Relaxin combined with transarterial chemoembolization achieved synergistic effects and inhibited liver cancer metastasis in a rabbit VX2 model.

OBJECTIVE: To explore the effect and mechanism of relaxin (RLX) in the growth and metastasis of livercancer after combination treatment with transarterial chemoembolization (TACE). MATERIALS AND METHODS: HCCLM3 and Huh-7 cells were adopted to evaluate the effect of tumor proliferation, migration, and invasion after RLX administration in vitro. The rabbit VX2 model was used to evaluate the biosafety, doxorubicin penetration, local tumor response, tumor metastasis, and survival benefit of RLX combined with TACE treatment. RESULTS: RLX did not affect the proliferation, migration, or invasion of HCCLM3 and Huh-7 cells, and the expression of E-cadherin and HIF-1α also remained unchanged while the MMP-9 protein was upregulated in vitro. In the rabbit VX2 model, compared to the normal saline group (NS), RLX group (RLX) and TACE mono-therapy group (TACE), the group that received TACE combined with RLX (TACE + RLX) showed an improved local tumor response and survival benefit. Furthermore, TACE combined with RLX was found to reduce tumor metastasis. This combination therapy reduced the fibrotic extracellular matrix in the tumor microenvironment, allowing for better penetration of doxorubicin, improved infiltration of CD8+ T cells and affected the secretion of cytokines. Additionally, RLX combined with TACE was able to decrease the expression of HIF-1α and PD-L1. The biosafety of TACE combined with RLX was also confirmed. CONCLUSION: RLX synergized with TACE by mitigating the fibrotic extracellular matrix and tumor hypoxic microenvironment, improving the therapeutic effect and inhibiting metastasis during the treatment of liver cancer.

Novel Relaxin Receptor RXFP1 Agonist AZD3427 in the Treatment of Heart Failure: A Phase 1a/b, First-in-Human, Randomized, Single-Blind, Placebo-Controlled Study.

BACKGROUND: Heart failure mortality remains high despite recent progress in pharmacological treatment. AZD3427 is a selective long-acting analog of relaxin, a vasodilatory hormone with antifibrotic effects. We assessed the safety, pharmacokinetics, and pharmacodynamics of AZD3427 in healthy volunteers and patients with heart failure on standard-of-care therapy. METHODS AND RESULTS: In this first-in-human, phase 1a/b, randomized, single-blind, placebo-controlled study, healthy volunteers were randomized 6:2 to receive a single dose of AZD3427 or placebo by subcutaneous injection in 5 mixed-ethnicity cohorts (5, 10, 30, 90, or 270 mg) and 1 Japanese-descent cohort (270 mg), or by intravenous injection in 1 cohort (15 mg). After confirming safety and tolerability in healthy volunteers, 3 cohorts of patients with heart failure and left ventricular ejection fraction ≤40% and 3 cohorts with ejection fraction ≥41% were randomized 6:2 to receive 5 weekly doses of AZD3427 (5, 15, or 45 mg) or placebo by subcutaneous injection. In total, 56 healthy volunteers and 48 patients with heart failure were randomized. AZD3427 was well tolerated at all doses. After subcutaneous administration, AZD3427 was absorbed slowly, and exposure was approximately linear across the dose range. In patients with heart failure, AZD3427 terminal half-life was 13 to 14 days and there were numerical increases in stroke volume and estimated glomerular filtration rate. No treatment-emergent antidrug antibodies were detected. CONCLUSIONS: AZD3427 had favorable safety and pharmacokinetic profiles. Hemodynamic changes in patients with heart failure were consistent with the anticipated effects of a relaxin analog. These findings support further development of AZD3427 as a novel long-term treatment for patients with heart failure. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique Identifier: NCT04630067.

Translational pharmacokinetic/pharmacodynamic model for mRNA-0184, an investigational therapeutic for the treatment of heart failure.

