Modulating cGMP levels as therapeutic drug targets …Modulating cGMP levels as therapeutic drug...

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FOR CITATION PURPOSES: Mnica FZ, Murad F, Bian K. Modulating cGMP levels as therapeutic drug targets in cardiovascular and non-cardiovascular diseases. OA Biochemistry 2014 Mar 11;2(1):3.

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Modulating cGMP levels as therapeutic drug targets in

cardiovascular and non-cardiovascular diseases

FZ Mónica1,2, F Murad2*, K Bian2*

Abstract Introduction Cyclic guanosine monophosphate (cGMP) signaling plays a critical role in physiological homeostatic processes such as smooth muscle tone in vascular and non-vascular tissues, platelet activity, cardiac contractility, renal function and fluid balance, as well as cell growth. The cGMP signaling consists of cGMP-generating guanylyl-cyclases, protein kinases, phosphodiesterases, endopeptidases, ion channel and efflux transporters and hence substances that increase cGMP levels are important targets for the treatment of cardiovascular and non-vascular-related diseases. Studies of the 90’s established endothelium dysfunction as one of the major causes of cardiovascular diseases and therapeutic strategies that benefit NO bioavailability have been applied in clinical medicine extensively. Recently, the basic and clinical studies of cGMP regulation through either activation of particulate guanylyl cyclase (pGC) and soluble guanylyl cyclase (sGC) or inhibition of cyclic nucleotide phosphodiesterase type 5 (PDE5) have resulted in effective therapies for congestive heart failure, pulmonary hypertension, erectile dysfunction and more recently for benign prostatic hyperplasia. This review highlights pharmacological aspects of sGC and pGC activation and PDE5 inhibition in the treatment of cardiovascular (heart failure and pulmonary hypertension) and vascular-related diseases (erectile dysfunction and lower urinary tract

symptoms secondary to benign prostatic hyperplasia). Conclusion The discovery of the NO-cGMP pathway revolutionized the comprehension of pathophysiological mechanisms involved on cardiovascular diseases. However, considering the expression “from bench to bedside”, the therapeutic alternatives that target NO-cGMP did not immediately follow the biochemical and pathophysiological revolution since few therapeutic options have been proven effective and released on the market for the treatment of cardiovascular disorders.

Introduction The second messenger cyclic 3’-5’ guanosine monophosphate (cGMP) is one of the key mediators of cell signaling and serves as an internal messenger to regulate a variety of physiological processes, including vascular1 and non-vascular smooth muscle relaxation2,3,4, natriuresis5, platelet function6, neutrophil adhesion7, sperm motility8, fluid and ion secretion9 and cancer cell proliferation10,11. The intracellular levels of cGMP are controlled by its formation mainly due to the activation of soluble guanylyl cyclase (sGC) and particulate guanylyl cyclase (pGC)12,13,14,15 and by its degradation by cyclic nucleotide phosphodiesterases (PDEs) activities 16. Cytoplasmatic levels of cGMP may also be modulated nonenzymatically by multi-drug resistant proteins type 4, 5 and 8 (MRP 4, MRP5, MRP 8, respectively), which pumps cGMP or cAMP out of the cell17. The physiological effects of cGMP are exerted through the activation of cGMP-dependent protein kinases (PKG), cyclic nucleotide-gate ion channels and the activation and/or

inhibition of PDEs. Alterations of cGMP-dependent pathways play an important role in cardiovascular and other related diseases, such as hypertension, pulmonary hypertension (PH), congestive heart failure (CHF), erectile dysfunction (ED) and lower urinary tract symptoms (LUTS) secondary to benign prostatic hyperplasia (LUTS-BPH). This review will focus on basic and clinical research, showing how sGC, pGC and PDE regulations lead to beneficial effects on cardiovascular (heart failure and pulmonary hypertension) and non-cardiovascular disorders. The use of nitrovasodilators to increase cGMP formation12 and their use in ischemic heart diseases and heart disease have been extensively revised and thus it will not be addressed in the present review.

Discussion The authors have referenced some of their own studies in this review. These referenced studies have been conducted in accordance with the Declaration of Helsinki (1964) and the protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed. All human subjects, in these referenced studies, gave informed consent to participate in these studies. Also animal care was in accordance with the institution guidelines. Clinical importance of the particulate guanylate cyclase activation in cardiovascular diseases Since our earliest studies in the 1970’s18, seven different isoforms of particulate guanylate cyclase (pGC) have been found to date in vertebrates, GC-A through GC-G. Among them, GC-A appears to be the most widely expressed throughout the body and was found in different cell types such as

*Corresponding author Email: [email protected]

1 Department of Pharmacology, Campinas,

Brazil

2 Department of Biochemistry and Molecular

Medicine, Washington, United States

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nerves, vessels, kidney, lung, intestine, testis, heart and adipose tissues15,19,20,21. GC-A is stimulated by atrial (ANP) and B-type natriuretic peptides (BNP), whereas GC-B is activated by C-type natriuretic peptide (CNP)14. ANP is a 28-amino acid with a disulfide bond between two cysteine residues at positions 7 and 23 and released by atrial granules. BNP is a 32-amino acid polypeptide, first isolated from brain homogenates, although a greater proportion of circulating BNP is thought to come from ventricules22. CNP is a 22-amino acid peptide and binds preferentially to GC-B, which is highly expressed in bone, lung, brain, heart and ovary23 and macrophage24. ANP, BNP and CNP are released as a pro hormone proANP1-126, proBNP1-108, proCNP1-126, respectively, which are then enzymatically cleaved into mature and more effective forms of ANP1-28, BNP1-32 and CNP1-22. Neprilysin is a metallaendo-endopeptidase and is involved in the metabolism of enkefalins, tachykinins, natriuretics and chemotactic peptides. The physiological effect of ANP, BNP and CNP can be attenuated by either increasing neprilysin activity or reducing their bioavailability. Hence increasing the concentration of natriuretic peptides through neprilysin inhibition represents a potential therapeutic approach for cardiac, vascular and renal protection25. ANP and BNP possess diuretic, natriuretic and hypotensive activity. Cardiomyocytes release ANP and BNP in response to volume and pressure overload, which increase cardiac transmural distending pressure. Circulating natriuretic peptide levels are highly correlated with the severity of systolic heart failure and both BNP and N-terminal fragment of BNP (NT-proBNP) concentrations are valuable biomarkers in the management of heart failure, since its levels are raised more than ANP in left ventricular overload26. NT-pro-BNP is derived from proteolysis of pro-BNP1-108 and consists of 76 amino acids. Both BNP and NT-proBNP are released in the plasma in equimolar quantities and

the latter is an alternative choice in monitoring heart failure due to its better stability (> 3 days) and longer half-life (60-120 min) when compared to BNP (24 hours and 21 min, respectively)27,28. In a prospective, multicenter trial, circulating BNP concentrations were determined to diagnose and evaluate the severity of patients with heart failure based on America Heart Association classification compared to the patients without cardiovascular symptom (BNP=9.29 pg/mL, n=473), the blood levels of BNP in heart failure class I through IV were 83.1 pg/mL (49.4-137 pg/mL, n=73), 235 pg/mL (137-391 pg/mL, n=135), 459 (200-871 pg/mL, n=141) and 1119 pg/mL (728>1300pg/mL,n=60), respectively, showing a high positive correlation29. It is important to emphasize that sex, age, body mass index, hypernatremia, myocardial ischemia, sympathetic nervous system activation, tachycardia and endocrinal substances (endothelin, angiotensin II, glucocorticoids and thryroid hormones) are conditions that can affect BNP/NT-proBNP synthesis and clearance30 and should be taken into consideration when BNP/NT-proBNP levels are used as diagnostic tools. Nesiritide, a 32-amino acid peptide, is an exogenous form of BNP manufactured by using recombinant DNA technology and approved by FDA in 2001 for use in the treatment of heart failure. However, despite its favorable cardiorenal properties, such as natriuresis, diuresis, inhibition of renin and aldosterone, BNP is a potent vasodilator and may decrease renal perfusion, thus compromising renal function in heart failure patients31. Thus, there is a need for novel natriuretic peptides that favors renal function. LCZ696 is a combination drug consisting of valsartan and AHU-377, which inhibits concomitantly neprilysin and the angiotensin receptor. In patients with chronic heart failure, LCZ696 increased plasma cGMP levels while decreasing NT-proBNP and plasma aldosterone levels24. The PARADIGM-HF trial of Novartis showed that chronic heart failure patients with reduced ejection

