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Randomised clinical trial: A phase 2 double‐blind study of namodenoson in non‐alcoholic fatty liver disease and steatohepatitis

Summary Background Namodenoson, an A3 adenosine receptor (A3AR) agonist, improved liver function/pathology in non‐alcoholic steatohepatitis (NASH) preclinical models. Aim To evaluate the efficacy and safety of namodenoson for the treatment of non‐alcoholic fatty liver disease (NAFLD) with or without NASH Methods This phase 2 study included 60 patients with NAFLD (ALT ≥60 IU/L) who were randomised (1:1:1) to oral namodenoson 12.5 mg b.d. (n = 21), 25 mg b.d. (n = 19), or placebo (n = 20) for 12 weeks (total follow‐up: 16 weeks). The main efficacy endpoint involved serum ALT after 12 weeks of treatment. Results Serum ALT decreased over time with namodenoson in a dose‐dependent manner. The difference between change from baseline (CFB) for ALT in the namodenoson 25 mg b.d. arm vs placebo trended towards significance at 12 weeks (P = 0.066). Serum AST levels also decreased with namodenoson in a dose‐dependent manner; at 12 weeks, the CFB for 25 mg b.d. vs placebo was significant (P = 0.03). At Week 12, 31.6% in the namodenoson 25 mg b.d. arm and 20.0% in the placebo arm achieved ALT normalisation (P = 0.405). At week 16, the respective rates were 36.8% and 10.0% (P = 0.038). A3AR expression levels were stable over time across study arms. Both doses of namodenoson were well tolerated with no drug‐emergent severe adverse events, drug‐drug interactions, hepatotoxicity, or deaths. Three adverse events were considered possibly related to study treatment: myalgia (12.5 mg b.d. arm), muscular weakness (25 mg b.d. arm), and headache (25 mg b.d. arm). Conclusion A3AR is a valid target; namodenoson 25 mg b.d. was safe and demonstrated efficacy signals (ClinicalTrials.gov #NCT02927314).


| INTRODUC TI ON
Non-alcoholic fatty liver disease (NAFLD) is associated with obesity, visceral adiposity, type 2 diabetes mellitus (T2DM), and the spectrum of metabolic syndrome. 1,2 Non-alcoholic steatohepatitis (NASH), the severe form of NAFLD, is characterised by lobular inflammation and hepatocellular ballooning apart from steatosis that is accompanied by fibrosis progression. 3 Long-lasting NASH may progress to cirrhosis and hepatocellular carcinoma (HCC). 4 The prevalence of NAFLD/NASH increased dramatically in the last decades and is highly correlated to the rise of obesity and T2DM. 5 NAFLD is currently the most common aetiology for liver disease in Western countries. 6 Better understanding of the pathophysiology of NAFLD/NASH facilitated the development of therapies for these diseases. 7 Many novel therapeutics are currently under clinical development; however, the US Food and Drug Administration (FDA) has approved none so far. [7][8][9][10][11] A3 adenosine receptor (A3AR) is one of four receptors that mediate extracellular adenosine signalling. It is overexpressed in liver cells derived from inflammatory and tumour tissues, but not in normal liver cells. [12][13][14] Namodenoson is a synthetic highly selective A3AR agonist with robust anti-inflammatory and anticancer effects as demonstrated in experimental animal models of autoimmune hepatitis and in orthotopic and xenograft liver cancer models. 13,14 In two clinical studies in advanced HCC, namodenoson showed anti-tumour activity with an excellent safety profile. 15,16 The effect of namodenoson on liver inflammation and fibrosis was demonstrated in NAFLD/NASH preclinical models. 17 Namodenoson significantly decreased the NAFLD activity score in the STAM model. It also improved alanine aminotransferase (ALT), adiponectin, and leptin values, as well as ameliorated liver inflammation/fibrosis in the carbon tetrachloride model. 17 Mechanistic studies suggest that namodenoson exerts this effect by de-regulation of the phosphoinositide 3-kinase (PI3K)/NF-κB/Wnt/β-catenin signalling pathway. 17 Namodenoson also acts as a protective agent in models of liver ischaemia and partial hepatectomy. 12,14 The anti-NAFLD/NASH effect of namodenoson observed in the preclinical models alongside its hepatoprotective and anti-cancer effects prompted its clinical development as a treatment for NAFLD/ NASH since these patients are at high risk of progression to cirrhosis and HCC. This phase 2 randomised, placebo-controlled, dosefinding study investigated the efficacy and safety of namodenoson in NAFLD (with/without NASH).

