Impact of age on NIS2+™ and other non-invasive blood tests for the evaluation of liver disease and detection of at-risk MASH.

Background & Aims: Robust performance of non-invasive tests (NITs) across ages is critical to assess liver disease among patients with metabolic dysfunction-associated liver disease (MASLD). We evaluated the impact of age on the performance of NIS2+™ vs. other NITs. Methods: An analysis cohort (N = 1,926) with biopsy-proven MASLD was selected among individuals screened for the phase III RESOLVE-IT clinical trial and divided into ≤45, 46-55, 56-64, and ≥65 years groups. To avoid potential confounding effects, a well-balanced cohort (n = 708; n = 177/age group) was obtained by applying a propensity score-matching algorithm to the analysis cohort. Baseline values of biomarkers and NITs were compared across age groups using one-way ANOVA, and the impact of age and histology were compared through three-way ANOVA. The impact of age on NIT performance for the detection of at-risk metabolic dysfunction-associated steatohepatitis (MASH; MASLD activity score [MAS] ≥4 and fibrosis stage [F] ≥2) was also evaluated. Results: Age did not affect the distributions of NIS2+™ and APRI (aspartate aminotransferase-to-platelet ratio index), but significantly (p <0.0001) impacted those of NFS (NAFLD fibrosis score), FIB-4 (Fibrosis-4 index), and Enhanced Liver Fibrosis (ELF™) score. NIS2+™ was the only NIT on which fibrosis and MAS exerted a moderate to large effect. While the impact of fibrosis on APRI was moderate, that of MAS was low. The impact of age on FIB-4 and NFS was larger than that of fibrosis. NIS2+™ exhibited the highest AUROC values for detecting at-risk MASH across age groups, with stable performances irrespective of cut-offs. Conclusions: NIS2+™ was not significantly impacted by age and was sensitive to both fibrosis and MAS grade, demonstrating a robust performance to rule in/out at-risk MASH with fixed cut-offs. Impact and Implications: While metabolic dysfunction-associated steatotic liver disease (MASLD) can affect individuals of all ages, patient age could represent an important confounding factor when interpreting non-invasive test (NIT) results, highlighting the need for reliable and efficient NITs that are not impacted by age and that could be interpreted with fixed cut-offs, irrespective of patient age. We report the impact of age on different well-established NITs - among those tested, only two panels, NIS2+™ and APRI, were not impacted by age and can be used and interpreted independently of patient age. NIS2+™ was also sensitive to both fibrosis and MAS, further confirming its efficiency for the detection of the composite endpoint of at-risk MASH and its potential as a valuable candidate for large-scale implementation in clinical practice and clinical trials.

Machine learning approaches to enhance diagnosis and staging of patients with MASLD using routinely available clinical information.

AIMS: Metabolic dysfunction Associated Steatotic Liver Disease (MASLD) outcomes such as MASH (metabolic dysfunction associated steatohepatitis), fibrosis and cirrhosis are ordinarily determined by resource-intensive and invasive biopsies. We aim to show that routine clinical tests offer sufficient information to predict these endpoints. METHODS: Using the LITMUS Metacohort derived from the European NAFLD Registry, the largest MASLD dataset in Europe, we create three combinations of features which vary in degree of procurement including a 19-variable feature set that are attained through a routine clinical appointment or blood test. This data was used to train predictive models using supervised machine learning (ML) algorithm XGBoost, alongside missing imputation technique MICE and class balancing algorithm SMOTE. Shapley Additive exPlanations (SHAP) were added to determine relative importance for each clinical variable. RESULTS: Analysing nine biopsy-derived MASLD outcomes of cohort size ranging between 5385 and 6673 subjects, we were able to predict individuals at training set AUCs ranging from 0.719-0.994, including classifying individuals who are At-Risk MASH at an AUC = 0.899. Using two further feature combinations of 26-variables and 35-variables, which included composite scores known to be good indicators for MASLD endpoints and advanced specialist tests, we found predictive performance did not sufficiently improve. We are also able to present local and global explanations for each ML model, offering clinicians interpretability without the expense of worsening predictive performance. CONCLUSIONS: This study developed a series of ML models of accuracy ranging from 71.9-99.4% using only easily extractable and readily available information in predicting MASLD outcomes which are usually determined through highly invasive means.

