Peptide hormone relaxin: from bench to bedside.

The peptide hormone relaxin has numerous roles both within and independent of pregnancy and is often thought of as a "pleiotropic hormone." Relaxin targets several tissues throughout the body, and has many functions associated with extracellular matrix remodeling and the vasculature. This review considers the potential therapeutic applications of relaxin in cervical ripening, in vitro fertilization, preeclampsia, acute heart failure, ischemia-reperfusion, and cirrhosis. We first outline the animal models used in preclinical studies to progress relaxin into clinical trials and then discuss the findings from these studies. In many cases, the positive outcomes from preclinical animal studies were not replicated in human clinical trials. Therefore, the focus of this review is to evaluate the various animal models used to develop relaxin as a potential therapeutic and consider the limitations that must be addressed in future studies. These include the use of human relaxin in animals, duration of relaxin treatment, and the appropriateness of the clinical conditions being considered for relaxin therapy.

Relaxin deficiency results in increased expression of angiogenesis- and remodelling-related genes in the uterus of early pregnant mice but does not affect endometrial angiogenesis prior to implantation.

BACKGROUND: Extensive uterine adaptations, including angiogenesis, occur prior to implantation in early pregnancy and are potentially regulated by the peptide hormone relaxin. This was investigated in two studies. First, we took a microarray approach using human endometrial stromal (HES) cells treated with relaxin in vitro to screen for target genes. Then we aimed to investigate whether or not relaxin deficiency in mice affected uterine expression of representative genes associated with angiogenesis and uterine remodeling, and also blood vessel proliferation in the pre-implantation mouse endometrium. METHODS: Normal HES cells were isolated and treated with recombinant human relaxin (10 ng/ml) for 24 h before microarray analysis. Reverse transcriptase PCR was used to analyze gene expression of relaxin and its receptor (Rxfp1) in ovaries and uteri; quantitative PCR was used to analyze steroid receptor, angiogenesis and extracellular matrix remodeling genes in the uteri of wild type (Rln+/+) and Rln-/- mice on days 1-4 of pregnancy. Immunohistochemistry localized endometrial endothelial cell proliferation and mass spectrometry measured steroid hormones in the plasma. RESULTS: Microarray analysis identified 63 well-characterized genes that were differentially regulated in HES cells after relaxin treatment. Expression of some of these genes was increased in the uterus of Rln+/+ mice by day 4 of pregnancy. There was significantly higher vascular endothelial growth factor A (VegfA), estrogen receptor 1 (Esr1), progesterone receptor (Pgr), Rxfp1, egl-9 family hypoxia-inducible factor 1 (Egln1), hypoxia inducible factor 1 alpha (Hif1α), matrix metalloproteinase 14 (Mmp14) and ankryn repeat domain 37 (Ankrd37) in Rln-/- compared to Rln+/+ mice on day 1. Progesterone receptor expression and plasma progesterone levels were higher in Rln-/- mice compared to Rln+/+ mice. However, endometrial angiogenesis was not advanced as pre-implantation endothelial cell proliferation did not differ between genotypes. CONCLUSIONS: Relaxin treatment modulates expression of a variety of angiogenesis-related genes in HES cells. However, despite accelerated uterine gene expression of steroid receptor, progesterone and angiogenesis and extracellular matrix remodeling genes in Rln-/- mice, there was no impact on angiogenesis. We conclude that although relaxin deficiency results in phenotypic changes in the pre-implantation uterus, endogenous relaxin does not play a major role in pre-implantation angiogenesis in the mouse uterus.

Recombinant human relaxin versus placebo for cervical ripening: a double-blind randomised trial in pregnant women scheduled for induction of labour.

