A randomized, placebo-controlled trial of emricasan in patients with NASH and F1-F3 fibrosis

Journal Pre-proof A randomized, placebo-controlled trial of emricasan in patients with NASH and F1-F3 fibrosis Stephen A. Harrison, Zachary Goodman, Abdul Jabbar, Ravi Vemulapalli, Ziad H. Younes, Bradley Freilich, Muhammad Y. Sheikh, Jörn M. Schattenberg, Zeid Kayali, Adam Zivony, Aasim Sheikh, Javier Garcia-Samaniego, Sanjaya K. Satapathy, George Therapondos, Edward Mena, Detlef Schuppan, James Robinson, Jean L. Chan, David T. Hagerty, Arun J. Sanyal PII:

S0168-8278(19)30758-5

DOI:

https://doi.org/10.1016/j.jhep.2019.11.024

Reference:

JHEPAT 7571

To appear in:

Journal of Hepatology

Received Date: 6 September 2019 Revised Date:

12 November 2019

Accepted Date: 27 November 2019

Please cite this article as: Harrison SA, Goodman Z, Jabbar A, Vemulapalli R, Younes ZH, Freilich B, Sheikh MY, Schattenberg JM, Kayali Z, Zivony A, Sheikh A, Garcia-Samaniego J, Satapathy SK, Therapondos G, Mena E, Schuppan D, Robinson J, Chan JL, Hagerty DT, Sanyal AJ, A randomized, placebo-controlled trial of emricasan in patients with NASH and F1-F3 fibrosis, Journal of Hepatology (2020), doi: https://doi.org/10.1016/j.jhep.2019.11.024. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V. on behalf of European Association for the Study of the Liver.

Title: A randomized, placebo-controlled trial of emricasan in patients with NASH and F1-F3 fibrosis. Authors: Stephen A. Harrison1, Zachary Goodman2, Abdul Jabbar3, Ravi Vemulapalli4, Ziad H. Younes5, Bradley Freilich6, Muhammad Y. Sheikh7, Jörn M. Schattenberg8, Zeid Kayali9, Adam Zivony10, Aasim Sheikh11, Javier Garcia-Samaniego12, Sanjaya K. Satapathy13, George Therapondos14, Edward Mena15, Detlef Schuppan8,16, James Robinson17, Jean L. Chan17, David T. Hagerty17, and Arun J. Sanyal18. 1

Pinnacle Clinical Research, San Antonio, TX

2

Inova Fairfax Hospital, Falls Church, VA

3

Gastroenterology of Southern Indiana, New Albany, IN

4

Iowa Digestive Disease Center‚ Clive, IA

5

Gastro One, Germantown, TN

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Kansas City Research Institute, Kansas City, MO

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Fresno Clinical Research Center, Fresno, CA

8

Department of Medicine, University Medical Center, Mainz, Germany

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Inland Empire Liver Foundation, Rialto, CA

10

Asheville Gastroenterology Associates, Asheville, NC

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Gastrointestinal Specialists of Georgia, Marietta, GA

12

Hospital Universitario La Paz, CIBERehd, IdiPAZ, Madrid, Spain

1

13

Methodist University Hospital, University of Tennessee Health Sciences Center,

Memphis, TN 14

Ochsner Medical Center, New Orleans, LA

15

California Liver Research Institute, Pasadena, CA

16

Division of Gastroenterology, Beth Israel Deaconess Medical Center, Boston, MA

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Conatus Pharmaceuticals, Inc., San Diego, CA

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Virginia Commonwealth University, Richmond, VA

Corresponding author: David T. Hagerty, M.D. Conatus Pharmaceuticals, Inc. 16745 W. Bernardo Dr, Suite 200, San Diego, CA 92127. [email protected] (858)228-6840 (cell) Keywords: Non-alcoholic steatohepatitis, liver fibrosis, emricasan, cleaved keratin-18 (cCK18), caspase 3/7, full length keratin-18 (flCK18) Word count: 6125 (after addressing Reviewer and Editor comments) Conflict of interest statement: James Robinson, Jean L. Chan and David T. Hagerty are employees of Conatus Pharmaceuticals. Financial support: The study was funded by Conatus Pharmaceuticals, Inc. and Novartis. Author contributions: Arun J. Sanyal, Detlef Schuppan, James Robinson, Jean L. Chan, and David T. Hagerty designed the study, analyzed the results and participated in writing the manuscript. Stephen A. Harrison was the lead study investigator and

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participated in the analysis and writing of the manuscript. Zachary Goodman read all study liver biopsies and participated in writing the manuscript. Abdul Jabbar, Ravi Vemulapalli, Ziad H. Younes, Bradley Freilich, Muhammad Y. Sheikh, Jörn M. Schattenberg, Zeid Kayali, Adam Zivony, Aasim Sheikh, Javier Garcia-Samaniego, Sanjaya K. Satapathy, George Therapondos, and Edward Mena were study investigators and participated in writing the manuscript. Clinical Trial Number: Clinical Trials.gov #NCT02686762. Abstract: Background and aims: Non-alcoholic steatohepatitis (NASH) is characterized by hepatocyte steatosis, ballooning, and lobular inflammation which may lead to fibrosis. Lipotoxicity activates caspases, which cause apoptosis and inflammatory cytokine (IL-1β and IL-18) production. Emricasan is a pan-caspase inhibitor that decreases serum aminotransferases and caspase activation in NASH patients. This study postulated that 72 weeks of emricasan treatment would improve liver fibrosis without worsening of NASH. Methods: This double-blind, placebo-controlled study randomized 318 subjects 1:1:1 to twice-daily treatment with emricasan (5 or 50 mg) or matching placebo for 72 weeks. Subjects had definite NASH and NASH CRN fibrosis stage F1-F3, as determined by a central reader, on a liver biopsy obtained within 6 months of randomization. Results: Emricasan treatment did not achieve the primary objective of fibrosis improvement without worsening of NASH (emricasan 5 mg: 11.2%; emricasan 50 3

mg: 12.3%; placebo: 19.0%; odds ratios vs. placebo 0.530 and 0.588, with p=0.972 and 0.972, respectively) or the secondary objective of NASH resolution without worsening of fibrosis (emricasan 5 mg: 3.7%; emricasan 50 mg: 6.6%; placebo: 10.5%; odds ratios vs. placebo 0.334 and 0.613, with p=0.070 and 0.335, respectively). In the small subset of subjects with consistent normalization of serum ALT over 72 weeks, emricasan may have improved histologic outcomes. Conclusions: Emricasan treatment did not improve liver histology in subjects with NASH fibrosis despite target engagement and may have worsened fibrosis and ballooning. Caspase inhibition lowered serum ALT in the short-term but may have directed cells to alternative mechanisms of cell death, resulting in more liver fibrosis and hepatocyte ballooning. Lay summary: Non-alcoholic steatohepatitis (NASH) is characterized by fat accumulation in liver cells, which leads to inflammation and fibrosis. Emricasan was previously shown to inhibit some of the liver enzymes which lead to liver inflammation and fibrosis. In this study, emricasan did not improve liver inflammation or fibrosis in patients with NASH and pre-existing liver fibrosis.

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Introduction Non-alcoholic fatty liver disease (NAFLD) is becoming a major epidemic. Up to 30% of Western populations have NAFLD, 10-20% of those patients will eventually develop non-alcoholic steatohepatitis (NASH) and fibrosis, and 10-20% of those NASH patients will develop cirrhosis [1, 2]. NASH with fibrosis is the most important risk factor for developing cirrhosis and liver-related morbidity [3]. Unlike hepatitis C infection, where the paradigm of hepatocellular carcinoma occurs typically via a cirrhotic pathway, newer data suggest that a significant number of new cases of hepatocellular carcinoma in NAFLD may arise outside of a cirrhotic phenotype [4, 5]. Consequently, NASH cirrhosis and hepatocellular carcinoma due to NASH are a leading indication for liver transplantation in the U.S. [6].