fraction who received LCZ696 lived longer without being hospitalized than those who received enalapril, a angiotensin-converting-enzyme (ACE) inhibitor, only32 (Figure 1). CNP mainly works in a paracrine manner and possess less diuretic and natriuretic effects since it lacks a C-terminus amino acid extension33. CNP plays a role in the suppression of neo-intima formation after intimal lesions of rat carotid arteries34. In cultured cardiac myocyte hypertrophy, CNP had anti-hypertrophic, anti-fibrotic and anti-proliferative effects and hence may be useful in preventing cardiac remodeling after myocardial infarction35. CD-NP (cenderitide) is a novel chimeric natriuretic peptide developed by Mayo Clinic consisting of full length CNP fused with the carboxyl tail Dendroaspis Natriuretic Peptide (DNP). DNP is an ANP analogue purified from the snake venom that activates GC-A. CD-NP is a dual activator of GC-A and GC-B, with higher affinity at GC-B than GC-A (Figure 1). In normal anaesthetized dogs CD-NP elevated cGMP, enhanced glomerular filtration, reduced cardiac filling pressures and suppresses the renin-angiotensin system36. In healthy volunteers CD-NP (17.5 ng/Kg/min) increased plasma cGMP, urinary cGMP excretion, urinary sodium excretion and urine flow within 4 hours of infusion37. In a randomized, double blind, placebo-controlled clinic trial with patients of chronic heart failure (ejection fraction≤40%), cenderitide (20 ng/Kg/min) increased cGMP levels without altering on blood pressure or heart rate. Aldosterone and ANP levels were reduced and glomerular filtration rate increased by using of cenderitide38. In a Phase II trial sponsored by Capricor Therapeutics, intravenous infusion at 1.25, 2.5 and 3.75 ng/Kg/min appeared well tolerated with a dose-dependent decrease on systolic blood pressure compared to placebo. Serum creatinine levels in the 1.25 and 2.5 groups improved relatively to the placebo. These findings led the authors to conclude that CD-NP was well tolerated and preserved renal function relative to placebo. However, more clinical studies need to be carried

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out to larger groups of patients in order to evaluate its effectiveness and safety. Guanylyl cyclase type C (GC-C) is localized almost exclusively in the epithelial cells of small intestine15,39,

40,41,42,43, but is also present in liver, uterus, kidney44 and pancreas45. GC-C serves as the receptor for guanylin, uroguanylin, lymphoguanylin and heat-stable enterotoxin (STa). In mammals, uroguanylin and guanylin are quite different in primary structures with uroguanylin possessing two acidic amino acids near N-terminus and an internal asparagine residue. Instead, guanylin have a conserved aromatic amino acid, tyrosine or phenylalanine. The bacterial STa peptide resembles uroguanylin more than guanylin in primary structure, because they possess an intern asparagine making them highly resistant to proteolytic attack by endoproteases such as chymotrypsin46,47. Since GC-C expression is presented mainly on intestinal mucosa, the majority of studies are focused on the role of GC-C pathway on cancer, diahrrea and obesity mechanisms (for review see 48). Clinical importance of the nitric oxide-soluble guanylate cyclase activation in cardiovascular diseases Nitric oxide (NO) can be considered as an intracellular second messenger with autocrine, paracrine, and endocrine effects49. Nitric oxide is biosynthesized by the action of nitric oxide synthases (NOS). Once formed, it diffuses into the cell to activate its intracellular receptor - soluble guanylate cyclase (sGC), which is a heterodimer protein consisting of two subunits, alpha (α) and beta (β). The most commonly studied isoform is the α1β1 protein, although α2β2 subunits have also been identified. Nitric oxide binds to heme of sGC to form a 6-coordinate complex, which is rapidly converted to a 5-coordinate ferrous nitrosyl complex and thus catalyzing the conversion of guanosine triphosphate (GTP) into cGMP50.

Recently, two classes of compounds that activate sGC have been approved by FDA and European Union Agency for treating two forms of pulmonary hypertension, thus, the therapeutic strategy by targeting sGC-cGMP signaling has again regained attention51 (Figure 2). It has been known from our studies in the 1970’s that nitroglycerin and other nitrovasodilators acted by forming nitric oxide and increasing sGC activity and cGMP formation52,53,54,55. The World Health Organization divides PH into five groups: 1) pulmonary arterial hypertension (PAH), that includes idiopathic PAH, familial PAH and PAH associated with other conditions such as connective tissue disease and congenital heart disease; 2) pulmonary hypertension with left heart failure, where mitral valve disease or long-term high blood pressure can cause left heart failure and PH; 3) PH associated with lung

diseases, such as chronic obstructive pulmonary disease and interstitial (IN-ter-STISH-al) lung diseases; 4) PH due to pulmonary embolism in the lungs or blood cloting disorders and 5) PH caused by other diseases such as blood disorders (polycythemia vera, essential thrombocythemia), systemic disorders (sarcoidosis, vasculitis), metabolic disorders (thyroid and glycogen storage diseases). In all groups, the average increased pressure in the pulmonary arteries is 25 mmHg or higher. The pressure in normal pulmonary arteries is 8–20 mmHg at rest. Inhlaled NO is used to treat newborn with PAH (WHO group 1). Low doses of inhaled NO at 20 ppm for 4 hours and then 6 ppm for 20 hours resulted in improved oxygenation without systemic effects and decreased systolic blood pressure in 9 newborn infants with persistent pulmonary hypertension56,57.

Figure 1: Pharmacological modulation of particulate guanylate cyclase (pGC) pathway. Atrial natriuretic peptide (ANP)/brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP) and uroguanylin/guanylin/STa are agonists for GC-A, GC-B and GC-C, respectively. Once activated, pGC leads to cyclic guanosine monophosphate (cGMP) accumulation, which can activate protein kinase G (PKG), multi-drug resistance protein (MRP), cyclic nucleotide gated-channel (CNG) and phosphodiesterases (PDEs). Neprylisin hydrolyzes ANP, BNP and CNP. Nesitiride is an exogenous form of BNP and activates GC-A. CD-NP (cenderitide) activates both GC-A and GC-B, whereas LCZ696 is a combination drug consisting of AHU-377 and valsartan, which inhibits concomitantly neprilysin and the angiotensin receptor type 1, respectively.

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The ability of inhaled NO to increase systemic oxygen levels was confirmed in multicenter randomized controlled studies of term and near-term babies with hypoxemia and PAH58,59. In adults with severe PAH, pulmonary vascular resistance was reduced significantly after inhaled NO and after prostacyclin (24 ug/h), whereas systemic vascular resistance was not affected by inhaled NO60. In an open, prospective, randomized, controlled trial, the combination of inhaled NO and oxygen improved pulmonary hemo-dynamics better than inhaled NO alone61. However, inhaled NO has only been formally approved by the FDA and the European agency (EMEA) for clinical use in the treatment in term and near-term neonates with hypoxemia and pulmonary hypertension. A significant proportion of adults with PAH do not respond to inhaled NO, likely because of sGC impairment62 or irreversible pulmonary disease such as fibrosis. Organic nitrates are not effective for PH because their long-term use leads to tolerance and upon discontinuation the development of serious rebound pulmonary hyper-tension. Furthermore, pulmonary smooth muscle is less sensitive to nitrovasodilators-induced relaxation than vascular smooth muscle63,64. It is worth noting that in patients with CTEPH (WHO 4), pulmonary endarterectomy (a surgery to remove old blood clots from the pulmonary arteries) is a therapeutic choice to restore pulmonary haemodynamics. However, in the cases of CTEPH it is deemed ineffective and those who develop persistent PH after surgery are eligible for using PAH-specific pharmacotherapy such as prostanoids, endothelin receptor antagonists, PDE5-inhbitors and more recently sGC stimulators. Three completed randomized controlled trials in patients with inoperable CTEPH have used prostanoids65, PDE-5 inhibitors, sildenafil66 and an endothelin-1 receptor antagonist, bosentan67. Only bosentan demonstrated a positive therapeutic effect on hemodynamics,