BACKGROUND: Nonclinical studies indicate that the hormone relaxin is a good candidate for a safe cervical ripening agent that does not cause uterine contractions. METHODS: This Phase II study (conducted November 2, 2005-October 20, 2006) was a randomised, double blind, placebo controlled trial testing 24-h intravenous infusion of serelaxin (recombinant human relaxin) or placebo for cervical ripening in 72 healthy, primiparous women. Eligible subjects had a singleton pregnancy ≥40 weeks, were planned for elective induction, had vertex presentation of the fetus, intact membranes and a Bishop score at screening ≤4. In Part A of the study, safety evaluation of three escalating doses of serelaxin (7.5, 25 or 75 µg/kg/day) or placebo was performed in 22 subjects admitted to the hospital 24 h prior to scheduled induction (n = 7, 4, 4, and 7 subjects, respectively). The highest safe dose from Part A and placebo were then tested in Part B for safety and cervical ripening (n = 25 subjects/arm). Planned randomisation ratio was of 4:2 (serelaxin:placebo) for each dose group in Part A and 1:1 for Part B. For analysis, subjects in Part B were pooled with those receiving the same dose in Part A and all subjects receiving placebo were pooled. The primary efficacy endpoint was change from baseline in Bishop score at 6, 12 and 24 h or end of study drug administration. Maternal safety evaluations included adverse events and vital signs through 4 weeks. Fetal assessments included serial heart rate monitoring and nonstress testing. Neonatal assessments included Apgar scores, NICU admissions, and adverse events through 4 weeks. RESULTS: Overall, 74 subjects were randomized and 72 were treated. There were no significant differences between the groups receiving the highest safe dose of serelaxin (75 µg/kg/day) and placebo in the primary or secondary efficacy endpoints. Changes from baseline in Bishop score at 24 h were 4.19 ± 1.9 and 3.26 ± 2.26 in the pooled placebo and serelaxin groups, respectively (p = 0.2507). Serelaxin was well tolerated and no anti-serelaxin antibodies were detected in either subjects or neonates. CONCLUSION: Serelaxin infusion at the end of pregnancy was well tolerated but did not advance cervical ripening. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT00259103 (15 November 2005).

Serelaxin in acute heart failure patients with preserved left ventricular ejection fraction: results from the RELAX-AHF trial.

AIMS: Serelaxin is effective in relieving dyspnoea and improving multiple outcomes in acute heart failure (AHF). Many AHF patients have preserved ejection fraction (HFpEF). Given the lack of evidence-based therapies in this population, we evaluated the effects of serelaxin according to EF in RELAX-AHF trial. METHODS AND RESULTS: RELAX-AHF randomized 1161 AHF patients to 48-h serelaxin (30 µg/kg/day) or placebo within 16 h from presentation. We compared the effects of serelaxin on efficacy endpoints, safety endpoints, and biomarkers of organ damage between preserved (≥50%) and reduced (<50%, HFrEF) EF. HFpEF was present in 26% of patients. Serelaxin induced a similar dyspnoea relief in HFpEF vs. HFrEF patients by visual analogue scale-area under the curve (VAS-AUC) through Day 5 [mean change, 461 (-195, 1117) vs. 397 (10, 783) mm h, P = 0.87], but had possibly different effects on the proportion of patients with moderately or markedly dyspnoea improvement by Likert scale at 6, 12, and 24 h [odds ratio for favourable response, 1.70 (0.98, 2.95) vs. 0.85 (0.62, 1.15), interaction P = 0.030]. No differences were encountered in the effect of serelaxin on short- or long-term outcome between HFpEF and HFrEF patients including cardiovascular death or hospitalization for heart/renal failure through Day 60, cardiovascular death through Day 180, and all-cause death through Day 180. Similar safety and changes in biomarkers (high-sensitivity troponin T, cystatin-C, and alanine/aspartate aminotransferases) were found in both groups. CONCLUSIONS: In AHF patients with HFpEF compared with those with HFrEF, serelaxin was well tolerated and effective in relieving dyspnoea and had a similar effect on short- and long-term outcome, including survival improvement.

Serelaxin, recombinant human relaxin-2, for treatment of acute heart failure (RELAX-AHF): a randomised, placebo-controlled trial.

