Supplementary appendix

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1 Supplementary appendix This appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors. Supplement to: Neuschwander-Tetri BA, Loomba R, Sanyal AJ, et al, for the NASH Clinical Research Network. Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebo-controlled trial. Lancet 2014; published online Nov 7. org/ /s (14) This online publication has been corrected. The corrected version first appeared at thelancet.com on March 13, The second corrected version first appeared at thelancet.com on April

2 SUPPLEMENTARY APPENDIX: Farnesoid-X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebocontrolled trial Table of contents Members of the Nonalcoholic Steatohepatitis Clinical Research Network (NASH CRN)... 2 Members of the NASH CRN Steering Committee... 3 Supplemental methods... 4 Detailed inclusion and exclusion criteria... 4 Interim analysis 1 based on ALT change at 24 weeks vanguard analysis... 4 Supplementary tables and figures... 6 Table S1. Sensitivity analyses of primary outcome of histologic improvement... 6 Table S2. Post-hoc analysis: Odds of histologic improvement between treatment groups by baseline and postrandomisation subgroups... 7 Table S3. Changes in liver enzymes, biochemical levels, metabolic factors, and quality of life from baseline to 96 weeks... 9 Table S4. Change from baseline to 72 weeks in detailed scoring of histologic features by treatment group Figure S1. Changes from baseline in serum lipids according to treatment group Figure S2. Changes from baseline in aspartate aminotransferase by treatment group... 18

3 2 Members of the Nonalcoholic Steatohepatitis Clinical Research Network Adult Clinical Centres Case Western Reserve University Clinical Centers: MetroHealth Medical Center, Cleveland, OH: Srinivasan Dasarathy, MD; Jaividhya Dasarathy, MD; Carol Hawkins, RN; Arthur J. McCullough, MD Cleveland Clinic Foundation, Cleveland, OH: Srinivasan Dasarathy, MD; Arthur J. McCullough, MD; Mangesh Pagadala, MD; Rish Pai, MD; Ruth Sargent, LPN Duke University Medical Center, Durham, NC: Manal F. Abdelmalek, MD, MPH; Mustafa Bashir, MD; Stephanie Buie; Anna Mae Diehl, MD; Cynthia Guy, MD; Christopher Kigongo; Yi-Ping Pan; Dawn Piercy, FNP Indiana University School of Medicine, Indianapolis, IN: Naga Chalasani, MD; Oscar W. Cummings, MD; Samer Gawrieh, MD; Marwan Ghabril, MD; Smitha Marri, MD; Linda Ragozzino, RN; Kumar Sandrasegaran, MD; Raj Vuppalanchi, MD Saint Louis University, St Louis, MO: Debra King, RN; Pat Osmack; Joan Siegner, RN; Susan Stewart, RN; Brent A. Neuschwander-Tetri, MD; Susan Torretta University of California San Diego, San Diego, CA: Brandon Ang; Cynthia Behling, MD, PhD; Archana Bhatt; Joel Lavine, MD, PhD (currently at Columbia University); Rohit Loomba, MD, MHSc; Michael Middleton, MD, PhD; Heather Patton, MD; Claude Sirlin, MD University of California San Francisco: San Francisco, CA: Bradley Aouizerat, PhD; Nathan M. Bass, MD, PhD ( ); Danielle Brandman, MD; Linda D. Ferrell, MD; Ryan Gill, MD, PhD; Bilal Hameed, MD; Claudia Ramos; Norah Terrault, MD, MPH; Ashley Ungermann Fresno, CA: Pradeep Atla, MD; Brandon Croft; Rebekah Garcia; Sonia Garcia; Muhammad Sheikh, MD; Mandeep Singh, MD Virginia Commonwealth University, Richmond, VA: Sherry Boyett, RN, BSN; Laura Carucci, MD; Melissa J. Contos, MD; Kenneth Kraft, PhD; Velimir AC Luketic, MD; Puneet Puri, MD; Arun J. Sanyal, MD; Jolene Schlosser, RN, BSN; Mohhamad S. Siddiqui, MD; Ben Wolford Virginia Mason Medical Center, Seattle, WA: Sarah Ackermann; Shannon Cooney; David Coy, MD, PhD; Katie Gelinas; Kris V. Kowdley, MD; Maximillian Lee, MD, MPH; Tracey Pierce; Jody Mooney, MS; James E. Nelson, PhD; Cheryl Shaw, MPH; Asma Siddique, MD; Chia Wang, MD Resource Centres Washington University, St. Louis, MO: Elizabeth M. Brunt, MD; Kathryn Fowler, MD National Cancer Institute, Bethesda, MD: David E. Kleiner, MD, PhD National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD: Sherry R. Brown, MS; Edward C. Doo, MD; Jay H. Hoofnagle, MD; Patricia R. Robuck, PhD, MPH ( ); Averell Sherker, MD; Rebecca J. Torrance, RN, MS Johns Hopkins University, Bloomberg School of Public Health (Data Coordinating Center), Baltimore, MD: Patricia Belt, BS; Jeanne M. Clark, MD, MPH; Michele Donithan, MHS; Erin Hallinan, MHS; Milana Isaacson, BS; Kevin P. May, MS; Laura Miriel, BS; Alice Sternberg, ScM; James Tonascia, PhD; Aynur Ünalp-Arida, MD, PhD; Mark Van Natta, MHS; Ivana Vaughn, MPH; Laura Wilson, ScM; Katherine Yates, ScM

