Serelaxin as a potential treatment for renal dysfunction in cirrhosis: Preclinical evaluation and results of a randomized phase 2 trial

<div><p>Background</p><p>Chronic liver scarring from any cause leads to cirrhosis, portal hypertension, and a progressive decline in renal blood flow and renal function. Extreme renal vasoconstriction characterizes hepatorenal syndrome, a functional and potentially reversible form of acute kidney injury in patients with advanced cirrhosis, but current therapy with systemic vasoconstrictors is ineffective in a substantial proportion of patients and is limited by ischemic adverse events. Serelaxin (recombinant human relaxin-2) is a peptide molecule with anti-fibrotic and vasoprotective properties that binds to relaxin family peptide receptor-1 (RXFP1) and has been shown to increase renal perfusion in healthy human volunteers. We hypothesized that serelaxin could ameliorate renal vasoconstriction and renal dysfunction in patients with cirrhosis and portal hypertension.</p><p>Methods and findings</p><p>To establish preclinical proof of concept, we developed two independent rat models of cirrhosis that were characterized by progressive reduction in renal blood flow and glomerular filtration rate and showed evidence of renal endothelial dysfunction. We then set out to further explore and validate our hypothesis in a phase 2 randomized open-label parallel-group study in male and female patients with alcohol-related cirrhosis and portal hypertension. Forty patients were randomized 1:1 to treatment with serelaxin intravenous (i.v.) infusion (for 60 min at 80 μg/kg/d and then 60 min at 30 μg/kg/d) or terlipressin (single 2-mg i.v. bolus), and the regional hemodynamic effects were quantified by phase contrast magnetic resonance angiography at baseline and after 120 min. The primary endpoint was the change from baseline in total renal artery blood flow.</p><p>Therapeutic targeting of renal vasoconstriction with serelaxin in the rat models increased kidney perfusion, oxygenation, and function through reduction in renal vascular resistance, reversal of endothelial dysfunction, and increased activation of the AKT/eNOS/NO signaling pathway in the kidney. In the randomized clinical study, infusion of serelaxin for 120 min increased total renal arterial blood flow by 65% (95% CI 40%, 95%; <i>p <</i> 0.001) from baseline. Administration of serelaxin was safe and well tolerated, with no detrimental effect on systemic blood pressure or hepatic perfusion. The clinical study’s main limitations were the relatively small sample size and stable, well-compensated population.</p><p>Conclusions</p><p>Our mechanistic findings in rat models and exploratory study in human cirrhosis suggest the therapeutic potential of selective renal vasodilation using serelaxin as a new treatment for renal dysfunction in cirrhosis, although further validation in patients with more advanced cirrhosis and renal dysfunction is required.</p><p>Trial registration</p><p>ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT01640964" target="_blank">NCT01640964</a></p></div

Breath Biopsy® to Identify Exhaled Volatile Organic Compounds Biomarkers for Liver Cirrhosis Detection.

BACKGROUND AND AIMS: The prevalence of chronic liver disease in adults exceeds 30% in some countries and there is significant interest in developing tests and treatments to help control disease progression and reduce healthcare burden. Breath is a rich sampling matrix that offers non-invasive solutions suitable for early-stage detection and disease monitoring. Having previously investigated targeted analysis of a single biomarker, here we investigated a multiparametric approach to breath testing that would provide more robust and reliable results for clinical use. METHODS: To identify candidate biomarkers we compared 46 breath samples from cirrhosis patients and 42 from controls. Collection and analysis used Breath Biopsy OMNI™, maximizing signal and contrast to background to provide high confidence biomarker detection based upon gas chromatography mass spectrometry (GC-MS). Blank samples were also analyzed to provide detailed information on background volatile organic compounds (VOCs) levels. RESULTS: A set of 29 breath VOCs differed significantly between cirrhosis and controls. A classification model based on these VOCs had an area under the curve (AUC) of 0.95±0.04 in cross-validated test sets. The seven best performing VOCs were sufficient to maximize classification performance. A subset of 11 VOCs was correlated with blood metrics of liver function (bilirubin, albumin, prothrombin time) and separated patients by cirrhosis severity using principal component analysis. CONCLUSIONS: A set of seven VOCs consisting of previously reported and novel candidates show promise as a panel for liver disease detection and monitoring, showing correlation to disease severity and serum biomarkers at late stage

Effect of acute serelaxin treatment on renal blood flow and tissue oxygenation in CCl₄ cirrhotic rats.

<p>Renal blood flow (RBF, A) and mean arterial pressure (MAP, B) responses to acute i.v. serelaxin (4 μg) or vehicle in 16-wk CCl₄ rats (<i>n =</i> 5–7). Measurement of velocity time integral (C) and renal resistive index (D) following acute i.v. serelaxin (4 μg) or vehicle (<i>n =</i> 6–8). Deoxygenated hemoglobin levels (R2*) in renal medulla in 8-wk (E) and 16-wk (F) CCl₄ rats at baseline, 30 min, and 60 min following acute i.v. serelaxin (4 μg) or vehicle (<i>n =</i> 5–8). Data presented as mean ± standard error of the mean, analyzed by two-way ANOVA (*<i>p <</i> 0.05; **<i>p <</i> 0.01; ***<i>p <</i> 0.001; NS, not significant) with post hoc Bonferroni correction to compare individual CCl₄ time points with respective vehicle controls (^#<i>p <</i> 0.05; ^##<i>p <</i> 0.01; ^###<i>p <</i> 0.001).</p

Baseline demographic and clinical characteristics of study participants at screening.

