COMBINATION DRUG THERAPY ALLOWS …...COMBINATION DRUG THERAPY ALLOWS SYNERGISTIC THERAPEUTIC DOSE REDUCTION IN NASH: A CASE STUDY OF ELAFIBRANOR (GFT505) AND AN FXR AGONIST COMBINATION

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[1] Lade A, Noon LA, Friedman SL. Contributions of metabolic dysregulation and inflammation to nonalcoholic steatohepatitis, hepatic fibrosis, and cancer. Curr Opin Oncol. 2014 Jan;26(1):100-107. ; [2] Paradis V, Zalinski S, Chelbi E, et al. Hepatocellular carcinomas in patients with metabolic syndrome often develop without significant liver fibrosis: a pathological analysis. Hepatology. 2009 Mar;49(3):851-9. ; [3] Adams LA, Anstee QM, Tilg H, Targher G. Non-alcoholic fatty liver disease and its relationship with cardiovascular disease and other extrahepatic diseases. Gut. 2017 Mar 17 . ; [4] Ratziu V, Harrison SA, Francque S, Bedossa P et al. Elafibranor, an Agonist of the Peroxisome Proliferator-Activated Receptor-α and -δ, Induces Resolution of Nonalcoholic Steatohepatitis Without Fibrosis Worsening. Gastroenterology. 2016 May; 150(5):1147-1159 . ; [5] Neuschwander-Tetri BA, Loomba R, Sanyal AJ, Lavine JE et al. Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebo-controlled trial. Lancet. 2015 Mar 14; 385 (9972):956-965. ; [6] Noel B, Rigou G, Degallaix N, Daix V et al. Elafibranor (GFT505) prevents NASH, hepatic fibrosis and hepatocarcinoma in a new disease model. AASLD LiverLearning®. Nov 15, 2015; 110218 . ; [7] Yadav B, Wennerberg K, Aittokallio T and Tang J. Searching for drug synergy in complex dose-response landscapes using an interaction potency model. Computational and Structural Biotechnology Journal. 2015; 13: 504-513. ; [8] Kleiner DE, Brunt EM, Van NM, Behling C, Contos MJ, Cummings OW, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005 Jun;41(6):1313-1321

INTRODUCTION HEPATIC GENE EXPRESSION AND BIOMARKER ANALYSISSYNERGY OF ELA AND OCA ON FIBROSIS DEVELOPMENT PREDOMINANT EFFECT OF ELA ON NASH HISTOLOGY

COMBINATION DRUG THERAPY ALLOWS SYNERGISTIC THERAPEUTIC DOSE REDUCTION IN NASH:A CASE STUDY OF ELAFIBRANOR (GFT505) AND AN FXR AGONIST COMBINATION IN A MODEL OF SEVERE NASH

Vlad Ratziu*1, 2, Stephen A. Harrison3, Benoit Noel4, Géraldine Rigou4, Nathalie Degallaix4, Frédéric Texier4, John Brozek4, Valérie Daix4,Carole Belanger4, Alice Roudot4, Sophie Mégnien4, Dean W. Hum4, Bart Staels5, Robert Walczak4, Arun Sanyal6

1Hôpital Pitié Salpêtrière, Paris, France, 2Institute of Cardiometabolism and Nutrition, Paris, France, 3Pinnacle Clinical Research Center, San Antonio, TX, United States,4GENFIT SA, Loos, France, 5Institut Pasteur de Lille, Lille, France, 6Virginia Commonwealth University, Richmond, VA, United States

Western lifestyle is invariably linked with high incidence rate of non-alcoholic steatohepatitis (NASH), a multifactorial, chronic liver disease that often progresses to liver fibrosis and cirrhosis and may ultimately lead to hepatocellular carcinoma [1, 2]. NASH has also been associated with increased occurrence of cardiovascular disease and other extrahepatic diseases [3].

Currently, there is no approved therapy for NASH. The treatment paradigm of drug combination has therefore emerged to provide the best possible therapy for the largest NASH population. Similar approaches were previously tested in other multifactorial systemic diseases, such as hypertension, dyslipidemia or type 2 diabetes, where drug combinations showed a better control of the underlying disease and better long term patient adherence to the treatment.

In recent phase 2B studies, both elafibranor (ELA, PPARα/δ agonist) and obeticholic acid (OCA; FXR agonist) have shown efficacy on NASH and fibrosis [4, 5]. Since these drugs have complementary mechanisms of action, we compared their effects on pathological features of NASH and liver fibrosis in a model of severe disease [6] to assess potential therapeutic benefits of such combination therapy.

