V Ratziu
email: vlad.ratziu@inserm.fr

L De Guevara

Investigator, Rifaat Safadi

Fred Poordad

Francisco Fuster

Jose Flores-Figueroa

Marco Arrese

Anna L Fracanzani

D Ben Bashat

Karoline Lackner

T Gorfine

Shaul Kadosh

Study Design, Data Review and Interpretation Ran Oren

Study Planning Maya Halperin

L Hayardeny

Investigator, Rohit Loomba

Scott Friedman

Arun J Sanyal

Laura Ladron

De Guevara

Investigator, Anna Ludovica Fracanzani

Dafna Ben Bashat

Design Liat Hayardeny

Data

Aramchol in patients with nonalcoholic steatohepatitis: a randomized, double-blind, placebo-controlled phase 2b trial

Keywords: Vlad Ratziu, Study Design, Patient Recruitment and Treatment, Data Analyses

Aramchol in patients with nonalcoholic steatohepatitis: a randomized, double-blind, placebo-controlled phase 2b trial

The placebo-corrected decrease in ALT for 600 mg was -29.1 IU/L, (95% CI:-41.6 to -16.5). Early termination due to adverse events (AE) was <5% and aramchol 600 mg and 400 mg were safe, well tolerated, without imbalance in serious or severe AE between arms. Although the primary endpoint of a reduction in liver fat did not meet the prespecified significance level with aramchol 600mg, the observed safety and changes in liver histology and enzymes provide a rationale for SCD1 modulation as a promising therapy for NASH and fibrosis and is being evaluated in an ongoing phase 3 program. Nonalcoholic fatty liver disease (NAFLD) is an increasingly common condition in the general population with a prevalence ranging from 13% in Africa to 32% in Latin America and the Middle-East, largely driven by rising rates of obesity and type 2 diabetes [START_REF] Powell | Non-alcoholic fatty liver disease[END_REF] . Nonalcoholic steatohepatitis (NASH), the progressive form of NAFLD, is characterized by liver fat accumulation coexisting with liver cell injury (hepatocyte ballooning) and hepatic inflammation. NASH leads to fibrosis progression and is a leading cause of cirrhosis, end-stage liver disease and liver transplantation. NASH is associated with overweight, obesity, type 2 diabetes (which are clinical features of the metabolic syndrome) and occurs in a context defined by insulin resistance and adipose tissue dysfunction [START_REF] Samuel | Nonalcoholic Fatty Liver Disease as a Nexus of Metabolic and Hepatic Diseases[END_REF] . Currently, there are no approved therapies for NASH. Ongoing latephase clinical trials are designed to test histological improvement, such as resolution of steatohepatitis or fibrosis regression, while long-term outcome trials will evaluate whether these histological surrogates will result in less progression to cirrhosis and liver related morbidity and mortality.

Patients with NASH have increased de novo lipogenesis and lipotoxic species generated by the increased flux of fatty acids in the liver are a major contributor to hepatic inflammation and liver cell death associated with steatohepatitis [START_REF] Lambert | Increased de novo lipogenesis is a distinct characteristic of individuals with nonalcoholic fatty liver disease[END_REF] . Several agents in development specifically inhibit key enzymes of lipogenesis such as acetyl-co-A or fatty acid synthase. Stearoyl-CoA desaturase 1 (SCD1) catalyzes the rate-limiting step in the biosynthesis of monounsaturated fatty acids [START_REF] Miyazaki | Stearoyl-CoA desaturase 1 gene expression is necessary for fructosemediated induction of lipogenic gene expression by sterol regulatory element-binding protein-1c-dependent and -independent mechanisms[END_REF] . In rodents, down regulation of SCD1 reduced body adiposity, increased energy expenditure, and upregulated expression of several genes encoding enzymes of fatty acid beta-oxidation in liver [START_REF] Gutierrez-Juarez | Critical role of stearoyl-CoA desaturase-1 (SCD1) in the onset of diet-induced hepatic insulin resistance[END_REF] .

Reduction of SCD1 is also known to elevate adenosine monophosphate-activated protein kinase (AMPK) activity and enhance insulin sensitivity [START_REF] Dobrzyn | Stearoyl-CoA desaturase 1 deficiency increases fatty acid oxidation by activating AMP-activated protein kinase in liver[END_REF] . In hepatic stellate cells direct SCD1 depletion downregulates their fibrogenic phenotype [START_REF] Bhattacharya | Aramchol downregulates stearoyl CoA-desaturase 1 in hepatic stellate cells to attenuate cellular fibrogenesis[END_REF] . Several small molecule complete SCD1 inhibitors have been discontinued because of skin and lachrymal gland toxicity [START_REF] Powell | An overview of patented small molecule stearoyl coenzyme-A desaturase inhibitors (2009 -2013)[END_REF] . 3β-arachidyl amido cholanoic acid (aramchol) is an oral, liver-targeted, fatty acid-bile acid conjugate [START_REF] Leikin-Frenkel | Fatty acid bile acid conjugate inhibits hepatic stearoyl coenzyme A desaturase and is non-atherogenic[END_REF] which partially inhibits hepatic SCD1 protein expression and reduces liver triglycerides [START_REF] Gilat | Prevention of diet-induced fatty liver in experimental animals by the oral administration of a fatty acid bile acid conjugate (FABAC)[END_REF][START_REF] Leikin-Frenkel | Treatment of preestablished diet-induced fatty liver by oral fatty acid-bile acid conjugates in rodents[END_REF] and fibrosis in animal models of steatohepatitis or fibrosis [START_REF] Golan-Gerstl | Anti-fibrotic effect of aramchol on fibrosis in TAA animal model[END_REF][START_REF] Iruarrizaga-Lejarreta | Role of Aramchol in steatohepatitis Commenté [TG4]: Shiri, note here as in the other legend. I cleaned to show only Shaul's 2 and fibrosis in mice[END_REF] . In hepatic stellate cells, aramchol down-regulates SCD1 and interferes with Wnt signaling to reduce cell proliferation, collagen and fibronectin production and -smooth-muscle actin expression [START_REF] Bhattacharya | Aramchol downregulates stearoyl CoA-desaturase 1 in hepatic stellate cells to attenuate cellular fibrogenesis[END_REF] . Direct SCD1 depletion using siRNA phenocopies the inhibitory effects of aramchol on HSC fibrogenesis [START_REF] Bhattacharya | Aramchol downregulates stearoyl CoA-desaturase 1 in hepatic stellate cells to attenuate cellular fibrogenesis[END_REF] . In a 12-week phase 2a trial aramchol at 300mg daily significantly reduced liver fat content as measured by MR spectroscopy (MRS) vs. placebo in a dose dependent manner [START_REF] Safadi | The Fatty Acid-bile Acid conjugate aramchol reduces liver fat content in patients with nonalcoholic Fatty liver disease[END_REF] . Aramchol was shown to be safe and well tolerated.

The results of the phase 2a study led to the initiation of a global phase 2b study to evaluate the effect of ARamchol for the REsolution of STeatohepatitis (ARREST) in patients with NASH confirmed by liver biopsy. Here we report the safety and efficacy results of 52 weeks of treatment with 400 mg and 600 mg doses of aramchol in patients with NASH.