BACKGROUND: Serelaxin, recombinant human relaxin-2, is a vasoactive peptide hormone with many biological and haemodynamic effects. In a pilot study, serelaxin was safe and well tolerated with positive clinical outcome signals in patients with acute heart failure. The RELAX-AHF trial tested the hypothesis that serelaxin-treated patients would have greater dyspnoea relief compared with patients treated with standard care and placebo. METHODS: RELAX-AHF was an international, double-blind, placebo-controlled trial, enrolling patients admitted to hospital for acute heart failure who were randomly assigned (1:1) via a central randomisation scheme blocked by study centre to standard care plus 48-h intravenous infusions of placebo or serelaxin (30 µg/kg per day) within 16 h from presentation. All patients had dyspnoea, congestion on chest radiograph, increased brain natriuretic peptide (BNP) or N-terminal prohormone of BNP, mild-to-moderate renal insufficiency, and systolic blood pressure greater than 125 mm Hg. Patients, personnel administering study drug, and those undertaking study-related assessments were masked to treatment assignment. The primary endpoints evaluating dyspnoea improvement were change from baseline in the visual analogue scale area under the curve (VAS AUC) to day 5 and the proportion of patients with moderate or marked dyspnoea improvement measured by Likert scale during the first 24 h, both analysed by intention to treat. This trial is registered at ClinicalTrials.gov, NCT00520806. FINDINGS: 1161 patients were randomly assigned to serelaxin (n=581) or placebo (n=580). Serelaxin improved the VAS AUC primary dyspnoea endpoint (448 mm × h, 95% CI 120-775; p=0·007) compared with placebo, but had no significant effect on the other primary endpoint (Likert scale; placebo, 150 patients [26%]; serelaxin, 156 [27%]; p=0·70). No significant effects were recorded for the secondary endpoints of cardiovascular death or readmission to hospital for heart failure or renal failure (placebo, 75 events [60-day Kaplan-Meier estimate, 13·0%]; serelaxin, 76 events [13·2%]; hazard ratio [HR] 1·02 [0·74-1·41], p=0·89] or days alive out of the hospital up to day 60 (placebo, 47·7 [SD 12·1] days; serelaxin, 48·3 [11·6]; p=0·37). Serelaxin treatment was associated with significant reductions of other prespecified additional endpoints, including fewer deaths at day 180 (placebo, 65 deaths; serelaxin, 42; HR 0·63, 95% CI 0·42-0·93; p=0·019). INTERPRETATION: Treatment of acute heart failure with serelaxin was associated with dyspnoea relief and improvement in other clinical outcomes, but had no effect on readmission to hospital. Serelaxin treatment was well tolerated and safe, supported by the reduced 180-day mortality. FUNDING: Corthera, a Novartis affiliate company.

Liver function, in-hospital, and post-discharge clinical outcome in patients with acute heart failure-results from the relaxin for the treatment of patients with acute heart failure study.

BACKGROUND: Elevated plasma concentrations of liver function tests are prevalent in patients with chronic heart failure (HF). Little is known about liver function in patients with acute HF. We aimed to assess the prevalence and prognostic value of serial measurements of liver function tests in patients admitted with acute decompensated HF. METHODS: We investigated liver function tests from all 234 patients from the Relaxin for the Treatment of Patients With Acute Heart Failure study at baseline and during hospitalization. The end points were worsening HF through day 5, 60-day mortality or rehospitalization, and 180-day mortality. RESULTS: Mean age was 70 ± 10 years, 56% were male, and most patients were in New York Heart Association functional class III/IV (73%). Abnormal liver function tests were frequently found for alanine transaminase (ALT; 12%), aspartate transaminase (AST; 21%), alkaline phosphatase (12%), and total bilirubin (19%), and serum albumin (25%) and total protein (9%) were decreased. In-hospital changes were very small. On a continuous scale, baseline ALT and AST were associated with 180-day mortality (hazard ratios [HRs; per doubling] 1.52 [P = .030] and 1.97 [P = .013], respectively) and worsening HF through day 5 (HRs [per doubling] 1.72 [P = .005] and 1.95 [P = .008], respectively). Albumin was associated with 180-day mortality (HR 0.86; P = .001) but not with worsening HF (HR 0.95; P = .248). Total protein was associated with only worsening HF (HR 0.91; P = .004). CONCLUSIONS: Abnormal liver function tests are often present in patients with acute HF and are associated with an increased risk for mortality, rehospitalization, and in-hospital worsening HF.