Hepatocyte steatosis accompanied by ballooning, lobular and portal inflammation, with or without fibrosis are histologic hallmarks of NASH [7]. NASH histologic activity is quantified by the non-alcoholic fatty liver disease activity score (NAS) and fibrosis is staged according to the NASH Clinical Research Network (CRN) criteria [8]. Improvements in NAS or fibrosis may be acceptable components of registrational endpoints for NASH studies [9].

Steatosis and toxic saturated fatty acids and lysophospholipids can activate hepatocyte cell membrane death receptors and cause endoplasmic reticulum and mitochondrial toxicity which activates caspases [10]. Caspases are intracellular proteases that 5

orchestrate apoptotic cell death by cleavage of cytoskeletal proteins such as keratin-18 (cCK18) contributing to hepatocyte ballooning, and activate proinflammatory cytokines such as IL-1β [11, 12]. Apoptosis is increased in patients with NASH [13] and levels of cleaved keratin-18 (cCK18) correlate with apoptosis and liver fibrosis [14]. Emricasan is a pan-caspase inhibitor that decreased caspase-3/7 activity, cCK18 and serum alanine aminotransferase (ALT) in patients with NASH [15], suggesting that pan-caspase inhibition could be therapeutically useful.

This randomized, double-blind, placebo-controlled study tested the hypothesis that emricasan treatment would decrease fibrosis without worsening NASH in patients with biopsy-proven NASH F1-F3 fibrosis.

Materials, Patients and Methods Patient population Subjects were male or female, 18 years or older and provided written informed consent. Subjects had definite NASH based on the NASH CRN histologic criteria [8], as determined by the central histopathologist (ZG) on a liver biopsy performed no more than 6 months prior to randomization. Inclusion/exclusion criteria are in Supplemental Table 1.

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Outpatient subjects were screened over ~6 weeks. Randomized subjects were treated for 72 weeks with emricasan 5 mg BID, emricasan 50 mg BID or matching placebo BID and then followed-up ~4 weeks after the last dose of blinded study medication. Study participants, site personnel and the Sponsor were all blinded to treatment group assignment.

The emricasan 5 mg and 50 mg doses were chosen to evaluate an active but submaximal dose and a dose that maximally decreased biomarkers. The dose response for emricasan was previously characterized using models based on reductions in serum ALT, AST, cCK18, and caspase-3/7 which indicated that emricasan doses >27.6 mg BID maximally lowered these biomarkers. Therefore, 50 mg BID was selected as the top dose in this Phase 2 study. However, 5 mg BID was nearly as active in lowering serum ALT in subjects with active hepatitis C virus infection.

The study was designed by expert clinicians experienced in NASH in conjunction with Sponsor representatives. The protocol was approved by the Quorum central institutional review board or the institutional review boards and ethics committees at each site prior to study-related procedures. The study was conducted according to the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines. All subjects provided written informed consent. An independent Data Monitoring Committee reviewed unblinded safety and efficacy data on a regular basis throughout the study. Data were collected by investigators and

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analyzed by the Sponsor. Authors had access to the data after unblinding, participated in data analysis and interpretation, and vouch for the accuracy of the results. All authors reviewed the manuscript and approved submission.

The primary efficacy measure was improvement of at least 1 NASH CRN fibrosis stage without worsening of NASH comparing the baseline and week 72 liver biopsies. This endpoint was chosen since preclinical studies showed that emricasan treatment had significant effects upon fibrosis resolution [16, 17]. The central pathologist read the week 72 liver biopsies without comparison to or rescoring of the baseline biopsies. Worsening of NASH was defined as a ≥1-point increase in ballooning score and ≥1point increase in inflammation score. NASH resolution was assessed 2 ways: as determined by the assessment of the central pathologist and examining the proportion of subjects with a NAS score of 0-1 for inflammation, 0 for ballooning, with any score for steatosis at week 72. Other endpoints included changes in the components of the NAS, changes in Mallory-Denk bodies and portal inflammation, changes in liver collagen content and steatosis, and improvement in serum aminotransferases and caspase-3/7, cCK18 and flCK18.

Laboratory chemistries and biomarker measurements Clinical laboratory tests and biomarker measurements were performed by PPD Labs (Highland Heights, KY, USA; Zaventem, Belgium). Keratin-18 is a major cytoplasmic intermediate filament protein in hepatocytes and epithelial cells that is cleaved by 8

executioner caspases-3, -6 and -7 during apoptosis and cell death. Full‐length keratin18 (flCK18) and cCK18 were quantified in sera using enzyme‐linked immunosorbent assays detecting the M65 epitopes (VLVbio, Sundbyberg, Sweden; measures both flCK18 and cCK18, reference range: 115‐413 U/L) and the M30 epitope (VLVbio, Sundbyberg, Sweden; measures cCK18 only, reference range: <260 U/L), respectively. Caspase-3/7 activity (Promega, Madison, WI, USA; reference range: 1429‐3908 relative light units or RLU) was measured in sera and detects activity of the executioner caspases-3 and -7.

Statistical considerations, outcomes measures, analyses and sample size estimation The number and proportion of subjects meeting the criteria in the composite primary endpoint of at least 1-stage improvement in NASH CRN fibrosis stage at Week 72 without worsening of NASH were summarized by treatment group.

The primary endpoint was analyzed using a logistic regression model comparing the response for each active treatment against placebo and adjusting for fibrosis stage at baseline and diabetes status.

A multiplicity adjustment for four primary efficacy comparisons due to two active doses (5 mg and 50 mg vs placebo) and two analysis populations (the overall group and the F2+F3 subgroup) was made. To account for the correlation among the four test

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statistics obtained from the logistic regression model, the graphical gate-keeping procedure [18] used weighted parametric tests to protect the overall type I error rate at 10% (1-sided). This study was to be deemed successful if at least one emricasan treatment group was statistically significantly better than placebo in either the F2+F3 subgroup or in the overall group. No multiplicity adjustment was applied for multiple comparisons within or across secondary endpoints. Descriptive statistics are presented for all other data.

Approximately 330 subjects were to be randomized 1:1:1 using a validated program in order to have 110 subjects per group. F1 subjects were limited to ~20% of subjects. Assuming 10% of subjects had missing biopsy data at Week 72, a sample size of 330 subjects with 1:1:1 randomization would provide approximately 80% power to detect a 40% difference in the primary endpoint between at least one of the 2 active treatment groups versus placebo for either the F2+F3 subgroup or the overall group (F1+F2+F3) with a family-wise type I error rate of 10% (1-sided). Power was estimated assuming simultaneous testing of the F1+F2+F3 total group and the F2+F3 subgroup taking into account that the F2+F3 subgroup was subset of the F1+F2+F3 total group. Separate power calculations for the 2 different subgroup tests were not conducted.

Results Disposition and participant flow diagram

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Disposition is shown in Supplemental Figure 1. Most excluded subjects did not meet study inclusion/exclusion criteria (78.6%). Fifty of 746 (6.7%) had cirrhosis based upon the screening biopsy.

Baseline demographics and clinical characteristics Baseline demographics (Supplemental Table 2) and clinical, histologic and laboratory characteristics (Table 1) showed that the treatment groups were generally balanced, including fibrosis stage, NAS, NAS components, proportion of subjects with diabetes mellitus and metabolic syndrome, and body mass index.

