sGC Stimulator Research Articles

Inhaled nitric oxide to control platelet hyper-reactivity in patients with acute submassive pulmonary embolism

BackgroundWe test if inhaled nitric oxide (NO) attenuates platelet functional and metabolic hyper-reactivity in subjects with submassive pulmonary embolism (PE). MethodsParticipants with PE were randomized to either 50 ppm NO + O2 or O2 only for 24 h with blood sampling at enrollment and after treatment; results were compared with healthy controls. Platelet metabolic activity was assessed by oxygen consumption (basal and uncoupled) and reactivity was assessed with agonist-stimulated thromboelastography (TEG) and fluorometric measurement of agonist-stimulated cytosolic [Ca++] without and with pharmacological soluble guanylate (sGC) modulation. ResultsParticipants (N = 38 per group) were well-matched at enrollment for PE severity, comorbidities as well as TEG parameters and platelet O2 consumption. NO treatment doubled the mean plasma [NO3-] (P < 0.001) indicating successful delivery, but placebo treatment produced no change. After 24 h, neither TEG nor O2 consumption parameters differed significantly between treatment groups. Platelet cytosolic [Ca++] was elevated with PE versus controls, and was decreased by treatment with cinaciguat (an sGC activator), but not riociguat (an sGC stimulator). Stimulated platelet lysate sGC activity was increased with PE compared with controls. ConclusionsIn patients with acute submassive PE, despite evidence of adequate drug delivery, inhaled NO had no major effect on platelet O2 consumption or agonist-stimulated parameters on TEG. Pharmacological activation, but not stimulation, of sGC effectively decreased platelet cytosolic [Ca++], and platelet sGC activity was increased with PE, confirming the viability of sGC as a therapeutic target.

Soluble guanylyl cyclase stimulation and phosphodiesterase-5 inhibition improve portal hypertension and reduce liver fibrosis in bile duct-ligated rats.

In cirrhosis, the nitric oxide-soluble guanylyl cyclase (sGC)-cyclic guanosine monophosphate (cGMP) pathway is impaired, which contributes to increased intrahepatic vascular resistance (IHVR) and fibrogenesis. We investigated if sGC stimulation (riociguat (RIO)), sGC activation (cinaciguat (CINA)) or phosphodiesterase (PDE)-5 inhibition (tadalafil (TADA)) improves portal hypertension (PHT) and liver fibrosis. Fifty male Sprague-Dawley rats underwent bile-duct ligation (BDL) or sham operation. RIO (0.5 mg/kg), CINA (1 mg/kg), TADA (1.5 mg/kg) or vehicle (VEH) was administered from weeks 2 to 4 after BDL. At week 4, invasive haemodynamic measurements were performed, and liver fibrosis was assessed by histology (chromotrope-aniline blue (CAB), Picro-Sirius red (PSR)) and hepatic hydroxyproline content. Cirrhotic bile duct-ligated rats presented with PHT (13.1 ± 1.0 mmHg) and increased IHVR (4.9 ± 0.5 mmHg⋅min/mL). Both RIO (10.0 ± 0.7 mmHg, p = 0.021) and TADA (10.3 ± 0.9 mmHg, p = 0.050) decreased portal pressure by reducing IHVR (RIO: -41%, p = 0.005; TADA: -21%, p = 0.199) while not impacting heart rate, mean arterial pressure and portosystemic shunting. Hepatic cGMP levels increased upon RIO (+239%, p = 0.006) and TADA (+32%, p = 0.073) therapy. In contrast, CINA dosed at 1 mg/kg caused weight loss, arterial hypotension and hyperlactataemia in bile duct-ligated rats. Liver fibrosis area was significantly decreased by RIO (CAB: -48%, p = 0.011; PSR: -27%, p = 0.121) and TADA (CAB: -21%, p = 0.342; PSR: -52%, p = 0.013) compared to VEH-treated bile duct-ligated rats. Hepatic hydroxyproline content was reduced by RIO (from 503 ± 20 to 350 ± 30 µg/g, p = 0.003) and TADA (282 ± 50 µg/g, p = 0.003), in line with a reduction of the hepatic stellate cell activation markers smooth-muscle actin and phosphorylated moesin. Liver transaminases decreased under RIO (AST: -36%; ALT: -32%) and TADA (AST: -24%; ALT: -27%) treatment. Hepatic interleukin 6 gene expression was reduced in the RIO group (-56%, p = 0.053). In a rodent model of biliary cirrhosis, the sGC stimulator RIO and the PDE-5 inhibitor TADA improved PHT. The decrease of sinusoidal vascular resistance was paralleled by a reduction in liver fibrosis and hepatic inflammation, while systemic haemodynamics were not affected.

EXPRESS: Switching to riociguat: A potential treatment strategy for the management of CTEPH and PAH.

