CDDO - International Dose

The importance of dose response for
the biology of synthetic triterpenoids
Karen T. Liby
Geisel School of Medicine at Dartmouth
Dose-Response 2013
Amherst, MA
April 24, 2013
Disease-specific death rates as a function of time
Cardiovascular
Cancer
Disease Death Rate
(per 100,000 population)
Alzheimer’s
Age Range (years)
Finkel, Nat Rev Mol Cell Biol, 2005
Non-resolving inflammation
Nathan Cell 140, 2010
Triterpenoids
Exist widely in nature, especially in higher plants
> 6,000 triterpenoids reported
Medicinal uses in Asian countries
Anti-inflammatory
Anti-tumorigenic
Poor potency
O
COOH
NC
Resemble steroids
Biosynthesis
Pleiotropic activities
O
Synthesized by cyclization of squalene
H
Oleanolic acid
CO2H
H
HO
H
Oleanolic Acid
• Weak anti-inflammatory agent
• Weak anti-carcinogenic agent
• Readily, cheaply available in kilogram quantities from
natural sources (e.g., olives, olive leaves)
Triterpenoids
O
O
CO2Me
COOH
NC
NC
O
O
CDDO
H
CDDO-Me
H
CDDO-Methyl ester
Phase I trial terminated
Phase III trial terminated
O
O
N
N
CONHCH2CH3
NC
NC
O
O
O
H
CDDO-Im
CDDO-Imidazolide
H
CDDO-EA
CDDO-Ethyl amide
Apoptosis
Triterpenoids
Differentiation
neuronal
adipogenic
.
Growth Suppression
iNOS
COX-2
MMPs
Anti-inflammation
Synthetic triterpenoids are
multifunctional molecules
hematopoietic
G1
S
M
G2
Oxidative Stress
Biological responses to triterpenoids
are strongly dependent on dose
nM
Increasing Concentration of Triterpenoid
PPARγ, Arp3 &
cytoskeleton
µM
Targeted Protein/
Network:
Keap/Nrf2/ARE
IKK and NF-ĸB,
JAK/STAT, ErbB2
Biological
response:
Induce phase 2
enzymes; Prevent
release of cytokines
Block DNA synthesis/
cell cycle progression
Activate
caspases
Final
Outcome:
Cytoprotection;
Anti-inflammatory
Induce differentiation;
Inhibit cell proliferation
Apoptosis
Low nM concentrations
of triterpenoids activate the
Nrf2 cytoprotective pathway
O
CO
CN2H
Me
NC
H
O
TP-46
TP-151
TP-155
TP-225
TP-82
H
(CDDO)
TP-46
10-4
10-5
10-6
TP-82
10-7
TP-151
10-8
10-9
TP-155
10-10
10-11
IC-50, Suppression of Induction of iNOS
TP-225
10-12
CDDO-Im increases expression of genes regulated by Nrf2
Fold Increase
CDDO
Gene
CDDO-Im
4 hrs
12 hrs
4 hrs
12 hrs
heme oxygenase (decycling) 1
19.70
17.15
90.51
111.43
ferritin, heavy polypeptide 1
2.64
6.06
2.64
6.96
NAD(P)H dehydrogenase, quinone 1
2.00
4.59
1.87
4.59
gamma-glutamylcysteine synthetase,
regulatory
2.83
4.00
2.30
4.29
epoxide hydrolase 1, microsomal (xenobiotic)
1.41
2.64
1.87
4.00
NAD(P)H dehydrogenase, quinone 2
1.15
1.41
1.52
4.00
thioredoxin reductase 1
2.46
3.48
2.30
3.48
UDP-glucose dehydrogenase
1.23
1.62
1.41
2.30
glutathione reductase
1.41
2.30
1.52
2.14
crystallin, zeta (quinone reductase)
1.00
1.15
1.32
2.14
gamma-glutamylcysteine synthetase, catalytic
1.32
1.32
1.52
2.00
glutathione S-transferase A4
1.15
1.23
1.62
2.00
The transcription factor Nrf2 activates a network of genes that
protect against oxidative and electrophilic stress
ROS
Electrophiles
MAPK
PKC
PI3K
P
Nrf2
Keap1
Keap1
P
Nrf2
Phase 2
Enzymes
P
Nrf2
Small Mafs
ARE
X
X=Transcription of
anti-oxidative and
cytoprotective genes
Target gene functions of the Nrf2-ARE pathway
•
•
•
•
•
•
•
•
•
Direct antioxidants
Free radical metabolism
Electrophile detoxification
Glutathione homeostasis
Generation of reducing equivalents
Solute transport
Inhibition of inflammation
DNA damage recognition
