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Fibrotic Diseases
New Targets and Model Development
Kenneth Zhang, Ph.D.
In Vivo Pharmacology
6/10/2015
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About GenScript
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Kenneth Zhang, Ph.D.
Associate Director, In vivo pharmacology
at GenScript
Tumor immunologist with expertise in
tumor microenvironment
• Published over 19 peer-reviewed
publications
Presenter Bio
3
Leads the development of:
• Hepatic, renal and pulmonary fibrosis models
• SC xenograft, orthotopic and syngeneic tumor models
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Outline
Fibrotic diseases
Anti-fibrotic therapeutic targets
GenScript fibrotic disease models
4
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Organ structure – parenchyma
and interstitium
Fibrotic diseases are the
replacement of organ
parenchyma by interstitium -
excessive extracellular matrix
(ECM) deposit during chronic
injury, which progressively leads
to impair or loss of organ
function.
Fibrotic Diseases
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Friedman, Sci Trans Med, 2013
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PubMed Indexed Publications on Fibrosis
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Nature
• A reversible wound-healing response characterized by the
accumulation of ECM following liver injury. Chronic injury and
inflammation leads to progressive substitution of liver parenchyma
by scar tissue (fibrosis) and finally end stage liver disease (cirrhosis).
Etiology
• The diseases can be induced by HBV/HCV infection, alcohol abuse,
nonalcoholic steatohepatitis (NASH), chronic cholestatic disorders
(including primary biliary cirrhosis) and autoimmune hepatitis etc.
Therapy
• Currently no drug approved
• Obeticholic acid designated Breakthrough Therapy by US FDA in
2015 (Farnesoid X receptor agonist).
Hepatic Fibrosis
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Development of Liver Fibrosis and Cirrhosis
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Gressner, Comparative Hepatology, 2007
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Hepatocyte injury and release of
reactive oxygen species (ROS) and
apoptotic bodies
Activation of immune-subset cells and
hepatic stellate cells (HSC), and their
cross-talk
• Immune infiltration and Kupffer cell
activation
• Hepatic stellate cell activation,
proliferation and trans-
differentiation into myofibroblasts
(α-SMA+)
Excessive ECM protein production by
myofibroblasts in the space of Disse,
blocking substance exchange between
hepatocytes and the blood
Major Cellular Events in Hepatic Fibrosis
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Bataller, J Clin Invest, 2005
Normal Liver Fibrosis
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TGF-β
• Secreted by Kupffer cells and activated HSCs
• Enhances the transition of HSCs into collagen-producing
myofibroblasts, stimulates the synthesis of ECM proteins and
over-expression of TIMP.
PDGF
• Secreted mainly by Kupffer cells
• Predominant mitogen of
activated HSCs.
MCP-1 (CCL2)
• Recruits monocytes
Key Soluble Factors Involved
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Schuppan, JCI, 2013
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Sources of Myofibroblasts in Liver Fibrosis
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Hernandez-Gea, Annu Rev Pathol Mech Dis, 2011
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The development of excessive connective tissue in the lung – most cases are
idiopathic pulmonary fibrosis (IPF)
Irreversible impairment of respiratory function
Etiology:
• Idiopathic; Secondary effect of Inhalation of pollutants or pathogens,
Cigarette smoking, Connective tissue disease, Infections, etc
Limited therapeutic:
• Pirfenidone and Nintedanib approved (Oct 2014) by US FDA
Pulmonary Fibrosis
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Wynn, Integrating mechanisms of pulmonary fibrosis, 2011
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Renal fibrosis is the common end point of virtually all
chronic (progressive) kidney diseases.