Effects of serelaxin in subgroups of patients with acute heart failure: results from RELAX-AHF.

AIM: Patients hospitalized for acute heart failure (AHF) differ with respect of many clinical characteristics which may influence their prognosis and response to treatment. We have assessed possible differences in the effects of serelaxin on dyspnoea relief, 60 Day outcomes and 180 Day mortality across patient subgroups in the RELAX-AHF trial. METHODS AND RESULTS: Subgroups were based on pre-specified covariates (age, sex, race, geographic region, estimated glomerular filtration rate, time from presentation to randomization, baseline systolic blood pressure, history of diabetes, atrial fibrillation, ischaemic heart disease, cardiac devices, i.v. nitrates at randomization). Other covariates which may modify the efficacy of AHF treatment were also analysed. Subgroup analyses did not show any difference in the effects of serelaxin vs. placebo on dyspnoea relief or on the incidence of cardiovascular death or rehospitalizations for heart failure or renal failure at 60 days. Nominally significant interactions between some patient subgroups and the effects of serelaxin on 180 days cardiovascular and all-cause mortality were noted but should be interpreted cautiously due to the number of comparisons and the low incidence of deaths in the subgroups at lower risk. CONCLUSION: The effects of serelaxin vs. placebo appeared to be similar across subgroups of patients in RELAX-AHF.

Design of the RELAXin in acute heart failure study.

BACKGROUND: Acute heart failure (AHF) remains a major public health burden with a high prevalence and poor prognosis. Relaxin is a naturally occurring peptide hormone that increases cardiac output, arterial compliance, and renal blood flow during pregnancy. The RELAX-AHF-1 study will evaluate the effect of RLX030 (recombinant form of human relaxin 2) on symptom relief and clinical outcomes in patients with AHF. METHODS: The protocol includes a completed phase 2 234-patient dose-finding study (Pre-RELAX-AHF) and an ongoing phase 3 1,160-patient trial (RELAX-AHF-1). Patients with AHF and systolic blood pressure >125 mm Hg are randomized within 16 hours of presentation to a 48-hour IV infusion of RLX030 or placebo. The 30 µg/kg per day dose of RLX030 was chosen for RELAX-AHF-1 based on effects on dyspnea, clinical outcomes, and safety observed in Pre-RELAX-AHF. Primary efficacy end points in RELAX-AHF-1 are (1) the area under the curve of change of the dyspnea Visual Analog Scale from baseline through day 5 and (2) whether the patient reports moderately to markedly better dyspnea at 6, 12, and 24 hours. Secondary efficacy end points include days alive and out of the hospital through day 60 and cardiovascular death or rehospitalization for heart failure or renal failure through day 60. Patients will be followed up through day 180 for mortality. As of September 19, 2011, 978 patients have been enrolled. CONCLUSIONS: Pre-RELAX-AHF results suggested that infusion of RLX030 may accelerate dyspnea relief and improve prognosis in patients hospitalized with AHF. RELAX-AHF-1 will further evaluate these effects.

Relaxin for the treatment of patients with acute heart failure (Pre-RELAX-AHF): a multicentre, randomised, placebo-controlled, parallel-group, dose-finding phase IIb study.

