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)

Per

cen

t C

on

tro

l

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

rag

e o

f F

ive

Co

un

tsat

Hig

h P

ow

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

Control

PNB-0718 .3 mg/pellet

PNB-0718 .03 mg/pellet

PNB-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)

Tu

mor

Vol

um

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)

Tu

mor

Volu

me (

mm

^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)

Tu

mor

Volu

me (

mm

^3)

Mean =/- SEM, N=8

12 13 14 15 16 170

1000

2000

3000Control

PNB-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

ontr

ol)

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

Pe

rce

nt

Co

ntr

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

Bo

dy

Wei

gh

t (g

r)

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

od

y F

at

Pathology

Effect of Chronic PNB-0718 on% Lean Body Mass in Mice

0

25

50

75LegendLegendLegend

Groups

% L

ean

Bo

dy

Mas

s *

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

Lea

n B

od

y M

ass

(gr)

New Question-are these compounds useful as anti-

obesity drugs?