Identification of Novel Series of Potent and Selective Relaxin Family Peptide Receptor 1 (RXFP1) Agonists.

Relaxin H2 is a clinically relevant peptide agonist for relaxin family peptide receptor 1 (RXFP1), but a combination of this hormone's short plasma half-life and the need for injectable delivery limits its therapeutic potential. We sought to overcome these limitations through the development of a potent small molecule (SM) RXFP1 agonist. Although two large SM HTS campaigns failed in identifying suitable hit series, we uncovered novel chemical space starting from the only known SM RXFP1 agonist series, represented by ML290. Following a design-make-test-analyze strategy based on improving early dose to man ranking, we discovered compound 42 (AZ7976), a highly selective RXFP1 agonist with sub-nanomolar potency. We used AZ7976, its 10 000-fold less potent enantiomer 43 and recombinant relaxin H2 to evaluate in vivo pharmacology and demonstrate that AZ7976-mediated heart rate increase in rats was a result of RXFP1 agonism. As a result, AZ7976 was selected as lead for continued optimization.

Discovery of Clinical Candidate AZD5462, a Selective Oral Allosteric RXFP1 Agonist for Treatment of Heart Failure.

Optimization of the highly potent and selective, yet metabolically unstable and poorly soluble hRXFP1 agonist AZ7976 led to the identification of the clinical candidate, AZD5462. Assessment of RXFP1-dependent cell signaling demonstrated that AZD5462 activates a highly similar panel of downstream pathways as relaxin H2 but does not modulate relaxin H2-mediated cAMP second messenger responsiveness. The therapeutic potential of AZD5462 was assessed in a translatable cynomolgus monkey heart failure model. Following 8 weeks of treatment with AZD5462, robust improvements in functional cardiac parameters including LVEF were observed at weeks 9, 13, and 17 without changes in heart rate or mean arterial blood pressure. AZD5462 was well tolerated in both rat and cynomolgus monkey and has successfully completed phase I studies in healthy volunteers. In summary, AZD5462 is a small molecule pharmacological mimetic of relaxin H2 signaling at RXFP1 and holds promise as a potential therapeutic approach to treat heart failure patients.

Suppression of endothelial P-selectin expression contributes to reduced cell trafficking in females: an effect independent of NO and prostacyclin.

OBJECTIVE: Sex hormones underlie the lower incidence of cardiovascular disease in premenopausal women. Vascular inflammation is involved in the pathogenesis of several cardiovascular diseases and it has been reported that sex hormones modulate inflammatory responses but mechanisms responsible for these effects are not yet fully established. Herein, we assessed whether sex differences in leukocyte recruitment might exist and investigated the underlying mechanisms involved in this response. METHODS AND RESULTS: Treatment with interleukin-1ß (IL-1ß) or tumor necrosis factor-α caused leukocyte rolling, adhesion, and emigration in mesenteric postcapillary venules in vivo that was substantially reduced in female mice compared with male mice; this difference was abolished by ovariectomy and partially restored by estrogen replacement. Deletion of endothelial nitric oxide (NO) synthase or cyclooxygenase-1 alone or in combination did not alter the leukocyte recruitment in IL-1ß-treated females but significantly enhanced this response in male mice. Treatment of murine pulmonary endothelial cells with IL-1ß increased expression of P-selectin in male but not female cells. CONCLUSIONS: We have demonstrated a profound estrogen-dependent and NO and prostacyclin-independent suppression of leukocyte recruitment in females.

Definitive role for natriuretic peptide receptor-C in mediating the vasorelaxant activity of C-type natriuretic peptide and endothelium-derived hyperpolarising factor.

OBJECTIVE: C-type natriuretic peptide (CNP) has recently been suggested to represent an endothelium-derived hyperpolarising factor (EDHF) in the mammalian resistance vasculature and, as such, important in the regulation of local blood flow and systemic blood pressure. Additionally, this peptide has been shown to protect against ischaemia-reperfusion injury and inhibits leukocyte and platelet activation. Herein, we use a novel, selective natriuretic peptide receptor-C (NPR-C) antagonist (M372049) to highlight the pivotal contribution of CNP/NPR-C signalling in the EDHF-dependent regulation of vascular tone and investigate the mechanism(s) underlying the release and biological activity of CNP. METHODS: In vitro pharmacological investigation was conducted in rat (Sprague-Dawley) aorta and mesenteric resistance arteries. Relaxant responses to CNP, atrial natriuretic peptide (ANP), the nitric oxide donor spermine-NONOate (SPER-NO) and the endothelium-dependent vasodilator, acetylcholine (ACh) were examined in the absence and presence of M372049 or inhibitor cocktails shown previously to block endothelium-dependent dilatation in the resistance vasculature. RT-PCR was employed to characterize the expression of NPR subtypes in the vessels studied. RESULTS: M372049 produced concentration-dependent inhibition of the vasorelaxant activity of CNP in rat isolated mesenteric resistance arteries but not aorta; in contrast, M372049 did not affect relaxations to ANP or SPER-NO in either vessel. M372049 or ouabain alone produced small, significant inhibition of EDHF-dependent relaxations in mesenteric arteries and in combination acted synergistically to abolish such responses. A combination of M372049 with established inhibitors of EDHF-dependent relaxation revealed that multiple, distinct pathways coordinate the bioactivity of EDHF in the resistance vasculature, and that CNP/NPR-C signalling represents a major component. CONCLUSIONS: These data substantiate CNP/NPR-C signalling as a fundamental pathway underlying EDHF-dependent regulation of vascular tone in the rat mesenteric resistance vasculature. An increased understanding of the physiological roles of CNP/NPR-C signalling in the vasculature (now facilitated by the identification of a selective NPR-C antagonist) should aid determination of the (patho)physiological importance of EDHF and might provide the rationale for the design of novel therapeutics.

