sGC Stimulator Research Articles

Pharmacological and transcriptomic characterization of the nitric oxide pathway in aortic rings isolated from the tortoise Chelonoidis carbonaria

In this study the nitric oxide (NO)-soluble guanylate cyclase (sGC) and phosphodiesterase-5 (PDE-5) pathway was characterized in tortoise Chelonoidis carbonaria aorta. Concentration response curves (CCR) to ATP, ADP, AMP, adenosine and histamine were performed in the presence and absence of L-NAME in aorta pre-contracted with ACh (3 μM). CCR to SNP, BAY 41-2272 (sGC stimulator), BAY 60-2770 (sGC activator) and tadalafil (PDE-5 inhibitor) were constructed in the presence and absence of ODQ (10 μM). ATP (pEC50 6.1 ± 0.1), ADP (pEC50 6.0 ± 0.2), AMP (pEC50 6.8 ± 0.1) and histamine (pEC50 6.8 ± 0.12) relaxed Chelonoidis aorta and the addition of L-NAME reduced their efficacy (p < .05). Adenosine effects (pEC50 6.6 ± 0.1) were not changed in the presence of L-NAME. SNP (pEC50 7.5 ± 0.7; Emax 102.2 ± 2.5%), BAY 41-2272 (pEC50 7.3 ± 0.2; Emax 130.3 ± 10.2%), BAY 60-2770 (pEC50 11.4 ± 0.1; Emax 130.3 ± 6.5%) and tadalafil (pEC50 6.7 ± 0.3; Emax 121.3 ± 15.3%) relaxed Chelonoidis aorta. The addition of ODQ reduced the SNP and tadalafil maximum response (p < .05) and promoted 63 fold right shift on BAY 41-2272 curve. In contrast, no alteration was observed on BAY 60-2770 response. Transcriptomic analysis for eNOS and sGC were found in aorta and brain libraries with high homology when compared with human transcripts. The NO-sGC-PDE-5 is functionally present in Chelonoidis aorta with a functional and genomic similarity to mammalian vessels. Unlike most of mammalian vessels, ACh did not cause endothelium-dependent relaxation in Chelonoidis carbonaria aortic rings.

Responsiveness of rat aorta and pulmonary artery to cGMP generators in the presence of thiol or heme oxidant

This study investigated the effects of thiol and heme oxidants on responsiveness to cGMP generators in isolated rat aorta and pulmonary artery using an organ chamber. The nitric oxide (NO) donor sodium nitroprusside (SNP)-induced relaxation was impaired by exposure to the thiol oxidant diamide in both the aorta and the pulmonary artery, whereas the soluble guanylate cyclase (sGC) stimulator BAY 41-2272- or the sGC activator BAY 60-2770-induced relaxation was not affected. The impairment by diamide of SNP-induced aortic and pulmonary arterial relaxation was completely restored by post-treatment with the thiol reductant dithiothreitol. However, regardless of the vessel type, the relaxant response to SNP or BAY 41-2272 was impaired by exposure to the heme oxidant ODQ, whereas the response to BAY 60-2770 was enhanced. The ODQ-induced effects were reversed partially by post-treatment with the heme reductant dithionite. These findings indicate that thiol oxidation attenuates only the vascular responsiveness to NO donors and that heme oxidation attenuates the responsiveness to NO donors and sGC stimulators but augments that to sGC activators. Therefore, under oxidative stress, the order of usability of the vasodilators is suggested to be: NO donors < sGC stimulators < sGC activators.

Synthesis and biological evaluation of pyrazolo[3,4-b]pyridine-3-yl pyrimidine derivatives as sGC stimulators for the treatment of pulmonary hypertension

A series of new pyrazolo[3,4-b]pyridin-3-yl pyrimidine derivatives were synthesized and evaluated for the activation of sGC. Compared with riociguat, compound 13a exhibited equivalent in vitro activity on preconstricted rat thoracic aorta rings and in Rat heart Langendorff preparation. Compound 13a also showed acceptable PK profiles, which might become a promising candidate for the treatment of pulmonary hypertension.

A Microdialysis Assay for Assessing sGC Stimulator Target Engagement in the Rat Liver

