Johannes-Peter Stasch

The Chemistry and Biology of Soluble Guanylate Cyclase Stimulators and Activators

The vasodilatory properties of nitric oxide (NO) have been utilized in pharmacotherapy for more than 130 years. Still today, NO-donor drugs are important in the management of cardiovascular diseases. However, inhaled NO or drugs releasing NO and organic nitrates are associated with noteworthy therapeutic shortcomings, including resistance to NO in some disease states, the development of tolerance during long-term treatment, and nonspecific effects, such as post-translational modification of proteins. The beneficial actions of NO are mediated by stimulation of soluble guanylate cyclase (sGC), a heme-containing enzyme which produces the intracellular signaling molecule cyclic guanosine monophosphate (cGMP). Recently, two classes of compounds have been discovered that amplify the function of sGC in a NO-independent manner, the so-called sGC stimulators and sGC activators. The most advanced drug, the sGC stimulator riociguat, has successfully undergone Phase III clinical trials for different forms of pulmonary hypertension.

NO independent stimulation or activation of soluble guanylyl cyclase during early reperfusion limits infarct size

Aims Guanylyl cyclase-cyclic guanosine monophosphate signalling plays an important role in endogenous cardioprotective signalling. The aim was to assess the potential of direct pharmacological activation and stimulation of soluble guanylyl cyclase, targeting different redox states of the enzyme, to limit myocardial necrosis during early reperfusion. Methods and results Rat isolated hearts were subjected to reversible left coronary artery occlusion (ischaemia-reperfusion) and infarct size was assessed by the tetrazolium staining technique. Administration during early reperfusion of BAY 41-2272, an NO-independent, haem-dependent stimulator of soluble guanylyl cyclase targeting the reduced state, or BAY 60-2770, an NO-independent, haem-independent activator targeting the oxidized state, significantly limited infarct size. Inhibition of NO synthesis did not abrogate this protection, but exogenous perfusion of NO with BAY 41-2272 produced a synergistic effect. The haem site oxidiser, ODQ abrogated the protection afforded by BAY 41-2272 but potentiated the protection afforded by BAY 60-2770. Targeting both the reduced and oxidized forms of sGC together did not afford additive protection. Conclusions Targeting either reduced or oxidized forms of sGC during early reperfusion affords cardioprotection, providing support for the concept that direct sGC manipulation at reperfusion has therapeutic potential for the management of acute myocardial infarction.

Riociguat: Mode of Action and Clinical Development in Pulmonary Hypertension

&NA; Pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH) are progressive and debilitating diseases characterized by gradual obstruction of the pulmonary vasculature, leading to elevated pulmonary artery pressure (PAP) and increased pulmonary vascular resistance (PVR). If untreated, they can result in death due to right‐sided heart failure. Riociguat is a novel soluble guanylate cyclase (sGC) stimulator that is approved for the treatment of PAH and CTEPH. We describe in detail the role of the nitric oxide‐sGC‐cyclic guanosine monophosphate (cGMP) signaling pathway in the pathogenesis of PAH and CTEPH and the mode of action of riociguat. We also review the preclinical data associated with the development of riociguat, along with the efficacy and safety data of riociguat from initial clinical trials and pivotal phase III randomized clinical trials in PAH and CTEPH.

Soluble guanylate cyclase as an alternative target for bronchodilator therapy in asthma

Significance Asthmatics depend on β-agonist bronchodilator drugs, but a majority develop resistance to these drugs in their lifetime, and new ways to bronchodilate are needed. We show that brochodilation can be triggered in normal human and asthmatic mouse airways through an alternative signaling pathway, using new pharmacologic agents that directly activate the soluble guanylate cyclase (sGC) enzyme. Because an sGC-based drug was recently approved to treat pulmonary arterial hypertension, our findings imply that such drugs could become new bronchodilators in asthma. Our work also provides insight on how the sGC signaling enzyme becomes desensitized toward NO in inflammatory asthma, and thus helps to explain why NO is an ineffective bronchodilator in this disease. Asthma is defined by airway inflammation and hyperresponsiveness, and contributes to morbidity and mortality worldwide. Although bronchodilation is a cornerstone of treatment, current bronchodilators become ineffective with worsening asthma severity. We investigated an alternative pathway that involves activating the airway smooth muscle enzyme, soluble guanylate cyclase (sGC). Activating sGC by its natural stimulant nitric oxide (NO), or by pharmacologic sGC agonists BAY 41–2272 and BAY 60–2770, triggered bronchodilation in normal human lung slices and in mouse airways. Both BAY 41–2272 and BAY 60–2770 reversed airway hyperresponsiveness in mice with allergic asthma and restored normal lung function. The sGC from mouse asthmatic lungs displayed three hallmarks of oxidative damage that render it NO-insensitive, and identical changes to sGC occurred in human lung slices or in human airway smooth muscle cells when given chronic NO exposure to mimic the high NO in asthmatic lung. Our findings show how allergic inflammation in asthma may impede NO-based bronchodilation, and reveal that pharmacologic sGC agonists can achieve bronchodilation despite this loss.

