pharmacokinetic characterization of angiotensin iv analogs with therapeutic potential for cancer and...

THESIS PROPOSAL DEFENSE

ALENE MCCOYTHESIS MENTOR: JOSEPH HARDING

APRIL 30, 2009

“Pharmacokinetic characterization of angiotensin IV analogs with therapeutic potential for cancer and

dementia.”

Overview

Background Cancer and Dementia Angiotensin IV and the AT4 receptor

Cancer Studies PNB-0718 Anti-Cancer Activity

PNB-0718 Pharmacokinetics PNB-0405 Anti-Cancer Activity

PNB-0405 Pharmacokinetics

Cognition Studies PNB-0408 Cognitive-Enhancing Activity

Plans for Future Studies

Age-related DementiaAge-related Dementia CancerCancer

Affects 1% at age 60, 35% at age 90.

Alzheimer’s – 7th leading cause of death in US.

No effective treatment available.

1 in 2 Men and 1 in 3 Women develop cancer.

2nd leading cause of death in US.

65% Survival rate. (51% survival in 1976.)

Background

Is there a molecular target for both, age related dementia

and cancer?

Angiotensins

Binds to AT1 & AT2:Blood pressureVasoconstriction

Binds to AT4:CognitionMemory

The AT4 Receptor

AT4 receptor discovered in 1992

Angiotensin IV binding

Binding sites widely distributed

Angiotensin IV–AT4 Activity in Cancer

AT4 Receptor Subtype Binding : Guinea Pig Heart

Aorta (Endothelial

Cells)

Effect of AT4 Antagonists on Human Endothelial Cell Growth

Control 10-6

10-8

10-10

10-12

0

50

100

150

PNB-0718 (M)

Ab

so

rba

nce

as

Pe

rcen

t o

f C

on

tro

l

HUVEC Cells

Cell number estimated by MTT

Mean +/- SEM, n=8

Effect of AT4 Antagonists on Human 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

HUVEC Cells

Cell Number Estimated by Visual Count

Mean +/- SEM, n=8

Inhibition of Angiogenesis in the Mouse Aortic Ring Assay

Con

trol

Tre

ated

0

2

4

6

Control PNB-07180.1 nM

An

giog

enes

is (area)

n=6n=8

+/- SEM* p=0.012

*

Angiogenesis in Cancer

Image obtained from roswellpark.org http://www.roswellpark.org/files/1_2_1/cancer_101/Lesson4/Lesson_4.pdf

Reduction of tumor growth.

PNB-0718 Inhibits the Growth of Established Melanoma Tumors

Time (Days)

Tu

mor

Volu

me (

mm

3)

Mean +/- SEM

N=8

12 13 14 15 16 170

1000

2000

3000

Control

PNB-0718 (2mg/kg/day)

PNB-0718 (0.2g/kg/day)

= Injection

AT4 Antagonists exhibit potent anti-cancer activity

Activity is much greater than for other anti-angiogenics

Direct effects on cancer cells?

Other known molecular targets that behave similarly?

c-Met

Receptor for hepatocyte growth factor (HGF).

Induces cellular proliferation and scattering.

Plays a major role in angiogenesis.

Over-expressed or mutated in many cancers.

HGF and Angiotensin IV share partial homology.

Are AT4 receptor ligands c-Met ligands?

Modulate c-Met autophosphorylation

Modulate Gab-1 association with c-Met

Modulate Gab-1 phosphorylation

Modulate HGF-dependent cell proliferation

Modulate HGF-dependant cell scattering

Blocked HGF binding to c-Met

Development of AT4 Ligand Molecules

Name Structure Activity

Angiotensin IV Val-Tyr-Ile-His-Pro-Phe +

PNB-0719 Nle-Tyr-Ile-His-Pro-Phe ++

PNB-0718“Norleual”

Nle-Tyr-Leu--His-Pro-Phe - -

PNB-0405 d-Nle-Tyr-Ile-CH-CH-CH-CH-CH-CH-NH2

OH-

PNB-0408 Hexanoic acid-Tyr-Ile-CH-CH-CH-CH-CH-CH-NH2

OH++

Development of AT4 Ligand Molecules

Name Structure Activity

Angiotensin IV Val-Tyr-Ile-His-Pro-Phe +

PNB-0719 Nle-Tyr-Ile-His-Pro-Phe ++

PNB-0718“Norleual”

Nle-Tyr-Leu--His-Pro-Phe - -

PNB-0405 d-Nle-Tyr-Ile-CH-CH-CH-CH-CH-CH-NH2

OH-

PNB-0408 Hexanoic acid-Tyr-Ile-CH-CH-CH-CH-CH-CH-NH2

OH++

PNB-0718 inhibits HGF-induced proliferation.

