Histopathology and imaging analysis-SMC’s CRO services in pharmacological R&DCONTENTS:- Company Profile (P2-)- NASH Services (P4-)- Basic Services (P12-)

SMC Laboratories, Inc.
www.smclab.co.jp

Ver. 2019.8

1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.

-2-

1

CRO expertise: Leading CRO in NASH/HCC■ SMC’s CRO services

Non-clinicalpharmacology study

Histological imaging- High-performance pharmacology services based on thecompany’s specialty in fibrosis research- In-house, high quality analyses; proven skills in Histology- Various lineup of disease models relating to fibrosis,- Elaborate and comprehensive reports by Ph.D. holders

inflammation, metabolic disorders and cancer- Professional support for data publication/IND application- Strategic study design proposed by experienced physicians andscientists

Performance

Region■ Over 500 clients worldwide■ Over 90 peer-reviewed papers and presentations■ 10 successful CTA packages

Japan
Asia

Number of clients
EuropeNorth
America

600
500
400
300
200
100
0

(% of the customers)-3-(year)

Histopathology and imaging services in NASH
■Histopathological parameters are important endpoints in nonclinical aswell as clinical studies in NASH and liver fibrosis.
SMC’s outstanding performance in NASH/liver fibrosis research:■As a leading CRO in NASH, SMC has accumulated know-how byassessing over 500 pharmacology studies and 50,000 slides of NASH andrelated diseases in mouse, rat and human.
- judgment of NAS (especially, ballooning), assessment ofpathological changes from disease control, discussion of clinicalrelevance… and more!
NAS; NAFLD Activity score

For…
■Assessment the grade of disease (NASH, fibrosis) in not only nonclinicalpharmacology studies but also clinical studies.
■Development of animal models (including Tg and KO mice) for drug
evaluation in the clients’ own laboratory.
■Achievement of high quality report/data package with objective
evaluation.
-4-NAS: “ballooning” or “pseudo-ballooning”?
NAS (including ballooning, inflammation, steatosis) is an important parameter to evaluate
the drug efficacy in NASH in both human and animal models.
In order to translate nonclinical results into clinical practice, it is essential to discriminate“true”- from “pseudo”-ballooning in disease models.

CV

PV

MCD mice

STAMTM NASH model mice: steatosis (fat droplet): inflammation: ballooning hepatocyte: “pseudo”-ballooning hepatocyte-5-PV; Portal vein, CV; Central vein

The grade of NAS: Achievement
An accurate scoring can evaluate the efficacy of the test substances on eachcomponents (steatosis, lobular inflammation, hepatocyte ballooning) of NAS.

Definition of NAS components-6-

Traber PG et al., PloS One. 2013;8:e83481

The grade of fibrosis (Method)
Threshold setting
after extraction

Image denoising

Steatosis

Original image

Steatohepatitis

Fibrosis area: 0.3%

-7-

Fibrosis area: 1.0%

The grade of fat (Method)
Threshold setting
after extraction

Image denoising

NASH-Vehicle

Original image

NASH-Treatment

Fat deposition area:12.9%-8-

Fat deposition area:4.9%

The grade of inflammation (Method)
Threshold setting
after extraction

Image denoising

NASH-Vehicle

Original image

NASH-Treatment

Inflammation area: 8.0%

-9-Inflammation area: 5.6%

Add interpretation to your slides by double/multi IHC~ Functional histopathology (quantitative/qualitative)

Double/ Multiple staining: M1(F4/80 + CD16/32) / M2(F4/80 + CD206)
ratio.
 Characterization of cytokine-producing cells.
 Identification of target molecule-expressing
cells (see the right picture).
 Investigation of proliferative cell types(insulin + BrdU, CD markers + BrdU or Ki-67).
 Cell-cell interaction (inflammatory cells +fibroblast, inflammatory cells + parenchymal
cells).
 Optimization of staining method with yourdeveloped antibodies and existing antibodies.
- Please ask us!
Traber PG et al., PloS One. 2013;8:e83481- 10 -