BACKGROUND: Most patients admitted for acute heart failure have normal or increase blood pressure. Relaxin is a natural human peptide that affects multiple vascular control pathways, suggesting potential mechanisms of benefit for such patients. We assessed the dose response of relaxin's effect on symptom relief, other clinical outcomes, and safety. METHODS: In a placebo-controlled, parallel-group, dose-ranging study, 234 patients with acute heart failure, dyspnoea, congestion on chest radiograph, and increased brain natriuretic peptide (BNP) or N-terminal prohormone of BNP, mild-to-moderate renal insufficiency, and systolic blood pressure greater than 125 mm Hg were recruited from 54 sites in eight countries and enrolled within 16 h of presentation. Patients were randomly assigned, in a double-blind manner via a telephone-based interactive voice response system, to standard care plus 48-h intravenous infusion of placebo (n=62) or relaxin 10 microg/kg (n=40), 30 microg/kg (n=43), 100 microg/kg (n=39), or 250 microg/kg (n=50) per day. Several clinical endpoints were explored to assess whether intravenous relaxin should be pursued in larger studies of acute heart failure, to identify an optimum dose, and to help to assess endpoint selection and power calculations. Analysis was by modified intention to treat. This study is registered with ClinicalTrials.gov, number NCT00520806. FINDINGS: In the modified intention-to-treat population, 61 patients were assessed in the placebo group, 40 in the relaxin 10 microg/kg per day group, 42 in the relaxin 30 microg/kg per day group, 37 in the relaxin 100 microg/kg per day group, and 49 in the relaxin 250 microg/kg per day group. Dyspnoea improved with relaxin 30 microg/kg compared with placebo, as assessed by Likert scale (17 of 42 patients [40%] moderately or markedly improved at 6 h, 12 h, and 24 h vs 14 of 61 [23%]; p=0.044) and visual analogue scale through day 14 (8214 mm x h [SD 8712] vs 4622 mm x h [9003]; p=0.053). Length of stay was 10.2 days (SD 6.1) for relaxin-treated patients versus 12.0 days (7.3) for those given placebo, and days alive out of hospital were 47.9 (10.1) versus 44.2 (14.2). Cardiovascular death or readmission due to heart or renal failure at day 60 was reduced with relaxin (2.6% [95% CI 0.4-16.8] vs 17.2% [9.6-29.6]; p=0.053). The number of serious adverse events was similar between groups. INTERPRETATION: When given to patients with acute heart failure and normal-to-increased blood pressure, relaxin was associated with favourable relief of dyspnoea and other clinical outcomes, with acceptable safety.

Low lymphocyte ratio as a novel prognostic factor in acute heart failure: results from the Pre-RELAX-AHF study.

BACKGROUND: Previous studies have suggested that a lower lymphocyte ratio (Ly%) in the white blood cell (WBC) differential count is related to worse outcomes in patients with acute heart failure (AHF) and other cardiovascular disorders. METHODS: In the Pre-RELAX-AHF study, 234 patients with AHF, systolic blood pressure >125 mm Hg and brain natriuretic peptide ≥350 pg/ml or equivalent were randomized to 1 of 4 intravenous doses of relaxin or placebo and followed up for 6 months following randomization. Complete blood count and differential were performed by a central laboratory at baseline and then daily to day 5 and on day 14. RESULTS: The WBC count by itself was not associated with measures of disease severity or outcome, and patients with Ly% <13% had similar baseline characteristics to patients with Ly% >13%, except for a higher baseline WBC count, elevated baseline glucose, older age and higher rates of peripheral vascular disease. However, patients with Ly% <13% had less improvement of dyspnea, greater worsening of heart failure, longer length of initial hospital stay and fewer days alive and out of hospital. Statistical significance was reached for all-cause death by days 60 and 180 (hazard ratio = 1.11 per percent decrease, 95% confidence interval 1.03-1.19; p = 0.0048). CONCLUSIONS: Despite no association with any baseline characteristic known to strongly predict outcome in AHF, low Ly% is associated with less symptom relief and worse in-hospital and postdischarge clinical outcomes.

Relaxin: review of biology and potential role in treating heart failure.

Relaxin is a naturally occurring human peptide initially identified as a reproductive hormone. More recently, relaxin has been shown to play a key role in the maternal hemodynamic and renal adjustments that accommodate pregnancy. An understanding of these physiologic effects has led to the evaluation of relaxin as a pharmacologic agent for the treatment of patients with acute heart failure. Preliminary results have been encouraging. In addition, the other known biologic properties of relaxin, including anti-inflammatory effects, extracellular matrix remodeling effects, and angiogenic and anti-ischemic effects, all may play a role in potential benefits of relaxin therapy. Ongoing, large-scale clinical testing will provide additional insights into the potential role of relaxin in the treatment of heart failure.

