Current Concepts In Therapeutic Angiogenesis

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 AHA Databank

3. Scope of Coronary Artery Disease • ~ 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

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

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

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

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

8. bFGF 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

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

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

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

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 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

14. Ischemic Angiogenesis 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 M. Cohen: J Mol Cell Cardiol; 1994

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

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 M. Fujita et al, Circulation; 1996

17. Ischemic Angiogenesis & Exogenous Growth Factors : Peripheral Models 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

18. Ischemic Angiogenesis & Exogenous Growth Factors : Peripheral Models 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

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

20. Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models 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

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

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

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

24. Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models 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

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

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

27. Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models 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

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

29. Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models 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

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

31. Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models 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

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

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

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

35. Ischemic Angiogenesis & Exogenous Growth Factors : Cardiac Models 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

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

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

39. Study Design • 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

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

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 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

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