Histologic response endpoints A summary of the efficacy responses is shown in Table 2. Greater fibrosis improvement without worsening of NASH was not achieved for either emricasan dose compared to placebo in either the overall group or the F2-F3 subgroup. Responses in the emricasan 5 mg (11.2%) and 50 mg (12.3%) groups were numerically less than that in the placebo (19.0%) group. Emricasan-treated subjects had numerically lower rates of NASH resolution (as assessed by the central pathologist) without worsening of fibrosis (emricasan 5 mg: 3.7%; emricasan 50 mg: 6.6%) when compared to placebo (10.5%, p=NS), resolution of fibrosis (emricasan 5 mg: 3.7%; emricasan 50 mg: 0.9%; and placebo: 4.8%), and NAS response (emricasan 5 mg: 10.3%; emricasan 50 mg: 9.4%; and placebo: 18.1%). Similar results for the proportion of subjects with NASH resolution were obtained when assessing changes in NAS scores with 1.9% (emricasan 5 mg), 11

2.8% (emricasan 50 mg) and 1.9% (placebo) of subjects achieving NASH resolution. None of these comparisons provided evidence of benefit for either emricasan dose compared to placebo. Since the primary endpoint was not met, nominal p-values are shown for informational purposes only.

Fibrosis, NAS and NAS component response categories regardless of NASH changes The proportion of subjects who were stable, improved or worse was examined. There was a trend for subjects treated with placebo to have improvement in fibrosis compared to the emricasan-treated subjects (21.5% vs. 13.7% and 13.4% in the emricasan 5 and 50 mg groups), although there was no effect of emricasan dose (Figure 1). The leastsquares mean (LSM) change from baseline in fibrosis stage for the emricasan 5 mg group was 0.405 (95% CI 0.237, 0.572; p=0.004), 0.357 (95% CI 0.191, 0.523; p=0.013) in the 50 mg group, and 0.061 (95% CI -0.108, 0.230) in the placebo group. Fewer placebo-treated subjects had worsening of fibrosis (20.4%) compared to the emricasantreated subjects (emricasan 5 mg: 41.1%; emricasan 50 mg: 38.1%).

Results for the NAS, NAS components (lobular inflammation, steatosis and ballooning), Mallory-Denk bodies and portal inflammation are shown in Figures 2A-F. More placebo-treated subjects had improvement in NAS (48.4%) compared to emricasan treatment (5 mg: 35.8%; 50 mg: 30.9%) (Figure 2A). The LSM changes from baseline were -0.265 (95% CI -0.545, 0.015; p=0.428) in the 5 mg group, -0.045 (95% CI 0.323,0.232; p=0.058) in the 50 mg group, and -0.423 (95% CI -0.706, -0.141) in 12

placebo subjects. More placebo-treated subjects also remained stable (26.9% vs 41.1% and 35.1% in the 5 and 50 mg groups) with a similar proportion of emricasan subjects with worsening (placebo: 24.7%; emricasan 5 mg: 23.2%; emricasan 50 mg: 34.0%).

Analysis of lobular inflammation suggested that the placebo and 5 mg groups were similar while the 50 mg group tended to be worse (Figure 2B). Fewer subjects improved in the emricasan 5 mg group (24.2%) compared to placebo (30.1%), more subjects in the emricasan 5 mg group were stable (61.1% vs. 48.4% in placebo) and fewer subjects worsened in the emricasan 5 mg group (14.7% vs. 21.5% in placebo). Fewer subjects in the emricasan 50 mg group improved compared to placebo (22.7% vs. 30.1%) and more worsened (30.9% vs. 21.5%). The LSM changes from baseline were -0.105 in the 5 mg group (95% CI -0.264, 0.053; p=0.898), 0.093 in the 50 mg group (95% CI -0.064, 0.250; p=0.060) and -0.120 in placebo subjects (95% CI 0.280,0.040).

There were numerical decreases in steatosis for both emricasan groups with LSM decreases of -0.197 (95% CI -0.293,-0.101; p=0.089) in the 5 mg group, -0.265 (95% CI -0.361,-0.170; p=0.007) in the 50 mg group, and -0.080 (95% CI -0.177, 0.017) in placebo subjects. More emricasan-treated subjects improved steatosis (5 mg: 20.0%; 50 mg: 25.8%) compared to placebo subjects (12.9%) (Figure 2C). Fewer emricasantreated subjects had worse steatosis compared to placebo (5 mg: 3.2%; 50 mg: 2.1%;

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pbo: 6.5%). There was no consistent effect of emricasan on levels of total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, hemoglobin A1c, insulin levels, Homeostatic Model Assessment of Insulin Resistance (HOMA-IR), or free fatty acids (data not shown). Mean body weight changes at week 72 in each treatment group were also relatively small (5 mg: -0.90%; 50 mg: -2.42%; placebo: -0.35%) and the differences in steatosis were not explained by changes in body weight since the same directional changes were present in subjects whose body weight remained within 5% of baseline (data not shown).

More placebo-treated subjects improved ballooning (29.0%) compared to emricasan subjects (5 mg: 13.7%; 50 mg: 8.2%) and less worsening of ballooning (11.8%) compared to emricasan (5 mg: 18.9%; 50 mg: 21.6%) (Figure 2D). The LSM changes from baseline in emricasan subjects were 0.037 (95% CI -0.094, 0.168; p=0.006) in the 5 mg group, 0.127 (95% CI -0.002, 0.257; p<0.001) in the 50 mg group, and -0.224 (95% CI -0.356,-0.092) in placebo subjects.

There was also a trend for more placebo-treated subjects to show improvement in the number of Mallory-Denk bodies (21.5%) compared to emricasan treatment (5 mg: 10.5%; 50 mg: 9.3%) and less worsening in the number of Mallory-Denk bodies (32.3% compared to 5 mg: 51.6%; 50 mg: 69.1%) (Figure 2E). There was a possible doseeffect, with the 50 mg dose having less improvement and greater worsening of MalloryDenk bodies compared to the 5 mg dose of emricasan. The LSM changes from

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baseline were 0.554 (5 mg, 95% CI 0.351, 0.757; p=0.007), 0.828 (50 mg, 95% CI 0.627,1.029; p<0.001) and 0.163 (placebo, 95% CI -0.042, 0.368).

Numerical differences in portal inflammation improvement/worsening also favored placebo treatment (Figure 2F) with LSM changes from baseline of 0.172 (5 mg, 95% CI 0.028, 0.316; p=0.039), 0.305 (50 mg, 95% CI 0.162,0.447; p<0.001) and -0.041 (placebo, 95% CI -0.186, 0.104). More placebo subjects improved portal inflammation (placebo: 23.7% vs. 5 mg: 15.8% and 50 mg: 7.2%) and had less worsening of portal inflammation (placebo: 17.2% vs. 5 mg: 28.4% and 50 mg: 33.0%) with a similar proportion of subjects who remained stable across all 3 treatment groups.

Liver fat and collagen content Liver fat and collagen content were quantified using digital image analysis. Consistent with changes in steatosis score and fibrosis stage, liver fat content decreased and collagen content tended to increase in the subjects treated with emricasan (Supplemental Table 3).

Changes in serum aminotransferases and biomarkers Serum ALT decreased in subjects treated with emricasan (Figure 3A) with the 50 mg dose being more efficacious (median change from baseline at week 72 -20.0 U/L; p<0.001) than the 5 mg dose (median change from baseline at week 72 -14.7 U/L;

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p=0.006). Average serum ALT values were lower by week 4 with both emricasan doses, with the 50 mg dose decreasing serum ALT to a greater degree and remaining consistently less than 40 U/mL through week 72. The decrease in ALT values without an effect upon fibrosis was not caused by the initiation of vitamin E or anti-diabetic treatment during the study since only 9 subjects in the 5 mg group, 11 subjects in the 50 mg group, and 6 subjects in the placebo group had at least 1 of these medications initiated.

Serum AST values decreased by week 4 with both emricasan doses (5 mg median change -9.7 U/L; 50 mg median change: -12.7 U/L; placebo median change: -2.6 U/L), but gradually increased toward baseline by weeks 24-36, and remained similar to placebo over the remainder of the study (Figure 3B).