Dysfunction of the nitric oxide (NO)‐soluble guanylate cyclase (sGC)‐cyclic guanosine monophosphate (cGMP) pathway has been implicated in numerous cardiovascular, metabolic, and fibrotic diseases. sGC stimulators are small molecules that bind sGC and synergize with NO to increase the enzymatic formation of cGMP and amplify downstream signaling. In preclinical models of liver disease, sGC stimulators have been shown to attenuate liver steatosis, inflammation, and fibrosis. To demonstrate their engagement in the sGC‐cGMP pathway in vivo, a unique microdialysis assay was developed to directly measure changes in cGMP levels in the rat liver.A midline laparotomy was performed on anesthetized male, Sprague Dawley rats (225–250 g) to expose the liver. A 25‐gauge needle was used to place a dialysis probe within the right medial lobe. The probe was perfused with saline at a rate of 2.5 μL/min and dialysate collected in one‐hour periods. Following baseline collection, test compound was administered intravenously, and dialysate collected for the following 2 hours. The NO donor sodium nitroprusside (SNP, 300 nM) was dosed via retrodialysis at study's end as a positive control. cGMP in the dialysate was measured using an enzyme immunoassay.In a study investigating our clinical‐stage sGC stimulator, praliciguat, an oral 3‐mg/kg dose produced a 3.9 ± 1.7‐fold increase (p&lt;.0001) in cGMP from baseline levels of 0.8 ± 0.1 nM. A 1‐mg/kg dose caused a 1.7 ± 0.1‐fold increase in cGMP (p&lt;.001), while the effect of a 0.3‐mg/kg dose was not significant. On average, SNP induced a 4.7 ± 1‐fold increase in cGMP (p&lt;.0001).In summary, our microdialysis assay enabled in vivo monitoring of sGC stimulation in the rat liver. Beyond demonstrating the effects of praliciguat, this method can be used to evaluate and characterize other sGC stimulators for their capacity to amplify liver cGMP production. Further, this assay helps to link previously observed hepatic effects of sGC stimulators in disease models with an enhancement of sGC signaling.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

FRI-296-Interaction between the soluble guanylate cyclase and the NLRP3 inflammasome in Kupffer cells: Implications for the anti-inflammatory actions of sGC stimulation in liver

FRI-296-Interaction between the soluble guanylate cyclase and the NLRP3 inflammasome in Kupffer cells: Implications for the anti-inflammatory actions of sGC stimulation in liver

Combined Activation of Guanylate Cyclase and Cyclic AMP in Lung Fibroblasts as a Novel Therapeutic Concept for Lung Fibrosis.

Remodelling of the peripheral lung tissue and fibrotic foci are the main pathologies of idiopathic pulmonary fibrosis (IPF), a disease that is difficult to treat. TGF-β activation of peripheral lung fibroblasts is indicated as the major cause of tissue remodelling in IPF and is resulting in fibroblast hyperplasia and deposition of extracellular matrix. Soluble guanylate cyclase (sGC) stimulators combined with cyclic AMP (cAMP) activators have been reported to reduce proliferation and matrix deposition in other conditions than IPF. Therefore, this drug combination may present a novel therapeutic concept for IPF. This study investigated the effect of BAY 41-2272 and forskolin on remodelling parameters in primary human lung fibroblasts. The study determined TGF-β induced proliferation by direct cell counts after 3 days; and deposition of collagen type-I, type III, and fibronectin. BAY 41-2272 significantly reduced TGF-β induced fibroblast proliferation, but did not reduce viability. This inhibitory effect was further supported by forskolin. Both BAY 41-2272 and forskolin alone reduced TGF-β induced collagen and fibronectin de novo synthesis as well as deposition. This effect was significantly stronger when the two compounds were combined. Furthermore, the TGF-β induced expression of fibrilar α-smooth muscle actin was reduced by BAY 41-2272 and this effect was strengthened by forskolin. In addition, BAY 41-2272 and forskolin reduced TGF-β induced β-catenin. All effects of BAY 41-2272 were concentration dependent. The findings suggest that BAY 41-2272 in combination with cAMP stimulation may present a novel therapeutic strategy to reduce tissue remodelling in IPF.

CGMP modulation therapeutics for sickle cell disease.

Sickle cell disease (SCD) is one of the most common inherited diseases and is associated with a reduced life expectancy and acute and chronic complications, including frequent painful vaso-occlusive episodes that often require hospitalization. At present, treatment of SCD is limited to hematopoietic stem cell transplant, transfusion, and limited options for pharmacotherapy, based principally on hydroxyurea therapy. This review highlights the importance of intracellular cGMP-dependent signaling pathways in SCD pathophysiology; modulation of these pathways with soluble guanylate cyclase (sGC) stimulators or phosphodiesterase (PDE) inhibitors could potentially provide vasorelaxation and anti-inflammatory effects, as well as elevate levels of anti-sickling fetal hemoglobin.

Quantitative mass balance, tissue distribution, pharmacokinetics and biotransformation pathway of praliciguat, a clinical-stage sgc stimulator, after oral administration in rats

Quantitative mass balance, tissue distribution, pharmacokinetics and biotransformation pathway of praliciguat, a clinical-stage sgc stimulator, after oral administration in rats

The Effect of the Soluble Guanylyl Cyclase Stimulator Olinciguat on ƴ-Globin Gene Induction in K562 Cells

