Study part one completed

In a randomized, doubleblind and placebo-controlled trial we assess both clinical and cellular effects of atorvastatin in patients with liver cirrhosis.

162 participants will be allocated to atorvastatin 10-20 mg or placebo for 18 months. Clinical outcomes of survival, hospitalizations and safety will be evaluated. Also, the trial will investigate cellular functions in the liver by mass spectrometry proteomics, and single cell transcriptomics as well as exploring atorvastatin effects on different fenotypes by metagenomics.

Introduction Several studies have demonstrated the beneficial effects of statins in vascular and heart disease. Statins have antithrombotic effects, decrease oxidative stress and inflammation at the vessel wall, and improve endothelial dysfunction by increasing Nitric Oxide (NO) production in endothelial cells.

Statins may also inhibit fibrogenesis in cirrhotic rats. In recent years, a series of pilot studies have assessed the effects of simvastatin on portal hypertension and risk of variceal bleeding.

Only a few studies have evaluated the efficacy of statins in cirrhosis of mixed etiology and decompensated cirrhosis. High quality clinical trials have focused on the hemodynamic effects of simvastatin on portal hypertension. Evidence supporting the use of statins in a real-world clinical setting, and data on the effects on inflammation and generation of fibrosis in the liver in humans is in high demand.

Study setting The trial will take place in university hospitals with tertiary referral from other hospitals, departments and general practice. Patients referred to the outpatient clinics or hospitalized in the Gastro Unit, Amager Hvidovre Hospital (AHH) and Department of Hepatology and Gastroenterology, Aarhus University Hospital (AUH), all Denmark are eligible for inclusion.

Study Part One

After enrolment of the first 48 participants an evaluation of the trial feasibility will performed on the following parameters:

i) recruitment rates, ii) allocation of adequate resources to the trial, iii) safety for the participants.

Study Part One will deliver data to conduct a pilot study on the exploratory endpoints of inflammation, proteomics and metabolomics.

The substudy will apply the following methodology:

Initial analyses of biological material are performed blinded based on studyID, without revealing allocation to atorvastatin or placebo.

Comparison between groups are performed by blinded allocation, meaning that analysis are performed as: group A versus group B, without prior knowledge of which group received the active drug.

Scientific evaluation and clinical translation of study results and data are only performed after revelation of which group received the active drug.

Study Part Two

If the steering committee find all three conditions: recruitment, resources and safety adequate, the trial will continue into Part Two, with enrolment up to 162 participants as required to balance the clinical endpoints.

Criteria for discontinuation of intervention:

Discontinuation of trial drug will be performed in case of:

• The participant withdraws informed consent.
• Participants reach a primary endpoint (death or liver transplantation).
• The participant experiences serious adverse events that cause a risk to the participant's life or viability.
• The participant develops toxicity, intolerance or allergy associated with intervention medication.
• The trial participant is admitted to hospital for other morbidity that is incompatible with further treatment with atorvastatin.
• Blinding is revealed.
• A trial participant will be classified as dropped out of the study if the participant has ingested less than 55 % of atorvastatin or placebo at days 180.

Statistical methods Data will be analyzed as repeated measures Analysis of variance (ANOVA). Independent groups will be compared using unpaired T-test or non-parametric tests depending on normality distribution of data. Survival analysis will be performed by Cox-regression analyses.

Statistical analyses will be performed as per protocol and as intention to treat analyses.

Missing data will not be replaced but left blank in statistical analysis. For intention to treat analyses on repeated measures, the last available value will be carried forward to last value.

Sample Size

Prior data may indicate that the median survival time on the control treatment is 35 months. If the true hazard ratio (relative risk) of control subjects relative to experimental subjects is 0.52, we will need to study 70 experimental subjects and 70 control subjects to reject the null hypothesis that the experimental and control survival curves are equal with probability (power) 0.85. The Type I error probability associated with the test of this null hypothesis is 0.05.

With an expected drop-out rate of fifteen percent, we plan to enroll in total 162 participants in Study Part One and Two, to reach the clinical endpoint of survival.

Auditing The trial will be approved by the Danish Authorities of Health and Medicine, and may be subject to auditing by this institution, independent of the sponsor and investigators. Investigator allows direct access to source data and documents (including patient records) used in the trial for monitoring, auditing and/or inspection from GCP-units and The Danish Medicines Agency.

Bio bank A biobank containing blood and liver tissue is established for the trial. The bio bank contains both samples for planned analyses, and in case of excess material, samples will be stored in a bio bank for future research.

All investigations and results derived from the bio bank for future research will serve the purpose of improving the life and health of patients with liver cirrhosis.

Research ethics approval Registration by the Danish Data protection Agency was conducted April 9, 2019. Application for approval at the Scientific Ethics Committees of the Capital Region of Denmark was approved by: September 3, 2019, with ammendments approved by: January 20, 2020 (protocol version 2) and August 7, 2020 (protocol verison 3).

Legal assistance Legal advice for contracts and agreements is provided by Centre for Innovation and Research, The Capital Region of Denmark c/o Cobis, Ole Maaløes vej 3, DK-2200 Copenhagen N.

Declaration of interests Authors of this protocol have no financial interests (holds no stakes, no ownership, and do not provide academic services) to any medical companies involved in producing or marketing statins.

Insurance Patients participating in this trial will be insured through the national patient insurance. Trial participants are informed of this relation in written information of trial participant's rights. Sponsor and investigators are covered by statutory insurance from AHH and AUH.

Dissemination policy This study will support an open access policy and aims to prepare all collected data to publication in anonymized form in an open access database after dissemination of the pre-defined trial outcomes.

Inclusion Criteria:

• Patients in the age of 18 to 80 years
• Patients with liver cirrhosis, diagnosed by liver biopsy or ultrasound or CT scan of the liver and clinical biochemistry compatible with cirrhosis within the past 3 months.
• In women, documented absence of pregnancy and unless in menopause commitment to use adequate contraception.
• Clinically significant portal hypertension with a hepatic venous pressure gradient measured by liver vein catheterization >10 mmHg.
• Ability to read and understand project information in Danish and give written, informed consent.

Exclusion Criteria:

• People treated with statins within the last year.
• People with liver cirrhosis, with a clinically verified infection (standard biochemistry, culture) within the last four weeks.
• Pregnancy or lactation.
• Hepatocellular carcinoma
• HIV infection and treatment with protease inhibitors
• People in whom the clinician and investigators may have reason to doubt compliance to trial medication
• Clinical and biochemical signs of hepato-renal syndrome defined by current guidelines (EASL) within the last 14 days
• A MELD score above 23, or Child-Pugh score higher than 13.
• Hepatic encephalopathy grade 2 or higher

IPD and supporting information are planned to be shared via data repository sites, such as UCPH data repository or other public data repository.

Data will be stored for up to 10 years after completion of the trial, depending on approval from Danish Health Authorities, Scientific Ethics Comittee and the Danish Data Protection Regulation.

upon request

Allocation and randomisation is blinded. Participants are only identified by randomisation number (no group names) Allocation ratio is 1:1 All personnel and participants are blinded through the study period. All outcome assessors are blinded to treatment, and initial data analysis is performed blinded.