The 50 mg dose of emricasan decreased the executioner caspases-3 and -7 more than the 5 mg dose, with the 5 mg dose decreasing median caspase-3/7 activity to levels only marginally less than placebo (Figure 3C). Emricasan 50 mg decreased caspase3/7 activity by week 4 (median change -1384.5 RLU) and levels remained lower throughout the study whereas the 5 mg dose had less of a decrease at week 4 (median change -852.5 RLU). Both the 5 mg and 50 mg doses were decreased (p=0.027 and p<0.001, respectively) at week 72 compared to placebo.

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Emricasan also decreased cCK18 levels compared to placebo (Figure 3D). The 50 mg dose of emricasan appeared to be superior to the 5 mg dose, with lower levels over time and more consistent average decreases in cCK18. At week 72, the median changes from baseline were -102.0 U/L (5 mg, p=0.165), -143.0 U/L (50 mg, p=0.006) and -34.0 U/L (placebo).

Values of flCK18 (Figure 3E) showed a similar pattern of reduction to that observed with serum AST. Both emricasan doses decreased flCK18 values by week 4 (5 mg median change -195.0 U/L; 50 mg median change -297.0 U/L; placebo median change 26.5 U/L), but values increased toward baseline by weeks 24-36 and remained similar to or slightly less than placebo through week 72.

Correlation of biomarker responses with histology endpoints To understand whether there was a potential relationship between treatment-associated decreases in serum ALT, cCK18, flCK18 and caspase-3/7 and histologic changes, subjects were categorized into “responders”, defined as a decrease to ≤ the upper limit of the normal range for that assay and by ≥30% of the baseline value, or nonresponders. Consistency of responses was defined 2 ways: meeting the responder criteria at all 3 visits between weeks 4 and 24, and at all 7 visits between weeks 4 and 72. Since there were few consistent responders, the emricasan-treated subjects were pooled and analyzed as a single group. The results by treatment are shown for subjects who did or did not have consistent responses for either 24 or 72 weeks for 17

serum ALT, cCK18, flCK18 or caspase 3/7 and achieved at least 1-point improvement from baseline in fibrosis score, NAS, lobular inflammation, ballooning and steatosis (Table 3). Also shown is the proportion of subjects who achieved at least 1-point improvement in fibrosis score without worsening of NASH.

Fifty-five (19.3%) and 23 (8.1%) subjects achieved consistent serum ALT responses at weeks 24 and 72, respectively. Only 1 placebo subject had a consistent serum ALT response at 24 or 72 weeks while 54 (18.9%) and 22 (7.7%) emricasan subjects responded at each time point, respectively. Although the number of emricasan subjects who achieved a consistent serum ALT response for 72 weeks was small, those subjects had greater histologic improvement when compared to those who did not, with the exception of steatosis, which improved to similar degrees in both serum ALT responders and non-responders. Additionally, subjects who had a consistent serum ALT response for all 72 weeks had better histologic responses than those who only had a consistent response between weeks 4-24. A related analysis examining subjects who had at least a 17 U/mL decrease in ALT showed similar results (Supplemental Table 4).

A similar trend for improvement in all histologic parameters was noted in the subjects with consistent cCK18 responses for 72 weeks (Supplemental Table 5). A total of 33 (11.6%) subjects (emricasan: 30; placebo: 3) achieved consistent cCK18 responses at 24 weeks while 14 (4.9%) subjects (emricasan: 12; placebo: 2) achieved consistent cCK18 responses for 72 weeks. Like the serum ALT analysis, subjects who had

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consistent cCK18 responses for all 72 weeks had better histologic responses than those who had consistent responses between weeks 4-24.

The relationship between flCK18 response (Supplemental Table 6) and caspase 3/7 response (Supplemental Table 7) and improvement in histologic measures was also assessed. Consistent improvement in flCK18 also appeared to be a good predictor of histologic response, however, the number of consistent responders over the entire 72 weeks was too small (5 and 6, respectively) to make firm conclusions.

Efficacy in the NASH CRN fibrosis stage F2 and F3 subpopulation All of the efficacy analyses shown above were in subjects with NASH CRN fibrosis stages F1 through F3. The F2-F3 subgroup was analyzed separately with similar results. Inclusion of fibrosis stage F1 subjects did not affect the efficacy conclusions of the study.

Safety Emricasan treatment was generally well-tolerated. Overall, the proportion of subjects with a treatment-emergent adverse event (TEAE) and the number of TEAEs were similar across all 3 treatment groups (Table 4). Related TEAEs and subjects with TEAEs leading to study discontinuation were also similar between treatment groups. There was a small numerical increase in serious TEAEs in subjects treated with

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emricasan. However, only 2 serious TEAEs (Table 5) occurred in more than 1 subject (cellulitis in 2 emricasan subjects and prostate cancer in 1 emricasan and 1 placebo subject). All other serious TEAEs were single events and distributed across multiple system organ classes. One placebo-treated subject developed a large hepatocellular carcinoma that was not present at baseline. The most frequent TEAEs (≥5% in total group) are shown in Table 4. There were occasional small numerical differences between the groups, with some favoring placebo and others emricasan. Overall, no adverse event was clearly associated with emricasan treatment.

Discussion Fat accumulation in hepatocytes and lipotoxic hepatocyte injury are important for the development of inflammation and fibrosis in NASH. Saturated fatty acids (such as palmitate) and lysophospholipids (such as lysophosphatidylcholine) can activate death receptors on the hepatocyte cell membrane, resulting in mitochondrial toxicity and activation of caspase-8, which then activates the executioner caspases-3, -6 and -7, leading to apoptosis which is characteristic of NASH [10, 13]. The executioner caspases cleave keratin-18 to cCK18 during apoptosis, which is detected by the M30 monoclonal antibody [19]. Total cell death can be assessed by the levels of flCK18/M65, which detects both cCK18 and intact keratin-18 [20]. Toxic lipids can also diffuse across the plasma membrane and directly cause endoplasmic reticulum stress and mitochondrial damage [10]. Palmitate has been shown to activate the inflammasome in hepatocytes, resulting in increased caspase-1 expression, and sensitization to LPS-induced IL-1β release in the methionine choline-deficient model of 20

NASH [21]. Mice genetically deficient in either caspase‐1 (which processes pro-IL-1β to IL-1β), −3, or −8 are resistant to diet‐induced NASH [22-24]. Additional evidence supporting the role of caspase-1 in NASH comes from studies in which NLRP3 inhibition or deletion ameliorated liver inflammation and fibrosis in NASH models [25, 26]. Emricasan had previously been shown to decrease caspase-3/7 activity, cCK18, flCK18 and serum ALT levels after 4 weeks of treatment with 25 mg BID in patients with NASH and elevated aminotransferases [15]. Thus, it was reasonable to hypothesize that pancaspase inhibition might decrease apoptosis, production of the pro-inflammatory cytokine IL-1β, and decrease liver inflammation and fibrosis in NASH patients.

The doses of emricasan chosen for this study (5 and 50 mg) had clear biological effects, with the 50 mg dose more active at decreasing average levels of serum ALT, caspase3/7 and cCK18 than the 5 mg dose. Even though the 50 mg emricasan dose clearly decreased serum ALT and biomarkers associated with apoptosis, analysis of the changes in histologic endpoints showed no improvement in fibrosis compared to placebo but rather suggested that pan-caspase inhibition may have worsened fibrosis in some subjects. More placebo-treated subjects had fibrosis improvement, and fewer had fibrosis worsening, compared to the emricasan-treated subjects. This semiquantitative assessment was supported by digitally-measured changes in liver collagen content. Although the increases in liver collagen content in the emricasan-treated subjects were numerically small, the 95% CI did not overlap with the placebo group and the nominal p-values were less than 0.05.