Induction of fetal hemoglobin (HbF: α2ƴ2) is a recognized mode of action of hydroxyurea, the sickle cell disease (SCD) standard of care in SCD, and has been shown to prevent red blood cell (RBC) sickling. Discovery of novel HbF inducers is underway and several therapeutics with the potential to increase HbF expression are currently at different stages of preclinical and clinical development. Soluble guanylyl cyclase (sGC) is a heterodimeric heme-containing enzyme whose catalytic activity is regulated by nitric oxide (NO). Binding of NO to heme activates the catalytic domain of sGC, enabling synthesis of the second messenger cyclic guanosine monophosphate (cGMP) from guanosine triphosphate. sGC stimulators are small molecules that synergize with NO to boost signaling via the NO-sGC-cGMP pathway. This signaling pathway is involved in the regulation of many physiologic processes including inflammation, fibrosis, and blood flow. Perhaps less well-known, cGMP-mediated signaling has also been implicated in the regulation of the gene encoding the ƴ-globin subunit of fetal hemoglobin (Modulation of NO signaling by sGC stimulation, therefore, has the therapeutic potential to target the complex pathology of SCD at multiple levels. In this study, we focused on one potential mode of action of sGC stimulation-increasing HbF expression. We characterized the effects of the sGC stimulator olinciguat on ƴ-globin gene expression. Olinciguat is currently being investigated for the treatment of patients with SCD in a Phase II STRONG-SCD study (NCT03285178).The effect of olinciguat treatment on ƴ-globin mRNA levels was studied in the K562 erythroleukemic cell line. For short-term (8 hours) treatment with olinciguat, K562 cells were maintained in a serum-free media. For long-term (4 and 7 days) treatment, cell culture media contained 1% fetal bovine serum. Hydroxyurea was used as a positive control. Levels of ƴ-globin mRNA were expressed relative to mRNA levels of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase.K562 cells were treated for 8 hours with increasing concentrations of olinciguat (0.01, 0.1, 1, and 10 µM). Treatment of K562 cells with 0.1, 1, and 10 µM of olinciguat increased ƴ-globin mRNA levels by 1.43±0.08-, 1.37±0.06-, and 1.47±0.06-fold (mean±SEM), respectively. For comparison, 8 hours of treatment with hydroxyurea (800 µM) increased ƴ-globin mRNA levels by 1.25±0.03-fold. When K562 cells were cultured in the presence of olinciguat for 4 days, significant (P<0.05) induction of ƴ-globin mRNA levels was observed at 1 and 10 µM (1.13±0.03- and 1.55±0.09-fold, respectively). Induction of ƴ-globin mRNA following 4 days of incubation with hydroxyurea (800 µM) was 2.38±0.2-fold. The effects of hydroxyurea and olinciguat on ƴ-globin mRNA levels were compared following 7 days of incubation with the compounds. After 7 days of treatment of K562 cells with 0.1, 1, 3, and 10 µM of olinciguat, ƴ-globin mRNA levels were increased by 1.83±0.19-, 1.66±0.09-, 2.4±0.06-, and 2.9±0.33-fold, respectively. Treatment with 50- and 800-µM hydroxyurea increased levels of ƴ-globin mRNA by 2.33±0.15- and 3.8±0.56-fold, respectively.In conclusion, the sGC stimulator olinciguat increased the expression of mRNA for the ƴ-globin subunit of fetal hemoglobin in the erythroleukemic K562 cell line. This finding indicates that amplifying NO signaling by stimulating sGC may increase HbF expression, thereby preventing pathologic RBC sickling; this extends the potential therapeutic utility of olinciguat in SCD. Finally, the ability of olinciguat to induce HbF in SCD patients will be assessed in the ongoing Phase II STRONG-SCD study (NCT03285178). DisclosuresMiyashiro:Ironwood Pharmaceuticals: Employment. Pant:Ironwood Pharmaceuticals: Employment. Tchernychev:Ironwood Pharmaceuticals: Employment, Equity Ownership. Milne:Ironwood Pharmaceutics, Inc: Employment. Currie:Ironwood Pharmaceuticals: Employment. Graul:Ironwood Pharmaceuticals, Inc: Employment. Masferrer:Ironwood Pharmaceuticals, Inc: Employment.

Synergistic Platelet Inhibitory Effects of Riociguat and Nitric Oxide Are Decreased By Protein Binding