Proteasome function
CDDO-Im inhibits NO production in Nrf2
WT cells but not in Nrf2 KO cells
120
Nrf2 Wild-type cells
Nrf2 Knockout cells
NO Produced
(Percent stimulated control)
100
80
60
40
20
0
0
0.3
1
3
10
30
0
CDDO-Im (nM)
0.3
1
3
10
30
CDDO-Imidazolide protects against ROS in
Nrf2 wild-type cells challenged with tBHP
Mean Fluorescence Intensity
150
120
90
60
30
0
DMSO
tBHP
250 µM
+ 0.01 nM
Im
+ 0.1 nM
Im
+ 1 nM
Im
+ 10 nM
Im
+ 100
nM Im
CDDO-Imidazolide does NOT protect
against ROS in Nrf2 knockout cells
Mean Fluorescence Intensity
120
90
60
30
0
DMSO
tBHP
250 µM
+ 0.01 nM + 0.1 nM
Im
Im
+ 1 nM
Im
+ 10 nM
Im
+ 100 nM
Im
CD, Quinone Reductase Induction
10-10
TP-225
10-9
TP-155
TP-151
O
10-8
CO
CN2H
Me
NC
H
10-7
O
TP-82
TP-46
TP-151
TP-82
TP-155
TP-225
H
(CDDO)
10-6
TP-46
10-5
10-4
TP-46
10-5
10-6
TP-82
10-7
TP-151
10-8
10-9
TP-155
10-10
10-11
IC-50, Suppression of Induction of iNOS
TP-225
10-12
CD, Quinone Reductase Induction
Activation of the phase 2 cytoprotective enzyme NQO1 and
inhibition of the inflammatory enzyme iNOS are tightly correlated
10-10
TP-225
TP-224
10-9
TP-155 , -223
TP-218
TP-151
TP-235
TP-226
TP-241
10-8
TP-230
TP-190
TP-222
TP-162
10-7
TP-233
TP-82
10-6
TP-156
TP-62
TP-46
10-5
10-4
10-5
10-6
10-7
10-8
10-9
10-10
10-11
IC-50, Suppression of Induction of iNOS
10-12
Activation of the Keap/Nrf2/ARE pathway
by the triterpenoids is cytoprotective
Consequences of Nrf2 activation in cancer
Higher concentrations (low µM) of
triterpenoids increase ROS and
induce apoptosis in cancer cells
Low concentrations of triterpenoids suppress formation of
ROS, but high concentrations increase ROS
600
+ 250 µM tBHP
60
Mean Fluorescence Intensity
Mean Fluorescence Intensity
70
50
40
30
20
500
400
300
200
100
10
0
+ 250 µM tBHP
0
0
0.1
1
10
nM CDDO-Imidazolide
100
0
0
0
200
300
nM CDDO-Imidazolide
400
Targeting the altered redox status
of cancer cells to preferentially kill
malignant cells
- Cancer Cell 10:241, 2006
- Nature Reviews Drug Discovery 8:579, 2009
Targeting cancer cells via ROS
Trachootham, Nat Rev Drug Discovery, 2009
CDDO-Im induces higher levels of ROS
in Brca-1 defective breast cancer cells
than in normal 3T3 cells
NIH3T3-control
NIH3T3-Im 1 μM
W780-1control
W780-Im 1 μM
Fold change mean fluorescence
4.0
3.5
3.0
2.5
NIH3T3
2.0
W780
1.5
1.0
0.5
0.0
cont
Im-1
CDDO-Im, 1 h treatment
MCF10 model of progressive breast disease
Malignancy
In vivo
A
Immortal
AT1
Ha-ras
transfected
Nontumorigenic
Premalignant
lesions
CA1a
Malignant
Poorly
differentiated
carcinomas
with
metastatic
potential
Mean Fluorescence Intensity
Cells transformed with activated Ras
generate more ROS when
challenged with tBHP
60
MCF-10 A
MCF-10 AT-1
50
40
30
20
10
0
No Rx
250 uM t-BHP
500 uM t-BHP
The production of ROS is dose-dependent
ROS levels in response to tBHP challenge is
dependent on triterpenoid dose and cell type
ROS Fluorescence
MCF10A cells
MCF10 AT-1 cells
Triterpenoids increase ROS
ROS Fluorescence
MCF10 AT-1 cells
MCF10 CA1a cells
Triterpenoids induce apoptosis
MCF10 CA1a cells
% apoptotic cells
MCF10 AT-1 cells
µM CDDO-Im
µM CDDO-Im
Prevention and treatment
of lung carcinogenesis
by triterpenoids
Prevention of lung carcinogenesis - A/J mouse model
14-20 weeks treatment
Latency period
8 weeks
of age
1 week after
carcinogen
Administer
Carcinogen
(vinyl carbamate)
Control diet
Heavy tumor