Renal fibrosis includes
• Glomerulosclerosis
• Tubulointerstitial fibrosis -> Renal failure
• Excessive production of ECM between microtubules
and peritubular vasculature
Renal Fibrosis
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Renal Fibrosis in Response to
Epithelial Injury
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Friedman, Sci Trans Med, 2013
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Epithelial injury and dysfunction
Accumulation of myofibroblasts
Deposition of fibrotic ECM
Inflammation and recruitment of immune infiltrate
Pro-fibrotic macrophages
Common Features of Fibrotic Diseases
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Target Agent
TGF-β expression and activation Pirfenidone; αvβ6 antibody; ATI and ATII receptor blockers; ACE inhibitors; CAT-192 (anti-TGFβ1 mAb)
TGF-β signaling pathways
TGFβR1 (ALK5) SM305
SMAD3 SIS3
CTGF Antibody
Tyrosine kinase inhibitors of non-canonical TGF-β and other signaling pathways
c-Abl, c-kit, PDGFR Imatinib
PDGFR, c-Abl, src Dasatinib
PDGFR, c-Abl, c-kit Nilotinib
PDGFR, VEGFR, FGFR Nintedanib (BIBF1120)
VEGFR, PDGFR Sorafenib
Transcription factors
FXR Agonist (Obeticholic acid)
PPARγ Agonist (Rosiglitazone)
AP-1 T-5524
Anti-fibrotic Therapeutic Targets
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Adapted from Rosenbloom, Biochimica et Biophysica Acta, 2013
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Target Agent
Recruitment of fibrocytes and monocytes/macrophages
CXCL12 (SDF-1) Antibody
CXCR4 AMD3100
MCP-1/CCR2 Inhibitors (PF-04136309)
Immune response regulator
IL-13 Antibody
IL-6 receptor Tocilizumab
TLR TLR inhibitors (E5564, TAK-242)
Other intracellular pathways
JAK2 TG101209
Wnt Dkk-1
RhoA Statins, GGT inhibitor
JNK CC-930
NOX4 (production of ROS) GKT136901
Endothelin-1 Bosentan, other ET receptor blockers
Matrix stiffness, collagen crosslinking D-penicillamine, clostridial collagenase
Anti-fibrotic Therapeutic Targets (cont’d)
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Adapted from Rosenbloom, Biochimica et Biophysica Acta, 2013
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Hepatic fibrosis
• CCl4-induced hepatic injury and fibrosis in rats
• CCl4-induced hepatic injury and fibrosis in mice
• BDL-induced hepatic injury and fibrosis in rats
• BDL-induced hepatic injury and fibrosis in mice
• TAA-induced hepatic fibrosis in mice (in development)
Renal fibrosis
• UUO-induced renal fibrosis in rats
• UUO-induced renal fibrosis in mice
Pulmonary fibrosis
• Bleomycin-induced pulmonary fibrosis in mice
Note:
BDL, Bile duct ligation
TAA, Thioacetamide
UUO, Unilateral ureteral obstruction
GenScript Fibrotic Disease Models
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CCl4-induced Liver Fibrosis in Mouse
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Animal: Balb/c mice
CCl4-olive oil mixed solution; i.p. injection
Induction of hepatocyte damage, necrosis, inflammation
and fibrosis
CCl4-induced Fibrosis (Intoxication)
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• Liver and body weight
• Serum biochemistry
• HE staining
• Sirius Red staining
• Masson trichrome staining
• IHC for myofibroblasts & macrophages
• Quantitative analysis of histol. images
• Hydroxyproline assay
• Profiling of markers by RT-qPCR
Available endpoints of measurement
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Disease Development at 6 Weeks
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Olive oil only
CCl4
H&E Sirius Red Myofibroblasts Macrophages
(α-SMA) (F4/80)
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A B
C
Disease Development at 6 Weeks (continued)
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O liv e o il C C l4
0
2 0 0
4 0 0
6 0 0
8 0 0A L T
* *
IU/L
O liv e o il C C l4
0
5 0 0
1 0 0 0
1 5 0 0A S T
* *
IU/L
Olive oil CCl4
0
100
200
300
400
500
Hydroxyproline
**
g
/gOlive oil CCl4
0
2
4
6
8Acta2
**
Fo
ld c
ha
ng
e
(mR
NA
/-a
cti
n)
O liv e o il C C l4
0
1 0
2 0
3 0
4 0
5 0C o l1 a 1
* *
Fo
ld c
ha
ng
e
(mR
NA
/-a
cti
n)
O liv e o il C C l4
0
1
2
3
4
5T g fb 1
* * *
Fo
ld c
ha
ng
e
(mR
NA
/-a
cti
n)
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A
B C
Case Study
Therapeutic Effect of Pirfenidone at 6 Weeks
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Olive oil CCl4 CCl4+PFD
S iriu s R e d
O liv e o il C C l4 C C l4 + P F D
0
5
1 0
1 5
* * ** * *
Re
lati
ve
Sir
ius
Re
d+
Are
a
Col1a1
Olive oil CCl4 CCl4+PFD0
50
100
150
200***
Fo
ld c
ha
ng
e
(mR
NA
/-a
cti
n)
N.S.