IntroductionNitric oxide (NO) released by endothelial cells interacts with platelets in which it stimulates soluble guanylate cyclase (sGC), thereby increasing platelet cyclic guanosine monophosphate (cGMP) and inhibiting platelet activation. Stimulation of sGC in other cells has been suggested as an attractive target for intervention in a range of diseases including pulmonary arterial hypertension, heart failure, and diabetes mellitus. Riociguat, the first FDA-approved sGC stimulator, potently increases platelet cGMP and inhibits platelet aggregation in washed platelets. Because riociguat binds to plasma proteins, higher concentrations of riociguat are required to inhibit platelet function in whole blood. However the potential synergistic inhibition of platelet function by riociguat and NO has not been well studied.GoalTo investigate the possible synergistic effects of riociguat and NO on platelet inhibition and to determine the effects of protein binding.MethodsPlatelet-rich plasma (PRP) was prepared from citrate (3.2%) anticoagulated whole blood collected from healthy donors following informed consent. Riociguat (10 mM) in DMSO and DETA-NONOate 10 mM (an NO donor) in 10 mM NaOH were stored at -80°C until use. PRP was diluted 10-fold in either HEPES-Tyrode's buffer or platelet poor plasma (PPP), then incubated with vehicle or riociguat 1, 10, or 100 µM, alone or in combination with DETA-NONOate 16, 31, or 250 µM for 30 minutes, then analyzed by flow cytometry. Platelet surface activated GPIIb-IIIa (detected by monoclonal antibody PAC1) and platelet surface P-selectin were measured with and without activation by ADP 5 µM or thrombin receptor activating peptide (TRAP) 5 µM. For light transmission platelet aggregation (LTA) and 96-well platelet aggregation, PRP was used without dilution.ResultsFor PRP diluted in buffer, riociguat and DETA-NONOate each produced concentration-dependent inhibition of ADP- and TRAP-stimulated platelet activation, as reported by platelet surface activated GPIIb-IIIa (Figure A) and P-selectin, and a synergistic inhibitory effect was observed when riociguat and DETA-NONOate were combined (for platelet surface activated GPIIb-IIIa, 40% inhibition with 1 µM riociguat alone; 30% inhibition with 31 µM DETA-NONOate alone; 90% inhibition with 1 µM riociguat and 31 µM DETA-NONOate combined). In contrast, when PRP was diluted in PPP, the concentrations of riociguat alone and DETA-NONOate alone needed to inhibit activation were dramatically increased and the combination of 1 µM riociguat and 31 µM DETA-NONOate produced less than 10% inhibition of platelet surface activated GPIIb-IIIa (Figure B). Synergistic inhibition in plasma was observed when DETA-NONOate was increased to 250 µM. Based on these results, a sub-threshold concentration of DETA-NONOate was chosen for investigation of the effects of riociguat on platelet aggregation. Using ADP 5 µM, TRAP, 2 µM, or collagen 2 µg/mL, riociguat alone at 10 µM (Figure C) and DETA-NONOate alone at 31 µM showed no inhibition of platelet aggregation. However, in the presence of 31 µM DETA-NONOate, riociguat showed a concentration-dependent inhibition of aggregation by each agonist (Figure C).ConclusionsPlatelets exposed to riociguat in combination with sub-threshold concentrations of NO, such as may occur in microvessels adjacent to the endothelial layer, are inhibited from undergoing platelet activation and aggregation. The presence of plasma proteins blunts the effects of riociguat and has even larger effects on the NO donor, DETA-NONOate. Taken together, these data suggest that NO potently synergizes with riociguat to inhibit platelet activation and aggregation, but in vivo this effect likely only occurs immediately adjacent to endothelial cells where NO concentrations are highest. DisclosuresMichelson:Alnylam, Instrumentation Laboratory, Surface Oncology: Consultancy; AstraZeneca, Chiesi, Dova, Janssen, LightIntegra, Megakaryon, Remora: Other: Scientific Advisory Board; Baxalta, Cellular Preservation Technologies, Ionis, Ironwood, Medtronic, Megakaryon, Pfizer, Sysmex: Research Funding. Frelinger:Surface Oncology: Consultancy; Cellular Preservation Technologies, Ironwood, Ionis, Medtronic, Megakaryon, Pfizer and Sysmex: Research Funding.

Soluble Guanylate Cyclase Modulators Effect Fetal Hemoglobin Levels through a Non-cGMP-Protein Kinase G Signaling Pathway