Assessment of human materials
Both human and animal samples can be evaluated by the same and standard methods.
Useful for discussion of clinical relevance.
Steatosis

Steatohepatitis

SR= 1.52%

SR= 5.11%

SR positive area

x 200

NAS= 2x 200

NAS= 6

NAFLD
Activity Score

x 200

Analysis of Collagen proportionate are (CPA) is also available!
- 11 -

x 200

Basic services
Processing & Embedding :• Embedding of human and animal tissues in paraffin (FFPE blocks) from fixed tissues.
• Embedding of human and animal tissues in OCT (frozen OCT embedded tissues) from fixed tissues.
• Instruction of fresh frozen block preparation for frozen sections.
Sectioning：• Cutting of sections (4-8 µm thick) from paraffin and O.C.T. embedded blocks.
• Preparation of serial thin sections.
Routine staining:
・ HE, Sirius red, Masson trichrome, PAS, Oil red, etc.
Immunohistochemistry (IHC): Inflammation-related molecules, fibrosis-related molecules etc.
・ Functional Immunohistology（Double/multiple staining).
・ New antibody staining protocol optimization & validation.
Imaging analysis:
・ Area, length, diameter, stained cell count, percentage positive area, shape etc.
・ Proliferation, Apoptosis, pathological grading etc.
Interpretation of results:
・ Discussion of the histological findings in view of pharmacology.
Reporting:- 12 -

Fibrosis in various disease models
Providing optimal disease/tissue-specific analytical methods to evaluate the grade of fibrosis inaccordance with the types of fibrotic/inflammatory diseases.
The number of
positive cells

Positive area

Qualitative score

Model:
DSS-induced colitis model

Model:
UUO-induced renal fibrosis model

Model:
BLM-induced lung fibrosis model

Organ: Colon

Organ: Kidney

Organ: Lung

Calculating the α-SMA positive
cells per fieldCalculating the Sirius red
positive area per field

Assigning a numerical scale of
the amount of fibrotic area (i.e.,
Ashcroft score)- 13 -

Inflammation in various disease models
Evaluation of the grade of inflammation in various tissues in various disease models by using
F4/80 as a macrophage markers.
Brain
Lung
Pancreas
F4/80

Gut

Liver

- 14 -

Adipose tissue

Proliferation and apoptosis in various disease models
Characterization, distribution and quantitative analysis of proliferative/apoptotic cells.
Gut

Liver
Control Ab

Liver

Fibroblast + Ki-67

αCXCL10 Ab

BrdU + Type IV collagen

Yoneyama H et al., Med Mol Morphol. 2007;40:191-7- 15 -TUNEL + Nucleus

Morphometric and functional analysis in diabetes
Insulin positive area, the number of insulin positive cells/proliferating β cells.
The number of BrdU+ β cells(after β cell injury by STZ)

The number of insulin positive cells

NormalInsulin + BrdU

NASH
Insulin- 16 -Morphometric analysis in kidney injury (Method)
PAS staining

Threshold setting
after extraction

Mesangial matrix area = PAS-positive area/tuft area

Okada S et al., Diabetes, 2003;52:2586-93- 17 -

PAS-positive area in the tuft
(excluding the nuclear region)

Target cell counts based on anatomical compartments
Quantitative analysis considering the distribution of cells of interests based on anatomical andfunctional compartment.
Ophthalmology: Retina

NormalVehicleImmunology : Lymph node

Treatment

The number of cells in GCL

GFAP

HEV

VehicleTreatment

GCL: Ganglion Cell Layer
INL: Inner Nuclear Layer
ONL: Outer Nuclear Layer

The number of target cells in GCL- 18 -

B220 + CD11C

HEV: High Endothelial Venule

The number of target cells in HEVPortfolio: non-clinical pharmacology models■ STAMTM: Premium preclinical platform for NASH-HCC
1. Pharmacology study- For efficacy evaluation of drug candidates/existing drugs- Histological review with in-depth knowledge of inflammationand fibrosis