Effects of Different Pulmonary Vasodilators on Arterial Saturation in a Model of Pulmonary Hypertension

Background Approved therapies for pulmonary arterial hypertension can induce oxygen desaturation when administered to patients with secondary forms of pulmonary hypertension (PH), probably due to an increase in ventilation/perfusion mismatch. Thus, so far these treatments have largely failed in secondary forms of PH. Methods We established an animal model of heterogeneous lung ventilation to evaluate the desaturation potential of mechanistically distinct vasoactive drugs launched or currently in clinical development for the treatment of PH. Single-lung ventilation was induced in five groups (Nu200a=u200a6) of anesthetized minipigs (7 weeks, 4 to 5 kg BW), and their hemodynamic parameters were monitored before and after intravenous injection of control (vehicle only), endothelin antagonist (bosentan; 0.3, 1, 3, 10 mg/kg), phosphodiesterase type 5 inhibitor (sildenafil; 3, 10, 30, 100 µg/kg), and soluble guanylate cyclase stimulators (BAY 41–8543 and riociguat; 1, 3, 10, 30 µg/kg). Cumulative doses were administered before successive unilateral ventilation cycles. The doses were chosen to achieve equal effect on blood pressure by the different pharmacologic principles. Results Single-lung ventilation resulted in transient increases in mean pulmonary artery pressure (mPAP) and desaturation. In contrast to control, all drugs dose-dependently decreased hypoxic mPAP (a positive treatment effect) and increased area under the arterial hemoglobin saturation curve (unwanted desaturation effect). Riociguat and bosentan reduced hypoxic mPAP to the greatest extent, while the soluble guanylate cyclase stimulators riociguat and BAY 41–8543 lowered arterial oxygen saturation of hemoglobin the least. Conclusions Future investigations will be required to confirm these findings in clinical settings.

Identification of acidic heterocycle-substituted 1H-pyrazolo[3,4-b]pyridines as soluble guanylate cyclase stimulators.

Novel guanylate cyclase stimulators are disclosed. Design, synthesis, SAR, and pharmacological profile of the compounds are discussed.

Pharmacotherapy of pulmonary hypertension

Preface.- Part I. Pulmonary hypertension: conceptual bases of the disease. Pulmonary hypertension: definition, classification, and epidemiology. Pulmonary hypertension: pathophysiology and signalling pathways. Pulmonary hypertension: pathology. Pulmonary hypertension: biomarkers. Pulmonary hypertension: animal models.- Part II. Pulmonary hypertension: established therapies. General supportive care. Calcium channel blockers. Prostacyclins. Endothelin receptor antagonists. Phosphodiesterase 5 inhibitors.- Part III. Pulmonary hypertension: novel pathways and emerging therapies Soluble guanylate cyclase stimulators. Therapeutics targeting dysregulated redox equilibrium and endothelial dysfunction (NOXs, sGC activators, GSNOR). Rho-kinase inhibitors. Serotonin transporters and serotonin receptors. PDGF inhibitors. Novel anti-proliferative strategies beyond imatinib. Immunosuppressive agents. Vasoactive peptides. Inhibitors of cGMP/cAMP metabolism (MRP4, MRP5, PDE1). Old targets re-visited. Stem cells and cell based therapy.- Part IV. Conclusions and Outlook. Pulmonary hypertension: evidence-based management in the modern era and future directions.