RESULTS

Study population

Between April 29th, 2015, and February 27th, 2017, 247 patients with NASH were randomized in a ratio of 2:2:1 to receive aramchol 400mg (n=101), aramchol 600mg (n=98) or placebo (n=48), once daily. The leading recruiting countries were Mexico (68 patients, 27% of study population) and the USA (64 patients, 26%). Thirty-two subjects (13%) were recruited in Israel (for the full list of countries, please see Methods). Figure 1 shows the disposition of the patients in the trial including reasons for trial discontinuation. The majority of study patients (219/247; 88.7%) completed 65 weeks in the study: 90/101 (89%), 88/98 (90%) and 41/48 (85%) in the aramchol 400mg, 600mg and placebo arms, respectively.

Baseline demographics and disease characteristics were balanced across study arms (Table 1).

Mean age was 54.4 years, 160/247 (65%) of trial participants were females, 156/247 (63%) White, 78/247 (32%) Hispanic and Latin. As per inclusion criteria, all patients were overweight or obese with a mean body mass index (BMI) of 32.7 kg/m 2 (median 32.8 kg/m2; min 25 kg/m2; max 42.7 kg/m 2 ). Drug-treated type 2 diabetes was present in 170/247 (69%) of participants, hypertension in 135/247 (55%) and dyslipidemia in 132/247 (53.4%) of them. Normal alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were seen in 107/247 (43.3%) and 138/247 (55.9%) of patients, respectively. At baseline, mean hemoglobin A1c (HbA1c) was similar across treatment arms (6.6%, 6.7% and 6.5% in the 400mg, 600mg and placebo arms, respectively). Most patients had histologically significant or advanced fibrosis with stage 2 and 3 (149/247, 60%) and active steatohepatitis (nonalcoholic fatty liver disease activity score [NAS] ≥5) in 173/247 (70%). Seven patients had fibrosis stage 0. Median NAS was 5.0 and median grades of steatosis, ballooning and inflammation were 2.0, 1.0 and 2.0, respectively. Baseline histological parameters were comparable between study arms except for a higher proportion of subjects with fibrosis stage 3 in the 400mg arm (Table 1). Mean baseline values for liver fat were comparable across study arms 27.3% ±11.8%, 30.2% ±12.4% and 27.5% ±9.3% in the aramchol 400mg, 600mg and placebo arms, respectively.

Efficacy Analyses

Hepatic fat reduction by imaging

214 subjects had paired MRS and were included in the FASMRI analysis set (n=90, 83 and 41 in the aramchol 400mg, 600mg and placebo arms, respectively (Figure 1). The primary endpoint analysis was performed in the FASMRI data set as prespecified. Hepatic triglyceride (%) measured by MRS was reduced in the aramchol 600mg [-3.2, 95% CI -5.2 to -1.2] vs. placebo [-0.1, 95% CI -2.8 to 2.6] with a mixed model for repeated measures (MMRM) difference between groups of -3.1 (95% CI -6.4 to 0.2, p=0.0655) (Table 2). Therefore, no further formal hierarchical statistical comparisons were performed. As prespecified, the remaining statistical comparisons of predefined key secondary and exploratory endpoints report the effects of aramchol with 95% confidence limits with nominal p-values. Hepatic fat was reduced in the aramchol 400 mg arm [-3.41; 95% CI -5.3 to -1.5] with an MMRM difference of -3.32 (95% CI 6.6 to -0.1; nominal p=0.045) (Table 2).

Liver histology

198 subjects had paired liver biopsies (n=80, 78 and 40 in the aramchol 400mg, 600mg and placebo arms, respectively; Figure 1) and were included in the pre-defined FASBiopsy analysis set. There was no statistical evidence of an imbalance across arms in the proportion of subjects without a pair of biopsies (21/101 (20.8%), 20/98 (20.4%) and 8/48 (16.7%) in the aramchol 400mg, 600mg and placebo arms, respectively). The effect of aramchol on key prespecified histological endpoints with corresponding nominal p values are shown in Table 2. NASH resolution without worsening of fibrosis was achieved in 16.7% (13/78) of the 600 mg arm vs. 5% (2/40) in the placebo arm (OR 4.74; 95% CI: 0.1-22.7; p=0.051; Figure 2). Improvement in fibrosis by one stage or more without worsening of steatohepatitis was observed in 29.5% (23/78) of the 600 mg arm vs. 17.5% (7/40) for the placebo arm (OR 1.88; CI: 0.7-5.0; p=0.21; Figure 2).

Biochemical and metabolic changes

Results for the prespecified key secondary endpoint of mean change from baseline in ALT (LSM) are shown in Table 3. ALT was reduced in the aramchol 600mg (-17.29±3.7 IU/L, 95% CI -24.6 to -10.0) vs. placebo (+11.77±5.2 IU/L, 95%CI: 1.4 to 22.1) with an MMRM difference between groups of -29.06 (95% CI -41.6 to -16.5; p< 0.0001). ALT was reduced in the in aramchol 400mg (-12.0±3.6 IU/L, 95% CI -19.1 to -4.8) with an MMRM difference between groups of -23.76 (95%CI: -36.2 to -11.3; p=0.0002).

Prespecified exploratory endpoints

At week 52, both doses of aramchol showed a decrease in HbA1c while placebo patients showed an increase, despite no notable changes in anti-diabetic medications in any of the three arms. HbA1c (%) was reduced in the in aramchol 600mg (-0.13±0.08, 95% CI -0.3 to 0.02) vs. placebo (+0.32, 95%CI: 0.1 to 0.5) with an MMRM difference between groups of -0.45±0.13 (95%CI:

-0.7 to -0.2; p=0.0008). HbA1c was reduced in the in aramchol 400mg (-0.04±0.08, 95%CI: -0.2 to 0.1) an MMRM difference between groups of -0.36±0.13 (95% CI -0.6 to -0.1; p=0.0061).

There was a numerical reduction in fasting serum glucose in the 600mg and 400mg arms vs. placebo but without changes in HOMA-IR. At Week 52, there were no discernible changes for other biochemical parameters including lipid parameters. Mean body weight did not change significantly: placebo-subtracted differences were -1.15 kg in the 400 mg arm and -0.41 kg in the 600 mg arm (Table 3).

The FIB-4 and NAFLD Fibrosis Score (NFS) scores, clinical and laboratory parameter-based scores associated with liver fibrosis in NASH, decreased at week 52 in the aramchol arms while placebo patients showed an increase. For FIB-4, placebo-subtracted differences were -0.27 in the 600 mg arm (95%CI: -0.5 to -0.1; p=0.008) and -0.21 in the 400 mg arm (95%CI: -0.4 to -0.02; p=0.033, respectively). For NFS, placebo-subtracted differences were -0.27 in the 600 mg arm (95%CI: -0.5 to -0.01; p=0.038) and -0.35 in the 400 mg arm (95% CI -0.6 to -0.1; p= 0.0080). There were no significant changes in fatty liver index (FLI), a marker of steatosis, fibrinogen, CRP, and adiponectin were not different between treatment arms.

Safety and tolerability

Aramchol was safe and well tolerated (Table 4). No deaths occurred during the study. (Table 4).

