CRO service specialized in NASH-HCC -Proprietary STAMTM mouse model-
SMC Laboratories, Inc. www.smclab.co.jp
Ver. 2019.8 1 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.
-3- 2 CRO expertise: Leading CRO in NASH/HCC
Over 500 clients worldwide
Over 90 peer-reviewed papers and presentations
10 successful CTA packages
The number of clients Region
550 500 450 Europe 400 Japan 350 US・ 300 Canada JPN 250 200 North Asia America 150 Europe 100 Asia・ 50 Oceania 0 2011 2012 2013 2014 (% of the customers) 2015 2016 2017 2018 (year)
-4- CRO capability:
■ 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 ■ Equipment: - CT system (In vivo) - Endoscopy (In vivo) - Confocal microscopy - Dry-chemistry analyzer - Real-time PCR - Multi-mode microplate reader - And more…
*MEXT: Ministry of Education, Culture, Sports, Science and Technology
-5- CRO portfolio: Nonclinical disease models
Available Services
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 Disease model lineup
■ STAMTM: Premium model for NASH-HCC (mice) ■ Other inflammation/fibrosis/cancer models (mice) CCl4 BLM
・Liver fibrosis: CCl4 model, BDL model
・Acute liver failure : CCl4 model, Concanavalin A model D-gal/LPS model, TAA model Sirius red CT (Lung) ・Pulmonary fibrosis: BLM-induced lung fibrosis model UUO DSS ・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 PAS Endoscopy ・Liver cancer: DEN-CCl4 liver cancer model ・Alzheimer’s disease: icv-STZ model -6- Overview
1 Company 2 Rationale: NASH 3 STAMTM: Proprietary model for NASH-HCC 4 Pharmacological study 5 CRO service
-7- Why focus on NASH?
■ 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 (NASH) ■ No established treatment
■ Economic loss -1 billion USD per year (US) Causes of Death in Japanese Diabetics ■ 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 -8- NASH: clinical process and diagnosis
Steatosis NASH Fibrosis HCC
10-30% 10-29%** 4-27%** (NASH to fibrosis) (NASH to HCC)
6.3 – 33% (med. 20%) 3 – 5% Similar to HCV in general population* in general population*
Screening/Initial evaluation
US Diagnosis -fatty change NAFL -chronic change NAFLD Liver biopsy NASH +/- fibrosis ALT↑ Imaging (CT, MR..) +/- HCC HBsAg (-) HCV (-) NAFLD fibrosis score ANA (-)~low Alcohol (-)~low
US: Ultra Sound, CT: Computed Tomography, MR: Magnetic Resonance
*CVD: Cardiovascular*Chalasani N., disease et al., Gastroenterology 142:1592, 2012 (AGA guideline), **Cohen J., et al., Science 332:1519, 2011
-9- No approved drugs: AGA guideline 2012
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
● Pioglitazone can be used to treat steatohepatitis in patients with biopsy-proven NASH Tiazolidinediones 1 B ● Long term safety and efficacy is not established ● α-tocopherol (800 IU/day) improves liver histology in non-diabetic adults with biopsy-proven 1 B Vitamin E NASH → First-line pharmacotherapy for this patient population ● 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 acids may be considered as first-line therapy for hypertriglycemia in patients Omega-3 fatty acid 1 B with NAFLD, but it is premature to recommend them ● Statins can be used to treat dyslipidemia in patients with NAFLD/NASH, but should not be Statin 1 B used to specifically treat NASH, pending evidence from RCTs
*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)
-10- Drug candidates in clinical trials
Company Drug Target Route Period Endpoint Stage
Intercept: OCA - 18 months 1)Histology P3 FXR REGENERATE study 2)Fibrosis and NASH
GENFIT: Elafibranor PPARα /δ Oral 72 weeks 1)Histology P3 RESOLVE-IT study 2)Fibrosis
Galmed Aramchol Synthetic fatty acid Oral 52 weeks 1)% change in liver triglyceride P3 bile conjugate 2)Fibrosis, NAS etc.
Allergan: Cenicriviroc (CVC) CCR5/CCR2 antagonist Oral 1 year 1)Fibrosis P3 AURORA study
Allergan: Cenicriviroc (CVC) CCR5/CCR2 antagonist Oral 1 year 1)NAS P2 CENTAUR study
Novartis/Allergan: Tropifexor (LJN452) FXR Oral 48 weeks 1) Number of participants with Adverse Events P2 TANDEM study Cenicriviroc (CVC) CCR5/CCR2 antagonist Oral 2) Fibrosis
Novo Nordisk: Liraglutide GLP-1 SC 48 weeks 1)Histology P2 LEAN study 2)NAS
Conatus: Emricasan Caspase inhibitor Oral 72 weeks 1)Fibrosis P2 ENCORE-NF study 2)NAS
Gilead GS-9674 FXR Oral 24 weeks plus 30 1)Overall safety profile P2 days
Gilead GS-0976 ACC Oral 24 weeks plus 30 1)Overall safety profile P2 days
Gilead Simtuzmab Loxl2 SC 96 weeks 1)Fibrosis P2 2)Safety
BMS BMS-986036 FGF21 - 16 weeks 1)Change in percent hepatic fat fraction P2 2) Average concentration
Galectin GR-MD-02 Galectin-3 IV 16 weeks 1)LiverMultiScan P2 Therapeutics 2)MR-elastography
NGM NGM282 FGF19 - 12 weeks 1)Change in absolute liver fat content P2 2)Change in percentage liver fat content
Cempra Solithromycin Ketolide antibiotic Oral 13 weeks 1)NAS P2 2)Changes in steatosis
Boehringer Ingelheim BI 1467335 SSAO/VAP-1 Oral 12 weeks 1)Target enzyme activity relative to baseline in P2 percent 2)Relative to ALT, AST, AP, GGT ,cleaved CK18, total CK18 change from baseline
-11- Source: clinicaltrial.gov Overview
1 Company 2 Rationale: NASH 3 STAMTM: Proprietary model for NASH-HCC 4 Pharmacological study 5 CRO service
-12- 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.