4 3 Nonalcoholic Steatohepatitis Clinical Research Network Steering Committee Members Joel Lavine, MD, PhD (co-chair); Arun Sanyal, MD (co-chair); Sarah Barlow, MD; Naga Chalasani, MD; Ed Doo, MD; Anna Mae Diehl, MD; Kris Kowdley, MD; Rohit Loomba, MD, MHSc; Arthur McCullough, MD; Jean Molleston, MD; Karen Murray, MD; Philip Rosenthal, MD; Claude Sirlin, MD; Jeffrey Schwimmer, MD; Norah Terrault, MD, MPH; Brent Neuschwander-Tetri, MD; James Tonascia, PhD; Peter Whitington, MD; Stavra Xanthakos, MD

5 4 Supplementary methods Detailed inclusion and exclusion criteria A total of 345 patients were evaluated for enrollment and 283 were randomised. Patients who met any of the following exclusion criteria were ineligible for enrollment: 1) Current or history of significant alcohol consumption for a period of more than 3 consecutive months within 1 year prior to screening (significant alcohol consumption was defined as more than 20 g/day in females and more than 30 g/day in males, on average); 2) Inability to reliably quantify alcohol consumption based upon site investigator s judgment; 3) Use of drugs historically associated with NAFLD for more than 2 weeks in the year prior to randomisation; 4) Prior or planned bariatric surgery or procedure; 5) Uncontrolled diabetes defined as HbA1c of 80 3 mmol/mol or higher within 60 days prior to enrollment; 6) Presence of cirrhosis on liver biopsy, 7) Platelet count < /L; 8) Clinical evidence of hepatic decompensation as defined by the presence of any of the following abnormalities: serum albumin less than 32 g/l, INR greater than 1 3, direct bilirubin greater than 22 2 µmol/l, or a history of esophageal varices, ascites, or hepatic encephalopathy; 9) Evidence of other forms of chronic liver disease: hepatitis B as defined by presence of hepatitis B surface antigen (HBsAg), hepatitis C as defined by presence of hepatitis C virus (HCV) RNA or positive hepatitis C antibody (anti- HCV), evidence of ongoing autoimmune liver disease as defined by compatible liver histology, primary biliary cirrhosis as defined by the presence of at least 2 criteria (biochemical evidence of cholestasis based mainly on alkaline phosphatase elevation, presence of anti-mitochondrial antibody [AMA], and histologic evidence of nonsuppurative destructive cholangitis and destruction of interlobular bile ducts), primary sclerosing cholangitis, Wilson s disease as defined by ceruloplasmin below the limits of normal and compatible liver histology, alpha-1- antitrypsin (A1AT) deficiency as defined by diagnostic features in liver histology (confirmed by alpha-1 antitrypsin level less than normal, exclusion at the discretion of the site investigator), history of hemochromatosis or iron overload as defined by presence of 3+ or 4+ stainable iron on liver biopsy, drug-induced liver disease as defined on the basis of typical exposure and history, known bile duct obstruction, suspected or proven liver cancer, or any other type of liver disease other than NASH; 10) Serum alanine aminotransferase (ALT) greater than 300 U/L; 11) Serum creatinine of µmol/l or greater; 12) Inability to safely obtain a liver biopsy; 13) History of biliary diversion; 14) Known positivity for Human Immunodeficiency Virus (HIV) infection; 15) Active, serious medical disease with likely life expectancy less than 5 years; 16) Active substance abuse including inhaled or injection drugs in the year prior to screening; 17) Pregnancy, planned pregnancy, potential for pregnancy and unwillingness to use effective birth control during the