2. Delivery of STAMTM Mice samples- For target (biomarker) discovery and validation- Provided as tissue and/or plasma (serum) samples■ Conventional disease models

CT-image of HCC (STAMTM)・Fatty liver/NAFLD: ob/ob model, MCD model・Diabetes mellitus : nSTZ model・Liver fibrosis: CCl4 model, BDL model・Acute liver failure: CCl4 model, Concanavalin A model

BLM

CCl4

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
・COPD: PPE-induced emphysema model・Alzheimer’s disease: icv-STZ model・Cancer: DEN- CCl4 liver cancer model
Xenograft tumor model

Sirius red

DSS

UUO

PAS

- 19 -CT (Lung)

EndoscopyContract Research Laboratory (CRL) 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- 20 -

Publications and Presentations■ Publications

42. International Journal of Gastroenterology, "Characterization of EDP-305, a Highly Potent andSelective 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 associatedwith 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, PXS5153A, reduces crosslinks and ameliorates fibrosis" (J Cell Mol Med., 23:1759-1770, 2019)38. Scientific Reports, "Connectivity mapping of angiotensin-PPAR interactions involved in theamelioration of non-alcoholic steatohepatitis by Telmisartan" (Sci Rep., Mar 8;9(1):4003, 2019)37. NPJ Precision Oncology, "Transcriptomic analysis of hepatocellular carcinoma reveals molecularfeatures of disease progression and tumor immune biology" (NPJ Precis Oncol., DOI: 10.1038/s41698018-0068-8, 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 andhaptoglobin as biomarker candidates in hepatocarcinogenesis of STAM mice” (Glycoconj J., Oct;35(5):467476, 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 andthe associated dysregulation of 1-carbon metabolism in nonalcoholic steatohepatitis and hepatocellular
carcinoma“ (FASEB J, DOI:10.1096/fj.201700866R, 2017)

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-alcoholicsteatohepatitis 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-alcoholicsteatohepatitis 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 ofLiver 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: aMechanism 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 nonalcoholic 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 nonalcoholicsteatohepatitis and subsequent fibrosis” (Sci Rep., 12466, 2015)
15. International Journal of Obesity, “Low cytochrome oxidase 4I1 links mitochondriazzzl dysfunction toobesity 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)

29. Oncotarget, “MicroRNA deregulation in nonalcoholic steatohepatitisassociated liver carcinogenesis”(Oncotarget, 8:88517-88528, 2017)

13. Oncology Reports, “Hepatic expression of the Sptlc3 subunit of serine palmitoyltransferase isassociated with the development of hepatocellular carcinoma in a mouse model of nonalcoholicsteatohepatitis” (Oncol Rep, 33:1657-66, 2015)

28. Oncotarget, “Peretinoin, an acyclic retinoid, suppresses steatohepatitis and tumorigenesis byactivating autophagy in mice fed an atherogenic high-fat diet” (Oncotarget, 8:39978-39993, 2017)12. Drug R D, “In Vivo Efficacy Study of Milk Thistle Extract (ETHIS-094TM) in STAMTM Model ofNonalcoholic Steatohepatitis” (Drugs R D, 14:291-9, 2014)27. Physiological Research, “Pathophysiological analysis of the progression of hepatic lesions in STAM
mice.” (Physiological Research, 66:791-799, 2017)11. PLoS One, “Photoacoustic Tomography of Human Hepatic Malignancies Using Intraoperative
Indocyanine Green Fluorescence Imaging” (PLoS One, 9:e112667, 2014)- 21 -

Publications and Presentations■ Publications (continued)