Sickle cell anemia results from a point mutation in both alleles of the β-globin gene. This homozygous mutation ultimately leads to a structural alteration of the hemoglobin protein that promotes polymerization of the mutant sickle hemoglobin tetramer (HbS) upon deoxygenation. HbS polymerization results in rigid, sickle-shaped RBCs with increased cell-to-cell adhesion properties. These sticky and rigid RBCs are prone to become trapped in small capillary networks leading to ischemia-reperfusion injury, endothelial damage and the hallmark pain crisis of sickle cell anemia. Neonates are protected from deoxy-HbS polymerization by high levels of fetal hemoglobin (HbF). HbF is composed of two α-globin and two γ-globin subunits(α2γ2), The γ-globin molecule cannot interact with deoxy-βS-globin polymers, which makes HbF an effective inhibitor of deoxy-HbS polymerization. The level of HbF required to reduce the symptoms of sickle cell anemia is 20-25%, but levels as low as 9% can prolong red cell survival. Treatment with hydroxyurea (HU) induces HbF and reduces the hematologic and clinical consequences of sickle cell anemia. Basal and inducible HbF levels are important in predicting the severity of sickle cell anemia and are highly phenotypically variable among patients, leading to varied responses to treatment. Patients are also variably susceptible to HU-induced cytopenias, which limits the use of HU in certain patients. HU is currently the only FDA-approved HbF-inducer for the treatment of sickle cell anemia and there is clearly a need for alternative HbF-inducers. Understanding the signaling pathways that regulate HbF induction will lead to novel therapeutic targets for sickle cell anemia. The soluble guanylate cyclase/cyclic guanosine monophosphate-dependent protein kinase (sGC/PKG) signaling pathway potentially links HU to the induction of HbF expression. In this study we investigated the direct role of sGC in HbF induction using novel pharmacologic modulators of sGC. Nitric oxide (NO) activates sGC by binding to the ferrous iron (Fe2+) in the active site heme moiety. Once activated, sGC converts GTP to cGMP, which in-turn activates PKG. Reactive oxygen species (ROS) oxidize the active site heme of sGC leading to NO-insensitivity. We tested the ability of a novel sGC activator, BAY 58-2667, to induce γ-globinin primary and immortalized (HUDEP-2) human erythroid progenitor cells. BAY 54-6544 binds to heme-free inactivated sGC to restore its guanylyl cyclase activity independent of NO. We also tested the ability of the sGC stimulator, BAY 41-2272, to induce γ-globinin primary and HUDEP-2 human erythroid progenitor cells. BAY 41-2272, binds to the ferrous iron at the active site of non-oxidized sGC to stimulate guanylyl cyclase activity in a synergistic manner with NO. We compared g-globin mRNA and protein expressionin the primary and immortalized human erythroid progenitors after treatment with different concentrations and combinations of BAY 54-6544, BAY 41-2667 and HU. We also evaluated g-globin induction in cellstreated with the pan-phosphodiesterase inhibitor IBMX and a synthetic cGMP analog. Although we see robust induction of cGMP and activation of PKG with all treatments, we only see significant induction of g-globin expression in the HU treated cells. This data suggests that the induction of HbF occurs through a non-sGC/PKG-dependent signaling pathway. These data demonstrate a very limited induction of γ-globin by BAY 54-6544 and BAY 41-2667 that appears to be disproportionate to, and independent of, cGMP/PKG signaling. These data also demonstrate, for the first time, that HU treatment of the immortalized HUDEP-2 cell line induces γ-globin expression more consistently than in primary erythroid progenitors. DisclosuresNo relevant conflicts of interest to declare.

The efficacy and safety of soluble guanylate cyclase stimulators in patients with heart failure: A systematic review and meta-analysis.

Background:Several randomized controlled trials (RCTs) have been investigated the benefits of soluble guanylate cyclase (sGC) stimulators in the treatment of heart failure, but a comprehensive evaluation is lacking. We performed a meta-analysis to evaluate the efficacy and safety of oral sGC stimulators (vericiguat and riociguat) in patients with heart failure.Methods:Studies were searched and screened in PubMed, Embase, and Cochrane Library. Eligible RCTs were included that reported mortality, the change of EuroQol Group 5-Dmensional Self-report Questionnaire (EQ-5D) US index, N-terminal pro-B-type natriuretic peptide (NT-proBNP), or serious adverse events (SAEs). Relative risk or weight mean difference (WMD) was estimated using fixed effect model or random effect model. Analysis of sensitivity and publication bias was conducted.Results:Five trials with a total of 1200 patients were included. sGC stimulators had no impact on the mortality (1.25; 95% confidence interval 0.50–3.11) and significantly improved EQ-5D US index (0.04; 95% confidence interval 0.020–0.05). Furthermore, in comparison with control group, NT-proBNP was statistically decreased in riociguat group (−0.78; 95% confidence interval −1.01 to −0.47), but not in vericiguat group (0.04, 95% confidence interval −0.18 to 0.25). There were not obverse differences in SAEs between sGC stimulators and control groups (0.90; 95% confidence interval 0.72–1.12).Conclusion:Our meta-analysis suggests that sGC stimulators could improve the quality of life in patients with heart failure with good tolerance and safety, but their long-term benefits need to be observed in the future. sGC stimulators are likely to be promising add-on strategies for the treatment of heart failure.

Abstract P146: Cytochrome B5 Reductase (CYB5R3) Function in Smooth Muscle Cells and Blood Pressure Regulation