AIMS

To test the hypothesis that elafibranor and FXR agonist combination has synergistic effects on NASH development and to assess potential therapeutic benefits of such a combination therapy

STUDY DESIGN

STATISTICAL ANALYSIS

Data are Mean ± SEM# p<0.05, ## p<0.01, ### p<0.001 using Student t-test*p<0.05, **p<0.01, ***p<0.001 vs CDAA/c, using One-way ANOVA and Bonferroni post-hoc § p<0.05, §§ p<0.01, §§§ p<0.001 vs CDAA/c, using Kruskal-Wallis test and Dunn’s post-hocDrug synergism was evaluated using the Highest Single Agent (HSA) model [7] and is represented in blue in the graphs or tables.

CONCLUSION

Wistar rats fed a CDAA/c diet developed severe NASH-related histology (advanced steatosis, lobular inflammation and ballooning) and fibrosis with high penetration rate.

Fibrosis development was significantly attenuated in animals that were administered with a high dose of Elafibranor (ELA) or Obeticholic Acid (OCA) as single agent.

A synergistic effect on fibrosis attenuation was observed in animals that received ELA/OCA combination treatment at submaximal doses.

In contrast to fibrosis, there was no synergistic effect of ELA/OCA combination therapy on either ballooning or lobular inflammation and the effect of elafibranor was predominant on NASH histology.

ELA revealed a predominant effect on transcriptomic NASH signature as compared to OCA, although there was a significant overlap in the hepatic transcripts that were modulated by each agent.

IPA analysis showed that pathways related to liver metastasis, damage and regeneration responded better to ELA/OCA combination in this study.

Our findings suggest that ELA and FXR agonist combination treatment would benefit a wider patient population and possibly at lower therapeutic doses.

A) SIRIUS RED STAINING OF LIVER SECTIONS (ORIGINAL MAGNIFICATION OF 50X)

B) RELATIVE FIBROTIC AREA (SIRIUS RED-STAINED)

Coadministration of ELA and OCA allows synergistic dose reduction of both compounds to treat fibrosis development.

The administration of ELA and OCA as single agents resulted in a dose-dependent attenuation of fibrosis development. Similarly, a significant reduction in fibrosis was also observed in animals that received both compounds and this effect was obtained at significantly lower doses as compared to single agents.

C) COLLAGEN CONTENT (HYDROXYPROLINE) combination

combination

A) LOBULAR INFLAMMATION SCORE

B) HEPATOCYTE BALLOONING SCORE

Coadministration of ELA and OCA did not result in any synergy on liver steatosis, inflammation or ballooning as defined by Kleiner.

In this study both drugs showed partial protection against histological NASH development but ELA was more potent in reducing the development of severe NASH pathology, i.e. in reducing hepatocyte ballooning and lobular inflammation. Steatosis was not affected by either ELA or OCA (not shown). No significant improvement in NASH diagnosis, was observed in animals that received both ELA and OCA as compared to any single agent.

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TRANSCRIPTOMIC AND FUNCTIONAL PATHWAY ANALYSIS OF HEPATIC GENE EXPRESSION

A) PHARMACOLOGICAL REVERSION OF THE NASH-RELATED SIGNATURE B) ELA SHOWED A PREDOMINANT EFFECT ON NASH SIGNATURE C) DIFFERENTIAL ANALYSIS OF GENES AND FUNCTIONAL PATHWAYS MODULATED BY ELA AND/OR OCA

The evidence of synergy between the actions of ELA and OCA was established for such processes as fibrosis, tissue remodeling and inflammation but not for the oxidative stress.

Several fibrosis and tissue remodeling markers but not inflammation markers were attenuated by either ELA or OCA as a single agent, especially at higher doses. The synergy between ELA and OCA is clearly visible on multiple markers of fibrosis, tissue remodeling and inflammation. In contrast, ELA was the only agent to attenuate oxidative stress markers in the liver.

CSAA CDAA/c OCA 10mpk OCA 30mpk ELA 1mpk ELA 3mpk ELA 10mpk ELA 1mpk ELA 10mpkELA 3mpk

+ OCA 10mpk

Transcriptomic signature (induced by the CDAA/c diet) was reversed by both ELA and OCA although the effect of ELA on hepatic gene expression was predominant in the conditions of this study.Functional pathways related to liver metastasis, damage and regeneration benefit from ELA and OCA coadministration.