although no improvement was observed in exercise capacity. Further trials are needed to ascertain the role of pharmacotherapy on patients with CTEPH. The role of the sGC stimulator riociguat (BAY 63-2521) in patients with CTEPH will be addressed below. In patients with PH-associated with left heart disease (WHO group 2) the role of PAH-specific drugs is controversial and not well studied in large randomized controlled trials. However, recent studies suggest that acute68,69 and chronic70,71 administration of oral sildenafil reduced pulmonary artery pressure and pulmonary vascular resistance without significant changes in systemic arterial pressure. It has been known for many years that PDE5 inhibitors can augment the accumulation of both cAMP and cGMP in most tissues including pulmonary tissues and blood vessels to increase their effects49,52,72,73. As for patients with PH-associated lung diseases (WHO 3) there exist few clinical evidences showing the efficacy of PDE-5 inhibitors. Sildenafil improved pulmonary hemodynamics at rest and during exercise in 20 patients with

chronic obstructive pulmonary disease (COPD)-associated PH74, whereas in another study sildenafil had no effect on stroke volume or exercise capacity in 9 patients with COPD-associated PH75. The phosphodiesterases (PDEs) are a family of enzymes that hydrolyze 3’, 5’-cyclic nucleotidemonophosphates into their respective 5’-monophosphates. PDE 5, PDE 6 and PDE 9 are selective for cGMP16 (Figure 2). An experimental ovine fetal model for perinatal pulmonary hypertension of the neonate was characterized by altered pulmonary vasoreactivity and structure. Infusion of PDE-5 inhibitors, dipyridamole and zaprinast lowered pulmonary resistance by 55 and 35%, respectively in hypertensive animals. Moreover, PDE5 activity was 150% higher in hypertensive fetal lambs in comparison with healthy animals76. In chronically hypoxia mice with disruption of gene encoding eNOS sildenafil reduced pulmonary arterial pressure less than in wild-type controls and failed to decrease right ventricular hypertrophy77. In the same model where GC-A was blunted, PDE5 inhibition reduced

Figure 2: Nitric oxide (NO) activates soluble guanylate cyclase (sGC), a heterodimer enzyme composed by alpha and beta subunits and a prosthetic heme group, which converts guanosine triphosphate (GTP) into cyclic guanosine monophosphate (cGMP). Nitric oxide and stimulators activate sGC in its reduced form (Fe2+) whereas activators act preferentially and effectively when sGC is oxidized (Fe3+). Phosphosdiesterase type 5 (PDE5) inhibitors lead to cGMP accumulation.

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pulmonary arterial pressure, right ventricular hypertrophy and pulmonary vascular remodeling with less efficacy than wild-type animals78, suggesting that NO or natriuretic peptides also influence to the vasodilator response to sildenafil by enhancing cGMP levels. Patients with idiopathic PH (n=9, WHO 1), PAH (n=2, WHO group 1) or PH due to left heart diseases (n=2, WHO 2) were given sildenafil, inhaled NO or sildenafil+inhaled NO. The decrease in pulmonary vascular resistance was similar with inhaled NO (-19 ± 5%) and sildenafil (27 ± 3%), whereas sildenafil+inhaled NO (-32 ± 5%, P<0.003) was more effective than inhaled NO. In addition, sildenafil and sildenafil+inhaled NO increased cardiac index79. Ten PAH patients with normal left ventricular function were included in a prospective randomized, placebo-controlled cross over study to evaluate the short-term effect of sildenafil. Patients in the sildenafil group have and improvement of 6-minute walk distance from 163.9 to 266.7 m (P<0.0005) and a decrease in the Borg dyspnea index (from 5.2 to 3.6, P<0.01) and pulmonary artery systolic pressure (from 80.8 to 55.3, P<0.05)80. In another double blind, placebo-controlled study, patients with PAH received placebo or sildenafil (20, 40 or 80 mg) for 12 weeks. The 6-minute walk increased from baseline in all sildenafil groups. All sildenafil doses reduced the mean pulmonary-artery pressure, showing the efficacy of sildenafil in improving exercise capacity and haemodynamics81. These observations provided the rationale to target PDE5 inhibition for the treatment of pulmonary hypertension with or without agents that enhance cGMP formation such as natriuretic peptides and/or nitro-vasodilators. As mentioned above, activating sGC has been approved as a new therapeutic method for pulmonary hypertension. sGC activation can be reached by two mechanisms: the sGC stimulators (BAY 41-2272, BAY 63-2521, BAY 41-8543) are dependent on the presence of the reduced (ferrous-

Fe2+) prosthetic on sGC heme and works synergically with NO, whereas sGC activators (BAY 58-2667, BAY 60-2770 and HMR 1766) preferentially and effectively activate sGC when it is oxidized (ferric-Fe3+) or in a heme-free state (Figure 2). Pulmonary arterial hypertension is associated with endothelial dysfunction and thus low bioavailability of NO, sGC stimulators or activators overcome such limitations, since these substances activate sGC in a NO-independent manner62. In a phase I study of 58 healthy male volunteers, oral riociguat (BAY 63-2521) was well tolerated. In a phase IIa study in 19 patients with diagnosis of PAH (WHO 1), distal chronic tromboembolic (WHO 4) or PH associated with mild to moderate interstitial lung disease (WHO 3) riociguat demonstrated hemodynamic efficacy and favorable tolerability to a greater extent than inhaled NO. After a 2.5 mg dose of riociguat, mean arterial pressure fell by 14%82. In patients with CTEPH (WHO 4) and PAH (WHO 1) mean arterial blood pressure decreased 4.5 mmHg from baseline after riociguat treatment (1 to 2.5 mg 3 times daily for three weeks). Dyspnea and a 6-minute walking distances showed significant improvement83. In a phase III, multicenter, randomized, double-blind, placebo-controlled study, 261 patients with CPTEPH (WHO group 4) or persistent pulmonary hypertension after surgery (WHO group 4) patients who received riociguat (n=173) had a mean increase of 39 m, whereas placebo (n=88) group a mean decrease of 6 m on 6-min walk distance test. Pulmonary vascular resistance decreased and increased in riociguat and placebo group, respectively. Riociguat also significantly reduced NT-proBNP levels84. In October 2013 riociguat was the first-in-class sGC stimulator approved by FDA for PAH and CTEPH, excluding the prior use of many nitrovasodilators. In an animal model of heart failure, PDE-5 inhibition showed beneficial effects after 4 weeks of treatment compared to control, which improved

contractility (end-systolic elastance 247 ± 68 vs 155 ± 71 mmHg/mL, P<0.05); prevented right ventricular dilatation (end-diastolic volume 733 ± 50 vs 874 ± 39 μL, P<0.05); reduced wall stress (peak wall stress 323 ± 46 vs 492 ±62 mmHg, P<0.05) and partially preserved exercise tolerance (running distance -33 ±15 vs -62 ± 12%, P<0.05), respectively85. In mice exposed to chronic pressure overload induced by transverse aortic constriction, sildenafil reduced chamber and myocyte hypertrophy and improved in vivo heart function86. In patients with congestive heart failure, sildenafil (10 mg) decreased pulmonary artery systolic (-21.6%) and diastolic (-31.8%) pressure and arteriolar resistance (-36.9%), without affecting ejection fraction, cardiac index, wedge pulmonary pressure and blood capillary volume, suggesting that sildenafil ameliorates pulmonary haemodynamics87. In a fixed-dose double-blind, randomized, placebo controlled, two-way crossover study, the effect of sildenafil (50 mg) was investigated in 23 men with chronic heart failure. Sildenafil reduced significantly heart rate, systolic and diastolic blood pressure before and after cardiopulmonary walking and exercise test88. Chronic treatment with sildenafil (50 mg, 8/8 hours) for 4 weeks improved maximal oxygen uptake, ventilator efficiency and oxygen uptake kinetics in patients with chronic heart failure with an ejection fraction of 28 ± 6% in comparison to placebo89. However, a multicenter, double-blind, placebo-controlled, parallel-group, randomized clinical trial of 216 outpatients with heart failure (ejection fraction ≥ 50%, elevated BNP and reduced exercise capacity), sildenafil (20 mg 8/8 hours for 12 weeks followed by 60 mg 8/8 hours for 12 weeks) did not result in significant improvement in exercise capacity or clinical status90. Soluble guanylate cyclase stimulator (riociguat, BAY 63-2521) and activator (cinaciguat, BAY 58-2667) are also being investigated in PH patients with left heart disease (WHO group 2). In patients with acute decompensated

