Ver. 2019.8 1
www.smclab.co.jp
SMC Laboratories, Inc.
CRO service specialized in NASH-HCC -Proprietary STAMTM mouse model-
-2-
Overview
1 Company
2 Rationale: NASH
3 STAMTM: Proprietary model for NASH-HCC
4 Pharmacological study
5 CRO service
2
Facts at a glance
■ Founded in October 2006
■ A privately-held non-clinical CRO based in Tokyo, Japan;
specialized in research on fibrosis and inflammation
■ CRO services
- Non-clinical pharmacology
- One of the leading CRO in liver research with Proprietary NASH-HCC (STAMTM) Model
- In vivo disease models for metabolic disorders, inflammation, fibrosis and tumor
- Histological imaging services
- Histological scoring: NAFLD activity score, fibrosis and inflammation scores etc.
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Over 500 clients worldwide
Over 90 peer-reviewed papers and presentations
10 successful CTA packages
JPNUS・Canada
Europe Asia・Oceania
CRO expertise: Leading CRO in NASH/HCC
Europe
North AmericaAsia
Japan
Region
(% of the customers)
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0
50
100
150
200
250
300
350
400
450
500
550
2011 2012 2013 2014 2015 20162017
2018
The number of clients
(year)
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■ Equipment:- CT system (In vivo)
- Endoscopy (In vivo)
- Confocal microscopy
- Dry-chemistry analyzer
- Real-time PCR
- Multi-mode microplate reader
- And more…
■ Facility- Accreditation by MEXT*
- Sponsor audit (QAU)
- Animal welfare audit by global
pharmaceuticals
■ SPF-grade animal room:- 2080 mice
■ CRO science team:- 10 full-time researchers
- 5 visiting scientists (MD, PhD)
- 3 external pathologists
*MEXT: Ministry of Education, Culture, Sports, Science and Technology
CRO capability:
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1. Pharmacology study
→ Efficacy of existing drugs/drug candidates
2. Delivery of mouse samples (organs, plasma/serum, fecal etc.)
→ Target discovery and validation
→ Biomarker discovery and validation
Available Services
Disease model lineup
CRO portfolio: Nonclinical disease models
■ STAMTM: Premium model for NASH-HCC (mice)
■ Other inflammation/fibrosis/cancer models (mice)
・Liver fibrosis: CCl4 model, BDL model
・Acute liver failure : CCl4 model, Concanavalin A model
D-gal/LPS model, TAA model
・Pulmonary fibrosis: BLM-induced lung fibrosis model
・Skin fibrosis: BLM-induced skin fibrosis model
・Renal diseases: UUO-induced renal fibrosis model
・Renal diseases:Adriamycin-induced nephropathy model
・IBD: DSS-induced colitis model
・Liver cancer: DEN-CCl4 liver cancer model
・Alzheimer’s disease: icv-STZ model
CCl4 BLM
UUO DSS
Sirius red
PAS
CT (Lung)
Endoscopy
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Overview
1 Company
2 Rationale: NASH
3 STAMTM: Proprietary model for NASH-HCC
4 Pharmacological study
5 CRO service
-8-
■ High prevalence
US: NAFLD 55-155 million
NASH 15-50 million
JP: NAFLD 10~ million*
NASH 2~ million*
■ Sharp increase in pediatric patients
■ Progression to HCC
■ No established treatment
■ Economic loss
-1 billion USD per year (US)
■ Comorbidity of diabetes
- 1 in 8 diabetes patients die of liver fibrosis/HCC.
- Over 30% of diabetes patients show liver injury.
■ NASH increases the risk of CVD
*Japan Study Group of NAFLD (JSG-NAFLD) 2008-2011 Hotta N., et al., Journal of the Japan Diabetes Society 50:47, 2007
Causes of Death in Japanese Diabetics
(NASH)
Why focus on NASH?
-9-
*CVD: Cardiovascular disease*Chalasani N., et al., Gastroenterology 142:1592, 2012 (AGA guideline), **Cohen J., et al., Science 332:1519, 2011
US
-fatty change
-chronic change
ALT↑
HBsAg (-) HCV (-)
ANA (-)~low
Alcohol (-)~low
NAFLD Liver biopsy
NAFL
NASH +/- fibrosis
+/- HCCImaging (CT, MR..)
NAFLD fibrosis score
Screening/Initial evaluation
Diagnosis
US: Ultra Sound, CT: Computed Tomography, MR: Magnetic Resonance
Fibrosis HCCNASHSteatosis
6.3 – 33% (med. 20%)
in general population*
3 – 5%
in general population*Similar to HCV
10-30% 10-29%**(NASH to fibrosis)
4-27%**(NASH to HCC)
NASH: clinical process and diagnosis
-10-
Intervention Recommendations GRADE*
S Q
Lifestyle ● Up to 10% weight loss may be needed to improve necroinflammation 1 B
Metformin ● Metformin is not recommended as a specific treatment in adults with NASH 1 A
Tiazolidinediones● Pioglitazone can be used to treat steatohepatitis in patients with biopsy-proven NASH
1 B
● Long term safety and efficacy is not established
Vitamin E
● α-tocopherol (800 IU/day) improves liver histology in non-diabetic adults with biopsy-proven
NASH → First-line pharmacotherapy for this patient population1 B
● NOT in other patient populations, pending further evidence supporting this efficacy 1 C
UDCA ● UDCA is not recommended for the treatment of NAFLD/NASH 1 B
Omega-3 fatty acid● Omega-3 fatty acids may be considered as first-line therapy for hypertriglycemia in patients
with NAFLD, but it is premature to recommend them1 B
Statin● Statins can be used to treat dyslipidemia in patients with NAFLD/NASH, but should not be
used to specifically treat NASH, pending evidence from RCTs1 B
*GRADE: Grading of Recommendations , Assessment, Development and Evaluation
S (Strength of recommendation): 1 = strong, 2 = weak
Q (Quality of evidence): A = high, B = moderate, C = low
Chalasani N., et al., Gastroenterology 142:1592, 2012 (AGA guideline)
No approved drugs: AGA guideline 2012
Source: clinicaltrial.gov
Drug candidates in clinical trials
Company Drug Target Route Period Endpoint Stage
Intercept:
REGENERATE study
OCAFXR
- 18 months 1)Histology
2)Fibrosis and NASH
P3
GENFIT:
RESOLVE-IT study
Elafibranor PPARα /δ Oral 72 weeks 1)Histology
2)Fibrosis
P3
Galmed Aramchol Synthetic fatty acid
bile conjugate
Oral 52 weeks 1)% change in liver triglyceride
2)Fibrosis, NAS etc.