Hypertension (HTN) is a major risk factor for cardiovascular-related morbidity and mortality. Dysregulation of nitric oxide (NO) signaling has been found in both human and mouse studies to impact the renin-angiotensin-aldosterone system (RAAS) and contribute to the vascular dysfunction leading to HTN. NO signals via soluble guanylyl cyclase (sGC) to generate cyclic guanosine monophosphate (cGMP) and downstream activation of protein kinase G (PKG) to induce vasodilation. However, the sGC heme iron needs to be in the reduced (Fe 2+ ) state for NO to bind as oxidized (Fe 3+ ) sGC is insensitive to NO signaling. We found that NADH cytochrome b5 reductase 3 (CYB5R3) is a sGC heme reductase that maintains sGC heme iron in its reduced state. Transient knockdown and pharmacological inhibition of CYB5R3 in smooth muscle cells (SMCs) blocks NO-mediated cGMP production and aortic relaxation. Taken together, we hypothesized that SMC CYB5R3 was important in blood pressure regulation. We created tamoxifen-inducible SMC-specific CYB5R3 knockout mice (SMC CYB5R3 KO) and found that they (n=8) had an ~5 mmHg increase in mean arterial blood pressure at baseline as compared to SMC CYB5R3 WT mice (n=9). In response to Angiotensin II (Ang II)-induced hypertension, SMC CYB5R3 KO mice (n=5) exhibited an ~14 mmHg increase in blood pressure and a significantly reduced heart rate as compared to SMC CYB5R3 WT mice (n=5). Lastly, ex vivo two-pin wire myography of Ang II-induced hypertensive SMC CYB5R3 KO (n= 6) mesenteric arteries treated with NO-independent sGC activator Bay 58-2667 showed increased vasodilation as compared to SMC CYB5R3 WT mice (n=10); Bay 58-2667 treatment at baseline showed no difference between groups. We predict that CYB5R3 regulation of SMC sGC heme iron redox state influences blood pressure in a NO-dependent manner. To test this, we will perform an intervention study where we will measure blood pressure in Ang II-induced hypertensive SMC CYB5R3 WT and SMC CYB5R3 KO mice treated with Bay 41-2272, a NO and heme dependent sGC stimulator, and Bay 58-2667. Also, we will assess the role of SMC CYB5R3 in protection against Ang II-induced vessel fibrosis and SMC dysfunction.

Pulmonary hypertension associated with left heart disease: Updated Recommendations of the Cologne Consensus Conference 2018

In the summer of 2016, delegates from the German Society of Cardiology (DGK), the German Respiratory Society (DGP), and the German Society of Pediatric Cardiology (DGPK) met in Cologne, Germany, to define consensus-based practice recommendations for the management of patients with pulmonary hypertension (PH). These recommendations were built on the 2015 European Pulmonary Hypertension guidelines, aiming at their practical implementation, considering country-specific issues, and including new evidence, where available. To this end, a number of working groups was initiated, one of which was specifically dedicated to PH associated with left heart disease. In this context, the European Guidelines point out that the drugs currently approved to treat patients with PAH (prostanoids, endothelin receptor antagonists, phosphodiesterase type 5 inhibitors, sGC stimulators) have not sufficiently been investigated in other forms of PH. However, despite the lack of respective efficacy data, an uncritical use of targeted PAH drugs in patients with PH associated with left heart disease is currently observed at an increasing rate. This development is a matter of concern. On the other hand, PH is a frequent problem that is highly relevant for morbidity and mortality in patients with left heart disease. In that sense, the distinction between isolated post-capillary pulmonary hypertension (IpcPH) and combined post- and pre-capillary pulmonary hypertension (CpcPH) and their proper definition may be of particular relevance. The detailed results and recommendations of the working group on PH associated with left heart disease, which were last updated in the spring of 2018, are summarized in this article.

Pulmonary hypertension due to lung diseases: Updated recommendations from the Cologne Consensus Conference 2018

The 2015 European Guidelines on Pulmonary Hypertension did not only cover pulmonary arterial hypertension (PAH) but also some aspects of pulmonary hypertension (PH) associated with chronic lung disease. The European Guidelines point out that the drugs currently used to treat patients with PAH (prostanoids, endothelin receptor antagonists, phosphodiesterase‑5 inhibitors, sGC stimulators) have not been sufficiently investigated in other forms of PH. Therefore, the European Guidelines do not recommend the use of these drugs in patients with chronic lung disease and PH. This recommendation, however, is not always in agreement with medical ethics as physicians sometimes feel inclined to treat other forms of PH which may affect quality of life and survival of these patients in a similar manner. To this end, it is crucial to consider the severity of both PH and the underlying lung disease. In June 2016, a Consensus Conference organized by the PH working groups of the German Society of Cardiology (DGK), the German Society of Respiratory Medicine (DGP) and the German Society of Paediatric Cardiology (DGPK) was held in Cologne, Germany, to discuss open and controversial issues surrounding the practical implementation of the European Guidelines. Several working groups were created, one of which was dedicated to the diagnosis and treatment of PH in patients with chronic lung disease. The 2018 updated recommendations of this working group are summarized in the present paper.

