Study participants
We enrolled male or female adult participants over the age of 18 with cirrhosis and PH. Full eligibility criteria are listed in Additional file 1. Main inclusion criteria included: clinically diagnosed or biopsy-proven liver cirrhosis of any aetiology; evidence of PH either on imaging or previous endoscopy; suspected HVPG ≥10 mmHg at baseline (if the baseline HVPG was subsequently found to be <10mmHg, the participant was withdrawn from the study). Main exclusion criteria included: pregnancy or breast-feeding; women of child-bearing potential not using highly effective methods of contraception; severe liver failure; history of variceal bleed within previous month; hepatocellular carcinoma or history of malignancy of any organ system (other than localised basal cell carcinoma of the skin); portal vein thrombosis; previous surgical shunt or TIPSS; current use of beta-blockers or nitrates, or any other drug therapy known to have an influence on portal pressure (diuretics were permitted provided patients had been on a stable dose for at least 30 days); history of active/recent drug or alcohol abuse; sitting systolic blood pressure <110 mmHg at screening visit or within 10 min prior to starting study drug infusion; significant arrhythmias, including prolonged QT interval; documented hypersensitivity to i.v. contrast agents and/or iodine; severe renal impairment; significant structural heart disease (including cardiomyopathy, valvular disease); major neurologic events, including cerebrovascular events, within previous month; clinical evidence of acute coronary syndrome currently or within previous month; pacemaker, cardiac resynchronisation device or implantable cardioverter-defibrillator in situ.
Data collection
This was a single-site study, undertaken at the Royal Infirmary of Edinburgh, Edinburgh, UK between 19th October 2017 and 15th August 2018.
Participants attended the RIE Clinical Research Facility for screening (visit 1) consisting of physical examination, blood tests (full blood count, coagulation and biochemistry), electrocardiogram (ECG), blood pressure measurement, and written informed consent. Randomisation was performed once it was known that the participant had passed screening, prior to the study visit.
On the study day (visit 2; ≤7 days after the screening visit), eligible participants attended for baseline haemodynamic measurements, following an overnight fast and the avoidance of caffeine for >8 h. After baseline evaluation and confirmation of HVPG ≥10 mmHg, participants received (in a double-blind fashion) either serelaxin or placebo. The haemodynamic measurements were repeated at specified time points. A peripheral blood sample was taken at baseline and after 2 h, processed, and stored for potential future analysis. After the post-treatment assessments, participants were observed for a recovery period of 4 h which included repeat physical examination, blood pressure, ECG measurement and routine laboratory blood tests. There was no follow-up visit. Participants were contacted by a member of the research team via telephone 24 h and again at 4 weeks after the study visit to collect information about potential AEs and concomitant medications.
Study design and randomisation
This study was a phase 2, double-blind, randomised controlled trial to investigate the effects of serelaxin on PH in patients with cirrhosis. The control group was used to maintain the blind and provide information to aid the design of future studies and no statistical comparison between treatment and control groups was planned.
Randomisation was carried out after it was confirmed that the participant had passed screening, prior to the study visit (visit 2). The randomisation service was carried out by the Edinburgh Clinical Trials Unit, allowing researchers and participants to remain blinded to treatment allocation. Random sequences of block sizes were generated by computer to achieve a 3:1 allocation ratio between serelaxin and placebo; there was no stratification to this allocation. Pharmacy prepared the appropriate treatment after randomisation.
Sample size
The primary efficacy endpoint was the decrease in fasting HVPG between baseline and 2 h post serelaxin treatment, targeting for a 20% reduction. The sample size calculation was based on a previous study in Edinburgh evaluating carvedilol13 and the data from the previous Novartis-sponsored serelaxin phase 2 study (ClinicalTrials.gov Identifier: NCT01640964). Assuming a mean baseline HVPG of 16.37 (SD=2.14) mmHg and post-baseline HVPG of 13.1 (SD=3.91) mmHg (20% decrease), the change from baseline in HVPG was estimated to be 3.3 (SD=4) mmHg. A sample size of 14 participants in the serelaxin group would provide 80% power to detect at least a 20% decrease from baseline in HVPG using a two-sided paired t-test with alpha level 0.05. A small number of placebo-treated patients were included in order to preserve double-blindness, not as a comparison group. Therefore, it was proposed that a total of 20 patients (15 serelaxin and 5 placebo) would be randomised in a 3:1 ratio.
Intervention
Recombinant human relaxin-2 (serelaxin (Novartis Pharmaceuticals, UK)) or placebo (20 mM sodium acetate buffer solution at pH 5.0 (Novartis Pharmaceuticals, UK)) were administered via peripheral i.v. infusion at two different infusion rates: 80 μg/kg/day for 60 min followed by 30 μg/kg/day for at least 60 min (until completion of the final HVPG/ICG measurements). This was achieved by a single infusion bag, prepared by the clinical trials pharmacist, with a uniform change in the administration rate. The placebo had an appearance identical to serelaxin to achieve blinding.
