The point on antiangiogenic drugs Francesco Bandello, Francesca Menchini Department of Ophthalmology University of Udine

1. The point on antiangiogenic drugs Francesco Bandello, Francesca Menchini Department of Ophthalmology University of Udine, Italy European Frontiers in Ocular Pharmacology Catania, January 18, 2008

2. History of neovascular AMD therapy 2006 2005 ‘60s 2000 2004 Laser Lucentis Avastin Photodynamic therapy (PDT) with verteporfin Macugen

3. Introduction • Limits of conventional therapies for exudative AMD: • Efficacy limited to selected CNV subtypes • Absent (laser photocoagulation) or limited (PDT) selectivity on the healthy surrounding retina • Efficacy on already formed new vessels (do not prevent recurrences) • Need for new treatment modalities that target not only established new vessels but prevent new vessels growth • Recent improved knowledge of biomolecular mechanisms at the basis of CNV development

4. VEGF: historical note (1) • “Factor X“ • diffusible factor responsible for retinal and iris neovascularization in proliferative retinopathy Michaelson. Trans Ophthalmol Soc UK, 1948 • “Tumor vascular permeability factor” • First isolated from guinea pig ascites • 50.000 times more potent than histamine in increasing vascular permeability Senger et al. Science 1983

5. VEGF: historical note (2) • VEGF was cloned and expressed in 1989 • Recognized as a potent stimulator of endothelial cell growth in vitro and neovascularization in vivo Leung et al. Science 1989 Keck et al. Science 1989 Ferrara et al, Biochem Biophys Res Commun 1989 • Hypothesis that VEGF might play a role in the regulation of physiological and pathological growth of blood vessels

6. VEGF family • Collection of related protein isoforms derived from the same gene • VEGF 121 • VEGF 165 • VEGF 189 • VEGF-B • VEGF-C • VEGF-D • VEGF-E • Placenta growth factor VEGF-A

7. VEGF family • VEGF isoforms have different affinity to heparine, that translates into an ability to interact with heparan sulfate proteoglycan on cell surface and basement membrane Houck et al. J Biol Chem 1992

8. VEGF isoforms • VEGF 121, the smallest isoform, does not bind heparin and is freely soluble • The largest isoform, VEGF 189, is found in cell surfaces and stored in basement membranes

9. Regulation of VEGF gene expression • Oxigen tension plays a key role in VEGF gene expression • Exposure to low PO2 in different circumstances induces VEGF m-RNA expression Semenza. Annu Rev Med, 2003 • Several major growth factors (TGF-α, TGF-β, IGF-I, PDGF) and inflammatory cytokines up-regulate VEGF m-RNA expression Frank et al. J Biol Chem 1995 Warren et al. J Biol Chem 1996 Cohen et al. J Biol Chem 1996

10. Role of VEGF in physiological conditions (1) • In vitro VEGF promotes growth of vascular endothelial cells derived from arteries, veins and lymphatics • In mice VEGF gene delection leads to early death in utero due to grossly deficient blood vessels Carmeliet et al. Nature 1996 • VEGF is essential in embryonic angiogenesis, including retinal vasculature • VEGF is required not only for proliferation but also for survival of endothelial cells Gerber et al. Development 1999 • VEGF is required for skeletal growth and endochondral bone formation

11. Role of VEGF in physiological conditions (2) • Embryogenesis • Ovulation • Bone growth • Wound healing • Collaterals in ischemic heart and limbs • Neurotrophysm

12. VEGF and the eye (1) • VEGF and its receptors are expressed in normal eye (particularly in the RPE) • Primary source of VEGF are neural cells, including ganglion, Muller and RPE cells Miller et al. Am J Pathol 1994 • VEGF is trophic for the choriocapillaris and required for the maintenance of the choriocapillaris fenestrae

13. Role of VEGF in pathologic conditions • VEGF m-RNA is expressed in the vast majority of human tumours • VEGF blockade with anti-VEGF antibodies decreases tumour perfusion, vascular volume, interstitial fluid pressure Kim et al. Nature 1993 • VEGF levels are elevated in ocular fluid in patients with diseases characterized by hyperpermeability and neovascularization

14. VEGF-A and angiogenesis Endothelial cells activation VEGF-A Basement membrane degradation Endothelial cells proliferation and migration Vessels formation, elongation, proliferation VEGF 165/120: 25:1 new vessels 2:1 normal New vessels-associated pericytes maturation Griffioen and Molema. PharmacolRev. 2000;52:237.