A Phase 3, Randomized, Controlled Trial of Resmetirom in NASH with Liver Fibrosis.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is a progressive liver disease with no approved treatment. Resmetirom is an oral, liver-directed, thyroid hormone receptor beta-selective agonist in development for the treatment of NASH with liver fibrosis. METHODS: We are conducting an ongoing phase 3 trial involving adults with biopsy-confirmed NASH and a fibrosis stage of F1B, F2, or F3 (stages range from F0 [no fibrosis] to F4 [cirrhosis]). Patients were randomly assigned in a 1:1:1 ratio to receive once-daily resmetirom at a dose of 80 mg or 100 mg or placebo. The two primary end points at week 52 were NASH resolution (including a reduction in the nonalcoholic fatty liver disease [NAFLD] activity score by ≥2 points; scores range from 0 to 8, with higher scores indicating more severe disease) with no worsening of fibrosis, and an improvement (reduction) in fibrosis by at least one stage with no worsening of the NAFLD activity score. RESULTS: Overall, 966 patients formed the primary analysis population (322 in the 80-mg resmetirom group, 323 in the 100-mg resmetirom group, and 321 in the placebo group). NASH resolution with no worsening of fibrosis was achieved in 25.9% of the patients in the 80-mg resmetirom group and 29.9% of those in the 100-mg resmetirom group, as compared with 9.7% of those in the placebo group (P<0.001 for both comparisons with placebo). Fibrosis improvement by at least one stage with no worsening of the NAFLD activity score was achieved in 24.2% of the patients in the 80-mg resmetirom group and 25.9% of those in the 100-mg resmetirom group, as compared with 14.2% of those in the placebo group (P<0.001 for both comparisons with placebo). The change in low-density lipoprotein cholesterol levels from baseline to week 24 was -13.6% in the 80-mg resmetirom group and -16.3% in the 100-mg resmetirom group, as compared with 0.1% in the placebo group (P<0.001 for both comparisons with placebo). Diarrhea and nausea were more frequent with resmetirom than with placebo. The incidence of serious adverse events was similar across trial groups: 10.9% in the 80-mg resmetirom group, 12.7% in the 100-mg resmetirom group, and 11.5% in the placebo group. CONCLUSIONS: Both the 80-mg dose and the 100-mg dose of resmetirom were superior to placebo with respect to NASH resolution and improvement in liver fibrosis by at least one stage. (Funded by Madrigal Pharmaceuticals; MAESTRO-NASH ClinicalTrials.gov number, NCT03900429.).

No correlation between MASLD and poor outcome of Atezolizumab-Bevacizumab therapy in patients with advanced HCC.

INTRODUCTION: It has been suggested that in patients with hepatocellular carcinoma (HCC) of metabolic aetiology, the efficacy of immunotherapy may be reduced. The aim was to investigate the impact of metabolic-associated steatotic liver disease (MASLD) and metabolic risk factors (MRF) on the outcomes of Atezolizumab-Bevacizumab (AtezoBev). METHODS: We collected data from 295 AtezoBev-treated patients, starting in 2020. MASLD was defined by the current/past presence of MRF, namely BMI ≥ 30 kg/m2, type 2 diabetes, arterial hypertension or dyslipidaemia and no other cause of liver disease (daily alcohol ≤30 g in males and ≤20 g in females). The influence of baseline characteristics on progression (PFS) and overall survival (OS) was assessed by uni/multivariate analysis using the Cox model. RESULTS: Risk factors for cirrhosis were viral infection in 47%, excessive alcohol consumption in 45% and MASLD in 13%. In the whole cohort, 27% had 1 MRF, 23% had 2 MRF, 15% had 3 MRF and 6% had 4 MRF. Median PFS and OS were 6.5 and 15.6 months, respectively, and similar in patients with or without MASLD in Log rank analysis. The number of MRF or MALSD was not associated with PFS or OS in the univariate analysis. Factors associated with PFS in multivariate analysis included ALBI grade 3 (HR = 1.60, p = .03), AFP (HR = 1.01, p = .01) and metastasis (HR = 1.77, p < .001). During follow-up, 10% of patients experienced immune-related adverse events, with age and female gender, but not MRF or MASLD, as independent predictors. CONCLUSION: Our study suggests that the presence of MASLD or the number of MRF did not lead to worse outcomes in advanced HCC patients treated with AtezoBev.