Only genes with FC≥2 and p value<0.01 were considered in this analysis. Analysis of hepatic PPAR and FXR target gene expression indicates that both molecular target classes (PPARα/δ and FXR) were engaged. Within the pool of genes that are modulated in the CDAA/c model, the transcriptional signature of the ELA+OCA combination is mainly driven by ELA (226 genes) as compared to OCA (68 genes) and 60 of these genes were modulated by both drugs. 93 genes were modulated exclusively by ELA/OCA administration.

(A) Heatmap representation of the CDAA/c diet induced pathology and the effect of pharmacologic treatments (NASH transcriptomic signature).(B) Analysis of the NASH transcriptomic signature in animals that received ELA+OCA combination as compared to any single agent. Examples of PPAR a/d and FXR target genes modulated by pharmacological treatments.(C) Differential analysis of genes and functional pathways (IPA) that are modulated (p value < 0.05) by any treatment within the NASH transcriptomic signature.

mRNA

Fibrosis and tissue remodeling markers Inflammation markers Oxidative stress markers

αSMA mNA Col1α1 mRNA TIMP1 mRNA TGFβ1 mRNA MMP2 mRNA MCP1 mRNA CCR2 mRNA CCR5 mRNA SOD1 mRNA Catalase mRNA

ELA 1 1,14 ± 0,17 0,94 ± 0,24 1,20 ± 0,14 1,33 ± 0,06 *** 1,00 ± 0,05 1,11 ± 0,08 1,51 ± 0,21 1,45 ± 0,17 1,21 ± 0,14 1,59 ± 0,15 *

ELA3 0,66 ± 0,09 0,31 ± 0,10 §§ 0,78 ± 0,08 0,94 ± 0,07 0,90 ± 0,11 0,65 ± 0,10 0,98 ± 0,12 1,03 ± 0,17 1,56 ± 0,13 §§§ 2,12 ± 0,15 ***

ELA 10 0,68 ± 0,07 0,10 ± 0,02 §§§ 0,50 ± 0,06 ** 0,76 ± 0,06 * 0,45 ± 0,08 0,61 ± 0,10 0,95 ± 0,12 0,69 ± 0,08 2,11 ± 0,12 §§§ 2,71 ± 0,24 ***

OCA 10 1,09 ± 0,23 0,72 ± 0,26 § 1,10 ± 0,14 1,10 ± 0,08 0,27 ± 0,03 0,95 ± 0,13 0,92 ± 0,15 0,81 ± 0,09 1,14 ± 0,09 1,06 ± 0,03

OCA 30 0,66 ± 0,07 0,25 ± 0,03 §§§ 0,92 ± 0,08 0,89 ± 0,07 0,90 ± 0,21 0,71 ± 0,06 0,73 ± 0,17 0,76 ± 0,12 1,00 ± 0,07 1,12 ± 0,06

ELA 1 + OCA 10 0,71 ± 0,11 0,28 ± 0,08 §§ 0,75 ± 0,12 0,92 ± 0,05 0,58 ± 0,07 0,72 ± 0,06 0,85 ± 0,11 0,76 ± 0,07 1,42 ± 0,18 1,77 ± 0,13 §§§

ELA3 + OCA 10 0,39 ± 0,06 §§§ 0,13 ± 0,02 §§§ 0,46 ± 0,04 §§§ 0,67 ± 0,05 *** 0,52 ± 0,09 §§ 0,58 ± 0,07 §§ 0,59 ± 0,06 * 0,56 ± 0,05 ** 1,73 ± 0,16 §§§ 2,04 ± 0,13 §§§

ELA 10 + OCA 10 0,32 ± 0,05 §§§ 0,05 ± 0,01 §§§ 0,29 ± 0,03 §§§ 0,48 ± 0,04 §§§ 0,28 ± 0,03 §§§ 0,30 ± 0,03 §§§ 0,37 ± 0,06 *** 0,42 ± 0,04 *** 1,87 ± 0,10 *** 2,58 ± 0,15 §§§

ELISA & biochemical assays

Fibrosis and tissue remodeling markers Inflammation markers Oxidative stress markers

α2-Macroglobulin PIIINP TIMP-1 CHI3L1 CK18-M30 Plasma LBP Plasma CXCL10 N/A hepatic TBARS level

hepatic Catalase activity

ELA 1 1,25 +/- 0,22 0,84 +/- 0,06 0,98 +/- 0,10 1,04 +/- 0,07 0,91 +/- 0,11 1,03 +/- 0,12 1,09 +/- 0,14 N/A 1,05 +/- 0,13 0,85 +/- 0,07

ELA3 0,32 +/- 0,04 §§ 0,62 +/- 0,04 *** 0,70 +/- 0,05 § 0,66 +/- 0,06 ** 0,59 +/- 0,07 ** 0,74 +/- 0,08 1,03 +/- 0,20 N/A 1,01 +/- 0,08 1,47 +/- 0,21

