The endothelin (ET) system has emerged as a novel target for hypertension treatment where a medical need persists despite availability of several pharmacological classes, including renin angiotensin system (RAS) blockers. ET receptor antagonism has demonstrated efficacy in preclinical models of hypertension, especially under low-renin conditions and in hypertensive patients. We investigated the pharmacology of aprocitentan (N-[5-(4-bromophenyl)-6-[2-[(5-bromo-2-pyrimidinyl)oxy]ethoxy]-4-pyrimidinyl]-sulfamide), a potent dual ETA/ETB receptor antagonist, on blood pressure (BP) in two models of experimental hypertension: deoxycorticosterone acetate (DOCA)-salt rats (low-renin model) and spontaneously hypertensive rats [(SHR), normal renin model]. We also compared the effect of its combination with RAS blockers (valsartan and enalapril) with that of the combination of the mineraloreceptor antagonist spironolactone with the same RAS blockers on BP and renal function in hypertensive rats. Aprocitentan was more potent and efficacious in lowering BP in conscious DOCA-salt rats than in SHRs. In DOCA-salt rats, single oral doses of aprocitentan induced a dose-dependent and long-lasting BP decrease and 4-week administration of aprocitentan dose dependently decreased BP (statistically significant) and renal vascular resistance, and reduced left ventricle hypertrophy (nonsignificant). Aprocitentan was synergistic with valsartan and enalapril in decreasing BP in DOCA-salt rats and SHRs while spironolactone demonstrated additive effects with these RAS blockers. In hypertensive rats under sodium restriction and enalapril, addition of aprocitentan further decreased BP without causing renal impairment, in contrast to spironolactone. In conclusion, ETA/ETB receptor antagonism represents a promising therapeutic approach to hypertension, especially with low-renin characteristics, and could be used in combination with RAS blockers, without increasing the risk of renal impairment.

Intratracheal administration of bleomycin induces fibrosis in the lung, which is mainly assessed by histopathological grading that is subjective. Current literature highlights the need of reproducible and quantitative pulmonary fibrosis analysis. If some quantitative studies looked at fibrosis parameters separately, none of them quantitatively assessed both aspects: lung tissue remodeling and collagenization. To ensure reliable quantification, support vector machine learning was used on digitalized images to design a fully automated method that analyzes two important aspects of lung fibrosis: (i) areas having substantial tissue remodeling with appearance of dense fibrotic masses and (ii) collagen deposition. Fibrotic masses were identified on low magnification images and collagen detection was performed at high magnification. To insure a fully automated application the tissue classifier was trained on several independent studies that were performed over a period of four years. The detection method generates two different values that can be used to quantify lung fibrosis development: (i) percent area of fibrotic masses and (ii) percent of alveolar collagen. These two parameters were validated using independent studies from bleomycin- and saline-treated animals. A significant change of these lung fibrosis quantification parameters- increased amount of fibrotic masses and increased collagen deposition- were observed upon intratracheal administration of bleomycin and subsequent significant beneficial treatments effects were observed with BIBF-1120 and pirfenidone.

Prostacyclin (PGI2) receptor (IP receptor) agonists, which are indicated for the treatment of pulmonary arterial hypertension (PAH), increase cytosolic cAMP levels and thereby inhibit pulmonary vasoconstriction, pulmonary arterial smooth muscle cell (PASMC) proliferation, and extracellular matrix synthesis. Selexipag (Uptravi, 2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide) is the first nonprostanoid IP receptor agonist, it is available orally and was recently approved for the treatment of PAH. In this study we show that the active metabolite of selexipag and the main contributor to clinical efficacy ACT-333679 (previously known as MRE-269) behaved as a full agonist in multiple PAH-relevant receptor-distal-or downstream-cellular assays with a maximal efficacy (Emax) comparable to that of the prototypic PGI2 analog iloprost. In PASMC, ACT-333679 potently induced cellular relaxation (EC50 4.3 nM) and inhibited cell proliferation (IC50 4.0 nM) as well as extracellular matrix synthesis (IC50 8.3 nM). In contrast, ACT-333679 displayed partial agonism in receptor-proximal-or upstream-cAMP accumulation assays (Emax 56%) when compared with iloprost and the PGI2 analogs beraprost and treprostinil (Emax ∼100%). Partial agonism of ACT-333679 also resulted in limited β-arrestin recruitment (Emax 40%) and lack of sustained IP receptor internalization, whereas all tested PGI2 analogs behaved as full agonists in these desensitization-related assays. In line with these in vitro findings, selexipag, but not treprostinil, displayed sustained efficacy in rat models of pulmonary and systemic hypertension. Thus, the partial agonism of ACT-333679 allows for full efficacy in amplified receptor-distal PAH-relevant readouts while causing limited activity in desensitization-related receptor-proximal readouts.