Artificial intelligence-assisted digital pathology for non-alcoholic steatohepatitis: current status and future directions.

The worldwide prevalence of non-alcoholic steatohepatitis (NASH) is increasing, causing a significant medical burden, but no approved therapeutics are currently available. NASH drug development requires histological analysis of liver biopsies by expert pathologists for trial enrolment and efficacy assessment, which can be hindered by multiple issues including sample heterogeneity, inter-reader and intra-reader variability, and ordinal scoring systems. Consequently, there is a high unmet need for accurate, reproducible, quantitative, and automated methods to assist pathologists with histological analysis to improve the precision around treatment and efficacy assessment. Digital pathology (DP) workflows in combination with artificial intelligence (AI) have been established in other areas of medicine and are being actively investigated in NASH to assist pathologists in the evaluation and scoring of NASH histology. DP/AI models can be used to automatically detect, localise, quantify, and score histological parameters and have the potential to reduce the impact of scoring variability in NASH clinical trials. This narrative review provides an overview of DP/AI tools in development for NASH, highlights key regulatory considerations, and discusses how these advances may impact the future of NASH clinical management and drug development. This should be a high priority in the NASH field, particularly to improve the development of safe and effective therapeutics.

NIS2+TM as a screening tool to optimize patient selection in metabolic dysfunction-associated steatohepatitis clinical trials.

BACKGROUND & AIMS: Strategies to reduce liver biopsy (LB) screen failures through better patient selection are needed for clinical trials. Standard fibrosis biomarkers were not derived to detect "at-risk" metabolic dysfunction-associated steatohepatitis (MASH; MASH with metabolic dysfunction-associated steatotic liver disease score ≥4 and fibrosis stage ≥2). We compared the performance of screening pathways that incorporate NIS2+™, an optimized version of the blood-based NIS4® technology designed to identify at-risk MASH, with those incorporating fibrosis (FIB)-4 within the RESOLVE-IT clinical trial (NCT02704403), aiming for optimized selection of patients for LB. METHODS: A retrospective simulation analysis was conducted in the RESOLVE-IT screening pathway (RSP) cohort. LB failure rate (LBFR), number of patients needed to screen, and overall cost estimations of different pathways were calculated for a range of NIS2+™ and FIB-4 cut-offs and compared with those of the RSP, which relied on investigators' local practices. An analysis of potential recruitment bias based on histology, sex, age, or comorbidities was performed. RESULTS: The analysis cohort included 1,929 patients, 765 (40%) with at-risk MASH. The NIS2+™ pathway resulted in a significantly lower LBFR (39%) compared with the FIB-4 pathway (58%) or the RSP (60%) when using cost-optimized cut-offs (NIS2+™, 0.53; FIB-4, 0.58). For every 1,000 inclusions, NIS2+™ significantly reduced unnecessary LBs (632 vs. 1,522; -58%) and screening costs (US$12.7 million vs. US$15.0 million) vs. the RSP, while the number of patients needed to screen increased moderately (3,220 to 4,033). NIS2+™ alone is better than FIB-4 alone or combined with FIB-4. CONCLUSIONS: This analysis demonstrated that patient selection for LB using NIS2+™ significantly reduced unnecessary biopsies and screening costs, which could greatly improve the feasibility of MASH clinical trials. IMPACT AND IMPLICATIONS: Simple and accurate non-invasive strategies to optimize the selection of patients who should be referred for liver biopsy for inclusion in MASH clinical trials is critical to reduce the high liver biopsy failure rates. While the use of the Fibrosis-4 index alone did not lead to a significant improvement of the screening process, selecting patients using NIS2+™, a recently developed optimization of the NIS4® technology for the detection of at-risk MASH, showed improved performance by simultaneously reducing liver biopsy failure rates and the overall cost of the trial, while maintaining the number of patients needed to screen at a manageable level and not generating any bias in included patients' characteristics. This makes NIS2+™ an accurate and reliable screening tool that could improve the recruitment of patients in future MASH clinical trials, and would lead to increased patient comfort and security, ensuring timely and cost-efficient trial completion.