-13- STAMTM: In vivo predictive pharmacology model
Steatosis NASH Fibrosis HCC
100% 100% 100%
① CHEMICAL ② DIET 1st hit Continuous 2nd hit - low dose streptozotocin - - high fat diet feeding -
Preparing pregnant C57BL/6J mice 4w 5w 6w 7w 9w 12w 16w
Birth Fatty liver evident 0w NASH evident Fibrosis* evident Nodule evident HCC evident
All mice at 6 weeks of age meet Fatty change (+) “baseline” criteria as in the case ALT↑ of clinical trial in human NAFLD Activity score↑
-14- * Perisinusoidal fibrosis resemble to human NASH 12 Mechanisms of STAMTM mice
2 day 4wks 5wks 7wks 9wks 12wks 16wks 20wks
Birth 0w 16 wks 〜HCC phase
12 wks 〜Nodule formation
9-12 wks: Fibrosis (to chronic fibrosis) phase
7-8 wks: Steatohepatitis phase
5-6 wks: Steatosis phase
2nd hit 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 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”).
-15- Examples of treatment period
4w 5w 7w 9w 12w 16w 20w
Birth 0w Steatosis-targeting study 5w 8w
NASH-targeting study 6w 9w
Fibrosis-targeting study 9w 12w
HCC-targeting study 16w 20w
HCC-prevention study 6w 9w 20w
-16- NAFLD spectrum of STAMTM mice
Macroscopic and histological appearance of STAMTM mice 6 wks 8 wks 12 wks 20 wks
(steatosis) (steatohepatitis) (chronic fibrosis) (HCC)
appearance
Macroscopic HE staining staining HE Original Magnification: x200 8 wks (steatohepatitis)
Ballooning degeneration Inflammatory foci (arrow head) (center) n=3-6 (Mean ± SD) Original Magnification: x400
■ STAMTM mice develop steatosis at 6 wks, steatohepatitis at 8 wks, chronic fibrosis at 12 wks and HCC at 20 wks of age. -17- General parameters in STAMTM mice
Body weight, liver weight and biochemistry
(mg/dL)
Body Body weight(g)
Liver weight (mg)weightLiver
Fastingblood glucose
Serum ALT ALT Serum (U/L)
Serum AST AST Serum (U/L) Liver TG (mg/gTGliver) Liver
n=3-6 (Mean ± SD) ■ 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).
ALT: Alanine aminotransferase, AST: Aspartate aminotransferase, TG: Triglyceride
-18- Metabolic parameters in STAMTM mice
Serum insulin and whole blood HbA1c at 9 weeks of age
Non-fasting Fasting
n=6 (Mean ± SD) ■ No changes were noted in the fasting serum insulin levels, but non-fasting serum insulin levels were decreased in STAMTM mice. TM ■ Whole blood HbA1c levels were increased in STAM mice compared to normal mice under fasting and non-fasting conditions. -19- Lipid parameters in STAMTM mice
Serum cholesterols and TG at 9 weeks of age
n=6 (Mean ± SD)
■ Serum cholesterol (VLDL, HDL, LDL and chylomicron) and triglyceride levels are increased in STAMTM mice with age.
VLDL: very low density lipoprotein, HDL: high-density lipoprotein, LDL: low-density lipoprotein -20- NASH and HCC biomarkers in STAMTM mice
Plasma CK-18 at 9 weeks of age and serum AFP at 20 weeks of age
n=6 (Mean ± SD)
■ 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 -21- Steatosis and inflammation in STAMTM mice
Oil-red staining and F4/80 immunostaining in STAMTM mice
6 wks 8 wks 12 wks 20 wks (steatosis) (steatohepatitis) (chronic fibrosis) (HCC)
* *
* * Oilred
** ** *
F4/80 F4/80 **
Original Magnification: x200 *: Central vein ■ 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.
-22- 14 Fibrosis in STAMTM mice
Sirius-red staining and ER-TR7 immunostaining in STAMTM mice
6 wks 8 wks 12 wks 20 wks (steatosis) (steatohepatitis) (chronic fibrosis) (HCC)
* *
Siriusred * * TR7 - *
ER * * *
Original magnification: upper panel (x 400), lower panel (x 200) *: Central vein ■ 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.
-23- Histological parameters in STAMTM mice
Quantitative analyses of % positive areas for F4/80, Sirius red and ER-TR7 in the liver
Macrophages Fibrosis Fibroblasts
*** *** ***
*** *** ** * *
** positive area (%) area positive
- *
TR7 positive area (%) TR7area positive
-
F4/80 positive area (%) area positive F4/80
ER Sirius red Sirius
*: p<0.05, **: p<0.01, ***: p<0.001 vs Normal, n=4 (Mean ± SD)
■ 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.
#: At 20 weeks of age, non-HCC lesions were analyzed. -24- Gene expressions in the liver of STAMTM mice
Real-time PCR assays in STAMTM mice
14 ** 18 *** 30 ** 16 25 *** 12 14 10 12 20 * 8
10 /36B4
g
1/36B4
/36B4 - - 15 a 6 8 - * **
6 10 IFN 4 MCP 4 TNF * 5 2 2
Inflammation 0 0 0 8w 6w 8w 12w 20w 8w 6w 8w 12w 20w 8w 6w 8w 12w 20w Normal STAM Normal STAM Normal STAM 30 ** 5 10 ** 25 4 8 * ** 20
3 /36B4
1/36B4 6
* b * -
15 - 2 *
10 4
TGF
TIMP Fibrosis 5 2 1 0 1/36B4 Type Collagen 0 0 8w 6w 8w 12w 20w 8w 6w 8w 12w 20w 8w 6w 8w 12w 20w Normal STAM Normal STAM Normal STAM
*: p<0.05, **: p<0.01, ***: p<0.001 vs Normal, n=3-4 (Mean ± SD) ■ 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.
