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AT4 Receptor Ligands as Angiogenic, Anti-angiogenic, and Anti-cancer Agents How do you translate basic science into a clinically useful technology? 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? How do you identify a function for a newly discovered receptor? Receptor Autoradiography of Tissue Slices AT4 Receptor Autoradiography-Guinea Pig Heart Aorta (Endothelial Cells?) Why were we immediately interested in Endothelial Cells? Presence of AT4 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! 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 Billion1 Largest growth segment is targeted therapies1 (1) Roche Analyst Presentation Angiogenesis Development, wound repair, tissue generation, and carcinogenesis Regulated by activators and inhibitors NC Angiogenesis and Tumor Growth 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 NCI 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 AT4 Receptor Ligand Library C-Met ligands ? ~300 Angiotensin IV analogs Agonists, partial agonists, antagonists Sub-picomolar to nanomolar affinities Peptidomimetics & peptides MW: 400-800 Angiogenesis requires that endothelial cells proliferate and migrate. Can AT4 receptor ligands affect these processes? Effect of PNB-0718 on Human Endothelial Cell Growth Percent Control 150 100 50 0 Control 10 -6 10 -8 10 -10 10 -12 PNB-0718 (M) Cell number estimated by MTT Mean +/- SEM, n=8 Average of Five Counts at High Power Effect of an PNB-0718 on Endothelial Cell Migration 100 control -8 -10 -12 -14 75 50 25 0 control -8 -10 -12 -14 Concentration (-log M) Mean +/- SEM, n=8 Do AT4 Receptor ligands affect angiogenesis? Aortic Ring Assay Rat Mouse Disc Assay Effects of an AT4 receptor antagonist on Angiogenesis in the Rat Aortic Ring Assay CONTROL PNB-0718 Rat Aortic Rings ( 1mm thick) imbedded in Matrigel & treated for 4 days with 10-12M PNB-0718 Can a AT4 Receptor Inhibit Tumor Angiogenesis and Growth? Can a AT4 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 Effects of PNB-0718 Treatment on Breast Cancer Growth In Vivo 6000 5000 Control 4000 PNB-0718 .3 mg/pellet PNB-0718 .03 mg/pellet 3000 PNB-0718 .003 mg/pellet 2000 1000 0 0 10 20 30 40 Time (Days) mean +/- SEM, n=6 Note: only 1/400 of pellet drug content is released per day PNB-0718 CONTROL Tumor Vascularization Following Treatment with PNB-0718 • +SA/ WAZ Murine Breast Cancer Tumor • 32 Days of Treatment with PNB-0718 (.75 mg/day/mouse) • Arrows and Red Staining (Von Willibrand’s Factor) Indicate Blood Vessels Can PNB-0718 Inhibit the Growth of other Primary Tumors? B16 Murine Melanoma IM Application of Elvax Pellet Inhibition of Melanoma In Vivo with SlowRelease Intramuscular Delivery Tumor Volume (mm^3) 2000 Control (n=7) PNB-0718 (n=6) 1500 Estimated Dose: 21 mg/kg/day 1000 500 0 9 10 11 12 13 14 15 16 Time (Days) Mean =/- SEM 17 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 Inhibition of Melanoma In Vivo following In Situ Injection 3000 Tumor Volume (mm^3) Control 2000 PNB-0718 (2mg/kg/day) PNB-0718 (0.2mg/kg/day) 1000 0 12 13 14 15 16 17 = Injection Time (Days) Mean =/- SEM, N=8 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 Inhibition of Lung Metastases • Metastatic tail vein assay – 2.5x105+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 Metastatic Burden ( Percent Control) 150 Tumor Control Uninjected Control Tumor Treated 100 50 mean+/- SEM, n = 8 0 Treatment Group Weights normalized to mean of tumor control group Why are AT4 Receptor Antagonists so Effective as Anti-cancer Agents? Are they Anti-angiogenic?—YES Can AT4 Receptor Antagonists Directly Effect Cancer Cells? Growth Migration Attachment Effect of PNB-0718 on Human (MDA-231) Breast Cancer Cell Growth 125 Control Percent Control 100 75 10 -8 10 -10 M 10 -12 M M 50 25 0 Control 10 -8 M 10 -10 M 10 -12 M PNB-0718 Concentration mean +/- SEM, n=6 Effect of PNB-0718 on Murine Breast Cancer Migration Area 750000 500000 250000 0 control -6 -10 -12 Concentration (-log M) +SA WAZ-2T Cells Summary of Actions of AT4 Receptor Antagonists Inhibit endothelial cell proliferation Inhibit primary tumor growth Inhibit endothelial cell migration Generally effective against solid tumors Inhibit angiogenesis Inhibit development of metastatic tumors Effective at very low doses What is the Molecular Target of these Molecules? I.E. What is the AT4 Receptor? What Molecular Target would produce both Anti-angiogenic Effects and direct Antitumor Activity when inhibited? c-Met Receptor for critical growth factor called Hepatocyte Growth Factor(HGF) 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 Toxicity? Effect of Chronic PNB-0718 on Body Weight in Mice Body Weight (gr) 50 Control Treated 40 30 20 10 0 0 25 50 75 100 125 Days 2.1 mg/kg/48 hours; i.m. injection Effect of PNB-0718 on Body Weight Control Treated Mouse DEXA Scan Effect of Chronic PNB-0718 on % Body Fat in Mice % Body Fat 50 40 Untreated Control Injection Control Treated * 30 20 10 0 Group Average dose: 8.3 mg/kg/day Dosing schedule: IM every 48 hrs mean +/- SEM, n= 6-11 * p<.0001; treated represents 22.6% decrease from control Pathology Effect of Chronic PNB-0718 on % Lean Body Mass in Mice % Lean Body Mass 75 * 50 Legend Legend Legend 25 0 Groups Average dose: 8.3 mg/kg/day Dosing schedule: IM every 48 hrs mean +/- SEM, n= 6-11 treated * p<.0001; represents 16.9% increase from control Effect of Chronic PNB-0718 on Absolute Lean Body Mass in Mice Lean Body Mass (gr) 30 Untreated Treated Control Treatment 20 10 0 Untreated Treated Control Treatment Group New Question-are these compounds useful as antiobesity drugs?