P3
Allergan:
AURORA study
Cenicriviroc (CVC) CCR5/CCR2 antagonist Oral 1 year 1)Fibrosis P3
Allergan:
CENTAUR study
Cenicriviroc (CVC) CCR5/CCR2 antagonist Oral 1 year 1)NAS P2
Novartis/Allergan:
TANDEM study
Tropifexor (LJN452)
Cenicriviroc (CVC)
FXR
CCR5/CCR2 antagonist
Oral
Oral
48 weeks 1) Number of participants with Adverse Events
2) Fibrosis
P2
Novo Nordisk:
LEAN study
Liraglutide GLP-1 SC 48 weeks 1)Histology
2)NAS
P2
Conatus:
ENCORE-NF study
Emricasan Caspase inhibitor Oral 72 weeks 1)Fibrosis
2)NAS
P2
Gilead GS-9674 FXR Oral 24 weeks plus 30
days
1)Overall safety profile P2
Gilead GS-0976 ACC Oral 24 weeks plus 30
days
1)Overall safety profile P2
Gilead Simtuzmab Loxl2 SC 96 weeks 1)Fibrosis
2)Safety
P2
BMS BMS-986036 FGF21 - 16 weeks 1)Change in percent hepatic fat fraction
2) Average concentration
P2
Galectin
Therapeutics
GR-MD-02 Galectin-3 IV 16 weeks 1)LiverMultiScan
2)MR-elastography
P2
NGM NGM282 FGF19 - 12 weeks 1)Change in absolute liver fat content
2)Change in percentage liver fat content
P2
Cempra Solithromycin Ketolide antibiotic Oral 13 weeks 1)NAS
2)Changes in steatosis
P2
Boehringer Ingelheim BI 1467335 SSAO/VAP-1 Oral 12 weeks 1)Target enzyme activity relative to baseline in
percent
2)Relative to ALT, AST, AP, GGT ,cleaved CK18,
total CK18 change from baseline
P2
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Overview
1 Company
2 Rationale: NASH
3 STAMTM: Proprietary model for NASH-HCC
4 Pharmacological study
5 CRO service
-13-
Advantages of STAMTM: Proprietary NASH-HCC model
■ Distinct from existing NAFL/diabetes models, STAMTM model represents the
patient population who develops HCC among NAFL/diabetic populations.
- By comparing with existing NAFL/diabetes models which never show fibrosis/HCC,
1) novel factors underlying worse prognosis can be investigated and
2) risk factor-modifying medicine/personalized medicine can be investigated in
diabetes/metabolic disease fields.
■ Clear onset of NAFL/NASH and 100% progression to fibrosis/HCC without exception.
- Both baseline and endpoint can be arranged according to researcher’s needs such
as clinical study design, mechanisms of tested molecules, etc.
■ Histological phenotype (including perisinusoidal fibrosis) similar to human NASH.
- Clinically equivalent endpoints (reduction of NAS, no increase of fibrosis, decrease of fibrosis)
can be evaluated.
- Major factors (①Inflammation, ②ballooning, ③fibrosis) and their relation with prognosis (HCC)
can be evaluated.
■ Virus-independent HCC pathway in steatohepatitis-background can be investigated.
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Birth
0w
Preparing
pregnant C57BL/6J mice
1st hit
- low dose streptozotocin -
4w 5w 7w 9w
Fatty liver evident
NASH evident
Fibrosis* evident
16w
HCC evident
12w
Nodule evident
* Perisinusoidal fibrosis resemble to human NASH 12
Fibrosis HCCNASHSteatosis
Fatty change (+)
ALT↑
NAFLD Activity score↑
All mice at 6 weeks of age meet
“baseline” criteria as in the case
of clinical trial in human
① CHEMICAL ② DIET
Continuous 2nd hit
- high fat diet feeding -
6w
100% 100% 100%
STAMTM: In vivo predictive pharmacology model
Birth
0w
4wks 5wks 7wks 9wks 16wks12wks 20wks
-15-
16 wks 〜HCC phase
Inhibition of O-GlcNAc-β-N-acetylglucosaminidase of β-cell (STZ)■ β cell-injury early after birth drives regenerative response with islet inflammation.
■ Accumulation of macrophages in the islet and adipose tissue.
■ Induction of mild diabetic condition.
■ Up-regulation of scavenger receptors and TNF-α in the liver (“priming”).
Continuous high fat diet feeding■ HFD augments fat deposition in the primed liver with increased lipogenesis.
■ Fatty acid oxidation induces ROS generation, lipid peroxidation, mitochondria dysfunction.
■ Recruitment and activation of inflammatory cells (macrophages followed by fibroblasts).
■ Proliferation of hepatocytes and formation of tumor.
1st hit
2nd hit
2 day
5-6 wks: Steatosis phase
7-8 wks: Steatohepatitis phase
9-12 wks: Fibrosis (to chronic fibrosis) phase
12 wks 〜Nodule formation
Mechanisms of STAMTM mice
Birth
0w
4w 5w 7w 9w 16w12w 20w
16w 20w
HCC-targeting study
6w 9w 20w
HCC-prevention study
6w 9w
NASH-targeting study
5w 8w
Steatosis-targeting study
9w 12w
Fibrosis-targeting study
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Examples of treatment period
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Macroscopic and histological appearance of STAMTM mice
■ STAMTM mice develop steatosis at 6 wks, steatohepatitis at 8 wks, chronic fibrosis at 12 wks and HCC at 20 wks of age.
Original Magnification: x400
n=3-6 (Mean ± SD)
NAFLD spectrum of STAMTM mice
HE
sta
inin
g
Macro
sco
pic
ap
peara
nce
6 wks
(steatosis)
8 wks
(steatohepatitis)
12 wks
(chronic fibrosis)
20 wks
(HCC)
8 wks (steatohepatitis)
Ballooning degeneration
(arrow head)
Inflammatory foci
(center)
Original Magnification: x200
-18-
Body weight, liver weight and biochemistry
■ Body weight and liver weight are increased in STAMTM mice with age.
■ Fasting blood glucose, serum AST, serum ALT levels are increased compared to normal mice.
■ Liver TG contents are increased at steatosis (6 wks) and steatohepatitis phase (8 wks), and slightly decreased at chronic fibrosis
phase (10-12 wks).
n=3-6 (Mean ± SD)
ALT: Alanine aminotransferase, AST: Aspartate aminotransferase, TG: Triglyceride
Bo
dy w
eig
ht
(g)
Liv
er
weig
ht
(mg
)
Fasti
ng
blo
od
glu
co
se
(mg
/dL
)
Seru
m A
LT
(U
/L)
Seru
m A
ST
(U
/L)
Liv
er
TG
(m
g/g
liv
er)
General parameters in STAMTM mice
-19-
■ No changes were noted in the fasting serum insulin levels, but non-fasting serum insulin levels were decreased in STAMTM mice.
■Whole blood HbA1c levels were increased in STAMTM mice compared to normal mice under fasting and non-fasting conditions.
n=6 (Mean ± SD)
Metabolic parameters in STAMTM mice
Non-fasting Fasting
Serum insulin and whole blood HbA1c at 9 weeks of age
-20-
■ Serum cholesterol (VLDL, HDL, LDL and chylomicron) and triglyceride levels are increased in STAMTM mice with age.
n=6 (Mean ± SD)
VLDL: very low density lipoprotein, HDL: high-density lipoprotein, LDL: low-density lipoprotein
Lipid parameters in STAMTM mice
Serum cholesterols and TG at 9 weeks of age
NASH and HCC biomarkers in STAMTM mice
■ Plasma CK-18 levels are increased in STAMTM mice compared to normal mice at steatohepatitis phase at 9 weeks of age.
■ Serum AFP levels are increased at HCC phase at 20 weeks of age.
CK-18: Cytokeratin 18 , AFP: Alpha-fetoprotein
n=6 (Mean ± SD)
Plasma CK-18 at 9 weeks of age and serum AFP at 20 weeks of age
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-22- 14
6 wks
(steatosis)
8 wks
(steatohepatitis)
12 wks
(chronic fibrosis)
20 wks
(HCC)
F4/8
0
Oil
red
■ Macro- and micro-vesicular fat deposition is observed at steatosis and steatohepatitis phases, and then fat deposition is
decreased at chronic fibrosis and HCC phases.
■ Macrophages (F4/80-positive cells) are accumulated in zone 3 and increased their number and size after the steatohepatitis
phase.