Tgfb1
Olive oil CCl4 CCl4+PFD0
1
2
3
4
5 *** *
Fo
ld c
ha
ng
e
(mR
NA
/-a
cti
n)
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Bile-duct Ligation (BDL)-induced Liver Fibrosis in Mouse and Rat
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Animal: C57BL/6 mice; Wistar rats
Surgery: Ligation of common bile duct (BDL)
Study conclusion at 2-4 weeks after surgery.
Bile-duct Ligation-induced Fibrosis
(Cholestasis)
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• Liver and body weight
• Serum biochemistry
• HE staining
• Sirius Red staining
• Masson trichrome staining
• IHC for myofibroblasts & macrophages
• Quantitative analysis of histol. images
• Hydroxyproline assay
• Profiling of markers by RT-qPCR
Available endpoints of measurement
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Hepatocellular injury and proliferation
Portal inflammatory response
• Neutrophils, B/T cells, Kupffer cells
Cholangiocellular proliferation
Fibrosis development
Development of the Experimental
Disease in Mouse
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Georgiev, Br J Surg., 2008
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Model Development in Mouse
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Sham
BDL
2 wks 4 wks 8 wks
ALT AST A
B
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Model Development in Rat – Serum Biochemistry
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A S T
2 3 14 280
5 0 0
1 0 0 0
1 5 0 0S h a m
B D L
* *
* *
N SN S
D a y s a f t e r s u r g e r y
AS
T
(I
U/
L)
A L T
2 3 14 280
5 0 0
1 0 0 0
1 5 0 0S h a m
B D L
* *
*
N SN S
D a y s a f t e r s u r g e r y
AL
T
(I
U/
L)
T B I L
2 3 14 280
5 0
1 0 0
1 5 0S h a m
B D L* * *
* * *
N S
* * *
D a y s a f t e r s u r g e r y
TB
IL
(¦
Ìm
ol
/L
)
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Model Development in Rat - Histology
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100x
Sham Day 14 Day 28 40x
100x
40x
H&
E
S
iriu
s R
ed
Hydroxyproline content
14 280
100
200
300
400
500 Sham
BDL
**
Days after surgery
Hyp
co
nte
nt
( g
/g)
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Unilateral Ureteral Obstruction (UUO)-induced Renal Fibrosis in Rat
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Animal: SD rats
Surgery: Ligation of the ureter of left kidney, leaving the
contralateral kidney as a control
Induction of chronic epithelial injury, inflammation and
tubulointerstitial fibrosis in left kidney
Method
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• Kidney and body weight
• Serum biochemistry
• HE staining
• Sirius Red staining
• Masson trichrome staining
• IHC staining
• Quantification analysis of histological
images
• Hydroxyproline assay
• RT-qPCR
Available endpoints of measurement
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A B
C
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Model Development
Hydroxyproline content
Sham 3 7 14 21 280
500
1000
1500
Days after surgery
****
***
Hyp
co
nte
nt
( g
/g)
R e n a l w e i g h t / b o d y w e i g h t
Sham 3 7 14 21 280
1 0
2 0
3 0
4 0
D a y s a f t e r s u r g e r y
* *
* * *
* ** *
*
le
ft
k
id
ne
y w
eig
ht
/b
od
y w
eig
ht
(
¡ë
)
CREA
Sham 3 7 14 21 280
10
20
30
40
50
Days after surgery
** ** *****
CR
EA
(u
mo
l/L
)
BUN
Sham 3 7 14 21 280
2
4
6
8
10
Days after surgery
** *** **
BU
N(m
mo
l/L
) *
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Model Development Shown by
Sirius Red Staining (100x)