| Study design and treatment
This multicentre randomised, double-blind, placebo-controlled study was conducted in three centres in Israel (Hadassah Hebrew University Medical Centre, Rabin Medical Centre, and the Holy Family Hospital). The study randomised patients with NAFLD and ALT ≥60 IU/L (1:1:1, using a central randomisation schedule generated by an independent biostatistician) to oral namodenoson 12.5 mg BD, 25 mg BD, or placebo BD for 12 weeks, and the total follow-up was 16 weeks. The randomisation was stratified by the presence/absence of T2DM. Patients were evaluated regularly for safety, and indicators of efficacy were measured at baseline and Week 12.
All relevant national regulatory authorities and local Ethics Committees/Institutional Review Boards approved the study. The study was conducted in accordance with the Declaration of Helsinki and written informed consent was obtained from all patients. All authors had access to the study data, and reviewed and approved the final manuscript. ClinicalTrials.gov #NCT02927314.

| Study participants
The study included patients aged ≥18 years with a diagnosis of NAFLD defined as hepatic steatosis ≥10% as determined by magnetic resonance imagining-determined proton-density fat-fraction (MRI-PDFF), and serum ALT ≥60 IU/L. Key inclusion criteria included ≥2 metabolic comorbidities out of (a) obesity defined as body mass index (BMI) ≥25 and ≤40 kg/m 2 or waist circumference >102 to <200 cm for men and >88 to <200 cm for women; (b) T2DM; (c) controlled hypertension; (d) hypertriglyceridemia defined as >150 mg/dL; and (e) reduced high-density lipoproteins cholesterol defined as <40 mg/dL in men or <50 mg/dL in women. Additionally, patients had to be with an acceptable hepatic metabolic/synthetic function (serum albumin ≥3.5 g/dL, international normalised ratio ≤1.4, and serum total bilirubin ≤2.0 mg/dL); absence of cirrhosis (FibroScan score of ≤F4 defined as liver stiffness measurement [LSM] of <12 kPa); absolute neutrophil count ≥1.0 × 10 9 /L; platelet count ≥100 × 10 9 /L; and serum creatinine ≤2.0 mg/dL. Key exclusion criteria included the presence of ascites; hepatic encephalopathy or other clinical evidence of cirrhosis; other active acute/chronic liver disease; familial dyslipidaemia; weight loss of >5% in the previous 6 months; bariatric surgery in the previous 5 years; type I diabetes; average daily alcohol intake >20 g/d for women and 30 g/d for men; haemoglobin A1c > 9.0%; treatment with vitamin E at ≥800-1000 IU daily; and treatment with certain anti-diabetic drugs (DPP-4 inhibitor, GLP-1 receptor agonists, pioglitazone, or SGLT2 inhibitors) unless dose and regimen have been stable for ≥3 months prior to screening.

| Assessments
The main efficacy endpoint involved serum ALT at Week 12. Other scores; controlled attenuation parameter (CAP) scores; FibroScanaspartate transaminase (FAST) scores, and body weight. Since liver biopsies were not performed, NASH presence was defined as having a FAST score >0.67. 18 Safety was also a main endpoint and was monitored through the assessment of adverse events (AEs) using the Common Terminology Criteria for Adverse Events (CTCAE) v4.0.
Changes from baseline in vital signs, laboratory parameters, and electrocardiograms were also assessed.

| Biomarkers
The peripheral blood expression of A3AR was measured at baseline and Week 12. A3AR mRNA expression in white blood cells was determined from blood collected to PAXgene RNA tubes (Qiagen), using the QuantiGene Plex 2.0 ® assay (Thermo Fisher). β-actin was used as a reference control, and the oligonucleotide probe sets were designed by Thermo Fisher. Luminescence from each specific probe set was captured by Glomax Multi (Promega). A3AR was expressed in units, where one unit was defined as the mean of A3AR expression in healthy subjects, as previously determined. 16

| Statistical analysis
Power calculation determined that assuming a standard deviation of 30% and a difference of 30% in percent change from baseline in serum ALT levels between either dose of namodenoson and placebo, 17 patients per group provide 80% power for a 2-sided test at a significance level of 0.05. Assuming a standardised between-treatment difference of 0.93, 20 patients/group provide 80% power for a 2sided test at level 0.05.
Efficacy analyses were performed on the intent-to-treat (ITT) population (all treated patients with ≥1 post-baseline efficacy assessment). Safety analysis was performed using the safety population (all patients who received at ≥1 dose of study medication). Descriptive statistics were used to summarise patient characteristics and safety.
Analyses of CFB for each of the variables were performed using ANCOVA by visit. Ad hoc analysis utilised t-tests for these analyses with the assumption of unequal variances. Ad hoc analysis of CFB in FAST score (within each arm) was performed on the ITT population using the sign test. All statistical tests were 2-sided and P ≤ 0.05 was considered statistically significant. Statistical analyses were conducted using SAS ® 9.4 (SAS Institute Inc). Two patients were withdrawn from the namodenoson 12.5 mg BD arm (one due to non-compliance and the other due to pregnancy of the patient's partner) and two from the 25 mg BD arm (one due to non-compliance and the other was lost to follow up) ( Figure 1). All 60 patients were included in the ITT and safety populations.