Serious adverse events (SAEs) were reported in 8.9% (9/101), 9.2% (9/98) and (6/48) 12.5% patients in the 400mg, 600mg and placebo arms, respectively. No clustering of event types was noted in the active-treatment arms. The overall incidence of early termination was low and slightly higher in the placebo than the two active-treatment arms (10.9% (11/101), 10.2% (10/98) and 14.6% (7/48) in the 400mg, 600mg and placebo arms, respectively). The leading causes for early termination were consent withdrawal and AEs. The incidence of early termination due to AEs was low and similar across study arms: (3%, 4.1% and 4.2% of subjects in the 400mg, 600mg and placebo arms, respectively. AEs were mainly mild and reversible. Headache was the most commonly reported AE in all study arms (13.9%, 15.3% and 12.5% in the 400mg, 600mg and placebo arms, respectively). A higher incidence of urinary tract infections (UTI) was noted in both aramchol arms, 14.9%, 13.3% and 6.3% in the 400mg, 600mg and placebo arms, respectively (p=0.13 and p=0.20 for 400mg and 600mg vs. placebo).

These were mostly single and mild events occurring in post-menopausal diabetic women. A numerical increase in pruritus was noted in the 600 mg arm, 11.2% compared to 6.9% and 6.3% in the 400 mg and placebo arms (p=0.34 for 600 mg vs. placebo). Pruritus events were mostly mild; none was severe and none leading to treatment discontinuation.

Post-hoc analyses

Post-hoc analyses for several proposed definitions of response [START_REF] Dulai | MRI and MRE for non-invasive quantitative assessment of hepatic steatosis and fibrosis in NAFLD and NASH: Clinical trials to clinical practice[END_REF][START_REF] Loomba | Multicenter Validation of Association Between Decline in MRI-PDFF and Histologic Response in NASH[END_REF] were performed to better understand the anti-steatotic effect of aramchol. The response rate for a ≥5% absolute reduction in liver fat content was 47.0% (39/83) for the 600 mg arm, 36.7% (33/90) for the 400 mg arm, and 24.4% (10/41) for the placebo arm (Table 2). Results for a 30% relative reduction were: 30.1% (25/83) in the 600mg arm, 25.6% (23/90) in the 400 mg arm and 14.6% (6/41) in the placebo arm.

Several additional post-hoc histological endpoints were analyzed to further characterize the effects of aramchol. Progression to cirrhosis occurred in only one patient (1.3%) in the 600 mg arm, six patients (7.5%) in the 400 mg arm and three patients (7.5%) in the placebo arm.

Hepatocyte ballooning improved by one grade or more in the 600 mg arm in 64% (50/78) of patients vs. 45% (18/40) in the placebo arm (OR 2.38 CI: 1.1 to 5.2; p=0.032) but not in the 400 mg arm (50% (40/80) of patients, OR 1.5 CI: 0.7 to 3.2; p=0.3). A larger proportion of subjects no longer had hepatocyte ballooning (grade 0) in the aramchol 600 mg arm vs. placebo [50% (39/78) vs. 35% (14/40), OR 2.3 CI: 1.0 to 5.2; p=0.0484] but not in the 400 mg arm (37.5% (30/80) OR 1.4 CI: 0.6 to 3.2; p=0.43).

Reductions in AST were documented in the aramchol 600mg arm (-10.83±2.5 IU/L, 95% CI -15.7 to -6.0) vs. placebo (+6.68±3.5 IU/L, 95% CI -0.2 to 13.6) with an MMRM difference between groups of -17.5 (95% CI -25.9 to -9.1; p< 0.0001). AST was also reduced in the aramchol 400mg arm (-7.21±2.4 IU/L, 95% CI -12.0 to -2.4), an MMRM difference between groups of -13.88 95% (CI -22.2 to -5.6; p=0.0011). Moreover 29% of patients in the 600 mg arm normalized ALT by the end of treatment vs. 21.9% in the 400 mg arm and 13.3% in the placebo arm. AST was normal at the end of treatment in 22.6% of patients in the 600 mg arm, 18.8% in the 400 mg arm and only 4.4% in the placebo arm. There were no meaningful changes in the enhanced liver fibrosis (ELF) score vs placebo (-0.049 in the 600 mg arm and -0.016 in the 400 mg arm; p=0.67 and 0.89 respectively).

DISCUSSION

This 52-week international, randomized, placebo-controlled trial demonstrated the antisteatotic potency of aramchol, building on a previous, smaller, lower dose, phase 2 study [START_REF] Safadi | The Fatty Acid-bile Acid conjugate aramchol reduces liver fat content in patients with nonalcoholic Fatty liver disease[END_REF] and confirmed its tolerability and safety in patients with NASH. In the principal analysis aramchol at the dose of 600 mg daily produced a placebo-corrected decrease in liver triglycerides without meeting the prespecified significance (p=0.06), while for aramchol 400 mg daily the nominal pvalue vs placebo was 0.045 reflecting the similarity in magnitude of treatment and heterogeneity of the study population.

The relevance of hepatic fat reduction as a predictor of histological improvement in NASH trials is a topic of great interest. Recent studies report that a 5% decrease in absolute liver fat content or a 30% relative fat reduction, measured by MRI-based methods, are associated with overall improvement in liver histology in several clinical trials [START_REF] Loomba | Multicenter Validation of Association Between Decline in MRI-PDFF and Histologic Response in NASH[END_REF][START_REF] Bril | Change in hepatic fat content measured by MRI does not predict treatment-induced histological improvement of steatohepatitis[END_REF][START_REF] Harrison | Resmetirom (MGL-3196) for the treatment of non-alcoholic steatohepatitis: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial[END_REF][START_REF] Middleton | Agreement Between Magnetic Resonance Imaging Proton Density Fat Fraction Measurements and Pathologist-Assigned Steatosis Grades of Liver Biopsies From Adults With Nonalcoholic Steatohepatitis[END_REF] . This might suggest that a responder analysis based on these thresholds could be more suitable to detect clinically meaningful anti-steatotic effects. A post-hoc responder analysis based on this cutoff demonstrated a stepwise increase in response from placebo to 400 mg to 600 mg aramchol. When considering the proportion of patients with a 30% or more reduction in liver fat, results for aramchol were slightly lower than that of pegbelfermin, a pegylated human fibroblast growth-factor 21 analogue [START_REF] Sanyal | Pegbelfermin (BMS-986036), a PEGylated fibroblast growth factor 21 analogue, in patients with non-alcoholic steatohepatitis: a randomised, double-blind, placebo-controlled, phase 2a trial[END_REF] and of firsocostat, an acetyl-CoA carboxylase inhibitor [START_REF] Loomba | Reduces Hepatic Steatosis and Fibrosis Markers in Patients With Nonalcoholic Fatty Liver Disease[END_REF] . Conversely, obeticholic acid, the only drug with confirmed histological efficacy in a phase 3 trial to date, reported an absolute reduction in liver fat of similar magnitude as aramchol [START_REF] Loomba | Multicenter Validation of Association Between Decline in MRI-PDFF and Histologic Response in NASH[END_REF] . Whether compounds with stronger effect on steatosis such as aldafermin [START_REF] Harrison | Efficacy and Safety of Aldafermin, an Engineered FGF19 Analog, in a Randomized, Double-Blind, Placebo-Controlled Trial of Patients With Nonalcoholic Steatohepatitis[END_REF] or resmetirom [START_REF] Harrison | Resmetirom (MGL-3196) for the treatment of non-alcoholic steatohepatitis: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial[END_REF] result in more marked histological improvement remains to be demonstrated in larger studies. These agents each have different mechanisms of action and the comparability of the clinical relevance of a specific change in liver fat measured by MRI on resolution of steatohepatitis and fibrosis improvement remains to be fully established while accounting for duration of exposure to the drug.