burden
Chemopreventive
agents in diet
Reduced tumor
burden
Vinyl carbamate induces highly
invasive carcinomas
High grade
carcinoma invading
into a bronchus
CDDO-methyl ester and CDDO-ethyl amide
prevent lung carcinogenesis
Control
CDDO-ME
60 mg/kg diet
CDDO-EA
400 mg/kg diet
CDDO-methyl ester and CDDO-ethyl
amide prevent lung cancer
Control CDDO-Me CDDO-EA
Ave tumor size, mm3
(% control)
2.2
(100%)
0.2 *
(9%)
0.2 *
(9%)
Ave tumor burden, mm3
(% control)
7.2
(100%)
0.1 *
(2%)
0.2 *
(2%)
*, P < 0.05 vs. control
Treatment of lung carcinogenesis - A/J mouse model
Treat 12 wks
8 weeks
of age
Control diet
Heavy tumor
burden
8 wks
C/P injections
Administer
Carcinogen
(vinyl carbamate)
Therapeutic
agents in diet
Reduced tumor
burden
Triterpenoids protect against
carboplatin/paclitaxel toxicity –
Survivors after 5 C/P injections
Treatment
C/P WITHOUT triterpenoid
C/P WITH triterpenoid
Survivors
3/8
(38%)
14/16
(88%)
Treatment of lung carcinogenesis - A/J mouse model
Treat 12 wks
8 weeks
of age
Control diet
Heavy tumor
burden
12 wks
C/P injections
Administer
Carcinogen
(vinyl carbamate)
Therapeutic
agents in diet
Reduced tumor
burden
Triterpenoids protect against
carboplatin/paclitaxel toxicity –
Survivors after 5 C/P injections
Treatment
Survivors
Carboplatin/paclitaxel alone
21/23
(91%)
Triterpenoids + C/P
31/32
(97%)
Triterpenoids increase NQO1 mRNA in PBMCs
NQO1 mRNA
(Fold induction vs. control)
16
12
8
4
0
control
CDDO-ME
CDDO-EA
Triterpenoids increase NQO1 enzyme activity
in A/J mice
NQO1 enzyme activity
∆ in Absorbance/mg of Protein
Liver
Lung
Control lungs
CDDO-Me + C/P
The combination of CDDO-Me and C/P
inhibits lung carcinogenesis in A/J mice
Control
CDDO-ME Carboplatin CDDO-Me
80
&
+
mg/kg diet Paclitaxel
C/P
Ave # of tumors/slide
(% control)
3.5
(100%)
2.5
(71%)
2.1 *
(62%)
1.5 *
(43%)
Ave tumor volume
(mm3) per tumor
(% control)
4.6
(100%)
1.6 *
(34%)
1.5 *
(33%)
1.0 *
(21%)
Ave tumor volume
(mm3) per slide
(% control)
15.9
(100%)
3.9 *
(25%)
3.3 *
(21%)
1.4 **
(10%)
* , P < 0.05 vs. control
**, P < 0.05 vs. Me and C/P
Histopathology of tumors in A/J Mice
CDDO-Me
Control
30%
45% *
55% *
70%
n = 52 slides, 180 tumors
Carboplatin/Paclitaxel
Low/med grade
High grade
n = 22 slides, 55 tumors
CDDO-Me + C/P
*, p < 0.001 vs control
36% *
43% *
57% *
n = 42 slides, 90 tumors
64% *
n = 32 slides, 45 tumors
High grade tumors in control group
100X
400X
Treatment – Carboplatin/Paclitaxel
100X
400X
Treatment – Triterpenoid + Carboplatin/Paclitaxel
100X
400X
Summary
• Low doses of triterpenoids activate the
Nrf2 cytoprotective pathway and reduce
inflammation and ROS
• High doses of triterpenoids increase ROS
and induce apoptosis in cancer cells
• Triterpenoids also reduce tumor burden
and enhance treatment with carboplatin
and paclitaxel in experimental lung cancer
Acknowledgements
Dartmouth
Michael Sporn
Renee Risingsong
Darlene Royce
Charlotte Williams
Ryan Collins
Andrew Place
Nanjoo Suh
Dept of Chemistry
Gordon Gribble
Tadashi Honda
DHMC
Ethan Dmitrovsky
Candice Black
Eric York
Johns Hopkins
Paul Talalay
Albena Dinkova-Kostova
Thomas Kensler
Melinda Yates
Duke Medical School
Thomas Sporn
Funding
Robert E. Gosselin
Fellowship
Sidney Kimmel Fdn for
Can Res
American Cancer Society
Breast Cancer Res Fdn
Komen for the Cure
NCI, NIH
Reata Pharmaceuticals

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