Currently, five classes of drug are approved for the treatment of pulmonary arterial hypertension (PAH): phosphodiesterase 5 inhibitors (PDE5i); endothelin receptor antagonists; prostacyclin analogs; the IP receptor agonist selexipag; and the soluble guanylate cyclase (sGC) stimulator riociguat. For patients with inoperable or persistent/recurrent chronic thromboembolic pulmonary hypertension (CTEPH), riociguat is currently the only approved pharmacotherapy. Despite the development of evidence-based guidelines on appropriate use of specific drugs, in clinical practice patients are often prescribed PAH-targeted therapies off label or at inadequate doses.PDE5i are the most often prescribed class of drugs as initial therapy, either alone or in combination with other drug classes. However, a proportion of patients receiving PAH therapies do not reach or maintain treatment goals. As PDE5i and riociguat target different molecules in the nitric oxide-sGC-cyclic guanosine monophosphate (NO-sGC-cGMP) signaling pathway, for patients with PAH without an initial or sustained response to PDE5i, there is a biological rationale for switching to riociguat. However, robust data from randomized controlled trials on the safety and efficacy of switching are lacking, as is formal guidance for clinicians. Here we review studies of sequential combination therapy, and trial data and case studies that have investigated switching between PAH-approved therapies, particularly from PDE5i to riociguat in patients with PAH with an insufficient response to PDE5i, and in patients with CTEPH who were receiving off-label treatment. These studies summarize the current evidence and practical real-life experience on the concept of switching treatments.

Soluble Guanylate Cyclase Agonists Induce Bronchodilation in Human Small Airways.

The soluble guanylyl cyclase (sGC)-cyclic guanosine monophosphate signaling pathway evokes vascular smooth muscle relaxation; whether this pathway mediates airway smooth muscle relaxation remains controversial. We posit that sGC activators are equi-effective as β-agonists in reversing contractile agonist-induced airway smooth muscle shortening. To provide clarity, we tested the efficacy of sGC stimulator and activator drugs, BAY 41-2272 and BAY 60-2270, respectively, in reversing bronchoconstriction of human small airways using human precision-cut lung slices (hPCLS). Both BAY drugs reversed carbachol-induced bronchoconstriction to a maximal degree comparable to that of formoterol. Moreover, the sGC drugs remained effective bronchodilators despite formoterol-induced desensitization of the airways. Analysis of the hPCLS after their activation by sGC or β2-adrenergic receptor agonist showed distinct cyclic nucleotide accumulation in the hPCLS. Collectively, these data suggest that cAMP and cyclic guanosine monophosphate pathways are equi-effective for reversing carbachol-induced bronchoconstriction in the human airway via separate and distinct second messenger pathways. This should open the door for future studies to test whether sGC-targeted drugs alone or in combination can serve as effective bronchodilators in asthma and chronic obstructive pulmonary disease.

Elucidation of cGMP-dependent induction of mitochondrial biogenesis through PKG and p38 MAPK in the kidney.

Previous studies have shown that cGMP increases mitochondrial biogenesis (MB). Our laboratory has determined that formoterol and LY344864, agonists of the β2-adrenergic receptor and 5-HT1F receptor, respectively, signal MB in a soluble guanylyl cyclase (sGC)-dependent manner. However, the pathway between cGMP and MB produced by these pharmacological agents in renal proximal tubule cells (RPTCs) and the kidney has not been determined. In the present study, we showed that treatment of RPTCs with formoterol, LY344864, or riociguat, a sGC stimulator, induces MB through protein kinase G (PKG), a target of cGMP, and p38, an associated downstream target of PKG and a regulator of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) expression in RPTCs. We also examined if p38 plays a role in PGC-1α phosphorylation in vivo. Administration of l-skepinone, a potent and specific inhibitor of p38α and p38β, to naïve mice inhibited phosphorylated PGC-1α localization in the nuclear fraction of the renal cortex. Taken together, we demonstrated a pathway, sGC/cGMP/PKG/p38/PGC-1α, for pharmacological induction of MB and the importance of p38 in this pathway.

195 Lipid profiling of bovine oocytes matured with different stimulation of cyclic GMP synthesis by multiple reaction monitoring profiling

Intrafollicular lipid metabolism is very important for production species such as cattle. Lipids are essential substrates to produce energy during growth, maturation, and acquisition of high competence for the development of oocytes. However, the quantity and distribution of these lipids has been identified as responsible for hindering the process of cryopreservation of oocytes and embryos produced invitro. Previous studies have indicated that the cyclic (c) GMP pathway may be involved in the lipid metabolism of bovine cumulus-oocyte complexes (COC). The synthesis of this nucleotide can be activated through guanylate cyclases (soluble, sGC; or membrane, mGC). Therefore, the objective of this study was to investigate the lipid profile of bovine oocytes matured invitro (IVM) when stimulated by specific stimulators of sGC (protoporphyrin IX) and mGC (NPPB: peptide natriuretic type B). Pools of ovum pickup (OPU) oocytes were matured invitro for 24h in TCM-199 medium, with 15% bovine serum (BS), 0.5µgmL−1 of FSH, 5µgmL−1 of LH, 0.8mM L-glutamine, and 50µgmL−1 of gentamicin at 38.5°C and 10−5 M protoporphyrin IX or 10−6 M NPPB. The control group was matured without NPs or protoporphyrin IX. After IVM, cumulus cells (CC) were removed and oocytes (OO) collected, washed in 1:3 methanol:water (v/v) and frozen at −80°C. The lipid extraction of the samples was performed based on a standard protocol (Bligh and Dyer 1959 Can. J. Biochem. Physiol. 37, 911-917) but adapted for small samples. The samples were diluted and analysed on an Agilent 6410 QQQ (Agilent Technologies) mass spectrometer and analysed according to the multiple reaction monitoring (MRM) method described (de Lima et al. 2018 J. Mass. Spectrom. 53, 1247-52). Data for 3 replicates/group were normalized and then submitted to ANOVA statistical analysis, followed by Tukey test and principal components analysis, by Metaboanalyst 4.0, with an α-level of 5%. The results, representing the analysis of 164 lipids, showed that the lipid profile was not affected when we used the cGMP synthesis stimulators protoporphyrin IX and NPPB, maintaining the same profile of lipid classes in control and treatments. In addition, the quantitative values of the major lipid classes, sphingomyelin, triglycerides, and phospholipids (phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol), were not altered in the treated groups. The values for each class (ng/oocyte) for control, NPPB, and protoporphyrin, respectively, were 0.89, 0.86, and 1.12 for sphingomyelin, 5.63, 5.66, and 6.90 for phosphatidylcholine, 7.34, 6.48, and 7.89 for triglycerides, 209.0, 244.0, and 207.4 for phosphatidylserine, 3.05, 3.0, and 2.35 for phosphatidylethanolamine, 3.40, 3.34, and 3.29 for phosphatidylglycerol, and 3.47, 3.52, and 3.51 for phosphatidylinositol (P&amp;lt;0.05). Further, the amount of these lipids per class was not affected by cGMP synthesis when stimulated by protoporphyrin IX and NPPB, showing that the relationship of this pathway with lipid metabolism needs additional study.