Improvements in key histological features such as resolution of steatohepatitis and improvement in fibrosis are considered likely surrogates of clinical events and therefore are being used as regulatory endpoints for conditional approval in NASH [START_REF]Reflection paper on regulatory requirements for the development of medicinal products for chronic non-infectious liver diseases[END_REF][START_REF] Anania | Nonalcoholic Steatohepatitis: Current Thinking From the Division of Hepatology and Nutrition at the Food and Drug Administration[END_REF] . A notable finding of this study is that resolution of steatohepatitis without worsening of fibrosis was achieved more frequently in the 600 mg arm than in the placebo arm. The low placebo rate noted in this trial is similar (6-12%) to that in some other trials (6-12% [START_REF] Harrison | Resmetirom (MGL-3196) for the treatment of non-alcoholic steatohepatitis: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial[END_REF][START_REF] Ratziu | an Agonist of the Peroxisome Proliferator-Activated Receptor-alpha and -delta, Induces Resolution of Nonalcoholic Steatohepatitis Without Fibrosis Worsening[END_REF][START_REF] Younossi | Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial[END_REF] ). In contrast, higher placebo responses were occasionally documented [START_REF] Newsome | A Placebo-Controlled Trial of Subcutaneous Semaglutide in Nonalcoholic Steatohepatitis[END_REF][START_REF] Harrison | Insulin sensitizer MSDC-0602K in non-alcoholic steatohepatitis: A randomized, double-blind, placebo-controlled phase IIb study[END_REF] , this heterogeneity in the placebo response possibly reflecting varying lifestyle choices, alcohol use, cross talk between liver disease and comorbid disease states, concomitant therapies and differences in biopsy interpretation. Other histological endpoints such as ballooning and fibrosis also favored the 600 mg dose. These findings may be relevant because ballooning is the hallmark of the steatohepatitic process and disease activity whereas fibrosis is the best histological marker of prognosis [START_REF] Taylor | Association Between Fibrosis Stage and Outcomes of Patients With Nonalcoholic Fatty Liver Disease: A Systematic Review and Meta-Analysis[END_REF] . Fibrosis improvement by one stage or more was numerically higher in the 600mg arm than in the placebo arm, without reaching statistical significance. This trial was not powered for histological endpoints, which were the key secondary endpoints.

Nonetheless, liver biopsy data suggest that key histological features related to disease progression may improve over a 52-week treatment. The numerical pattern of response for the 400 and 600 mg arms for both histological endpoints suggests that aramchol may improve NASH. Also, these results argue that while the dose of 400 mg may be sufficient for fat reduction and improvement in ALT and HbA1c, a higher dose may be needed for histological improvement. The current ongoing phase 3 trial (NCT04104321) is adequately powered to detect differences of the magnitude observed in this study and patients are receiving a different regimen (aramchol 300mg BID) to achieve higher exposure. Some biochemical parameters suggestive of histological improvement were also affected by the study drug. There was an early, dose-related reduction in ALT, that was maintained throughout the treatment period. Aminotransferase reduction has been observed each time histology improved in placebo-controlled trials of NASH [START_REF] Harrison | Resmetirom (MGL-3196) for the treatment of non-alcoholic steatohepatitis: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial[END_REF][START_REF] Ratziu | an Agonist of the Peroxisome Proliferator-Activated Receptor-alpha and -delta, Induces Resolution of Nonalcoholic Steatohepatitis Without Fibrosis Worsening[END_REF][START_REF] Younossi | Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial[END_REF][START_REF] Neuschwander-Tetri | Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebocontrolled trial[END_REF][START_REF] Sanyal | Vitamin E, or Placebo for Nonalcoholic Steatohepatitis[END_REF] . The mean absolute change of -17 IU/L in the 600 mg arm is similar to the mean value that independently predicted histological improvement in a smaller phase 2 trial of obeticholic acid [START_REF] Loomba | Factors Associated With Histologic Response in Adult Patients With Nonalcoholic Steatohepatitis[END_REF] . Gamma-glutamyl transferase declined in a pattern similar to ALT.

Two well-validated serum fibrosis markers, FIB-4 and NFS, although not ELF, were also reduced in the high dose aramchol arm vs. placebo. However, AST and ALT levels are part of the FIB4 and NFS and changes in the short term may reflect changes in these parameters and not changes in fibrogenesis. Despite this, improvements in FIB4 have been associated with improved liver histology both in the contexts of clinical trials and clinical practice [START_REF] Chalasani | Relationship between three commonly used non-invasive fibrosis biomarkers and improvement in fibrosis stage in patients with non-alcoholic steatohepatitis[END_REF][START_REF] Siddiqui | Diagnostic Accuracy of Noninvasive Fibrosis Models to Detect Change in Fibrosis Stage[END_REF] . The utility of FIB4, NFS and other biomarkers as surrogates of histological response are currently under active investigation in the fully powered phase 3 trial of aramchol for NASH.

Several studies have documented a reduction in SCD1 activity, without complete inhibition, upon aramchol administration both in vitro [START_REF] Leikin-Frenkel | Fatty acid bile acid conjugate inhibits hepatic stearoyl coenzyme A desaturase and is non-atherogenic[END_REF] and in vivo [START_REF] Leikin-Frenkel | Treatment of preestablished diet-induced fatty liver by oral fatty acid-bile acid conjugates in rodents[END_REF] . In the methionine-choline deficient model of steatohepatitis, aramchol reduced SCD1 protein content, liver monounsaturated fatty acid concentration and the desaturation index [START_REF] Iruarrizaga-Lejarreta | Role of Aramchol in steatohepatitis Commenté [TG4]: Shiri, note here as in the other legend. I cleaned to show only Shaul's 2 and fibrosis in mice[END_REF] . Multiple lines of evidence suggest that modulating SCD-1 activity is an attractive pharmacological target in metabolic diseases associated with obesity, including NAFLD [START_REF] Brown | Stearoyl-coenzyme A desaturase 1 inhibition and the metabolic syndrome: considerations for future drug discovery[END_REF] . In humans, obesity is associated with increased surrogates of SCD1 activity, such as desaturation indexes or palmitoleate concentrations, both in plasma and in adipose tissue [START_REF] Warensjo | Fatty acid composition and estimated desaturase activities are associated with obesity and lifestyle variables in men and women[END_REF] .