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heart failure, infusion of cinaciguat (50-400 mg/h, 6 hours) led to significant reductions in pulmonary capillary wedge pressure, mean right arterial pressure, mean pulmonary artery pressure, pulmonary vascular resistance and systemic vascular resistance, while increasing heart rate and cardiac output91. In patients with heart failure with preserved left ventricular ejection fraction and pulmonary hypertension, riociguat (2 mg) significantly increased stroke volume, decreased systolic blood pressure and right ventricular end-diastolic area92. Whether sGC stimulators or activators are effective in heart failure requires more clinical trials with a larger number of patients. Clinical applications of NO-cGMP regulators through vascular-regulated mechanisms Lower urinary tract symptoms (LUTS), benign prostatic hyperplasia (BPH) and erectile dysfunction (ED) are common conditions in elderly males and the prevalence increases with aging. Clinical and basic research data suggest that the incidence of LUTS, BPH and ED are higher in individuals with vascular risk, such as hypertension, diabetes, hyper-lipidaemia and obesity. Besides, many clinicians consider ED as an early symptom of vascular damage and should be used as a diagnosis for the identification of cardiovascular diseases93. Alterations of the NO-cGMP pathway, enhancement of Rho-associated protein kinase (ROCK), contractile signaling, autonomic adrenergic hyperactivity and pelvic atherosclerosis might decrease pelvic arterial blood flow, leading to arterial insufficiency, smooth muscle relaxation and contractility alterations and thus bladder94, prostate95 and penis96 dysfunctions. Since there exist clinical evidence of the interrelation between ED and BPH-related LUTS, substances that alter the NO-cGMP signaling pathway are an alternative for the treatment of LUTS-BPH-ED. Nitric oxide is the principal mediator in penile erection and it can be released from the action of endothelial

or neuronal nitric oxide synthase in “nitrinergic neurons”. Rajfer et al.97 was the first group to demonstrate that electrical field stimulation (EFS) induced frequency dependent relaxation, which was inhibited and enhanced, respectively, in the presence of inhibitors of NOS (N-nitro-L-arginine and N-amino-L-arginine) or sGC inbibitors (methylene blue) and PDE (M&B 22,948), suggesting that NO is an important mediator of nonadrenergic noncholinergic neurotransmittion. Few years latter studies have shown that the magnitude of relaxation induced by EFS98 or by NO-donors99 was reduced in men with vascular impotence in comparison with controls, thus providing evidence that lower levels of NO may be responsible for erectile dysfunction. Since 1990 many studies have been published showing that alterations on NO-pathway, such as low NO-bioavailability and/or oxidation of sGC is the main cause of erectile dysfunction. The first-in-class selective phosphodiesterase type 5 (PDE5) inhibitor, was zaprinast introduced in the early 1980’s. Subsequently, Viagra (sildenafil citrate) was introduced in 1998 for the treatment of ED. The impact of sildenafil has stimulated academic, clinical and industrial research to better understand the nature of sexual function and develop better treatment and management for sexual dysfunctions such as ED. However, patients with ED and high cardiovascular risk do not respond to PDE5 inhibitor treatment. Nonresponding patients showed a significant higher severity and duration of ED, higher level of arterial insufficiency and higher endothelial apoptosis100. Thus, new treatments options are required to fulfill the medical need in those men with greater endothelial damage. Soluble guanylate cyclase stimulators and activator constitute a new therapeutic option for ED, when NO formation or bioavailability is decreased by oxidative stress or when PDE-inhibitors are no longer effective. Soluble guanylate cyclase stimulator,

BAY 41-2272 relaxed pre-contracted corpus cavernosum from rabbit101,102, human101,102 and mice103, which was reduced in the presence of L-NAME, a NOS inhibitor or ODQ, a sGC inhibitor. In human corpus cavernosum from PDE5 inhibitor nonresponders co-incubation with BAY 60-4552 (sGC stimulator) and vardenafil (PDE-5 inhibitor) induced significantly greater relaxation compared with either compound alone104 In rats with cavernous nerve injury both vardenafil (0.03 mg/Kg) and BAY 60-4552 (0.03 and 0.3 mg/Kg) increased intracavernous pressure (ICP) and their combination provided synergic response on ICP105. Intracavernous (ic) administration with sGC activator BAY 60-2770 increased ICP and this response was enhanced by ODQ (2 mg/Kg, ic)106. To date sGC stimulators has been approved for the treatment of pulmonary hypertension. Whether sGC stimulators and activators have any role on erectile dysfunction, controlled, randomized, placebo-control studies should be carried out in order to ascertain the safety, efficacy and cost-effectiveness of these drugs in urologic disorders. Lower urinary tract symptoms (LUTS) secondary to benign prostatic hyperplasia (BPH) is a condition that impacts the quality of life in elderly men and characterized by increased frequency of urination and urgency with or without nocturia. Therapeutic management for LUTS-BPH is aimed to relax bladder and/or prostate smooth muscle and to inhibit prostatic cell growth. There is an increasing interest in agents that increase cGMP levels for the treatment of LUTS-BPH, since there exist a dense nitrinergic innervation in the transition zone of the human prostate, fibromuscular stroma, glandular epithelium and blood vessels107. Recently, FDA approved tadalafil for the treatment of LUTS-BPH. PDE5 is expressed in vessels, bladder, urethra, and prostate smooth muscle, but not in epithelium. In human prostate, SNP induced concentration-dependent relaxation and was significantly potentiated by tadalafil (30-60 nM). The combination

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of tadalafil and tansulosin, an alpha1-adrenoceptor blocker, reduced significantly EFS-induced contraction in prostate and bladder neck108. In prostatic smooth muscle, but not in bladder cells, NO-donor, sodium nitroprusside (SNP) and sGC stimulator, BAY 41-8543 showed a concentration-dependent antiproliferative effect, which was enhanced in the presence of vardenafil109. The efficacy of tadalafil as monotherapy110,111,112,113 or in combination with alpha-adrenoceptor blockers114,115 or 5-alpha reductase inhibitors116 for the treatment of LUTS-BPH has been demonstrated in several randomized, placebo controlled clinical trials.

Conclusion The discovery of the NO-cGMP pathway by our lab in the 1970’s revolutionized the comprehension of pathophysiological mechanisms involved on cardiovascular diseases. However, considering the expression “from bench to bedside” we concluded that therapeutic alternatives that target NO-cGMP did not immediately follow the biochemical and pathophysiological revolution, since few therapeutic options have been proven effective and released on the market for the treatment of cardiovascular disorders such as recombinant brain natriuretic peptide (nesiritide), inhaled NO, PDE-5 inhibitors (sildenafil, tadalafil and vardenafil) and more recently sGC stimulator (riociguat). Initially, the scientific community was reluctant to accept nitric oxide as a free radical and gas to be an important intracellular messenger, autacoid, paracrine substance and hormone as we have predicted.

Acknowledgement Fabiola Zakia Mónica acknowledges Fundacao de Amparo a Pesquisa Estado de Sao Paulo (FAPESP 2013/02246-7) and State University of Campinas for research support.

Abbreviations NO, nitric oxide; GTP, guanosine triphosphate; cGMP,cyclic 3’-5’ guanosine monophosphate; sGC, soluble guanylyl cyclase; pGC, particulate guanylyl cyclase; PDE’s, phosphodiesterases; PKG, protein kinase G; ANP, atrial natriuretip peptide; BNP, brain natriuretic peptide; CNP, C-natriuretic; NT-BNP, N-terminal fragment of BNP; CD-NP, chimeric natriuretic peptide; GC-A, guanylyl cyclase A; GC-B, guanylyl cyclase B; GC-C, guanylyl cyclase C; ROCK, Rho-associated protein kinase; PH, pulmonary hypertension; PAH, pulmonary arterial hypertension; CHF, chronic heart failure; CTEPH, chronic thromboembolic pulmonary hypertension; COPD, chronic obstructive pulmonary disease; LUTS, lower urinary tract symptoms; BPH, benign prostatic hyperplasia; ED, erectile dysfunction; EFS, electrical field stimulation

References 1. Bian K, Doursout MF, Murad F. Vascular system: role of nitric oxide in cardiovascular diseases. Clin Hypertens. 2008 Apr; 10(4):304-10. 2. Mónica FZ, Reges R, Cohen D, Silva FH, De Nucci G, D'Ancona CA, Antunes E. Long-term administration of BAY 41-2272 prevents bladder dysfunction in nitric oxide-deficient rats. Neurourol Urodyn. 2011 Mar; 30(3):456-60. 3. Báu FR, Mónica FZ, Priviero FB, Baldissera L Jr, de Nucci G, Antunes E. Evaluation of the relaxant effect of the nitric oxide-independent soluble guanylyl cyclase stimulator BAY 41-2272 in isolated detrusor smooth muscle. Eur J Pharmacol. 2010 Jul; 637(1-3):171-7. 4. Alexandre EC, Leiria LO, Silva FH, Mendes-Silvério CB, Calmasini FB, Davel AP, Mónica FZ, De Nucci G, Antunes E. Soluble guanylyl cyclase (sGC) degradation and impairment of nitric oxide-mediated responses in urethra from obese mice: reversal by the sGC activator BAY 60-2770. J Pharmacol Exp Ther. 2014 Apr; 349 (1):2-9.