A randomized, double-blind, placebo-controlled trial of aldafermin in patients with NASH and compensated cirrhosis.

BACKGROUND AND AIMS: Aldafermin, an engineered analog of the human hormone FGF19, improves liver histology in patients with noncirrhotic NASH; however, its efficacy and safety in compensated cirrhosis is unknown. No drug has yet to demonstrate benefit in the compensated NASH population. APPROACH AND RESULTS: In this multicenter, double-blind, placebo-controlled, phase 2b trial, 160 patients with compensated NASH cirrhosis were randomized to aldafermin 0.3 mg (n = 7), 1 mg (n = 42), 3 mg (n = 55), or placebo (n = 56) for 48 weeks. The 0.3 mg group was discontinued to limit exposure to suboptimal doses. The primary end point was a change in Enhanced Liver Fibrosis from baseline to week 48. The analyses were performed in the intention-to-treat population. At week 48, the least-squares mean difference in the change in Enhanced Liver Fibrosis was -0.5 (95% CI, -0.7 to -0.2; p = 0.0003) between the 3 mg group and the placebo group. 15%, 21%, and 23% of patients in the placebo, 1 mg, and 3 mg group, respectively, achieved fibrosis improvement ≥ 1 stage; and 13%, 16%, and 20% achieved fibrosis improvement ≥ 1 stage without NASH worsening. Improvement in alanine aminotransferase, aspartate aminotransferase, neoepitope-specific N-terminal pro-peptide of type III collagen, and liver stiffness favored aldefermin groups over placebo. Diarrhea was the most frequent adverse event, occurring at 26% and 40% in the 1 mg and 3 mg groups, respectively, compared to 18% in the placebo group. Overall, 0%, 2%, and 9% of patients in the placebo, 1 mg, and 3 mg group, respectively, discontinued due to treatment-related adverse events. CONCLUSIONS: Aldafermin 3 mg resulted in a significant reduction in Enhanced Liver Fibrosis in patients with compensated NASH cirrhosis.

A multisociety Delphi consensus statement on new fatty liver disease nomenclature.

The principal limitations of the terms NAFLD and NASH are the reliance on exclusionary confounder terms and the use of potentially stigmatising language. This study set out to determine if content experts and patient advocates were in favor of a change in nomenclature and/or definition. A modified Delphi process was led by three large pan-national liver associations. The consensus was defined a priori as a supermajority (67%) vote. An independent committee of experts external to the nomenclature process made the final recommendation on the acronym and its diagnostic criteria. A total of 236 panelists from 56 countries participated in 4 online surveys and 2 hybrid meetings. Response rates across the 4 survey rounds were 87%, 83%, 83%, and 78%, respectively. Seventy-four percent of respondents felt that the current nomenclature was sufficiently flawed to consider a name change. The terms "nonalcoholic" and "fatty" were felt to be stigmatising by 61% and 66% of respondents, respectively. Steatotic liver disease was chosen as an overarching term to encompass the various aetiologies of steatosis. The term steatohepatitis was felt to be an important pathophysiological concept that should be retained. The name chosen to replace NAFLD was metabolic dysfunction-associated steatotic liver disease. There was consensus to change the definition to include the presence of at least 1 of 5 cardiometabolic risk factors. Those with no metabolic parameters and no known cause were deemed to have cryptogenic steatotic liver disease. A new category, outside pure metabolic dysfunction-associated steatotic liver disease, termed metabolic and alcohol related/associated liver disease (MetALD), was selected to describe those with metabolic dysfunction-associated steatotic liver disease, who consume greater amounts of alcohol per week (140-350 g/wk and 210-420 g/wk for females and males, respectively). The new nomenclature and diagnostic criteria are widely supported and nonstigmatising, and can improve awareness and patient identification.