Oil-red staining and F4/80 immunostaining in STAMTM mice
Original Magnification: x200
* * **
*: Central vein
Steatosis and inflammation in STAMTM mice
* * *
***
*
*
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ER
-TR
7S
iriu
s r
ed
■ Collagen deposition around hepatocytes (chicken-wire pattern) is observed from the steatohepatitis phase and persists through
chronic fibrosis and HCC phases.
■ Accumulation of fibroblasts (ER-TR7-positive cells) is observed in zone 3 from steatohepatitis phase, and whose distribution is
correlated with collagen deposition.
6 wks
(steatosis)
8 wks
(steatohepatitis)
12 wks
(chronic fibrosis)
20 wks
(HCC)
Original magnification: upper panel (x 400), lower panel (x 200)
Sirius-red staining and ER-TR7 immunostaining in STAMTM mice
*: Central vein
**
**
Fibrosis in STAMTM mice
***
*
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Quantitative analyses of % positive areas for F4/80, Sirius red and ER-TR7 in the liver
■ The F4/80 positive areas (Macrophages) are increased in all stages of NAFLD-HCC.
■ The Sirius red-positive areas (Fibrosis) are increased at steatohepatitis, chronic fibrosis and HCC# phases.
■ The ER-TR7 positive areas (Fibroblasts) are increased after steatohepatitis phase.
Macrophages Fibrosis Fibroblasts
*: p<0.05, **: p<0.01, ***: p<0.001 vs Normal, n=4 (Mean ± SD)
*
***
***
***
***
***
*
**
***
#: At 20 weeks of age, non-HCC lesions were analyzed.
F4/8
0 p
osit
ive a
rea (
%)
Sir
ius r
ed
-p
osit
ive a
rea (
%)
ER
-TR
7 p
osit
ive a
rea (
%)
Histological parameters in STAMTM mice
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■ The expressions of pro-inflammation-related genes (MCP-1,TNF-a and IFN-γ) are increased in steatosis and steatohepatitis
phases.
■ The expressions of fibrosis-related genes (TIMP-1, Collagen Type 1 and TGF-b) are increased prior to histological evidence of
collagen deposition.
Real-time PCR assays in STAMTM mice
TN
F-a
/36B
4
0
5
10
15
20
25
30
* **
***
Normal
6w 12w8w 20w8w
STAM
MC
P-1
/36B
4
0
2
46
8
10
12
14
16
18 ***
*
Normal
6w 12w8w 20w
STAM
8w
**
STAM
Colla
gen T
ype 1
/36B
4
0
2
4
6
8
10
*
*
Normal
6w 12w8w 20w8w
TIM
P-1
/36B
4
0
5
10
15
20
25
30
Normal
6w 12w8w 20w8w
STAM
Inflam
mation
Fib
rosis
*: p<0.05, **: p<0.01, ***: p<0.001 vs Normal, n=3-4 (Mean ± SD)
IFN
-g/3
6B
4
0
Normal
6w 12w8w 20w8w
STAM
2
4
6
10
12
14
*
**
8
TG
F-b
/36B
4
Normal
6w 12w8w 20w8w
STAM
0
1
2
3
4
5**
**
**
**
Gene expressions in the liver of STAMTM mice
-26- 18
Tumor growth
rate
Mean tumor
diameter
Individual
tumor diameter
■ STAMTM mice develop multiple tumor in the liver.
■ ”Early wash-in and late wash-out”.
■ Individual tumor diameter gradually increases with age.
Multiple tumors in STAMTM mice (20 wks)
Dynamic CT in STAMTM mice (20 wks)
Histological images of STAMTM mice (20 wks)
HE staining GS immunostaining
HCC in STAMTM mice
-27-
Kaplan-Meier survival curve of STAMTM mice (no intervention)
■ The mortality rate at 16 and 20 weeks of age are 30% and 45%, respectively.
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 220
10
20
30
40
50
60
70
80
90
100
weeks of age
Su
rviv
al ra
te (
%)
Fibrosis
phase
Nodule
formation
HCC
phase
55%
(~20 wks)
70%
(~16 wks)
95%
(~12 wks)
Steatohepatitis
phase
Steatosis
phase
100%
(~8 wks)
Survival curve in STAMTM mice
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Overview
1 Company
2 Rationale: NASH
3 STAMTM: Proprietary model for NASH-HCC
4 Pharmacological study
5 CRO service
■ Study design
4 wks 5 wks 9 wks6 wks 8 wks7 wks
STAMTM-Vehicle
STAMTM-Telmisartan 10 mg/kg (n=6)
(n=6)
Sacrifice
Normal (n=6)
Oral, QD for 3 weeks
Oral: oral administration, QD: once-daily, NAS: NAFLD Activity Score, TNF: Tumor Necrosis Factor, MCP: Monocyte Chemotactic Protein, SMA: Smooth Muscle Actin,
TIMP: Tissue Inhibitors of Metalloproteinase
Oral, QD for 3 weeks
Histopathological assay
HE staining (NAS score)
Sirius red staining (Fibrosis area)
General
Body weight
Liver weight
Liver-to-body weight ratio
Biochemistry
Serum ALT
Liver TG
■ Analyses
Gene expression assays
TNF-a
MCP-1
• α-SMA
• TIMP-1
Birth
(0 wks)
Day 2:
Injection of streptozotocin
High fat diet feeding
■ Study aim: To investigate potential effects of ARB telmisartan on NASH/fibrosis
Rationale:
• Telmisartan is an angiotensin receptor blocker (ARB).
• Dysregulation of renin-angiotensin system has been implicated in the fibrogenic activation of HSC
Clinical relevance of the study:
• Telmisartan improved serum ALT, NAS and hepatic collagen deposition in STAMTM mice, similarly to clinical studies
[Georgescu EF., et al., WJ Gastroenterol.15:942, 2009].
Normal diet feeding
Pharmacological study-1: Study design
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-30-
Effects of telmisartan on general conditions and biochemistry
Figure 1. Body weight changes
Figure 2. Liver weight
■ Telmisartan significantly inhibited the increase in liver weight and liver TG levels, and tended to decrease the serum ALT levels.
Mean ± SD
Mean ± SD
Serum ALT
Figure 3. Biochemistry (serum ALT and liver TG)
Mean ± SDMean ± SD
Liver TG
Pharmacological study-1: Results
-31-
Figure 4. Effects of telmisartan on
NAFLD Activity score
x200
24
Mean ± SD
TelmisartanNormal Vehicle
Steatosis score
Inflammation score
Ballooning score
***
NAFLD Activity score
TelmisartanNormal Vehicle
Mean ± SD
Figure 5. Effects of telmisartan on liver fibrosis
Fibrosis area
x400
Effects of telmisartan on histopathological analysis
■ Telmisartan significantly inhibited the increase NAFLD Activity score and fibrosis area.
HE-staining Sirius red-staining
: central vein*
Pharmacological study-1: Results
-32-
■ Telmisartan significantly decreased the gene expressions of inflammatory related gene (TNF-a and MCP-1) and fibrosis related
gene (a-SMA and TIMP-1) in the liver.
Figure 6. The expression of inflammatory and fibrosis related genes in the liver Mean ± SD
MCP-1TNF-a a-SMA TIMP-1
Effects of telmisartan on inflammation and fibrosis related gene expressions
Pharmacological study-1: Results
Disclaimer: The data here are to show a representative example of study results. Gene expression patterns may change depending on study settings.
We make no representations or warranties as to the accuracy, reproducibility or completeness of the information provided.
