Effects of systemic and renal intramedullary endothelin-1 receptor blockade on tissue NO and intrarenal hemodynamics in normotensive and hypertensive rats.

Endothelin 1 (ET-1) seems essential in salt-dependent hypertension, and activation of ETA receptors causes renal vasoconstriction. However, the response in the renal medulla and the role of tissue NO availability has never been adequately explored in vivo. We examined effects of ETA and ETB receptor blockade (atrasentan and BQ788) on blood pressure (MAP), medullary blood flow (MBF) and medullary tissue NO. Effects of systemic and intramedullary blocker application were compared in anesthetized normotensive ET-1-pretreated Sprague-Dawley rats (S-D), in salt-dependent hypertension (HS/UNX) and in spontaneously hypertensive rats (SHR). Total renal blood flow (RBF) was measured using a Transonic renal artery probe, MBF as laser-Doppler flux, and tissue NO signal using selective electrodes. In normotensive rats ET-1 significantly increased MAP, decreased RBF (-20%) and renal medullary NO. In HS/UNX rats atrasentan decreased MAP and increased medullary NO, earlier and more profoundly with intravenous infusion. In SHR atrasentan decreased MAP, more effectively with intravenous infusion; the increase in tissue NO (∼10%) was similar with both routes; however, only intramedullary atrasentan increased MBF. No consistent responses to BQ788 were seen. We confirmed dominant role of ETA receptors in regulation of blood pressure and renal hemodynamics in normotensive and hypertensive rats and provided novel evidence for the role of ETA in control of intrarenal NO bioavailability in salt-dependent and spontaneous hypertension. Under conditions of activation of the endothelin system ETB stimulation preserved medullary perfusion.

Endothelin A and B Receptors: Potential Targets for Microcirculatory-Mitochondrial Therapy in Experimental Sepsis.

The hypoxia-sensitive endothelin (ET) system plays an important role in circulatory regulation through vasoconstrictor ETA and ETB2 and vasodilator ETB1 receptors. Sepsis progression is associated with microcirculatory and mitochondrial disturbances along with tissue hypoxia. Our aim was to investigate the consequences of treatments with the ETA receptor (ETA-R) antagonist, ETB1 receptor (ETB1-R) agonist, or their combination on oxygen dynamics, mesenteric microcirculation, and mitochondrial respiration in a rodent model of sepsis. Sprague Dawley rats were subjected to fecal peritonitis (0.6 g kg i.p.) or a sham operation. Septic animals were treated with saline or the ETA-R antagonist ETR-p1/fl peptide (100 nmol kg i.v.), the ETB1-R agonist IRL-1620 (0.55 nmol kg i.v.), or a combination therapy 22 h after induction. Invasive hemodynamic monitoring and blood gas analysis were performed during a 90-min observation, plasma ET-1 levels were determined, and intestinal capillary perfusion (CPR) was detected by intravital videomicroscopy. Mitochondrial Complex I (CI)- and CII-linked oxidative phosphorylation (OXPHOS) was evaluated by high-resolution respirometry in liver biopsies. Septic animals were hypotensive with elevated plasma ET-1. The ileal CPR, oxygen extraction (ExO2), and CI-CII-linked OXPHOS capacities decreased. ETR-p1/fl treatment increased ExO2 (by >45%), CPR, and CII-linked OXPHOS capacity. The administration of IRL-1620 countervailed the sepsis-induced hypotension (by >30%), normalized ExO2, and increased CPR. The combined ETA-R antagonist-ETB1-R agonist therapy reduced the plasma ET-1 level, significantly improved the intestinal microcirculation (by >41%), and reversed mitochondrial dysfunction. The additive effects of a combined ETA-R-ETB1-R-targeted therapy may offer a tool for a novel microcirculatory and mitochondrial resuscitation strategy in experimental sepsis.