Heart failure (HF) is a complex, progressive disorder that is associated with substantial morbidity and mortality on a global scale. Relaxin-2 is a naturally occurring hormone that may have potential therapeutic benefit for patients with HF. To investigate the therapeutic potential of relaxin in the treatment of patients with HF, mRNA-0184, a novel, investigational, lipid nanoparticle (LNP)-encapsulated mRNA therapy that encodes for human relaxin-2 fused to variable light chain kappa (Rel2-vlk) was developed. A translational semi-mechanistic population pharmacokinetic (PK)/pharmacodynamic (PD) model was developed using data from non-human primates at dose levels ranging from 0.15 to 1 mg/kg. The PK/PD model was able to describe the PK of Rel2-vlk mRNA and translated Rel2-vlk protein in non-human primates adequately with relatively precise estimates. The preclinical PK/PD model was then scaled allometrically to determine the human mRNA-0184 dose that would achieve therapeutic levels of Rel2-vlk protein expression in patients with stable HF with reduced ejection fraction. Model-based simulations derived from the scaled PK/PD model support the selection of 0.025 mg/kg as an appropriate starting human dose of mRNA-0184 to achieve average trough relaxin levels between 1 and 2.5 ng/mL, which is the potential exposure for cardioprotective action of relaxin.

R2R01: A long-acting single-chain peptide agonist of RXFP1 for renal and cardiovascular diseases.

BACKGROUND: The therapeutic potential of relaxin for heart failure and renal disease in clinical trials is hampered by the short half-life of serelaxin. Optimization of fatty acid-acetylated single-chain peptide analogues of relaxin culminated in the design and synthesis of R2R01, a potent and selective RXFP1 agonist with subcutaneous bioavailability and extended half-life. EXPERIMENTAL APPROACH: Cellular assays and pharmacological models of RXFP1 activation were used to validate the potency and selectivity of R2R01. Increased renal blood flow was used as a translational marker of R2R01 activity. Human mastocytes (LAD2 cells) were used to study potential pseudo-allergic reactions and CD4+ T-cells to study immunogenicity. The pharmacokinetics of R2R01 were characterized in rats and minipigs. KEY RESULTS: In vitro, R2R01 had comparable potency and efficacy to relaxin as an agonist for human RXFP1. In vivo, subcutaneous administration of R2R01 increased heart rate and renal blood flow in normotensive and hypertensive rat and did not show evidence of tachyphylaxis. R2R01 also increased nipple length in rats, used as a chronic model of RXFP1 engagement. Pharmacokinetic studies showed that R2R01 has a significantly extended terminal half-life. The in vitro assays with LAD2 cells and CD4+ T-cells showed that R2R01 had low potential for pseudo-allergic and immunogenic reactions, respectively. CONCLUSION AND IMPLICATIONS: R2R01 is a potent RXFP1 agonist with an extended half-life that increases renal blood flow in various settings including normotensive and hypertensive conditions. The preclinical efficacy and safety data supported clinical development of R2R01 as a potential new therapy for renal and cardiovascular diseases.

Inhaled B7 alleviates bleomycin-induced pulmonary fibrosis in mice.

Treatment options for the progression of pulmonary fibrosis (PF), which ultimately causes respiratory failure, are limited. According to recent studies, recombinant human relaxin is potentially therapeutic against fibrosis and contraction during pulmonary damage. However, the production of recombinant H2 relaxin is laborious and expensive, limiting its extensive application. Thankfully, alternative research has revealed that treatment with a single-chain peptide of relaxin attenuates organ fibrosis in rodent models too, with the production of a single-chain peptide of relaxin simple and cheap; it could be therapeutic against idiopathic pulmonary fibrosis. Here, we explored the probable inhibiting effects of B7, a B chain of recombinant human relaxin, on bleomycin-induced pulmonary inflammation. Inhaled B7 efficiently reduced the number of inflammatory leukocytes and neutrophils in the bronchoalveolar lavage fluid of mice with bleomycin-induced PF, significantly improved the structure of the damaged alveolar, reduced collagen deposition, suppressed the main pathological features of idiopathic pulmonary fibrosis, i.e. the expression of both pulmonary α-smooth muscle actin and pulmonary vimentin, and inhibited the transcription of inflammation and collagen deposition-related mRNAs, including fibronectin, α-smooth muscle actin (α-SMA), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and alpha-1 type 1 collagen (Col-1a), and the expression of inflammation-related proteins, such as IL-1ß, IL-6, chemokines (KC), TIMP metallopeptidase inhibitor 1 (TIMP-1), and hydroxyproline (Hyp). Overall, our findings suggest that inhaled B7 exerts beneficial effects against pulmonary fibrosis via attenuating inflammation. It could be developed into a simple, highly effective therapeutic approach for pulmonary fibrosis.