CLINICAL CASE OF REPLACEMENT OF ENDOTHELIN RECEPTOR ANTAGONIST (BOSENTAN) BY SOLUBLE GUANYLATE CYCLASE STIMULATOR (RIOCIGUAT) IN THE PATIENT WITH PULMONARY ARTERIAL HYPERTENSION

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive increase in pulmonary vascular resistance (PVR), higher pulmonary arteries mean pressure (mPAP), decrease in cardiac minute output (CMO) and other hemodynamic, functional and biochemical disorders. Existing PAH therapeutic algorithm, described in the European and Russian clinical guidelines, mainly suggest strategy of sequential combination therapy in the case of inadequate response to initial monotherapy. At this, switching from one PAH-specific drug to another is studied to a lesser extent. At the present similar approach (switching) has already been tested, for example, in switching from one endothelin receptor antagonist (ERA) to another, or from phosphodiesterase type 5 inhibitor (iPDE-5) to soluble guanylate cyclase stimulator (sGC). There are virtually no data about change of monotherapy when drugs target different molecular pathways of vascular tone regulation, in particular, in the pulmonary circulation vessels. This article describes clinical case of female patient with FC III pulmonary arterial hypertension (WHO) switched from therapy by endothelin-1 receptor antagonist (bosentan) to soluble guanylate cyclase stimulator (riociguat). Female, 37 years old, with verified by the right heart catheterization (RHC) diagnosis of idiopathic PAH (iPAH) received PAH-specific monotherapy of bosentan. Due to condition deterioration on the background of bosentan dose increase, medical conference decided to switch this patient to another class of PAH-specific therapy (sGC stimulator riociguat) with consequent follow-up. As a result of such approach positive changes in patient's condition, improvement of hemodynamic parameters, increased tolerance of physical exercise was demonstrated in comparison with previous therapy. This clinical case demonstrates safe and successful transfer from bosentan to riociguat in the patient with idiopathic PAH. Similar tactic for the change of therapy should be studied in further clinical trials.

Praliciguat, a Clinical-Stage sGC Stimulator, Improved Glucose Tolerance and Insulin Sensitivity and Lowered Triglycerides in a Mouse Diet-Induced Obesity Model

Background: Praliciguat (IW-1973) is a soluble guanylate cyclase (sGC) stimulator in clinical development. In animal models, praliciguat has broad tissue distribution and enhances nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signaling, which has been shown to elicit hemodynamic, anti-inflammatory, and anti-fibrotic effects. This study assessed the metabolic effects of praliciguat in a mouse DIO model. Methods: Male C57BL/6J mice (12 weeks old at the start of treatment) were either maintained on regular chow (lean mice) or switched to a 60% high fat diet (HFD) starting at age 6 weeks (obese mice). At the start of treatment, obese mice received either praliciguat (6 mg/kg) or vehicle in the HFD. The lean mice remained on regular chow. On day 28, an oral glucose tolerance test (OGTT) was performed after a 3-h fast. On day 29, organs and terminal blood were collected for analysis after a 3-h fast. Results: Praliciguat treatment lowered OGTT glucose area under the curve (-22%, P&amp;lt;0.05), fasting plasma insulin (-53%, P&amp;lt;0.01) and HOMA-IR, an index of insulin resistance (-53%, P&amp;lt;0.01) vs. vehicle treatment. Praliciguat-treated mice also had lower liver (-36%, P&amp;lt;0.05) and fasting circulating triglycerides (-30%, P&amp;lt;0.01) vs. vehicle-treated mice. Furthermore, several fasting biomarkers did not change in treated vs. vehicle including beta-hydroxybutyrate, free fatty acids, plasma glucose, cholesterol, leptin, and adiponectin. Conclusions: Praliciguat improved insulin sensitivity, glucose tolerance, and yielded lower circulating and liver triglycerides in a mouse diet-induced obesity model. Disclosure C.D. Schwartzkopf: None. J. Hadcock: None. J.E. Jones: None. M. Currie: Employee; Self; Ironwood Pharmaceuticals, Inc. G.T. Milne: Employee; Self; Ironwood Pharmaceuticals, Inc.. Stock/Shareholder; Self; Ironwood Pharmaceuticals, Inc.. Employee; Spouse/Partner; Catabasis Pharmaceuticals. Stock/Shareholder; Spouse/Partner; Catabasis Pharmaceuticals. J. Masferrer: Employee; Self; Ironwood Pharmaceuticals, Inc..

Rapid Dose Escalation Study of Praliciguat, a Soluble Guanylate Cyclase Stimulator, in Patients with Diabetes and Hypertension

