Oleg V. Evgenov

NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential

Soluble guanylate cyclase (sGC) is a key signal-transduction enzyme activated by nitric oxide (NO). Impaired bioavailability and/or responsiveness to endogenous NO has been implicated in the pathogenesis of cardiovascular and other diseases. Current therapies that involve the use of organic nitrates and other NO donors have limitations, including non-specific interactions of NO with various biomolecules, lack of response and the development of tolerance following prolonged administration. Compounds that activate sGC in an NO-independent manner might therefore provide considerable therapeutic advantages. Here we review the discovery, biochemistry, pharmacology and clinical potential of haem-dependent sGC stimulators (including YC-1, BAY 41-2272, BAY 41-8543, CFM-1571 and A-350619) and haem-independent sGC activators (including BAY 58-2667 and HMR-1766).

Soluble guanylate cyclase as an emerging therapeutic target in cardiopulmonary disease.

Soluble guanylate cyclase (sGC), a key enzyme of the nitric oxide (NO) signaling pathway, is attracting rapidly growing interest as a therapeutic target in cardiopulmonary disease, with several sGC agonists currently in clinical development. On binding of NO to a prosthetic heme group on sGC, the enzyme catalyzes synthesis of the second messenger cGMP, which produces vasorelaxation and inhibits smooth muscle proliferation, leukocyte recruitment, and platelet aggregation through a number of downstream mechanisms.1,2 Impaired NO and cGMP signaling has been implicated in the pathogenesis of cardiovascular disease, including systemic arterial and pulmonary hypertension (PH), coronary artery disease, peripheral vascular disease (including erectile dysfunction), and atherosclerosis.1,3,–,5 Organic nitrates that target the NO signaling pathway have been used to treat cardiovascular disease for >150 years. More recently, gaseous NO administered by inhalation has been approved for the treatment of persistent PH of the newborn.3,6 These agents nonetheless have several important limitations. Cardiovascular disease is associated with resistance to NO and organic nitrates.7 This may be due to the oxidative stress–induced alteration of the redox state of the prosthetic heme on sGC (from ferrous to ferric) that weakens the binding of heme to the enzyme and renders sGC unresponsive to NO.1,8 Furthermore, the long-term efficacy of organic nitrates is limited by the development of tolerance.9 Nitric oxide may also have numerous cytotoxic effects, mostly attributed to the reactive oxidant peroxynitrite (formed from the diffusion-controlled reaction of NO with superoxide).3,10 Peroxynitrite interacts with proteins and lipids, altering cellular signaling, disrupting mitochondrial function, and damaging DNA, which can eventually culminate in cellular dysfunction and/or death.3 Because the beneficial effects of NO appear to be …

Infusion of methylene blue in human septic shock: A pilot, randomized, controlled study

ObjectiveTo evaluate the effects of continuous infusion of methylene blue (MB), an inhibitor of the nitric oxide pathway, on hemodynamics and organ functions in human septic shock. DesignProspective, randomized, controlled, open-label, pilot study. SettingMultidisciplinary intensive care unit of a university hospital. PatientsTwenty patients with septic shock diagnosed <24 hrs before randomization. InterventionsPatients were randomized 1:1 to receive either MB (MB group, n = 10) or isotonic saline (control group, n = 10), adjunctive to conventional treatment. MB was administered as an intravenous bolus injection (2 mg/kg), followed 2 hrs later by infusion at stepwise increasing rates of 0.25, 0.5, 1, and 2 mg/kg/hr that were maintained for 1 hr each. During infusion, mean arterial pressure was maintained between 70 and 90 mm Hg, while attempting to reduce concurrent adrenergic support. Measurements and Main Results Hemodynamics and organ function variables were assessed over a 24-hr period, and the survival rate at day 28 was noted. Infusion of MB prevented the stroke volume and the left-ventricular stroke work indexes from falling and increased mean arterial pressure. Compared with the control group, MB reduced the requirement for norepinephrine, epinephrine, and dopamine by as much as 87%, 81%, and 40%, respectively. Oxygen delivery remained unchanged in the MB group and decreased in the control group. MB also reduced the body temperature and the plasma concentration of nitrates/nitrites. Leukocytes and organ function variables such as bilirubin, alanine aminotransferase, urea, and creatinine were not significantly affected. Platelet count decreased in both groups. Five patients treated with MB survived vs. three patients receiving conventional treatment. ConclusionsIn human septic shock, continuously infused MB counteracts myocardial depression, maintains oxygen transport, and reduces concurrent adrenergic support. Infusion of MB appears to have no significant adverse effects on the selected organ function variables.