Ligand-activated RXFP1 gene therapy ameliorates pressure overload-induced cardiac dysfunction.

Recurrent episodes of decompensated heart failure (HF) represent an emerging cause of hospitalizations in developed countries with an urgent need for effective therapies. Recently, the pregnancy-related hormone relaxin (RLN) was found to mediate cardio-protective effects and act as a positive inotrope in the cardiovascular system. RLN binds to the RLN family peptide receptor 1 (RXFP1), which is predominantly expressed in atrial cardiomyocytes. We therefore hypothesized that ventricular RXFP1 expression might exert potential therapeutic effects in an in vivo model of cardiac dysfunction. Thus, mice were exposed to pressure overload by transverse aortic constriction and treated with AAV9 to ectopically express RXFP1. To activate RXFP1 signaling, RLN was supplemented subcutaneously. Ventricular RXFP1 expression was well tolerated. Additional RLN administration not only abrogated HF progression but restored left ventricular systolic function. In accordance, upregulation of fetal genes and pathological remodeling markers were significantly reduced. In vitro, RLN stimulation of RXFP1-expressing cardiomyocytes induced downstream signaling, resulting in protein kinase A (PKA)-specific phosphorylation of phospholamban (PLB), which was distinguishable from ß-adrenergic activation. PLB phosphorylation corresponded to increased calcium amplitude and contractility. In conclusion, our results demonstrate that ligand-activated cardiac RXFP1 gene therapy represents a therapeutic approach to attenuate HF with the potential to adjust therapy by exogenous RLN supplementation.

Investigation of molecular mechanisms of experimental compounds in murine models of chronic allergic airways disease using synchrotron Fourier-transform infrared microspectroscopy.

The ovalbumin-induced (OVA) chronic allergic airways murine model is a well-established model for investigating pre-clinical therapies for chronic allergic airways diseases, such as asthma. Here, we examined the effects of several experimental compounds with potential anti-asthmatic effects including resveratrol (RV), relaxin (RLN), L-sulforaphane (LSF), valproic acid (VPA), and trichostatin A (TSA) using both a prevention and reversal model of chronic allergic airways disease. We undertook a novel analytical approach using focal plane array (FPA) and synchrotron Fourier-transform infrared (S-FTIR) microspectroscopic techniques to provide new insights into the mechanisms of action of these experimental compounds. Apart from the typical biological effects, S-FTIR microspectroscopy was able to detect changes in nucleic acids and protein acetylation. Further, we validated the reduction in collagen deposition induced by each experimental compound evaluated. Although this has previously been observed with conventional histological methods, the S-FTIR technique has the advantage of allowing identification of the type of collagen present. More generally, our findings highlight the potential utility of S-FTIR and FPA-FTIR imaging techniques in enabling a better mechanistic understanding of novel asthma therapeutics.

Serelaxin activates eNOS, suppresses inflammation, attenuates developmental delay and improves cognitive functions of neonatal rats after germinal matrix hemorrhage.

Germinal matrix hemorrhage (GMH) is a detrimental form of neonatal CNS injury. Following GMH-mediated eNOS inhibition, inflammation arises, contributing to GMH-induced brain injury. We investigated the beneficial effects of Serelaxin, a clinical tested recombinant Relaxin-2 protein, on brain injury after GMH in rats. We investigated whether effects of Serelaxin are mediated by its ability to activate the GMH-suppressed eNOS pathway resulting in attenuation of inflammatory marker overproduction. GMH was induced by intraparenchymal injection of bacterial collagenase (0.3U). Seven day old Sprague-Dawley rat pups (P7) were used (n = 63). GMH animals were divided in vehicle or serelaxin treated (3 µg once, 30 µg once, 30 µg multiple, i.p., starting 30 after GMH and then daily). Sham operated animals were used. We monitored the developmental profile working memory and spatial function (T-maze and open field test respectively). At day 28, all rats underwent MRI-scans for assessment of changes in cortical thickness and white matter loss. Effects of Serelaxin on eNOS pathway activation and post-GMH inflammation were evaluated. We demonstrated that Serelaxin dose-dependently attenuated GMH-induced developmental delay, protected brain and improved cognitive functions of rats after GMH. That was associated with the decreased post-GMH inflammation, mediated at least partly by amelioration of GMH-induced eNOS inhibition.