-25- HCC in STAMTM mice
Multiple tumors in STAMTM mice (20 wks) Individual Mean tumor Tumor growth tumor diameter diameter rate
Dynamic CT in STAMTM mice (20 wks)
Histological images of STAMTM mice (20 wks)
HE staining GS immunostaining
■ STAMTM mice develop multiple tumor in the liver. ■ ”Early wash-in and late wash-out”. ■ Individual tumor diameter gradually increases with age. -26- 18 Survival curve in STAMTM mice
Kaplan-Meier survival curve of STAMTM mice (no intervention)
100% 95% (~8 wks) (~12 wks)
70% 100 (~16 wks) 90 55% 80 (~20 wks) 70 60 50 40 Steatosis Steatohepatitis phase phase
Survival rate (%) rate Survival 30 20 Fibrosis Nodule HCC phase formation phase 10 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 weeks of age
■ The mortality rate at 16 and 20 weeks of age are 30% and 45%, respectively.
-27- Overview
1 Company 2 Rationale: NASH 3 STAMTM: Proprietary model for NASH-HCC 4 Pharmacological study 5 CRO service
-28- Pharmacological study-1: Study design
■ 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].
■ Study design Birth (0 wks) 4 wks 5 wks 6 wks 7 wks 8 wks 9 wks
Normal diet feeding Normal (n=6) High fat diet feeding Oral, QD for 3 weeks STAMTM-Vehicle (n=6) Oral, QD for 3 weeks STAMTM-Telmisartan 10 mg/kg (n=6)
Day 2: Sacrifice Injection of streptozotocin ■ Analyses
General Biochemistry Histopathological assay Gene expression assays Body weight Serum ALT HE staining (NAS score) TNF-a • α-SMA Liver weight Liver TG Sirius red staining (Fibrosis area) MCP-1 • TIMP-1 Liver-to-body weight ratio
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 -29- Pharmacological study-1: Results
Effects of telmisartan on general conditions and biochemistry Serum ALT
Mean ± SD
Mean ± SD Figure 1. Body weight changes
Liver TG
Mean ± SD Mean ± SD Figure 2. Liver weight Figure 3. Biochemistry (serum ALT and liver TG)
■ Telmisartan significantly inhibited the increase in liver weight and liver TG levels, and tended to decrease the serum ALT levels.
-30- Pharmacological study-1: Results
Effects of telmisartan on histopathological analysis HE-staining Sirius red-staining
Normal Vehicle Telmisartan Normal Vehicle Telmisartan
* * *
x200 x400 : central vein NAFLD Activity score Fibrosis area Steatosis score *
Inflammation score
Ballooning score
Mean ± SD
Mean ± SD
Figure 4. Effects of telmisartan on Figure 5. Effects of telmisartan on liver fibrosis NAFLD Activity score ■ Telmisartan significantly inhibited the increase NAFLD Activity score and fibrosis area. -31- 24 Pharmacological study-1: Results
Effects of telmisartan on inflammation and fibrosis related gene expressions
TNF-a MCP-1 a-SMA TIMP-1
Figure 6. The expression of inflammatory and fibrosis related genes in the liver Mean ± SD
■ 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.
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.
-32- Pharmacological study-2: Study design
■ Study aim: To investigate potential effects on NASH-HCC with sorafenib ■ Design Birth (0 wks) 4 wks 16 wks 17 wks 18 wks 19 wks
High fat diet feeding Oral, QD for 3 weeks STAMTM-Vehicle (n=10)
Oral, QD for 3 weeks STAMTM-Sorafenib 30 mg/kg (n=3)
CT evaluation for CT evaluation Day 2: selection and Sacrifice Injection of streptozotocin randomization (Vehicle = 6, Sorafenib = 3) Oral: oral administration, QD: once-daily Survival rate (%) Vehicle: 60.0% Sorafenib: 100% ■ Analyses
General Survival rate Tumor analysis Body weight Kaplan-Meier survival curve Assessment of visible tumor number Liver weight Assessment of visible tumor size Liver-to-body weight ratio Assessment of SOL volume Assessment of SOL volume growth ratio -33- Pharmacological study-2: Results
Effects of Sorafenib on survival and body weight
28 100 90 24 80 20 70 60 16 50 12 40 30 8 Vehicle (n=10) Vehicle
Body (g) weight Percentsurvival 20 4 10 Sorafenib (n=3) Sorafenib 0 0 0 7 14 21 0 7 14 21 Days after the start of treatment Days after the start of treatment
Figure 1. Survival curves from Figure 2. Body weight changes from 16 to 19 weeks of age 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.
-34- Pharmacological study-2: Results
Effects of Sorafenib on liver weight and visible tumors on the surface of liver
n.s.
4000
3000
2000
1000
Liver weight (mg)Liver weight Vehicle
0 Vehicle Sorafenib
Figure 4. Liver weight
n.s. n.s.
