1. Current Concepts In Therapeutic Angiogenesis C. Michael Gibson M.S., M.D.

2. Scope of Coronary Artery Disease ~ 15 - 20 million Americans have a history of MI, angina, or both Cardiovascular disease is the number one cause of death in the US ~ 500,000 deaths per year 1,500,000 new or recurrent MI’s per year ~ 15 - 20 million Americans have a history of MI, angina, or both Cardiovascular disease is the number one cause of death in the US ~ 500,000 deaths per year 1,500,000 new or recurrent MI’s per year AHA Databank

3. ~ 10 million Americans have angina ~ 350,000 new cases of angina diagnosed each year ~ 1.5 million coronary angiograms per year ~ 500,000 PTCA per year ~ 500,000 CABGs per year ~ 10 million Americans have angina ~ 350,000 new cases of angina diagnosed each year ~ 1.5 million coronary angiograms per year ~ 500,000 PTCA per year ~ 500,000 CABGs per year AHA Databank Scope of Coronary Artery Disease

4. )Therapeutic Angiogenesis Current Therapeutic Options Medical Antianginals Antiplatelet Agents Lipid Lowering Agents Anticoagulants Vasodilators Medical Antianginals Antiplatelet Agents Lipid Lowering Agents Anticoagulants Vasodilators Interventional PTCA CABG TMR/PMR Cardiac Transplantation PVD: Amputation Interventional PTCA CABG TMR/PMR Cardiac Transplantation PVD: Amputation New Therapeutic Option

5. Therapeutic Angiogenesis: Definitions Angiogenesis The formation of new capillary blood vessels from existing microvessels by sprouting, i.e. cellular outgrowth Vasculogenesis The formation of new blood vessels of all types from blood islands, i.e. committed stem cells, in early embryogenesis Growth Factor Polypeptide which acts as a regulator of cellular function, including proliferation, migration, differentiation, and survival/apoptosis Angiogenesis The formation of new capillary blood vessels from existing microvessels by sprouting, i.e. cellular outgrowth Vasculogenesis The formation of new blood vessels of all types from blood islands, i.e. committed stem cells, in early embryogenesis Growth Factor Polypeptide which acts as a regulator of cellular function, including proliferation, migration, differentiation, and survival/apoptosis

6. Angiogenic Growth Factors Basic fibroblast growth factor (bFGF) Acidic fibroblast growth factor (aFGF) Angiogenin Angiotropin Insulin-like growth factor Interleukin-8 Platelet activating factor (PAF) Basic fibroblast growth factor (bFGF) Acidic fibroblast growth factor (aFGF) Angiogenin Angiotropin Insulin-like growth factor Interleukin-8 Platelet activating factor (PAF) J. Battegay: J. Mol Med; 1995 Platelet-derived growth factor (PDGF) Proliferin Transforming growth factor-  Transforming growth factor-  Tumor necrosis factor-  Vascular endothelial growth factor (VEGF) Platelet-derived growth factor (PDGF) Proliferin Transforming growth factor-  Transforming growth factor-  Tumor necrosis factor-  Vascular endothelial growth factor (VEGF)

7. Basic Fibroblast Growth Factor (bFGF) 154 amino acids, MW 18 kD Additional higher MW forms exist Post-translational modification may yield a shorter form Present in almost all cells Present from embryogenesis to adult cells Released from extracellular sites by heparin and various proteolytic enzymes 154 amino acids, MW 18 kD Additional higher MW forms exist Post-translational modification may yield a shorter form Present in almost all cells Present from embryogenesis to adult cells Released from extracellular sites by heparin and various proteolytic enzymes

8. b FGF Receptors The cell receptor is a transmembrane tyrosine kinase Found on numerous cell types Receptor expression upregulated by injury (PTCA & ischemia) bFGF Binds to heparin which protects it from degradation Seperate binding sites for bFGF receptor and heparin The cell receptor is a transmembrane tyrosine kinase Found on numerous cell types Receptor expression upregulated by injury (PTCA & ischemia) bFGF Binds to heparin which protects it from degradation Seperate binding sites for bFGF receptor and heparin