Altered Cerebral Blood Flow and Potential Neuroprotective Effect of Human Relaxin-2 (Serelaxin) During Hypoxia or Severe Hypovolemia in a Sheep Model.

Specific neuroprotective strategies to minimize cerebral damage caused by severe hypoxia or hypovolemia are lacking. Based on previous studies showing that relaxin-2/serelaxin increases cortical cerebral blood flow, we postulated that serelaxin might provide a neuroprotective effect. Therefore, we tested serelaxin in two emergency models: hypoxia was induced via inhalation of 5% oxygen and 95% nitrogen for 12 min; thereafter, the animals were reoxygenated. Hypovolemia was induced and maintained for 20 min by removal of 50% of the total blood volume; thereafter, the animals were retransfused. In each damage model, the serelaxin group received an intravenous injection of 30 µg/kg of serelaxin in saline, while control animals received saline only. Blood gases, shock index values, heart frequency, blood pressure, and renal blood flow showed almost no significant differences between control and treatment groups in both settings. However, serelaxin significantly blunted the increase of lactate during hypovolemia. Serelaxin treatment resulted in significantly elevated cortical cerebral blood flow (CBF) in both damage models, compared with the respective control groups. Measurements of the neuroproteins S100B and neuron-specific enolase in cerebrospinal fluid revealed a neuroprotective effect of serelaxin treatment in both hypoxic and hypovolemic animals, whereas in control animals, neuroproteins increased during the experiment. Western blotting showed the expression of relaxin receptors and indicated region-specific differences in relaxin receptor-mediated signaling in cortical and subcortical brain arterioles, respectively. Our findings support the hypothesis that serelaxin is a potential neuroprotectant during hypoxia and hypovolemia. Due to its preferential improvement of cortical CBF, serelaxin might reduce cognitive impairments associated with these emergencies.

A phase 2 randomised controlled trial of serelaxin to lower portal pressure in cirrhosis (STOPP).

BACKGROUND: In preclinical models, recombinant human relaxin-2 (serelaxin) had anti-fibrotic effects and ameliorated portal hypertension (PH). A small exploratory study in patients with cirrhosis also suggested that serelaxin could reduce portal pressure. METHODS: In a phase 2, double-blind, randomised controlled study conducted in a single centre (Royal Infirmary of Edinburgh, UK), male and female adult participants with cirrhosis and clinically significant PH (CSPH; hepatic venous pressure gradient (HVPG) > 10 mmHg) were enrolled. Participants were allocated to serelaxin or placebo in a 3:1 ratio. The placebo was matched to serelaxin on appearance and administration protocol to create and maintain blinding. The primary endpoint was the change from baseline in fasting HVPG after 2 h of peripheral i.v. serelaxin infusion (80 µg/kg/day for 60 min followed by 30 µg/kg/day for at least 60 min). Secondary endpoints included the change from baseline in hepatic blood flow and systemic haemodynamics (cardiac index, systemic vascular resistance index and aortic pulse wave velocity). Short-term safety and tolerability of serelaxin were assessed. RESULTS: A total of 17 participants were screened, 15 were randomised and 11 completed the study (n = 9 serelaxin, n = 2 placebo). Reasons for withdrawal were baseline HVPG < 10 mmHg (n = 2) and technical failure (n = 2). The trial ended early due to manufacturer discontinuation of the study drug. The median age was 56 (range 43-69) years and 73% of participants were male. Alcohol was the commonest cirrhosis aetiology (n = 10). Participants had a median Model for End-Stage Liver Disease score of 10 (range 6-14). The mean baseline HVPG was 16.3 (range 10.3-21.7) mmHg. Individual responses were variable, but overall there was no statistically significant change in HVPG after 2 h of i.v. serelaxin (arithmetic mean of difference ± SD was 0.4 ± 3.5 mmHg (95% CI -2.3, 3.1; p = 0.76)). There were also no substantial changes from baseline in hepatic or systemic haemodynamics. We recorded 12 adverse events in 7 participants treated with serelaxin; none were significant, and most were unrelated to the investigational medicinal product. There were no serious adverse events. CONCLUSION: In a small randomised, phase 2, proof-of-concept study in patients with cirrhosis and CSPH, serelaxin infusion was safe and well-tolerated but had a neutral effect on HVPG. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02669875. Registered on 1 February 2016.