PNB-0718 (“Norleual”) inhibits c-Met signaling.

PNB-0718 reduces tumor growth.

Pharmacokinetic Characterization

• Absorption

• Distribution

• Metabolism

• Elimination

Modeling of Physicochemical Properties

ADMET Predictor --Absorption, Distribution, Metabolism, Elimination, Toxicity

•Physico-chemical properties:

pKa

Solubility

Gut permeability

Plasma protein binding

•Toxicity

Carcinogenicity

Specific Receptors

•Metabolism

Enzymatic reactions

Modeling of Physicochemical Properties of PNB-0718

Physicochemical Property Predicted Value Interpretation

logP 0.68 slightly hydrophobic

Fraction Unbound (to plasma proteins)

13.32 mostly bound to plasma proteins

Effective Human Jejunal Permeability

0.28

not well absorbed in the gut (oral administration)

Average Intestinal Permeability 0.08

Blood Stability Study

Pharmacokinetic Characterization

Adult male Sprague Dawley rats

Jugular vein catheter

Metabolic cages

Blood and urine collected

Analyzed by LC-MS

HPLC-MS System

Column- Reverse Phase C183µm

Shimadzu High-Sensitivity Liquid Chromatograph Mass Spectrometer Single Quadrupole

Shimadzu LC Solution Software

Pharmacokinetic Data

89 mg/kg IV bolus doses

PNB-0719 internal standard

Elimination Half Life

Area Under the Curve (Extent of Exposure)

Volume of Distribution (Extent of Distribution into

Tissues)

Clearance (Rate of Removal from Blood)

Intravenous Bolus Dose Study

Intravenous Bolus Dose Study

PNB-0718 89 mg/kg

Pharmacokinetic Parameter Mean ± SEM

AUC0-∞ (min.ng/mL) 1,863,051.1 ± 530,304.7

Vd (L/kg) 1.8 ± 0.4

Cp0 (ng/mL) 827,803.2 ± 247,039.1

t1/2 (min) 28.0 ± 10.8

KE (min-1) 0.044 ± 0.021

CL (L/min/kg) 0.057 ± 0.013

Development of AT4 Ligand Molecules

Name Structure Activity

Angiotensin IV Val-Tyr-Ile-His-Pro-Phe +

PNB-0719 Nle-Tyr-Ile-His-Pro-Phe ++

PNB-0718“Norleual”

Nle-Tyr-Leu--His-Pro-Phe - -

PNB-0405 d-Nle-Tyr-Ile-CH-CH-CH-CH-CH-CH-NH2

OH-

PNB-0408 Hexanoic acid-Tyr-Ile-CH-CH-CH-CH-CH-CH-NH2

OH++

Development of AT4 Ligand Molecules

Name Structure Activity

Angiotensin IV Val-Tyr-Ile-His-Pro-Phe +

PNB-0719 Nle-Tyr-Ile-His-Pro-Phe ++

PNB-0718“Norleual”

Nle-Tyr-Leu--His-Pro-Phe - -

PNB-0405 d-Nle-Tyr-Ile-CH-CH-CH-CH-CH-CH-NH2

OH-

PNB-0408 Hexanoic acid-Tyr-Ile-CH-CH-CH-CH-CH-CH-NH2

OH++

PNB-0405 inhibits HGF-induced migration.

-150-125-100

-75-50-25

0255075

100125150

Control10 ng/ml HGF

HGF+10-10M PNB-0405

HGF+10-11M PNB-0405

HGF+10-10M PNB-0412

HGF+10-11M PNB-0412

HGF+10-10M PNB-0414

HGF+10-11M PNB-0413

HGF+10-10M PNB-0413

HGF+10-11M PNB-0414

n=6Mean +/- SEM

Flu

ore

scen

ce(%

HG

F R

esp

on

se)

PNB-0405 inhibits HGF-induced migration.

-150-125-100

-75-50-25

0255075

100125150

Control10 ng/ml HGF

HGF+10-10M PNB-0405

HGF+10-11M PNB-0405

HGF+10-10M PNB-0412

HGF+10-11M PNB-0412

HGF+10-10M PNB-0414

HGF+10-11M PNB-0413

HGF+10-10M PNB-0413

HGF+10-11M PNB-0414

n=6Mean +/- SEM

Flu

ore

scen

ce(%

HG

F R

esp

on

se)

PNB-0405 inhibits HGF-induced scattering.