Flagellin from gram-negative bacteria is a potent mediator of acute pulmonary inflammation in sepsis.

Flagellin is a recently identified bacterial product that elicits immune response via toll-like receptor 5. Here, we demonstrate that flagellin is an extraordinarily potent proinflammatory stimulus in the lung during sepsis. In vitro, flagellin triggers the production of interleukin (IL)-8 by human lung epithelial (A549) cells, with 50% of the maximal response obtained at a concentration of 2 × 10−14 M. Flagellin also induces the expression of ICAM-1 in vitro. Intravenous administration of flagellin to mice elicited a severe acute lung inflammation that was significantly more pronounced than following lipopolysaccharide (LPS) administration. Flagellin induced a local release of proinflammatory cytokines, the accumulation of inflammatory cells, and the development of pulmonary hyperpermeability. These effects were associated with the nuclear translocation of the transcription NF-&kgr;B in the lung. Flagellin remained active in inducing pulmonary inflammation at doses as low as 10 ng/mouse. In the plasma of patients with sepsis, flagellin levels amounted to 7.1 ± 0.1 ng/mL. Plasma flagellin levels showed a significant positive correlation with the lung injury score, with the alveolar-arterial oxygen difference as well as with the duration of the sepsis. Flagellin emerges as a potent trigger of acute respiratory complications in gram-negative bacterial sepsis.

Soluble Guanylate Cyclase Activator Reverses Acute Pulmonary Hypertension and Augments the Pulmonary Vasodilator Response to Inhaled Nitric Oxide in Awake Lambs

Background—Inhaled nitric oxide (NO) is a potent and selective pulmonary vasodilator, which induces cGMP synthesis by activating soluble guanylate cyclase (sGC) in ventilated lung regions. Carbon monoxide (CO) has also been proposed to influence smooth muscle tone via activation of sGC. We examined whether direct stimulation of sGC by BAY 41-2272 would produce pulmonary vasodilation and augment the pulmonary responses to inhaled NO or CO. Methods and Results—In awake, instrumented lambs, the thromboxane analogue U-46619 was intravenously administered to increase mean pulmonary arterial pressure to 35 mm Hg. Intravenous infusion of BAY 41-2272 (0.03, 0.1, and 0.3 mg · kg−1 · h−1) reduced mean pulmonary arterial pressure and pulmonary vascular resistance and increased transpulmonary cGMP release in a dose-dependent manner. Larger doses of BAY 41-2272 also produced systemic vasodilation and elevated the cardiac index. N&ohgr;-nitro-l-arginine methyl ester abolished the systemic but not the pulmonary vasodilator effects of BAY 41-2272. Furthermore, infusing BAY 41-2272 at 0.1 mg · kg−1 · h−1 potentiated and prolonged the pulmonary vasodilation induced by inhaled NO (2, 10, and 20 ppm). In contrast, inhaled CO (50, 250, and 500 ppm) had no effect on U-46619–induced pulmonary vasoconstriction before or during administration of BAY 41-2272. Conclusions—In lambs with acute pulmonary hypertension, BAY 41-2272 is a potent pulmonary vasodilator that augments and prolongs the pulmonary vasodilator response to inhaled NO. Direct pharmacological stimulation of sGC, either alone or in combination with inhaled NO, may provide a novel approach for the treatment of pulmonary hypertension.

CB1 cannabinoid receptors promote oxidative stress and cell death in murine models of doxorubicin-induced cardiomyopathy and in human cardiomyocytes

AIMS Here we investigated the mechanisms by which cardiovascular CB1 cannabinoid receptors may modulate the cardiac dysfunction, oxidative stress, and interrelated cell death pathways associated with acute/chronic cardiomyopathy induced by the widely used anti-tumour compound doxorubicin (DOX). METHODS AND RESULTS Both load-dependent and -independent indices of left-ventricular function were measured by the Millar pressure-volume conductance system. Mitogen-activated protein kinase (MAPK) activation, cell-death markers, and oxidative/nitrosative stress were measured by molecular biology/biochemical methods and flow cytometry. DOX induced left-ventricular dysfunction, oxidative/nitrosative stress coupled with impaired antioxidant defense, activation of MAPK (p38 and JNK), and cell death and/or fibrosis in hearts of wide-type mice (CB1(+/+)), and these effects were markedly attenuated in CB1 knockouts (CB1(-/-)). In human primary cardiomyocytes expressing CB1 receptors (demonstrated by RT-PCR, western immunoblot, and flow cytometry) DOX, likewise the CB1 receptor agonist HU210 and the endocannabinoid anandamide (AEA), induced MAPK activation and cell death. The DOX-induced MAPK activation and cell death were significantly enhanced when DOX was co-administered with CB1 agonists AEA or HU210. Remarkably, cell death and MAPK activation induced by AEA, HU210, and DOX +/- AEA/HU210 were largely attenuated by either CB1 antagonists (rimonabant and AM281) or by inhibitors of p38 and JNK MAPKs. Furthermore, AEA or HU210 in primary human cardiomyocytes triggered increased reactive oxygen species generation. CONCLUSION CB1 activation in cardiomyocytes may amplify the reactive oxygen/nitrogen species-MAPK activation-cell death pathway in pathological conditions when the endocannabinoid synthetic or metabolic pathways are dysregulated by excessive inflammation and/or oxidative/nitrosative stress, which may contribute to the pathophysiology of various cardiovascular diseases.