9. Functions of Growth Factors Embryogenesis: Stimulates proliferation & differentiation of a variety of cells Wound Healing: Stimulates migration and proliferation of connective tissue Cytoprotection: CNS, vascular smooth muscle and endothelial cells Angiogenesis: Ischemic & Non-Ischemic Active at 0.1 to 1.0 ng/ml Embryogenesis: Stimulates proliferation & differentiation of a variety of cells Wound Healing: Stimulates migration and proliferation of connective tissue Cytoprotection: CNS, vascular smooth muscle and endothelial cells Angiogenesis: Ischemic & Non-Ischemic Active at 0.1 to 1.0 ng/ml

10. Augustin-Voss et al. Passage Number Endothelial Cell Migration (  m 72 hours) Bovine Endothelial Cell Migration

11. Proliferation % / 48 hr Augustin-Voss et al. Impact of bFGF on Bovine Endothelial Cell Proliferation

12. Functions of bFGF Non-ischemic Angiogenesis Promotes endothelial cell migration and tube formation Stimulates the production of collagenases and plasminogen activator necessary for basement membrane remodeling Non-ischemic Angiogenesis Promotes endothelial cell migration and tube formation Stimulates the production of collagenases and plasminogen activator necessary for basement membrane remodeling M. Klagsbrun: Progress Growth Factor Research; 1989

13. Ischemic Angiogenesis Endogenous bFGF production in the presence of ischemia Impact of exogenous bFGF on ischemic tissues Ischemic Angiogenesis Endogenous bFGF production in the presence of ischemia Impact of exogenous bFGF on ischemic tissues

14. Ischemic Angiogenesis M. Cohen: J Mol Cell Cardiol; 1994 Longitudinal Changes in Myocardial Basic Fibroblast Growth Factor (FGF-2) Activity Following Coronary Artery Ligation in the Dog Michael V. Cohen et al. Albert Einstein College of Medicine Longitudinal Changes in Myocardial Basic Fibroblast Growth Factor (FGF-2) Activity Following Coronary Artery Ligation in the Dog Michael V. Cohen et al. Albert Einstein College of Medicine

15. Demonstration of Endogenous Tissue Production of bFGF: Canine LAD Occlusion Model Time Following Canine LAD Occlusion Ischemic/Normal Myocardial bFGF ratio * * * * Cohen et al: J Mol Cell Cardiol; 1994 ) Endogenous bFGF production assayed in ischemic and adjacent normal cardiac tissue ) bFGF production rose as early as 2 hours ) Endogenous bFGF production assayed in ischemic and adjacent normal cardiac tissue ) bFGF production rose as early as 2 hours

16. Production of Growth Factors in Ischemia: Other Indirect Evidence ) Fujita et al measured the bFGF levels in the pericardial fluid of patients undergoing open heart surgery for unstable angina (CABG) versus those undergoing surgery for non-ischemic causes )Elevated bFGF levels found in the pericardial fluid of patients with unstable angina ) Fujita et al measured the bFGF levels in the pericardial fluid of patients undergoing open heart surgery for unstable angina (CABG) versus those undergoing surgery for non-ischemic causes )Elevated bFGF levels found in the pericardial fluid of patients with unstable angina M. Fujita et al, Circulation; 1996

17. Baffour et al. Rabbit model of hind limb ischemia (ligated main arteries in staged procedure over 2 weeks) Compared two weeks of IM bFGF to saline Results: bFGF groups had angiographically improved collaterals bFGF groups had greater capillary density (per mm and per muscle fiber) bFGF groups had greater muscle viability Baffour et al. Rabbit model of hind limb ischemia (ligated main arteries in staged procedure over 2 weeks) Compared two weeks of IM bFGF to saline Results: bFGF groups had angiographically improved collaterals bFGF groups had greater capillary density (per mm and per muscle fiber) bFGF groups had greater muscle viability R. Baffour et al, J Vasc Surg; 1992 Ischemic Angiogenesis & Exogenous Growth Factors : Peripheral Models