Relaxin enhances bone regeneration with BMP-2-loaded hydroxyapatite microspheres.

Our aims were to 1) evaluate the capacity of hollow hydroxyapatite (HA) microspheres (212-250 µm) to serve as a delivery system for controlled release of BMP-2 in vitro and 2) examine relaxin as an enhancer of BMP-2 for bone regeneration. Hollow HA microspheres were converted from borate glass microspheres and characterized using X-ray diffraction, Fourier-transform infrared spectroscopy, scanning electron microscopy, and the Brunauer-Emmett-Teller method. The microspheres loaded with BMP-2 and relaxin were implanted for 6 weeks in Sprague Dawley rats with calvarial defects. BMP-2 alone in the range up to 1 µg per defect exhibited dose-dependent bone regeneration while relaxin alone in the range up to 0.25 µg per defect did not promote bone regeneration. When compared with BMP-2 alone (1 µg per defect), a 50% reduction in the BMP-2 dose was achieved with the addition of 0.05, 0.1, or 0.25 µg of relaxin per defect. These results show that loading HA microspheres with a combination of relaxin and BMP-2 can significantly reduce the BMP-2 dose required to regenerate an equivalent amount of bone.

Relaxin reverses maladaptive remodeling of the aged heart through Wnt-signaling.

Healthy aging results in cardiac structural and electrical remodeling that increases susceptibility to cardiovascular diseases. Relaxin, an insulin-like hormone, suppresses atrial fibrillation, inflammation and fibrosis in aged rats but the mechanisms-of-action are unknown. Here we show that relaxin treatment of aged rats reverses pathological electrical remodeling (increasing Nav1.5 expression and localization of Connexin43 to intercalated disks) by activating canonical Wnt signaling. In isolated adult ventricular myocytes, relaxin upregulated Nav1.5 (EC50 = 1.3 nM) by a mechanism inhibited by the addition of Dickkopf-1. Furthermore, relaxin increased the levels of connexin43, Wnt1, and cytosolic and nuclear ß-catenin. Treatment with Wnt1 or CHIR-99021 (a GSK3ß inhibitor) mimicked the relaxin effects. In isolated fibroblasts, relaxin blocked TGFß-induced collagen elevation in a Wnt dependent manner. These findings demonstrate a close interplay between relaxin and Wnt-signaling resulting in myocardial remodeling and reveals a fundamental mechanism of great therapeutic potential.

Relaxin inhibits patellar tendon healing in rats: a histological and biochemical evaluation.

BACKGROUND: Female patients are more likely to have tendon injuries than males, especially those who has a higher concentration of relaxin. Previous studies have demonstrated that relaxin attenuates extracellular matrix (ECM) formation. However, the mechanism of relaxin on tendon repair remains unclear. We hypothesize that relaxin inhibits tendon healing by disrupting collagen synthesis. METHODS: A patellar tendon window defect model was established using Sprague-Dawley rats. The center of the patellar tendon was removed from the patella distal apex and inserted to the tibia tuberosity in width of 1 mm. Then, the rats were injected with saline (0.2 µg/kg/day) or relaxin (0.2 µg/kg/day) for two and four weeks, which was followed by biomechanical analysis and histological and histochemical examination. RESULTS: Mechanical results indicated that relaxin induces a significant decrease in tear resistance, stiffness, and Young's modulus compared to those rats without relaxin treatment. In addition, it was shown that relaxin activates relaxin family peptide receptor 1(RXFP1), disturbs the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteases (TIMPs), and reduces the deposition of collagen in injury areas. CONCLUSIONS: Relaxin impairs tendon healing in rats. Also, relaxin might lead to tendon injury more commonly for females than males.