trial, or breast feeding; 18) Participation in an IND trial in the 30 days before randomisation; 19) Any other condition which, in the opinion of the site investigator, would impede compliance or hinder completion of the study; or 20) failure to give informed consent. Interim analysis 1 based on ALT change at 24 weeks vanguard analysis An unmasked interim look, which we termed a vanguard analysis in the FLINT protocol, was conducted prior to performing any post-treatment liver biopsies. This interim analysis compared the obetichoic acid group with placebo using a surrogate outcome measure: change in ALT (U/L) from baseline to the 24 week follow-up visit. This unmasked analysis was conducted approximately 65 weeks after the first randomised patient was randomised and was reviewed by the DSMB in order to advise the NIDDK on whether, based on ALT reduction favoring obeticholic acid, the FLINT trial should continue and begin the 72-week post-treatment patient biopsies. This interim analysis was planned so that it occurred, depending on the pace of enrollment, when between 70 (25% of total sample size) and 112 (40%) would have a 24 week ALT measure, but before the first patient (or any other patient) had undergone a 72 week post-treatment liver biopsy. The pre-specified guideline for continuing the FLINT trial with follow-up liver histology needed for the primary histological improvement was evidence of net (vs. baseline) reduction of ALT in the obetichoic acid group compared to placebo of at least 20 percent. The statistical criterion was met if the 95% confidence limit for the net percentage reduction in mean ALT (BL-24 weeks) favoring the obeticholic acid group vs. placebo 20% or greater. The method for calculating the net reduction and 95% upper confidence interval was calculated from a multiple linear regression model with patient level changes in ALT (U/L) at baseline minus ALT (U/L) at 24 weeks as the response variable (y) and independent variables: obeticholic acid indicator (x2=1, if obeticholic acid, 0, if placebo) and baseline ALT (U/L) (x1): E(y i)=β 0+ β 1x 1+ β 2x i2+ε i, i=1,2,,n; n=number of patients in the interim (vanguard) analysis and ε i are i.i.d. Gaussian(0,σ 2 ). The estimated between group (obeticholic acid vs. placebo) difference in mean change in ALT from BL to 24 weeks is b 2 with 95% CI: b 2 ± t n 3 σ (b 2 ) where t n 3 is the 97 5th percentile of the t-distribution with n-3 degrees of

6 5 freedom and σ (b 2 ) is the estimated standard error of b 2 from the regression analysis. The estimated net reduction in ALT in the obeticholic acid group, expressed as percentage of the overall baseline mean ALT, ALT, is 100 b 2/(ALT) with upper 95% confidence limit: 100 [b 2 + t n 3 σ (b 2 )] / (ALT). SAS PROC REG was used to fit the regression model and calculate the upper 95% confidence limit to determine whether its value is a 20% or more net reduction in ALT. The result of this analysis was: Net change in 24-week ALT: -24% (Favor obeticholic acid) 95% CI: (-3, -45%) (45% is greater than 20%) Since the 95% confidence limit on net reduction (-45%) favoring obeticholic acid was greater than 20%, the guideline for continuing the trial was met. The interim analysis was accompanied by a detailed safety data report to assist the DSMB in determining whether any emergent safety issues were present. This report included counts of adverse events (AEs) and serious adverse events (SAEs) by treatment group with associated P-values based on Fisher's exact tests. No emergent issues were identified.