18. Yang et al Rat model of hind limb ischemia Compared one to four weeks of continuous intra- arterial bFGF (1  g/day) to heparinized saline control Demonstrated improvement in: Collateral blood flow by microspheres Capillary density Muscle performance by stimulated tension Yang et al Rat model of hind limb ischemia Compared one to four weeks of continuous intra- arterial bFGF (1  g/day) to heparinized saline control Demonstrated improvement in: Collateral blood flow by microspheres Capillary density Muscle performance by stimulated tension H. Yang et al, Circ. Res 1996 Ischemic Angiogenesis & Exogenous Growth Factors : Peripheral Models

19. Time Following Hind Limb Arterial Occlusion Collateral Flow (ml / min / 100g) * * * * H. Yang et al, Circ. Res.; 1996 Ischemic Angiogenesis & Exogenous Growth Factors : Peripheral Models

20. Uchida et al Occluded canine LAD model Compared intrapericardial bFGF, heparin, or both to saline control (drug given 30 minutes after occlusion) Measured: Ejection fraction Capillary density Infarct size Uchida et al Occluded canine LAD model Compared intrapericardial bFGF, heparin, or both to saline control (drug given 30 minutes after occlusion) Measured: Ejection fraction Capillary density Infarct size Y. Uchida et al, Am Heart J; 1995 Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models

21. Time Following Arterial Occlusion Ejection Fraction * * * * Y. Uchida: Am Heart J; 1995 Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models

22. Infarcted Weight (% of LV) ‡ ‡ * * Y. Uchida et al, Am Heart J; 1995 Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models

23. Capillary Number per 200  m ‡ ‡ Y. Uchida et al, Am Heart J; 1995 * * Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models

24. Ameroid constriction of porcine circumflex Compared intrapericardial bFGF and/or heparin to saline control Measured: Angiographic collaterals Microsphere blood flow MRI Cardiac function MRI collateral flow Ameroid constriction of porcine circumflex Compared intrapericardial bFGF and/or heparin to saline control Measured: Angiographic collaterals Microsphere blood flow MRI Cardiac function MRI collateral flow M. Simmons, personal communication; 1997 Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models

25. Circumflex Flow (ml / min / g) * * * Time Post Drug Administration M. Simmons, personal communication; 1997 Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models

26. Delayed Contrast Arrival Extent (%) * * M. Simmons, personal communication; 1997 * * * * Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models

27. Lazarous et al. Ameroid constriction of porcine circumflex Daily systemic bFGF (4 to 9 weeks) vs saline control Measured microsphere determinations of collateral blood flow Lazarous et al. Ameroid constriction of porcine circumflex Daily systemic bFGF (4 to 9 weeks) vs saline control Measured microsphere determinations of collateral blood flow D. Lazarous et al, Circulation; 1995 Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models

28. D. Lazarous et al, Circulation; 1995 Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models

29. Lazarous et al. Short Infusion Model Ameroid constriction of porcine circumflex Shorter duration of systemic bFGF (7 days) or VEGF to saline control Measured microsphere determinations of collateral blood flow Lazarous et al. Short Infusion Model Ameroid constriction of porcine circumflex Shorter duration of systemic bFGF (7 days) or VEGF to saline control Measured microsphere determinations of collateral blood flow D. Lazarous et al, Circulation; 1996 Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models

30. D. Lazarous: Circulation; 1996 Lazarous et al: Data Following 7 Day Infusions

31. Yanagisawa-Miwa et al. Acute occlusion of canine LAD Compared two bolus circumflex injections of bFGF vs saline Measured: LV Function Infarct size Histologic assessment of collateral growth Yanagisawa-Miwa et al. Acute occlusion of canine LAD Compared two bolus circumflex injections of bFGF vs saline Measured: LV Function Infarct size Histologic assessment of collateral growth A. Yanagisawa-Miwa et al, Science; 1992 Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models

32. Ejection Fraction * * A. Yanagisawa-Miwa: Science; 1992 Time post Infarction Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models

33. Infarct weight / LV weight (%) * * A. Yanagisawa-Miwa et al, Science; 1992 Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models

34. Capillary Number / Unit Area * * A. Yanagisawa-Miwa et al, Science; 1992 * * Arteriole Number / Unit Area Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models Control bFGF