7 45 6 40 35 5 30 4 25 3 20 15 2
Tumor number 10
1 5 Sorafenib
0 Total tumor diameter (mm) 0 Vehicle Sorafenib Vehicle Sorafenib
Figure 3. Macroscopic appearance of livers Figure 5. The number Figure 6. The sum of of visible tumor (>2 mm) tumor diameters
■ 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. -35- Pharmacological study-2: Results
Effects of Sorafenib on tumor growth assessed by contrast-enhanced CT 16 wks (before) 19 wks (after)
600% increase P<0.05
1400
Vehicle 1200 1000 800 600 Volume: 47.3 mm3 Volume: 284.5 mm3 400 200
Mean SOL growth (%) rate Mean 0 Vehicle Sorafenib
36% decrease Figure 8. Comparison of the
growth rate of SOL Sorafenib
Volume: 54.2 mm3 Volume: 34.9 mm3 SOL: space occupied lesion Figure 7. Contrast-enhanced CT images of liver
■ Sorafenib significantly decreased the growth rate of SOL. CT evaluation enables accurate assessment of tumor development.
-36- Pharmacological study-2: Study design
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.
Birth 0w HCC-targeting study 16w 20w
HCC-prevention study 6w 9w 20w
-37- Publications and Presentations
■ Publications 42. International Journal of Gastroenterology, "Characterization of EDP-305, a Highly Potent and 26. Molecular Cancer Research, “Inhibition of the cell death pathway in non-alcoholic steatohepatitis Selective Farnesoid X Receptor Agonist, for the Treatment of Non-alcoholic Steatohepatitis" (International (NASH)-related hepatocarcinogenesis is associated with histone H4 lysine 16 deacetylation” (Molecular Journal of Gastroenterology, DOI: 10.11648/j.ijg.20190301.12, 2019) Cancer Research, DOI:10.1158/1541-7786.MCR-17-0109, 2017) 41. Experimental Animals, "Analysis of amino acid profiles of blood over time and biomarkers associated 25. Magnetic Resonance Imaging, “The natural history of streptozotocin-stimulated non-alcoholic with non-alcoholic steatohepatitis in STAM mice" (Exp Anim., DOI: 10.1538/expanim.18-0152, 2019) steatohepatitis mice followed by Gd-EOB-DTPA-enhanced MRI: Comparison with simple steatosis mice.” (Magn Reson Imaging, 38:123-128, 2017) 40. Frontiers in Genetics, "Gene Expression and DNA Methylation Alterations During Non-alcoholic Steatohepatitis-Associated Liver Carcinogenesis" (Front Genet., May 29;10:486, 2019) 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) 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) 23. Oncotarget, “Distinctly altered gut microbiota in the progression of liver disease” (Oncotarget, 7: 19355-19366, 2016) 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) 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 37. NPJ Precision Oncology, "Transcriptomic analysis of hepatocellular carcinoma reveals molecular steatohepatitis and diabetes“ (Diabetology & Metabolic Syndrome, 8:45, 2016) features of disease progression and tumor immune biology" (NPJ Precis Oncol., DOI: 10.1038/s41698- 018-0068-8, 2018) 21. Journal of Immunology, Infection & Inflammatory Diseases, “Solithromycin Diminishes Steatohepatitis by Modulating Gluconeogenesis and Inhibits Tumor Growth in a Diabetic Mouse Model of 36. Cellular and Molecular Gastroenterology and Hepatology, "Dipeptidyl Peptidase 4 inhibitors Non-Alcoholic Steatohepatitis” (J Immunol Infect Inflam Dis, 1:004, 2016) Reduce Hepatocellular Carcinoma by Activating Lymphocyte Chemotaxis in Mice" (CMGH, DOI: 10.1016/j.jcmgh.2018.08.008, 2018) 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) 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- 19. Cell Reports, “Cancer-Associated Fibroblasts Regulate Tumor-Initiating Cell Plasticity in 476, 2018) Hepatocellular Carcinoma through c-Met/FRA1/HEY1 Signaling” (Cell Press, 15:1175-1189, 2016) 34. Proc Natl Acad Sci U S A, “Integrative genomic analysis of mouse and human hepatocellular 18. International Journal of Medical Sciences, “Palmitate-induced Regulation of PPARγ via PGC1α: a carcinoma” (Proc Natl Acad Sci U S A, DOI: 10.1073/pnas.1811029115, 2018) Mechanism for Lipid Accumulation in the Liver in Nonalcoholic Fatty Liver Disease” (Int. J. Med. Sci, 13:169-178, 2016) 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, 17. European Journal of Pharmacology, “Lipid-lowering agents inhibit hepatic steatosis in a non- 2018) alcoholic steatohepatitis-derived hepatocellular carcinoma mouse model” (Eur J Pharmacol, 772:22-32, 2016) 32. PLoS One, “Gemcabene downregulates inflammatory, lipid-altering and cell-signaling genes in the STAM™ model of NASH” (PLoS One, 13(5): e0194568 , 2018) 16. Scientific Reports, “Characterization of hepatic lipid profiles in a mouse model with nonalcoholic steatohepatitis and subsequent fibrosis” (Sci Rep., 12466, 2015) 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, 15. International Journal of Obesity, “Low cytochrome oxidase 4I1 links mitochondriazzzl dysfunction to 2018) obesity and type 2 diabetes in humans and mice” (Int J Obes, 39:1254-63, 2015) 30. The FASEB Journal, “Epigenetically mediated inhibition of S-adenosylhomocysteine hydrolase and 14. Proc Natl Acad Sci U S A, “Immunomodulatory spherical nucleic acids” (Proc Natl Acad Sci U S A, the associated dysregulation of 1-carbon