Phase I study of the anti-endothelin B receptor antibody-drug conjugate DEDN6526A in patients with metastatic or unresectable cutaneous, mucosal, or uveal melanoma.

Background Endothelin B receptor (ETBR) is involved in melanoma pathogenesis and is overexpressed in metastatic melanoma. The antibody-drug conjugate DEDN6526A targets ETBR and is comprised of the humanized anti-ETBR monoclonal antibody conjugated to the anti-mitotic agent monomethyl auristatin E (MMAE). Methods This Phase I study evaluated the safety, pharmacokinetics, pharmacodynamics, and anti-tumor activity of DEDN6526A (0.3-2.8 mg/kg) given every 3 weeks (q3w) in patients with metastatic or unresectable cutaneous, mucosal, or uveal melanoma. Results Fifty-three patients received a median of 6 doses of DEDN6526A (range 1-49). The most common drug-related adverse events (>25% across dose levels) were fatigue, peripheral neuropathy, nausea, diarrhea, alopecia, and chills. Three patients in dose-escalation experienced a dose-limiting toxicity (infusion-related reaction, increased ALT/AST, and drug-induced liver injury). Based on cumulative safety data across all dose levels, the recommended Phase II dose (RP2D) for DEDN6526A was 2.4 mg/kg intravenous (IV) q3w. The pharmacokinetics of antibody-conjugated MMAE and total antibody were dose-proportional at doses ranging from 1.8-2.8 mg/kg. A trend toward faster clearance was observed at doses of 0.3-1.2 mg/kg. There were 6 partial responses (11%) in patients with metastatic cutaneous or mucosal melanoma, and 17 patients (32%) had prolonged stable disease ≥6 months. Responses were independent of BRAF mutation status but did correlate with ETBR expression. Conclusion DEDN6526A administered at the RP2D of 2.4 mg/kg q3w had an acceptable safety profile and showed evidence of anti-tumor activity in patients with cutaneous, mucosal, and uveal melanoma. ClinicalTrials.gov identifier: NCT01522664.

Intravitreal administration of endothelin type A receptor or endothelin type B receptor antagonists attenuates hypertensive and diabetic retinopathy in rats.

Hypertension is an independent risk factor for diabetic retinopathy, yet anti-hypertensive medications such as blockade of angiotensin II do not completely protect against vision-threatening vascular disease. We hypothesized that the potent vasoactive factor, endothelin (ET), is up-regulated in diabetic retinopathy and antagonism of the ET type A receptor (ETRA) or ET type B receptor (ETRB) ameliorates retinal vascular leakage independently of any blood pressure lowering effects. Spontaneously hypertensive rats (SHR) and their normotensive and genetic controls, Wistar Kyoto rats, were randomized to become diabetic or non-diabetic and studied for 8 weeks. Rats were further randomized to receive by intravitreal injection the ETRA antagonist, BQ123, the ETRB antagonist, BQ788, or vehicle, 5 days after the induction of streptozotocin diabetes and 4 weeks later. The treatments had no effect on systolic blood pressure which remained elevated in SHR. ET-1, ET-2, ETRA and ETRB were expressed in retina and retinal pigment epithelium (RPE)/choroid and increased by hypertension or diabetes. BQ123 reduced ET-1 and ET-2 expression in retina and RPE/choroid, while BQ788 had a similar effect but did not influence the mRNA levels of ET-1 in retina. Retinal vascular leakage and Müller cell stress as well as vascular endothelial growth factor (VEGF) expression in retina and RPE/choroid, were increased by hypertension or diabetes and there was an additive effect of these conditions. Treatment with BQ123 or BQ788 effectively reduced these events as well as the elevated levels of inflammatory factors in the retina. Our findings indicate that local ET systems exist in the retina and RPE/choroid that are up-regulated by hypertension and diabetes. The ability of locally delivered ET receptor antagonists to supress these overactive ET systems and reduce retinal vascular leakage and VEGF in the presence of hypertension indicate the potential of these approaches for the treatment of diabetic retinopathy.