15. VEGF: proinflammatory effect • VEGF receptors are expressed on inflammatory cells1 • Inflammatory cells produce and release VEGF2-5 • Inflammatory cells are involved in BRB break and neovascularization5 Leucocytes recruitment 1. Barleon B et al. Blood. 1996;87:3336-3343. 2. Gaudry M et al. Blood. 1997;90:4153-4161. 3. Iijima K et al. Kidney Int. 1993;44:959-966. 4. Mohle R et al. ProcNatl Acad Sci USA. 1997;94:663-668 . 5. Ishida S et al. J Exp Med. 2003;198:483-489.

16. The angiogenic cascade Basement membrane Proliferation Proteolysis Migration Increased VEGF expression Other angiogenic growth factors e.g. bFGF Hypoxia Migrating endothelial cells form new blood vessels in formerly avascular space Vascular endothelial cell 1. Aiello LP et al. N Engl J Med. 1994;331:1480-1487. 2. Campochiaro PA et al. Mol Vis. 1999;5:34. 3. Dvorak HF et al. Am J Pathol. 1995;146:1029-1039. 4. Ferrara N. Recent Prog Horm Res. 2000;55:15-35. 5. Miller JW. Am J Pathol. 1997;151:13-23. 6. Miller JW et al. Am J Pathol. 1994;145:574-584. 7. Pe’er J et al. Lab Invest. 1995;72:638-645. 8. Spilsbury K et al. Am J Pathol. 2000;157:135-144.

17. Pathogenesis of CNV • Initial stimulus: • Choriocapillaris flow reduction • Lipofuscin accumulation • Oxidative stress • Bruch’s membrane alteration VEGF Ambati et al. Surv Ophthalmol. 2003 Witmer et al. Prog Retin Eye Res. 2003

18. VEGF164(165) blockage inhibits pathologic retinal neovascularization in animal model p<0,01 0,6 Murin VEGF 164 is equivalent to human VEGF165 0,4 Area (mm2) 0,2 0 Control Selective VEGF164 block Non selective VEGF block Ishida S et al, J Exp Med 2003

19. VEGF164(165) blockage has no effect on physiologic retinal revascularization in animal model Murin VEGF 164 is equivalent to human VEGF165 p<0,01 30 20 Area (mm2) 10 0 Control Selective VEGF164 block Non selective VEGF block Ishida S et al, J Exp Med 2003

20. Principal anti-VEGF compounds • Selective: • Macugen (Pegaptanib sodium) • Non selective: • Ranibizumab (Lucentis) • Bevacizumab (Avastin)

21. Macugen: Mechanism of action Basement membrane Proliferation Proteolysis Migration VEGF165 Other angiogenic growth factors Hypoxia VEGF Pegaptanib acts here • Binds to VEGF165 • Inhibits permeability • Inhibits neovascularization Vascular endothelial cell 1. Ambati J et al. Surv Ophthalmol. 2003;48:257-293. 2. Miller DW et al. In: Holz FG et al. Age-Related Macular Degeneration. Springer-Verlag; 2004. 3. Singerman LJ, Miller DG. Pharmacological treatments for AMD [Review of Ophthalmology Web site]. Available at: www.revophth.com/index.asp?page=1_422.htm. Accessed July 20, 2004. 4. Sorbera LA et al. Drugs of the Future. 2002;27:841-845.

22. Macugen • Aptamer, selective inhibitor of VEGF 165 • VISION (VEGF Inhibition Study In Ocular Neovascularization) • Efficacy for any lesion subtypes • Mean number of injection at 24 months: 16 • Therapeutic efficacy comparable to PDT • Mean visual loss at 2 year: 9-10 ETDRS letters • Visual improvement: 6%

23. Macugen: 24 months results Mean visual acuity 45% Relative benefitP<0.01* Visual acuity (letters) Year 2 Year 1 Weeks DC=discontinued, *Nominal P Value

24. Macugen: 24 months resultsVisual loss 30 26.5% MACUGEN 0.3 mg-DC (n=132) 67% relative difference P<0.05* MACUGEN 0.3 mg-0.3 mg (n=133) 20 % of patients with visual loss ≥15 letters 15.8% 10 0 54 60 66 72 78 84 90 96 102 End of year 1 End of year 2 Weeks DC=discontinued, *Nominal P Value