-33-
(Vehicle = 6, Sorafenib = 3)
General
Body weight
Liver weight
Liver-to-body weight ratio
■ Analyses
Survival rate (%)
Vehicle: 60.0%
Sorafenib: 100%
■ Design
4 wks 19 wks16 wks 18 wks17 wks
STAMTM-Vehicle
STAMTM-Sorafenib 30 mg/kg (n=3)
(n=10)
Sacrifice
Oral, QD for 3 weeks
Oral, QD for 3 weeks
Birth
(0 wks)
Day 2:
Injection of streptozotocin
High fat diet feeding
■ Study aim: To investigate potential effects on NASH-HCC with sorafenib
Tumor analysis
Assessment of visible tumor number
Assessment of visible tumor size
Assessment of SOL volume
Assessment of SOL volume growth
ratio
Survival rate
Kaplan-Meier survival curve
Oral: oral administration, QD: once-daily
CT evaluation for
selection and
randomization
CT evaluation
Pharmacological study-2: Study design
-34-
Effects of Sorafenib on survival and body weight
Figure 1. Survival curves from
16 to 19 weeks of age
Figure 2. Body weight changes from
16 to 19 weeks of age
■ No animal died in the Sorafenib group. The general condition was not affected for most of the treatment period.
0 7 14 210
4
8
12
16
20
24
28
Vehicle
Sorafenib
Days after the start of treatment
Bo
dy w
eig
ht
(g)
0 7 14 210
10
20
30
40
50
60
70
80
90
100
Vehicle (n=10)
Sorafenib (n=3)
Days after the start of treatment
Pe
rcen
t s
urv
ival
Pharmacological study-2: Results
-35-
Effects of Sorafenib on liver weight and visible tumors on the surface of liver
Figure 5. The number
of visible tumor (>2 mm)
Figure 6. The sum of
tumor diametersFigure 3. Macroscopic appearance of livers
Figure 4. Liver weight
■ Sorafenib demonstrated macroscopic improvement in the liver. There were no significant differences in the number and diameter of
visible tumor on the surface of liver compared to the Vehicle.
Ve
hic
leS
ora
fen
ib
Vehicle Sorafenib0
1000
2000
3000
4000
n.s.
Liv
er
weig
ht
(mg
)
Vehicle Sorafenib0
1
2
3
4
5
6
7
n.s.
Tu
mo
r n
um
be
r
Vehicle Sorafenib0
5
10
15
20
25
30
35
40
45
n.s.
To
tal tu
mo
r d
iam
ete
r (m
m)
Pharmacological study-2: Results
-36-
Effects of Sorafenib on tumor growth assessed by contrast-enhanced CT
Vehicle Sorafenib0
200
400
600
800
1000
1200
1400
P<0.05
Mean
SO
L g
row
th r
ate
(%
)
Ve
hic
leS
ora
fen
ib
Figure 7. Contrast-enhanced CT images of liver
16 wks (before) 19 wks (after)
Volume: 47.3 mm3 Volume: 284.5 mm3
Volume: 54.2 mm3 Volume: 34.9 mm3
Figure 8. Comparison of the
growth rate of SOL
■ Sorafenib significantly decreased the growth rate of SOL. CT evaluation enables accurate assessment of tumor development.
SOL: space occupied lesion
600% increase
36% decrease
Pharmacological study-2: Results
-37-
HCC-targeting study with Sorafenib:• Molecular targeting therapy by Sorafenib demonstrated significant suppression of tumor growth rate,
showing efficacy of the drug as well as clinical relevance of the model.
• STAMTM model allows a flexible treatment design for HCC therapeutics, including a
preventive regimen against HCC development.
<Examples of treatment period>
16w 20w
HCC-targeting study
6w 9w 20w
HCC-prevention study
Birth
0w
4w 5w 7w 9w 16w12w 20w
Pharmacological study-2: Study design
■ Publications
Publications and Presentations
26. Molecular Cancer Research, “Inhibition of the cell death pathway in non-alcoholic steatohepatitis
(NASH)-related hepatocarcinogenesis is associated with histone H4 lysine 16 deacetylation” (Molecular
Cancer Research, DOI:10.1158/1541-7786.MCR-17-0109, 2017)
25. Magnetic Resonance Imaging, “The natural history of streptozotocin-stimulated non-alcoholic
steatohepatitis mice followed by Gd-EOB-DTPA-enhanced MRI: Comparison with simple steatosis mice.”
(Magn Reson Imaging, 38:123-128, 2017)
24. Journal of Pharmacology and Experimental Therapeutics, “Selective Inhibition of Autotaxin Is
Efficacious in Mouse Models of Liver Fibrosis” (J Pharmacol Exp Ther, 360:1-13, 2017)
23. Oncotarget, “Distinctly altered gut microbiota in the progression of liver disease” (Oncotarget, 7:
19355-19366, 2016)
22. Diabetology & Metabolic Syndrome, “Empagliflozin (an SGLT2 inhibitor), alone or in combination
with linagliptin (a DPP-4 inhibitor), prevents steatohepatitis in a novel mouse model of non-alcoholic
steatohepatitis and diabetes“ (Diabetology & Metabolic Syndrome, 8:45, 2016)
21. Journal of Immunology, Infection & Inflammatory Diseases, “Solithromycin Diminishes
Steatohepatitis by Modulating Gluconeogenesis and Inhibits Tumor Growth in a Diabetic Mouse Model of
Non-Alcoholic Steatohepatitis” (J Immunol Infect Inflam Dis, 1:004, 2016)
20. PLoS One, “Antifibrotic Effects of the Dual CCR2/CCR5 Antagonist Cenicriviroc in Animal Models of
Liver and Kidney Fibrosis“ (PLoS One, 11:e0158156, 2016)
19. Cell Reports, “Cancer-Associated Fibroblasts Regulate Tumor-Initiating Cell Plasticity in
Hepatocellular Carcinoma through c-Met/FRA1/HEY1 Signaling” (Cell Press, 15:1175-1189, 2016)
18. International Journal of Medical Sciences, “Palmitate-induced Regulation of PPARγ via PGC1α: a
Mechanism for Lipid Accumulation in the Liver in Nonalcoholic Fatty Liver Disease” (Int. J. Med. Sci,
13:169-178, 2016)
17. European Journal of Pharmacology, “Lipid-lowering agents inhibit hepatic steatosis in a non-
alcoholic steatohepatitis-derived hepatocellular carcinoma mouse model” (Eur J Pharmacol, 772:22-32,
2016)
16. Scientific Reports, “Characterization of hepatic lipid profiles in a mouse model with nonalcoholic
steatohepatitis and subsequent fibrosis” (Sci Rep., 12466, 2015)
15. International Journal of Obesity, “Low cytochrome oxidase 4I1 links mitochondriazzzl dysfunction to
obesity and type 2 diabetes in humans and mice” (Int J Obes, 39:1254-63, 2015)
14. Proc Natl Acad Sci U S A, “Immunomodulatory spherical nucleic acids” (Proc Natl Acad Sci U S A,
31;112:3892-7, 2015)
13. Oncology Reports, “Hepatic expression of the Sptlc3 subunit of serine palmitoyltransferase is
associated with the development of hepatocellular carcinoma in a mouse model of nonalcoholic
steatohepatitis” (Oncol Rep, 33:1657-66, 2015)
12. Drug R D, “In Vivo Efficacy Study of Milk Thistle Extract (ETHIS-094TM) in STAMTM Model of
Nonalcoholic Steatohepatitis” (Drugs R D, 14:291-9, 2014)
11. PLoS One, “Photoacoustic Tomography of Human Hepatic Malignancies Using Intraoperative
Indocyanine Green Fluorescence Imaging” (PLoS One, 9:e112667, 2014)