Design of the phase 3 MAESTRO clinical program to evaluate resmetirom for the treatment of nonalcoholic steatohepatitis.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is a progressive form of non-alcoholic fatty liver disease (NAFLD) associated with steatosis, hepatocellular injury, inflammation and fibrosis. In a Phase 2 trial in adults with NASH (NCT02912260), resmetirom, an orally administered, liver-targeted thyroid hormone receptor-ß selective agonist, significantly reduced hepatic fat (via imaging) and resolved NASH without worsening fibrosis (via liver biopsy) in a significant number of patients compared with placebo. AIMS: To present the design of the Phase 3 MAESTRO clinical programme evaluating resmetirom for treatment of NASH (MAESTRO-NAFLD-1 [NCT04197479], MAESTRO-NAFLD-OLE [NCT04951219], MAESTRO-NASH [NCT03900429], MAESTRO-NASH-OUTCOMES [NCT05500222]). METHODS: MAESTRO-NASH is a pivotal serial biopsy trial in up to 2000 adults with biopsy-confirmed at-risk NASH. Patients are randomised to a once-daily oral placebo, 80 mg resmetirom, or 100 mg resmetirom. Liver biopsies are conducted at screening, week 52 and month 54. MAESTRO-NAFLD-1 is a 52-week safety trial in ~1400 adults with NAFLD/presumed NASH (based on non-invasive testing); ~700 patients from MAESTRO-NAFLD-1 are enrolled in MAESTRO-NAFLD-OLE, a 52-week active treatment extension to further evaluate safety. MAESTRO-NASH-OUTCOMES is enrolling 700 adults with well-compensated NASH cirrhosis to evaluate the potential for resmetirom to slow progression to hepatic decompensation events. Non-invasive tests (biomarkers, imaging) are assessed longitudinally throughout, in addition to validated patient-reported outcomes. CONCLUSION: The MAESTRO clinical programme was designed in conjunction with regulatory authorities to support approval of resmetirom for treatment of NASH. The surrogate endpoints, based on week 52 liver biopsy, serum biomarkers and imaging, are confirmed by long-term clinical liver-related outcomes in MAESTRO-NASH (month 54) and MAESTRO-NASH-OUTCOMES (time to event).

Utility of pathologist panels for achieving consensus in NASH histologic scoring in clinical trials: Data from a phase 3 study.

BACKGROUND: Liver histopathologic assessment is the accepted surrogate endpoint in NASH trials; however, the scoring of NASH Clinical Research Network (CRN) histologic parameters is limited by intraobserver and interobserver variability. We designed a consensus panel approach to minimize variability when using this scoring system. We assessed agreement between readers, estimated linear weighted kappas between 2 panels, compared them with published pairwise kappa estimates, and addressed how agreement or disagreement might impact the precision and validity of the surrogate efficacy endpoint in NASH trials. METHODS: Two panels, each comprising 3 liver fellowship-trained pathologists who underwent NASH histology training, independently evaluated scanned whole slide images, scoring fibrosis, inflammation, hepatocyte ballooning, and steatosis from baseline and month 18 biopsies for 100 patients from the precirrhotic NASH study REGENERATE. The consensus score for each parameter was defined as agreement by ≥2 pathologists. If consensus was not reached, all 3 pathologists read the slide jointly to achieve a consensus score. RESULTS: Between the 2 panels, the consensus was 97%-99% for steatosis, 91%-93% for fibrosis, 88%-92% for hepatocyte ballooning, and 84%-91% for inflammation. Linear weighted kappa scores between panels were similar to published NASH CRN values. CONCLUSIONS: A panel of 3 trained pathologists independently scoring 4 NASH CRN histology parameters produced high consensus rates. Interpanel kappa values were comparable to NASH CRN metrics, supporting the accuracy and reproducibility of this method. The high concordance for fibrosis scoring was reassuring, as fibrosis is predictive of liver-specific outcomes and all-cause mortality.

