Transcript AT4 Receptor Ligands as Angiogenic, Anti

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?