Non Alcoholic Fatty Liver Disease NAFLD Summary of current knowledge NAFLD January 2018

Natural History of Nonalcoholic Fatty Liver Disease (NAFLD) RISK FACTORS Overweight Obesity Metabolic syndrome Dyslipidemia Type 2 diabetes Polycystic ovary syndrome ? HCC ? > 1-2%/year 25% NAFLD NASH F0 F1 F2 F3 F4 Regression 25%, with progression of one stage fibrosis per decade ? Isolated liver steatosis Decompensation NAFLD Adapted from Diehl AM. NEJM 2017;377:2063-72

NAFLD: progression of disease Healthy Liver NAFLD NASH Cirrhosis Reversible Reversible Irreversible Large droplets of fat Bloated hepatocyte Collagen fibers Scarring Hepatocyte Nucleus Inflamed dying hepatocyte Remnant of dead cells Small droplets of fat Displaced nucleus

Liver histology in NAFLD NASH Cirrhosis Hepatic steatosis ≥ 5% Current or previous Lobular and portal inflammation ± Yes Hepatocellular injury (ballooning) No Fibrosis F0 to F3 F4 Histology scoring NAS (NAFLD Activity Score), reflects disease activity: unweighted composite of Steatosis (0 to 3) Lobular inflammation (0 to 3) Ballooning (0 to 2) SAF Score: semi-quantitative score of Activity (lobular inflammation + ballooning) = NAS (0 to 8) Fibrosis (0 to 4) NAFLD Adapted from Chalasani N. Practice Guidance from the AASLD;Hepatology 2018;67:328-357

Liver-related symptoms or signs ? Abnormal liver biochemistries ? Screening for NAFLD Incidentally discovered Hepatic Steatosis (Imaging) Liver-related symptoms or signs ? or Abnormal liver biochemistries ? YES NO Evaluation for potential NAFLD Assess for metabolic risk factors Obesity Diabetes Dyslipidemia Alternate causes Significant alcoholism (> 21 standard drinks/week in men, > 14 in women) Medications Type 2 diabetes Exclude other etiologies for steatosis, chronic liver diseases NAFLD Fibrosis Score (NFS) FIB-4 index Elastography NAFLD Adapted from Chalasani N. Practice Guidance from the AASLD; Hepatology 2018;67:328-357

Non-invasive Scores of NAFLD NAFLD Fibrosis Score (NFS) * FIB-4 ** Age + AST ALT Platelet count BMI Albumin Impaired fasting glucose or diabetes * NFS: www.mdcalc.com/nafld-non-alcoholic-fatty-liver-disease-fibrosis-score or gihep.com/calculators/hepatology/nafld-fibrosis-score ** FIB-4: www.mdcalc.com/fibrosis-4-fib-4-index-liver-fibrosis or gihep.com/calculators/hepatology/fibrosis-4-score NAFLD

Assessment of NAFLD Non invasive evaluation Presence of metabolic syndrome: predictive of steatosis LIVER BIOPSY NFS*, FIB-4, vibration controlled transient elastography, magnetic resonance elastography: predictive of advanced fibrosis  LIVER BIOPSY Serum ferritin and iron saturation: if , marker of possible hepatic injury and/or hemochromatosis (homozygote or heterozygote C282Y HFE mutation)  LIVER BIOPSY High serum titers of autoantibodies in association with other features suggestive of autoimmune liver disease (> 5 ULN aminotransferases, high globulins, or high total protein to albumin ratio)  work-up for autoimmune liver disease Consider presence of commonly associated comorbidities (central obesity, hypertension, dyslipidemia, diabetes or insulin resistance, hypothyroidism, polycystic ovary syndrome, obstructive sleep apnea) * NFS is based on 6 variables (age, BMI, hyperglycemia, platelet count, albumin, and AST/ALT ratio) and is calculated using the published formula http://gihep.com/calculators/hepatology/nafld-fibrosis-score/ NAFLD Adapted from Chalasani N. Practice Guidance from the AASLD ; Hepatology 2018;67:328-357