metabolism in nonalcoholic steatohepatitis and hepatocellular 31;112:3892-7, 2015) carcinoma“ (FASEB J, DOI:10.1096/fj.201700866R, 2017) 13. Oncology Reports, “Hepatic expression of the Sptlc3 subunit of serine palmitoyltransferase is 29. Oncotarget, “MicroRNA deregulation in nonalcoholic steatohepatitisassociated liver carcinogenesis” associated with the development of hepatocellular carcinoma in a mouse model of nonalcoholic (Oncotarget, 8:88517-88528, 2017) steatohepatitis” (Oncol Rep, 33:1657-66, 2015) 28. Oncotarget, “Peretinoin, an acyclic retinoid, suppresses steatohepatitis and tumorigenesis by 12. Drug R D, “In Vivo Efficacy Study of Milk Thistle Extract (ETHIS-094TM) in STAMTM Model of activating autophagy in mice fed an atherogenic high-fat diet” (Oncotarget, 8:39978-39993, 2017) Nonalcoholic Steatohepatitis” (Drugs R D, 14:291-9, 2014) 27. Physiological Research, “Pathophysiological analysis of the progression of hepatic lesions in STAM 11. PLoS One, “Photoacoustic Tomography of Human Hepatic Malignancies Using Intraoperative mice.” (Physiological Research, 66:791-799, 2017) Indocyanine Green Fluorescence Imaging” (PLoS One, 9:e112667, 2014) -38- Publications and Presentations
■ Publications (continued) 10. Cancer Science, “Silencing of microRNA-122 is an early event during hepatocarcinogenesis from non- 66. AASLD 2018, “Dipeptidyl Peptidase 4 Inhibitors Reduce the Progression of Hepatocellular Carcinoma alcoholic steatohepatitis” (Cancer Sci, 105:1254-60, 2014) By Activating T Cell and Natural Killer Cell Chemotaxis in Mice” Kawasaki Medical School 9. Anticancer Research, “Characterization of non-alcoholic steatohepatitis-derived hepatocellular 65. AASLD 2018, “Effects of a DPP4 Inhibitor on Progression of Nash-Related Hepatoma and DNA carcinoma as a human stratification model in mice” (Anticancer Res, 34:4849-4856, 2014) Synthesis Pathway Via p62/Keap1/Nrf2 in a Mouse Model: A Metabolomic Analysis” Kurume University 8. PLoS One, “L-carnitine prevents progression of non-alcoholic steatohepatitis in a mouse model with School of Medicine upregulation of mitochondrial pathway.” (PLoS One, 9:e100627, 2014) 64. AASLD 2018, “Gemcabene Regulates Hepatic Genes Associated with Inflammation and Fibrosis with 7. Medical Molecular Morphology, “Linagliptin alleviates hepatic steatosis and inflammation in a mouse Impact on Non-Alcoholic Fatty Liver Disease” Gemphire Therapeutics Inc. model of non-alcoholic steatohepatitis” (Med Mol Morph, 47:137-149) 63. AASLD 2018, “CM101, a Novel CCL24 Blocking Antibody, Suppresses Hepatic Injury and Fibrosis In 6. PLoS One, “Therapy of Experimental NASH and Fibrosis with Galectin Inhibitors” (PLoS One, Experimental Models of Nash and Liver Fibrosis” ChemomAb Ltd. 8:e83481, 2013) 62. AASLD 2018, “Unexpected Antidiabetic Effects Combined with Antifibrotic Activities of LXR Inverse 5. International Journal of Oncology, “Identification of an H2-Kb or H2-Db restricted and glypican-3- Agonists in Mouse Models of NAFLD/Nash” Phenex Pharmaceuticals AG derived cytotoxic T-lymphocyte epitope peptide” (Int J Oncol, 42:831-838, 2013) 61. The 78th Scientific Sessions ADA, 2018, “Canagliflozin, an SGLT2 Inhibitor, Prevents Development 4. International Journal of Experimental Pathology, “Inhibition of Glutaminyl Cyclases alleviates CCL2- of Hepatocellular Carcinoma (HCC) from Nonalcoholic Steatohepatitis (NASH) in a Mouse Model of NASH- mediated inflammation of non-alcoholic fatty liver disease in mice” (Int J Exp Pathol, 94: 217-225, 2013) HCC Under Diabetic State” Dokkyo Medical University 3. Medical Molecular Morphology, “A murine model for non-alcoholic steatohepatitis showing evidence 60. The 78th Scientific Sessions ADA, 2018, “Combination of SGLT2 Inhibitor and Novel Selective of association between diabetes and hepatocellular carcinoma” (Med Mol Morph, 46:141-152, 2013) PPARα Modulator, Tofogliflozin (Tofo) and Pemafibrate (Pema), Improves Survival Rate in STAM Mice as a 2. Hepatology, “Hydrogen-rich water prevents progression of non-alcoholic steatohepatitis and Diabetic NASH Model” Kowa Company Ltd. accompanying hepatocarcinogenesis in mice” (Hepatology, 56:912-921, 2012) 59. EASL the International Liver CongressTM 2018, “Interfering with local fibrotic platelet activation 1. Journal of Nutritional Science and Vitaminology, “Effects of Dietary Supplementation with Betaine significantly inhibits fibrosis in multiple animal models: suggestions of the importance of the platelet-wound on a Nonalcoholic Steatohepatitis (NASH) Mouse Model” (J Nutr Sci Vitaminol, 58:371–375, 2012) healing axis for fibrosis” Symic Bio, Inc. ■ Presentations 58. EASL the International Liver CongressTM 2018, “BMS-986036, a PEGylated fibroblast growth factor 73. DDW 2019, “Change of Gut Microbiome after Treatment with the Traditional Japanese Medicine 21 analogue, reduces fibrosis and PRO-C3 in a mouse model of non-alcoholic steatohepatitis” Bristol-Myers Daisaikoto is Associated with Improved Liver Steatosis in a Non-alcoholic Fatty Liver Mouse Model” Squibb Company TSUMURA & Co. 57. EASL the International Liver CongressTM 2018, “LJN452 (tropifexor) attenuates