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Hepatocellular ballooning scores also tended to worsen in the emricasan-treated subjects. More placebo-treated subjects improved ballooning, and fewer worsened, compared to emricasan treatment. Although the numerical differences were small, the nominal p-values were significant. An increase in ballooning score in the emricasan subjects was supported by an increase in Mallory-Denk bodies, both of which had nominal p-values different from placebo. Mallory-Denk bodies are predominantly filamentous cytoplasmic inclusions present in ballooned hepatocytes and contain keratins-8 and -18 [27] along with other proteins involved in protein folding and degradation which are produced as a result of endoplasmic reticulum stress and activation of the unfolded protein response [28, 29].

The effect of emricasan treatment on lobular inflammation scores was less clear, but there may have been an inverse dose-effect. The placebo and 5 mg groups both had similar small decreases from baseline in lobular inflammation (-0.120 and -0.105, respectively) while the 50 mg group had a small increase in lobular inflammation.

Steatosis scores tended to decrease in the emricasan treatment groups. Baseline scores were 1.2 in all 3 treatment groups and decreased in all 3 groups, but the decreases were larger in the 5 mg (-0.117) and 50 mg groups (-0.186). The nominal pvalues approached significance in the 5 mg group (p=0.089) and were significant in the 50 mg group (p=0.007). The explanation for the larger decreases in the emricasan 22

groups is not clear. There were no consistent changes in metabolic factors that might be expected to decrease steatosis and changes in body weight also did not appear to be responsible. One possible explanation is that the decrease in steatosis scores was related to increases in fibrosis. Steatosis is known to decrease as fibrosis scores increase [30]. Decreases in steatosis scores in emricasan-treated subjects were also supported by decreases in digitally-measured liver fat content.

While there were consistent trends in the overall data suggesting that pan-caspase inhibition made fibrosis and ballooning worse in NASH fibrosis patients, the mechanistic explanation is less clear. One potential explanation is that caspase inhibition did not block but instead re-directed cells toward other mechanisms of cell death such as necrosis. While cCK18 levels (a marker of apoptotic cell death) generally decreased, flCK18 levels (a marker of apoptotic and necrotic cell death) [20] decreased transiently but returned to near-baseline values by weeks 24-36 and remained there through the end of the study, consistent with ongoing cell death. It could be speculated that apoptotic cell death could have shifted toward other more inflammatory forms of cell death. Emricasan has previously been shown to shift cell death from apoptosis toward necroptosis in acute myeloid leukemia cells treated with the second mitochondrialderived activator of caspases (SMAC) mimetic birinapant, which enhances cancer cell apoptosis [31].

23

Experimental data indicate that the types of cells undergoing apoptosis, and the cells responsible for apoptotic cell removal, may have a differential effect upon fibrogenesis and fibrolysis [32]. It is possible that inhibition of apoptosis could shift the balance between fibrogenesis and fibrolysis toward fibrogenesis. Thus, apoptotic profibrogenic cholangiocytes during spontaneous resolution of biliary fibrosis are engulfed by Kupffer cells and elicit programs of fibrolysis, including release and activation of certain matrix metalloproteinases [33]. Activated hepatic stellate cells and (portal) myofibroblasts are the primary producer of extracellular matrix, and apoptosis of hepatic stellate cells has been shown to promote fibrosis resolution [34]. In this line, induction of apoptosis in hepatic stellate cells, either by ursodeoxycholic acid [35] or by the administration of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) [36] ameliorated fibrosis in rodent models.

There was a small subgroup of subjects treated with emricasan in whom consistent normalization of serum ALT or cCK18 was associated with improvement in liver inflammation and fibrosis. Previous studies with metabolic therapies showed that vitamin E and pioglitazone decreased serum ALT, and that these decreases were associated with improvement in inflammation, but not fibrosis [37, 38]. In the PIVENS trial, over 80% of patients treated with vitamin E whose serum ALT levels fell to 40 U/L or less and by at least 30% of baseline, had histologic improvement, and none of those patients had evidence of worsening. Using a similar definition of response that required normalization of serum ALT and at least a 30% decrease from baseline, emricasan subjects who achieved a serum ALT or cCK18 response for the entire 72 weeks had 24

improvement in liver fibrosis and other measures of liver histology. Consistent achievement of a serum ALT or cCK18 response was important, since a shorter requirement for consistent improvement (24 weeks) had much less of a beneficial effect upon liver histology.

Further supporting the idea that emricasan may have made fibrosis worse in a subgroup of subjects, those who did not achieve a consistent serum ALT or cCK18 response tended to have worse histology after 72 weeks of treatment compared to placebo nonresponders. While there appeared to be a small subgroup of subjects who benefitted from emricasan, there was a much larger subgroup in whom inhibition of apoptosis may have led to worse liver histology.

Acknowledgements We would like to thank the patients and investigators who participated in our study: United States--M. Abdalmalek (Duke University), J. Ackermann (PMG Research of Charleston), V. Ankoma-Sey (Liver Associates of Texas), M. Ashfaq (Baylor All Saints Medical Center), J. Baber (Preferred Research Partners), A. Barritt (UNC Liver Center), M. Cave (University of Louisville), V. Clark (University of Florida), K. Corey (Massachusetts General Hospital), A. deLemos (Carolinas Medical Center), J. Fenkel (Thomas Jefferson University), B. Freilich (Kansas City Research Center), M. Fuchs (Mcguire VA), J. Galati (Research Specialists of Texas), R. Ghalib (Texas Clinical Research Institute), F. Gordon (Lahey Clinic Medical Center), S. Gordon (Henry Ford Health System), S. Harrison (Pinnacle Clinical Research), D. Hillebrand (Center for Liver Disease), A. Jabbar (Gastroenterology of Southern Indiana), L. Jeffers (Miami VA Healthcare System), K. Johnson (IResearch Atlanta), M. Jonas (Consultants for Clinical Research), Z. Kayali (Inland Empire Liver Foundation), N. Kemmer (Tampa General Medical Group), K. Korenblat (Washington University), A. Koteish (Florida Hospital Transplant Institute), S. Kumar (Weill Cornell Medical College), M. Lai (Beth Israel Deaconess Medical Center), C. Landis (University of Washington), J. Laurin (Sibley 25

Memorial Hospital), E. Lawitz (Texas Liver Institute), W. Lee (UTSW), S. Lidofsky (University of Vermont), J. Lim (Yale University School of Medicine), R. Looma (UCSD Medical Center), H. Malhi (Mayo Clinic), O. Massoud (University of Alabama, Birmingham), M. McKenzie (ClinSearch), E. Mena (California Liver Research Institute), M. Noureddin (Cedars Sinai Medical Center), J. Pan (University of Arizona), P. Pandya (Kansas City VA Medical Center), A. Paredes (Brooke Army Medical Center), J. Provenza (Louisiana Research Center), N. Pyrsopoulos (Rutgers-NJMS), R. Reddy (University of Pennsylvania), D. Rockey (Medical University South Carolina), S. Rossi (Albert Einstein Medical Center), M. Ryan (Digestive & Liver Disease Specialists), S. Saab (UCLA – Pfleger Liver Institute), S. Sarkar (UC Davis Medical Center), S. Satapathy (Methodist University Hospital), A. Scanga (Vanderbilt University), E. Schiff (University of Miami), T. Sepe (University Gastroenterology), N. Shah (Rush University Medical Center), A. Sheikh (Gastrointestinal Specialists of Georgia), M. Sheikh (Fresno Clinical Research Center), M. Shiffman (Bon Secours Richmond Health System), H. Tatum (Options Health Research), H. Te (University of Chicago Medical Center), G. Therapondos (Ochsner Health System), I. Thomason (University of Utah Health Sciences), P. Thuluvath (Mercy Medical Center), D. Torres (Walter Reed Medical Center), R. Vemulapalli (Iowa Digestive Disease Center), J. Vierling (Baylor College of Medicine), I. Weisberg (Beth Israel Medical Center), Z. Younes (Gastro One), A. Zivony (Ashevelle Gastroenterology Associates) Spain--A. Albillos (Hospital Ramon y Cajal), J. Arenas Ruiz-Tapiador (Hospital de Donostia), S. Augustin (Hospital Valle d’Hebron), R. Banares (Hospital General Universitario Gregorio Maranon), J. Calleja (Hospital Puerta de Hierro), J. Crespo Garcia (Hospital Marques de Valdecilla), M. de los Desamparados Escudero (Universitario de Valencia), L. Garcia Buey (Hospital de la Princesa), J. GarciaSamaniego (Hospital Universitario La Paz, CIBERehd) Germany--H. Heinzow (Universitätsklinikum Münster), J. Kluwe (Universitätsklinikum Hamburg Eppendorg), T. Mueller (Charite – Universitatsmedizin Berlin), J. Rasenack (Universitatsklinikum Freiburg), J. Schattenberg (University Medical Center, Mainz), I. Schiefke (Eugastro GmbH), C. Trautwein (Universitätsklinikum der RWTH Aachen), A. Zipprich (Universitätsklinikum Halle)

