal Stem Cell Therapy Post MI CP1264711-4 Hare J: ACC, 2007 RC trial 34 pt MSC 19 pt Placebo No change in EF Adverse arrhythmic events Adverse arrhythmic events Mean no. of rehosp – 0.26 MSC – 0.37 placebo 6-month outcomes Pt (%) Pt (%)

11. Evaluation of Antiarrhythmic Therapy Using Holter Monitoring Morganroth: AJC, 1988 CP1264989-1 “Ventricular arrhythmias undergo a high degree of spontaneous variability” >75% PVC frequency   90% NSVT frequency  Definition of a therapeutic drug effect

12. 8-9 Avg VPDs for 3 consecutive days (000s) Morganroth: AJC, 1988 Mean Extent of Variability in PVCs CP1265174-2 3-day monitoring periods 3 mo apart 3-day monitoring periods 3 mo apart 10-11 12-1 2-34-56-78-9 10-11 12-1 2-34-56-7 9-10 11-12 1-23-45-67-8 9-10 11-12 1-23-45-67-8 am pm am Scans 1/77 4/77 7/77 Scans 1/77 4/77 7/77

13. Improved LV function/ perfusion Clinical trials Mechanisms unexplained Regeneration – unlikely Mechanisms unexplained Regeneration – unlikely CP1237887-1 Murry: JACC, 2006 FrustratingEncouraging Opportunity for basic mechanistic research Predominance of benefit Current Status: Discouraging or Reality?

14. Are clinical trials premature since basic questions are unresolved ? Aspirin Statins ACE-inhibitors Aldosterone antagonists Aspirin Statins ACE-inhibitors Aldosterone antagonists CP1214543-3 Trials provide some answers to preconceived hypotheses but also generate new questions..

15. Power Calculations – 5-Year Death/CHF Sample Sizes 80% Power: 5-Year Death/CHF CP1182813-3 No. of cases/ group 50 Magnitude of benefit (% reduction) 30 15 10- and 30-day survivors 5-year Death/ CHF – 40% VALIANT Trial Treatment Arm

16. Use of Stem Cells for Cardiac Repair Tip of iceberg Ongoing specific questions? Which patients-age/ comorbities What cells, in what numbers and in which conditions Methods and sites of isolation, expression and delivery What cells, in what numbers and in which conditions Methods and sites of isolation, expression and delivery Clinical impact Mechanisms of benefit Early Late Safety Cells Adjunctive cytokine therapies Not yet ready for clinical application outside a research/trial setting Timing of administration CP1264606-1

17. Morphology/Histology Microarray gene expression Molecular imaging Tissue Hierarchial Endpoints in Trials LV function Remodeling Infarct size Electro- physiologic milieu Biomarkers Proteomics Cell tracking Mortality/CHF Perfusion/metabolism Gersh and Simari: Nature CV Med, 2006 CP1264606-4 The pivotal rate of imaging

18. CP1177150-2 Milrinone in Severe CHF 1,088 Patients PROMISE Trial Cumulative Survival (All Pt) Placebo Milrinone Placebo Milrinone Survival probability Milrinone – actions  intracellular cyclic AMP  Improved contractility Packer: NEJM, 1991 Month of study Cumulative Survival (Pt with NYHA Class IV) Survival probability Month of study  28% 53%  P=0.038

19. Cell Repair Therapy – the Future Enthusiasm and allure ahead of the science? Lessons to be learned from developments in angiogenesis and gene therapy Close collaboration between “bench and bedside” Use of control experiments and blinding Flourish Founder ? CP1237887-7

20. Stone age man MI vulnerable Cardiac Cell Repair The Magnitude of the Task Mortality would have little impact on survival of the species CP1244912-1 Leapfrog 15,000 yr of evolutionary biology Leapfrog 15,000 yr of evolutionary biology

21. Stem Cells Applications and Mechanisms Myocyte regeneration Angiogenesis Inflammation  Apoptosis Angiogenesis Inflammation  Apoptosis Autocrine Paracrine Endocrine ? Stem cells * Vectors * Vectors * Vectors * Vectors * Vectors for delivery of drugs, growth factors, gene therapy Gene transduction Gene transduction CP1236917-5 Modification

22. Louis Pasteur 1822-1895 Keep your enthusiasm but let verification be it,s constant companion CP1176240-1

24. CP1216048-2 “If you are not confused by this – you are not thinking clearly.” Pogo

25. CP1094005-1 “It is common sense to make a method and try it; if it fails admit it frankly and try another. But above all, try something.” Address at University, May 22, 1932 Franklin D. Roosevelt Address at Oglethorpe University, May 22, 1932 Franklin D. Roosevelt

26. CP1181571-1 Prometheus Bound Paul Rubens Philadelphia Museum of Art

27. *No protocol angiography Pt (%) TrialF-U (mo) BOOST (n=60)18 Janssens (n=67) 4 ASTAMI (n=100)12 REPAIR MI (n=204)12 TrialF-U (mo) BOOST (n=60)18 Janssens (n=67) 4 ASTAMI (n=100)12 REPAIR MI (n=204)12 Clinical Events in BMSC Trials CP1266653-1 50 40 30 20 10 0 Treated Arm Placebo Arm * *

28. Cardiac Cell Repair Therapy Can evolve into all tissues Electrophysiologic integration Immune responses Social/ethical issues Can evolve into all tissues Electrophysiologic integration Immune responses Social/ethical issues Embryonic stem cells Can be isolated and expressed Stable engraftment Electrophysiologic coupling Current role in cardiac homeostasis and injury response – unknown Can be isolated and expressed Stable engraftment Electrophysiologic coupling Current role in cardiac homeostasis and injury response – unknown Resident cardiac progenitors Improve cardiac function/  infarct size Electrophysiologic integration? Cell regeneration? Paracrine effects? Improve cardiac function/  infarct size Electrophysiologic integration? Cell regeneration? Paracrine effects? BM cells Skeletal myoblasts EPCMSC CP1252593-9 Myocytes Tumors Myocytes Tumors

29. In Vivo Effects of Conditioned Medium Subjected To Hypoxia Gnecchi: Nature, 2005 (correspondence) CP1210424-1 Infarct size (% LV) Infarct Size Apoptotic index Apoptotic Index * * * *

30. CP1182450-2 “Surrogate end points in clinical trials Are we being misled?” Fleming and de Mets: Ann Intern Med, 1996 Surrogates Apparently logical Convenient May provide unexpected surprises Apparently logical Convenient May provide unexpected surprises “A surrogate does not necessarily a correlate make”

31. CP1213477-2 Profound Thoughts About the Future Dan QuayleDan Quayle Predictions are risky especially about the future Tony BlairTony Blair "I don't like making predictions, I never have and I never will!" (1997) Dan QuayleDan Quayle Predictions are risky especially about the future Tony BlairTony Blair "I don't like making predictions, I never have and I never will!" (1997)

32. Power Calculations (80% Probability) Mortality Differences 50 5-Year Mortality Trend No. pt/ group Recent MI (6-40 days) EF <0.35  HRV CP1196890-3 DINAMIT Trial 18% mortality No ICD benefit 2 ½ mean F-U 3015 Magnitude of reduction in mortality (%) 1977

33. Are large randomized trials needed? Are such trials premature? CP1188110-10