5. Sindić A, Hirsch JR, Velic A, Piechota H, Schlatter E. Guanylin and uroguanylin regulate electrolyte transport in isolated human cortical collecting ducts. Kidney Int. 2005 Apr; 67(4):1420-7. 6. Mendes-Silverio CB, Leiria LO, Morganti RP, Anhê GF, Marcondes S, Mónica FZ, De Nucci G, Antunes E. Activation of haem-oxidized soluble guanylyl cyclase with BAY 60-2770 in human platelets lead to overstimulation of the cyclic GMP signaling pathway. PLoS One. 2012; 7(11):e47223. 7. Jenei V, Deevi RK, Adams CA, Axelsson L, Hirst DG, Andersson T, Dib K. Nitric oxide produced in response to engagement of beta2 integrins on human neutrophils activates the monomeric GTPases Rap1 and Rap2 and promotes adhesion. J Biol Chem. 2006 Nov; 281(46):35008-20. 8. Willipinski-Stapelfeldt B, Lübberstedt J, Stelter S, Vogt K, Mukhopadhyay AK, Müller D. Comparative analysis between cyclic GMP and cyclic AMP signalling in human sperm. Mol Hum Reprod. 2004 Jul;10(7):543-52. 9. Arshad N, Visweswariah SS. The multiple and enigmatic roles of guanylyl cyclase C in intestinal homeostasis. FEBS Lett. 2012 Aug; 586(18):2835-40. 10. Zhu H, Li JT, Zheng F, Martin E, Kots AY, Krumenacker JS, Choi BK, McCutcheon IE, Weisbrodt N, Bögler O, Murad F, Bian K. Restoring soluble guanylyl cyclase expression and function blocks the aggressive course of glioma. Mol Pharmacol. 2011 Dec; 80(6):1076-84. 11. Bian K, Murad F. sGC-cGMP Signaling: Target for Anticancer Therapy. Adv Exp Med Biol. 2014; 814:5-13. 12. Katsuki S, Arnold WP, Murad F. Effects of sodium nitroprusside, nitroglycerin, and sodium azide on levels of cyclic nucleotides and mechanical activity of various tissues. J Cyclic Nucleotide Res. 1977 Aug; 3(4):239-47. 13. Waldman SA, Rapoport RM, Fiscus RR, Leitman DC, Chang LY, Murad F. Regulation of particulate guanylate cyclase by atriopeptins: relation between peptide structure, receptor binding, and enzyme kinetics. Biochim

of 12



Critical review

Co

mp

etin

g in

tere

sts:

No

ne

dec

lare

d.

Co

nfl

ict

of

inte

rest

s: N

on

e d

ecla

red

. A

ll a

uth

ors

co

ntr

ibu

ted

to

co

nce

pti

on

an

d d

esig

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an

usc

rip

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rep

ara

tio

n, r

ead

an

d a

pp

rove

d t

he

fin

al m

an

usc

rip

t.

All

au

tho

rs a

bid

e b

y th

e A

sso

cia

tio

n f

or

Med

ica

l Eth

ics

(AM

E) e

thic

al r

ule

s o

f d

iscl

osu

re.

Biophys Acta. 1989 Nov; 999(2):157-62. 14. Waldman SA, Rapoport RM, Murad F. Atrial natriuretic factor selectively activates particulate guanylate cyclase and elevates cyclic GMP in rat tissues. J Biol Chem. 1984 Dec; 259(23): 14332-4. 15. Kuno T, Andresen JW, Kamisaki Y, Waldman SA, Chang LY, Saheki S, Leitman DC, Nakane M, Murad F. Co-purification of an atrial natriuretic factor receptor and particulate guanylate cyclase from rat lung. J Biol Chem. 1986 May; 261(13):5817-23. 16. Maurice DH, Ke H, Ahmad F, Wang Y, Chung J, Manganiello VC. Advances in targeting cyclic nucleotide phosphodiesterases. Nat Rev Drug Discov. 2014 Apr; 13(4):290-314. 17. Sager G. Cyclic GMP transporters. Neurochem Int. 2004 Nov; 45(6):865-73. 18. Kimura H, Murad F. Two forms of guanylate cyclase in mammalian tissues and possible mechanisms for their regulation. Metabolism. 1975 Mar;24(3):439-45. 19. Chinkers M, Garbers DL, Chang MS, Lowe DG, Chin HM, Goeddel DV, Schulz S. A membrane form of guanylate cyclase is an atrial natriuretic peptide receptor. Nature. 1989 Mar;338(6210):78-83. 20. Drewett JG, Garbers DL. The family of guanylyl cyclase receptors and their ligands. Endocr Rev. 1994 Apr; 15(2):135-62. 21. Kiemer AK, Vollmar AM. Autocrine regulation of inducible nitric-oxide synthase in macrophages by atrial natriuretic peptide. J Biol Chem. 1998 May; 273(22):13444-51. 22. Potter LR, Yoder AR, Flora DR, Antos LK, Dickey DM. Natriuretic peptides: their structures, receptors, physiologic functions and therapeutic applications. Handb Exp Pharmacol. 2009;(191): 341-66. 23. Chen HH, Burnett JC Jr. C-type natriuretic peptide: the endothelial component of the natriuretic peptide system. J Cardiovasc Pharmacol. 1998; 32 Suppl 3:S22-8. 24. Ishizaka Y, Kangawa K, Minamino N, Ishii K, Takano S, Eto T, Matsuo H. Isolation and identification of C-type natriuretic peptide in human

monocytic cell line, THP-1. Biochem Biophys Res Commun. 1992 Dec; 189 (2):697-704. 25. Ruilope LM, Dukat A, Böhm M, Lacourcière Y, Gong J, Lefkowitz MP. Blood-pressure reduction with LCZ696, a novel dual-acting inhibitor of the angiotensin II receptor and neprilysin: a randomised, double-blind, placebo-controlled, active comparator study. Lancet. 2010 Apr; 375(9722):1255-66. 26. Gluba A, Bielecka-Dabrowa A, Mikhailidis DP, Wong ND, Franklin SS, Rysz J, Banach M. An update on biomarkers of heart failure in hypertensive patients. J Hypertens. 2012 Sep; 30(9): 1681-9. 27. de Lemos JA, McGuire DK, Drazner MH. B-type natriuretic peptide in cardiovascular disease. Lancet. 2003 Jul; 362(9380):316-22. 28. Omland T, Hagve TA. Natriuretic peptides: physiologic and analytic considerations. Heart Fail Clin. 2009 Oct: 5(4):471-87. 29. Wieczorek SJ, Wu AH, Christenson R, Krishnaswamy P, Gottlieb S, Rosano T, Hager D, Gardetto N, Chiu A, Bailly KR, Maisel A. A rapid B-type natriuretic peptide assay accurately diagnoses left ventricular dysfunction and heart failure: a multicenter evaluation. Am Heart J. 2002 Nov; 144(5):834-9. 30. Zois NE, Bartels ED, Hunter I, Kousholt BS, Olsen LH, Goetze JP. Natriuretic peptides in cardiometabolic regulation and disease. Nat Rev Cardiol. 2014 Jul; 11(7):403-12. 31. Tate S, Griem A, Durbin-Johnson B, Watt C, Schaefer S. Marked elevation of B-type natriuretic peptide in patients with heart failure and preserved ejection fraction. J Biomed Res. 2014 Jul; 28(4):255-61. 32. McMurray JJ, Packer M, Desai AS, Gong J, Lefkowitz M, Rizkala AR, Rouleau JL, Shi VC, Solomon SD, Swedberg K, Zile MR; PARADIGM-HF Committees Investigators. Baseline characteristics and treatment of patients in Prospective comparison of ARNI with ACEI to Determine Impact on Global Mortality and morbidity in Heart Failure trial (PARADIGM-HF).