Soluble Guanylate Cyclase Stimulators and Activators: Where are We and Where to Go?

Soluble Guanylate Cyclase (sGC) is the intracellular receptor of Nitric Oxide (NO). The activation of sGC results in the conversion of Guanosine Triphosphate (GTP) to the secondary messenger cyclic Guanosine Monophosphate (cGMP). cGMP modulates a series of downstream cascades through activating a variety of effectors, such as Phosphodiesterase (PDE), Protein Kinase G (PKG) and Cyclic Nucleotide-Gated Ion Channels (CNG). NO-sGC-cGMP pathway plays significant roles in various physiological processes, including platelet aggregation, smooth muscle relaxation and neurotransmitter delivery. With the approval of an sGC stimulator Riociguat for the treatment of Pulmonary Arterial Hypertension (PAH), the enthusiasm in the discovery of sGC modulators continues for broad clinical applications. Notably, through activating the NO-sGC-cGMP pathway, sGC stimulator and activator potentiate for the treatment of various diseases, such as PAH, Heart Failure (HF), Diabetic Nephropathy (DN), Systemic Sclerosis (SS), fibrosis as well as other diseases including Sickle Cell Disease (SCD) and Central Nervous System (CNS) disease. Here, we review the preclinical and clinical studies of sGC stimulator and activator in recent years and prospect for the development of sGC modulators in the near future.

Cyclic nucleotide-dependent relaxation in human umbilical vessels.

Umbilical vessels have a low sensitivity to dilate, and this property is speculated to have physiological implications. We aimed to investigate the different relaxing responses of human umbilical arteries (HUAs) and veins (HUVs) to agonists acting through the cAMP and cGMP pathways. Vascular rings were suspended in organ baths for isometric force measurement. Following precontraction with the thromboxane prostanoid (TP) receptor agonist U44069, concentration-response curves to the nitric oxide (NO) donor sodium nitroprusside (SNP), the soluble guanylate cyclase (sGC) stimulator BAY 41-2272, the adenylate cyclase (AC) activator forskolin, the β-adrenergic receptor agonists isoproterenol (ADRB1), salmeterol (ADRB2), and BRL37344 (ADRB3), and the phosphodiesterase (PDE) inhibitors milrinone (PDE3), rolipram (PDE4), and sildenafil (PDE5) were performed. None of the tested drugs induced a relaxation higher than 30% of the U44069-induced tone. Rings from HUAs and HUVs showed a similar relaxation to forskolin, SNP, PDE inhibitors, and ADRB agonists. BAY 41-2272 was significantly more efficient in relaxing veins than arteries. ADRB agonists evoked weak relaxations (< 20%), which were impaired in endothelium-removed vessels or in the presence of the NO synthase inhibitor L-NAME, sGC inhibitor ODQ. PKA and PKG inhibitors impaired ADBR1-mediated relaxation but did not affect ADRB2-mediated relaxation. ADRB3-mediated relaxation was impaired by PKG inhibition in HUAs and by PKA inhibition in HUVs. Although HUA and HUV rings were relaxed by BRL37344, immunohistochemistry and RT-qPCR analysis showed that, compared to ADRB1 and ADRB2, ADRB3 receptors are weakly or not expressed in umbilical vessels. In conclusion, our study confirmed the low relaxing capacity of HUAs and HUVs from term infants. ADRB-induced relaxation is partially mediated by endothelium-derived NO pathway in human umbilical vessels.

The Effect of the Soluble Guanylate Cyclase Stimulator Olinciguat on Renal Function and Biomarkers of Inflammation and Renal Injury in Sickle Cell Mice