ELA 10 0,31 +/- 0,07 §§ 0,50 +/- 0,02 *** 0,45 +/- 0,03 §§§ 0,45 +/- 0,04 *** 0,41 +/- 0,04 *** 0,46 +/- 0,05 *** 0,68 +/- 0,05 § N/A 0,86 +/- 0,04 1,83 +/- 0,23 §

OCA 10 0,70 +/- 0,16 0,73 +/- 0,07 ** 0,83 +/- 0,09 0,91 +/- 0,10 0,83 +/- 0,08 0,82 +/- 0,05 0,68 +/- 0,08 * N/A 3,47 +/- 0,72 §§ 0,94 +/- 0,07

OCA 30 0,37 +/- 0,07 ** 0,64 +/- 0,03 *** 0,74 +/- 0,07 * 0,90 +/- 0,08 0,67 +/- 0,07 ** 0,80 +/- 0,08 0,70 +/- 0,10 * N/A 5,95 +/- 1,04 §§§ 1,14 +/- 0,09

ELA 1 + OCA 10 0,31 +/- 0,03 * 0,59 +/- 0,04 *** 0,66 +/- 0,07 § 0,85 +/- 0,09 0,66 +/- 0,09 0,64 +/- 0,05 ** 0,61 +/- 0,04 §§ N/A 2,23 +/- 0,14 §§ 1,46 +/- 0,43

ELA3 + OCA 10 0,23 +/- 0,03 §§§ 0,54 +/- 0,03 *** 0,51 +/- 0,05 §§§ 0,51 +/- 0,08 *** 0,58 +/- 0,08 *** 0,84 +/- 0,15 0,64 +/- 0,05 §§ N/A 1,77 +/- 0,25 § 1,96 +/- 0,61

ELA 10 + OCA 10 0,20 +/- 0,01 §§§ 0,47 +/- 0,01 §§§ 0,35 +/- 0,03 §§§ 0,32 +/- 0,03 §§§ 0,37 +/- 0,04 *** 0,32 +/- 0,03 *** 0,47 +/- 0,03 *** N/A 1,20 +/- 0,13 1,83 +/- 0,20

mRNA

Fibrosis and tissue remodeling markers Inflammation markers Oxidative stress markers

αSMA mNA Col1α1 mRNA TIMP1 mRNA TGFβ1 mRNA MMP2 mRNA MCP1 mRNA CCR2 mRNA CCR5 mRNA SOD1 mRNA Catalase mRNA

ELA 1 1,14 ± 0,17 0,94 ± 0,24 1,20 ± 0,14 1,33 ± 0,06 *** 1,00 ± 0,05 1,11 ± 0,08 1,51 ± 0,21 1,45 ± 0,17 1,21 ± 0,14 1,59 ± 0,15 *

ELA3 0,66 ± 0,09 0,31 ± 0,10 §§ 0,78 ± 0,08 0,94 ± 0,07 0,90 ± 0,11 0,65 ± 0,10 0,98 ± 0,12 1,03 ± 0,17 1,56 ± 0,13 §§§ 2,12 ± 0,15 ***

ELA 10 0,68 ± 0,07 0,10 ± 0,02 §§§ 0,50 ± 0,06 ** 0,76 ± 0,06 * 0,45 ± 0,08 0,61 ± 0,10 0,95 ± 0,12 0,69 ± 0,08 2,11 ± 0,12 §§§ 2,71 ± 0,24 ***

OCA 10 1,09 ± 0,23 0,72 ± 0,26 § 1,10 ± 0,14 1,10 ± 0,08 0,27 ± 0,03 0,95 ± 0,13 0,92 ± 0,15 0,81 ± 0,09 1,14 ± 0,09 1,06 ± 0,03

OCA 30 0,66 ± 0,07 0,25 ± 0,03 §§§ 0,92 ± 0,08 0,89 ± 0,07 0,90 ± 0,21 0,71 ± 0,06 0,73 ± 0,17 0,76 ± 0,12 1,00 ± 0,07 1,12 ± 0,06

ELA 1 + OCA 10 0,71 ± 0,11 0,28 ± 0,08 §§ 0,75 ± 0,12 0,92 ± 0,05 0,58 ± 0,07 0,72 ± 0,06 0,85 ± 0,11 0,76 ± 0,07 1,42 ± 0,18 1,77 ± 0,13 §§§