10. Cancer Science, “Silencing of microRNA-122 is an early event during hepatocarcinogenesis from nonalcoholic steatohepatitis” (Cancer Sci, 105:1254-60, 2014)

66. AASLD 2018, “Dipeptidyl Peptidase 4 Inhibitors Reduce the Progression of Hepatocellular Carcinoma

9. Anticancer Research, “Characterization of non-alcoholic steatohepatitis-derived hepatocellular
carcinoma as a human stratification model in mice” (Anticancer Res, 34:4849-4856, 2014)

65. AASLD 2018, “Effects of a DPP4 Inhibitor on Progression of Nash-Related Hepatoma and DNA

8. PLoS One, “L-carnitine prevents progression of non-alcoholic steatohepatitis in a mouse model withupregulation of mitochondrial pathway.” (PLoS One, 9:e100627, 2014)
7. Medical Molecular Morphology, “Linagliptin alleviates hepatic steatosis and inflammation in a mousemodel 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-3derived cytotoxic T-lymphocyte epitope peptide” (Int J Oncol, 42:831-838, 2013)
4. International Journal of Experimental Pathology, “Inhibition of Glutaminyl Cyclases alleviates CCL2mediated 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 evidenceof association between diabetes and hepatocellular carcinoma” (Med Mol Morph, 46:141-152, 2013)
2. Hepatology, “Hydrogen-rich water prevents progression of non-alcoholic steatohepatitis andaccompanying 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)■ Presentations
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.

By Activating T Cell and Natural Killer Cell Chemotaxis in Mice” Kawasaki Medical School
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 Developmentof Hepatocellular Carcinoma (HCC) from Nonalcoholic Steatohepatitis (NASH) in a Mouse Model of NASHHCC 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-woundhealing 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

72. DDW 2019, “Influence of the O-GlcNAc Modification in Hepatic Carcinogenesis by Non-alcoholic Fatty
Liver Disease” Osaka Medical College

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

71. EASL the International Liver CongressTM 2018, “LXR inverse agonists reduce steatosis and fibrosisin the STAM mouse model but also improve insulin sensitivity in a high fat diet mouse clamp study” Phenex
Pharmaceuticals AG

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 throughimmunomodulation” Promethera Biosciences LLC

70. 3rd Annual World Preclinical Congress Europe 2018, “LXR Inverse Agonists for the Treatment of
NASH” Phenex Pharmaceuticals AG

55. First EASL NAFLD Summit 2017, “Dual CCR2/5 antagonist decreases hepatic inflammation in acuteliver injury and NASH metabolic animal models” Pfizer Inc.

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.

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 nonalcoholicsteathepatitis (NASH)” Axcella Health, Inc.

68. AASLD 2018, “AXA1125, a Novel Composition of Amino Acids Reprograms the Multifactorial
Pathophysiology in NAFLD” Axcella Health Inc.

53. AASLD 2017, “The Anti-Fibrogenic and Liver Protective Effects of Namodenoson (CF102) in a NonAlcoholic 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- 22 -

Publications and Presentations■ Presentations (continued)
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

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

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.

35. AASLD 2015, “Anti-Fibrotic Effect of Autotaxin and LPA1R Antagonists in a Rodent Model of NASH”
Bristol-Myers Squibb Company

50. AASLD 2017, “Combination Treatment of LJN452 and Cenicriviroc Snows Additive Effects in a DietInduced NASH Model” Genomics Institute of the Novartis Research Foundation/Allergan plc/Novartis
Institutes for BioMedical Research, Inc.

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

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.

33. AASLD 2015, “Reduction of Hepatic 27-Hydroxycholesterol in Steatohepatitis Model Mice with Insulin
Resistance” Tokyo Medical University Ibaraki Medical Center

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

32. AASLD 2015, “Disturbance of regulatory T cells, MDSCs and NK cells is involved in NASH and mousemodel 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.