steatohepatitis, 72. DDW 2019, “Influence of the O-GlcNAc Modification in Hepatic Carcinogenesis by Non-alcoholic Fatty inflammation, and fibrosis in dietary mouse models of nonalcoholic steatohepatitis” Genomics Institute of Liver Disease” Osaka Medical College the Novartis Research Foundation 71. EASL the International Liver CongressTM 2018, “LXR inverse agonists reduce steatosis and fibrosis 56. EASL the International Liver CongressTM 2018, “Clinical-grade human liver mesenchymal stem cells in the STAM mouse model but also improve insulin sensitivity in a high fat diet mouse clamp study” Phenex reduce NAS score and fibrosis progression in advanced stage NASH pre-clinical model through Pharmaceuticals AG immunomodulation” Promethera Biosciences LLC 70. 3rd Annual World Preclinical Congress Europe 2018, “LXR Inverse Agonists for the Treatment of 55. First EASL NAFLD Summit 2017, “Dual CCR2/5 antagonist decreases hepatic inflammation in acute NASH” Phenex Pharmaceuticals AG liver injury and NASH metabolic animal models” Pfizer Inc. 69. 3rd Annual World Preclinical Congress Europe 2018, “MTBL0036, a Promising, New Anti-NASH 54. First EASL NAFLD Summit 2017, “AXA1125, a novel defined amino acid composition (DAAC), and Antifibrotic Candidate: MTBL0036 showed a decrease in NAFLD Activity score in the STAM model” improves NAFLD activity score (NAS) and reduces fibrosis in two rodent models of nonalcoholic Metabolys, Inc. steathepatitis (NASH)” Axcella Health, Inc. 68. AASLD 2018, “AXA1125, a Novel Composition of Amino Acids Reprograms the Multifactorial 53. AASLD 2017, “The Anti-Fibrogenic and Liver Protective Effects of Namodenoson (CF102) in a Non- Pathophysiology in NAFLD” Axcella Health Inc. Alcoholic Steatohepatitis Model” Can-Fite BioPharma Ltd. 67. AASLD 2018, “Treatment of Hepatocellular Carcinoma Using 2-Deoxy-D-Glucose Encapsulated in PLGA Nanoparticles in Mice” Kawasaki Medical School -39- Publications and Presentations
■ Presentations (continued) 52. AASLD 2017, “DPP4 Inhibitor Suppressed Progression of NASH-related Hepatocellular Carcinoma 36. WDC 2015, “Empagliflozin (an SGLT2 inhibitor), alone or in combination with linagliptin (a DPP-4 with Inhibition of Metabolic Reprograming in p62-Keap 1-Nrf2-pentose Phosphate Pathway in a Mouse inhibitor), prevents steatohepatitis in a novel mouse model of non-alcoholic steatohepatitis and diabetes” Model: A Metabolomic Analysis” Kurume University School of Medicine Dokkyo Medical University 51. AASLD 2017, “CB4209 and CB4211 Reduce the NAFLD Activity Score in the STAM Model of NASH, 35. AASLD 2015, “Anti-Fibrotic Effect of Autotaxin and LPA1R Antagonists in a Rodent Model of NASH” Reduce Triglyceride Levels, and Induce Selective Fat Mass Loss in DIO Mice” CohBar, Inc. Bristol-Myers Squibb Company 50. AASLD 2017, “Combination Treatment of LJN452 and Cenicriviroc Snows Additive Effects in a Diet- 34. AASLD 2015, “Sitagliptin, a Dipeptidyl Peptidase 4 inhibitor, Suppressed Tumor Progression with Induced NASH Model” Genomics Institute of the Novartis Research Foundation/Allergan plc/Novartis Down-regulation of Nrf Nuclear Expression in a Mouse Model of Non-alcoholic Steatohepatitis-related Institutes for BioMedical Research, Inc. Hepatocellular Carcinoma” Kurume University School of Medicine 49. AASLD 2017, “Gemcabene Attenuates the NAFLD Activity and Fibrosis Scores, and Downregulates 33. AASLD 2015, “Reduction of Hepatic 27-Hydroxycholesterol in Steatohepatitis Model Mice with Insulin Hepatic Inflammatory Genes in the STAMTM Murine Model of NASH-HCC” Gemphire Therapeutics Inc. Resistance” Tokyo Medical University Ibaraki Medical Center 48. DDW 2017, “A HMG-CoA Reductase Inhibitor, Rosuvastatin, as a Potential Preventive Drug for The 32. AASLD 2015, “Disturbance of regulatory T cells, MDSCs and NK cells is involved in NASH and mouse Development of Hepatocellular Carcinoma Associated With Non-alcoholic Fatty Liver Disease in Mice” model of NASH” Tohoku University Hospital. Osaka Medical College 31. AASLD 2015, “Mechanism of Action of the Anti-NASH effects of Solithromycin in a Predictive NASH TM 47. EASL the International Liver Congress 2017, “Anti-fibrotic effect of NV556,a sanglifehrin-based HCC Mouse Model” Cempra Pharmaceuticals, Inc. cyclophilin inhibitor,in a preclinical model of non-alcoholic steatohepatitis” Neuro Vive Pharmaceutical AB 30. DDW 2015, “Effects of Sitagliptin, a Dipeptidyl Peptidase 4 Inhibitor, on Tumor Progression and 46. AACR 2017, “Inhibition of gene expression during non-alcoholic steatohepatitis (NASH)-related p62/SQSTM1 Subcellular Localization in a Mouse Model of Non-Alcoholic Steatohepatitis-Related hepatocarcinogenesis is mediated by histone H4 lysine 16 deacetylation” FDA-National Center for Hepatocellular Carcinoma” Kurume University Toxicological Research. 