ELA3 + OCA 10 0,39 ± 0,06 §§§ 0,13 ± 0,02 §§§ 0,46 ± 0,04 §§§ 0,67 ± 0,05 *** 0,52 ± 0,09 §§ 0,58 ± 0,07 §§ 0,59 ± 0,06 * 0,56 ± 0,05 ** 1,73 ± 0,16 §§§ 2,04 ± 0,13 §§§

ELA 10 + OCA 10 0,32 ± 0,05 §§§ 0,05 ± 0,01 §§§ 0,29 ± 0,03 §§§ 0,48 ± 0,04 §§§ 0,28 ± 0,03 §§§ 0,30 ± 0,03 §§§ 0,37 ± 0,06 *** 0,42 ± 0,04 *** 1,87 ± 0,10 *** 2,58 ± 0,15 §§§

ELISA & biochemical assays

Fibrosis and tissue remodeling markers Inflammation markers Oxidative stress markers

α2-Macroglobulin PIIINP TIMP-1 CHI3L1 CK18-M30 Plasma LBP Plasma CXCL10 N/A hepatic TBARS level

hepatic Catalase activity

ELA 1 1,25 +/- 0,22 0,84 +/- 0,06 0,98 +/- 0,10 1,04 +/- 0,07 0,91 +/- 0,11 1,03 +/- 0,12 1,09 +/- 0,14 N/A 1,05 +/- 0,13 0,85 +/- 0,07

ELA3 0,32 +/- 0,04 §§ 0,62 +/- 0,04 *** 0,70 +/- 0,05 § 0,66 +/- 0,06 ** 0,59 +/- 0,07 ** 0,74 +/- 0,08 1,03 +/- 0,20 N/A 1,01 +/- 0,08 1,47 +/- 0,21

ELA 10 0,31 +/- 0,07 §§ 0,50 +/- 0,02 *** 0,45 +/- 0,03 §§§ 0,45 +/- 0,04 *** 0,41 +/- 0,04 *** 0,46 +/- 0,05 *** 0,68 +/- 0,05 § N/A 0,86 +/- 0,04 1,83 +/- 0,23 §

OCA 10 0,70 +/- 0,16 0,73 +/- 0,07 ** 0,83 +/- 0,09 0,91 +/- 0,10 0,83 +/- 0,08 0,82 +/- 0,05 0,68 +/- 0,08 * N/A 3,47 +/- 0,72 §§ 0,94 +/- 0,07

OCA 30 0,37 +/- 0,07 ** 0,64 +/- 0,03 *** 0,74 +/- 0,07 * 0,90 +/- 0,08 0,67 +/- 0,07 ** 0,80 +/- 0,08 0,70 +/- 0,10 * N/A 5,95 +/- 1,04 §§§ 1,14 +/- 0,09

ELA 1 + OCA 10 0,31 +/- 0,03 * 0,59 +/- 0,04 *** 0,66 +/- 0,07 § 0,85 +/- 0,09 0,66 +/- 0,09 0,64 +/- 0,05 ** 0,61 +/- 0,04 §§ N/A 2,23 +/- 0,14 §§ 1,46 +/- 0,43

ELA3 + OCA 10 0,23 +/- 0,03 §§§ 0,54 +/- 0,03 *** 0,51 +/- 0,05 §§§ 0,51 +/- 0,08 *** 0,58 +/- 0,08 *** 0,84 +/- 0,15 0,64 +/- 0,05 §§ N/A 1,77 +/- 0,25 § 1,96 +/- 0,61

ELA 10 + OCA 10 0,20 +/- 0,01 §§§ 0,47 +/- 0,01 §§§ 0,35 +/- 0,03 §§§ 0,32 +/- 0,03 §§§ 0,37 +/- 0,04 *** 0,32 +/- 0,03 *** 0,47 +/- 0,03 *** N/A 1,20 +/- 0,13 1,83 +/- 0,20

mRNA

Fibrosis and tissue remodeling markers Inflammation markers Oxidative stress markers

αSMA mNA Col1α1 mRNA TIMP1 mRNA TGFβ1 mRNA MMP2 mRNA MCP1 mRNA CCR2 mRNA CCR5 mRNA SOD1 mRNA Catalase mRNA

ELA 1 1,14 ± 0,17 0,94 ± 0,24 1,20 ± 0,14 1,33 ± 0,06 *** 1,00 ± 0,05 1,11 ± 0,08 1,51 ± 0,21 1,45 ± 0,17 1,21 ± 0,14 1,59 ± 0,15 *