Dual Endothelin-A/Endothelin-B Receptor Blockade and Cardiac Remodeling in Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Despite the increasing prevalence of heart failure with preserved ejection fraction (HFpEF) in humans, there remains no evidence-based therapies for HFpEF. Endothelin-1 (ET-1) antagonists are a possibility because elevated ET-1 levels are associated with adverse cardiovascular effects, such as arterial and pulmonary vasoconstriction, impaired left ventricular (LV) relaxation, and stimulation of LV hypertrophy. LV hypertrophy is a common phenotype in HFpEF, particularly when associated with hypertension. METHODS AND RESULTS: In the present study, we found that ET-1 levels were significantly elevated in patients with chronic stable HFpEF. We then sought to investigate the effects of chronic macitentan, a dual ET-A/ET-B receptor antagonist, on cardiac structure and function in a murine model of HFpEF induced by chronic aldosterone infusion. Macitentan caused LV hypertrophy regression independent of blood pressure changes in HFpEF. Although macitentan did not modulate diastolic dysfunction in HFpEF, it significantly reduced wall thickness and relative wall thickness after 2 weeks of therapy. In vitro studies showed that macitentan decreased the aldosterone-induced cardiomyocyte hypertrophy. These changes were mediated by a reduction in the expression of cardiac myocyte enhancer factor 2a. Moreover, macitentan improved adverse cardiac remodeling, by reducing the stiffer cardiac collagen I and titin n2b expression in the left ventricle of mice with HFpEF. CONCLUSIONS: These findings indicate that dual ET-A/ET-B receptor inhibition improves HFpEF by abrogating adverse cardiac remodeling via antihypertrophic mechanisms and by reducing stiffness. Additional studies are needed to explore the role of dual ET-1 receptor antagonists in patients with HFpEF.

Safety, efficacy, and clinical utility of macitentan in the treatment of pulmonary arterial hypertension.

Pulmonary arterial hypertension is a progressive, debilitating disease caused by a dysregulation of the pulmonary vascular tone that inevitably leads to right heart failure and death without treatment. Until relatively recently, the treatment options for those afflicted by pulmonary arterial hypertension were limited; today, a greater understanding of the pathophysiology behind this disease has led to several evidence-based therapies that can improve pulmonary function and quality of life for these patients. One of the primary mediators of pulmonary vascular tone is endothelin-1, which is a potent and long-lasting vasoconstrictor. Macitentan is a second-generation endothelin receptor antagonist that acts selectively as a pulmonary vasodilator without the significant side effects noted with previous endothelin receptor antagonists. This review focuses on the mechanism of action and pharmacokinetics of macitentan, as well as the adverse effects, efficacy, and clinical uses of macitentan in the clinical trials to date. In addition, the authors briefly review clinical trials currently underway to illustrate possible future directions for the use of macitentan.

Central mediators involved in the febrile response induced by polyinosinic-polycytidylic acid: lack of involvement of endothelins and substance P.

The present study evaluated the involvement of interleukin(IL)-1ß, tumor necrosis factor-α (TNF-α), IL-6, interferon(IFN)-γ, prostaglandins of the E2 series, endothelins, substance P and opioids within the central nervous system in polyinosinic:polycytidylic acid (Poly I:C)-induced fever in rats. Poly I:C injection induced a febrile response which was reduced by intracerebroventricular administration of the antibodies against TNF-α, IL-6, or IFN-γ, or by IL-1 or µ receptor antagonists. Intraperitoneal injection of indomethacin or oral administration of celecoxib also reduced Poly I:C-induced fever. Poly I:C increased prostaglandin E2 levels in the cerebrospinal fluid of the animals which was also reduced by indomethacin. The intracerebroventricular injection of ETB or NK1 receptor antagonists did not alter Poly I:C-induced fever. These data suggest the involvement of IL-1ß, TNF-α, IL-6, IFN-γ, prostaglandin E2, and opioids but not endothelins and substance P on Poly I:C-induced fever.