Eur J Heart Fail. 2014 Jul; 16(7):817-25. 33. Clavell AL, Stingo AJ, Wei CM, Heublein DM, Burnett JC Jr. C-type natriuretic peptide: a selective cardiovascular peptide. Am J Physiol. 1993 Feb; 264(2 Pt 2):R290-5. 34. Furuya M, Miyazaki T, Honbou N, Kawashima K, Ohno T, Tanaka S, Kangawa K, Matsuo H. C-type natriuretic peptide inhibits intimal thickening after vascular injury. Ann N Y Acad Sci. 1995 Jan; 748:517-23. 35. Tokudome T, Horio T, Soeki T, Mori K, Kishimoto I, Suga S, Yoshihara F, Kawano Y, Kohno M, Kangawa K. Inhibitory effect of C-type natriuretic peptide (CNP) on cultured cardiac myocyte hypertrophy: interference between CNP and endothelin-1 signaling pathways. Endocrinology. 2004 May; 145(5):2131-40. 36. Lisy O, Huntley BK, McCormick DJ, Kurlansky PA, Burnett JC Jr. Design, synthesis, and actions of a novel chimeric natriuretic peptide: CD-NP. J Am Coll Cardiol. 2008 Jul; 52(1):60-8. 37. Lee CY, Chen HH, Lisy O, Swan S, Cannon C, Lieu HD, Burnett JC Jr. Pharmacodynamics of a novel designer natriuretic peptide, CD-NP, in a first-in-human clinical trial in healthy subjects. J Clin Pharmacol. 2009 Jun; 49(6):668-73. 38. Costello-Boerrigter LC, Schirger JA, Miller WL, Boerrigter G, Chen HH, Ruth Kempf R, Lee CY, Burnett Jr JC. Cenderitide (CD-NP), a novel peptide designed to activate both guanylyl cyclase B and A, activates the second messenger cGMP, suppresses aldosterone, and preservers GFR without reducing blood pressure in a proof-of-concept study in patients with chronic heart failure. BMC Pharmacology. 2011 Jun; 11(Suppl 1):P18. 39. Murad F, Arnold WP, Mittal CK, Braughler JM Properties and regulation of guanylate cyclase and some proposed functions for cyclic GMP.. Adv Cyclic Nucleotide Res. 1979; 11:175-204. 40. Guerrant RL, Hughes JM, Chang B, Robertson DC, Murad F Activation of intestinal guanylate cyclase by heat-stable enterotoxin of Escherichia coli: studies of tissue specificity, potential

of 12



Critical review

Co

mp

etin

g in

tere

sts:

No

ne

dec

lare

d.

Co

nfl

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of

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rest

s: N

on

e d

ecla

red

. A

ll a

uth

ors

co

ntr

ibu

ted

to

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nce

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on

an

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an

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rep

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ead

an

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rove

d t

he

fin

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an

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rip

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rs a

bid

e b

y th

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cia

tio

n f

or

Med

ica

l Eth

ics

(AM

E) e

thic

al r

ule

s o

f d

iscl

osu

re.

receptors, and intermediates.J Infect Dis. 1980 Aug; 142(2):220-8. 41. Waldman SA, Lewicki JA, Brandwein HJ, Murad F. Partial purification and characterization of particulate guanylate cyclase from rat liver after solubilization with trypsin. J Cyclic Nucleotide Res. 1982; 8(6):359-70. 42. Waldman SA, Rapoport RM, Murad F Atrial natriuretic factor selectively activates particulate guanylate cyclase and elevates cyclic GMP in rat tissues. J Biol Chem. 1984 Dec; 259(23):14332-4. 43. Fiscus RR, Rapoport RM, Waldman SA, Murad F Atriopeptin II elevates cyclic GMP, activates cyclic GMP-dependent protein kinase and causes relaxation in rat thoracic aorta. Biochim Biophys Acta. 1985 Jul; 846(1):179-84. 44. Bakre MM, Sopory S, Visweswariah SS. Expression and regulation of the cGMP-binding, cGMP-specific phosphodiesterase (PDE5) in human colonic epithelial cells: role in the induction of cellular refractoriness to the heat-stable enterotoxin peptide. J Cell Biochem. 2000 Feb; 77(1):159-67. 45. Kloeters O1, Friess H, Giese N, Buechler MW, Cetin Y, Kulaksiz H. Uroguanylin inhibits proliferation of pancreatic cancer cells. Scand J Gastroenterol. 2008;43(4):447-55. 46. Carpick BW, Gariépy J. The Escherichia coli heat-stable enterotoxin is a long-lived superagonist of guanylin. Infect Immun. 1993 Nov; 61(11):4710-5. 47. Hamra FK, Fan X, Krause WJ, Freeman RH, Chin DT, Smith CE, Currie MG, Forte LR. Prouroguanylin and proguanylin: purification from colon, structure, and modulation of bioactivity by proteases. Endocrinology. 1996 Jan;137(1):257-65. 48. Lin JE, Valentino M, Marszalowicz G, Magee MS, Li P, Snook AE, Stoecker BA, Chang C, Waldman SA. Bacterial heat-stable enterotoxins: translation of pathogenic peptudes into novel targetes diagnostics and therapeutics. Toxins (Basel). 2010 Aug;2(8):2028-54.

49. Murad F. J The 1996 Albert Lasker Medical Research Awards. Signal transduction using nitric oxide and cyclic guanosine monophosphate. JAMA. 1996 Oct;276(14):1189-92. 50. Stone JR, Marletta MA. Spectral and kinetic studies on the activation of soluble guanylate cyclase by nitric oxide. Biochemistry. 1996 Jan; 35(4):1093-9. 51. Guha M. First-in-class guanhlate cyclase stimulator approved for PAH. Nat Biotechnol. 2013 Dec; 31 (12):1064. 52. Murad F, Mittal CK, Arnold WP, Katsuki S, Kimura H. Guanylate cyclase: activation by azide, nitro compounds, nitric oxide, and hydroxyl radical and inhibition by hemoglobin and myoglobin. Adv Cyclic Nucleotide Res. 1978; 9:145-58. 53. Rapoport RM, Draznin MB, Murad F. Sodium nitroprusside-induced protein phosphorylation in intact rat aorta is mimicked by 8-bromo cyclic GMP. Proc Natl Acad Sci U S A. 1982 Nov; 79(21):6470-4. 54. Lewicki JA, Brandwein HJ, Mittal CK, Arnold WP, Murad F. Properties of purified soluble guanylate cyclase activated by nitric oxide and sodium nitroprusside. J Cyclic Nucleotide Res. 1982;8(1):17-25. 55. Murad F. Nitric oxide signaling: would you believe that a simple free radical could be a second messenger, autacoid, paracrine substance, neurotransmitter, and hormone? Recent Prog Horm Res. 1998;53:43-59; discussion 59-60. 56. Kinsella JP, Neish SR, Shaffer E, Abman SH. Low-dose inhlatation nitric oxide in persistent pulmonary hypertension of the newborn. Lancet. 1992 Oct 3;340(8823):819-20. 57. Kinsella JP, Neish SR, Ivy DD, Shaffer E, Abman SH. Clinical responses to prolonged treatment of persistent pulmonary hypertension of the newborn with low doses of inhaled nitric oxide. J Pediatr. 1993 Jul;123 (1):103-8. 58. Roberts JD Jr, Fineman JR, Morin FCr, Shaul PW, Rimar S, Schreiber MD, et al. Inhaled nitric oxide and persistent pulmonary hypertension of the newborn. N Engl J Med. 1997 Feb; 336(9):605–10.