Introduction: Nephropathy, one of the most common complications of sickle cell disease (SCD), shortens life expectancy and accounts for 16-18% of SCD-associated deaths. SCD nephropathy is caused by recurrent vaso-occlusions (Am. J. Hematol. 2014;89:907) and by hemolytic anemia, which result in chronic tissue hypoxia, inflammation, and ultimately organ damage (Haematologica. 2012;97(2):201; Pediatr. Nephrol. 2014;29(10):1997). Reduced bioavailability of the gasotransmitter nitric oxide (NO) is one of the consequences of red blood cell hemolysis in SCD. NO insufficiency dysregulates the NO-soluble guanylyl cyclase (sGC)- cyclic guanosine monophosphate (cGMP) signaling pathway, promoting vasoconstriction, inflammation, and assembly of hetero cellular aggregates on the vascular wall, which occlude venous microcirculation. Olinciguat, a stimulator of soluble guanylate cyclase, increases NO-induced synthesis of the second messenger cGMP. In preclinical models, olinciguat has been shown to induce vasorelaxation and elicit anti-inflammatory and reno-protective effects. In an SCD mouse model of vaso-occlusive crisis (VOC), olinciguat reduced leukocyte/endothelial cell interactions, improved blood flow, and increased survival time (EHA 2019, PS1521). Here we evaluated the effect of chronic olinciguat treatment on biomarkers of inflammation and renal injury in an SCD mouse model. Methods: 18-week-old Townes non-disease control (HbAA) and SCD mice (HbSS) were studied. SCD mice were twice daily orally administered with either vehicle or olinciguat (20 mg/kg/day) for 8 weeks. Animals were placed in metabolic cages at 8 weeks for 24-hour urine collection. At the end of the study, plasma and kidneys were collected for biomarker analysis. Results: Plasma concentrations of the inflammatory biomarkers serum amyloid P component (SAP), serum amyloid A (SAA), interleukin 6 (IL-6), and plasminogen activator inhibitor 1 (PAI-1) were higher in SCD mice compared to non-disease control mice. SCD mice treated with olinciguat had significantly (p&amp;lt;0.05) lower levels of SAP, SAA, IL-6, and PAI-1 as compared to SCD control mice. Plasma levels of soluble adhesion receptors were also higher in SCD mice than non-disease control mice which indicate endothelial activation. SCD mice that were treated with olinciguat had significantly lower levels of plasma sE-selectin and sP-selectin compared to SCD control mice. There was no effect of treatment on sICAM-1. Cystatin C, a plasma biomarker of renal function, was significantly higher in SCD mice as compared to non-disease control mice; while mice treated with olinciguat had significantly lower levels compared to SCD control mice. Compared to non-disease control mice, SCD mice had proteinuria and albuminuria along with increased urine output and water consumption. Urine protein and urine albumin were lower in mice treated with olinciguat yet these data did not reach statistical significance (p=0.053 and p=0.065, respectively). Urine excretion was significantly lower in SCD mice treated with olinciguat as compared to SCD control mice. Olinciguat-treated mice also consumed significantly less water than mice from the SCD control mice. As compared to healthy control mice, SCD mice had significantly lower urine osmolality indicating tubular dysfunction while olinciguat treatment had no effect on urine osmolality. Analysis of biomarkers of kidney injury demonstrated that, relative to healthy control mice, urinary excretion of NAG, NGAL and KIM-1 was significantly higher in SCD mice. SCD mice treated with olinciguat had significantly less urinary excretion of NAG and NGAL as compared to SCD control mice. KIM-1 was not affected by olinciguat treatment. Kidney weight was significantly increased in sickle cell mice as compared to healthy control mice. Olinciguat-treated SCD mice had significantly lower kidney weight than SCD mice from vehicle control group. Conclusions: In summary, treatment of SCD mice with the sGC stimulator olinciguat elicited lower levels of plasma biomarkers of inflammation and endothelial cell activation and biomarkers of kidney injury. Disclosures Tchernychev: Cyclerion Therapeutics: Employment, Equity Ownership. Liu:Cyclerion Therapeutics: Employment, Equity Ownership. Hall:Cyclerion Therapeutics: Employment, Equity Ownership. Graul:Cyclerion Therapeutics: Employment, Equity Ownership. Masferrer:Cyclerion Therapeutics: Equity Ownership.

SGC Stimulators and Activators

Nitric oxide (NO)-soluble guanylate cyclase(sGC)-cGMP signalling is impaired in HF syndromes, which could predispose to vascular oxidative stress. Nitrates directly stimulate cGMP, but are limited by tolerance. Therapeutic targets that aim at increasing cGMP concentrations have therefore been explored. Recently, two classes of drugs have been discovered, the sGC activators and the sGC stimulators, which target two different redox states of sGC: the NO-sensitive reduced (ferrous) sGC and NO-insensitive oxidized (ferric) sGC, respectively. Cinaciguat is an activator and riociguat and vericiguat are sGC stimulators. Vericiguat is the most advanced agent in its clinical trial programme with two completed phase IIb studies, SOCRATES -REDUCED in HFrEF and SOCRATES-PRESERVED in HFpEF, with mixed results on NT-proBNP. The ongoing VICTORIA trial in HFrEF will study 4,872 participants with a mortality/morbidity end-point and VITALITY HFpEF trial will study 735 participants, with a quality of life end-point.

Soluble guanylate cyclase stimulator praliciguat attenuates inflammation, fibrosis, and end-organ damage in the Dahl model of cardiorenal failure.

Reduced nitric oxide (NO) and a decrease in cGMP signaling mediated by soluble guanylate cyclase (sGC) has been linked to the development of several cardiorenal diseases. Stimulation of sGC is a potential means for enhancing cGMP production in conditions of reduced NO bioavailability. The purpose of our studies was to determine the effects of praliciguat, a clinical-stage sGC stimulator, in a model of cardiorenal failure. Dahl salt-sensitive rats fed a high-salt diet to induce hypertension and organ damage were treated with the sGC stimulator praliciguat to determine its effects on hemodynamics, biomarkers of inflammation, fibrosis, tissue function, and organ damage. Praliciguat treatment reduced blood pressure, improved cardiorenal damage, and attenuated the increase in circulating markers of inflammation and fibrosis. Notably, praliciguat affected markers of renal damage at a dose that had minimal effect on blood pressure. In addition, liver fibrosis and circulating markers of tissue damage were attenuated in praliciguat-treated rats. Stimulation of the NO-sGC-cGMP pathway by praliciguat attenuated or normalized indicators of chronic inflammation, fibrosis, and tissue dysfunction in the Dahl salt-sensitive rat model. Stimulation of sGC by praliciguat may present an effective mechanism for treating diseases linked to NO deficiency, particularly those associated with cardiac and renal failure. Praliciguat is currently being evaluated in patients with diabetic nephropathy and heart failure with preserved ejection fraction.

