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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 • Balance of these = on/off • In a mature healthy adult, vasculature is relatively quiescent (little endothelial cell turnover) • Tumor cells alter balance = “angiogenic switch” NCI
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 150 100 Percent Control 50 0 -6 -8 -10 -12 Control 10 10 10 10 PNB-0718 (M) Cell number estimated by MTT Mean +/- SEM, n=8
100 control -8 75 -10 -12 Average of Five Counts at High Power 50 -14 25 0 control -8 -10 -12 -14 Concentration (-log M) Effect of an PNB-0718 on Endothelial Cell Migration Mean +/- SEM, n=8
Do AT4 Receptor ligands affect angiogenesis? • Aortic Ring Assay • Rat • Mouse • Disc Assay
CONTROL PNB-0718 Effects of an AT4 receptor antagonist on Angiogenesisin the Rat Aortic Ring Assay Rat Aortic Rings ( 1mm thick) imbedded in Matrigel & treated for 4 days with 10-12M PNB-0718
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
6000 5000 4000 Tumor Volume (mm3) 3000 mean +/- SEM, n=6 2000 Note: only 1/400 of pellet drug content is released per day 1000 0 0 10 20 30 40 Time (Days) PNB-0718 CONTROL Effects of PNB-0718 Treatment on Breast Cancer Growth In Vivo Control PNB-0718 .3 mg/pellet PNB-0718 .03 mg/pellet PNB-0718 .003 mg/pellet
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
2000 Control (n=7) PNB-0718 (n=6) 1500 1000 500 0 9 10 11 12 13 14 15 16 17 Inhibition of Melanoma In Vivo with Slow-Release Intramuscular Delivery Estimated Dose: 21 mg/kg/day Tumor Volume (mm^3) Time (Days) Mean =/- SEM
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 Control PNB-0718 (2mg/kg/day) PNB-0718 (0.2mg/kg/day) 2000 Tumor Volume (mm^3) 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 – 2.5x10 +SA cells injected into lateral tail vein of BALB/c mice 5 – 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 150 Tumor Control Uninjected Control Tumor Treated 100 ( Percent Control) Metastatic Burden 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
but that table is now empty. Effect of PNB-0718 on Human (MDA-231) Breast Cancer Cell Growth 125 Control -8 100 10 M -10 10 M 75 -12 10 M Percent Control 50 25 0 -8 -10 -12 Control 10 M 10 M 10 M PNB-0718 Concentration mean +/- SEM, n=6 This graph will plot data from
Effect of PNB-0718 on Murine Breast Cancer Migration 750000 500000 Area 250000 0 control -6 -10 -12 Concentration (-log M) +SA WAZ-2T Cells
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 Summary of Actions of AT4 Receptor Antagonists
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 AT4 Receptor? 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
Effect of PNB-0718 on Body Weight Control Treated