ELA3 0,66 ± 0,09 0,31 ± 0,10 §§ 0,78 ± 0,08 0,94 ± 0,07 0,90 ± 0,11 0,65 ± 0,10 0,98 ± 0,12 1,03 ± 0,17 1,56 ± 0,13 §§§ 2,12 ± 0,15 ***

ELA 10 0,68 ± 0,07 0,10 ± 0,02 §§§ 0,50 ± 0,06 ** 0,76 ± 0,06 * 0,45 ± 0,08 0,61 ± 0,10 0,95 ± 0,12 0,69 ± 0,08 2,11 ± 0,12 §§§ 2,71 ± 0,24 ***

OCA 10 1,09 ± 0,23 0,72 ± 0,26 § 1,10 ± 0,14 1,10 ± 0,08 0,27 ± 0,03 0,95 ± 0,13 0,92 ± 0,15 0,81 ± 0,09 1,14 ± 0,09 1,06 ± 0,03

OCA 30 0,66 ± 0,07 0,25 ± 0,03 §§§ 0,92 ± 0,08 0,89 ± 0,07 0,90 ± 0,21 0,71 ± 0,06 0,73 ± 0,17 0,76 ± 0,12 1,00 ± 0,07 1,12 ± 0,06

ELA 1 + OCA 10 0,71 ± 0,11 0,28 ± 0,08 §§ 0,75 ± 0,12 0,92 ± 0,05 0,58 ± 0,07 0,72 ± 0,06 0,85 ± 0,11 0,76 ± 0,07 1,42 ± 0,18 1,77 ± 0,13 §§§

ELA3 + OCA 10 0,39 ± 0,06 §§§ 0,13 ± 0,02 §§§ 0,46 ± 0,04 §§§ 0,67 ± 0,05 *** 0,52 ± 0,09 §§ 0,58 ± 0,07 §§ 0,59 ± 0,06 * 0,56 ± 0,05 ** 1,73 ± 0,16 §§§ 2,04 ± 0,13 §§§

ELA 10 + OCA 10 0,32 ± 0,05 §§§ 0,05 ± 0,01 §§§ 0,29 ± 0,03 §§§ 0,48 ± 0,04 §§§ 0,28 ± 0,03 §§§ 0,30 ± 0,03 §§§ 0,37 ± 0,06 *** 0,42 ± 0,04 *** 1,87 ± 0,10 *** 2,58 ± 0,15 §§§

ELISA & biochemical assays

Fibrosis and tissue remodeling markers Inflammation markers Oxidative stress markers

α2-Macroglobulin PIIINP TIMP-1 CHI3L1 CK18-M30 Plasma LBP Plasma CXCL10 N/A hepatic TBARS level

hepatic Catalase activity

ELA 1 1,25 +/- 0,22 0,84 +/- 0,06 0,98 +/- 0,10 1,04 +/- 0,07 0,91 +/- 0,11 1,03 +/- 0,12 1,09 +/- 0,14 N/A 1,05 +/- 0,13 0,85 +/- 0,07

ELA3 0,32 +/- 0,04 §§ 0,62 +/- 0,04 *** 0,70 +/- 0,05 § 0,66 +/- 0,06 ** 0,59 +/- 0,07 ** 0,74 +/- 0,08 1,03 +/- 0,20 N/A 1,01 +/- 0,08 1,47 +/- 0,21

ELA 10 0,31 +/- 0,07 §§ 0,50 +/- 0,02 *** 0,45 +/- 0,03 §§§ 0,45 +/- 0,04 *** 0,41 +/- 0,04 *** 0,46 +/- 0,05 *** 0,68 +/- 0,05 § N/A 0,86 +/- 0,04 1,83 +/- 0,23 §

OCA 10 0,70 +/- 0,16 0,73 +/- 0,07 ** 0,83 +/- 0,09 0,91 +/- 0,10 0,83 +/- 0,08 0,82 +/- 0,05 0,68 +/- 0,08 * N/A 3,47 +/- 0,72 §§ 0,94 +/- 0,07

OCA 30 0,37 +/- 0,07 ** 0,64 +/- 0,03 *** 0,74 +/- 0,07 * 0,90 +/- 0,08 0,67 +/- 0,07 ** 0,80 +/- 0,08 0,70 +/- 0,10 * N/A 5,95 +/- 1,04 §§§ 1,14 +/- 0,09

ELA 1 + OCA 10 0,31 +/- 0,03 * 0,59 +/- 0,04 *** 0,66 +/- 0,07 § 0,85 +/- 0,09 0,66 +/- 0,09 0,64 +/- 0,05 ** 0,61 +/- 0,04 §§ N/A 2,23 +/- 0,14 §§ 1,46 +/- 0,43