P1614Soluble guanylate cyclase as a therapeutic target in heart failure: myocardial gene expression in response to sGC stimulation in pressure overload

Abstract Background Heart Failure (HF) is associated with endothelial dysfunction and reduced bioavailability of NO with insufficient stimulation of sGC and reduced production of cGMP. Therefore, the impairment of the NO-sGC-cGMP pathway results in vasoconstriction, platelet aggregation, inflammation, fibrosis and most importantly maladaptive cardiac hypertrophy. The restoration of the NO-sGC -cGMP pathway is an attractive pharmacological target for HF therapy. Purpose Riociguat is an NO independent stimulator of the sGC that sensitizes the sGC to endogenous NO and directly stimulates sGC to produce cGMP. We therefore hypothesized that Riociguat prevents pathological effects occurring during HF. Methods Pressure overload was induced by transverse aortic constriction (TAC) in 8 weeks old male C57Bl6/N mice. Three weeks after TAC when cardiac hypertrophy has developed either Riociguat (RIO; 3 mg/kg) or a Solvent was administered daily for 5 more weeks (n=12 per group). Animals with sham surgery and same drug regime served as controls. The heart function in all groups was evaluated weekly by small animal echocardiography. Eight weeks after surgery, the transcriptome of the left ventricles (LV) of sham and TAC mice were analysed by RNA Sequencing. Differentially expressed genes (DEG) were categorised using Ingenuity Pathway Analysis (IPA). Results TAC resulted in a steady decrease of left ventricular fractional shortening (FS) in the mice until week 3. When Riociguat treatment commenced, the systolic LV function of the TAC+Rio group recovered significantly whereas the solvent group showed a further decline until week 8 (FS 21.4±3.4% vs. 9.5±2%, p&lt;0.001). Both sham groups (Sham+Sol and Sham+Rio) showed no changes in the heart function over timer. Regarding the hypertrophic response to LV pressure overload, Riociguat treatment attenuated significantly the increase of the left ventricular mass (LVM 208.3±15.8mg vs. 148.9±11.8mg, p&lt;0.001) after TAC. In line with the reduced LVM, histological staining showed a significantly reduced fibrosis and myocyte cross sectional area in the TAC+Rio group compared to TAC+Sol group. Regarding the myocardial transcriptome, the treatment with Riociguat resulted in less changes of gene expression pattern after TAC (TAC+Sol vs. Sham+Sol 3160 DEG; TAC+Rio vs. Sham+Rio 2237 DEG). The expression of heart failure marker genes like ANP (Nppa), BNP (Nppb), β-Myosin Heavy Chain (Myh7) and the Collagens 1 and 3 (Col1a1, Col1a2, Col3a1) were significantly decreased in TAC+Rio, when compared to TAC+Sol. IPA analysis revealed that the activation of biological pathways in response to TAC, like actin cytoskeleton- and Integrin signalling, renin-angiotensin or cardiac hypertrophy signalling was attenuated when Riociguat was administered. Conclusion Riociguat attenuates pressure overload induced LV remodelling resulting in less hypertrophy, improved heart function and less alteration of gene expression pattern.