42. International Journal of Gastroenterology, "Characterization of EDP-305, a Highly Potent and
Selective Farnesoid X Receptor Agonist, for the Treatment of Non-alcoholic Steatohepatitis" (International
Journal of Gastroenterology, DOI: 10.11648/j.ijg.20190301.12, 2019)
41. Experimental Animals, "Analysis of amino acid profiles of blood over time and biomarkers associated
with non-alcoholic steatohepatitis in STAM mice" (Exp Anim., DOI: 10.1538/expanim.18-0152, 2019)
40. Frontiers in Genetics, "Gene Expression and DNA Methylation Alterations During Non-alcoholic
Steatohepatitis-Associated Liver Carcinogenesis" (Front Genet., May 29;10:486, 2019)
39. Journal of Cellular and Molecular Medicine, "The lysyl oxidase like 2/3 enzymatic inhibitor, PXS-
5153A, reduces crosslinks and ameliorates fibrosis" (J Cell Mol Med., 23:1759-1770, 2019)
38. Scientific Reports, "Connectivity mapping of angiotensin-PPAR interactions involved in the
amelioration of non-alcoholic steatohepatitis by Telmisartan" (Sci Rep., Mar 8;9(1):4003, 2019)
37. NPJ Precision Oncology, "Transcriptomic analysis of hepatocellular carcinoma reveals molecular
features of disease progression and tumor immune biology" (NPJ Precis Oncol., DOI: 10.1038/s41698-
018-0068-8, 2018)
36. Cellular and Molecular Gastroenterology and Hepatology, "Dipeptidyl Peptidase 4 inhibitors
Reduce Hepatocellular Carcinoma by Activating Lymphocyte Chemotaxis in Mice" (CMGH, DOI:
10.1016/j.jcmgh.2018.08.008, 2018)
35. Glycoconjugate Journal, “Identification of unique glycoisoforms of vitamin D-binding protein and
haptoglobin as biomarker candidates in hepatocarcinogenesis of STAM mice” (Glycoconj J., Oct;35(5):467-
476, 2018)
34. Proc Natl Acad Sci U S A, “Integrative genomic analysis of mouse and human hepatocellular
carcinoma” (Proc Natl Acad Sci U S A, DOI: 10.1073/pnas.1811029115, 2018)
33. Liver Cancer, “Effects of a DPP4 Inhibitor on Progression of NASH-related HCC and the p62/
Keap1/Nrf2-Pentose Phosphate Pathway in a Mouse Model” (Liver Cancer, DOI: 10.1159/000491763,
2018)
32. PLoS One, “Gemcabene downregulates inflammatory, lipid-altering and cell-signaling genes in the
STAM™ model of NASH” (PLoS One, 13(5): e0194568 , 2018)
31. World Journal of Gastroenterology, “Mouse models for investigating the underlying mechanisms of
nonalcoholic steatohepatitis-derived hepatocellular carcinoma” (World J Gastroenterol, 24(18):1989-1994,
2018)
30. The FASEB Journal, “Epigenetically mediated inhibition of S-adenosylhomocysteine hydrolase and
the associated dysregulation of 1-carbon metabolism in nonalcoholic steatohepatitis and hepatocellular
carcinoma“ (FASEB J, DOI:10.1096/fj.201700866R, 2017)
29. Oncotarget, “MicroRNA deregulation in nonalcoholic steatohepatitisassociated liver carcinogenesis”
(Oncotarget, 8:88517-88528, 2017)
28. Oncotarget, “Peretinoin, an acyclic retinoid, suppresses steatohepatitis and tumorigenesis by
activating autophagy in mice fed an atherogenic high-fat diet” (Oncotarget, 8:39978-39993, 2017)
27. Physiological Research, “Pathophysiological analysis of the progression of hepatic lesions in STAM
mice.” (Physiological Research, 66:791-799, 2017)-38-
66. AASLD 2018, “Dipeptidyl Peptidase 4 Inhibitors Reduce the Progression of Hepatocellular Carcinoma
By Activating T Cell and Natural Killer Cell Chemotaxis in Mice” Kawasaki Medical School
65. AASLD 2018, “Effects of a DPP4 Inhibitor on Progression of Nash-Related Hepatoma and DNA
Synthesis Pathway Via p62/Keap1/Nrf2 in a Mouse Model: A Metabolomic Analysis” Kurume University
School of Medicine
64. AASLD 2018, “Gemcabene Regulates Hepatic Genes Associated with Inflammation and Fibrosis with
Impact on Non-Alcoholic Fatty Liver Disease” Gemphire Therapeutics Inc.
63. AASLD 2018, “CM101, a Novel CCL24 Blocking Antibody, Suppresses Hepatic Injury and Fibrosis In
Experimental Models of Nash and Liver Fibrosis” ChemomAb Ltd.
62. AASLD 2018, “Unexpected Antidiabetic Effects Combined with Antifibrotic Activities of LXR Inverse
Agonists in Mouse Models of NAFLD/Nash” Phenex Pharmaceuticals AG
61. The 78th Scientific Sessions ADA, 2018, “Canagliflozin, an SGLT2 Inhibitor, Prevents Development
of Hepatocellular Carcinoma (HCC) from Nonalcoholic Steatohepatitis (NASH) in a Mouse Model of NASH-
HCC Under Diabetic State” Dokkyo Medical University
60. The 78th Scientific Sessions ADA, 2018, “Combination of SGLT2 Inhibitor and Novel Selective
PPARα Modulator, Tofogliflozin (Tofo) and Pemafibrate (Pema), Improves Survival Rate in STAM Mice as a
Diabetic NASH Model” Kowa Company Ltd.
59. EASL the International Liver CongressTM 2018, “Interfering with local fibrotic platelet activation
significantly inhibits fibrosis in multiple animal models: suggestions of the importance of the platelet-wound
healing axis for fibrosis” Symic Bio, Inc.
58. EASL the International Liver CongressTM 2018, “BMS-986036, a PEGylated fibroblast growth factor
21 analogue, reduces fibrosis and PRO-C3 in a mouse model of non-alcoholic steatohepatitis” Bristol-Myers
Squibb Company
57. EASL the International Liver CongressTM 2018, “LJN452 (tropifexor) attenuates steatohepatitis,
inflammation, and fibrosis in dietary mouse models of nonalcoholic steatohepatitis” Genomics Institute of
the Novartis Research Foundation
56. EASL the International Liver CongressTM 2018, “Clinical-grade human liver mesenchymal stem cells
reduce NAS score and fibrosis progression in advanced stage NASH pre-clinical model through
immunomodulation” Promethera Biosciences LLC
55. First EASL NAFLD Summit 2017, “Dual CCR2/5 antagonist decreases hepatic inflammation in acute
liver injury and NASH metabolic animal models” Pfizer Inc.
54. First EASL NAFLD Summit 2017, “AXA1125, a novel defined amino acid composition (DAAC),
improves NAFLD activity score (NAS) and reduces fibrosis in two rodent models of nonalcoholic
steathepatitis (NASH)” Axcella Health, Inc.
53. AASLD 2017, “The Anti-Fibrogenic and Liver Protective Effects of Namodenoson (CF102) in a Non-
Alcoholic Steatohepatitis Model” Can-Fite BioPharma Ltd.
73. DDW 2019, “Change of Gut Microbiome after Treatment with the Traditional Japanese Medicine
Daisaikoto is Associated with Improved Liver Steatosis in a Non-alcoholic Fatty Liver Mouse Model”
TSUMURA & Co.