59. Clark RH, Kueser TJ, Walker MW, Southgate WM, Huckaby JL, Perez JA, et al. Low-dose nitric oxide therapy for persistent pulmonary hypertension of the newborn. Clinical Inhaled Nitric Oxide Research Group. N Engl J Med. 2000 Feb;342(7):469–74. 60. Pepke-Zaba J, Higenbottam TW, Dinh-Xuan AT, Stone D, Wallwork J. Inhaled nitric oxide as a cause of selective pulmonary vasodilatation in pulmonary hypertension. Lancet. 1991 Nov; 338(8776):1173-4. 61. Barst RJ, Agnoletti G, Fraisse A, Baldassarre J, Wessel DL. NO Diagnostic Study Group. Vasodilator testing with nitric oxide and/or oxygen in pediatric pulmonary hypertension. Pediatr Cardiol. 2010 Jul; 31(5):598-606. 62. Montani D, Chaumais MC, Guignabert C, Günther S, Girerd B, Jaïs X1, Algalarrondo V, Price LC, Savale L, Sitbon O, Simonneau G, Humbert M. Targeted therapies in pulmonary arterial hypertension. Pharmacol Ther. 2014 Feb;141(2):172-91. 63. Boerrigter G, Burnett JC Jr. Soluble guanylate cyclase: not a dull enzyme. Circulation. 2009 Jun; 119(21):2752-4. 64. Ghofani HA, Grimminger F. Soluble guanylate cyclase stimulation: an emerging option in pulmonary hypertension therapy. Eur Respir Rev. 2009 Mar;18(111):35-41. 65. Olschewski H1, Simonneau G, Galiè N, Higenbottam T, Naeije R, Rubin LJ, Nikkho S, Speich R, Hoeper MM, Behr J, Winkler J, Sitbon O, Popov W, Ghofrani HA, Manes A, Kiely DG, Ewert R, Meyer A, Corris PA, Delcroix M, Gomez-Sanchez M, Siedentop H, Seeger W. Aerosolized Iloprost Randomized Study Group. Inhaled iloprost for severe pulmonary hypertension. N Engl J Med. 2002 Aug; 347(5):322-9. 66. Suntharalingam J, Treacy CM, Doughty NJ, Goldsmith K, Soon E, Toshner MR, Sheares KK, Hughes R, Morrell NW, Pepke-Zaba J. Long-term use of sildenafil in inoperable chronic thromboembolic pulmonary hypertension. Chest. 2008 Aug; 134 (2):229-36. 67. Jaïs X, D'Armini AM, Jansa P, Torbicki A, Delcroix M, Ghofrani HA, Hoeper MM, Lang IM, Mayer E, Pepke-Zaba J, Perchenet L, Morganti A, Simonneau G, Rubin LJ. Bosentan

of

12



Critical review

Co

mp

etin

g in

tere

sts:

No

ne

dec

lare

d.

Co

nfl

ict

of

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rest

s: N

on

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red

. A

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ors

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rove

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or

Med

ica

l Eth

ics

(AM

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thic

al r

ule

s o

f d

iscl

osu

re.

Effects in iNopErable Forms of chronIc Thromboembolic pulmonary hypertension Study Group. Bosentan for treatment of inoperable chronic thromboembolic pulmonary hypertension: BENEFiT (Bosentan Effects in iNopErable Forms of chronIc Thromboembolic pulmonary hypertension), a radomized, placebo-controlled trial. J Am Coll Cardiol. 2008 Dec; 52(25):2127-34. 68. Alaeddini J, Uber PA, Park MH, Scott RL, Ventura HO, Mehra MR. Efficacy and safety of sildenafil in the evaluation of pulmonary hypertension in severe heart failure. Am J Cardiol. 2004 Dec 1; 94(11):1475-7. 69. Guazzi M, Tumminello G, Di Marco F, Guazzi MD. Influences of sildenafil on lung function and hemodynamics in patients with chronic heart failure. Clin Pharmacol Ther. 2004 Oct; 76(4):371-8. 70. Lewis GD, Lachmann J, Camuso J, Lepore JJ, Shin J, Martinovic ME, Systrom DM, Bloch KD, Semigran MJ. Sildenafil improves exercise hemodynamics and oxygen uptake in patients with systolic heart failure. Circulation. 2007 Jan;115(1):59-66. 71. Behling A, Rohde LE, Colombo FC, Goldraich LA, Stein R, Clausell N. Effects of 5'-phosphodiesterase four-week long inhibition with sildenafil in patients with chronic heart failure: a double-blind, placebo-controlled clinical trial. J Card Fail. 2008 Apr; 14(3):189-97. 72. Murad F. Cyclic guanosine monophosphate as a mediator of vasodilation.J Clin Invest. 1986 Jul; 78(1):1-5. 73. Murad F. Nitric oxide signaling: would you believe that a simple free radical could be a second messenger, autacoid, paracrine substance, neurotransmitter, and hormone? Recent Prog Horm Res. 1998; 53:43-59; discussion 59-60. 74. Gimeno E, Munoz PA, Pizarro S, Gistau C, Rodriguez-Roisin R, Roca J, Barberà JA. Hemodynamic and gas exchange effects of sildenafil in patients with chronic obstructive pulmonary disease and pulmonary hypertension. Am J Respir Crit Care Med. 2010 Feb;181(3):270-8.

75. Rietema H, Holverda S, Bogaard HJ, Marcus JT, Smit HJ, Westerhof N, Postmus PE, Boonstra A, Vonk-Noordegraaf A. Sildenafil treatment in COPD does not affect stroke volume or exercise capacity. Eur Respir J. 2008 Apr; 31(4):759-64. 76. Hanson KA, Ziegler JW, Rybalkin SD, Miller JW, Abman SH, Clarke WR. Chronic pulmonary hypertension increases fetal lung cGMP phosphodiesterase activity. Am J Physiol. 1998 Nov; 275(5 Pt 1):L931-41. 77. Zhao L, Mason NA, Morrell NW, Kojonazarov B, Sadykov A, Maripov A, Mirrakhimov MM, Aldashev A, Wilkins MR. Sildenafil inhibits hypoxia-induced pulmonary hypertension. Circulation. 2001 Jul: 104(4): 424-8. 78. Zhao L, Mason NA, Strange JW, Walker H, Wilkins MR. Beneficial effects of phosphodiesterase 5 inhibition in pulmonary hypertension are influenced by natriuretic Peptide activity. Circulation. 2003 Jan; 107(2):234-7. 79. Michelakis E, Tymchak W, Lien D, Webster L, Hashimoto K, Archer S. Oral sildenafil is an effective and specific pulmonary vasodilator in patients with pulmonary arterial hypertension: comparison with inhaled nitric oxide. Circulation. 2002 May; 105(20):2398-403. 80. Bharani A, Mathew V, Sahu A, Lunia B. The efficacy and tolerability of sildenafil in patients with moderate-to-severe pulmonary hypertension. Indian Heart J. 2003 Jan-Feb:55 (1):55-9. 81. Galiè N, Ghofrani HA, Torbicki A, Barst RJ, Rubin LJ, Badesch D, Fleming T, Parpia T, Burgess G, Branzi A, Grimminger F, Kurzyna M, Simonneau G. Sildenafil Use in Pulmonary Arterial Hypertension (SUPER) Study Group. Sildenafil citrate therapy for pulmonary arterial hypertension. N Engl J Med. 2005 Nov;353(20):2148-57. 82. Grimminger F, Weimann G, Frey R, Voswinckel R, Thamm M, Bölkow D, Weissmann N, Mück W, Unger S, Wensing G, Schermuly RT, Ghofrani HA. First acute haemodynamic study of soluble guanylate cyclase stimulator riociguat in pulmonary