Sphingosine‐1‐phosphate receptor 1 (S1P 1 ) modulators sequester circulating lymphocytes within lymph nodes, thereby preventing potentially pathogenic autoimmune cells from exiting into the blood stream and reaching inflamed tissues. S1P 1 receptor modulation may thus offer potential to treat various autoimmune diseases. The first nonselective S1P 1‐5 receptor modulator FTY 720/fingolimod/Gilenya ® has successfully demonstrated clinical efficacy in relapsing forms of multiple sclerosis. However, cardiovascular, hepatic, and respiratory side‐effects were reported and there is a need for novel S1P 1 receptor modulators with better safety profiles. Here, we describe the discovery of cenerimod, a novel, potent and selective S1P 1 receptor modulator with unique S1P 1 receptor signaling properties and absence of broncho‐ and vasoconstrictor effects ex vivo and in vivo. Cenerimod dose‐dependently lowered circulating lymphocyte counts in rats and mice after oral administration and effectively attenuated disease parameters in a mouse experimental autoimmune encephalitis ( EAE ) model. Cenerimod has potential as novel therapy with improved safety profile for autoimmune diseases with high unmet medical need.

The P2Y 12 receptor is a validated target for prevention of major adverse cardiovascular events in patients with acute coronary syndrome. The aim of this study was to compare two direct‐acting, reversible P2Y 12 antagonists, ACT ‐246475 and ticagrelor, in a rat thrombosis model by simultaneous quantification of their antithrombotic efficacy and surgery‐induced blood loss. Blood flow velocity was assessed in the carotid artery after FeCl 3 ‐induced thrombus formation using a Doppler flow probe. At the same time, blood loss after surgical wounding of the spleen was quantified. Continuous infusions of ACT ‐246475 and ticagrelor prevented the injury‐induced reduction of blood flow in a dose‐dependent manner. High doses of both antagonists normalized blood flow and completely abolished thrombus formation as confirmed by histology. Intermediate doses restored baseline blood flow to ≥65%. However, ACT ‐246475 caused significantly less increase of blood loss than ticagrelor; the difference in blood loss was 2.6‐fold ( P < 0.01) at high doses and 2.7‐fold ( P < 0.05) at intermediate doses. Potential reasons for this unexpected difference were explored by measuring the effects of ACT ‐246475 and ticagrelor on vascular tone. At concentrations needed to achieve maximal antithrombotic efficacy, ticagrelor compared with ACT ‐246475 significantly increased carotid blood flow velocity in vivo ( P = 0.003), induced vasorelaxation of precontracted rat femoral arteries, and inhibited contraction of femoral artery induced by electrical field stimulation or by phenylephrine. Overall, ACT ‐246475 showed a significantly wider therapeutic window than ticagrelor. The absence of vasodilatory effects due to high selectivity of ACT ‐246475 for P2Y 12 provides potential arguments for the observed safety advantage of ACT ‐246475 over ticagrelor.

This article has not been copyedited and formatted. The final version may differ from this version. Abstract Endothelin receptor antagonists have been associated with fluid retention. It has been suggested that, of the two endothelin receptor subtypes, ET B receptors should not be blocked, because of their involvement in natriuresis and diuresis. Surprisingly, clinical data suggest that ET A-selective antagonists pose a greater risk of fluid overload than dual antagonists. The purpose of this study was to evaluate the contribution of each endothelin receptor to fluid retention and vascular permeability in rats. Sitaxentan and ambrisentan as ET A-selective antagonists, bosentan and macitentan as dual antagonists were used as representatives of each class, respectively. ET A-selective antagonism caused dose-dependent haematocrit/haemoglobin decrease that was prevented by ET B-selective receptor antagonism. ET A-selective antagonism led to a significant blood pressure reduction, plasma volume expansion, and more vascular permeability increase than dual antagonism. Isolated vessels experiments showed that ET A-selective antagonism increased vascular permeability via ET B receptor overstimulation. Acutely, ET A-selective but not dual antagonism activated sympathetic activity and increased plasma arginine vasopressin and aldosterone concentrations. Haematocrit/haemoglobin decrease induced by ET A-selective antagonism was reduced in Brattleboro rats and in Wistar rats treated with an arginine vasopressin receptor antagonist. Finally haematocrit/haemoglobin decrease was larger in the venous than in the arterial side, suggesting fluid redistribution. In conclusion, endothelin receptor antagonists, and particularly ET A-selective antagonists, by activating ET B receptors, favour edema formation by causing 1) fluid retention resulting from arginine vasopressin and aldosterone activation secondary to vasodilation, 2) vascular permeability increase. Plasma volume redistribution may explain the clinical observation of haematocrit/haemoglobin decrease even in the absence of signs of fluid retention.