Characterisation of preproendothelin-1 derived peptides identifies Endothelin-Like Domain Peptide as a modulator of Endothelin-1.

Endothelin-1 (ET-1) is involved in the pathogenesis of cardiac and renal diseases, and in the progression of tumour growth in cancer, but current diagnosis and treatment remain inadequate. Peptides derived from the 212 amino acid precursor preproendothelin-1 (ppET-1) may have utility as biomarkers, or cause biological effects that are unaffected by endothelin receptor antagonists. Here, we used specific immunoassays and LC-MS/MS to identify NT-proET-1 (ppET-1[18-50]), Endothelin-Like Domain Peptide (ELDP, ppET-1[93-166]) and CT-proET-1 (ppET-1[169-212]) in conditioned media from cultured endothelial cells. Synthesis of these peptides correlated with ET-1, and plasma ELDP and CT-proET-1 were elevated in patients with chronic heart failure. Clearance rates of NT-proET-1, ELDP and CT-proET-1 were determined after i.v. injection in anaesthetised rats. CT-proET-1 had the slowest systemic clearance, hence providing a biological basis for it being a better biomarker of ET-1 synthesis. ELDP contains the evolutionary conserved endothelin-like domain sequence, which potentially confers biological activity. On isolated arteries ELDP lacked direct vasoconstrictor effects. However, it enhanced ET-1 vasoconstriction and prolonged the increase in blood pressure in anaesthetised rats. ELDP may therefore contribute to disease pathogenesis by augmenting ET-1 responses.

Quercetin downregulates NADPH oxidase, increases eNOS activity and prevents endothelial dysfunction in spontaneously hypertensive rats.

BACKGROUND AND OBJECTIVE: Several studies have found that chronic treatment with the dietary flavonoid quercetin lowers blood pressure and restores endothelial dysfunction in hypertensive animal models. We hypothesized that increased endothelial nitric oxide synthase (eNOS) and/or decreased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase protein expression and activity, and reduced reactive oxygen species might be involved in the improvement of endothelial function induced by quercetin in spontaneously hypertensive rats (SHR). DESIGN AND METHODS: Male SHR and Wistar-Kyoto (WKY) rats (5 weeks old) were treated with quercetin (10 mg/kg) or vehicle for 13 weeks. Changes in vascular expression of eNOS, caveolin-1 and p47 were analysed by Western blot, eNOS activity by conversion of [H]arginine to L-[H]citrulline, and NADPH oxidase activity by NADPH-enhanced chemoluminescence of lucigenin. RESULTS: In SHR, quercetin reduced the increase in blood pressure and heart rate and enhanced the endothelium-dependent aortic vasodilation induced by acetylcholine, but had no effect on the endothelium-independent response induced by nitroprusside. However, quercetin had no effect on endothelium-dependent vasoconstriction and aortic thromboxane B2 production. Compared to WKY, SHR showed upregulated eNOS and p47 protein expression, downregulated caveolin-1 expression, increased NADPH-induced superoxide production but, paradoxically, eNOS activity was reduced. Chronic quercetin treatment prevented all these changes in SHR. In WKY, quercetin had no effect on blood pressure, endothelial function or the expression or activity of the proteins analysed. CONCLUSIONS: Enhanced eNOS activity and decreased NADPH oxidase-mediated superoxide anion (O2) generation associated with reduced p47 expression appear to be essential mechanisms for the improvement of endothelial function and the antihypertensive effects of chronic quercetin.

Functional pharmacological characterization of SER100 in cardiovascular health and disease.