Role of soluble guanylate cyclase in renal hemodynamics and autoregulation in the rat

We studied the influence of soluble guanylate (sGC) on renal blood flow (RBF), glomerular filtration rate (GFR), and RBF autoregulation and its role in mediating the hemodynamic effects of endogenous nitric oxide (NO). Arterial pressure (AP), heart rate (HR), RBF, GFR, urine flow (UV), and the efficiency and mechanisms of RBF autoregulation were studied in anesthetized rats during intravenous infusion of sGC activator cinaciguat before and (except GFR) also after inhibition of NO synthase (NOS) by Nω-nitro-L-arginine methyl ester. Cinaciguat (0.1, 0.3, 1, 3, 10 μg·kg(-1)·min(-1), n=7) reduced AP and increased HR, but did not significantly alter RBF. In clearance experiments (FITC-sinistrin, n=7) GFR was not significantly altered by cinaciguat (0.1 and 1 μg·kg(-1)·min(-1)), but RBF slightly rose (+12%) and filtration fraction (FF) fell (-23%). RBF autoregulatory efficiency (67 vs. 104%) and myogenic response (33 vs. 44 units) were slightly depressed (n=9). NOS inhibition (n=7) increased AP (+38 mmHg), reduced RBF (-53%), and greatly augmented the myogenic response in RBF autoregulation (97 vs. 35 units), attenuating the other regulatory mechanisms. These changes were reversed by 77, 78, and 90% by 1 μg·kg(-1)·min(-1) cinaciguat. In vehicle controls (n=3), in which cinaciguat-induced hypotension was mimicked by aortic compression, the NOS inhibition-induced changes were not affected. We conclude that sGC activation leaves RBF and GFR well maintained despite hypotension and only slightly impairs autoregulation. The ability to largely normalize AP, RBF, RBF autoregulation, and renovascular myogenic response after NOS inhibition indicates that these hemodynamic effects of NO are predominantly mediated via sGC.

Receptor Binding Assay for NO-Independent Activators of Soluble Guanylate Cyclase

The characterization of the interaction between a ligand and its receptor is crucial for a broad variety of applications in academia as well as in the pharmaceutical industry. Although various sophisticated high-throughput technologies have been established to investigate the binding of ligands to their receptors, classical filtration-based receptor binding assays still have some advantages when smaller number of samples need to be tested. Here we describe a technically easy, cheap, and reliable receptor binding assay that was successfully applied to determine the binding constant of the NO-independent activator of soluble guanylate cyclase, cinaciguat, and the impact of other small molecules on its interaction with the enzyme.

Improvement of right heart structure and function by BAY 41-8543 in pulmonary artery banded mice

Background Receptors for the natriuretic peptides (NPs) ANP, BNP and CNP are highly expressed in lung, suggesting that this organ is an important physiological target for NP signalling. By stimulating vasoand bronchodilation and inhibiting cell proliferation and fibrotic processes, NPs may have therapeutic potential in various lung diseases. Mechanisms responsible for the relatively short half-life (few min) of NPs and the control of their local concentrations include (i) receptor-mediated internalization by the so called clearance receptor (NPR-C) and (ii) degradation by a membrane metalloprotease, called neutral endopeptidase (NEP) or neprilysin [1]. The velocity of peptide degradation/inactivation differs between ANP, BNP and CNP. Interestingly, cleavage of NPs by insulindegrading enzyme (IDE) [2] may have a particular role in NP signalling by generating peptide fragments that are hyperactive in receptor stimulation [3]. The physiological significance of NEP was supported by several studies in rodents showing that NEP inhibition leads to increased NP concentrations and activity. Moreover, we found that NEP inhibition is necessary and sufficient for detection of GC-A and GC-B by affinity labelling experiments with radioactive ANP or CNP in mouse and rat lung membrane preparations. Analogous assays, however, failed to label these receptors in human lung membranes, suggesting potent NP-degrading activity of NEP inhibitor-insensitive proteases. Methods and results ANP degradation by lung membranes in either the absence or presence of NEP inhibitors was analyzed by thin-layer-chromatography and mass spectrometry [2]. We found that NEP inhibition strongly reduces ANP degradation by rat and mouse but not human membranes. ANP-degrading activity in human lung membranes under conditions of NEP inhibition was very potent and even detectable with 1 ng of membrane protein. Like ANP, CNP was rapidly hydrolyzed. In both peptides, initial cleavage occurred at the same position within the conserved peptide ring structure being essential for biological activity. A second cleavage each was localized to the amino-terminus (behind Arg-4 or Lys-4, respectively). The cleavage sites are unrelated to those by NEP and IDE and indicate trypsin-like enzyme activity. Unlike ANP and CNP, BNP is a poor substrate and shows a completely different and complex cleavage pattern after prolonged incubation. The NEP inhibitor-insensitive protease was also detectable, albeit at much lower levels, in membranes from human aorta and mesenteric arteries, but not at all in placenta. Studies with various protease inhibitors revealed that leupeptin exposure potently inhibits NP degradation by this activity.