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Sham Day 3 Day 7
Day 14 Day 21 Day 28
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A
B C
Case Study
Therapeutic Effect of Pirfenidone at 4 Weeks
Sham UUO UUO+PFD
Sirius
Red
100x
H&E
200x
Sirius Red
0
5
10
15
20 ** *
Sir
ius R
ed
+ A
rea (
%)
Sham
UUO
UUO+PFD
H y d ro x y p ro lin e c o n te n t
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0* * *
g
/g*
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Bleomycin-induced Lung Fibrosis in Mouse
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Animal: C57BL/6 mice
Disease induction: Intratracheal instillation of bleomycin (BLM)
• Induction of lung epithelial injury, inflammation and fibrosis
Model Development
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• Lung and body weight
• BALF cell count
• HE staining
• Masson trichrome staining
• IHC staining
• Quantification analysis of histological
images
• Ashcroft score
• Hydroxyproline assay
• RT-qPCR
Available endpoints of measurement
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Histopathological Changes 3 Days after
Bleomycin Instillation (HE Staining)
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Saline Bleomycin dose gradient
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A B C
D E F
Model Development
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BAL total cells
Vehicle BLM0
20
40
60
80
*
To
tal cells (
×10
4)
3 days after BLM treatment
Animal Survival
0 10 20 300
20
40
60
80
100
BLM
Vehicle
Days after BLM treatment
Perc
en
t su
rviv
al (%
)
Body weight
0 5 10 15 20 25 3015
20
25
30
35 Vehicle
BLM
Days after BLM treatment
Bo
dy w
eig
ht
(g)
Mean
SE
M
L u n g w e ig h t/b o d y w e ig h t
V e h ic le 3 7 1 4 2 1
0
5
1 0
1 5
2 0
2 5
D a y s a f te r B L M tre a tm e n t
*
* * *
* * * * * *
Lu
ng
we
igh
t/b
od
y w
eig
ht(
‰)
Me
an
±S
EM
Hydroxyproline content
Vehicle 7 14 21 280
100
200
300
400
Days after BLM treatment
*
*** ******
Hyp
co
nte
nt
( g
/lu
ng
)
A s h c ro ft s c o re
V e h ic le 7 1 4 2 1 2 8
0
2
4
6
8
D a y s a f te r B L M tre a tm e n t
As
hc
ro
ft s
co
re
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Model Development - Histology
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H&E staining (40x)
Masson’s trichrome staining (40x)
Vehicle Day 7 Day 14 Day 21 Day 28
Bleomycin
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There is huge unmet clinical need for effective and potent
therapeutics of fibrotic diseases.
Properly developed animal models that mimic diseases in
human are critical for efficacy evaluation of leads.
GenScript has developed multiple fibrotic disease models
and will develop more to facilitate drug discovery and
development for fibrotic diseases.
Concluding Remarks
40
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Discovery Biology Services
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Antibody and Protein Engineering
•Single domain antibody generation
•Antibody sequencing
•Affinity maturation and humanization
In-vitro Pharmacology
•CellPower™ custom stable cell line for assays
•GenCRISPR™ custom knock-out or knock-in cell lines
•Cell-based assays and Ion channel and GPCR assays
In-vivo Pharmacology
•Tumor models including SC xenograft, orthotopic and syngeneic
•Bioluminescence imaging of tumors
•Fibrosis models
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Thank you for your participation
We wish you all success in your research
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