25. Efficacy: CNV subtypes Mean visual acuity change at 1 year Pegaptanib 0,3 mg (n=294) -4 -20 Sham injection (n=296) p<0,01 -3 -15 p<0,05 p<0,001 -2 -10 VA change in letters VA change in lines -1 -5 n=72 n=76 n=110 n=101 n=112 n=119 0 0 Predominantly classic Minimally classic Occult

26. V.I.S.I.O.N. Early lesions vs all patients All patients Early lesion Pegaptanib 0,3 mg (n=30) Pegaptanib 0,3 mg (n=294) 80 Usual care (n=35) Usual care (n=296) 80 70 57 55 60 47 % pazienti 33 40 23 20 20 20 6 2 0 0 <15 lettere ≥0 lettere ≥15 lettere <15 lettere ≥0 lettere ≥15 lettere

27. The bevacizumab revolution • Recombinant, humanized full-leght antibody, against all VEGF isoforms • FDA-approved for metastatic colorectal cancer in combination with 5FU (off-label) • Systemic dose 400 x intravitreal dose • Anecdotal reports (case series) on safety and efficacy • Anatomical and function improvement on the short-term • Different doses used (1 mg-2,5 mg) • Cost: 50-100$ vs 995$ (Macugen) and 2120$ (Lucentis)

28. The bevacizumab revolution • Toxicity studies done in rabbit, pig, cow, and monkey • No toxicity at standard dose • Some evidence of toxicity at 5x-10x standard dose (probably pH related) • Bevacizumab detected in 11.8% of patients’ serum following IVT injection • May be significant given 20-day half life in serum (compared with 0.5-day half life of ranibizumab)

29. Lucentis® and Avastin™: different and developed on parallel timelines Lucentis (48 kDa) (E. coli vector to mass produce) Insertion ofmurineanti-VEGF-Asequences into a humanFAb framework rhu Fab v1 Anti-VEGF-AMurine MAb(~150 kDa) Affinitymaturation (140x) Humanization Humanization Insertion of murineanti-VEGF-A sequences into a full-length human IgG Avastin (149 kDa) (CHO cell vector to mass produce)

30. Bevacizumab: CATT Trial • Comparison of AMD Treatment Trials (CATT) supported by National Eye Institute • 40 centers • 1200 patients (enrollment starting in January 2008) • 300 pts: monthly ranibizumab • 300 pts: monthly bevacizumab • 300 pts: PRN ranibizumab • 300 pts: PRN bevacizumab

31. Lucentis MARINA / ANCHOR = New Gold Standard

32. Ranibizumab: Mechanism of action Basement membrane Proliferation Proteolysis Migration VEGF110, VEGF121, VEGF165 Other angiogenic growth factors Hypoxia VEGF Ranibizumab acts here • Acts early in the cascade1 • Attacks disease in two ways • Inhibits permeability2 • Blocks angiogenesis3 • Penetrates retina to block all VEGF isoforms4 Vascular endothelial cell 1. Lowe J et al. Invest Ophthalmol Vis Sci. 2003; vol. 44. E-ARVO abstract 1828. 2. Gaudreault J et al. Invest Ophthalmol Vis Sci. 2003; vol. 44. E-ARVO abstract 3942. 3. Krzystolik MG et al. Arch Ophthalmol. 2002;120:338-346.4. Mordenti J et al. Toxicol Pathol. 1999;27:536-544.

33. MARINA and ANCHOR study design Investigator identifiespotential subjects Investigator identifiespotential patients Reading center confirmsangiographic eligibility Reading center confirmsangiographic eligibility Predominantly classic lesions (n=423) Minimally classic oroccult with no classic lesions (n=716) Randomized 1:1:1 Randomized 1:1:1 Visudyne Sham PDT Sham PDT Ranibizumab 0.5 mg(n=240) Sham (n=238) Ranibizumab 0.3 mg(n=238) Sham injection(n=143) Ranibizumab 0.3 mg(n=140) Ranibizumab 0.5 mg(n=140) MARINA ANCHOR Rosenfeld et al, N Engl J Med 2006; 355(14): 1419 Brown et al, N Engl J Med 2006; 355(14): 1432

34. MARINA and ANCHOR :Patients losing <15 letters from baseline Visudyne Sham Lucentis 0.5 mg MARINA ANCHOR Patients (%) *** *** *** *** 96.4 95 100 90 89.9 65.7 64.3 62 53 0 Month 12 Month 24 Month 12 Month 24 Rosenfeld et al, N Engl J Med 2006 Brown et al, N Engl J Med 2006 ***p<0.0001 vs. sham