FibroScan-aspartate aminotransferase (FAST) score for monitoring histological improvement in non-alcoholic steatohepatitis activity during semaglutide treatment: post-hoc analysis of a randomised, double-blind, placebo-controlled, phase 2b trial.

Background: Currently, assessment of candidate pharmacotherapies in patients with non-alcoholic steatohepatitis (NASH) involves invasive liver biopsy. Non-invasive scores, such as the FibroScan-aspartate aminotransferase (FAST) score, are used to identify candidates for therapy, but their ability to assess disease progression or treatment effect is unknown. We aimed to assess the association between FAST score and histological endpoints. Methods: We conducted a post-hoc analysis using data from a prior randomised, double-blind, placebo-controlled, phase 2b trial at 143 sites across 16 countries. Patients (aged 18-75 years) with biopsy-confirmed NASH, fibrosis stage 1-3, and a Non-alcoholic fatty liver disease Activity Score (NAS) ≥4 were enrolled between January 2017 and September 2018, and randomly assigned to receive once-daily subcutaneous semaglutide 0.1, 0.2, or 0.4 mg or placebo for 72 weeks. A subgroup analysis of patients with FAST score and histological data in the pooled semaglutide treatment and placebo arms at baseline and week 72 was performed. The original trial is registered at ClinicalTrials.gov, NCT02970942. Findings: A total of 122 patients were included in this post-hoc analysis (93 received semaglutide and 29 received placebo). FAST score reduction was associated with achieving the primary endpoint of NASH resolution without worsening of fibrosis in the pooled semaglutide group (area under the receiver operating curve 0.69; 95% confidence interval [CI] 0.58, 0.81). Mean FAST score reduction from baseline to week 72 was greatest in patients who met the primary endpoint vs those who did not in both the semaglutide (-0.40 [95% CI -0.84, 0.04] vs -0.22 [95% CI -0.74, 0.30] points; p = 0.002) and placebo groups (-0.25 [95% CI -0.72, 0.23] vs 0.00 [95% CI -0.50, 0.50] points; p = 0.047). Similarly, mean reductions in FAST score at week 72 were greater in those with NAS improvement vs those without in the semaglutide and placebo groups (≥1 point, -0.36 [95% CI -0.82, 0.11] vs -0.08 [95% CI -0.53, 0.38] points [p < 0.001] and -0.25 [95% CI -0.64, 0.14] vs -0.06 [95% CI -0.40, 0.53] points [p = 0.001]; ≥2 points, -0.40 [95% CI -0.86, 0.06] vs -0.14 [95% CI -0.56, 0.28] points [p < 0.001] and -0.29 [95% CI -0.67, 0.09] vs -0.05 [95% CI -0.40, 0.50] points; [p < 0.001]). A FAST score reduction of more than 0.22 points after semaglutide treatment was associated with meeting the primary endpoint (sensitivity 78%; specificity 60%; positive likelihood ratio 1.26; negative likelihood ratio 0.25; odds ratio 4.93). Interpretation: The potential of the FAST score as a non-invasive monitoring tool to identify histological changes following treatment requires further evaluation and validation. Funding: Novo Nordisk A/S.

Artificial intelligence scoring of liver biopsies in a phase II trial of semaglutide in nonalcoholic steatohepatitis.