29. Keystone Symposia 2015, “DGAT2 Inhibition Prevents NAFLD and Fibrosis in the STAM Mouse 45. AACR 2017, “Alterations in the chromatin accessibility in nonalcoholic steatohepatitis-associated Model of NASH“ Pfizer Inc. hepatocellular carcinoma” FDA-National Center for Toxicological Research 28. Keystone Symposia 2015, “Oxidized-Phospholipid Small Molecule Inhibits Non-Alcoholic 44. AACR 2017, “Role of miRNAome deregulation in the pathogenesis of non-alcoholic steatohepatitis Steatohepatitis (NASH) and Liver Fibrosis“ Vascular Biogenics Ltd (NASH)-derived hepatocellular carcinoma” FDA-National Center for Toxicological Research 27. AASLD 2014, “L-carnitine prevents progression of non-alcoholic steatohepatitis in a mouse model with 43. AASLD 2017, Emerging Trends Conference: Emerging Trends in Non-Alcoholic Fatty Liver upregulation of mitochondrial pathway“ Department of Gastroenterology and Hepatology, Okayama Disease, “The Novel Antidiabetic Candidate MTBL0036 Greatly Diminishes The NAFLD Activity Score in University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences The STAM Mouse Model of NASH” Metabolys Inc. 26. AASLD 2014, “MN-001 (tipelukast), a novel, orally bioavailable drug, reduces fibrosis and inflammation 42. AASLD 2017, Emerging Trends Conference: Emerging Trends in Non-Alcoholic Fatty Liver and down-regulates TIMP-1, collagen Type 1 and LOXL2 mRNA overexpression in an advanced NASH Disease, “DUR-928, An Endogenous Regulatory Molecule, Exhibits Anti-Inflammatory and Antifibrotic (nonalcoholic steatohepatitis) model“ MediciNova, Inc. Activity in a Mouse Model of NASH” DURECT Corporation 25. ICLAF 2014, “MN-001 (tipelukast), a nonselective phosphodiesterase, 5-lipoxygenase, leukotriene, 41. AASLD 2016, “A Phase 2 study of BMS-986036 (Pegylated FGF21) in Obese Adults with Type 2 phospholipase C and thromboxane A2 inhibitor, demonstrates anti-fibrotic effects in the bleomycin-induced Diabetes and a High Prevalence of Fatty Liver” Bristol-Myers Squibb Company idiopathic pulmonary fibrosis mouse model“ MediciNova, Inc. 40. AASLD 2016, “Effects of BMS-986036 (pegylated fibroblast growth factor 21) on hepatic steatosis and 24. ADA 2014, “Liraglutide prevents steatohepatitis, liver fibrosis, and accompanying carcinogenesis in a fibrosis in a mouse model of nonalcoholic steatohepatitis” Bristol-Myers Squibb Company . diabetes and nonalcoholic steatohepatitis mouse model treated with STZ-HFD“ Saga University 39. DDW 2016, “Inhibition of the Ileal Bile Acid Transporter (IBAT) by A4250 Reduces Hepatic Damage in 23. ATS 2014, “Solithromycin Reduces Inflammation In Mice Caused By Bleomycin-Induced Lung Injury“ a Mouse Model of Non-Alcoholic Steatohepatitis (NASH)” Albireo AB Cempra, Inc. 38. EASL the International Liver CongressTM 2016, “DPP4 Inhibitor Suppresses Steatohepatitis and 22. DDW 2014, “Anti-NASH Effects of Solithromycin in NASH-HCC Mouse Model“ Cempra, Inc. HCC Progression with Glucose Re-Programing in a Mouse Model of NASH” Kurume University School of 21. AACR 2014, “Clinicopathological characterization of non-alcoholic Steatohepatitis (NASH)-derived Medicine Hepatocellular carcinoma (HCC) as a patient stratification model in mice)” The Jikei University School of 37. HEP DART 2015, “The Cyclophilin Inhibitor, CPI-431-32, is a Hepatitis B Oral Drug Candidate with Medicine Antiviral and Antifibrotic Activities” Ciclofilin Pharmaceuticals Inc. -40- Publications and Presentations
■ Presentations (continued) 20. Keystone Symposia 2014, “The NADPH Oxidase (NOX) Inhibitor GKT137831 Alleviates Liver 5. EASL The International Liver CongressTM 2012 - 47th Annual Meeting of the European Inflammation and Fibrosis in a Mouse Model of Non-Alcoholic Steatohepatitis (NASH)” Genkyotex S.A. Association for the Study of the Liver, “FXR agonists prevent steatosis, hepatocyte death and 19. 15th International Workshop on Co-morbidities and Adverse Drug Reactions in HIV, “Anti-fibrotic progression of NASH towards HCC in a hypoinsulinaemic mouse model of progressive liver disease” and anti-inflammatory activity of the dual CCR2 and CCR5 antagonist cenicriviroc in a mouse model of Phenex Pharmaceuticals AG NASH” Tobira Therapeutics Inc. 4. AASLD 2011, “The Dipeptidyl Peptidase-4 Inhibitor Linagliptin is an Effective Therapeutic for Metabolic 18. AASLD 2013, “Anti-fibrotic and anti-inflammatory activity of the dual CCR2 and CCR5 antagonist Liver Disease in Several Rodent Models of Non-Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic cenicriviroc in a mouse model of NASH” Tobira Therapeutics Inc. Steatohepatitis (NASH)” Boehringer Ingelheim GmbH & Co. KG 17. AASLD 2013, “L-carnitine prevents progression of non-alcoholic steatohepatitis with regulation of 3. EASL Special Conference - Liver Transplantation 2011, “Improvement of steatosis, inflammation, mitochondrial β-oxidation and redox system in NASH model Mice” Department of Gastroenterology and and fibrosis in a mouse model of steatohepatitis following treatment with galectin inhibitor” Galectin Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Therapeutics Inc. 16. FASEB SRC 2013, Lysophospholipid and Other Related Mediators - From Bench to Clinic, “ATX 2. EASL The International Liver CongressTM 2011 - 46th Annual Meeting of the European inhibition prevents progression of non-alcoholic steatohepatitis (NASH) in a hypoinsulinemic mouse model Association for the Study of the Liver, “Novel FXR agonists with