In rodents, SCD1 genetic inactivation results in resistance to diet-induced weight gain, fat accumulation and dyslipidaemia [START_REF] Ntambi | Loss of stearoyl-CoA desaturase-1 function protects mice against adiposity[END_REF] . Specifically, SCD1 is strongly induced in the liver upon high carbohydrate feeding and controls a rate-limiting step of hepatic de novo lipogenesis. Liver specific inhibition of SCD1 consequently protects against high carbohydrate diet-induced adiposity and steatosis, reduces lipogenesis, hepatic triglyceride secretion and white adipose tissue weight. In addition to controlling the rate of lipogenesis and triglyceride synthesis and excretion, changes in SCD1 activity also modulate fatty acid disposal thus further promoting liver fat loss [START_REF] Ntambi | Loss of stearoyl-CoA desaturase-1 function protects mice against adiposity[END_REF] . Inactivation of SCD1 activity in rodents results in upregulation of lipid oxidation genes including, carnitine palmitoyl 1, a major regulator of mitochondrial oxidation of fatty acids [START_REF] Ntambi | Loss of stearoyl-CoA desaturase-1 function protects mice against adiposity[END_REF] . SCD1 activity also promotes AMPK activation which in turn down-regulates Acetyl-CoA Carboxylase activity [START_REF] Dobrzyn | Stearoyl-CoA desaturase 1 deficiency increases fatty acid oxidation by activating AMP-activated protein kinase in liver[END_REF] . Thus, SCD1 inhibition promotes both fatty acid disposal and reduces triglyceride synthesis.

In the current trial, aramchol induced an improvement in HbA1c levels. This change is clinically relevant as study participants had either type 2 diabetes or pre-diabetes [START_REF]Classification and Diagnosis of Diabetes:Standards of Medical Care in Diabetes-2018[END_REF] . Because type 2 diabetes is a major comorbidity associated with more severe forms of NASH and higher potential for disease progression, optimal control of their diabetes and other metabolic comorbidities is essential. Drugs that contribute to the control of these comorbidities or, at a minimum are neutral, are highly anticipated. Despite no changes in insulin levels, HbA1c improvement induced by aramchol without hypoglycemic episodes is supported by experimental data in rodents demonstrating both in vitro and in vivo an increase in AMPK activity with subsequent reduction in gluconeogenesis [START_REF] Fernández-Ramos | Aramchol improves liver glucose and lipid homeostasis in NASH via AMPK and mTOR regulation[END_REF] . Other data linked SCD1 inactivation with an improved insulin sensitivity. Whole-body SCD1 knock-out rodents display improved insulin signalling [START_REF] Rahman | Stearoyl-CoA desaturase 1 deficiency elevates insulin-signaling components and down-regulates protein-tyrosine phosphatase 1B in muscle[END_REF] and increased GLUT-4 and GLUT-2 expression in skeletal muscle and hepatocytes [START_REF] Poletto | Oleic and linoleic fatty acids downregulate Slc2a4/GLUT4 expression via NFKB and SREBP1 in skeletal muscle cells[END_REF] mainly mediated through a reduction in palmitoleate and oleate and in ceramide synthesis [START_REF] Dobrzyn | Stearoyl-CoA desaturase-1 deficiency reduces ceramide synthesis by downregulating serine palmitoyltransferase and increasing beta-oxidation in skeletal muscle[END_REF] .

Larger studies in humans are however necessary to confirm a beneficial effect of aramchol on glycemic regulation and insulin sensitivity.

Although largely metabolically beneficial, SCD1 inactivation can theoretically also result in inflammatory tissue damage. Accumulation of SCD1 substrates such as palmitate and stearate can induce apoptosis [START_REF] Li | Hepatic lipid partitioning and liver damage in nonalcoholic fatty liver disease: role of stearoyl-CoA desaturase[END_REF] and endoplasmic reticulum stress [START_REF] Liu | Hepatic oleate regulates liver stress response partially through PGC-1alpha during high-carbohydrate feeding[END_REF] thus contributing to lipotoxic liver injury.

Mice treated with aramchol were protected from oxidative stress by an increased glutathione and glutathione/glutathione disulphide ratio and displayed less inflammation and also less fibrosis [START_REF] Iruarrizaga-Lejarreta | Role of Aramchol in steatohepatitis Commenté [TG4]: Shiri, note here as in the other legend. I cleaned to show only Shaul's 2 and fibrosis in mice[END_REF] . Differences between genetically-driven total suppression of SCD1 activity and partial pharmacological inhibition as induced by aramchol could explain the observed differences in the overall net effects. Similar data of reduced inflammation and prevention of fibrosis onset have been reported with other preclinical SCD1 inhibitor compounds [START_REF] Kurikawa | A novel inhibitor of stearoyl-CoA desaturase-1 attenuates hepatic lipid accumulation, liver injury and inflammation in model of nonalcoholic steatohepatitis[END_REF] . Other fibrosis models have confirmed an antifibrotic potency of aramchol that parallels SCD1 inhibition in hepatic stellate cells [START_REF] Bhattacharya | Aramchol downregulates stearoyl CoA-desaturase 1 in hepatic stellate cells to attenuate cellular fibrogenesis[END_REF] . Direct SCD1 depletion using siRNA results in down-regulation of fibrogenesis i.e., reduction of collagen 1A1 and alpha smooth muscle actin production by hepatic stellate cells [START_REF] Bhattacharya | Aramchol downregulates stearoyl CoA-desaturase 1 in hepatic stellate cells to attenuate cellular fibrogenesis[END_REF] . Importantly, the ARREST study is the first biopsy-based clinical trial of aramchol and demonstrates no adverse impact on liver cell injury or inflammation. These findings support the safe use of this agent in a phase 3 trial.

Several attempts to develop small molecule SCD1 inhibitors for the treatment of metabolic diseases have failed due to severe skin and lachrymal gland toxicity in animals [START_REF] Powell | An overview of patented small molecule stearoyl coenzyme-A desaturase inhibitors (2009 -2013)[END_REF] . Owing to its different molecular structure which possibly targets tissue distribution preferentially to the liver and only partial SCD1 inhibition no particular side effects were noted with aramchol. There were a few cases of uncomplicated lower UTI in post-menopausal women. While there is no apparent explanation for this finding, given the inclusion criteria of pre-diabetes or type 2 diabetes and the occurrence of UTI mainly in post-menopausal women, this event is not considered atypical for the study population. There was a small numerical increase in mostly single and mild pruritus events in the 600 mg arm that did not lead to treatment discontinuation. This study has several limitations. Histological outcomes were only key secondary endpoints, and the trial was not powered to show histological benefit. Patients from Israel did not have week 52 biopsy assessments because of restrictions imposed by the Ministry of Health.

Strengths of trial include centralized assessment of biochemical parameters and blinded central review of liver biopsies by an expert hepatopathologist. A strength of this study is the inclusion of a large proportion of Hispanic patients, who have a higher prevalence of a disease-associated variant (I1 48M) of the PNPLA3 gene, which is associated with higher risk of progression to NASH [START_REF] Trepo | Update on NAFLD genetics: From new variants to the clinic[END_REF] . In addition, the population studied in ARREST was an enriched targeted population where all patients were overweight or obese and had pre-diabetes or type 2 diabetes.