There is an urgent unmet medical need for novel antibiotics that are effective against a broad range of bacterial species, especially multidrug resistant ones. Tetrahydropyran-based inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) display potent activity against Gram-positive pathogens and no target-mediated cross-resistance with fluoroquinolones. We report our research efforts aimed at expanding the antibacterial spectrum of this class of molecules toward difficult-to-treat Gram-negative pathogens. Physicochemical properties (polarity and basicity) were considered to guide the design process. Dibasic tetrahydropyran-based compounds such as 6 and 21 are potent inhibitors of both DNA gyrase and topoisomerase IV, displaying antibacterial activities against Gram-positive and Gram-negative pathogens (Staphylococcus aureus, Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii). Compounds 6 and 21 are efficacious in clinically relevant murine infection models.

Endothelin (ET) receptor antagonists have been associated with fluid retention. It has been suggested that, of the two endothelin receptor subtypes, ETB receptors should not be blocked, because of their involvement in natriuresis and diuresis. Surprisingly, clinical data suggest that ETA-selective antagonists pose a greater risk of fluid overload than dual antagonists. The purpose of this study was to evaluate the contribution of each endothelin receptor to fluid retention and vascular permeability in rats. Sitaxentan and ambrisentan as ETA-selective antagonists and bosentan and macitentan as dual antagonists were used as representatives of each class, respectively. ETA-selective antagonism caused a dose-dependent hematocrit/hemoglobin decrease that was prevented by ETB-selective receptor antagonism. ETA-selective antagonism led to a significant blood pressure reduction, plasma volume expansion, and a greater increase in vascular permeability than dual antagonism. Isolated vessel experiments showed that ETA-selective antagonism increased vascular permeability via ETB receptor overstimulation. Acutely, ETA-selective but not dual antagonism activated sympathetic activity and increased plasma arginine vasopressin and aldosterone concentrations. The hematocrit/hemoglobin decrease induced by ETA-selective antagonism was reduced in Brattleboro rats and in Wistar rats treated with an arginine vasopressin receptor antagonist. Finally, the decrease in hematocrit/hemoglobin was larger in the venous than in the arterial side, suggesting fluid redistribution. In conclusion, by activating ETB receptors, endothelin receptor antagonists (particularly ETA-selective antagonists) favor edema formation by causing: 1) fluid retention resulting from arginine vasopressin and aldosterone activation secondary to vasodilation, and 2) increased vascular permeability. Plasma volume redistribution may explain the clinical observation of a hematocrit/hemoglobin decrease even in the absence of signs of fluid retention.

In a previous communication we reported on the discovery of alkylamino pyridine derivatives (e.g. 1) as a new class of potent, selective and efficacious S1P1 receptor (S1PR1) agonists. However, more detailed profiling revealed that this compound class is phototoxic in vitro. Here we describe a new class of potent S1PR1 agonists wherein the exocyclic nitrogen was moved away from the pyridine ring (e.g. 11c). Further structural modifications led to the identification of novel alkylaminomethyl substituted phenyl and thienyl derivatives as potent S1PR1 agonists. These new alkylaminomethyl aryl compounds showed no phototoxic potential. Based on their in vivo efficacy and ability to penetrate the brain, the 5-alkyl-aminomethyl thiophenes appeared to be the most interesting class. Potent and selective S1PR1 agonist 20e, for instance, maximally reduced the blood lymphocyte count (LC) for 24 h after oral administration of 10 mg/kg to rat and its brain concentrations reached >500 ng/g over 24 h.