Intraarticular injection of relaxin-2 alleviates shoulder arthrofibrosis.

Arthrofibrosis is a prevalent condition affecting greater than 5% of the general population and leads to a painful decrease in joint range of motion (ROM) and loss of independence due to pathologic accumulation of periarticular scar tissue. Current treatment options are limited in effectiveness and do not address the underlying cause of the condition: accumulation of fibrotic collagenous tissue. Herein, the naturally occurring peptide hormone relaxin-2 is administered for the treatment of adhesive capsulitis (frozen shoulder) and to restore glenohumeral ROM in shoulder arthrofibrosis. Recombinant human relaxin-2 down-regulates type I collagen and α smooth muscle actin production and increases intracellular cAMP concentration in human fibroblast-like synoviocytes, consistent with a mechanism of extracellular matrix degradation and remodeling. Pharmacokinetic profiling of a bolus administration into the glenohumeral joint space reveals the brief systemic and intraarticular (IA) half-lives of relaxin-2: 0.96 h and 0.62 h, respectively. Furthermore, using an established, immobilization murine model of shoulder arthrofibrosis, multiple IA injections of human relaxin-2 significantly improve ROM, returning it to baseline measurements collected before limb immobilization. This is in contrast to single IA (sIA) or multiple i.v. (mIV) injections of relaxin-2 with which the ROM remains constrained. The histological hallmarks of contracture (e.g., fibrotic adhesions and reduced joint space) are absent in the animals treated with multiple IA injections of relaxin-2 compared with the untreated control and the sIA- and mIV-treated animals. As these findings show, local delivery of relaxin-2 is an innovative treatment of shoulder arthrofibrosis.

Efficacy and safety of serelaxin when added to standard of care in patients with acute heart failure: results from a PROBE study, RELAX-AHF-EU.

AIM: Serelaxin is a recombinant human relaxin-2 hormone, which confers receptor-mediated vasodilatation in a tissue-specific fashion. The RELAX-AHF-EU study assessed the effect of serelaxin when added to standard-of-care (SoC) therapy on worsening heart failure (WHF)/all-cause death through Day 5 in patients hospitalised for acute heart failure (AHF) in Europe. METHODS AND RESULTS: This multicentre, prospective, randomised, open-label, blinded-endpoint validation study enrolled hospitalised AHF patients and randomised (2:1) eligible patients (mild-to-moderate renal impairment and systolic blood pressure ≥ 125 mmHg) within 16 h of presentation with signs/symptoms of AHF, to receive 48 h intravenous infusion of 30 µg/kg/day serelaxin + SoC or SoC alone. The primary endpoint was adjudicated WHF/all-cause death through Day 5. Of 3183 patients targeted, 2666 were randomised when the study was terminated early by the sponsor due to the neutral results of the pivotal RELAX-AHF-2 study. Adjudicated WHF/all-cause death through Day 5 was significantly reduced in the serelaxin + SoC vs. SoC group (5.0% vs. 6.9%; hazard ratio 0.71; 95% confidence interval 0.51-0.98; P = 0.0172) (absolute risk reduction 1.9%, number needed to treat 53). The difference between treatment groups was not significant for WHF/all-cause death/heart failure rehospitalisation through Day 14 and length of hospital stay. A significantly smaller proportion of patients in the serelaxin + SoC vs. SoC group experienced persistent heart failure signs/symptoms at each visit until Day 4, or renal deterioration through Day 5 (all P ≤ 0.01). Overall incidence of treatment-emergent adverse events was comparable between treatment groups. Hypotension and decrease in haemoglobin/haematocrit were more frequent in the serelaxin + SoC group. CONCLUSION: When added to SoC, serelaxin reduced adjudicated WHF or all-cause death through Day 5 in AHF patients. The results from this open-label study should be considered in the context of the totality of the double-blind, randomised evidence on serelaxin in AHF.

Nano-targeted relaxin impairs fibrosis and tumor growth in pancreatic cancer and improves the efficacy of gemcitabine in vivo.