In conclusion, in this randomized, placebo-controlled, global trial of aramchol, a partial SCD1 inhibitor, the reduction in liver fat did not meet the prespecified primary endpoint for statistical significance. However, the totality of the data based on prespecified key secondary endpoints, exploratory analyses and post-hoc analyses suggest a potential for improving liver histology in patients with type 2 diabetes or prediabetes with histologically confirmed steatohepatitis and with high disease activity and pre-cirrhotic stages of fibrosis. These are corroborated by the observed biochemical improvement in liver enzymes. This will be further tested in an ongoing large, international phase 3 trial (NCT 04104321). Data is presented as % of subjects meeting endpoint or as mixed model derived least squares means ± standard error; p values beyond the primary endpoint are nominal; * Post-Hoc analysis: responder is defined according to ≥5% absolute improvement from baseline

TABLES

Randomization and blinding

Eligible patients were randomly assigned in a 2:2:1 ratio (48 blocks), to receive either daily aramchol 400mg, aramchol 600mg or placebo, orally, for 52 weeks. The randomization ratio was 2:2:1 stratified by country. The randomization list was generated prior to the study initiation, using a computer-generated randomization list, and done using an interactive web response system. Treatment assignments were masked to patients, investigators, site personnel, Sponsor, and central readers of biopsy and MRS data. Aramchol and matching placebo were of identical appearance.

Dose selection

Dose selection was based on clinical pharmacology considerations and corroborating evidence from the Phase 2a study. Aramchol is a biopharmaceutics classification system Class IV compound with low solubility and low permeability. Data from Phase 1 pharmacokinetics studies in healthy volunteers evaluating single doses up to 900mg aramchol and repeat daily doses of 600mg once daily showed sub-proportional increases in exposure with dose where once daily doses of >600 mg are not expected to result in higher exposures. None of the studies raised safety concerns and dose response data in the Phase 2a suggested that a higher Commenté [TG5]: Shiri I changed here adding the new sentence after the old to encompass all ECs and not just the ones mentioned.

Procedures and assessments

Following randomization, patients were evaluated at 9 scheduled visits: weeks 2, 4, 8, 12, 24, 32, 40, 52 (end of treatment) and 65 (follow-up). Body weight and waist circumference were measured at Screening, Baseline, Week 24, Termination/Early Termination and at Week 65.

During study visits, subjects were counseled on the importance of diet and exercise in proper weight management and asked if any change took place in their lifestyle between visits. Blood samples were obtained at these visits for routine biochemical and hematology tests and measured centrally (Clinical Research Laboratory (CRL)). Based on CRL cut-offs, normal alanine aminotransferase (ALT) was <45 IU/L and normal AST<41IU/L. Data was collected using the electronic data capture system DSG eCaseLink V8.0.

MRS Evaluation

Patients were required to perform two MRS scans, at Screening and at week 52. MRS evaluation was also recommended for patients with early study termination at week 24 or beyond. MRS scans were read centrally at the Tel Aviv Sourasky Medical Center (Tel Aviv, Israel) by a specialized radiologist masked to treatment allocation (DBB).

Liver Biopsy

Biopsies were performed at Screening (if not available within 6 months prior) and at week 52.

In case of early study termination, a biopsy was recommended if patients completed at least 40 weeks in the study. Patients from Israel (n=24) were not allowed to undergo an end-ofstudy liver biopsy as per Israeli Ministry of Health regulatory restrictions at the time the study was submitted. Liver biopsies were centrally read by a single pathologist (CL) masked to treatment allocation. Analyses used the initial, baseline qualifying read and the end-oftreatment read for assessing histological changes. Steatohepatitis was diagnosed based on the presence of steatosis, inflammation, and ballooning. Biopsy specimens were graded according 0

Primary efficacy endpoint and principal statistical analysis

The primary endpoint of the study was the absolute % change from baseline to end of study in liver triglycerides to water ratio (fat/water+fat) as measured by MRS. The FAS was used as the primary analysis set for efficacy analysis and inference. The statistical model was a Mixed Model (SAS ® MIXED procedure) with random intercept subcommand and REML estimation method was used and degrees of freedom were adjusted using the Kenward-Roger method.

The model included the following covariates: treatment group, Country and Geographical Region (CGR), age, sex, Baseline liver fat and Baseline BMI.

Other endpoints analyses

Analyses of biopsy derived endpoints used the baseline adjusted logistic regression (SAS ® LOGISTIC procedure) stratified by CGR using the STRATA subcommand with the following effects: treatment group, Baseline CRN Fibrosis Score and NAS score was used to test the between the active groups and placebo contrasts.

The statistical model used for the analyses of change in baseline for laboratory derived endpoints was a Mixed Model for Repeated Measures (MMRM) (SAS ® MIXED procedure with REPEATED subcommand). The model included the following fixed effects: categorical week in trial by treatment interaction, CGR and Baseline value using the unstructured covariance structure and the REML estimation method and degrees of freedom were adjusted using the Kenward-Roger method. When there were no repeated measures, analysis of covariance was used (NFS, FLI, Adiponectin, hs-CRP and ELF) 1 are not publicly available due to patients' right of privacy and confidentiality as well as ethical and commercial limitations imposed on Galmed. Upon request, Galmed will consider sharing certain data sets, in accordance with applicable local laws as well as patient consent. The data sharing request should include the type of data requested, the reason the data is requested and the intended use of the data.

Role of the funding source

This study was sponsored by Galmed Pharmaceuticals. The protocol was written by a panel of experts and sponsor representatives. Authors participated actively in drafting and reviewing Commenté [TG7]: gap again the manuscript. The corresponding author had full access to all data in the study and had final responsibility for the decision to submit for publication.

Figure 1 .Figure 2 .

 12 Figure 1. Patient disposition in the trial

3 6 dose

 36 may result in better efficacy.

Table 1 : Demographic and Baseline Characteristics

 1

		Placebo	Aramchol 400	Aramchol 600
		N*=48	N*=101	N*=98
	Demographics
	Age (yrs.)	54.4±10.3	53.9±10.9	54.9±9.8
	Sex
	Male	23 (48%)	36 (36%)	28 (29%)
	Race/ Ethnicity
	White	30 (63%)	63 (62%)	63 (64%)
	Hispanic/Latin/Latin American	16 (33%)	33 (33%)	29 (30%)
	Other	2 (4%)	5 (5%)	6 (6%)
	Comorbidities
	Hypertension	24 (50%)	53 (52.5%)	58 (59.2%)
	Dyslipidemia	30 (62.5%)	57 (56.4 %)	45 (45.9%)
	Drug-treated type 2 diabetes (%)	72.9	68.3	67.3
	Metabolic Factors
	BMI (kg/m 2 )	32.6±4.9	32.4±4.5	33±4.2
	Weight (Kg)	88.6±18.2	88.1±17.4	86.9±15.5
	Waist Circumference (cm)	107.5±12.1	108.7±13.8	107.6±11.2
	Glycemic Parameters
	Serum glucose (mmol/L)	6.55±1.9	6.56±1.5	6.94±2.4
	Hemoglobin A1c (%)	6.53±1.0	6.56±0.9	6.65±1.0
	HOMA-IR (Units)	10.0±8.7	9.1±6.5	9.6±6.5