7 6 Appendix Table S1. Sensitivity analyses of primary outcome of histologic improvement Panel A. Number of completed and missing week 72 biopsies Obeticholic Acid (n=141) Placebo (n=142) Completed week 72 biopsy Missing week 72 biopsy Week 72 window closed before 6 Jan Week 72 window open but completed Missed Visit form for Week 72 visit before 6 Jan Week 72 window open Panel B. Sensitivity analysis: Histologic improvement by treatment using various missing data scenarios Obeticholic Acid vs. Placebo No. of patients Odds Missing data scenario Ratio 95% CI P-value OCA Plbo Per modified protocol All randomised patients Delete missing Multiple imputation for missing Worst case scenario for OCA Best case scenario for OCA < Observations with missing week 72 biopsy imputed as no improvement among patients at risk of week 72 biopsy Observations with missing week 72 biopsy imputed as no improvement among all randomised patients Observations with missing week 72 biopsy were imputed using 50 datasets. Imputation model included the following baseline variables: treatment group indicator, NAFLD activity score, fibrosis score; One observation was dropped due to missing both baseline and followup biopsy Observations with missing week 72 biopsy were imputed as improvement for patients assigned to Placebo and no improvement for patients assigned to OCA Observations with missing week 72 biopsy imputed as no improvement for patients assigned to Placebo and improvement for patients assigned to OCA Panel C. Post-hoc sensitivity analysis: Potential mediation of the Obeticholic Acid histologic improvement effect by weight change or ALT change Obeticholic Acid vs. Placebo No. of patients Odds Model Ratio 95% CI P-value OCA Plbo No adjustment Adjusted for change in weight (kg) at 72 weeks Adjusted for change in ALT (U/L) at 72 weeks Adjusted for change in weight (kg) and change in ALT (U/L) at 72 weeks Complete case analysis

8 7 Appendix Table 2. Post-hoc analysis: Odds of histologic improvement between treatment groups by baseline and post-randomisation subgroups Histologic Improvement Odds Ratio Obeticholic Acid Placebo (95% CI) Subgroup % x/n % x/n OCA vs. Plbo Overall 45% 50/110 21% 23/ (1 7, 5 6) Treatment by Subgroup P- value Stratification variables Diabetes at baseline 0 16 No 37% 19/51 23% 12/ (0 8, 4 7) Yes 53% 31/59 19% 11/ (2 0, 10 6) Clinic 0 46 A 69% 9/13 15% 2/ (1 8, 83 7) B 40% 4/10 10% 1/ (0 5, 67 6) C 62% 5/8 22% 2/9 5 8 (0 7, 48 9) D 18% 2/11 27% 3/ (0 1, 4 5) E 45% 5/11 0% 0/ (1 3, ) F 43% 6/14 36% 5/ (0 3, 6 2) G 60% 6/10 44% 4/9 1 9 (0 3, 11 6) H 50% 9/18 29% 5/ (0 6, 9 7) I 27% 4/15 7% 1/ (0 5, 48 8) Enrollment date 0 95 < 1 Jan % 29/67 20% 14/ (1 4, 6 5) 1 Jan % 21/43 23% 9/ (1 2, 8 3) Demographics Gender 0 58 Female 47% 36/77 20% 14/ (1 7, 7 3) Male 42% 14/33 23% 9/ (0 9, 6 8) Age at baseline yrs 0 09 < 55 41% 26/63 25% 17/ (1 0, 4 4) 55 51% 24/47 14% 6/ (2 2, 17 6) Race 0 67 Non-white 60% 6/10 38% 5/ (0 4, 13 0) White 43% 42/98 17% 16/ (1 8, 7 0) Ethnicity 0 60 Non-Hispanic 45% 43/96 19% 18/ (1 8, 6 5) Hispanic 50% 7/14 31% 5/ (0 5, 9 7) Liver enzymes ALT at baseline U/L 0 44 < 60 40% 19/47 13% 6/ (1 6, 12 5) 60 49% 31/63 27% 17/ (1 3, 5 6) Metabolic features Body mass index kg/m < 35 46% 26/56 27% 20/ (1 1, 4 8) 35 44% 23/52 8% 3/ (2 4, 32 1) Insulin resistance at baseline 0 38 Insulin sensitive 14% 1/7 10% 1/ (0 1, 28 9) Insulin resistant intact beta cells 42% 14/33 31% 11/ (0 6, 4 5) Insulin resistant early beta cell loss 44% 4/9 21% 3/ (0 5, 18 3) Insulin resistant advanced beta cell loss 52% 31/60 17% 8/ (2 1, 13 3) Glucose at baseline mmol/l 0 09 < % 35/69 18% 11/ (2 1, 10 7) % 15/41 26% 12/ (0 7, 4 2)