potent lipid lowering, insulin sensitising, of progressive liver disease” F. Hoffmann-La Roche, Ltd anti-inflammatory and anti-fibrotisation effects in mouse models of metabolic syndrome and NASH” Phenex Pharmaceuticals AG 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 1. The 9th JSH SingleTopic Conference “NASH 2010”, “Strong Anti-steatotic and Anti-fibrotic Effects of and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Novel FXR Agonists in a Murine NASH Model that Resembles Human NASH” Phenex Pharmaceuticals AG Sciences ■ Patents 14. DDW 2013, “Rosuvastatin Prevents Liver Tumorigenesis in High-Fat Diet-Fed Mice“ 2nd Department of Internal Medicine Osaka Medical College • International publication No.: WO2011/013247 Title of the invention: "Steatohepatitis-Liver Cancer Model Animal” 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 • Publication No. (JP) : 2009-178143 Title of the invention: "Steatohepatitis-Liver Cancer Model Animal (EN)” 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 ・ List of presentations in domestic meeting is available only in Japanese version. 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 -41- Overview
1 Company 2 Rationale: NASH 3 STAMTM: Proprietary model for NASH-HCC 4 Pharmacological study 5 CRO service
-42- Utilizing the STAMTM model
■ Drugs with proven efficacy against NASH in STAMTM Mice
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
■ Possible types of studies using STAMTM Mice
1 Target discovery/validation 2 Candidate screening 4 Preclinical evaluation ・ Evaluation of approved drugs ・ Seed/Lead selection ・ Dose-dependency ・ Gene silencing study ・ PK/PD ・ Knock out mice study 3 Biomarker discovery ・ Data for clinical trial design ・ Proof of principle experiment ・ Disease/ Efficacy/ Mechanism biomarkers
-43- List of analysis items
■ The assays listed below will allow assessment of pharmacological effect and clarification of mechanism of action in drug candidates. ■ Biochemistry Blood biochemistry Liver biochemistry ・ Glucose ・ TG
・ HbA1c ・ FFA ・ AST ・ Cholesterol ・ ALT ・ Hydroxyproline (collagen) ・ TG ・ ELISAs (TIMP-1, PDGF ligands, ) ・ Total cholesterol ・ Lipoprotein profiling (HDL, LDL, VLDL, CM) ・ ELISAs (HA, Insulin, Leptin, Adiponectin,...)
■ Histopathological assay Evaluation of fat deposition Evaluation of inflammation Evaluation of fibrosis ・ Oil-red staining (steatosis area) ・ HE staining (NAFLD Activity score) • Sirius-red staining (fibrosis area) ・ IHC for macrophage marker • Masson trichrome staining Evaluation of hepatocyte (inflammation area) • IHC for a-SMA proliferation/damage ・ IHC for mononuclear cell marker • IHC for Collagen Type 1 ・ IHC for proliferation marker ・ IHC for scavenger receptor marker • IHC for Collagen Type 3 ・ Staining for apoptosis marker
■ Gene expression assay Metabolic gene Inflammation-related gene Fibrosis-related gene • SREBP-1c • TNF-a • TGF-b • FAS • IFN-g • TIMP-1 • ACC • IL-10 • a-SMA • CPT-1 • MCP-1 • MMP-9 • PPAR-a • CCR2 • CTGF • ChREBP • SOCS3 • PAI-1
-44- Process
■ Standard process of our CRO service
1-2 months 1 month 3-6* months
Discussion Contract Preparation Study & Report ・Non-disclosure ・Study protocol ・Test compound ・Treatment agreement ・Quote ・Pregnant mice ・Analyses (If needed) ・Contract ・Disease induction ・Report
Service Agreement Test Customer Work Order Compound
Quote 1st Interim Final 2nd SMC Service Agreement Invoice report report Invoice
*depending on the treatment duration and/or analysis items
-45- 32 Feedback from customers
■ Feedback from customers
Below are examples of feedbacks we received from our customers regarding our service:
“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.” Speed “We would like to thank you very much again for sending us this nice report in a timely manner. The report is well organized.”
“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.” Quality “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.”
“In addition to quality service providers, we view the group at SMC as scientific collaborators and colleague”
Support “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.”
-46- Appendix 1: Comparison of murine NAFL/NASH models
High-calorie Model STAMTM MCDD feeding Ob/ob KK-Ay PTEN null mice diet feeding
Blood glucose >300 mg/dL 60 -100 mg/dL >200 mg/dL >200 mg/dL >200 mg/dL Not changed
Blood lipid Increase Decrease Increase Increase Increase Increase parameter
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
・Telmisartan ・DPP-4 ・FXR agonist Effective drugs ・Telmisartan inhibitor ・Galectin-3 ・GLP-1 agonist ・Pioglitazone No drug tested for the model ・FXR agonist ・CB1 inhibitor antagonist ・DPP-4 inhibitor
MCDD: methionine choline deficient diet Takahashi Y., et al., World J Gastroenterol 18:2300, 2012, Nagarajan P., et al., World J Gastroenterol 18:1141, 2012
-47- 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.
Dow M., et al., Proc Natl Acad Sci U S A. 10:1073, 2018 -48- *;partial modification9 Appendix 2: Clinical relevance of HCC models and STAM tumors
■ 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.
Dow M., et al., Proc Natl Acad Sci U S A. 10:1073, 2018 -49- 9