Intravenous recombinant human relaxin in compensated heart failure: a safety, tolerability, and pharmacodynamic trial.

BACKGROUND: Relaxin is upregulated in human heart failure (HF). Animal and clinical data suggest beneficial hemodynamic and renal effects from vasodilation. We determined safety, tolerability, and pharmacodynamic effects of human Relaxin in stable HF. METHODS AND RESULTS: Sixteen patients were treated with open-label intravenous Relaxin in 3 dose-escalation cohorts and monitored hemodynamically for 24-hour infusion and postinfusion periods and followed until Day 30. The safety demonstrated in Group A (8-hour sequential infusions at dose levels of 10, then 30, and then 100 microg x kg x day equivalents) allowed escalation to Group B (240, 480, and 960 microg x kg x day). The highest safe dose, 960 microg x kg x day, was selected for a 24-hour infusion in Group C. Relaxin showed no adverse effects; produced hemodynamic effects consistent with vasodilation (ie, trends toward increases in cardiac index, decreases in pulmonary wedge pressure, and decreases in circulating NT-pro BNP without inducing hypotension; improved markers of renal function [creatinine, blood urea nitrogen]). The highest dose caused a transient elevation in creatinine and blood urea nitrogen at Day 9 that was without apparent clinical significance. CONCLUSIONS: Relaxin was safe and well-tolerated in patients with stable HF, and preliminary pharmacodynamic responses suggest it causes vasodilation. Further evaluation of the safety and efficacy of this drug in HF appears warranted.

Relaxin, a pleiotropic vasodilator for the treatment of heart failure.

Relaxin is a naturally occurring peptide hormone that plays a central role in the hemodynamic and renovascular adaptive changes that occur during pregnancy. Triggering similar changes could potentially be beneficial in the treatment of patients with heart failure. The effects of relaxin include the production of nitric oxide, inhibition of endothelin, inhibition of angiotensin II, production of VEGF, and production of matrix metalloproteinases. These effects lead to systemic and renal vasodilation, increased arterial compliance, and other vascular changes. The recognition of this has led to the study of relaxin for the treatment of heart failure. An initial pilot study has shown favorable hemodynamic effects in patients with heart failure, including reduction in ventricular filling pressures and increased cardiac output. The ongoing RELAX-AHF clinical program is designed to evaluate the effects of relaxin on the symptoms and outcomes in a large group of patients admitted to hospital for acute heart failure. This review will summarize both the biology of relaxin and the data supporting its potential efficacy in human heart failure.

Relaxin family peptide receptors--from orphans to therapeutic targets.

The relaxin family peptides have distinct expression profiles and physiological functions. Several of them are the cognate ligands for 4 G-protein-coupled relaxin family peptide receptors (RXFPs; formerly LGR7, LGR8, GPCR135, GPCR142). The relaxin/RXFP1 system has roles in reproductive physiology but is also involved in fibrosis, wound healing and responses to infarction. Relaxin has a potential use in congestive heart failure where fibrosis plays an important role in organ failure. The INSL3/RXFP2 system has biological roles in reproductive biology that may have limited therapeutic potential. However, the recently characterized relaxin-3/RXFP3 system is important in stress/anxiety and body composition. RXFP3 receptor antagonists are potentially novel anti-anxiety and anti-obesity drugs.

Serelaxin in clinical development: past, present and future.

The availability of highly purified recombinant human relaxin, serelaxin, has allowed clinical trials to be conducted in several indications and the elucidation of its pharmacology in human subjects. These studies have demonstrated that serelaxin has unique haemodynamic properties that are likely to contribute to organ protection and long-term outcome benefits in acute heart failure. Clinical observations support its consideration for therapeutic use in other patient populations, including those with chronic heart failure, coronary artery disease, portal hypertension and acute renal failure. LINKED ARTICLES: This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.

Relaxin modulates fibroblast function, collagen production, and matrix metalloproteinase-2 expression by cardiac fibroblasts.