9 8 Insulin at baseline pmol/l 0 68 < 72 48% 45/93 23% 19/ (1 7, 6 2) 72 31% 5/16 17% 4/ (0 5, 10 3) Histology NAFLD Activity Score at baseline 2-3 0% 0/7 0% 0/12 n/c % 50/103 24% 23/ (1 7, 5 6) n/c Steatohepatitis at baseline 0 71 Not NASH 22% 2/9 18% 2/ (0 1, 11 5) Borderline NASH 27% 3/11 10% 1/ (0 3, 39 3) Definite NASH 50% 45/90 23% 20/ (1 8, 6 4) Ballooning at baseline 0 79 None 20% 3/15 12% 2/ (0 3, 13 1) Few 39% 12/31 14% 5/ (1 2, 13 3) Many 55% 35/64 30% 16/ (1 4, 6 2) Fibrosis at baseline 0 85 Stage % 18/45 18% 7/ (1 1, 8 1) Stage % 32/65 23% 16/ (1 6, 7 0) Biopsy length at baseline mm 0 84 < 15 36% 12/33 17% 5/ (0 9, 9 4) 15 49% 38/77 23% 18/ (1 7, 6 6) Biopsy length at week 72 mm 0 36 < 15 41% 18/44 13% 6/ (1 6, 13 2) 15 48% 32/66 27% 17/ (1 2, 5 3) Concomitant medications Vitamin E use at baseline 0 39 No 42% 37/88 15% 13/ (1 9, 8 2) Yes 59% 13/22 40% 10/ (0 7, 7 0) Aspirin use at baseline 0 44 No 42% 33/79 17% 14/ (1 7, 7 4) Yes 55% 17/31 36% 9/ (0 7, 6 4) Statin use at baseline 0 13 No 44% 24/54 27% 17/ (1 0, 4 7) Yes 46% 26/56 13% 6/ (2 1, 15 8) Post-randomisation subgroups Change in weight at 72 weeks kg 0 98 Decrease 2 or more 60% 27/45 33% 10/ (1 1, 7 9) Change < 2 35% 13/37 17% 6/ (0 9, 7 9) Increase 2 or more 43% 10/23 20% 7/ (1 0, 9 9) Change in ALT at 72 weeks 0 34 Decrease 53% 49/92 29% 20/ (1 4, 5 3) No change or increase 8% 1/13 9% 3/ (0 1, 8 8) The primary outcome of histologic improvement required a decrease of 2 or more points in the total NAFLD activity score and no worsening in the fibrosis score in 219 patients at risk for week 72 biopsies. The odds ratio and 95% confidence interval were derived from a logistic regression of the odds of histologic improvement by treatment group within each stratum of the subgroup. The treatment by subgroup p-value was derived from Wald s test of one or more indicator variables of the interaction of treatment group and subgroup from a logistic regression of the odds of histologic improvement. Excludes observations with missing subgroup data. Based on cross-classifying glucose (<5 55 vs mmol/l) with insulin (<72 vs. 72 pmol/l)