We also gratefully thank Dr. Rohit Loomba for providing helpful input and advice during the early design of the study.

26

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caspase 3/7 activation in subjects with non-alcoholic fatty liver disease. Aliment Pharmacol Ther 2019;49:64-73. [16] Barreyro FJ, Holod S, Finocchietto PV, Camino AM, Aquino JB, Avagnina A, et al. The pan-caspase inhibitor Emricasan (IDN-6556) decreases liver injury and fibrosis in a murine model of non-alcoholic steatohepatitis. Liver Int 2015;35:953-966. [17] Canbay A, Feldstein A, Baskin-Bey E, Bronk SF, Gores GJ. The caspase inhibitor IDN-6556 attenuates hepatic injury and fibrosis in the bile duct ligated mouse. J Pharmacol Exp Ther 2004;308:1191-1196. [18] Bretz F, Posch M, Glimm E, Klinglmueller F, Maurer W, Rohmeyer K. Graphical approaches for multiple comparison procedures using weighted Bonferroni, Simes, or parametric tests. Biom J 2011;53:894-913. [19] Caulin C, Salvesen GS, Oshima RG. Caspase cleavage of keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis. J Cell Biol 1997;138:1379-1394. [20] Kramer G, Erdal H, Mertens HJ, Nap M, Mauermann J, Steiner G, et al. Differentiation between cell death modes using measurements of different soluble forms of extracellular cytokeratin 18. Cancer research 2004;64:1751-1756. [21] Csak T, Ganz M, Pespisa J, Kodys K, Dolganiuc A, Szabo G. Fatty acid and endotoxin activate inflammasomes in mouse hepatocytes that release danger signals to stimulate immune cells. Hepatology 2011;54:133-144. [22] Dixon LJ, Flask CA, Papouchado BG, Feldstein AE, Nagy LE. Caspase-1 as a central regulator of high fat diet-induced non-alcoholic steatohepatitis. PLoS One 2013;8:e56100. [23] Hatting M, Zhao G, Schumacher F, Sellge G, Al Masaoudi M, Gabetaler N, et al. Hepatocyte caspase-8 is an essential modulator of steatohepatitis in rodents. Hepatology 2013;57:2189-2201. [24] Thapaliya S, Wree A, Povero D, Inzaugarat ME, Berk M, Dixon L, et al. Caspase 3 inactivation protects against hepatic cell death and ameliorates fibrogenesis in a dietinduced NASH model. Dig Dis Sci 2014;59:1197-1206. [25] Mridha AR, Wree A, Robertson AAB, Yeh MM, Johnson CD, Van Rooyen DM, et al. NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice. J Hepatol 2017;66:1037-1046. [26] Cai C, Zhu X, Li P, Li J, Gong J, Shen W, et al. NLRP3 Deletion Inhibits the Nonalcoholic Steatohepatitis Development and Inflammation in Kupffer Cells Induced by Palmitic Acid. Inflammation 2017;40:1875-1883. [27] Denk H, Stumptner C, Zatloukal K. Mallory bodies revisited. J Hepatol 2000;32:689-702. [28] Puri P, Mirshahi F, Cheung O, Natarajan R, Maher JW, Kellum JM, et al. Activation and dysregulation of the unfolded protein response in nonalcoholic fatty liver disease. Gastroenterology 2008;134:568-576. [29] Malhi H, Kaufman RJ. Endoplasmic reticulum stress in liver disease. J Hepatol 2011;54:795-809. [30] Yeh MM, Brunt EM. Pathology of nonalcoholic fatty liver disease. American journal of clinical pathology 2007;128:837-847. [31] Brumatti G, Ma C, Lalaoui N, Nguyen NY, Navarro M, Tanzer MC, et al. The caspase-8 inhibitor emricasan combines with the SMAC mimetic birinapant to induce 28

necroptosis and treat acute myeloid leukemia. Science translational medicine 2016;8:339ra369. [32] Schuppan D, Kim YO. Evolving therapies for liver fibrosis. J Clin Invest 2013;123:1887-1901. [33] Popov Y, Sverdlov DY, Bhaskar KR, Sharma AK, Millonig G, Patsenker E, et al. Macrophage-mediated phagocytosis of apoptotic cholangiocytes contributes to reversal of experimental biliary fibrosis. Am J Physiol Gastrointest Liver Physiol 2010;298:G323334. [34] Iredale JP, Benyon RC, Pickering J, McCullen M, Northrop M, Pawley S, et al. Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors. J Clin Invest 1998;102:538-549. [35] Wang X, Ikejima K, Kon K, Arai K, Aoyama T, Okumura K, et al. Ursolic acid ameliorates hepatic fibrosis in the rat by specific induction of apoptosis in hepatic stellate cells. J Hepatol 2011;55:379-387. [36] Oh Y, Park O, Swierczewska M, Hamilton JP, Park JS, Kim TH, et al. Systemic PEGylated TRAIL treatment ameliorates liver cirrhosis in rats by eliminating activated hepatic stellate cells. Hepatology 2016;64:209-223. [37] Belfort R, Harrison SA, Brown K, Darland C, Finch J, Hardies J, et al. A placebocontrolled trial of pioglitazone in subjects with nonalcoholic steatohepatitis. N Engl J Med 2006;355:2297-2307. [38] Hoofnagle JH, Van Natta ML, Kleiner DE, Clark JM, Kowdley KV, Loomba R, et al. Vitamin E and changes in serum alanine aminotransferase levels in patients with non-alcoholic steatohepatitis. Aliment Pharmacol Ther 2013;38:134-143.

29

Figure Legends Figure 1. Fibrosis response, regardless of NASH response, was scored according to NASH CRN criteria. A fibrosis score decrease of 1 point was considered as improvement.

Figure 2. Changes in histologic categories (improved, stable, or worse) for NAS, individual NAS components, Mallory-Denk bodies and portal inflammation regardless of fibrosis response. Any increase or decrease in score was considered a change in category. A) NAS, B) Lobular inflammation, C) Steatosis, D) Ballooning, E) MalloryDenk bodies and F) Portal inflammation. The percent of subjects in each category are shown numerically within each category of the bar graph. Blue= improved, Brown=stable, and Green=worse.

Figure 3. Changes in average biomarker values by study visit week. Values shown are the median and inter-quartile range. A) serum ALT, B) serum AST, C) caspase 3/7, D) cleaved keratin-18, and E) full-length keratin-18. Placebo subjects are in black. Emricasan 5 mg subjects are in green and 50 mg subjects are in blue.