Cardiac fibrosis is a hallmark of heart disease and involves recruitment, proliferation, and differentiation of extracellular matrix-producing fibroblasts, leading to overproduction of collagen within the myocardium. In this study, the effects of relaxin in inhibiting these processes were investigated. We used neonatal rat atrial and ventricular fibroblasts, which respond to pro-fibrotic stimuli (i.e., transforming growth factor-beta and angiotensin II) and naturally express the relaxin receptor LGR7. Relaxin significantly inhibited TGF-beta- and angiotensin II-mediated fibroblast function and collagen production over a 72-h period, while increasing MMP-2 expression and activity in the presence of both profibrotic factors (all P < .05). These studies demonstrate that relaxin may have therapeutic potential in diseased states characterized by cardiac fibrosis.

Relaxin modulates cardiac fibroblast proliferation, differentiation, and collagen production and reverses cardiac fibrosis in vivo.

Cardiac fibrosis is a key component of heart disease and involves the proliferation and differentiation of matrix-producing fibroblasts. The effects of an antifibrotic peptide hormone, relaxin, in inhibiting this process were investigated. We used rat atrial and ventricular fibroblasts, which respond to profibrotic stimuli and express the relaxin receptor (LGR7), in addition to two in vivo models of cardiac fibrosis. Cardiac fibroblasts, when plated at low density or stimulated with TGF-beta or angiotensin II (Ang II), accelerated fibroblast differentiation into myofibroblasts, as demonstrated by significantly increased alpha-smooth muscle actin expression, collagen synthesis, and collagen deposition (by up to 95% with TGF-beta and 40% with Ang II; all P < 0.05). Fibroblast proliferation was significantly increased by 10(-8) m and 10(-7) m Ang II (63-75%; P < 0.01) or 0.1-1 microg/ml IGF-I (27-40%; P < 0.05). Relaxin alone had no marked effect on these parameters, but it significantly inhibited Ang II- and IGF-I-mediated fibroblast proliferation (by 15-50%) and Ang II- and TGF-beta-mediated fibroblast differentiation, as detected by decreased expression of alpha-smooth muscle actin (by 65-88%) and collagen (by 60-80%). Relaxin also increased matrix metalloproteinase-2 expression in the presence of TGF-beta (P < 0.01) and Ang II (P < 0.05). Furthermore, relaxin decreased collagen overexpression when administered to two models of established fibrotic cardiomyopathy, one due to relaxin deficiency (by 40%; P < 0.05) and the other to cardiac-restricted overexpression of beta2-adrenergic receptors (by 58%; P < 0.01). These coherent findings indicate that relaxin regulates fibroblast proliferation, differentiation, and collagen deposition and may have therapeutic potential in diseased states characterized by cardiac fibrosis.

Effect of serelaxin on mode of death in acute heart failure: results from the RELAX-AHF study.

BACKGROUND: Little is known about mode of death after acute heart failure (AHF) hospitalization. In the RELAX-AHF (Efficacy and Safety of Relaxin for the Treatment of Acute Heart Failure) study, serelaxin, the recombinant form of human relaxin-2, reduced post-discharge mortality at 180 days in selected patients with AHF. OBJECTIVES: The goal of this study was to assess the effect of serelaxin on specific modes of death in patients with AHF. METHODS: The RELAX-AHF study randomized 1,161 patients with AHF to 48 h of therapy with intravenous serelaxin or placebo. Patients were followed for vital status through 180 days. A blinded clinical events committee reviewed all deaths and adjudicated a cause of death on the basis of pre-specified criteria. Cox proportional hazard models were used to assess the effect of serelaxin on each mode of death, on the basis of pre-specified groupings of mode of death. RESULTS: There were 107 deaths (9.3%): 37 (35%) due to HF, 25 (23%) due to sudden death, 15 (14%) due to other cardiovascular (CV) causes, 19 (18%) due to non-CV causes, and 11 (10%) classified as unknown. The treatment effect of serelaxin was most pronounced on other CV deaths (hazard ratio [HR]: 0.29; 95% CI: 0.12 to 0.73; p = 0.005) and sudden death (HR: 0.46; 95% CI: 0.20 to 1.07; p = 0.065). There was no apparent impact of serelaxin treatment on HF deaths or non-CV deaths. CONCLUSIONS: In the RELAX-AHF study, the effects of serelaxin on mortality were primarily driven by reduction in mortality from other CV causes and sudden death, without apparent impact on HF deaths. (Efficacy and Safety of Relaxin for the Treatment of Acute Heart Failure [RELAX-AHF]; NCT00520806).