Study Endpoints
The primary endpoint was the change from baseline in fasting HVPG after 2 h serelaxin infusion. HVPG was measured as previously described.13 The procedure was performed after overnight fast and at the same time of day due to circadian variation in HVPG measurements. Prior to catheter insertion, participants were offered low dose (≤ 0.02mg/kg) midazolam to reduce any anxiety. A 7F venous introducer was inserted into the right femoral vein using the Seldinger technique under ultrasound guidance. A balloon-tipped catheter was then advanced into a hepatic vein using fluoroscopy. The free hepatic venous pressure (FHVP) was measured with the balloon deflated and floating freely in the hepatic vein close to its junction with the inferior vena cava (IVC). The wedged hepatic venous pressure (WHVP) was measured with the balloon inflated until the branch of hepatic vein was completely occluded. HVPG was obtained by subtracting the FHVP from the WHVP. All measurements were performed in triplicate and permanent tracings were printed, stored, and read blindly at the end of the study prior to the opening of the randomisation codes. HVPG was measured at baseline, then repeated after 60 min and 120 min of the IMP (either serelaxin or placebo). Baseline HVPG ≥ 10mmHg confirmed the presence of CSPH. If an HVPG < 10mmHg was obtained the study participant was withdrawn. The IVC pressure (IVCP) was measured at baseline and after the final HVPG measurement.
Secondary endpoints included: the change from baseline in fasting HVPG after 1 h serelaxin infusion; the change from baseline in fasting hepatic blood flow (HBF) after 2 h serelaxin infusion (measured from the concentration of indocyanine green (ICG) in the hepatic venous blood vs peripheral venous blood using the Fick Principle); the change from baseline in IVCP after 2 h serelaxin infusion; the change from baseline in cardiac index (CI) after 2 h serelaxin infusion; the change from baseline in systemic vascular resistance index (SVRI) after 2 h serelaxin infusion; the change from baseline in aortic pulse wave velocity after 2 h serelaxin infusion; safety and tolerability of serelaxin infusion (as assessed throughout the study by monitoring AEs, clinical laboratory blood tests, heart rate, blood pressure and ECG); change from baseline in exploratory blood biomarker measurements after 2 h serelaxin infusion (if a demonstrable effect on HVPG was observed).
The total HBF was calculated using the ICG constant infusion method14,15 and derived from measurements of ICG clearance and extraction. Baseline serum samples were taken prior to each ICG infusion. Thereafter, 10 mg of ICG (10 mL) was given as a slow i.v. bolus via a peripheral cannula, followed by an infusion of 0.2 mg/min (0.2 mL/min or 12 mL/h) by accurate infusion pump (Alaris Asena, Becton Dickinson, USA). After an equilibrium period of at least 40 min, samples were taken simultaneously from the right hepatic vein (via the catheter tip) and the femoral vein (via the side port of the introducer). Paired samples (hepatic and femoral) were taken in triplicate, 2 min apart in order to confirm equilibrium. The HBF was measured in this way both at baseline and after 120 min of IMP infusion.
Cardioscreen 1000 (Medis, Germany) was used for non-invasive measurement of cardiac output (CO = heart rate x stroke volume/1000, L/min), CI (L/min/m2) and SVRI (dyne · s · cm5 · m2) by the bio-impedence technique before and after 120 min of IMP infusion. Similarly, arterial function was measured using an Arteriograph device (TensioMed™, Hungary). Arterial stiffness and central haemodynamics were assessed by the application of an inflatable cuff to the upper arm. Aortic pulse wave velocity (APWV, m/s) was calculated as the distance the pulse wave travels in the aorta (as measured from the suprasternal notch to pubic bone) divided by the measured transit time.
Participants were monitored for 4 h after IMP discontinuation and removal of the catheter and introducer from the femoral vein. Vital signs were recorded every 30 min throughout the infusion and recovery periods, with regular ECG monitoring. Adverse events (AEs) were collected during visit 2 and by follow-up telephone calls at 24 h and 4 weeks. The severity, expectedness and causality of AEs in relation to the study medication were noted by the study team.
Statistical analysis
Analysis was per protocol. Summary statistics (n, mean, SD, median, min, max, Q1 and Q3) were generated over time for the baseline, post-baseline and change from baseline measurements for the primary endpoint in the serelaxin and placebo group. The geometric mean was calculated for the baseline value, post-baseline values, and for the ratio to the baseline values. Confidence intervals were calculated for both the arithmetic and geometric means. Paired t-tests were used to test the mean change from baseline measurements. The secondary endpoints were subjected to the same analysis as the HVPG.
The placebo control group was used to maintain the blind. We present the baseline to 2 h change in the same way as the primary outcome although as this has not been powered for, no direct statistical comparison was made between serelaxin and placebo.
All participants were analysed in the group to which they were originally assigned irrespective of the treatment received with the exception of AEs which are presented according to allocated treatment and also treatment received. For all analysis Unless otherwise specified statistical significance was taken to be p<0.05.
Any missing data as a consequence of the participant not having post-baseline measurement was not imputed and participants with missing post-baseline data were excluded from the analysis at that time point. The number of participants who withdrew during the course of the study is presented broken down by treatment allocation and presented with reasons for withdrawal where available.