Table 2 : Change in MRS and histology-based endpoints after 52 weeks of treatment Difference as Compared to Placebo Odds Ratio and 95% Confidence Interval

 2

		Placebo	Aramchol 400mg	Aramchol 600mg	Aramchol 400mg	Aramchol 600mg	Aramchol 400mg	Aramchol 600mg
	Primary outcome
	Number of patients with paired MRI evaluations	41	90	83
	Absolute % Change from Baseline in Mean Liver Fat	-0.09%±1.38%	-3.41%±0.96%	-3.18%±1.01%	-3.32%±1.65% P=0.045	-3.09%±1.67% P=0.066
	% of MRS Responders*	24.4%	36.7%	47.0%			2.20 (0.89-5.46) P=0.088	2.77 (1.12-6.89) P=0.028
	Changes from baseline in
	histopathological
	parameters
	Number of patients with paired biopsies	40	80	78
	NASH Resolution Without Worsening of Fibrosis	5.0%	7.5%	16.7%			1.79 (0.33-9.62) P=0.50	4.74 (0.99-22.66) P=0.051
	Fibrosis Improvement Without Worsening of NASH	17.5%	21.3%	29.5%			1.11 (0.40-3.05) P=0.84	1.88 (0.7-5.04) P=0.21
	≥2 points improvement in
	NAS contributed by at least
	two of: steatosis, inflammation, ballooning	17.5%	20.0%	25.6%			1.36 (0.49-3.80) P=0.56	1.68 (0.62-4.57) P=0.31
	Without Worsening of
	Fibrosis
	≥2 points improvement in SAF activity score Without Worsening of Fibrosis	25.0%	25.0%	35.9%			1.08 (0.44-2.63) P=0.86	1.84 (0.78-4.35) P=0.16

Table 4 : Safety and tolerability data

 4 AEs with an incidence ≥10% in any treatment arm are presented by system organ class and preferred term. No deaths were reported during the study. 1

		Placebo	Aramchol 400	Aramchol 600
		(N=48)	(N=101)	(N=98)
	Overall treatment withdrawal rate	7 (14.6%)	11 (10.9%)	10 (10.2%)
	Treatment withdrawal due to adverse event	2 (4.2%)	3 (3%)	4 (4.1%)
	Participants with serious adverse event	6 (12.5%)	9 (8.9%)	9 (9.2%)
	Participants with severe adverse event	5 (10.4%)	7 (6.9%)	6 (6.1%)
	Participants with any adverse event	33 (68.8%)	75 (74.3%)	77 (78.6%)
	Gastrointestinal disorders
	Constipation	6 (12.5%)	5 (5%)	8 (8.2%)
	Nausea	6 (12.5%)	10 (9.9%)	9 (9.2%)
	Nervous system disorders
	Headache	6 (12.5%)	14 (13.9%)	15 (15.3%)
	Skin disorders
	Pruritus	3 (6.3%)	7 (6.9%)	11 (11.2%)
	Infections
	Urinary tract infection	3 (6.3%)	15 (14.9%)	13 (13.3%)

Commenté [TG2]: Shiri, note here I removed my proposal and left as Shaul edited.I copied also into the author list

Commenté [TG3]: please fix font size

Acknowledgments

Financial support for medical editorial assistance was provided by Galmed Pharmaceuticals. We thank medical writer Shiri Diskin, Ph.D. (Bioforum Group) for editorial assistance with this manuscript.

Data Availability

The data that supports the findings of this study are owned by Galmed Research and Development Ltd. ("Galmed") and contains potentially identifying or sensitive patient information as it includes, among others, human research participant data. Therefore, the data

Author Contributions

Competing Interests Statement V.R. and R.L. are Galmed consultants and Investigators in the Galmed-sponsored study described in the article; S.F. and A.J.S are Galmed consultants, T.G., M.H., R.O. and L.H. are current or former Galmed employees, D. BB. was responsible for central lab services for the Galmed-sponsored study described in the article; K.L. was responsible for histological analysis services for the Galmed-sponsored study described in the article; S.K. is the Galmed statistician; L.L.de-G., R.S., F.P., F.F., J. F-F., M.A., and A.L.F were Investigators in the Galmed-sponsored study described in the article. The ARREST investigator study group members were investigators or sub-investigators in the Galmed-sponsored study described in the article. Means (LSM) ± standard error of absolute changes from baseline by treatment group; When there were no repeated measures, analysis of baseline adjusted covariance was used; Two-sided nominal p -values beyond the primary endpoint testing the between active groups and placebo contrasts to the NASH Clinical Research Network (CRN) scoring system 2 for steatosis (scored 0-3), Commenté [TG6]: please remove gap from here. I tried but it messed the lines… 3 8 inflammation (scored 0-3) and hepatocellular ballooning (scored 0-2). Fibrosis was evaluated using the NASH CRN fibrosis staging system (stages 1-4) 2 . Biopsies were also scored based on the steatosis, activity, and fibrosis (SAF) algorithm [START_REF] Lambert | Increased de novo lipogenesis is a distinct characteristic of individuals with nonalcoholic fatty liver disease[END_REF] .

NMED-A108256D

Outcomes

The primary endpoint of the study was the absolute change from Baseline to end of study in liver fat content assessed by MRS and measured as a triglyceride-to water-ratio (fat/water+fat, %). Key secondary endpoints were: proportion of subjects with NASH resolution at week 52 (no evidence of steatohepatitis with ballooning score of 0 and an inflammation score of 0 or Safety and tolerability were evaluated based on treatment emergent serious adverse events (SAEs); adverse events (AEs); safety laboratory; vital signs; 12-Lead electrocardiograms (ECG); physical examinations and the proportion of patients who prematurely discontinued from the study. AEs were graded for severity. An independent data monitoring committee reviewed safety data during the study.

Several post-hoc analyses were performed to further describe the effects of aramchol regarding: liver de-fattening as measured by MRS (responder analyses), histological changes

Statistical analysis

Sample size and power considerations

The planned sample size was 215 patients, 86 in each of the two active groups and 43 in the placebo group. Sample size calculation was based on an effect size of 0.6 for the primary endpoint between the active groups and placebo with a 5% significance level and 89% power.

Based on an expected drop-out rate of 10%, the total sample size was 240.

Significance level and multiplicity adjustment

One primary endpoint and five secondary endpoints were pre-defined. The overall experiment-wise significance level was 5% using two-tailed tests with the hierarchical gatekeeping approach to control the overall Type-I error rate for multiple contrasts and multiple endpoints (refer to supplementary Table 2; Order of testing for Contrasts). According to the gate-keeping approach, the 1 st contrast (600mg vs. placebo in the primary endpoint) was tested using a two-tailed 5% significance level. If the first contrast fails to reach statistical significance, all p-values reported, as per SAP, are nominal p-values.

Pre-defined Analyses sets:

Full Analysis Set (FAS): included all patients randomized and who had baseline and at least one post-baseline efficacy assessment. The FAS analysis set includes efficacy observations that were collected up to and including Week 52. FAS for MRS (FASMRI) included all subjects that had a paired MRS with pre-and post-treatment measurements. FAS for Liver Biopsy Data (FASBiopsy): included all patients randomized who underwent the baseline and week 52 biopsies.