Soluble guanylate cyclase stimulators in pulmonary hypertension.

Soluble guanylate cyclase (sGC) is a key enzyme in the nitric oxide (NO) signalling pathway. On binding of NO to its prosthetic haem group, sGC catalyses the synthesis of the second messenger cyclic guanosine monophosphate (cGMP), which promotes vasodilation and inhibits smooth muscle proliferation, leukocyte recruitment, platelet aggregation and vascular remodelling through a number of downstream mechanisms. The central role of the NO-sGC-cGMP pathway in regulating pulmonary vascular tone is demonstrated by the dysregulation of NO production, sGC activity and cGMP degradation in pulmonary hypertension (PH). The sGC stimulators are novel pharmacological agents that directly stimulate sGC, both independently of NO and in synergy with NO. Optimisation of the first sGC stimulator, YC-1, led to the development of the more potent and more specific sGC stimulators, BAY 41-2272, BAY 41-8543 and riociguat (BAY 63-2521). Other sGC stimulators include CFM-1571, BAY 60-4552, vericiguat (BAY 1021189), the acrylamide analogue A-350619 and the aminopyrimidine analogues. BAY 41-2272, BAY 41-8543 and riociguat induced marked dose-dependent reductions in mean pulmonary arterial pressure and vascular resistance with a concomitant increase in cardiac output, and they also reversed vascular remodelling and right heart hypertrophy in several experimental models of PH. Riociguat is the first sGC stimulator that has entered clinical development. Clinical trials have shown that it significantly improves pulmonary vascular haemodynamics and increases exercise ability in patients with pulmonary arterial hypertension (PAH), chronic thromboembolic PH and PH associated with interstitial lung disease. Furthermore, riociguat reduces mean pulmonary arterial pressure in patients with PH associated with chronic obstructive pulmonary disease and improves cardiac index and pulmonary vascular resistance in patients with PH associated with left ventricular systolic dysfunction. These promising results suggest that sGC stimulators may constitute a valuable new therapy for PH. Other trials of riociguat are in progress, including a study of the haemodynamic effects and safety of riociguat in patients with PH associated with left ventricular diastolic dysfunction, and long-term extensions of the phase 3 trials investigating the efficacy and safety of riociguat in patients with PAH and chronic thromboembolic PH. Finally, sGC stimulators may also have potential therapeutic applications in other diseases, including heart failure, lung fibrosis, scleroderma and sickle cell disease.

Soluble Guanylate Cyclase Stimulation Prevents Fibrotic Tissue Remodeling and Improves Survival in Salt-Sensitive Dahl Rats

Background A direct pharmacological stimulation of soluble guanylate cyclase (sGC) is an emerging therapeutic approach to the management of various cardiovascular disorders associated with endothelial dysfunction. Novel sGC stimulators, including riociguat (BAY 63-2521), have a dual mode of action: They sensitize sGC to endogenously produced nitric oxide (NO) and also directly stimulate sGC independently of NO. Little is known about their effects on tissue remodeling and degeneration and survival in experimental malignant hypertension. Methods and Results Mortality, hemodynamics and biomarkers of tissue remodeling and degeneration were assessed in Dahl salt-sensitive rats maintained on a high salt diet and treated with riociguat (3 or 10 mg/kg/d) for 14 weeks. Riociguat markedly attenuated systemic hypertension, improved systolic heart function and increased survival from 33% to 85%. Histological examination of the heart and kidneys revealed that riociguat significantly ameliorated fibrotic tissue remodeling and degeneration. Correspondingly, mRNA expression of the pro-fibrotic biomarkers osteopontin (OPN), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and plasminogen activator inhibitor-1 (PAI-1) in the myocardium and the renal cortex was attenuated by riociguat. In addition, riociguat reduced plasma and urinary levels of OPN, TIMP-1, and PAI-1. Conclusions Stimulation of sGC by riociguat markedly improves survival and attenuates systemic hypertension and systolic dysfunction, as well as fibrotic tissue remodeling in the myocardium and the renal cortex in a rodent model of pressure and volume overload. These findings suggest a therapeutic potential of sGC stimulators in diseases associated with impaired cardiovascular and renal functions.