Background: Impaired nitric oxide (NO) signaling through soluble guanylate cyclase (sGC) has been associated with microvascular complications of diabetes. Praliciguat (IW-1973), an sGC stimulator that enhances NO signaling, reduced fasting plasma glucose and proteinuria in an animal model of diabetic nephropathy. In a Phase 1 clinical study in healthy adults, doses of 15-40 mg were tolerated and lowered blood pressure (BP). Methods: This Phase 2a open-label in-clinic study assessed the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics of oral praliciguat in 11 patients with type 2 diabetes mellitus (T2DM) and hypertension on stable glycemic and BP therapy. Patients received sequential 3-day dosing cycles escalating through placebo (PBO), 10, 20, 30, 40 and 50 mg praliciguat QD. Assessments included adverse events (AEs), blood chemistry, ambulatory BP and heart rate, platelet function, and reactive hyperemia index (RHI). Results: The escalating dose regimen was generally well tolerated; there were no serious or severe AEs. Escalation in 2 patients was stopped due to asymptomatic BP lowering. Overall PK showed a median Tmax of 1-3 h, low to moderate variability, and ∼2-fold accumulation supporting QD dosing. Average 12-h daytime mean arterial BP decreased by 2 to 9 mmHg relative to time-matched PBO baseline across praliciguat dose levels, while heart rate increased 4 to 7 bpm. Notable changes from PBO baseline [mean (95% CI)] at the end of treatment were seen for fasting serum glucose [-26.5 mg/dL (-43.4, -9.5], HbA1c [-0.4% (-0.8, -0.04)], cholesterol [-43 mg/dL (-67, -20)], and body weight [-3.5 kg (-4.9, -2.0)]. Praliciguat had no effect on RHI or platelet function. Conclusion: T2DM patients with hypertension on standard therapy showed improvements in hemodynamics and metabolic parameters in this small in-clinic study. These results support further evaluation of praliciguat in T2DM patients including those with complications. Disclosure J.P. Hanrahan: Employee; Self; Ironwood. J.D. Wakefield: Employee; Self; Ironwood Pharmaceuticals, Inc.. Stock/Shareholder; Self; Ironwood Pharmaceuticals, Inc.. P.J. Wilson: None. D.P. Zimmer: Employee; Self; Ironwood Pharmaceuticals, Inc.. Stock/Shareholder; Self; Ironwood Pharmaceuticals, Inc.. M. Mihova: None. J. Chickering: None. D. Ruff: None. M.L. Hall: Employee; Self; Ironwood Pharmaceuticals, Inc.. Stock/Shareholder; Self; Ironwood Pharmaceuticals, Inc. M. Currie: Employee; Self; Ironwood Pharmaceuticals, Inc. G. Milne: Employee; Self; Ironwood Pharmaceuticals, Inc.. Stock/Shareholder; Self; Ironwood Pharmaceuticals, Inc.. Employee; Spouse/Partner; Catabasis Pharmaceuticals. Stock/Shareholder; Spouse/Partner; Catabasis Pharmaceuticals. A.T. Profy: Other Relationship; Self; Ironwood Pharmaceuticals.

The soluble guanylate cyclase stimulator riociguat reduces fibrogenesis and portal pressure in cirrhotic rats

In cirrhotic patients, portal hypertension (PHT) deteriorates survival, yet treatment options are limited. A major contributor to increased intrahepatic vasoconstriction in PHT is dysfunctional nitric-oxide signaling. Soluble guanylate cyclase (sGC) is the receptor of nitric-oxide and can be stimulated by riociguat. Riociguat is approved for pulmonary hypertension but has not been studied in liver cirrhosis. In this study we assessed the effects of riociguat on PHT and liver fibrosis in cholestatic (bile duct ligation, BDL) and toxic (carbon-tetrachloride, CCl4) rat models. In cirrhotic livers sGC expression was upregulated. In BDL rats, riociguat reduced liver fibrosis and decreased portal pressure without affecting systemic hemodynamics. In an early BDL disease stage, riociguat decreased bile duct proliferation, improved sinusoidal vascular dysfunction and inhibited angiogenesis. In advanced BDL riociguat exhibited anti-inflammatory effects. In CCl4 rats the beneficial effects of riociguat treatment were less pronounced and confined to an early disease stage. Similarly, in patients with cholestatic cirrhosis and PHT nitrates (that induce sGC activity) decreased portal pressure more effectively than in patients with non-cholestatic etiology. We also found an improvement of transaminases in patients with pulmonary hypertension receiving riociguat. Our findings support the clinical development of sGC stimulators in patients with cirrhotic PHT.

The Impact of the Nitric Oxide (NO)/Soluble Guanylyl Cyclase (sGC) Signaling Cascade on Kidney Health and Disease: A Preclinical Perspective

Chronic Kidney Disease (CKD) is a highly prevalent disease with a substantial medical need for new and more efficacious treatments. The Nitric Oxide (NO), soluble guanylyl cyclase (sGC), cyclic guanosine monophosphate (cGMP) signaling cascade regulates various kidney functions. cGMP directly influences renal blood flow, renin secretion, glomerular function, and tubular exchange processes. Downregulation of NO/sGC/cGMP signaling results in severe kidney pathologies such as CKD. Therefore, treatment strategies aiming to maintain or increase cGMP might have beneficial effects for the treatment of progressive kidney diseases. Within this article, we review the NO/sGC/cGMP signaling cascade and its major pharmacological intervention sites. We specifically focus on the currently known effects of cGMP on kidney function parameters. Finally, we summarize the preclinical evidence for kidney protective effects of NO-donors, PDE inhibitors, sGC stimulators, and sGC activators.