<p>Baseline demographic and clinical characteristics of study participants at screening.</p

Effect of serelaxin infusion on pharmacokinetics and plasma biomarkers in patients with cirrhosis and portal hypertension.

<p>Serum serelaxin concentration measured by ELISA pre-dose (0 min), at 60 min and 120 min post-initiation of serelaxin infusion, in recovery period (~60 min after cessation of serelaxin), and at 4-wk follow-up visit (A). Data presented as mean ± standard deviation (<i>n =</i> 20). Plasma nitrate (B), endothelin-1 (ET-1) (C), and matrix metalloproteinase-9 (MMP-9) (D) measured by ELISA pre-dose (0 min) and at 120 min after initiation of serelaxin infusion. Data presented as geometric mean ± 95% CI (<i>n =</i> 20). NS, not significant.</p

Effect of acute serelaxin treatment on renal blood flow and tissue oxygenation in CCl₄ cirrhotic rats.

<p>Renal blood flow (RBF, A) and mean arterial pressure (MAP, B) responses to acute i.v. serelaxin (4 μg) or vehicle in 16-wk CCl₄ rats (<i>n =</i> 5–7). Measurement of velocity time integral (C) and renal resistive index (D) following acute i.v. serelaxin (4 μg) or vehicle (<i>n =</i> 6–8). Deoxygenated hemoglobin levels (R2*) in renal medulla in 8-wk (E) and 16-wk (F) CCl₄ rats at baseline, 30 min, and 60 min following acute i.v. serelaxin (4 μg) or vehicle (<i>n =</i> 5–8). Data presented as mean ± standard error of the mean, analyzed by two-way ANOVA (*<i>p <</i> 0.05; **<i>p <</i> 0.01; ***<i>p <</i> 0.001; NS, not significant) with post hoc Bonferroni correction to compare individual CCl₄ time points with respective vehicle controls (^#<i>p <</i> 0.05; ^##<i>p <</i> 0.01; ^###<i>p <</i> 0.001).</p

Rat models of advanced cirrhosis, portal hypertension, and renal dysfunction.

<p>Portal pressure (PP; A), renal blood flow (RBF; B), and glomerular filtration rate (GFR; C) in 16-wk CCl₄ and olive oil (OO) control rats (<i>n =</i> 6–11). Representative H&E-stained kidney (scale bar 50 μm) showing minor tubular epithelial cell vacuolation (arrows) without significant necrosis after 16 wk of CCl₄ (D). PP (E), RBF (F), and GFR (G) in bile duct ligation (BDL) and sham-operated (sham) control rats (<i>n =</i> 4–8). Representative H&E-stained kidney (scale bar 50 μm) showing necrotic cells within the tubule lumen and loss of the normal circumferential epithelial cell population (arrows) 4 wk after BDL (H). Data presented as mean ± standard error of the mean, analyzed by one-way ANOVA with post hoc Bonferroni correction (*<i>p <</i> 0.05; **<i>p <</i> 0.01; ***<i>p <</i> 0.001).</p

Effect of sustained serelaxin infusion on AKT/eNOS/NO signaling in cirrhotic rat kidney.

<p>Quantification of p-eNOS/eNOS and p-AKT/AKT in whole kidney extracts from 72-h serelaxin- or vehicle-treated 16-wk CCl₄ and 4-wk bile duct ligation (BDL) rats (<i>n =</i> 4) (A–F). Data presented as mean ± standard error of the mean (SEM), analyzed by unpaired <i>t</i>-test (*<i>p <</i> 0.05; **<i>p <</i> 0.01; ***<i>p <</i> 0.001). NOS activity in whole kidney extracts from CCl₄ (G) and BDL (H) rats treated with serelaxin or vehicle (<i>n =</i> 6–8). Data presented as mean ± SEM, analyzed by unpaired <i>t</i>-test (*<i>p <</i> 0.05; **<i>p <</i> 0.01). Renal blood flow (RBF; I) and glomerular filtration rate (GFR; J) in 16-wk CCl₄ rats co-treated with L-N^G-nitroarginine methyl ester (L-NAME) (<i>n =</i> 4–8). Data presented as mean ± SEM, analyzed by one-way ANOVA with post hoc Bonferroni correction (*<i>p <</i> 0.05; **<i>p <</i> 0.01; ***<i>p <</i> 0.001). OO, olive oil.</p

Effect of 72-h serelaxin infusion on renal perfusion and renovascular responses in cirrhotic rats.

<p>Relative <i>Rxfp1</i> transcripts (normalized to 18S rRNA) in whole kidney extracts from 16-wk CCl₄ (A) and 4-wk bile duct ligation (BDL) (E) rats (<i>n =</i> 3–6). Renal blood flow (RBF; B and F), mean arterial pressure (MAP; D and H), and glomerular filtration rate (GFR; C and G) in CCl₄ and BDL rats after 72-h s.c. serelaxin or vehicle (<i>n =</i> 5–8). Data presented as mean ± standard error of the mean (SEM), analyzed by unpaired <i>t</i>-test (*<i>p <</i> 0.05; **<i>p <</i> 0.01; ***<i>p <</i> 0.001; NS, not significant). Concentration–response curves to acetylcholine (ACh; I), phenylephrine (PE; J), and sodium nitroprusside (SNP; K) in the presence of serelaxin or vehicle in 16-wk CCl₄ rats (<i>n =</i> 5–8). Data presented as mean ± SEM, analyzed by two-way ANOVA with post hoc Bonferroni correction (*<i>p <</i> 0.05; **<i>p <</i> 0.01; ***<i>p <</i> 0.001). OO, olive oil.</p