35. Horrigan et al. Four hour balloon occlusion of canine LAD Compared two bolus LM injections of bFGF or vehicle Measured: Infarct size Histologic assessment of collateral growth Horrigan et al. Four hour balloon occlusion of canine LAD Compared two bolus LM injections of bFGF or vehicle Measured: Infarct size Histologic assessment of collateral growth M. Horrigan et al, Circulation; 1996 Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models

36. Infarct Size (% area at risk) * * M. Horrigan et al, Circulation; 1996 Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models

37. Chiron Study Multi-Center, Single-Blind, Dose Escalation, Safety and Tolerability Study of Recombinant Fibroblast Growth Factor-2 (rFGF-2) in Subjects with Advanced Coronary Artery Disease

38. Study Objectives Evaluate safety, tolerability and pharmacokinetics of short-term (20 min) intracoronary (IC) and intravenous (IV) single infusions of ascending doses of rFGF-2 Determine maximum tolerated IC and IV doses Measure preliminary efficacy data using nuclear stress imaging and cardiac MRI Evaluate safety, tolerability and pharmacokinetics of short-term (20 min) intracoronary (IC) and intravenous (IV) single infusions of ascending doses of rFGF-2 Determine maximum tolerated IC and IV doses Measure preliminary efficacy data using nuclear stress imaging and cardiac MRI

39. Study Agent Recombinant protein produced in yeast Differs from native human bFGF by only two amino acids Essentially identical angiogenic properties Enrollment Screening physical exam and labs Exercise or dipyridamole stress test with dual isotope imaging Cardiac MRI with cardiac function and collateral flow determinations Ophthalmologic exam Quality of Life questionnaire Study Agent Recombinant protein produced in yeast Differs from native human bFGF by only two amino acids Essentially identical angiogenic properties Enrollment Screening physical exam and labs Exercise or dipyridamole stress test with dual isotope imaging Cardiac MRI with cardiac function and collateral flow determinations Ophthalmologic exam Quality of Life questionnaire Study Design

40. Inclusion Criteria Severe CAD with inducible ischemia on stress test No optimal revascularization option Normal routine laboratory screening Willingness and ability to complete all components of the study and its follow-up Signed informed consent Severe CAD with inducible ischemia on stress test No optimal revascularization option Normal routine laboratory screening Willingness and ability to complete all components of the study and its follow-up Signed informed consent

41. Class IV CHF or EF < 20% MI < 3 months New or unstable angina < 3 wks CABG < 6 months PTCA or TMR < 6 months Significant arrhythmias Pacemaker or AICD LBBB Class IV CHF or EF < 20% MI < 3 months New or unstable angina < 3 wks CABG < 6 months PTCA or TMR < 6 months Significant arrhythmias Pacemaker or AICD LBBB Severe valvular heart disease Restrictive or hypertrophic cardiomyopathy Known AVMs TIA or CVA < 6 mths DM with end-organ damage Cr Cl < 80 ml/min or proteinuria Cancer within 10 years Severe valvular heart disease Restrictive or hypertrophic cardiomyopathy Known AVMs TIA or CVA < 6 mths DM with end-organ damage Cr Cl < 80 ml/min or proteinuria Cancer within 10 years Exclusion Criteria

42. Drug Administration Intracoronary Infusion Left and Right heart catheterizations 10 minute infusions of bFGF into the RCA and LM (or the major supplying bypass graft) Intravenous Infusion Left heart catheterization (if not done within 6 mths) 10 minute infusion into a peripheral vein Intracoronary Infusion Left and Right heart catheterizations 10 minute infusions of bFGF into the RCA and LM (or the major supplying bypass graft) Intravenous Infusion Left heart catheterization (if not done within 6 mths) 10 minute infusion into a peripheral vein

43. Follow-Up Protocol Clinic visits with routine blood tests at day 6, day 14 and at 1, 2, 6 and 12 months Repeat stress test and MRI scans at 1 month Subsequent exams only if indicated Repeat Eye exams at 2 and 12 months Repeat Quality of Life questionnaire at 2 months Clinic visits with routine blood tests at day 6, day 14 and at 1, 2, 6 and 12 months Repeat stress test and MRI scans at 1 month Subsequent exams only if indicated Repeat Eye exams at 2 and 12 months Repeat Quality of Life questionnaire at 2 months