In a previous communication we reported on the discovery of aminopyridine 1 as a potent, selective and orally active S1P1 receptor agonist. More detailed studies revealed that this compound is phototoxic in vitro. As a result of efforts aiming at eliminating this undesired property, a series of alkoxy substituted pyridine derivatives was discovered. The photo irritancy factor (PIF) of these alkoxy pyridines was significantly lower than the one of aminopyridine 1 and most compounds were not phototoxic. Focused SAR studies showed, that 2-, 3-, and 4-pyridine derivatives delivered highly potent S1P1 receptor agonists. While the 2-pyridines were clearly more selective against S1PR3, the corresponding 3- or 4-pyridine analogues showed significantly longer oral half-lives and as a consequence longer pharmacological duration of action after oral administration. One of the best compounds, cyclopentoxy-pyridine 45b lacked phototoxicity, showed EC50 values of 0.7 and 140 nM on S1PR1 and S1PR3, respectively, and maximally reduced the blood lymphocyte count for at least 24 h after oral administration of 10 mg/kg to Wistar rats.

The bleomycin-induced rodent lung fibrosis model is commonly used to study mechanisms of lung fibrosis and to test potential therapeutic interventions, despite the well recognized dissimilarities to human idiopathic pulmonary fibrosis (IPF). Therefore, in this study, we sought to identify genomic commonalities between the gene expression profiles from 100 IPF lungs and 108 control lungs that were obtained from the Lung Tissue Research Consortium, and rat lungs harvested at Days 3, 7, 14, 21, 28, 42, and 56 after bleomycin instillation. Surprisingly, the highest gene expression similarity between bleomycin-treated rat and IPF lungs was observed at Day 7. At this point of maximal rat-human commonality, we identified a novel set of 12 disease-relevant translational gene markers (C6, CTHRC1, CTSE, FHL2, GAL, GREM1, LCN2, MMP7, NELL1, PCSK1, PLA2G2A, and SLC2A5) that was able to separate almost all patients with IPF from control subjects in our cohort and in two additional IPF/control cohorts (GSE10667 and GSE24206). Furthermore, in combination with diffusing capacity of carbon monoxide measurements, four members of the translational gene marker set contributed to stratify patients with IPF according to disease severity. Significantly, pirfenidone attenuated the expression change of one (CTHRC1) translational gene marker in the bleomycin-induced lung fibrosis model, in transforming growth factor-β1-treated primary human lung fibroblasts and transforming growth factor-β1-treated human epithelial A549 cells. Our results suggest that a strategy focused on rodent model-human disease commonalities may identify genes that could be used to predict the pharmacological impact of therapeutic interventions, and thus facilitate the development of novel treatments for this devastating lung disease.

Chemical evolution of mibefradil resulted in the identification of novel bridged tetrahydronaphthalene derivatives as potent T/L-type calcium channel blockers. A SAR study, in vitro and in vivo DMPK properties as well as the in vivo antihypertensive effect in rats are presented.

Recent post hoc analyses of several clinical trials with P2Y(12) antagonists showed the need for new molecules being fully efficacious as antiplatelet agents and having a reduced propensity to cause major bleeding. We have previously reported the discovery of the 2-phenylpyrimidine-4-carboxamide analogs as P2Y(12) antagonists with nanomolar potency in the disease-relevant platelet aggregation assay in human plasma. Herein we present the optimization steps that led to the discovery of clinical candidate ACT-246475 (30d). The key step was the replacement of the carboxylic acid functionality by a phosphonic acid group which delivered the most potent molecules of the program. In addition, low in vivo clearance in rat and dog was achieved for the first time. Since the bioavailability of 30d was low in rat and dog, we developed the bis((isopropoxycarbonyl)oxy)methyl ester prodrug (ACT-281959, 45). Compound 30d showed efficacy in the rat ferric chloride thrombosis model when administered intravenously as parent or orally as its prodrug 45. Moreover, 30d displays a wider therapeutic window as compared to clopidogrel in the rat surgical blood loss model.

INTRODUCTION:The goal of this study was to characterize the role of Endothelin (ET) type B receptors (ETB) on vascular function in healthy and diseased conditions and demonstrate how it affects the pharmacological activity of ET receptor antagonists (ERAs).METHODS:The contribution of the ETB receptor to vascular relaxation or constriction was characterized in isolated arteries from healthy and diseased rats with systemic (Dahl-S) or pulmonary hypertension (monocrotaline). Because the role of ETB receptors is different in pathological vis-à-vis normal conditions, we compared the efficacy of ETA-selective and dual ETA/ETB ERAs on blood pressure in hypertensive rats equipped with telemetry.RESULTS:In healthy vessels, ETB receptors stimulation with sarafotoxin S6c induced vasorelaxation and no vasoconstriction. In contrast, in arteries of rats with systemic or pulmonary hypertension, endothelial ETB-mediated relaxation was lost while vasoconstriction on stimulation by sarafotoxin S6c was observed. In hypertensive rats, administration of the dual ETA/ETB ERA macitentan on top of a maximal effective dose of the ETA-selective ERA ambrisentan further reduced blood pressure, indicating that ETB receptors blockade provides additional benefit.CONCLUSIONS:Taken together, these data suggest that in pathology, dual ETA/ETB receptor antagonism can provide superior vascular effects compared with ETA-selective receptor blockade.

