Anti-angiogenic therapy and other solid tumours Alfredo Zurlo Roche, Basel, Switzerland.
AT 4 Receptor Ligands as Angiogenic, Anti-angiogenic, and Anti-cancer Agents
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Transcript of AT 4 Receptor Ligands as Angiogenic, Anti-angiogenic, and Anti-cancer Agents
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
NCI
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”
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
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
Control 10-6 10-8 10-10 10-12 0
50
100
150
PNB-0718 (M)
Perc
ent C
ontr
ol
Cell number estimated by MTT
Mean +/- SEM, n=8
Effect of an PNB-0718 on Endothelial Cell Migration
control -8 -10 -12 -140
25
50
75
100control-8-10-12-14
Concentration (-log M)
Ave
rage
of F
ive
Cou
nts
at H
igh
Pow
er
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 Angiogenesis in 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 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
ControlPNB-0718 .3 mg/pelletPNB-0718 .03 mg/pelletPNB-0718 .003 mg/pellet
Note: only 1/400 of pellet drugcontent is released per day
mean +/- SEM, n=6
Effects of PNB-0718 Treatment on Breast Cancer Growth In Vivo
0 10 20 30 400
1000
2000
3000
4000
5000
6000
Time (Days)
Tum
or V
olum
e ( m
m3)
PNB-0718 CONTROL
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
Can PNB-0718 Inhibit the Growth of other Primary Tumors?
B16 Murine Melanoma
IM Application of Elvax Pellet
Time (Days)
Tum
or V
olum
e (m
m^
3)Inhibition of Melanoma In Vivo with Slow-
Release Intramuscular Delivery
Mean =/- SEM
9 10 11 12 13 14 15 16 170
500
1000
1500
2000Control (n=7)PNB-0718 (n=6)
Estimated Dose: 21 g/kg/day
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
Time (Days)
Tum
or V
olum
e (m
m^
3)
Mean =/- SEM, N=8
12 13 14 15 16 170
1000
2000
3000ControlPNB-0718 (2mg/kg/day)PNB-0718 (0.2g/kg/day)
= Injection
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
0
50
100
150Tumor ControlUninjected ControlTumor Treated
Treatment Group
mean+/- SEM, n = 8
Weights normalized to meanof tumor control group
Met
asta
tic B
urde
n( P
erce
nt C
ontro
l)
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
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
Control 10 -8 M 10 -10 M 10 -12 M0
25
50
75
100
125Control
10 -8 M
10 -10 M
10 -12 M
mean +/- SEM, n=6PNB-0718 Concentration
Perc
ent C
ontr
ol
Effect of PNB-0718 on Murine Breast Cancer Migration
control -6 -10 -120
250000
500000
750000
Concentration (-log M)
Are
a
+SA WAZ-2T Cells
Summary of Actions of AT4 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
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
Toxicity?
Effect of Chronic PNB-0718 onBody Weight in Mice
0 25 50 75 100 1250
10
20
30
40
50ControlTreated
Days
Bod
y W
eigh
t (gr
)
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
0
10
20
30
40
50Untreated ControlInjection ControlTreated
mean +/- SEM, n= 6-11
*
* p<.0001; treatedrepresents 22.6%decrease from control
Average dose: 8.3 g/kg/dayDosing schedule: IM every 48 hrs
Group
% B
ody
Fat
Pathology
Effect of Chronic PNB-0718 on% Lean Body Mass in Mice
0
25
50
75LegendLegendLegend
Groups
% L
ean
Bod
y M
ass *
mean +/- SEM, n= 6-11
* p<.0001; treatedrepresents 16.9%increase from control
Average dose: 8.3 g/kg/dayDosing schedule: IM every 48 hrs
Effect of Chronic PNB-0718 onAbsolute Lean Body Mass in
Mice
Untreated Treated ControlTreatment0
10
20
30UntreatedTreated ControlTreatment
Group
Lean
Bod
y M
ass
(gr)
New Question-are these compounds useful as anti-
obesity drugs?