Likelihood of NASH and fibrosis in patients with NAFLD Low Intermediate High Age < 40 > 40 > 50 Type 2 diabetes No Yes, well-controlled Yes Obesity Hypertension ± Fibroscan kPa < 7 > 7 > 8.5 AST < 20 > 20 and AST/ALT ratio ≥ 1 NFS (NAFLD Fibrosis Score) < - 1.455 - > 0.676 FIB-4 < 1.3 > 2.67 Patients at risk for clinical progression Steatosis ≥ 1 Ballooning ≥ 1 Inflammation ≥ 1 NAS (NAFLD Activity Score) ≥ 4 ≥ F2 NAFLD Adapted from Konerman MA. J Hepatol 2018 (Epub ahead of print)

Comorbidities of NAFLD CV diseases Metabolic comorbidities More frequent More severe Cancer Liver complications Cirrhosis End-stage liver disease Liver (HCC)* GI malignancies Colon (men)** Esophagus Stomach Pancreas Non-GI malignancies Kidney (men) Breast (women)** HR of * 17 ; ** 2 ; *** 1.9 Adapted from Kim GE. J Hepatol 2018;68:140-6 ; Chalasani N. Practice Guidance from the AASLD; Hepatology 2018;67:328-357 NAFLD

Management of patients with NASH and fibrosis Interventions recommended Objective/restrictions Benefit Weight loss (Hypocaloric Diet [ by 500-1,000 kcal/day]) 3-5% 7-10%  > 10% Improve steatosis Improve NASH, including fibrosis NASH resolution and  fibrosis Moderate-intensity exercise (30 min/day x 3-5 times/week) If isolated Prevent or reduce steatosis Limit alcohol consumption (≤ 2 drinks/day men, ≥ 1 drink women) All patients Coffee consumption (≥ 2 cups/day)  Inflammation and fibrosis Modification of CVD risk factors CV morbidity and mortality Statins If dyslipidemia Interventions to consider Pioglitazone In patients with or without type 2 diabetes Improves liver histology Vitamin E (rrr a-tocopherol) In non-diabetic patients Foregut bariatric surgery In otherwise eligible obese Omega-3 fatty acids If hypertriglyceridemia Improve hypertriglyceridemia Not recommended : Metformin, GLP-1 agonists (liraglutide), Systematic bariatric surgery, Ursodeoxycholid acid Adapted from Chalasani N. Practice Guidance from the AASLD ; Hepatology 2018;67:328-357 ; Diehl AM. NEJM 2017;377:2063-72 NAFLD

Endpoints in clinical trials of NAFLD and NASH Outcomes Hard endpoints Surrogate endpoints Clinical All cause mortality Liver-related mortality Child-Pugh and MELD scores Hepatic venous portal gradient Hepatic decompensation Progression to cirrhosis Fibroscan (kPa) and magnetic resonance elastography Biomarkers (pro-C3, FIB-4, NFS, enhanced liver fibrosis) Metabolic  in hepatic fat content MRI-derived proton density fraction, Multiparametric MRI, Controlled attenuation parameter  in insulin resistance HbA1c, fasting glucose, HOMA-IR Impact on lipids  in weight/BMI Inflammatory  in necro-inflammation Multiparametric MRI, Liver enzymes  in hepatocyte ballooning Fibrosis  in fibrosis stage NAFLD Adapted from Konerman MA. J Hepatol 2018 (Epub ahead of print)

NASH-new serum non-invasive biomarkers Not yet fully validated Could diagnose NASH and its severity Could identify patients likely to respond to therapy Chitinase-3-like protein 1(CHI3L1) – YKL-40 Enhanced Liver Fibrosis (ELF) Test Comprised of TIMP-1 (tissue inhibitor of matrix metalloproteinase-1), PIII-NP (mino-terminal peptide of type III procollagen), and hyaluronic acid Pro C3 Derived from synthesis of type III collagen FIB-C3 Score HepQuant STAT Quantitative liver function test 60 min after oral administration of 4 mg tetra-deuterated cholic acid Non-invasive score-4 (NIS4) Detect patients with NAS ≥ 4 and F ≥ 2 Comprised of Chitinase-3-like protein 1(CHI3L1), alpha2-macroglobulin, microRNA 34a (MIR34a), and HbA1C NAFLD Adapted from Harrison SA. AASLD 2017