10 9 Appendix Table S3. Changes in liver enzymes, biochemical levels, metabolic factors, and quality of life from baseline to 96 weeks Variable Mean±SD change from Adjusted net baseline at 96 weeks change from baseline Obeticholic Obeticholic Acid Acid Placebo - Placebo (N=122) (N=120) (95% CI) P value Liver enzymes Alanine aminotransferase U/L -27±49-21±34-4 (-13, 5) 0 36 Asparate aminotransferase U/L -20±39-11±26-4 (-10, 3) 0 24 Alkaline phosphatase U/L 0±21-2±17 2 (-2, 7) 0 31 γ-glutamyl transpeptidase U/L -15±51-6±37-4 (-14, 6) 0 40 Total bilirubin μmol/l 0 5± ± (-0 8, 1 3) 0 66 Lipids Cholesterol Total mmol/l -0 3± ± (-0 2, 0 2) 0 89 High-density lipoprotein mmol/l 0 0± ± (0 0, 0 0) 0 80 Low-density lipoprotein mmol/l -0 3± ± (-0 2, 0 2) 0 86 Trigylcerides mmol/l 0 0± ± (-0 2, 0 4) 0 58 Haematology Haemoglobin g/l 0 5± ± (-1 3, 3 6) 0 36 Haematocrit proportion of ± ± (-0 01, 0 01) 0 77 Mean corpuscular volume fl -0 5± ± (-1 2, 0 0) 0 07 White blood cell count 10 9 /L 0 1± ± (-0 2, 0 6) 0 24 Platelet count 10 9 /L -3±34-4±36 1 (-7, 10) 0 80 Chemistries Bicarbonate mmol/l -0 4± ± (-0 9, 0 3) 0 30 Calcium mmol/l 0 01± ± (-0 01, 0 07) 0 16 Phosphate mmol/l 0 01± ± (-0 04, 0 05) 0 77 Creatinine ìmol/l 3 7± ± (-3 0, 5 7) 0 53 Uric acid ìmol/l -10±65-10±66 2 (-13, 16) 0 84 Albumin g/l 0 1± ± (-0 6, 0 8) 0 76 Total protein g/l -0 3± ± (-0 6, 1 4) 0 45 Other laboratory results Prothrombin time sec n/a n/a n/a n/a International normalised ratio n/a n/a n/a n/a Metabolic factors Fasting serum glucose mmol/l 0 1± ± (-1 0, 0 1) 0 14 Insulin pmol/l 24±189 36±136-1 (-43, 41) 0 95 HOMA-IR glucose [mmol/l] insulin [pmol/l] / ±86 16±64-5 (-24, 15) 0 63 Haemoglobin AlC mmol/mol -1 1± ± (-4 6, 0 2) 0 07 Weight kg -1 3± ± (-3 2, 0 3) 0 10 Body-mass index kg/m 2-0 3± ± (-1 0, 0 2) 0 16 Waist circumference cm -0 9± ± (-2 1, 1 7) 0 85 Waist to hip ratio 0 00± ± (-0 01, 0 02) 0 55 Systolic blood pressure mmhg 0±18-2±16 2 (-1, 5) 0 26 Diastolic blood pressure mmhg 1±11 1±11-1 (-3, 1) 0 34 SF-36 Quality of life Physical component summary 0±8-1±7 1 (-1, 3) 0 19 Mental component summary 0±9-1±10 2 (-1, 4) 0 14 Adjusted for baseline value Not collected at week 96 visit Homeostasis Model Assessment-estimated Insulin Resistance

11 10 Appendix Table 4. Change from baseline to 72 weeks in detailed scoring of histologic features by treatment group A. Fibrosis stage (0-4) Treatment Group Baseline (stage) None (Stage 0) Perisinusoidal or Periportal ( Stage 1) Week 72 (stage) Perisinusoidal and Periportal ( Stage 2) Bridging Fibrosis ( Stage 3) Cirrhosis ( Stage 4) Mean Change in Stage Improve Stage (%) OCA None ( Stage 0) Perisinusoidal or Periportal ( Stage 1) Perisinusoidal and Periportal ( Stage 2) Bridging Fibrosis (Stage 3) Cirrhosis (Stage 4) Total % Plbo None (Stage 0) Perisinusoidal or Periportal (Stage 1) Perisinusoidal and Periportal (Stage 2) Bridging Fibrosis (Stage 3) Cirrhosis (Stage 4) Total % P-value Based on ANCOVA model regressing change on baseline value and treatment group for change and Cochran-Mantel-Haenszel chi-square test stratified by clinic and diabetes status for improvement. Includes 1a=Mild, zone 3 perisinusoidal, 1b=Moderate, zone 3, perisinusoidal and 1c=Portal/periportal only

12 11 B. Steatohepatitis categories Treatment Group Baseline Category Not NAFLD NAFLD, Not NASH Week 72 Category Borderline NASH Definite NASH Resolve (%) OCA Not NAFLD NAFLD, Not NASH Borderline NASH Definite NASH Total 22% Plbo Not NAFLD NAFLD, Not NASH Borderline NASH Definite NASH Total 13% P-value 0 08 Defined as Not NAFLD or NAFLD, Not NASH on 72 week biopsy Based on Cochran-Mantel-Haenszel chi-square test stratified by clinic and diabetes status

