1. AT 4 Receptor Ligands as Angiogenic, Anti-angiogenic, and Anti-cancer Agents How do you translate basic science into a clinically useful technology?

2. You’ve discovered a new membrane protein. What’s next? Does it do anything that is physiologically meaningful? If so, what does it do? Is it a receptor? If so, might it have relevance to clinical conditions? What tools do you need to study this system? If you can make them, might they also have therapeutic value?

3. How do you identify a function for a newly discovered receptor? Receptor Autoradiography of Tissue Slices

4. AT 4 Receptor Autoradiography-Guinea Pig Heart Aorta (Endothelial Cells?)

5. Why were we immediately interested in Endothelial Cells? Presence of AT 4 receptors on endothelial cells Importance of endothelial cells in regulating blood vessel growth (angiogenesis) Numerous pathologies with aberrant blood vessel growth (too much or too little) Cancer!

6. Why cancer ? Cancer responsible for 12% of deaths worldwide Overall survival 50-60% in developed world, 30-40% world wide Estimated global market (2007) $52 Billion 1 Largest growth segment is targeted therapies 1 (1) Roche Analyst Presentation

7. Angiogenesis NC I  Development, wound repair, tissue generation, and carcinogenesis  Regulated by activators and inhibitors Balance of these = on/off In a mature healthy adult, vasculature is relatively quiescent (little endothelial cell turnover) Tumor cells alter balance = “angiogenic switch”

8. Angiogenesis and Tumor Growth NCI  Uncontrolled growth  Require more nutrients, oxygen and removal of waste due to higher metabolic demand  New blood vessels fulfill all these requirements  Without angiogenesis Tumor growth is restricted Ability to metastasize is reduced

9. Tools/ Therapeutics What kind of tools do we need? Angiotensin IV analogs [ Val-Tyr-Ile-His-Pro-Phe] What part of the angiotensin IV molecule is critical for activity? What properties do we want our analogs to have? Specificity High affinity Bioavailability

10. AT 4 Receptor Ligand Library C-Met ligands ? ~300 Angiotensin IV analogs Agonists, partial agonists, antagonists Sub-picomolar to nanomolar affinities Peptidomimetics & peptides MW: 400-800

11. Angiogenesis requires that endothelial cells proliferate and migrate. Can AT 4 receptor ligands affect these processes?

12. Effect of PNB-0718 on Human Endothelial Cell Growth Control10 -6 10 -8 10 -10 10 -12 0 50 100 150 PNB-0718 (M) Percent Control Cell number estimated by MTT Mean +/- SEM, n=8

13. Effect of an PNB-0718 on Endothelial Cell Migration Mean +/- SEM, n=8

14. Do AT 4 Receptor ligands affect angiogenesis? Aortic Ring Assay Rat Mouse Disc Assay

15. CONTROLPNB-0718 Effects of an AT 4 receptor antagonist on Angiogenesis in the Rat Aortic Ring Assay Rat Aortic Rings ( 1mm thick) imbedded in Matrigel & treated for 4 days with 10 -12 M PNB-0718

16. Can a AT 4 Receptor Inhibit Tumor Angiogenesis and Growth?

17. Can a AT 4 Receptor Antagonist Inhibit Primary Tumor Growth In Vivo? Murine mammary cancer model Female BALB/c +SA WAZ-2T mice Cells injected into the thoracic mammary fat pad Simultaneous insertion of Elvax pellet containing drug into the fat pad

18. Control PNB-0718.3 mg/pellet PNB-0718.03 mg/pellet PNB-0718.003 mg/pellet Note: only 1/400 of pellet drug content is released per day mean +/- SEM, n=6 Effects of PNB-0718 Treatment on Breast Cancer Growth In Vivo PNB-0718 CONTROL

19. Tumor Vascularization Following Treatment with PNB-0718 +SA/ WAZ Murine Breast Cancer Tumor 32 Days of Treatment with PNB-0718 (.75  g/day/mouse) Arrows and Red Staining (Von Willibrand’s Factor) Indicate Blood Vessels

20. Can PNB-0718 Inhibit the Growth of other Primary Tumors? B16 Murine Melanoma IM Application of Elvax Pellet

21. Time (Days) Tumor Volume (mm^3) Inhibition of Melanoma In Vivo with Slow- Release Intramuscular Delivery Mean =/- SEM 91011121314151617 0 500 1000 1500 2000 Control (n=7) PNB-0718 (n=6) Estimated Dose: 21  g/kg/day

22. Can PNB-0718 Inhibit the Growth of an Already Established Tumor? B16 Murine Melanoma Tumor Established and Palpable after 13 Days Daily In Situ Application

23. Inhibition of Melanoma In Vivo following In Situ Injection Time (Days) Tumor Volume (mm^3) Mean =/- SEM, N=8 121314151617 0 1000 2000 3000 Control PNB-0718 (2mg/kg/day) PNB-0718 (0.2  g/kg/day) = Injection

24. Can PNB-0718 Inhibit the Development of Lung Metastases? +SA/WAZ-2T cells were injected into the tail vein Simultaneous im. Implantation of an Elvax pellet containing PNB-0718 7 weeks later lungs were weighed to determine tumor burden

25. 0 50 100 150 Tumor Control Uninjected Control Tumor Treated Treatment Group mean+/- SEM, n = 8 Weights normalized to mean of tumor control group Metastatic Burden ( Percent Control) Inhibition of Lung Metastases Metastatic tail vein assay –2.5x10 5 +SA cells injected into lateral tail vein of BALB/c mice –PNB-0718-containing Elvax pellets implanted intramuscularly into left and right Gluteus maximus. Dose=.75ug/day/mouse – Lungs removed 7 weeks following tumor cell injection

26. Why are AT 4 Receptor Antagonists so Effective as Anti-cancer Agents? Are they Anti-angiogenic?—YES Can AT 4 Receptor Antagonists Directly Effect Cancer Cells? Growth Migration Attachment

27. This graph will plot data from but that table is now empty. Effect of PNB-0718 on Human (MDA-231) Breast Cancer Cell Growth Control10 -8 M10 -10 M10 -12 M 0 25 50 75 100 125 Control 10 -8 M 10 -10 M 10 -12 M mean +/- SEM, n=6 PNB-0718 Concentration Percent Control

28. Effect of PNB-0718 on Murine Breast Cancer Migration control-6-10-12 0 250000 500000 750000 Concentration (-log M) Area +SA WAZ-2T Cells

29. Summary of Actions of AT 4 Receptor Antagonists Inhibit endothelial cell proliferation Inhibit endothelial cell migration Inhibit angiogenesis Inhibit primary tumor growth Generally effective against solid tumors Inhibit development of metastatic tumors Effective at very low doses

30. What Molecular Target would produce both Anti-angiogenic Effects and direct Anti- tumor Activity when inhibited? What is the Molecular Target of these Molecules? I.E. What is the AT 4 Receptor? c-Met Receptor for critical growth factor called Hepatocyte Growth Factor(HGF)

31. What is c-Met and Why is an Attactive Cancer Therapeutic Target? First described as an oncogene Many human cancers either have a c-Met mutation or over-express c-Met c-Met is, in large part, responsible for the ability of both endothelial and cancer cells to: Migrate Lose cell-to-cell adhesions (scattering) To survive in suspension Proliferate

32. Toxicity?

34. Effect of PNB-0718 on Body Weight ControlTreated

35. Mouse DEXA Scan

37. Pathology

40. New Question-are these compounds useful as anti- obesity drugs?