Effect of serelaxin on cardiac, renal, and hepatic biomarkers in the Relaxin in Acute Heart Failure (RELAX-AHF) development program: correlation with outcomes.

OBJECTIVES: The aim of this study was to assess the effects of serelaxin on short-term changes in markers of organ damage and congestion and relate them to 180-day mortality in patients with acute heart failure. BACKGROUND: Hospitalization for acute heart failure is associated with high post-discharge mortality, and this may be related to organ damage. METHODS: The Pre-RELAX-AHF (Relaxin in Acute Heart Failure) phase II study and RELAX-AHF phase III study were international, multicenter, double-blind, placebo-controlled trials in which patients hospitalized for acute heart failure were randomized within 16 h to intravenous placebo or serelaxin. Each patient was followed daily to day 5 or discharge and at days 5, 14, and 60 after enrollment. Vital status was assessed through 180 days. In RELAX-AHF, laboratory evaluations were performed daily to day 5 and at day 14. Plasma levels of biomarkers were measured at baseline and days 2, 5, and 14. All-cause mortality was assessed as a safety endpoint in both studies. RESULTS: Serelaxin reduced 180-day mortality, with similar effects in the phase II and phase III studies (combined studies: N = 1,395; hazard ratio: 0.62; 95% confidence interval: 0.43 to 0.88; p = 0.0076). In RELAX-AHF, changes in markers of cardiac (high-sensitivity cardiac troponin T), renal (creatinine and cystatin-C), and hepatic (aspartate transaminase and alanine transaminase) damage and of decongestion (N-terminal pro-brain natriuretic peptide) at day 2 and worsening heart failure during admission were associated with 180-day mortality. Serelaxin administration improved these markers, consistent with the prevention of organ damage and faster decongestion. CONCLUSIONS: Early administration of serelaxin was associated with a reduction of 180-day mortality, and this occurred with fewer signs of organ damage and more rapid relief of congestion during the first days after admission.

Permutation criteria to evaluate multiple clinical endpoints in a proof-of-concept study: lessons from Pre-RELAX-AHF.

BACKGROUND: Clinically relevant endpoints cannot be routinely targeted with reasonable power in a small study. Hence, proof-of-concept studies are often powered to a primary surrogate endpoint. However, in acute heart failure (AHF) effects on surrogates have not translated into clinical benefit in confirmatory studies. Although observing an effect on one of many endpoints due to chance is likely, observing concurrent positive trends across several outcomes by chance is usually unlikely. METHODS: Pre-RELAX-AHF, which compared 4 relaxin doses with placebo in AHF, has shown favourable trends versus placebo (one-sided P < 0.10) on six of nine clinical endpoints in the 30 µg/kg/day group. To illustrate evaluation of multiple, correlated clinical endpoints for evidence of efficacy and for dose selection, a permutation method was applied retrospectively. By randomly re-assigning the treatment group to the actual data for each of the 229 subjects, 20,000 permutation samples were constructed. RESULTS: The permutation P value for at least six favourable trends among nine endpoints in any dose groups was 0.0073 (99.9% CI 0.0053-0.0093). This is higher than would be expected if the endpoints were uncorrelated (0.00026), but much lower than the probability of observing one of nine comparisons significant at the traditional two-sided P < 0.05 (0.74). Thus, the result was unlikely due to correlated endpoints or to chance. CONCLUSIONS: Examining consistency of effect across multiple clinical endpoints in a proof-of-concept study may identify efficacious therapies and enable dose selection for confirmatory trials. The merit of the approach described requires confirmation through prospective application in designing future studies.