Pharmacokinetic and pharmacodynamic evaluation of macitentan , a novel endothelin receptor antagonist for the treatment of pulmonary arterial hypertension.

INTRODUCTION: Pulmonary arterial hypertension (PAH) is a chronic disorder of the pulmonary vasculature characterized by elevated mean pulmonary arterial pressure eventually leading to right-sided heart failure and premature death. Macitentan is an oral, once-daily, dual endothelin (ET)A and ETB receptor antagonist with high affinity and sustained receptor binding that was approved in the USA, Europe, Canada, and Switzerland for the treatment of PAH. AREAS COVERED: This review discusses the pharmacokinetics (PK) and pharmacodynamics (PD) of macitentan and its drug interaction potential based on preclinical and clinical data. EXPERT OPINION: Up to date, macitentan is the only registered treatment for PAH that significantly reduced morbidity and mortality as a combined endpoint in a long-term event-driven study. The safety profile of macitentan is favorable with respect to hepatic safety and edema/fluid retention and may be better than that of other ET receptor antagonists such as bosentan and ambrisentan. The PK profile supports a once-a-day dosing regimen. Macitentan has limited interactions with other drugs. Based on these characteristics macitentan is an important new addition to the treatment of PAH.

Antiallodynic effect through spinal endothelin-B receptor antagonism in rat models of complex regional pain syndrome.

Complex regional pain syndrome (CRPS) is a very complicated chronic pain disorder that has been classified into two types (I and II). Endothelin (ET) receptors are involved in pain conditions at the spinal level. We investigated the role of spinal ET receptors in CRPS. Chronic post-ischemia pain (CPIP) was induced in male Sprague-Dawley rats as a model for CRPS-I by placing a tourniquet (O-ring) at the ankle joint for 3h, and removing it to allow reperfusion. Ligation of L5 and L6 spinal nerves to induce neuropathic pain was performed as a model for CRPS-II. After O-ring application and spinal nerve ligation, the paw withdrawal threshold was significantly decreased at injured sites. Intrathecal administration of the selective ET-B receptor antagonist BQ 788 dose-dependently increased the withdrawal threshold in both CRPS-I and CRPS-II. In contrast, ET-A receptor antagonist BQ 123 did not affect the withdrawal threshold in either CRPS type. The ET-1 levels of plasma and spinal cord increased in both CRPS types. Intrathecal BQ 788 decreased the spinal ET-1 level. These results suggest that ET-1 is involved in the development of mechanical allodynia in CRPS. Furthermore, the ET-B receptor appears to be involved in spinal cord-related CRPS.

Endothelin-a receptor antagonism after renal angioplasty enhances renal recovery in renovascular disease.

Percutaneous transluminal renal angioplasty/stenting (PTRAS) is frequently used to treat renal artery stenosis and renovascular disease (RVD); however, renal function is restored in less than one half of the cases. This study was designed to test a novel intervention that could refine PTRAS and enhance renal recovery in RVD. Renal function was quantified in pigs after 6 weeks of chronic RVD (induced by unilateral renal artery stenosis), established renal damage, and hypertension. Pigs with RVD then underwent PTRAS and were randomized into three groups: placebo (RVD+PTRAS), chronic endothelin-A receptor (ET-A) blockade (RVD+PTRAS+ET-A), and chronic dual ET-A/B blockade (RVD+PTRAS+ET-A/B) for 4 weeks. Renal function was again evaluated after treatments, and then, ex vivo studies were performed on the stented kidney. PTRAS resolved renal stenosis, attenuated hypertension, and improved renal function but did not resolve renal microvascular rarefaction, remodeling, or renal fibrosis. ET-A blocker therapy after PTRAS significantly improved hypertension, microvascular rarefaction, and renal injury and led to greater recovery of renal function. Conversely, combined ET-A/B blockade therapy blunted the therapeutic effects of PTRAS alone or PTRAS followed by ET-A blockade. These data suggest that ET-A receptor blockade therapy could serve as a coadjuvant intervention to enhance the outcomes of PTRAS in RVD. These results also suggest that ET-B receptors are important for renal function in RVD and may contribute to recovery after PTRAS. Using clinically available compounds and techniques, our results could contribute to both refinement and design of new therapeutic strategies in chronic RVD.