35. MARINA and ANCHOR:Mean change in VA over time Lucentis 0.3 mg (n=238) MARINA Sham (n=238) Lucentis 0.5 mg (n=240) ANCHOR Visudyne (n=143) Lucentis 0.3 mg (n=238) Lucentis 0.5 mg (n=240) Rosenfeld et al, N Engl J Med 2006 Brown et al, N Engl J Med 2006 ***p<0.0001 vs. sham or Visudyne

36. MARINA and ANCHOR: Patients gaining ≥15 letters from baseline *** *** 33 34 4 5 Visudyne Sham Lucentis 0.5 mg Patients (%) 100 MARINA ANCHOR *** *** 41.0 40.3 5.6 6.3 0 Month 12 Month 24 Month 12 Month 24 Rosenfeld et al, N Engl J Med 2006; 355(14): 1419 Brown et al, N Engl J Med 2006; 355(14): 1432 ***p<0.0001 vs. sham

37. MARINA and ANCHOR : Maintain baseline VA or gain ≥0 letters Visudyne Sham Lucentis 0.5 mg Patients (%) MARINA ANCHOR 100 *** 85 *** *** 71 70 Data to be added when available from CSR 34 29 25 0 Month 12 Month 24 Month 12 Month 24 Rosenfeld et al, N Engl J Med 2006; 355(14): 1419 Brown et al, N Engl J Med 2006; 355(14): 1432 ***p<0.0001 vs. sham

38. MARINA and ANCHOR: Incidence of ocular serious AEs at 24 months MARINA ANCHOR

39. MARINA and ANCHOR: Arterial thromboembolic events (ATEs) ANCHOR MARINA

40. Efficacy conclusions • Lucentis (ranibizumab 0.5 mg dose), on average rapidly improves VA and sustains improvement over 24 months in patients with CNV lesions of all types: • over 70% of Lucentis-treated patients gained more than 0 letters in VA compared with baseline • over 33% of Lucentis-treated patients gained 15 letters or more in VA compared with baseline

41. Safety conclusions • Lucentis is well tolerated in studies for up to 24 months in duration • Low rate of ocular and non-ocular serious adverse events in both clinical trials • APTC ATEs occurred in a small number of patients in Lucentis and control groups across both trials

42. Lucentis posology:overview of rationale MARINA & ANCHOR  Monthly injections • Limits of monthly regimen: • Cost • Time consuming for pts and • medical personnel • Long-term side effects (?)

43. How can we use Anti-VEGFs most optimally in clinical practice? • It is easy to decide when to treat, but: • How do we determine when to stop treating? • Evidence? • Functional changes? • Anatomical changes?

44. How can we use Anti-VEGFs most optimally in clinical practice? • How do we determine when to stop treating? EVIDENCE • Subanalysis of MARINA and ANCHOR reveals that 30% of treated pts who did not gain vision at 4 months, at 12 months are 3 line gainers if treatment is continued

45. How can we use Anti-VEGFs most optimally in clinical practice? • How do we determine when to stop treating? FUNCTION • ETDRS refraction impractical in clinical practice • AMD vision variability (SRF, RPE detachment) • Vision gain and loss lags anatomy

46. How can we use Anti-VEGFs most optimally in clinical practice? • How do we determine when to stop treating? ANATOMY • How does anti-VEGFs work? • Anti-VEGF essentially dry out fluid (usually CNV remains perfused) • What is the most sensitive tool to assess the anatomy? • OCT? • FA?

47. PIER study design 0 0 1 2 3 4 5 6 7 8 9 10 11 12 23 24 Month Lucentis® 0.5 mg Sham Primaryendpoint Finalvisit PIER 12 months

48. PIER: Primary End PointMean change in VA over 12 months 10 5 0 -5 -10 -15 0 ETDRS letters -0.2 1 2 3 4 5 6 7 8 9 10 11 12 16.1 letterdifference*** -16.3 Month Sham (n=63) Lucentis® 0.5 mg (n=61) PIER 12 months ***p<0.0001 vs sham

49. Proportion sustaining gain* (%) Sustain the gain*: Monthly and quarterly regimen 100 • High rate of 40%of patients in PIER still sustained vision improvement with quarterly dosing suggesting that not all patients need monthly dosing 69 64 40 0 ANCHOR MARINA PIER *Visual acuity change from month 3 at each monitoring visits ≥-5 letters

50. Observation from phase III trials: a biphasic treatment effect Maintain the gain Mean change from baseline (letters) Gain Vision