Cancer-associated fibroblasts (CAFs), are the key effector cells in pancreatic ductal adenocarcinoma (PDAC), known to induce tumor growth and progression. Pancreatic stellate cells (PSCs) are the precursors of CAFs in PDAC that secrete abundant extracellular matrix, growth factors and cytokines. In this study, we targeted human relaxin-2 (RLX), an endogenous hormone, to PSCs to inhibit their differentiation into CAF-like myofibroblasts. RLX significantly inhibited TGF-ß induced PSCs differentiation by inhibiting pSmad2 signaling pathway. In vitro in primary human PSCs (hPSCs), treatment with RLX dose-dependently inhibited the migration, contraction, and protein expression of alpha smooth muscle actin and collagen I These data demonstrate that RLX can regulate hPSCs activation in vitro. However, RLX has several drawbacks i.e. poor pharmacokinetics and systemic vasodilation, that limits its preclinical and clinical application. Thus, we designed and successfully synthesized a nanoparticle system by chemically conjugating RLX to superparamagnetic iron oxide nanoparticle (SPION) to improve its pharmacokinetics. Interestingly, we found RLX-SPION to be more efficacious compared to free RLX in vitro. Significantly, we observed RLX-SPION retarded the tumor growth by itself and also potentiated the effect of gemcitabine in a subcutaneous co-injection (Panc1 and hPSCs) tumor model. The treatment resulted in significant inhibition in tumor growth, which was attributed to reduced collagen I (ECM), desmin (hPSC marker) and CD31 (endothelial marker) expression. In contrast, free RLX showed no significant effects. Altogether, this study presents a novel therapeutic approach against tumor stroma using RLX-SPION to achieve an effective treatment against pancreatic tumor.

Population pharmacokinetics of serelaxin in patients with acute or chronic heart failure, hepatic or renal impairment, or portal hypertension and in healthy subjects.

AIMS: Serelaxin is a recombinant human relaxin-2 peptide being developed for the treatment of acute heart failure (AHF). The present analyses aimed to evaluate serelaxin pharmacokinetics following intravenous administration and to identify covariates that may explain pharmacokinetic variability in healthy subjects and patients. METHODS: Serum concentration-time data for 613 subjects from nine phase I and II studies were analysed using a nonlinear mixed-effects model to estimate population pharmacokinetics and identify significant covariates. A quantile regression analysis was also conducted to assess the relationship between clearance and covariates by including sparse data from a phase III study. RESULTS: A three-compartment disposition model was established to describe serelaxin pharmacokinetics. Three out of 23 covariates, including baseline body mass index (BMI) and estimated glomerular filtration rate (eGFR) and study A1201, were identified as significant covariates for clearance but with a moderate impact on steady-state concentration, reducing the intersubject variability from 44% in the base model to 41% in the final model with covariates. The steady-state volume of distribution (Vss) was higher in patients with AHF (544 ml kg-1 ) or chronic heart failure (434 ml kg-1 ), compared with typical nonheart failure subjects (347 ml kg-1 ). Quantile regression analysis showed that a 20% increase in BMI or a 20% decrease in eGFR decreased serelaxin clearance by 9.2% or 5.2%, respectively. CONCLUSIONS: Patients with HF showed higher Vss but similar clearance (and therefore steady-state exposure) vs. non nonheart failure subjects. BMI and eGFR were identified as the main covariates explaining intersubject variability in clearance; however, the impact of these covariates on steady-state concentration was moderate and therefore unlikely to be clinically relevant.

Site enrollment rate, outcomes, and study drug effects in a multicenter trial. Results from RELAX-AHF.

BACKGROUND: Site selection is critical in acute heart failure trials. We assessed whether the enrollment rate per site affects patients' characteristics, outcomes and treatment response. METHODS AND RESULTS: A total of 1161 patients enrolled at 96 sites in the RELAX-AHF trial (serelaxin vs placebo) were included. Annualized enrollment rate was calculated as the total number of patients enrolled at each site divided by time that the site was open (patients per year). Sites were classified in low (<10), medium (10-20) and high enrolling sites (>20 patients per site/year) and were compared for prognosis and serelaxin effect. High enrolling sites were more prevalent in Eastern Europe and Israel. Time from hospital admission to randomization was shorter in high enrolling sites (6.3±4.4h>20 patients sites versus 8.7±4.5h for <10 patients sites; p<0.0001). Patients had slightly fewer comorbidities, lower levels of natriuretic peptides and creatinine and more severe pulmonary congestion in high enrolling sites. Use of evidence-based therapies was higher in high enrolling sites. The rates of worsening heart failure to day 5, 180-day cardiovascular and all-cause mortality and 60-day heart failure/renal failure rehospitalization or cardiovascular death, were similar across study groups even after adjustment for covariates. The effects of serelaxin on these outcomes did not differ by enrollment rate. CONCLUSIONS: Characteristics of RELAX-AHF study patients enrolled in high versus low enrolling sites differed only slightly and there were no differences in outcomes. Differences in serelaxin effects by enrollment rate were not discernible.