Late dual endothelin receptor blockade with bosentan restores impaired cerebrovascular function in diabetes.

AIMS: Up-regulation of the endothelin (ET) system in type-2 diabetes increases contraction and decreases relaxation in basilar artery. We showed that 1) ET-receptor antagonism prevents diabetes-mediated cerebrovascular dysfunction; and 2) glycemic control prevents activation of the ET-system in diabetes. Here, our goal is to determine whether and to what extent glycemic control or ET-receptor antagonism reverses established cerebrovascular dysfunction in diabetes. MAIN METHODS: Non-obese type-2 diabetic Goto-Kakizaki rats were administered either vehicle, metformin (300 mg/kg/day) or dual ET-receptor antagonist bosentan (100mg/kg) for 4-weeks starting at 18-weeks after established cerebrovascular dysfunction (n=5-6/group). Control group included vehicle-treated aged-matched Wistar rats. Blood glucose and pressure were monitored weekly. At termination, basilar arteries were collected and cumulative dose-response curves to ET-1 (0.1-500 nM), 5-HT (1-1000 nM) and acetylcholine (Ach, 0.1 nM-5 µM) were studied by wire myograph. Middle cerebral artery (MCA) myogenic reactivity and tone were measured using pressurized arteriograph. KEY FINDINGS: There was no difference in ET-1 and 5-HT-mediated constrictions. Endothelium-dependent relaxation was impaired in diabetes. Bosentan improved sensitivity to Ach as well as the maximum relaxation. Myogenic-tone is decreased over the course of the disease. Both treatments improved the ability of MCAs to develop tone at 80 mm Hg and only bosentan improved the tone at higher pressures. SIGNIFICANCE: These results suggest that contractile response is not affected by glycemic control or ET-receptor antagonism. Meanwhile, dual ET-receptor blockade is effective in partially improving endothelium-dependent relaxation and myogenic response in a blood pressure-independent manner even after established cerebrovascular dysfunction and offers therapeutic potential.

Disparate effects of single endothelin-A and -B receptor blocker therapy on the progression of renal injury in advanced renovascular disease.

We hypothesized that chronic specific endothelin-A (ET-A) receptor blockade therapy would reverse renal dysfunction and injury in advanced experimental renovascular disease. To test this, unilateral renovascular disease was induced in 19 pigs, and after 6 weeks, single-kidney hemodynamics and function was quantified in vivo using computed tomography. All pigs with renovascular disease were divided such that seven were untreated, seven were treated with ET-A blockers, and five were treated with ET-B blockers. Four weeks later, all pigs were restudied in vivo, and then killed and ex vivo studies performed on the stenotic kidney to quantify microvascular density, remodeling, renal oxidative stress, inflammation, and fibrosis. Renal blood flow, glomerular filtration rate, and redox status were significantly improved in the stenotic kidney after ET-A but not ET-B blockade. Furthermore, only ET-A blockade therapy reversed renal microvascular rarefaction and diminished remodeling, which was accompanied by a marked decreased in renal inflammatory and fibrogenic activity. Thus, ET-A but not ET-B blockade ameliorated renal injury in pigs with advanced renovascular disease by stimulating microvascular proliferation and decreasing the progression of microvascular remodeling, renal inflammation, and fibrosis in the stenotic kidney. These effects were functionally consequential as ET-A blockade improved single kidney microvascular endothelial function, renal blood flow, and glomerular filtration rate, and decreased albuminuria.