LESSONS LEARNED FROM TRIPLE COMBINATION THERAPY IN PULMONARY ARTERIAL HYPERTENSION

SESSION TITLE: Monday Fellow Case Report Posters SESSION TYPE: Fellow Case Report Posters PRESENTED ON: 10/21/2019 02:30 PM - 03:15 PM INTRODUCTION: Current guidelines published by Chest in 2019 suggest that patients who present with low or intermediate risk pulmonary arterial hypertension (PAH) should be initiated on oral therapy with endothelin receptor antagonists (ERAs) and phosphodiesterase-5 inhibitors (PDE-5i) or soluble guanylate cyclase stimulators (sGCs) (1). They suggest using triple combination therapy including subcutaneous or IV prostacyclin analogues in high-risk patients (1). The following is a series of three patients with PAH who dramatically improved on triple combination therapy. CASE PRESENTATION: A 49 year old male with Sjogrens disease and associated PAH (APAH) was found to have clinical improvement on an ERA, PDE-5i, and prostacyclin analogue combination. With the switch of the PDE-5i to a sGCs based on the Respite trial and switch of ERA choice within the class due to side effect (nasal congestion and lower extremity edema), the patient had near normalization of his echocardiogram and right heart catherization (RHC) findings as well as an increase in his 6 minute walk distance (6MWD) (2) (see Table 1, Patient 1). Similarly, a 75 year-old female with scleroderma APAH was treated with an ERA, PDE-5 inhibitor, and a prostacyclin analogue. Significant hemodynamic improvement was noted when ERA choice was changed within its class and sildenafil was changed to a SGCs (2) (See Table 1, Patient 2). Finally, a 44 year-old female with PAH secondary to HIV was noted to have improvement in 6MWD and decrease in mean pulmonary artery pressure and pulmonary vascular resistance with the addition of an ERA and PDE-5 inhibitor to IV epropostenol. Interestingly, when IV epropostenol was switched to subcutaneous (SC) trepostinil due to recurrent line infections, the patient’s hemodynamics worsened. When the patient developed angioedema to ambrisentan, she was switched to macitentan with subsequent improvement in 6MWD and hemodynamics (see Table 1, Patient 3). DISCUSSION: Classically, it is considered of limited efficacy to switch ERAs within the class and there is limited information in added benefit of switching PDE-5 inhibitors to Riociguat, a sGC stimulator. In each of these patient cases, changes were made in medication regimens due to progression of disease or medication side effects and patterns of clinical improvement were subsequently noted. This suggests that by using triple drug combination therapy, including parental prostacyclines, and switching medications within the ERA class and between nitric oxide pathway agents (PDE-5 inhibitors to sGC stimulators), physicians may further optimize a patient’s clinical status by hemodynamic parameters and 6MWD. CONCLUSIONS: Triple combination therapy may lead to dramatic clinical and hemodynamic improvement. Further studies need to be performed to determine if triple therapy might lead to better long-term survival. Reference #1: Klinger, J. R. (2019). Therapy for Pulmonary Arterial Hypertension in Adults. Chest Journal, 155(3), 565-586. doi:https://doi.org/10.1016/j.chest.2018.11.030 Reference #2: Hoeper, M. M., Corris, P. A., Klinger, J. R., Langleben, D., Naeije, R., Simonneau, G., & Benza, R. L. (2016). RESPITE: Riociguat in pulmonary arterial hypertension patients with an inadequate response to phosphodiesterase type 5 inhibitors. 4.3 Pulmonary Circulation and Pulmonary Vascular Diseases. https://doi.org/10.1183/13993003.congress-2016.oa263 DISCLOSURES: No relevant relationships by Sheeja Schuster, source=Web Response Consultant relationship with Actelion Please note: $5001 - $20000 Added 03/15/2019 by Roxana Sulica, source=Web Response, value=Consulting fee Consultant relationship with Bayer Please note: $5001 - $20000 Added 03/15/2019 by Roxana Sulica, source=Web Response, value=Consulting fee Consultant relationship with United Therapeutics Please note: $5001 - $20000 Added 03/15/2019 by Roxana Sulica, source=Web Response, value=Consulting fee research relationship with Reata Please note: $5001 - $20000 Added 03/15/2019 by Roxana Sulica, source=Web Response, value=insitution was paid for resear

Abstract P3047: Soluble Guanylyl Cyclase Stimulators And Activators Inhibit Hypoxia-induced Placental Sflt-1 Levels

Preeclampsia (PE) is a pregnancy-specific disease characterized by placental ischemia, maternal endothelial dysfunction, and hypertension. The ischemic placenta releases anti-angiogenic factors, such as sFlt-1, which antagonizes the angiogenic factors, Placental Growth Factor (PlGF) and Vascular Endothelial Growth Factor (VEGF), causing endothelial dysfunction and impaired nitric oxide (NO) signaling. During normal pregnancy, NO would bind soluble guanylyl cyclase (sGC) increasing cGMP levels leading to vasodilation. However, in PE this signaling is reduced. sGC stimulators and activators are a novel class of drug that directly stimulate sGC despite low levels of NO. Our preliminary data suggest that sGC modulators reduce hypertension in an animal model of PE, but the mechanisms are not known. Therefore, this study tested the hypothesis that sGC stimulators (Riociguat) and activators (Cinaciguat) attenuate sFlt-1 production and increase PlGF levels in the placenta. Placentas were obtained from normal pregnant rats on gestational day 19. Isolated placental villi were plated (1 per well) and coated with Matrix Matrigel Basement Membrane. The villi were treated with various doses of sGC modulators (0.2μM, 1μM, 10μM, 30μM) and then exposed to a hypoxic environment (1% oxygen) for 48 hours. Levels of sFlt-1, PlGF, and VEGF were determined using an Enzyme-Linked Immunosorbent Assay kit.sFlt-1 concentration was significantly reduced in both groups treated with the stimulator or activator compared to the untreated group. The greatest attenuation was observed with the 30μM sGC activator treatment (Untreated, 5117±1313 pg/mL; 30μM sGC stimulator, 3975±621pg/mL, 30μM sGC activator, 3811±712 pg/mL, P&lt;0.05). PlGF was not significantly different between groups, however, VEGF concentrations were significantly increased at higher doses (Untreated, 3099±100 pg/mL; 30μM sGC stimulator, 3871±449 pg/mL 30μM sGC activator, 3952±252 pg/mL; p&lt;0.05). These data suggest that sGC modulators significantly attenuate sFlt-1 and thus, improve the sFlt-1/PlGF and sFlt-1/VEGF ratios. Therefore, a potential mechanism whereby sGC modulators reduce blood pressure in PE is by inhibiting hypoxia-induced placental sFlt-1 levels.

Protein Kinase G Is Involved in Acute but Not in Long-Term Regulation of Renin Secretion.