72. DDW 2019, “Influence of the O-GlcNAc Modification in Hepatic Carcinogenesis by Non-alcoholic Fatty
Liver Disease” Osaka Medical College
71. EASL the International Liver CongressTM 2018, “LXR inverse agonists reduce steatosis and fibrosis
in the STAM mouse model but also improve insulin sensitivity in a high fat diet mouse clamp study” Phenex
Pharmaceuticals AG
70. 3rd Annual World Preclinical Congress Europe 2018, “LXR Inverse Agonists for the Treatment of
NASH” Phenex Pharmaceuticals AG
69. 3rd Annual World Preclinical Congress Europe 2018, “MTBL0036, a Promising, New Anti-NASH
and Antifibrotic Candidate: MTBL0036 showed a decrease in NAFLD Activity score in the STAM model”
Metabolys, Inc.
68. AASLD 2018, “AXA1125, a Novel Composition of Amino Acids Reprograms the Multifactorial
Pathophysiology in NAFLD” Axcella Health Inc.
67. AASLD 2018, “Treatment of Hepatocellular Carcinoma Using 2-Deoxy-D-Glucose Encapsulated in
PLGA Nanoparticles in Mice” Kawasaki Medical School
Publications and Presentations
■ Presentations
10. Cancer Science, “Silencing of microRNA-122 is an early event during hepatocarcinogenesis from non-
alcoholic steatohepatitis” (Cancer Sci, 105:1254-60, 2014)
9. Anticancer Research, “Characterization of non-alcoholic steatohepatitis-derived hepatocellular
carcinoma as a human stratification model in mice” (Anticancer Res, 34:4849-4856, 2014)
8. PLoS One, “L-carnitine prevents progression of non-alcoholic steatohepatitis in a mouse model with
upregulation of mitochondrial pathway.” (PLoS One, 9:e100627, 2014)
7. Medical Molecular Morphology, “Linagliptin alleviates hepatic steatosis and inflammation in a mouse
model of non-alcoholic steatohepatitis” (Med Mol Morph, 47:137-149)
6. PLoS One, “Therapy of Experimental NASH and Fibrosis with Galectin Inhibitors” (PLoS One,
8:e83481, 2013)
5. International Journal of Oncology, “Identification of an H2-Kb or H2-Db restricted and glypican-3-
derived cytotoxic T-lymphocyte epitope peptide” (Int J Oncol, 42:831-838, 2013)
4. International Journal of Experimental Pathology, “Inhibition of Glutaminyl Cyclases alleviates CCL2-
mediated inflammation of non-alcoholic fatty liver disease in mice” (Int J Exp Pathol, 94: 217-225, 2013)
3. Medical Molecular Morphology, “A murine model for non-alcoholic steatohepatitis showing evidence
of association between diabetes and hepatocellular carcinoma” (Med Mol Morph, 46:141-152, 2013)
2. Hepatology, “Hydrogen-rich water prevents progression of non-alcoholic steatohepatitis and
accompanying hepatocarcinogenesis in mice” (Hepatology, 56:912-921, 2012)
1. Journal of Nutritional Science and Vitaminology, “Effects of Dietary Supplementation with Betaine
on a Nonalcoholic Steatohepatitis (NASH) Mouse Model” (J Nutr Sci Vitaminol, 58:371–375, 2012)
■ Publications (continued)
-39-
52. AASLD 2017, “DPP4 Inhibitor Suppressed Progression of NASH-related Hepatocellular Carcinoma
with Inhibition of Metabolic Reprograming in p62-Keap 1-Nrf2-pentose Phosphate Pathway in a Mouse
Model: A Metabolomic Analysis” Kurume University School of Medicine
51. AASLD 2017, “CB4209 and CB4211 Reduce the NAFLD Activity Score in the STAM Model of NASH,
Reduce Triglyceride Levels, and Induce Selective Fat Mass Loss in DIO Mice” CohBar, Inc.
50. AASLD 2017, “Combination Treatment of LJN452 and Cenicriviroc Snows Additive Effects in a Diet-
Induced NASH Model” Genomics Institute of the Novartis Research Foundation/Allergan plc/Novartis
Institutes for BioMedical Research, Inc.
49. AASLD 2017, “Gemcabene Attenuates the NAFLD Activity and Fibrosis Scores, and Downregulates
Hepatic Inflammatory Genes in the STAMTM Murine Model of NASH-HCC” Gemphire Therapeutics Inc.
48. DDW 2017, “A HMG-CoA Reductase Inhibitor, Rosuvastatin, as a Potential Preventive Drug for The
Development of Hepatocellular Carcinoma Associated With Non-alcoholic Fatty Liver Disease in Mice”
Osaka Medical College
47. EASL the International Liver CongressTM 2017, “Anti-fibrotic effect of NV556,a sanglifehrin-based
cyclophilin inhibitor,in a preclinical model of non-alcoholic steatohepatitis” Neuro Vive Pharmaceutical AB
46. AACR 2017, “Inhibition of gene expression during non-alcoholic steatohepatitis (NASH)-related
hepatocarcinogenesis is mediated by histone H4 lysine 16 deacetylation” FDA-National Center for
Toxicological Research.
45. AACR 2017, “Alterations in the chromatin accessibility in nonalcoholic steatohepatitis-associated
hepatocellular carcinoma” FDA-National Center for Toxicological Research
44. AACR 2017, “Role of miRNAome deregulation in the pathogenesis of non-alcoholic steatohepatitis
(NASH)-derived hepatocellular carcinoma” FDA-National Center for Toxicological Research
43. AASLD 2017, Emerging Trends Conference: Emerging Trends in Non-Alcoholic Fatty Liver
Disease, “The Novel Antidiabetic Candidate MTBL0036 Greatly Diminishes The NAFLD Activity Score in
The STAM Mouse Model of NASH” Metabolys Inc.
42. AASLD 2017, Emerging Trends Conference: Emerging Trends in Non-Alcoholic Fatty Liver
Disease, “DUR-928, An Endogenous Regulatory Molecule, Exhibits Anti-Inflammatory and Antifibrotic
Activity in a Mouse Model of NASH” DURECT Corporation
41. AASLD 2016, “A Phase 2 study of BMS-986036 (Pegylated FGF21) in Obese Adults with Type 2
Diabetes and a High Prevalence of Fatty Liver” Bristol-Myers Squibb Company
40. AASLD 2016, “Effects of BMS-986036 (pegylated fibroblast growth factor 21) on hepatic steatosis and
fibrosis in a mouse model of nonalcoholic steatohepatitis” Bristol-Myers Squibb Company .
39. DDW 2016, “Inhibition of the Ileal Bile Acid Transporter (IBAT) by A4250 Reduces Hepatic Damage in
a Mouse Model of Non-Alcoholic Steatohepatitis (NASH)” Albireo AB
38. EASL the International Liver CongressTM 2016, “DPP4 Inhibitor Suppresses Steatohepatitis and
HCC Progression with Glucose Re-Programing in a Mouse Model of NASH” Kurume University School of
Medicine
37. HEP DART 2015, “The Cyclophilin Inhibitor, CPI-431-32, is a Hepatitis B Oral Drug Candidate with
Antiviral and Antifibrotic Activities” Ciclofilin Pharmaceuticals Inc.