Novel endothelin receptor antagonist attenuates endotoxin-induced lung injury in sheep.

ObjectiveTo evaluate the cardiopulmonary effects of the novel endothelin receptor antagonist tezosentan in endotoxin-induced lung injury in sheep and to assess the dose response to tezosentan and endothelin-1 in healthy sheep. DesignProspective, randomized, controlled experimental study. SettingUniversity animal laboratory. SubjectsTwenty-one yearling sheep. InterventionsSeventeen awake, chronically instrumented sheep were subjected to intravenous infusion of Ringer’s lactate for 24 hrs. The animals were randomly assigned to a sham-operated group (n = 3), a lipopolysaccharide group (n = 7) receiving an intravenous infusion of Escherichia coli lipopolysaccharide 15 ng·kg−1·min−1, and a tezosentan group (n = 7) subjected to lipopolysaccharide and, from 4 hrs, an intravenous injection of tezosentan 3 mg/kg followed by infusion of 1 mg·kg−1·hr−1. In addition, four healthy sheep, exposed to an intravenous infusion of endothelin-1 at 20 ng·kg−1·min−1, after 1 hr received tezosentan in stepwise increasing doses of 0.5, 1, and 2 mg·kg−1·hr−1 that were maintained for 1 hr each. After a 4-hr recovery, the sheep received infusions of tezosentan at the same dose rates as a pretreatment to endothelin-1. Measurements and Main ResultsIn the sham-operated sheep, all cardiopulmonary variables remained unchanged. Lipopolysaccharide caused pulmonary hypertension, increased extravascular lung water index, and induced arterial hypoxemia. Tezosentan decreased the increments in pulmonary vascular resistance and extravascular lung water index by as much as 60% and 70%, respectively. In parallel, tezosentan ameliorated arterial hypoxemia, increased cardiac index, attenuated the decrease in stroke volume index, and reduced systemic vascular resistance. Compared with the lipopolysaccharide group, tezosentan further increased plasma concentrations of endothelin-1. In healthy animals, the administration of endothelin-1 induced systemic and pulmonary hypertension, increased extravascular lung water index, and evoked bradycardia and a decrease in cardiac index. These changes were attenuated by tezosentan infused at 1 and 2 mg·kg−1·hr−1. ConclusionsIn an ovine model of endotoxin-induced lung injury, tezosentan ameliorates pulmonary hypertension, lung edema, cardiac dysfunction, and arterial hypoxemia. Tezosentan counteracts the hemodynamic effects of endothelin-1 in a dose-dependent manner.

Methylene blue reduces lung fluid filtration during the early phase of endotoxemia in awake sheep.

ObjectiveTo determine whether methylene blue (MB), an inhibitor of soluble guanylate cyclase and nitric oxide synthase, alters lung hemodynamics and fluid filtration after endotoxin in sheep. DesignProspective, randomized, controlled experimental study with repeated measurements. SettingUniversity animal laboratory. SubjectsEight yearling, awake sheep. InterventionsSheep were instrumented for a chronic study with vascular and lung lymph catheters. In two experiments, separated by 1 wk of recovery, the animals received intravenously either an injection of MB 10 mg/kg or a corresponding volume of 0.9% sodium chloride as pretreatment. Thirty minutes later, sheep received a bolus injection of Escherichia coli endotoxin 1 &mgr;g/kg, followed by either an infusion of MB 2.5 mg/kg/hr or a corresponding volume of 0.9% sodium chloride for 5 hrs. Measurements and Main Results MB decreased the early phase endotoxin-induced rises in pulmonary capillary pressure and pulmonary vascular resistance. MB also reduced the increments in lung lymph flow (&OV0422;L) and protein clearance (CL) as well as the rightward shift of the permeability-surface area product (PS). In addition, MB diminished the decrease in cardiac output, stabilized mean arterial pressure, and precluded the rise in plasma and lung lymph cyclic guanosine 3′-5′ monophosphate. However, during the late phase, MB-treated sheep presented with a faster rise in &OV0422;L with no difference in CL and PS from the endotoxemic controls. ConclusionsDuring the early phase of endotoxemia in sheep, MB attenuates lung injury by decreasing the enhanced lung fluid filtration as a result of reduced pulmonary capillary pressure and permeability. However, MB does not counteract the late phase increase in lung fluid filtration.