ELA3 + OCA 10 0,23 +/- 0,03 §§§ 0,54 +/- 0,03 *** 0,51 +/- 0,05 §§§ 0,51 +/- 0,08 *** 0,58 +/- 0,08 *** 0,84 +/- 0,15 0,64 +/- 0,05 §§ N/A 1,77 +/- 0,25 § 1,96 +/- 0,61

ELA 10 + OCA 10 0,20 +/- 0,01 §§§ 0,47 +/- 0,01 §§§ 0,35 +/- 0,03 §§§ 0,32 +/- 0,03 §§§ 0,37 +/- 0,04 *** 0,32 +/- 0,03 *** 0,47 +/- 0,03 *** N/A 1,20 +/- 0,13 1,83 +/- 0,20

Disclosure: V.Ratziu: Consultant: GENFIT SA, S. A.Harrison: : Consultant: GENFIT SA, B.Noel: Employee: GENFIT SA, G.Rigou: Employee: GENFIT SA, N.Degallaix: Employee: GENFIT SA, V.Daix: Employee: GENFIT SA, C.Belanger: Employee: GENFIT SA, A.Roudot: Employee: GENFIT SA, S.Mégnien: Employee: GENFIT SA, D. W. Hum: Employee: GENFIT SA, B.Staels: Consultant: GENFIT SA, R.Walczak: Employee: GENFIT SA, A.Sanyal: Consultant: GENFIT SA - corresponding author: [email protected] - EASL ILC 2017

All data are expressed as fold change vs levels found in CDAA/c control rats. Drug synergism was evaluated using the Highest Single Agent (HSA) model [7] and is represented in blue in the tables.

Male Wistar rats were randomly divided into 11 groups.

Control rats were fed either a control CSAA (choline supplemented) diet or a pathogenic, choline deficient diet that was supplemented with 1% cholesterol (CDAA/c) for 12 weeks.

Starting on day1 and up to the end of the study, at the end of week12, drug-treated rats were fed a CDAA/c diet supplemented with the active substances:

elafibranor (ELA): 1mg/kg/day, 3mg/kg/day or 10mg/kg/dayobeticholic acid (OCA): 10mg/kg/day or 30mg/kg/dayELA/OCA combination at 1mpk/10mpk, 3mpk/10mpk or 10mpk/10mpk

Animals treated with ELA, OCA or combination of both drugs were compared to control rats that received a CDAA/c diet and developped NASH pathology. NASH pathology and fibrosis development were scored by histology as described by Kleiner [8]. Additional biochemical and gene expression studies were also run to corroborate the histological outcomes.

Hepatic gene expression was studied by Affymetrix microarrays.

C) DISTRIBUTION OF NASH/NO NASH SCORE no NASH NASH

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0

1

2

3

0

2 5

5 0

7 5

1 0 0

0

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2

3

0

2 5

5 0

7 5

1 0 0

0

1

2

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0

2 5

5 0

7 5

1 0 0

0

1

2

3

CSAA Ct

rl

1mg/

kg

3mg/

kg

10m

g/kg

ELA

CDAA/c

% o

f aff

ecte

dan

imal

s

0

2 5

5 0

7 5

1 0 0

CSAA Ct

rl

10m

g/kg

30m

g/kg

OCA

CDAA/c

% o

f aff

ecte

dan

imal

s

0

2 5

5 0

7 5

1 0 0

% o

f aff

ecte

dan

imal

s

0

2 5

5 0

7 5

1 0 0

% o

f aff

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dan

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0

2 5

5 0

7 5

1 0 0

CSAA Ct

rl

ELA

1

CDAA/c

OCA

10

ELA

+ O

CA

CSAA Ct

rl

ELA

1

CDAA/c

OCA

10

ELA

+ O

CA

0

2 5

5 0

7 5

1 0 0

0

1

2

% o

f aff

ecte

dan

imal

s

CSAA Ct

rl

1mg/

kg

3mg/

kg

10m

g/kg

ELA

0

2 5

5 0

7 5

1 0 0

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1

2

CSAA Ct

rl

10m

g/kg

30m

g/kg

OCA

% o

f aff

ecte

dan

imal

s

0

2 5

5 0

7 5

1 0 0

0

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2

% o

f aff

ecte

dan

imal

s

0

2 5

5 0

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1 0 0

0

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2

% o

f aff

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dan

imal

s

CSAA Ct

rl

ELA

1

CDAA/c

OCA

10

ELA

+ O

CA

CSAA Ct

rl

ELA

1

CDAA/c

OCA

10

ELA

+ O

CA

IPA PATHWAY OR FUNCTION ANNOTATION ELA OCA ELA / OCA

hepatocellular carcinoma YES NO YESliver metastasis YES NO YES

activation of hepatic stellate cells NO NO YESproliferation of hepatic stellate cells YES YES YES