■ Presentations (continued)
36. WDC 2015, “Empagliflozin (an SGLT2 inhibitor), alone or in combination with linagliptin (a DPP-4
inhibitor), prevents steatohepatitis in a novel mouse model of non-alcoholic steatohepatitis and diabetes”
Dokkyo Medical University
35. AASLD 2015, “Anti-Fibrotic Effect of Autotaxin and LPA1R Antagonists in a Rodent Model of NASH”
Bristol-Myers Squibb Company
34. AASLD 2015, “Sitagliptin, a Dipeptidyl Peptidase 4 inhibitor, Suppressed Tumor Progression with
Down-regulation of Nrf Nuclear Expression in a Mouse Model of Non-alcoholic Steatohepatitis-related
Hepatocellular Carcinoma” Kurume University School of Medicine
33. AASLD 2015, “Reduction of Hepatic 27-Hydroxycholesterol in Steatohepatitis Model Mice with Insulin
Resistance” Tokyo Medical University Ibaraki Medical Center
32. AASLD 2015, “Disturbance of regulatory T cells, MDSCs and NK cells is involved in NASH and mouse
model of NASH” Tohoku University Hospital.
31. AASLD 2015, “Mechanism of Action of the Anti-NASH effects of Solithromycin in a Predictive NASH
HCC Mouse Model” Cempra Pharmaceuticals, Inc.
30. DDW 2015, “Effects of Sitagliptin, a Dipeptidyl Peptidase 4 Inhibitor, on Tumor Progression and
p62/SQSTM1 Subcellular Localization in a Mouse Model of Non-Alcoholic Steatohepatitis-Related
Hepatocellular Carcinoma” Kurume University
29. Keystone Symposia 2015, “DGAT2 Inhibition Prevents NAFLD and Fibrosis in the STAM Mouse
Model of NASH“ Pfizer Inc.
28. Keystone Symposia 2015, “Oxidized-Phospholipid Small Molecule Inhibits Non-Alcoholic
Steatohepatitis (NASH) and Liver Fibrosis“ Vascular Biogenics Ltd
27. AASLD 2014, “L-carnitine prevents progression of non-alcoholic steatohepatitis in a mouse model with
upregulation of mitochondrial pathway“ Department of Gastroenterology and Hepatology, Okayama
University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences
26. AASLD 2014, “MN-001 (tipelukast), a novel, orally bioavailable drug, reduces fibrosis and inflammation
and down-regulates TIMP-1, collagen Type 1 and LOXL2 mRNA overexpression in an advanced NASH
(nonalcoholic steatohepatitis) model“ MediciNova, Inc.
25. ICLAF 2014, “MN-001 (tipelukast), a nonselective phosphodiesterase, 5-lipoxygenase, leukotriene,
phospholipase C and thromboxane A2 inhibitor, demonstrates anti-fibrotic effects in the bleomycin-induced
idiopathic pulmonary fibrosis mouse model“ MediciNova, Inc.
24. ADA 2014, “Liraglutide prevents steatohepatitis, liver fibrosis, and accompanying carcinogenesis in a
diabetes and nonalcoholic steatohepatitis mouse model treated with STZ-HFD“ Saga University
23. ATS 2014, “Solithromycin Reduces Inflammation In Mice Caused By Bleomycin-Induced Lung Injury“
Cempra, Inc.
22. DDW 2014, “Anti-NASH Effects of Solithromycin in NASH-HCC Mouse Model“ Cempra, Inc.
21. AACR 2014, “Clinicopathological characterization of non-alcoholic Steatohepatitis (NASH)-derived
Hepatocellular carcinoma (HCC) as a patient stratification model in mice)” The Jikei University School of
Medicine
Publications and Presentations
-40-
■ Patents
・ List of presentations in domestic meeting is available only in Japanese version.
• International publication No.: WO2011/013247 Title of the invention: "Steatohepatitis-Liver Cancer
Model Animal”
• Publication No. (JP) : 2009-178143 Title of the invention: "Steatohepatitis-Liver Cancer Model Animal
(EN)”
20. Keystone Symposia 2014, “The NADPH Oxidase (NOX) Inhibitor GKT137831 Alleviates Liver
Inflammation and Fibrosis in a Mouse Model of Non-Alcoholic Steatohepatitis (NASH)” Genkyotex S.A.
19. 15th International Workshop on Co-morbidities and Adverse Drug Reactions in HIV, “Anti-fibrotic
and anti-inflammatory activity of the dual CCR2 and CCR5 antagonist cenicriviroc in a mouse model of
NASH” Tobira Therapeutics Inc.
18. AASLD 2013, “Anti-fibrotic and anti-inflammatory activity of the dual CCR2 and CCR5 antagonist
cenicriviroc in a mouse model of NASH” Tobira Therapeutics Inc.
17. AASLD 2013, “L-carnitine prevents progression of non-alcoholic steatohepatitis with regulation of
mitochondrial β-oxidation and redox system in NASH model Mice” Department of Gastroenterology and
Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences
16. FASEB SRC 2013, Lysophospholipid and Other Related Mediators - From Bench to Clinic, “ATX
inhibition prevents progression of non-alcoholic steatohepatitis (NASH) in a hypoinsulinemic mouse model
of progressive liver disease” F. Hoffmann-La Roche, Ltd
15. DDW 2013, “Vitamin E and L-Carnitine Prevents Progression of Non-Alcoholic Steatohepatitis With
Regulation of Intestinal Inflammasome Activation in NASH Model Mice” Department of Gastroenterology
and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical
Sciences
14. DDW 2013, “Rosuvastatin Prevents Liver Tumorigenesis in High-Fat Diet-Fed Mice“ 2nd Department of
Internal Medicine Osaka Medical College
13. AASLD 2012, “Comparative proteomic analysis of the liver in a murine model of non- alcoholic
steatohepatitis” Third Department of Internal Medicine, Niigata University Medical School
12. AASLD 2012, “Inhibition of endoplasmic reticulum stress by 4-phenylbutyrate prevents steatohepatitis
progression and tumorigenesis in NASH-HCC model mice” Department of Gastroenterology, Juntendo
University School of Medicine
11. AASLD 2012, “Galectin-3 targeting drugs inhibit multiple pathological pathways leading to improvement
of non-alcoholic steatohepatitis (NASH)” Galectin Therapeutics Inc.
10. AASLD 2012, “Hepatic gene expression of the SPTLC3 subunit of serine palmitoyltransferase is
associated with the development of liver cancer in a NASH mouse model” Department of Human and
Environmental Sciences, Kagoshima University Graduate School of Medicine and Dental Sciencesq
9. The 72th Scientific Sessions ADA, 2012, “Linagliptin is an Effective Therapeutic for Non-alcoholic
Fatty Liver Disease (NAFLD) and Non-alcoholic Steatohepatitis (NASH)” Boehringer Ingelheim GmbH &
Co. KG
8. DDW 2012, “A Novel Murine Model Recapitulates the Pathogenesis of Human Non-alcoholic
steatohepatitis (NASH) and NASH-related Hepatocellular Carcinoma”
7. DDW 2012, “Effects of Telmisartan on a Murine Model of Non-alcoholic Steatohepatitis (NASH) and
NASH-related Hepatocellular Carcinoma”
6. DDW 2012, “The Chemical Chaperon 4-Phenylbutyrate Inhibits Liver Fibrosis and Tumorigenesis in
High-Fat Diet With N-acetyl-β-D-glucosaminedase Inhibitor-Induced NASH Model Mice” Department of
Gastroenterology, Juntendo University School of Medicine
■ Presentations (continued)
5. EASL The International Liver CongressTM 2012 - 47th Annual Meeting of the European
Association for the Study of the Liver, “FXR agonists prevent steatosis, hepatocyte death and
progression of NASH towards HCC in a hypoinsulinaemic mouse model of progressive liver disease”
Phenex Pharmaceuticals AG
4. AASLD 2011, “The Dipeptidyl Peptidase-4 Inhibitor Linagliptin is an Effective Therapeutic for Metabolic
Liver Disease in Several Rodent Models of Non-Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic
Steatohepatitis (NASH)” Boehringer Ingelheim GmbH & Co. KG
3. EASL Special Conference - Liver Transplantation 2011, “Improvement of steatosis, inflammation,
and fibrosis in a mouse model of steatohepatitis following treatment with galectin inhibitor” Galectin
Therapeutics Inc.