Relaxin-mediated renal vasodilation in the rat is associated with falls in glomerular blood pressure.

Relaxin (RLX) is a pleiotropic peptide hormone with marked renal vasodilatory actions that are physiologically important during pregnancy. RLX also has potent antifibrotic actions and is being tested therapeutically in various fibrotic diseases, including chronic kidney disease (CKD). Since renal vasodilation may expose the glomerulus to increased blood pressure [glomerular capillary pressure (PGC)], which exacerbates progression of CKD, we assessed the glomerular hemodynamic actions of acute (0.89 µg·100 g body wt-1·h-1 iv over 75 min) and chronic (1.5 µg·100 g body wt-1·h-1 sc) administration of RLX. Both acute and chronic RLX produced marked renal vasodilation and increased renal plasma flow (RPF) in euvolemic, anesthetized male rats. Glomerular filtration rate also increased with RLX, but the magnitude of the rise was much less than the increase in RPF due to concomitant decreases in filtration fraction. The fall in filtration fraction was the result of significant decreases in PGC, despite a slight increase in mean arterial blood pressure (MAP) with acute RLX and no net change in MAP with chronic RLX. This fall in PGC occurred because of the "in-series" arrangement of the afferent and efferent arteriolar resistance vessels, which can regulate PGC independently of MAP. With both acute and chronic RLX, efferent arteriolar resistance vessels relaxed to a greater extent than afferent arteriolar resistance vessels, thus producing falls in PGC. Based on this finding, RLX has a beneficial hemodynamic impact on the kidney, which, together with the antifibrotic actions of RLX, suggests a strong therapeutic potential for use in CKD.

Chronic lower-dose relaxin administration protects from arrhythmia in experimental myocardial infarction due to anti-inflammatory and anti-fibrotic properties.

BACKGROUND: The peptide hormone relaxin-2 (RLX) exerts beneficial effects during myocardial ischemia, but functional data on lower-dose RLX in myocardial infarction (MI) is lacking. Therefore, we investigated the impact of 75µg/kg/d RLX treatment on electrical vulnerability and left ventricular function in a mouse model of MI. METHODS AND RESULTS: Standardized cryoinfarction of the left anterior ventricular wall was performed in mice. A two week treatment period with vehicle or RLX via subcutaneously implanted osmotic minipumps was started immediately after MI. The relaxin receptor RXFP1 was expressed on ventricular/atrial cardiomyocytes, myofibroblasts, macrophages and endothelial but not vascular smooth muscle cells of small coronary vessels. RLX treatment resulted in a significant reduction of ventricular tachycardia inducibility (vehicle: 91%, RLX: 18%, p<0.0001) and increased epicardial conduction velocity in the left ventricle and borderzone. Furthermore, left ventricular function following MI was improved in RLX treated mice (left ventricular ejection fraction; vehicle: 41.1±1.9%, RLX: 50.5±3.5%, p=0.04). Interestingly, scar formation was attenuated by RLX with decreased transcript expression of connective tissue growth factor. Transcript levels of the pro-inflammatory cytokines interleukin-6 and interleukin-1ß were upregulated in hearts of vehicle treated animals compared to mice without MI. Application of RLX attenuated this inflammatory response. In addition, macrophage infiltration was reduced in the borderzone of RLX treated mice. CONCLUSION: Treatment with lower-dose RLX in mice prevents post-infarction ventricular tachycardia due to attenuation of scar formation and cardiac inflammation. Therefore, RLX could be evaluated as new therapeutic option in the treatment of MI.