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