Current Status of Pharmacologic Treatments for NASH No approved therapies for NASH Currently available therapeutics with (some) proven efficacy Vitamin E Pioglitazone NAFLD

NASH: targets for therapeutics Insulin resistance Cell stress apoptosis Inflammation Fibrogenic remodeling Insulin resistance modifiers Cell stress modifiers Anti-inflammatory agents Anti-fibrotic agents Exemples of drugs in development PPAR FXR agonist (obeticholic acid, GS-9674) GLP-1 FABAC FGF-21 (BMS-986036) Thyroxine analog Vitamin E ASK-1 inhibitor (selonsertib) PPAR-γ agonsit FXR agonist Dual PPAR-/δ agonist FGF-21 FGF-19-like agent CCR2-CCR5 antagonist ASK-1 inhibitor Galectin 3 Lysyl oxidase-like 2 inhibitor Endpoints for clinical trials Disease Activity (steatohepatitis) = NAFLD Activity Score (NAS) Disease Progression = Fibrosis Stage Clinical outocmes: Cirrhosis (MELD, Portal hypertension), Liver-related outcome, death NAFLD Adapted from Konerman MA. J Hepatol 2018 (Epub ahead of print) ; Diehl AM. NEJM 2017;377:2063-72

NASH – Pharmacological agents in development Drug Class Mechanism of action Objective Phase 2b Aramchol Synthetic fatty acid/bile acid conjugate Impacts lipogenesis by upregulates the ABCA1 reverse cholesterol transporter and functions  liver fat and NAFLD activity score without  of fibrosis Emricasan Pan-caspase inhibitor Blockage of apoptotic and inflammatory caspase activation involved in hepatocyte cell death  fibrosis and portal hypertension GR-MD-02 Galectin-3 protein inhibitor Anti-fibrogenesis Phase 3 Obeticholic acid Semi-synthetic derivative of acid chenodeoxycholic acid Agonist of the farnesoid X receptor : downregulates hepatic glucose and lipid metabolism. May also  portal pressure and have anti-inflammatory and antifibrotic activity  fibrosis without  NASH ; NASH resolution without  of fibrosis Elafibranor Dual PPAR-a/d agonist Regulation of metabolic homeostasis, inflammation, cellular growth, and differentiation NASH resolution without  of fibrosis ; Clinical outcomes Cenicriviroc dual CCR2/CCR5 antagonist Reduction of hepatic inflammation and fibrosis  NAFLD activity score without  of fibrosis ;  fibrosis without  NASH Selonsertib (GS-4997) Apoptosis signal regulating kinase 1 (ASK1) inhibitor Reduction in oxidative stress-related cell death, fibrosis and inflammation NAFLD Adapted from Konerman MA. J Hepatol 2018 (Epub ahead of print)

Improvement of NASH with pharmacologic agents Results of trials for individual treatment agents * Vitamin E Placebo Pioglitazone Obeticholic acid Elafibranor Cenicriviroc Selonsertib Simtuzumab 20 40 60 80 100 Improvement in hepatic steatosis p = 0.005 p < 0.001 p = 0.10 p = 0.001 54 31 69 61 38 35 18 72 70 102 98 39 Resolution of NASH p = 0.05 p = 0.01 p = 0.08 36 21 47 22 13 29 5 Improvement in fibrosis p = 0.24 p = 0.12 p = 0.02 p = 0.004 41 44 19 10 145 144 57 37 p = ns % * Enrollment criteria and durations of therapy differed between studies, and the primary endpoint definitions were not identical NAFLD Adapted from Konerman MA. J Hepatol 2018 (Epub ahead of print)

Combination of metabolic damages NASH Combination of metabolic damages to the liver Prevention and Lifestyle Modifications are key ! NAFLD