Pharmacological inhibition of the renin–angiotensin–aldosterone system (RAAS) is, in combination with diuretics, the first-choice treatment for hypertension, although 10–20% of patients do not respond adequately. Next to the RAAS, the nitric oxide/cGMP/protein kinase G (PKG) system is the second fundamental blood pressure regulator. Whether both systems influence each other is not well-studied. It has been shown that nitric oxide (NO) supports renin recruitment via activation of soluble guanylate cyclase (sGC) and subsequent generation of cGMP. Whether this leads to an ensuing activation of PKGs in this context is not known. PKGIα, as well as PKGII, is expressed in renin-producing cells. Hence, we analyzed whether these enzymes play a role regarding renin synthesis, secretion, or recruitment. We generated renin-cell-specific PKGI-knockout mice and either stimulated or inhibited the renin system in these mice by salt diets. To exclude the possibility that one kinase isoform can compensate the lack of the other, we also studied double-knockout animals with a conditional knockout of PKGI in juxtaglomerular cells (JG cells) and a ubiquitous knockout of PKGII. We analyzed blood pressure, renin mRNA and renal renin protein content as well as plasma renin concentration. Furthermore, we stimulated the cGMP system in these mice using BAY 41-8543, an sGC stimulator, and examined renin regulation either after acute administration or after 7 days (application once daily). We did not reveal any striking differences regarding long-term renin regulation in the studied mouse models. Yet, when we studied the acute effect of BAY 41-8543 on renin secretion in isolated perfused kidneys as well as in living animals, we found that the administration of the substance led to a significant increase in plasma renin concentration in control animals. This effect was completely abolished in double-knockout animals. However, after 7 days of once daily application, we did not detect a persistent increase in renin mRNA or protein in any studied genotype. Therefore, we conclude that in mice, cGMP and PKG are involved in the acute regulation of renin release but have no influence on long-term renin adjustment.

Treatment of pediatric pulmonary arterial hypertension: A focus on the NO-sGC-cGMP pathway.

ObjectiveWhile pulmonary arterial hypertension (PAH) is rare in infants and children, it results in substantial morbidity and mortality. In recent years, prognosis has improved, coinciding with the introduction of new PAH‐targeted therapies, although much of their use in children is off‐label. Evidence to guide the treatment of children with PAH is less extensive than for adults. The goal of this review is to discuss the treatment recommendations for children with PAH, as well as the evidence supporting the use of prostanoids, endothelin receptor antagonists (ERAs), and phosphodiesterase type 5 inhibitors (PDE5i) in this setting.Data SourcesNonsystematic PubMed literature search and authors’ expertise.Study SelectionArticles were selected concentrating on the nitric oxide (NO)‐soluble guanylate cyclase (sGC)‐cyclic guanosine monophosphate (cGMP) pathway in PAH. The methodology of an ongoing study evaluating the sGC stimulator riociguat in children with PAH is also described.ResultsDespite recent medical advances, improved therapeutic strategies for pediatric PAH are needed. The efficacy and tolerability of riociguat in adults with PAH have been well trialed.ConclusionThe pooling of data across trials, supplemented by registry data, will help to confirm the safety and tolerability of prostanoids, ERAs, and PDE5i in children. Ongoing studies will clarify the place of sGC stimulators in the treatment strategy for pediatric PAH.

Protective role of endothelial progenitor cells stimulated by riociguat in chronic thromboembolic pulmonary hypertension

BackgroundPulmonary endothelial damage has a negative impact on the maintenance of normal pulmonary vascular function. Such damage results in delayed thrombus dissolution and vascular remodeling in chronic thromboembolic pulmonary hypertension (CTEPH). Although endothelial progenitor cells (EPCs) may be incorporated into neovasculature during vascular repair, their function in CTEPH remains unclarified, especially under the augmentation of soluble guanylate cyclase (sGC) activity. Methods and resultsWe evaluated the effect of EPCs on endothelial function and compared the effect of riociguat, a sGC stimulator, on the number and function of circulating EPCs in two groups of CTEPH patients. The two groups consisted 16 CTEPH patients who were treatment naïve (Naïve group), and 14 CTEPH patients who were being treated with riociguat, a sGC stimulator (Riociguat group). The number of circulating EPCs in the Riociguat group was significantly higher than that in the Naïve group. Gene expression levels associated with angiogenesis were significantly higher in EPCs of the Riociguat group. EPC-stimulated tube formation and migration of human pulmonary microvascular endothelial cell (hPMVEC) in the Riociguat group exceeded that in the Naïve group. The angiogenic ability of hPMVECs stimulated by EPCs in the Riociguat group was enhanced compared to that of the sGC stimulator, BAY 41–2272. ConclusionThese findings indicate that riociguat may induce EPCs to play a protective role via modulation of endothelial functions associated with CTEPH. Translation aspect of the workEndothelial dysfunction exacerbates CTEPH. Riociguat enhanced the protective role of EPCs via neovascularization, which prevented vascular remodeling and alleviated CTEPH.

Therapeutic effects of soluble guanylate cyclase stimulation on pulmonary hemodynamics and emphysema development in guinea pigs chronically exposed to cigarette smoke.