2. EASL The International Liver CongressTM 2011 - 46th Annual Meeting of the European
Association for the Study of the Liver, “Novel FXR agonists with potent lipid lowering, insulin sensitising,
anti-inflammatory and anti-fibrotisation effects in mouse models of metabolic syndrome and NASH” Phenex
Pharmaceuticals AG
1. The 9th JSH SingleTopic Conference “NASH 2010”, “Strong Anti-steatotic and Anti-fibrotic Effects of
Novel FXR Agonists in a Murine NASH Model that Resembles Human NASH” Phenex Pharmaceuticals AG
Publications and Presentations
-41-
-42-
Overview
1 Company
2 Rationale: NASH
3 STAMTM: Proprietary model for NASH-HCC
4 Pharmacological study
5 CRO service
-43-
■ Drugs with proven efficacy against NASH in STAMTM Mice
■ Possible types of studies using STAMTM Mice
1 Target discovery/validation
3 Biomarker discovery
4 Preclinical evaluation2 Candidate screening
・ Disease/ Efficacy/ Mechanism biomarkers
・ Evaluation of approved drugs
・ Gene silencing study
・ Knock out mice study
・ Dose-dependency
・ PK/PD
・ Data for clinical trial design
・ Seed/Lead selection
・ Proof of principle experiment
Drugs Effect on NAS Effect on Fibrosis Effect on Tumor
Telmisartan Improved Improved - Served as positive control in STAMTM mice
Pioglitazone Not improved Not improved Not improved Published in Hepatology 2012.
Disclosable under CDA
CB1 Improved Improved - Disclosable under CDA
Vitamin E Improved - Improved PLoS One, 9:e100627, 2014,
Presented in 67th Annual Meeting of Japan
Society of Nutrition and Food Science
CCL2/CCR2 inhibitor Improved Improved - Published in Int J Exp Pathol 2013
Linagliptin Improved Improved-
Med Mol Morph, DOI 10.1007/s00795-013-
0053-9
Imatinib Improved Improved - Disclosable under CDA
Utilizing the STAMTM model
-44-
■ The assays listed below will allow assessment of pharmacological effect and clarification of mechanism of
action in drug candidates.
■ Gene expression assay
Blood biochemistry・ Glucose
・ HbA1c
・ AST
・ ALT
・ TG
・ Total cholesterol
・ Lipoprotein profiling (HDL, LDL, VLDL, CM)
・ ELISAs (HA, Insulin, Leptin, Adiponectin,...)
Evaluation of inflammation・ HE staining (NAFLD Activity score)
・ IHC for macrophage marker
(inflammation area)・ IHC for mononuclear cell marker
・ IHC for scavenger receptor marker
Liver biochemistry・ TG
・ FFA
・ Cholesterol
・ Hydroxyproline (collagen)
・ ELISAs (TIMP-1, PDGF ligands, )
Evaluation of fibrosis
• Sirius-red staining (fibrosis area)
• Masson trichrome staining
• IHC for a-SMA
• IHC for Collagen Type 1
• IHC for Collagen Type 3
Evaluation of fat deposition・ Oil-red staining (steatosis area)
Inflammation-related gene
• TNF-a
• IFN-g
• IL-10
• MCP-1
• CCR2
• SOCS3
Fibrosis-related gene
• TGF-b
• TIMP-1
• a-SMA
• MMP-9
• CTGF
• PAI-1
Metabolic gene
• SREBP-1c
• FAS
• ACC
• CPT-1
• PPAR-a
• ChREBP
Evaluation of hepatocyte
proliferation/damage・ IHC for proliferation marker
・ Staining for apoptosis marker
■ Histopathological assay
■ Biochemistry
List of analysis items
-45- 32
1-2 months 1 month
Preparation
・Test compound
・Pregnant mice
・Disease induction
Contract
・Study protocol
・Quote
・Contract
Discussion
・Non-disclosure
agreement
(If needed)
Quote
Service Agreement
■ Standard process of our CRO service
Customer
SMC
Study & Report
・Treatment
・Analyses
・Report
Test
CompoundService Agreement
Work Order
Interim
report
*depending on the treatment duration and/or analysis items
3-6* months
Process
Final
report
2nd
Invoice
1st
Invoice
-46-
Below are examples of feedbacks we received from our customers regarding our service:
“In addition to quality service providers, we view the group at SMC as scientific collaborators and
colleague”
“SMC team was always willing to adapt to certain needs in terms of study design and we were very
satisfied with their performance”
“Thanks for the work and professional follow up on this study. I am very pleased with the interactions
and the deliverables.”
“SMC’s CRO service is excellent in terms of quality and timely delivery.”
“Thank you very much for the well prepared report. It was comprehensive and sound.”
“Many thanks any for your efficient and professional work on our project.”
“All their interactions and work have been professional, efficient, and of outstanding quality.”
“Thanks for sending the last interim report as promised! We are highly satisfied with the way and the
quality this study was performed.”
“Thanks for the timely delivery of the final report documentation! It was a pleasure working with you. ”
“Thank you very much for these interim results of our NASH study. Certainly these data look good.”
“We would like to thank you very much again for sending us this nice report in a timely manner. The
report is well organized.”
Support
Speed
Quality
■ Feedback from customers
Feedback from customers
Model STAMTM MCDD feedingHigh-calorie
diet feedingOb/ob KK-Ay PTEN null mice
Blood glucose >300 mg/dL 60 -100 mg/dL >200 mg/dL >200 mg/dL >200 mg/dL Not changed
Blood lipid
parameterIncrease Decrease Increase Increase Increase Increase
Steatosis Yes Yes Yes Yes Yes Yes
Steatohepatitis Yes Yes No No No Yes
Fibrosis Yes Yes No No No Yes
HCC Yes No Yes No No Yes
Effective drugs
for the model
・Telmisartan
・FXR agonist
・Galectin-3
inhibitor
・DPP-4 inhibitor
・Telmisartan
・FXR agonist
・DPP-4
inhibitor
・CB1
antagonist
・GLP-1 agonist ・Pioglitazone No drug tested
Takahashi Y., et al., World J Gastroenterol 18:2300, 2012, Nagarajan P., et al., World J Gastroenterol 18:1141, 2012
MCDD: methionine choline deficient diet
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Appendix 1: Comparison of murine NAFL/NASH models
9
Appendix 2: Clinical relevance of HCC models and STAM tumors
Table 1. Description of mouse models
■ Among the four HCC mouse models (Table 1*), STAM model is the only model that closely
recapitulated the molecular characteristics of human HCC.
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Dow M., et al., Proc Natl Acad Sci U S A. 10:1073, 2018*;partial modification
9
Appendix 2: Clinical relevance of HCC models and STAM tumors
Dow M., et al., Proc Natl Acad Sci U S A. 10:1073, 2018
■ Most molecularly similar to human HCC, with frequent mutations in Ctnnb1, similar alterations of
Wnt cell-cycle and chondroitin-modification pathways.
■ High transcriptomic similarity to high-grade, proliferative human tumors with poor prognosis.
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