migration of hepatic stellate cells YES NO YEShepatic steatosis YES NO YES

liver inflammation YES NO YESliver fibrosis YES YES YES

Cirrhosis YES NO YESliver metastasis by cancer stem cells NO NO YES

nonalcoholic steatohepatitis YES NO YESbleeding of liver parenchyma YES YES YES

liver necrosis NO NO YESliver hyperplasia NO YES YES

liver regeneration NO YES YESproliferation of hepatocytes NO NO YES

apoptosis of liver cells NO NO YESInhibition of Matrix Metalloproteases YES NO YES

Agranulocyte Adhesion and Diapedesis YES NO YESGranulocyte Adhesion and Diapedesis YES NO YES

Fatty Acid α-oxidation YES NO YESLeukocyte Extravasation Signaling YES NO YES

Wnt/β-catenin Signaling YES NO YESBile Acid Biosynthesis, Neutral Pathway NO YES NO

Integrin-like Kinase Signaling YES YES YES

CDAA/cCDAA/c

CDAAvsCSAA

OCA10vsCDAA

GFTvsCDAA

GFTOCAvsCDAA

0

1

2log10(FC)

327 genes

CDAA/cvs CSAA

OCA10mpkvs CDAA/c

ELA3mpkvs CDAA/c

ELA3+OCA10vs CDAA/c

FC comparison plot betweenELA+OCA vs ELA

Log2

(FC)

: ELA

+OCA

vs

CDA

A/c

Log2(FC): ELA vs CDAA/c

FC comparison plot betweenELA+OCA vs OCA

Log2(FC): OCA vs CDAA/c

Fibr

otic

area

(%)

0

2

4

6

******

***

# # #

CSAA Ct

rl

1mg/

kg

3mg/

kg

10m

g/kg

ELA

CDAA/c

Fibr

otic

area

(%)

0

2

4

6

**

# # #

CSAA Ct

rl

10m

g/kg

30m

g/kg

OCA

CDAA/c

Fibr

otic

area

(%)

CSAA Ct

rl

ELA

3

CDAA/c

OCA

10

ELA

+ O

CA

0

2

4

6

§§

§ § §

# # ## # #

#

Fibr

otic

area

(%)

CSAA Ct

rl

ELA

1

CDAA/c

OCA

10

ELA

+ O

CA

0

2

4

6

§ § §

# # ###

##

0 .0

0 .5

1 .0

1 .5

**

***

*

##

### # #

mg c

olla

gen/

g liv

er

CSAA Ct

rl

ELA

1

CDAA/c

OCA

10

ELA

+ O

CA

0 .0

0 .5

1 .0

1 .5

§§§ § §

# # #

## # #

mg c

olla

gen/

g liv

er

CSAA Ct

rl

ELA

3

CDAA/c

OCA

10

ELA

+ O

CA

0 .0

0 .5

1 .0

1 .5

# # #

**

***

***

mg c

olla

gen/

g liv

er

0 .0

0 .5

1 .0

1 .5

**

***

# # #

mg c

olla

gen/

g liv

er

CSAA Ct

rl

1mg/

kg

3mg/

kg

10m

g/kg

ELA

CDAA/c

CSAA Ct

rl

10m

g/kg

30m

g/kg

OCA

CDAA/c

PPARα/δ target genes expressionELA3mpk vs CDAA/c

gene symbol Fold change p valueCPT1B 14,83 1,95E-15EHHADH 5,42 2,87E-15UCP3 5,23 4,54E-08CROT 4,03 3,60E-07ACOX1 2,37 3,36E-14

FXR target genes expressionOCA10mpk vs CDAA/c

gene symbol Fold change pvalueNR0B2 2,96 1,95E-05ABCB11 1,89 7,12E-07CYP7A1 -2,40 1,11E-02CYP8B1 -14,01 1,18E-11

DRUG TARGET ENGAGEMENTOCA10

Vs CDAA/c(68)

ELA3vs CDAA/c

(226)ELA+OCAvs CDAA/c

(301)

3

5

59

1

93

144

22

Log2

(FC)

: ELA

+OCA

vs

CDA

A/c

+ OCA 10mpk

ELA 1mpk ELA 3mpk ELA 10mpk ELA 1mpk ELA 10mpkELA 3mpk

+ OCA 10mpk

ELA3

ELA3

ELA3