PNB-0405 inhibits c-Met signaling.

0718

0405

0407

0410

0413

0416

0415

0408

0409

0406

0411

0412

-100

0

100

200

300

HGF

Compound (1pM)

Per

cen

t H

GF

Eff

ect

c-Met-Gab-1 Association in HEK Cells (Immunoprecipitation)

PNB-0405 inhibits c-Met signaling.

0718

0405

0407

0410

0413

0416

0415

0408

0409

0406

0411

0412

-100

0

100

200

300

HGF

Compound (1pM)

Per

cen

t H

GF

Eff

ect

c-Met-Gab-1 Association in HEK Cells (Immunoprecipitation)

PNB-0405 reduces tumor growth.

PNB-0405 Inhibits MurineBreast Cancer Tumor

Growth

0 10 20 300

100

200

300

400ControlTreated

+SA Murine Breast Cancer CellsPNB-0405 delivered in ElvaxPellets- estimated deliveryrate=0.7g/day Mean+/- SEM,N=8

Days

Tu

mo

r V

olu

me

(mm

3)

Pharmacokinetic Characterization

• Absorption

• Distribution

• Metabolism

• Elimination

Modeling of Physicochemical Properties

Physicochemical Property Predicted Value Interpretation

logP 1.45 hydrophobic

Fraction Unbound (to plasma proteins)

42.68 ~50% bound to plasma proteins

Effective Human Jejunal Permeability

1.53

moderately well absorbed in the gut (oral administration)

Average Intestinal Permeability 0.39

Blood Stability Study

Pharmacokinetic Data

24 mg/kg IV bolus doses

PNB-0413 internal standard

Elimination Half Life

Area Under the Curve (Extent of Exposure)

Volume of Distribution (Extent of Distribution into Tissues)

Clearance (Rate of Removal from Blood)

Intravenous Bolus Dose Study

Intravenous Bolus Dose Study

PNB-0405 24 mg/kg

Pharmacokinetic Parameter Mean ± SEM

AUC0-∞ (min.ng/mL) 692.5 ± 293.2

Vd (L/kg) 104,186.8 ± 65034.3

Cp0 (ng/mL) 68.2 ± 32.2

t1/2 (min) 1012.0 ± 391.4

KE (min-1) 0.001 ± 0.0002

CL (L/min/kg) 58.3 ± 15.6

Pharmacokinetic Data

24 mg/kg Subcutaneous bolus doses

PNB-0413 internal standard

Elimination Half Life

Area Under the Curve (Extent of Exposure)

Volume of Distribution (Extent of Distribution into Tissues)

Clearance (Rate of Removal from Blood)

Subcutaneous Bolus Dose Study

Subcutaneous Bolus Dose Study

PNB-0405 24 mg/kg

Pharmacokinetic Parameter Mean ± SEM

AUC0-∞ (min.ng/mL) 178.0 ± 41.7

Vd (L/kg) 84,850.2 ± 30,560.7

Cp0 (ng/mL) 0.83 ± 0.24

t1/2 (min) 376.2 ± 132.9

KE (min-1) 0.003 ± 0.0007

CL (L/min/kg) 172.8 ± 43.6

F (% Bioavailablility) 25.7

Angiotensin IV–AT4 Activity in Cognition

AT4 Receptor Subtype Binding : Guinea Pig Neocortex and Hippocampus

Angiotensin IV reverses cognitive dysfunction in many models:

Scopolamine Muscarinic cholinergic receptor blockade

Cerebral Arterial Occlusion Cerebral ischemia model

Kainic acid Lesion of Hippocampus Eliminates ~85% of neurons in hippocampus

Perforant Pathway Cut Blocks hippocampal input ~65%

Development of AT4 Ligand Molecules

Name Structure Activity

Angiotensin IV Val-Tyr-Ile-His-Pro-Phe +

PNB-0719 Nle-Tyr-Ile-His-Pro-Phe ++

PNB-0718“Norleual”

Nle-Tyr-Leu--His-Pro-Phe - -

PNB-0405 d-Nle-Tyr-Ile-CH-CH-CH-CH-CH-CH-NH2

OH-

PNB-0408 Hexanoic acid-Tyr-Ile-CH-CH-CH-CH-CH-CH-NH2

OH++

Development of AT4 Ligand Molecules

Name Structure Activity

Angiotensin IV Val-Tyr-Ile-His-Pro-Phe +

PNB-0719 Nle-Tyr-Ile-His-Pro-Phe ++

PNB-0718“Norleual”

Nle-Tyr-Leu--His-Pro-Phe - -

PNB-0405 d-Nle-Tyr-Ile-CH-CH-CH-CH-CH-CH-NH2

OH-

PNB-0408 Hexanoic acid-Tyr-Ile-CH-CH-CH-CH-CH-CH-NH2

OH++

PNB-0408 induces c-Met signaling.