O Bourgeois
email: olivier.bourgeois@grenoble.cnrs.fr.

C Macovei

E André

J-L Garden

J Chaussy

D Givord

A new sensor for thermodynamic measurements of magnetization reversal in magnetic nanomaterials

Keywords: Type pacs here, separated by semicolons, Type your keywords here, separated by semicolons, Specific heat measurements, microsystems

A sensor for thermal and thermodynamic measurements of small magnetic systems have been designed and built. It is based on a 5µm-thick suspended polymer membrane, which has a very low heat capacity (≈ 10 -6 J/K at nitrogen temperature), and on which a heater and a highly sensitive thermometer are deposited. The sensor properties have been characterized as a function of temperature and frequency. Energy exchanges as small as 1 picojoule (10 -12 Joule) were detected in the 40K-300K temperature range. Such values correspond to those required for measuring the thermal signatures occurring during magnetization reversal in very thin samples (typically 10 nm thick), which would be deposited on the membrane. It is expected that this method will constitute a powerful tool in view of analyzing magnetization reversal processes in magnetic nanosystems, e.g. exhibiting the exchange-spring and exchange-bias phenomena.

Introduction

The exchange-spring [1] and the exchange-bias [2] phenomena constitute specific magnetization processes occurring in magnetic nanosystems. Both these phenomena originate from interfacial exchange coupling and they manifest themselves when the crystallite size becomes of the order of the domain wall width. Recent focus went from layered systems to systems containing nanoparticles or clusters [3].

From a thermodynamic point of view, it is well known that the progressive magnetization change under an external magnetic field leads to a change in the system magnetic entropy, in particular through the field dependence of the spontaneous magnetization. Since the total entropy is constant under adiabatic conditions, heat releases or changes in the specific heat occur which can be detected by a proper sensor [4,[START_REF] Chikazumi | Physics of Magnetism[END_REF].

In this work an adiabatic sensor dedicated to the measurement of the specific heat of small systems (less than 1 microgram) was designed and built and its thermal properties were characterized.

Sensor fabrication

In such experiment, where small mass samples will be used, the expected thermal signals are very weak, of the order of magnitude of some picojoules. The detection of such small signals at temperatures above 50 K, requires the development of a new type of sensors.

The calorimetric sensor designed for this purpose is based on a suspended polymer membrane. Two thin film transducers (a heater and a thermometer) are deposited on each side of the membrane. Using an adiabatic calorimetric method (ac calorimetry), the specific heat can be measured provided that the membrane is sufficiently thermally isolated. This principle of suspended sensor was already applied to different areas of solid state physics [START_REF] Riou | [END_REF]7], and it has been demonstrated recently that such highly sensitive technique can be fruitfully applied to the detection of small thermal signals of mesoscopic objects in magnetism or superconductivity [8,9].

The sensor fabrication procedure is made of a series of steps (see Fig. 1): a 5µm thick para-xylylen C (parylen C) film is first deposited on a copper disk. This film which allows all contacts and connections to be electrically isolated from the copper substrate, is eliminated at the end of the process. The electrical leads and the above-mentioned transducers are made of niobium nitride (NbN x ). The heater and associated leads, which are made of a low nitrogen concentration alloy (NbN 1.1 ), are sputtered on the 5-µm polymer film and lithographed. On top of these, a second parylen layer, 500 nm to 800 nm thick, is deposited. The function of this layer is to hold the transducers at final stage. The thermometer and the current leads are sputtered. Next, the membrane is freed from the copper substrate to thermally isolate the center of the sensor from the heat bath. Finally, the first thick layer of parylen is removed by oxygen plasma etching. In its final configuration, the sensor is composed of two transducers and of the very thin polymer membrane. Note that the thermometer is made of a highly nitrogen doped niobium nitride thin film: NbN 1.7 . At such nitrogen concentration, niobium nitride has a high-temperature metal to insulator transition. As a result, it constitutes a very sensitive thermometer between 40K and 300K.

For thermodynamic measurements, it is expected that the magnetic sample will be either sputtered directly onto the sensor or glued on the thermometer. In all cases, the quality of the thermal contact between the sample and the sensor will constitute an important parameter determining the quality of the measurements.

Thermal characterization

The specific heat is measured by ac calorimetry [10]. An alternating current is applied to the heater at a frequency f creating a temperature oscillation which is detected at 2f by measuring the thermometer response. In order to thermally characterize the sensor, the electrical response at 2f of the thermometer was measured between 0.5 Hz and 15 Hz. The optimum working frequency, f opt , corresponds to the maximum in the variation of ωT ac versus f (Figure 2) where ω = 2πf and T ac is the amplitude of the temperature oscillation.

The frequency f opt is determined by two characteristic response times of the sensor: on the one hand, the heat diffusion time in the sensor which gives the frequency high limit and, on the other hand, the relaxation time characterizing heat transfer from the sensor to the heat bath (given by C/K, where C is the sensor heat capacity and K is the thermal conductance of the thermal link) which gives a lower bound for the frequency of the measurements. As seen in Fig. 2, the optimum frequency can be chosen between 1Hz and 5Hz typically.

The measurements of the heat capacity of different sensors (S2, A7 and S15) are given in Fig. 3. As expected, the heat capacity of these systems is very low, around 10 -6 Joule/K at liquid nitrogen temperature with a resolution of at least 10 -4 . Considering that the temperature oscillation is typically of the order of magnitude of 1mK, the sensitivity in energy is significantly better than 1 picojoule. processes of 3d metals (Fe, Co, Ni) are of the order of 10 3 J/m3 [4]. For a sample in the form of a 1 mm x 1mm square and 10 nm thick, this corresponds to 10 -11 J approximately. Such values will be accessible to the measurements with the developed sensor. Heat releases will be measured by applying a modulated magnetic field at the frequency corresponding to the adiabatic (typically from 1 to 10 Hz, see Fig. 2). Any reversible heat release occurring along the hysteresis cycle can be measured on the thermometer at the same frequency. Temperature oscillations, as small as 5µK, can be detected with the present device. Knowing the heat capacity from previous measurements deriving of the heat release is straightforward. Such measurements are currently underway.

Conclusions

An original sensor has been developed using microfabrication technology for thermal measurements of magnetic systems. This sensor is based on suspended membranes. The specific heat is measured using an ac calorimetry technique. Due to its very low heat capacity (few microjoule/K), this sensor is adapted to the measurement of energy variations as small as 1picojoule at liquid nitrogen temperature. This will enable the measurement of the thermal signals associated to the magnetization processes occurring along the hysteresis cycles of magnetic systems.

Fig. 3 .Fig. 1 .Fig. 2 .

 312 Fig. 3. Measurement of the heat capacity of different sensors at various temperatures between 2Hz and 4Hz. Due to the high fragility of the membrane it has been difficult to vary the temperature on a given sensor. A typical heat capacity of 1microjoule/K is measured at nitrogen temperature.Typical values for the heat released during magnetization

The authors would like to thank P. Gandit, R. Morel, A. Brenac, P. Brosse-Maron, P. Lachkar for fruitful help and discussion and the Institut de Physique de la Matière Condensée (IPMC) for financial support.