BACKGROUND AND PURPOSE: SER100 is a selective nociceptin (NOP) receptor agonist with sodium-potassium-sparing aquaretic and anti-natriuretic activity. This study was designed to characterize the functional cardiovascular pharmacology of SER100 in vitro and in vivo, including experimental models of cardiovascular disease. EXPERIMENTAL APPROACH: Haemodynamic, ECG parameters and heart rate variability (HRV) were determined using radiotelemetry in healthy, conscious mice. The haemodynamic and vascular effects of SER100 were also evaluated in two models of cardiovascular disease, spontaneously hypertensive rats (SHR) and murine hypoxia-induced pulmonary hypertension (PH). To elucidate mechanisms underlying the pharmacology of SER100, acute blood pressure recordings were performed in anaesthetized mice, and the reactivity of rodent aorta and mesenteric arteries in response to electrical- and agonist-stimulation assessed. KEY RESULTS: SER100 caused NOP receptor-dependent reductions in mean arterial blood pressure and heart rate that were independent of NO. The hypotensive and vasorelaxant actions of SER100 were potentiated in SHR compared with Wistar Kyoto. Moreover, SER100 reduced several indices of disease severity in experimental PH. Analysis of HRV indicated that SER100 decreased the low/high frequency ratio, an indicator of sympatho-vagal balance, and in electrically stimulated mouse mesenteric arteries SER100 inhibited sympathetic-induced contractions. CONCLUSIONS AND IMPLICATIONS: SER100 exerts a chronic hypotensive and bradycardic effects in rodents, including models of systemic and pulmonary hypertension. SER100 produces its cardiovascular effects, at least in part, by inhibition of cardiac and vascular sympathetic activity. SER100 may represent a novel therapeutic candidate in systemic and pulmonary hypertension.

Distinct endothelial pathways underlie sexual dimorphism in vascular auto-regulation.

BACKGROUND AND PURPOSE: Pre-menopausal females have a lower incidence of cardiovascular disease compared with age-matched males, implying differences in the mechanisms and pathways regulating vasoactivity. In small arteries, myogenic tone (constriction in response to raised intraluminal pressure) is a major determinant of vascular resistance. Endothelium-derived dilators, particularly NO, tonically moderate myogenic tone and, because the endothelium is an important target for female sex hormones, we investigated whether NO-mediated moderation of myogenic tone differed between the sexes. EXPERIMENTAL APPROACH: Pressure-diameter or relaxation concentration-response curves to the NO donor spermine-NO or soluble guanylate cyclase (sGC) stimulation (BAY41-2272) were constructed before and following drug intervention in murine mesenteric resistance arteries. Hypotensive responses to activators of the NO-sGC pathway were determined. Quantitative PCR and Western blotting were used for expression analysis. KEY RESULTS: NO synthase inhibition enhanced myogenic tone of arteries of both sexes while block of endothelium-derived hyperpolarizing factor (EDHF) enhanced responses in arteries of females only. Spermine-NO concentration-dependently relaxed mesenteric arteries isolated from either sex. However, while inhibition of sGC activity attenuated responses of arteries from male mice only, endothelial denudation attenuated responses of arteries from females only. BAY41-2272 and spermine-NO-induced vasodilatation and hypotension were greater in males than in females. CONCLUSIONS AND IMPLICATIONS: NO moderated myogenic tone in arteries of male mice by a sGC-dependent pathway while EDHF was the predominant endothelial regulator in arteries of females. This is a potentially important sexual dimorphism in NO-mediated reactivity and further implicates EDHF as the predominant endothelial vasodilator in female resistance arteries.

Sex differences in vascular function: implication of endothelium-derived hyperpolarizing factor.

The vascular endothelium plays a crucial role in the regulation of vascular homeostasis by controlling vascular tone, coagulation, and inflammatory responses. These actions are exerted by endothelial factors including nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF). The greater incidence of cardiovascular disease (CVD) in men and postmenopausal women compared with premenopausal women implies a vasoprotective phenotype of females, which may be influenced by sex hormones. These hormones, particularly estrogen, have modulatory effects on the endothelium and circulating cells that have been implicated in vascular inflammation and in the development of CVD. EDHF seems to be the predominant endothelial factor in the resistance vasculature of females and this mediator could afford the beneficial cardiovascular risk profile observed in premenopausal woman. In this review, we discuss sex differences in EDHF biology and how sex hormones can modulate EDHF responses. We also review the implication of sex hormone-dependent regulation of EDHF in inflammatory processes, platelet function, and repair after vascular damage, each of which have a critical role in several aspects of the pathogenesis of CVD.

Quercetin and isorhamnetin prevent endothelial dysfunction, superoxide production, and overexpression of p47phox induced by angiotensin II in rat aorta.

The dietary flavonoid quercetin reduces blood pressure and improves endothelial function in several rat models of hypertension. We analyzed the effects of quercetin and its methylated metabolite isorhamnetin on the aortic endothelial dysfunction induced by incubation with angiotensin II (AngII) in vitro for 6 h. AngII diminished the relaxant responses to acetylcholine in phenylephrine-contracted aorta. Coincubation with quercetin or isorhamnetin, or addition of superoxide (O(2)(-)) dismutase or apocynin to the assay medium, prevented these inhibitory effects. At 6 h, AngII induced a marked increase in O(2)(-) production as measured by dihydroethidium fluorescence, which was prevented by quercetin and isorhamnetin. AngII also increased the expression of p47(phox), a regulatory subunit of the membrane NADPH oxidase. Immunohistochemical analysis revealed that overexpression of p47(phox) occurred mainly in the medial layer. p47(phox) overexpression was also prevented by quercetin and isorhamnetin. Taken together, these results show for the first time, to our knowledge, that quercetin and isorhamnetin prevent AngII-induced endothelial dysfunction by inhibiting the overexpression of p47(phox) and the subsequent increased O(2)(-) production, resulting in increased nitric oxide bioavailability.