13 12 C. Steatosis scores (0-3) Treatment Group Baseline (score) <5% (0) 5-33% (1) Week 72 (score) 34-66% (2) >66% (3) Mean Change in Score Improve Score (%) OCA <5% (0) % (1) % (2) >66% (3) Total % Plbo <5% (0) % (1) % (2) >66% (3) Total % P-value Based on ANCOVA model regressing change on baseline value and treatment group for change and Cochran-Mantel-Haenszel chi-square test stratified by clinic and diabetes status for improvement.

14 13 D. Hepatocellular ballooning scores (0-2) Treatment Group Baseline (score) None (0) Week 72 (score) Few (1) Many (2) Mean Change in Score Improve Score (%) OCA None (0) Few (1) Many (2) Total % Plbo None (0) Few (1) Many (2) Total % P-value Based on ANCOVA model regressing change on baseline value and treatment group for change and Cochran-Mantel-Haenszel chi-square test stratified by clinic and diabetes status for improvement.

15 14 E. Lobular inflammation scores (0-3) Treatment Group Baseline (score) 0 (0) <2 (1) Week 72 (score) 2-4 (2) >4 (3) Mean Change in Score Improve Score (%) OCA 0 (0) <2 (1) (2) >4 (3) Total % Plbo 0 (0) <2 (1) (2) >4 (3) Total % P-value Based on ANCOVA model regressing change on baseline value and treatment group for change and Cochran-Mantel-Haenszel chi-square test stratified by clinic and diabetes status for improvement.

16 15 F. NAFLD Activity Scores (1-8) Treatment Group Baseline NAS Week 72 NAS Mean Change in Score Improve Score (%) OCA Total % Plbo Total % P-value < Based on ANCOVA model regressing change on baseline value and treatment group for change and Fisher s exact test for improvement.

17 16 G. Portal inflammation scores (0-2) Treatment Group Baseline (score) None (0) Week 72 (score) Mild (1) > Mild (2) Mean Change in Score Improve Score (%) OCA None (0) Mild (1) > Mild (2) Total % Plbo None (0) Mild (1) > Mild (2) Total % P-value Based on ANCOVA model regressing change on baseline value and treatment group for change and Cochran-Mantel-Haenszel chi-square test stratified by clinic and diabetes status for improvement.

18 17 Appendix Figure S1. Changes from baseline in serum lipids according to treatment group Mean (95% CI) change - mmol/l Total Cholesterol Obeticholic Acid Placebo Median (95% CI) change - mmol/l Triglycerides Placebo Obeticholic Acid Weeks Weeks p<0 05 p<0 05 Mean (95% CI) change - mmol/l High-density Lipoprotein Placebo Obeticholic Acid Weeks Mean (95% CI) change - mmol/l Low-density Lipoprotein Obeticholic Acid Placebo Weeks p<0 05 p<0 05 Analysis of serum lipids demonstrated that the total cholesterol and LDL cholesterol were significantly increased by 12 weeks in obeticholic acid treated patients. A small but statistically significant decrease in HDL cholesterol levels also occurred in obeticholic acid treated patients within 12 weeks. Fasting serum triglyceride levels decreased early during treatment but subsequently increased over time and the levels were not significantly different from placebo treated patients by the end of the treatment period at 72 weeks.

19 18 Appendix Figure S2. Changes from Baseline in Aspartate Aminotransferase by Treatment Group Mean (95% CI) change - U/L Aspartate Aminotransferase Placebo Obeticholic Acid Weeks p<0 05 Mean values of changes from baseline during treatment with obeticholic acid (141 subjects) or placebo (142 subjects) for up to 72 weeks follow by a 24 week post treatment period without treatment. Error bars indicate 95% confidence intervals. Aspartate aminotransferase levels dropped during treatment with obeticholic acid, reaching a reduced baseline 48 weeks after initiating treatment, whereas levels in placebo treated subjects remained unchanged. The aminotransferase levels in obeticholic acid-treated subjects reverted back to being indistinguishable from placebo-treated subjects 24 weeks after treatment discontinuation.