We have analyzed the effect of the soluble guanylate cyclase (sGC) stimulator BAY 41-2272 in a therapeutic intervention in guinea pigs chronically exposed to cigarette smoke (CS). The effects of sGC stimulation on respiratory function, pulmonary hemodynamics, airspace size, vessel remodeling, and inflammatory cell recruitment to the lungs were evaluated in animals that had been exposed to CS for 3 mo. CS exposure was continued for an additional 3 mo in half of the animals and withdrawn in the other half. Animals that stopped CS exposure had slightly lower pulmonary artery pressure (PAP) and right ventricle (RV) hypertrophy than those who continued CS exposure, but they did not recover from the emphysema and the inflammatory cell infiltrate. Conversely, oral BAY 41-2272 administration stopped progression or even reversed the CS-induced emphysema in both current and former smokers, respectively. Furthermore, BAY 41-2272 produced a reduction in the RV hypertrophy, which correlated with a decrease in the PAP values. By contrast, the degree of vessel remodeling induced by CS remained unchanged in the treated animals. Functional network analysis suggested perforin/granzyme pathway downregulation as an action mechanism capable of stopping the progression of emphysema after sGC stimulation. The pathway analysis also showed normalization of the expression of cGMP-dependent serine/kinases. In conclusion, in guinea pigs chronically exposed to CS, sGC stimulation exerts beneficial effects on the lung parenchyma and the pulmonary vasculature, suggesting that sGC stimulators might be a potential alternative for chronic obstructive pulmonary disease treatment that deserves further evaluation.

1924-P: Praliciguat, a Clinical-Stage sGC Stimulator, Improves Insulin Sensitivity, Lipid Tolerance, and Energy Utilization in a Mouse Diet-Induced Obesity Model Housed at Thermoneutrality

Background: Praliciguat (IW-1973) is a soluble guanylate cyclase (sGC) stimulator under clinical investigation as a potential treatment for diabetic nephropathy. In animal models, praliciguat distributes broadly to tissues and elicits hemodynamic, anti-inflammatory, anti-fibrotic, and metabolic effects. Here, we assessed the metabolic effects of praliciguat in a mouse diet-induced obesity (DIO) model when housed at thermoneutrality. Methods: Six week old male C57BL/6N mice were maintained on a low-fat diet (LFD, lean mice) or put on a 60% high-fat diet (HFD, obese mice). At 14 weeks of age, one group of obese mice was maintained on HFD (obese controls) while another group of obese mice was switched to HFD formulated with praliciguat to achieve a Cmax similar to a daily oral administration of 6 mg/kg (praliciguat treatment). A group of lean mice were also included (lean controls). Indirect calorimetry was performed on days 8, 9, 20, 21, 32 and 33. On day 35, an oral lipid tolerance test (LTT) was conducted. In a second study under the same dosing paradigm, overnight fasted blood and organs were collected on day 28. Results: Compared to obese controls, energy expenditure was higher in praliciguat-treated mice throughout a 24h recording period as assessed by AUC (Day 8: +13%, Day 20: +9%, Day 21: +7%, Day 32: +5%, P&amp;lt;0.05) and LTT triglycerides were lower as assessed by AUC (-34%, P&amp;lt;0.01). In the second study, praliciguat-treated mice had lower fasting insulin (-28%, P&amp;lt;0.05), c-peptide (-20%, P&amp;lt;0.05), HOMA-IR (-26%, P&amp;lt;0.05) and triglycerides (-16%, P&amp;lt;0.01) compared to obese controls. Caloric intake, body weight and composition were unaffected by praliciguat treatment. Conclusions: In a mouse DIO model housed at thermoneutrality, praliciguat increased energy utilization, improved triglycerides in response to an oral lipid challenge, improved insulin sensitivity, and lowered plasma triglycerides. Disclosure C. Schwartzkopf: Employee; Self; Ironwood Pharmaceuticals, Inc. J. Hadcock: Employee; Self; Ironwood Pharmaceuticals, Inc. J. Roux: None. G. Liu: None. C. Shea: Employee; Self; Ironwood Pharmaceuticals, Inc. M. Currie: Employee; Self; Ironwood Pharmaceuticals, Inc. G. Milne: Employee; Spouse/Partner; Catabasis Pharmaceuticals. Employee; Self; Ironwood Pharmaceuticals, Inc. Stock/Shareholder; Spouse/Partner; Catabasis Pharmaceuticals. Stock/Shareholder; Self; Ironwood Pharmaceuticals, Inc. J. Masferrer: Employee; Self; Ironwood Pharmaceuticals, Inc. J.E. Jones: None.

SGC stimulator praliciguat suppresses stellate cell fibrotic transformation and inhibits fibrosis and inflammation in models of NASH

Endothelial dysfunction and reduced nitric oxide (NO) signaling are a key element of the pathophysiology of nonalcoholic steatohepatitis (NASH). Stimulators of soluble guanylate cyclase (sGC) enhance NO signaling; have been shown preclinically to reduce inflammation, fibrosis, and steatosis; and thus have been proposed as potential therapies for NASH and fibrotic liver diseases. Praliciguat, an oral sGC stimulator with extensive distribution to the liver, was used to explore the role of this signaling pathway in NASH. We found that sGC is expressed in hepatic stellate cells and stellate-derived myofibroblasts, but not in hepatocytes. Praliciguat acted directly on isolated hepatic stellate cells to inhibit fibrotic and inflammatory signaling potentially through regulation of AMPK and SMAD7. Using in vivo microdialysis, we demonstrated stimulation of the NO-sGC pathway by praliciguat in both healthy and fibrotic livers. In preclinical models of NASH, praliciguat treatment was associated with lower levels of liver fibrosis and lower expression of fibrotic and inflammatory biomarkers. Praliciguat treatment lowered hepatic steatosis and plasma cholesterol levels. The antiinflammatory and antifibrotic effects of praliciguat were recapitulated in human microtissues in vitro. These data provide a plausible cellular basis for the mechanism of action of sGC stimulators and suggest the potential therapeutic utility of praliciguat in the treatment of NASH.