Table 1. Baseline clinical, histologic and laboratory characteristics

Clinical characteristics: Weight, median(kg) Body Mass Index, median(kg/m2) Clinical diagnoses: Metabolic Syndrome n (%) Type 2 diabetes mellitus n (%) Obesity n (%) Hyperlipidemia - elevated LDL n (%) High triglycerides n (%) Low HDL n (%) Gout n (%) Polycystic ovary syndrome n (%) Coronary artery disease n (%) Angina n (%) Myocardial infarction n (%) Stroke n (%) Liver histology: NASH CRN Fibrosis Stage n (%) F1 F2 F3 NAS, mean (SD) Steatosis, mean (SD) Lobular Inflammation, mean (SD) Ballooning, mean (SD) Mallory-Denk, mean (SD) Portal inflammation, mean (SD) Liver collagen content (%), mean (SD) Liver fat content (%) Laboratory values: Cholesterol (mg/dL) Mean (SD) Median Min, Max LDL Cholesterol (mg/dL) Mean (SD) Median Min, Max HDL Cholesterol (mg/dL) Mean (SD) Median Min, Max Triglycerides (mg/dL) Mean (SD) Median

Emricasan 5 mg (N=107)

Emricasan 50 mg (N=106)

Placebo (N=105)

102.33 34.80

91.40 33.30

95.80 33.90

71 (66.4) 55 (51.4) 87 (81.3) 60 (56.1) 39 (36.4) 32 (29.9) 8 (7.5) 6 (5.6) 10 (9.3) 4 (3.7) 3 (2.8) 2 (1.9)

62 (58.5) 55 (51.9) 81 (76.4) 59 (55.7) 43 (40.6) 20 (18.9) 5 (4.7) 8 (7.5) 8 (7.5) 6 (5.7) 3 (2.8) 2 (1.9)

57 (54.3) 51 (48.6) 75 (71.4) 64 (61.0) 44 (41.9) 34 (32.4) 8 (7.6) 3 (2.9) 7 (6.7) 6 (5.7) 2 (1.9) 0

23 (21.5) 45 (42.1) 39 (36.4) 5.6 (0.95) 1.2 (0.48) 2.7 (0.50) 1.7 (0.47) 0.8 (0.75) 1.7 (0.55) 2.8 (2.40)

22 (20.8) 45 (42.5) 39 (36.8) 5.5 (0.93) 1.2 (0.44) 2.6 (0.52) 1.7 (0.45) 0.9 (0.72) 1.7 (0.56) 2.8 (2.21)

23 (21.9) 44 (41.9) 38 (36.2) 5.5 (0.97) 1.2 (0.42) 2.6 (0.57) 1.7 (0.47) 0.8 (0.72) 1.8 (0.47) 2.9 (2.80)

17.8 (8.09)

16.8 (7.51)

16.5 (7.68)

n=107 178.9 (40.98) 184.0 66, 273 n=105 99.9 (33.91) 99.0 14, 173 n=107 44.5 (16.80) 42.0 21, 152 n=107 181.2 (102.78) 153.0

n=106 187.3 (42.09) 185.0 84, 308 n=105 104.0 (33.30) 101.0 16, 180 n=106 44.0 (11.23) 43.0 24, 80 n=106 201.5 (104.37) 175.5

n=105 180.7 (44.02) 179.0 92, 303 n=105 102.4 (38.89) 97.0 6, 215 n=105 45.3 (11.59) 44.0 23, 80 n=105 165.0 (77.87) 144.0

Min, Max Hemoglobin A1C (%) Mean (SD) Median Min, Max Alanine Aminotransferase (U/L) Mean (SD) Median Min, Max Aspartate Aminotransferase (U/L) Mean (SD) Median Min, Max Caspase 3/7 activity Mean (SD) Median Min, Max cCK18 Mean (SD) Median Min, Max flCK18 Mean (SD) Median Min, Max

43, 641 n=107 6.28 (0.900) 6.00 4.4, 8.8 n=106 66.7 (37.48) 59.0 11, 260 n=106 51.8 (27.39) 45.8 10, 152 n=107 4169.01 (2529.327) 3454.00 200.0, 15312.0 n=106 1315.87 (6457.394) 381.00 83.0, 64000.0 n=107 863.79 (674.003) 691.00 106.0, 4300.0

65, 608 n=106 6.26 (1.028) 6.10 4.2, 8.9 n=106 63.1 (32.44) 58.8 13, 183 n=106 47.1 (23.32) 41.0 15, 150 n=106 4010.33 (3070.618) 3389.50 1215.0, 28677.0 n=106 451.81 (276.003) 380.50 78.0, 1148.0 n=106 754.44 (421.871) 688.00 158.0, 2254.0

28, 371 n=105 6.23 (0.967) 6.00 4.4, 8.8 n=105 63.3 (34.35) 55.7 15, 206 n=105 48.6 (25.67) 41.3 17, 155 n=105 3878.26 (2499.263) 3165.00 1125.0, 18824.0 n=105 455.96 (334.920) 364.00 31.0, 2034.0 n=105 818.28 (617.853) 611.00 92.0, 3923.0

Table 2. Summary of histologic responses by treatment group Endpoint (N=318) Improvement (≥1-stage) in NASH CRN Fibrosis Stage without Worsening of Steatohepatitis (≥1-stage increase in ballooning and lobular inflammation)

NASH Resolution (histopathologist’s assessment) without Worsening of Fibrosis (≥1-stage increase using NASH CRN)

Resolution of Fibrosis (NASH CRN fibrosis score of 0)

NAS Response (at least a 2-point decrease in the NAS score)

a

Treatment effect Emricasan 5 mg (N=107) Emricasan 50 mg (N=106) Placebo (N=105) Emricasan 5 mg (N=107) Emricasan 50 mg (N=106) Placebo (N=105) Emricasan 5 mg (N=107) Emricasan 50 mg (N=106) Placebo (N=105) Emricasan 5 mg (N=107) Emricasan 50 mg (N=106) Placebo (N=105)

Hypothesis testing: Emricasan versus a Placebo 5mg 50 mg

11.2% 0.972

0.972

0.070

0.335

0.703

0.131

0.108

0.072

12.3% 19.0% 3.7% 6.6% 10.5% 3.7% 0.9% 4.8% 10.3% 9.4% 18.1%

One-sided p-values were calculated from a flexible graphical gate-keeping procedure in accordance with the statistical analysis plan for the primary endpoint; otherwise two-sided p-values are presented.

Table 3. Subjects with histologic improvement based upon consistent ALT responses, defined as maintaining ALT levels ≤ 40 U/L (upper limit of normal) or less and at least a 30% decrease from the baseline value at each visit between either weeks 4-24 (all 3 visits) or for all on-treatment visits between weeks 4-72 (7 visits). ALT improvement Weeks 424*

ALT improvement Weeks 472*

No (N=230)

Yes (N=55)

No (N=262)

Yes (N=23)

PRIMARY ENDPOINT: Fibrosis improvement without worsening of steatohepatitis n=45

30/230 =13.0

15/55 = 27.3

34/262 = 13.0

11/23 = 47.8

All Emricasan (N=192)

10/138 = 7.2

15/54 = 27.8

14/170 = 8.2

11/22 = 50.0

Placebo (N=93)

20/92 = 21.7

0/1 = 0

20/92 = 21.7

0/1 = 0

30/230 =13.0

16/55 = 29.1

35/262 = 13.4

11/23 = 47.8

All Emricasan (N=192)

10/138 = 7.2

16/54 = 29.6

15/170 = 8.8

11/22 = 50.0

Placebo (N=93)

20/92 = 21.7

0/1 = 0

20/92 = 21.7

0/1 = 0

81/230 =35.2

28/55 = 50.9

93/262 = 35.5

16/23 = 69.6

All Emricasan (N=192)

36/138 =26.1

28/54 = 51.8

48/170 = 28.2

16/22 = 72.7

Placebo (N=93)