BACKGROUND AND AIMS: Artificial intelligence-powered digital pathology offers the potential to quantify histological findings in a reproducible way. This analysis compares the evaluation of histological features of NASH between pathologists and a machine-learning (ML) pathology model. APPROACH AND RESULTS: This post hoc analysis included data from a subset of patients (n=251) with biopsy-confirmed NASH and fibrosis stage F1-F3 from a 72-week randomized placebo-controlled trial of once-daily subcutaneous semaglutide 0.1, 0.2, or 0.4 mg (NCT02970942). Biopsies at baseline and week 72 were read by 2 pathologists. Digitized biopsy slides were evaluated by PathAI's NASH ML models to quantify changes in fibrosis, steatosis, inflammation, and hepatocyte ballooning using categorical assessments and continuous scores. Pathologist and ML-derived categorical assessments detected a significantly greater percentage of patients achieving the primary endpoint of NASH resolution without worsening of fibrosis with semaglutide 0.4 mg versus placebo (pathologist 58.5% vs. 22.0%, p < 0.0001; ML 36.9% vs. 11.9%; p =0.0015). Both methods detected a higher but nonsignificant percentage of patients on semaglutide 0.4 mg versus placebo achieving the secondary endpoint of liver fibrosis improvement without NASH worsening. ML continuous scores detected significant treatment-induced responses in histological features, including a quantitative reduction in fibrosis with semaglutide 0.4 mg versus placebo ( p =0.0099) that could not be detected using pathologist or ML categorical assessment. CONCLUSIONS: ML categorical assessments reproduced pathologists' results of histological improvement with semaglutide for steatosis and disease activity. ML-based continuous scores demonstrated an antifibrotic effect not measured by conventional histopathology.

A randomised Phase IIa trial of amine oxidase copper-containing 3 (AOC3) inhibitor BI 1467335 in adults with non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is a progressive, inflammatory liver disease with no approved pharmacological treatment. This Phase IIa, double-blind, placebo-controlled, multicentre trial (ClinicalTrials.gov: NCT03166735) investigated pharmacodynamics and safety of BI 1467335, an amine oxidase copper-containing 3 (AOC3) inhibitor, in adults with NASH from Europe and North America. Participants from 44 centres across the US, Germany, Spain, Belgium, the UK, Netherlands, Canada, France and Ireland were randomised (2:1:1:1:2; 27 July 2017 to 14 June 2019) to daily oral BI 1467335 1 mg (n = 16), 3 mg (n = 16), 6 mg (n = 17), 10 mg (n = 32) or placebo (n = 32) for 12 weeks, with follow-up to Week 16. Primary endpoint was AOC3 activity relative to baseline (%), 24 hours post-dose after 12 weeks' treatment. Secondary biomarker endpoints included changes from baseline at Week 12 in alanine aminotransferase (ALT) and caspase-cleaved cytokeratin 18 (CK-18 caspase). Mean AOC3 activities relative to baseline at Week 12: 90.4% (placebo; n = 32), 26.5% (1 mg; n = 16), 10.4% (3 mg; n = 16), 5.0% (6 mg; n = 16), 3.3% (10 mg; n = 32). These changes indicated that BI 1467335 dose-dependently inhibited AOC3 activity; ≥3 mg doses achieved >80% inhibition ( < 20% activity) at Week 4. At Week 12 following doses of BI 1467335 ≥ 3 mg, ALT and CK-18 caspase decreased dose-dependently. All tested BI 1467335 doses were well tolerated, with no clinically relevant treatment-emergent safety signals. BI 1467335 strongly inhibited AOC3 in participants with NASH, with doses ≥3 mg dose-dependently reducing the levels of liver injury biomarkers, ALT and CK-18. This trial was registered with ClinicalTrials.gov (NCT03166735) and the European Union Drug Regulating Authorities Clinical Trials Database (EudraCT 2016-000499-83).