0718

0405

0407

0410

0413

0416

0415

0408

0409

0406

0411

0412

-100

0

100

200

300

HGF

Compound (1pM)

Per

cen

t H

GF

Eff

ect

c-Met-Gab-1 Association in HEK Cells (Immunoprecipitation)

PNB-0408 induces c-Met signaling.

0718

0405

0407

0410

0413

0416

0415

0408

0409

0406

0411

0412

-100

0

100

200

300

HGF

Compound (1pM)

Per

cen

t H

GF

Eff

ect

c-Met-Gab-1 Association in HEK Cells (Immunoprecipitation)

Hippocampal long-term potentiation (LTP) is the most prevalent model for synaptic plasticity

In-Vitro Model for Synaptic Plasticity

stimulating recording

Schaffer collateral (CA3)Dendritic field (apical) of CA1

400 μM

Hippocampal Slice

Long-Term Potentiation (LTP)

Baseline stimulation(.1Hz) 100 Hz (burst)

5 Hz (1 theta-train)

Theta burst stimulation (TBS)

tetanus

-10 0 10 20 30 40 50 60-25

0

25

50

75

100

ControlsTime (min)

fEP

SP

Am

plit

ud

e (%

Ch

ang

efr

om

Bas

elin

e)

LTP timecourse (% change from baseline)

Induction

Maintenance

4-trains

PNB-0408 (“Hex”) induces long-term potentiation (LTP).

LTP in Hippocampal Neurons

-20 -10 0 10 20 30-50

-25

0

25

50

75

100

125

Control

Hex -8M

Hex-Y-I-6AH

TBS

Hex -6M

Time (min)

fEP

SP

Am

pli

tud

e (

% C

han

ge f

rom

Ba

seli

ne

)

Morris Water Maze Task Morris Water Maze Task of Spacial Memoryof Spacial Memory

Morris Water Maze Task Morris Water Maze Task of Spacial Memoryof Spacial Memory

Extra-maze cues

hidden pedestal

4 - trials per day

120 seconds per trial

20 second on-pedestal rest between trials

1-2 Trials1-2 Trials 3-4 Trials3-4 Trials 13+ Trials13+ Trials5-12 Trials5-12 Trials

Day 1Day 1 Day 2-3Day 2-3 Day 4Day 4

Morris Water MazeMorris Water MazeRepresentative Swim PatternsRepresentative Swim Patterns

PNB-0408 reverses a chemically-induced learning deficit.

Rat Performance in the Morris Water Maze

Acquisition (days)

0 2 4 6 8

Late

ncy

to f

ind

plat

form

(se

c)

0

20

40

60

80

100

120

140

icv Scop/Oral d.w. icv Scop/Oral Hex(1.25mg/kg) icv Scop/Oral Hexa in d.w. (2.0 mg/kg)

PNB-0408 improves spacial learning in aged animals.

Rat Performance in the Morris Water Maze

Old male rats (19-21 months) Delivered by gavage

Days of testing0 2 4 6 8

Lata

ency

to

find

the

plat

form

(se

c)

0

20

40

60

80

100

Hex (2 mg/kg) d.w. (2 ml/kg)

Future Aims for PNB-0408

Determination of Pharmacokinetic Profile

Demonstrate Blood-Brain Barrier Penetration

Future Aims for PNB-0408

Determination of

physicochemical

properties of PNB-0408 by

the use of prediction

software

Future Aims for PNB-0408

In-vitro blood stability study

Future Aims for PNB-0408

Recovery

Selectivity

Precision

Accuracy

Linearity

Limit of Detection

Limit of Quantitation

Ruggedness

Validation of methods for

processing and analysis of

PNB-0408 in blood and urine

Future Aims for PNB-0408

Intravenous bolus dose study with analysis of blood and urine

Elimination Half Life

Extent of Exposure (Area Under the Curve)

Volume of Distribution

Clearance

Future Aims for PNB-0408

Blood-brain barrier study

In-situ brain perfusion study

3H-PNB-0408

14C-inulin standard for vascular compartment

Joe Harding

Jay Wright

Neal Davies

Ray Quock

Heiko Jansen

Brent Yamamoto

Bryan Hudson

Pete Meighan

Starla Meighan

Patrick Elias

Caroline Benoist

Acknowledgements

Questions?