Aims: We compared the efficacy of macitentan, a novel dual endothelin A/endothelin B receptor antagonist, with that of another dual endothelin receptor antagonist, bosentan, in a rat model of non-vasoreactive pulmonary hypertension (PH) with particular emphasis on right ventricular (RV) remodeling. Methods and Results: Unlike monocrotaline or hypoxic/sugen rats, bleomycin-treated rats presented a non-vasoreactive PH characterized by the absence of pulmonary dilatation to adenosine. We therefore chose the bleomycin rat model to compare the effects of the maximally effective doses of macitentan and bosentan on pulmonary vascular and RV remodeling. Macitentan (100 mg·kg−1·d−1), but not bosentan (300 mg·kg−1·d−1), significantly prevented pulmonary vascular remodeling, RV hypertrophy, and cardiomyocyte diameter increase. Cardiac protection by macitentan was associated with a significant attenuation of genes related to cell hypertrophy and extracellular matrix remodeling. Microautoradiography and high performance liquid chromatography analysis showed greater distribution of macitentan than bosentan in the RV and pulmonary tissue. Conclusions: Macitentan was more efficacious than bosentan in preventing the development of pulmonary and RV hypertrophies in a model of non-vasoreactive PH. Greater ability to distribute into the tissue could contribute to the greater structural improvement by macitentan compared with bosentan.

Selexipag, an orally available, selective prostacyclin (PGI2) receptor (IP receptor) agonist, significantly decreased pulmonary vascular resistance in patients with pulmonary arterial hypertension (PAH) in a phase 2 clinical trial. Selexipag is structurally different from PGI2. Reduced clinical

Novel antibacterial drugs that are effective against infections caused by multidrug resistant pathogens are urgently needed. In a previous report, we have shown that tetrahydropyran-based inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) display potent antibacterial activity and exhibit no target-mediated cross-resistance with fluoroquinolones. During the course of our optimization program, lead compound 5 was deprioritized due to adverse findings in cardiovascular safety studies. In the effort of mitigating these findings and optimizing further the pharmacological profile of this class of compounds, we have identified a subseries of tetrahydropyran-based molecules that are potent DNA gyrase and topoisomerase IV inhibitors and display excellent antibacterial activity against Gram positive pathogens, including clinically relevant resistant isolates. One representative of this class, compound 32d, elicited only weak inhibition of hERG K(+) channels and hNaV1.5 Na(+) channels, and no effects were observed on cardiovascular parameters in anesthetized guinea pigs. In vivo efficacy in animal infection models has been demonstrated against Staphylococcus aureus and Streptococcus pneumoniae strains.

The endothelin (ET) system is a tissular system, as the production of ET isoforms is mostly autocrine or paracrine. Macitentan is a novel dual ETA/ETB receptor antagonist with enhanced tissue distribution and sustained receptor binding properties designed to achieve a more efficacious ET receptor blockade. To determine if these features translate into improved efficacy in vivo, a study was designed in which rats with either systemic or pulmonary hypertension and equipped with telemetry were given macitentan on top of maximally effective doses of another dual ETA/ETB receptor antagonist, bosentan, which does not display sustained receptor occupancy and shows less tissue distribution.After establishing dose-response curves of both compounds in conscious, hypertensive Dahl salt-sensitive and pulmonary hypertensive bleomycin-treated rats, macitentan was administered on top of the maximal effective dose of bosentan.In hypertensive rats, macitentan 30 mg/kg further decreased mean arterial blood pressure (MAP) by 19 mm Hg when given on top of bosentan 100 mg/kg (n=9, p<0.01 vs. vehicle). Conversely, bosentan given on top of macitentan failed to induce an additional MAP decrease. In pulmonary hypertensive rats, macitentan 30 mg/kg further decreased mean pulmonary artery pressure (MPAP) by 4 mm Hg on top of bosentan (n=8, p<0.01 vs. vehicle), whereas a maximal effective dose of bosentan given on top of macitentan did not cause any additional MPAP decrease.The add-on effect of macitentan on top of bosentan in two pathological models confirms that this novel compound can achieve a superior blockade of ET receptors and provides evidence for greater maximal efficacy.