45/92 = 48.9

0/1 = 0

45/92 = 48.9

0/1 = 0

51/230 =22.2

22/55 = 40.0

60/262 = 22.9

13/23 = 56.5

All Emricasan (N=192)

23/138 =16.7

22/54 = 40.7

32/170 = 18.8

13/22 = 59.1

Placebo (N=93)

28/92 = 30.4

0/1 = 0

28/92 = 30.4

0/1 = 0

35/230 =15.2

13/55 = 23.6

38/262 = 14.5

10/23 = 43.5

All Emricasan (N=192)

8/138 = 5.8

13/54 = 24.1

11/170 = 6.5

10/22 = 45.4

Placebo (N=93)

27/92 = 29.3

0/1 = 0

27/92 = 29.3

0/1 = 0

44/230 =19.1

12/55 = 21.8

51/262 = 19.5

5/23 = 21.7

All Emricasan (N=192)

32/138 =23.2

12/54 = 22.2

39/170 = 22.9

5/22 = 22.7

Placebo (N=93)

12/92 = 13.0

0/1 = 0

12/92 = 13.0

0/1 = 0

Fibrosis improvement

NAS improvement

n=46

n=109

Lobular inflammation improvement n=73

Ballooning improvement n=48

Steatosis improvement

n=56

*ALT improvement was defined as maintaining ALT levels ≤ 40 U/L (upper limit of normal) or less and at least a 30% decrease from the baseline value at each visit between either weeks 4-24 (all 3 visits) or for all on-treatment visits between weeks 4-72 (7 visits).

Table 4. Safety Summary and Most Frequent (>5%) TEAEs

Summary Subjects with TEAEs (n [%]) Number of TEAE events (n) Subjects with serious TEAEs (n [%]) Subjects with TEAEs related to study drug (n [%]) Subjects with TEAEs leading to study discontinuation (n [%]) Subjects with TEAEs leading to study drug discontinuation (n [%]) Most Frequent by Preferred Term Diarrhoea Upper respiratory tract infection Nausea Sinusitis Back pain Fatigue Abdominal pain upper Abdominal pain Arthralgia Diabetes mellitus Headache Nasopharyngitis Urinary tract infection Vomiting Gastrooesophageal reflux disease Hypertension

Emricasan 5 mg (N=107)

Emricasan 50 mg (N=106)

Placebo (N=105)

93 (86.9) 440 9 (8.4) 26 (24.3)

99 (93.4) 488 15 (14.2) 34 (32.1)

91 (86.7) 475 7 (6.7) 30 (28.6)

1 (0.9)

3 (2.8)

2 (1.9)

4 (3.7)

3 (2.8)

1 (1.0)

n (%) 10 (9.3) 11 (10.3) 9 (8.4) 8 (7.5) 6 (5.6) 9 (8.4) 6 (5.6) 7 (6.5) 9 (8.4) 7 (6.5) 5 (4.7) 8 (7.5) 7 (6.5) 6 (5.6) 5 (4.7) 4 (3.7)

n (%) 17 (16.0) 12 (11.3) 15 (14.2) 10 (9.4) 10 (9.4) 8 (7.5) 6 (5.7) 8 (7.5) 4 (3.8) 8 (7.5) 6 (5.7) 4 (3.8) 4 (3.8) 8 (7.5) 5 (4.7) 3 (2.8)

n (%) 11 (10.5) 12 (11.4) 10 (9.5) 15 (14.3) 11 (10.5) 10 (9.5) 12 (11.4) 8 (7.6) 9 (8.6) 7 (6.7) 8 (7.6) 6 (5.7) 7 (6.7) 4 (3.8) 7 (6.7) 9 (8.6)

Table 5. Serious adverse events

Preferred Term Any Treatment Emergent Serious Adverse Event Cellulitis Prostate cancer Abdominal pain upper Arthralgia Biliary dyskinesia Breast cancer Colitis Colon adenoma Deep vein thrombosis Dermal cyst Diabetes mellitus Drug hypersensitivity Enteritis Gouty arthritis Haematochezia Hepatic failure Hypersensitivity Hypoaesthesia oral Inguinal hernia Intestinal perforation Liposarcoma Musculoskeletal chest pain Myoclonus Non-cardiac chest pain Peritonitis bacterial Pneumonia Pneumonia aspiration Postoperative wound infection Pyelonephritis Retinal detachment Retinal vein occlusion Sepsis Suicidal ideation Transient ischaemic attack Urosepsis Wound dehiscence

Emricasan 5 mg (N=107) n (%) 9 (8.4)

Emricasan 50 mg (N=106) n (%) 15 (14.2)

Placebo (N=105) n (%) 7 (6.7)

0 0 0 1 (0.9) 0 1 (0.9) 0 0 0 1 (0.9) 0 0 0 1 (0.9) 0 0 0 1 (0.9) 0 0 0 0 0 0 0 0 0 1 (0.9) 0 1 (0.9) 1 (0.9) 0 0 1 (0.9) 0 0

2 (1.9) 1 (0.9) 0 0 1 (0.9) 0 1 (0.9) 0 1 (0.9) 0 0 1 (0.9) 1 (0.9) 0 0 0 1 (0.9) 0 1 (0.9) 1 (0.9) 1 (0.9) 0 1 (0.9) 1 (0.9) 0 0 1 (0.9) 0 0 0 0 1 (0.9) 1 (0.9) 0 1 (0.9) 1 (0.9)

0 1 (1.0) 1 (1.0) 0 0 0 0 1 (1.0) 0 0 1 (1.0) 0 0 0 1 (1.0) 1 (1.0) 0 0 0 0 0 1 (1.0) 0 0 1 (1.0) 1 (1.0) 0 0 1 (1.0) 0 0 0 0 0 0 0

A. ALT 90 80 70

ALT (U/L)

60 50 40 30 20 10 0 Baseline

Wk 4

Wk 12

Wk 24

Wk 36

Wk 48

Wk 60

Analysis Visit

Emricasan 5 mg

Emricasan 50 mg

Placebo

Wk 72

Follow-Up

B. AST 80 70 60

AST (U/L)

50 40 30 20 10 0 Baseline

Wk 4

Wk 12

Wk 24

Wk 36

Wk 48

Wk 60

Analysis Visit

Emricasan 5 mg

Emricasan 50 mg

Placebo

Wk 72

Follow-Up

C. Caspase 3/7 7400 6800 6200

Caspase 3/7 (RLU)

5600 5000 4400 3800 3200 2600 2000 1400 800 Baseline

Wk 4

Wk 12

Wk 24

Wk 36

Wk 48

Wk 60

Analysis Visit

Emricasan 5 mg

Emricasan 50 mg

Placebo

Wk 72

Follow-Up

D. Cleaved Cytokeratin-18 800

700

cCK18/M30 (U/L)

600

500

400

300

200

100 Baseline

Wk 4

Wk 12

Wk 24

Wk 36

Wk 48

Wk 60

Analysis Visit

Emricasan 5 mg

Emricasan 50 mg

Placebo

Wk 72

Follow-Up

E. Full-length Cytokeratin-18 1300 1200 1100

flCK18/M65 (U/L)

1000 900 800 700 600 500 400 300 200 Baseline

Wk 4

Wk 12

Wk 24

Wk 36

Wk 48

Wk 60

Analysis Visit

Emricasan 5 mg

Emricasan 50 mg

Placebo

Wk 72

Follow-Up

Highlights: • • • •

Pan-caspase inhibition with emricasan did not improve liver histology in NASH patients and may have worsened fibrosis and ballooning Caspase inhibition lowered average serum alanine aminotransferase values in the overall population, but did not consistently normalize values in individual patients over the study Emricasan was generally well-tolerated over 72 weeks of treatment While speculative, pan-caspase inhibition may have shifted cells from apoptotic to more inflammatory kinds of cell death