| Patient disposition and characteristics
Baseline demographics and disease characteristics were generally similar between the groups (Table 1). Overall, the mean (SD) age was 45 (12) years, the majority of patients were males (77%), and a quarter (25%) had T2DM. All patients except for one (in the placebo group) were White/Caucasian. At Screening, three patients (18.8%) in the 12.5 mg BD arm, four patients (28.6%) in the 25 mg BD arm, and none in the placebo arm had NASH, as defined in the current study (FAST score >0.67). BD arms, respectively) and slightly higher in the placebo arm (93.6%).

F I G U R E 1 Patient disposition
The mean duration of treatment was similar between the namodenoson 12.5 mg BD and the placebo arms (86.0 and 88.4 days, respectively).
One patient in the 25 mg BD was lost to follow up; after excluding him, the mean duration of treatment for the 25 mg BD was also similar at 81.1 days.  Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; BD, twice a day; MRI-PDFF, magnetic resonance imagining-determined proton-density fat-fraction; NAFLD, nonalcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; SD, standard deviation. a Data were missing for 5, 4, and 3 patients in the 12.5 mg, 25 mg, and placebo arms, respectively. CFB was also numerically higher in the 25 mg BD arm compared to placebo (mean, 220 ng/mL vs −78 ng/mL); however, the difference was not statistically significant (P = 0.216) ( Figure 2D). ALT: Normalisation rate over time at Week 12 compared to screening (from 50% to 31% and from 43% to 14%, respectively), in contrast to the placebo arm where the proportion of such patients increased in this timeframe (from 33% to 40%), although the differences between the treatment arms and the placebo arm were not statistically significant ( Figure 3B). Liver

| Safety
Namodenoson was well-tolerated at both doses. No patient withdrew due to toxicity. No drug-drug interactions were reported. No severe treatment-related adverse events (TEAEs) were reported and three patients (all in the 12.5 mg BD group) had moderate severity TEAEs (Table 2). In the namodenoson arms, three TEAEs (7.5%) were considered as possibly related to treatment, the remaining TEAEs were considered not-related to study treatment ( Table 2). The three TEAEs considered possibly related to study treatment included myalgia in the 12.5 mg BD arm, and one case each of muscular weakness and headache in the 25 mg BD arm (Table 2). No hepatotoxicity was observed and no deaths occurred during the study.  13,14 Our safety results are also consistent with findings from clinical studies investigating namodenoson in advanced HCC. 15 As in prior studies, no clinically meaningful drug-drug interactions were reported, which is key, as NAFLD/NASH patients typically receive additional therapies for the abovementioned prevalent comorbidities such as T2DM.

| D ISCUSS I ON
At present, no therapeutic has been FDA-approved for NAFLD/ NASH. The therapies that are currently under clinical development vary with respect to mechanism of action, and no other drug candidate targets A3AR. [8][9][10][11] Namodenoson is also different than some of the other investigated drugs as it is an orally administered drug (unlike the GLP-1 inhibitor liraglutide that is administered via subcutaneous injection 22 ).
Notably, since A3AR belongs to a family of Gi protein-associated receptors that were shown to undergo desensitisation/re-sensitisation in response to agonist treatment, 23 we examined A3AR expression levels before and after treatment. Consistent with our previous clinical study in HCC, 16 we found that A3AR expression remained stable after 12 weeks of treatment suggesting that A3AR is a valid target that is not desensitised by chronic exposure to namodenoson.
The study is limited by the relatively small number of patients in each study arm and the homogenous patient population (all patients except one were White). The study is also limited by the difference in the proportion of patients with FAST score >0.67 at randomisation and the paucity of data in some measurements (eg, adiponectin levels at Week 12). Another limitation is the short duration of active treatment (12 weeks), which impeded our ability to observe changes in liver stiffness (eg, changes observed in FAST likely stemmed from changes in AST levels). Also, in the current study, patients did not undergo post-treatment liver biopsy to determine the impact of the study drug on fibrosis or NASH. A longer-term study with a larger sample size, a more diverse patient population, and that includes post-treatment liver biopsy is required to fully assess the potential clinical utility of

AUTH O R S H I P
Guarantor of the article: PF.
Author contributions: RS and PF contributed to the study concept and design. RS, MB, AF, YM, MM, DH, WH, AI and NL were investigators, supervised study-related activities at their study sites, and contributed to data acquisition, analysis and interpretation of data.
ZH, MF, II, ABS, MHS and PF contributed to analysis and interpretation of data. All authors contributed to the drafting, critical revision for intellectual content, and final approval of the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.