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

30. DDW 2015, “Effects of Sitagliptin, a Dipeptidyl Peptidase 4 Inhibitor, on Tumor Progression andp62/SQSTM1 Subcellular Localization in a Mouse Model of Non-Alcoholic Steatohepatitis-Related
Hepatocellular Carcinoma” Kurume University

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.

29. Keystone Symposia 2015, “DGAT2 Inhibition Prevents NAFLD and Fibrosis in the STAM Mouse
Model of NASH“ Pfizer Inc.

45. AACR 2017, “Alterations in the chromatin accessibility in nonalcoholic steatohepatitis-associatedhepatocellular carcinoma” FDA-National Center for Toxicological Research

28. Keystone Symposia 2015, “Oxidized-Phospholipid Small Molecule Inhibits Non-Alcoholic
Steatohepatitis (NASH) and Liver Fibrosis“ Vascular Biogenics Ltd

44. AACR 2017, “Role of miRNAome deregulation in the pathogenesis of non-alcoholic steatohepatitis(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 withupregulation of mitochondrial pathway“ Department of Gastroenterology and Hepatology, Okayama
University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences

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.

26. AASLD 2014, “MN-001 (tipelukast), a novel, orally bioavailable drug, reduces fibrosis and inflammationand down-regulates TIMP-1, collagen Type 1 and LOXL2 mRNA overexpression in an advanced NASH
(nonalcoholic steatohepatitis) model“ MediciNova, 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

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.

40. AASLD 2016, “Effects of BMS-986036 (pegylated fibroblast growth factor 21) on hepatic steatosis andfibrosis in a mouse model of nonalcoholic steatohepatitis” Bristol-Myers Squibb Company .

24. ADA 2014, “Liraglutide prevents steatohepatitis, liver fibrosis, and accompanying carcinogenesis in adiabetes 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 ina Mouse Model of Non-Alcoholic Steatohepatitis (NASH)” Albireo AB

23. ATS 2014, “Solithromycin Reduces Inflammation In Mice Caused By Bleomycin-Induced Lung Injury“
Cempra, Inc.

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.

22. DDW 2014, “Anti-NASH Effects of Solithromycin in NASH-HCC Mouse Model“ Cempra, Inc.

- 23 -

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■ Presentations (continued)
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.

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 andprogression of NASH towards HCC in a hypoinsulinaemic mouse model of progressive liver disease”
Phenex Pharmaceuticals AG

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.

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

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 andHepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences

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.

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 modelof progressive liver disease” F. Hoffmann-La Roche, Ltd

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

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

1. The 9th JSH SingleTopic Conference “NASH 2010”, “Strong Anti-steatotic and Anti-fibrotic Effects ofNovel FXR Agonists in a Murine NASH Model that Resembles Human NASH” Phenex Pharmaceuticals AG■ Patents

14. DDW 2013, “Rosuvastatin Prevents Liver Tumorigenesis in High-Fat Diet-Fed Mice“ 2nd Department ofInternal Medicine Osaka Medical College
13. AASLD 2012, “Comparative proteomic analysis of the liver in a murine model of non- alcoholicsteatohepatitis” 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

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)”・ List of presentations in domestic meeting is available only in Japanese version.

11. AASLD 2012, “Galectin-3 targeting drugs inhibit multiple pathological pathways leading to improvementof non-alcoholic steatohepatitis (NASH)” Galectin Therapeutics Inc.
10. AASLD 2012, “Hepatic gene expression of the SPTLC3 subunit of serine palmitoyltransferase isassociated with the development of liver cancer in a NASH mouse model” Department of Human andEnvironmental 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-alcoholicsteatohepatitis (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•- 24 -

Slide gallery (Functional histopathology)

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B
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Dendritic cells efflux from the liver through lymph

Clustering between dendritic cells and proliferating T cells in the liver

Distribution of proliferating B cells during the second challenge of immune responses

Fat-laden Kupffer cells in NASH liver

Clustering between HSCs and dividing hepatocytes