hypertension. Eur Respir J. 2009 Apr; 33(4):785-92. 83. Ghofrani HA, Hoeper MM, Halank M, Meyer FJ, Staehler G, Behr J, Ewert R, Weimann G, Grimminger F. Riociguat for chronic thromboembolic pulmonary hypertension and pulmonary arterial hypertension: a phase II study. Eur Respir J. 2010 Oct;36(4):792-9. 84. Ghofrani HA, Galiè N, Grimminger F, Grünig E, Humbert M, Jing ZC, Keogh AM, Langleben D, Kilama MO, Fritsch A, Neuser D, Rubin LJ. PATENT-1 Study Group. Riociguat for the treatment of pulmonary arteria hypertension. N Engl J Med. 2013 Jul 25; 369(4):330-40. 85. Borgdorff MA, Bartelds B, Dickinson MG, Boersma B, Weij M, Zandvoort A, Silljé HH, Steendijk P, de Vroomen M, Berger RM. Sildenafil enhances systolic adaptation, but does not prevent diastolic dysfunction, in the pressure-loaded right ventricle. Eur J Heart Fail. 2012 Sep; 14(9):1067-74. 86. Takimoto E, Champion HC, Li M, Belardi D, Ren S, Rodriguez ER, Bedja D, Gabrielson KL, Wang Y, Kass DA. Chronic inhibition of cyclic GMP phosphodiesterase 5A prevents and reverses cardiac hyperthrophy. Nat Med. 2005 Feb; 11(2):214-22. 87. Guazzi M, Reina G, Tumminello G, Guazzi MD. Exercise ventilation inefficiency and cardiovascular mortality in heart failure: the critical independent prognostic value of the arterial CO2 partial pressure. Eur Heart J. 2005 Mar; 26(5):472-80. 88. Bocchi EA, Guimarães G, Mocelin A, Bacal F, Bellotti G, Ramires JF. Sildenafil effects on exercise, neurohormonal activation, and erectile dysfunction in congestive heart failure: a double-blind, placebo-controlled, randomized study followed by a prospective treatment for erectile dysfunction. Circulation. 2002 Aug; 106(9):1097-103. 89. Behling A, Rohde LE, Colombo FC, Goldraich LA, Stein R, Clausell N. Effects of 5'-phosphodiesterase four-week long inhibition with sildenafil in patients with chronic heart failure: a double-blind, placebo-controlled clinical trial. J Card Fail. 2008 Apr; 14(3):189-97. 90. Redfield MM, Chen HH, Borlaug BA, Semigran MJ, Lee KL, Lewis G, LeWinter MM, Rouleau JL, Bull DA, Mann DL, Deswal A, Stevenson LW, Givertz MM,

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Ofili EO, O'Connor CM, Felker GM, Goldsmith SR, Bart BA, McNulty SE, Ibarra JC, Lin G, Oh JK, Patel MR, Kim RJ, Tracy RP, Velazquez EJ, Anstrom KJ, Hernandez AF, Mascette AM, Braunwald E. RELAX Trial. Effect of phosphodiesterase-5 inhibition on exercise capacity and clinical status in heart failure with preserved ejection fraction: a randomized clinical trial. JAMA. 2013 Mar; 309(12):1268-77. 91. Lapp H, Mitrovic V, Franz N, Heuer H, Buerke M, Wolfertz J, Mueck W, Unger S, Wensing G, Frey R. Cinaciguat (BAY 58-2667) improves cardiopulmonary hemodynamics in patients with acute decompensated heart failure. Circulation. 2009 Jun; 119(21):2781-8. 92. Bonderman D, Pretsch I, Steringer-Mascherbauer R, Jansa P, Rosenkranz S, Tufaro C, Bojic A, Lam CS, Frey R, Ochan Kilama M, Unger S, Roessig L, Lang IM. Acute Hemodynamic Effects of Riociguat in Patients with Pulmonary Hypertension Associated with Diastolic Heart Failure (DILATE-1): A Randomized, Double-Blind, Placebo-Controlled, Single-Dose Study. Chest. 2014 Jul. 93. Foresta C, Caretta N, Aversa A, Bettocchi C, Corona G, Mariani S, Rossato M. Erectile dysfunction: symptom or disease? J Endocrinol Invest. 2004 Jan; 27(1):80-95. 94. Azab S. The impact of atherosclerosis on lower urinary tract function. Aging Male. 2013 Sep; 16(3): 108-11. 95. Berger AP, Bartsch G, Deibl M, Alber H, Pachinger O, Fritsche G, Rantner B, Fraedrich G, Pallwein L, Aigner F, Horninger W, Frauscher F. Atherosclerosis as a risk factor for benign prostatic hyperplasia. BJU Int. 2006 Nov; 98(5):1038-42. 96. Miner M, Nehra A, Jackson G3, Bhasin S4, Billups K5, Burnett AL6, Buvat J7, Carson C8, Cunningham G9, Ganz P10, Goldstein I11, Guay A12, Hackett G13, Kloner RA14, Kostis JB15, LaFlamme KE16, Montorsi P17, Ramsey M16, Rosen R18, Sadovsky R19, Seftel A20, Shabsigh R21, Vlachopoulos C22, Wu F23. All men with vasculogenic erectile dysfunction require a cardiovascular workup. Am J Med. 2014 Mar; 127(3):174-82.

97. Rajfer J, Aronson WJ, Bush PA, Dorey FJ, Ignarro LJ. Nitric oxide as a mediator of relaxation of the corpus cavernosum in response to nonadrenergic, noncholinergic neurotransmission. N Engl J Med. 1992 Jan; 326(2):90-4. 98. Pickard RS, Powell PH, Zar MA. Nitric oxide and cyclic GMP formation following relaxant nerve stimulation in isolated human corpus cavernosum. Br J Urol. 1995 Apr; 75(4):516-22. 99. Christ GJ, Kim DC, Taub HC, Gondré CM, Melman A. Characterization of nitroglycerine-induced relaxation in human corpus cavernosum smooth muscle: implications to erectile physiology and dysfunction.Can J Physiol Pharmacol. 1995 Dec; 73(12): 1714-26. 100. Condorelli RA, Calogero AE, Favilla V, Morgia G, Johnson EO, Castiglione R, Salemi M, Mongioí L, Nicoletti C, Duca Y, Di Mauro M, Vicari E, La Vignera S. Arterial erectile dysfunction: different severities of endothelial apoptosis between diabetic patients "responders" and "non-responders" to sildenafil. Eur J Intern Med. 2013 Apr; 24(3):234-40. 101. Kalsi JS1, Rees RW, Hobbs AJ, Royle M, Kell PD, Ralph DJ, Moncada S, Cellek S. BAY41-2272, a novel nitric oxide independent soluble guanylate cyclase activator, relaxes human and rabbit corpus cavernosum in vitro. J Urol. 2003 Feb; 169(2):761-6. 102. Baracat JS, Teixeira CE, Okuyama CE, Priviero FB, Faro R, Antunes E, De Nucci G. Relaxing effects induced by the soluble guanylyl cyclase stimulator BAY 41-2272 in human and rabbit corpus cavernosum. Eur J Pharmacol. 2003 Sep; 477(2):163-9. 103. Teixeira CE, Priviero FB, Webb RC. Effects of 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4-ylamine (BAY 41-2272) on smooth muscle tone, soluble guanylyl cyclase activity, and NADPH oxidase activity/expression in corpus cavernosum from wild-type, neuronal, and endothelial nitric-oxide synthase null mice. J Pharmacol Exp Ther. 2007 Sep; 322(3):1093-102.

104. Albersen M, Linsen L, Tinel H, Sandner P, Van Renterghem K. Synergistic effects of BAY 60-4552 and vardenafil on relaxation of corpus cavernosum tissue of patients with erectile dysfunction and clinical phosphodiesterase type 5 inhibitor failure. J Sex Med. 2013 May; 10(5): 1268-77. 105. Oudot A, Behr-Roussel D, Poirier S, Sandner P, Bernabé J, Alexandre L, Giuliano F. Combination of BAY 60-4552 and vardenafil exerts proerectile facilitator effects in rats with cavernous nerve injury: a proof of concept study for the treatment of phosphodiesterase type 5 inhibitor failure. Eur Urol. 2011 Nov; 60(5):1020-6. 106. Lasker GF, Pankey EA, Frink TJ, Zeitzer JR, Walter KA, Kadowitz PJ. The sGC activator BAY 60-2770 has potent erectile activity in the rat. Am J Physiol Heart Circ Physiol. 2013 Jun; 304(12):H1670-9. 107. Kedia GT, Uckert S, Jonas U, Kuczyk MA, Burchardt M. The nitric oxide pathway in the human prostate: clinical implications in men with lower urinary tract symptoms. World J Urol. 2008 Dec;26(6):603-9. 108. Angulo J, Cuevas P, Fernández A, La Fuente JM, Allona A, Moncada I, Sáenz de Tejada I. Tadalafil enhances the inhibitory effects of tamsulosin on neurogenic contractions of human prostate and bladder neck. J Sex Med. 2012 Sep; 9(9):2293-306. 109. Fibbi B, Morelli A, Vignozzi L, Filippi S, Chavalmane A, De Vita G, Marini M, Gacci M, Vannelli GB, Sandner P, Maggi M. Characterization of phosphodiesterase type 5 expression and functional activity in the human male lower urinary tract. J Sex Med. 2010 Jan; 7(1 Pt 1):59-69. 110. Roehrborn CG, McVary KT, Elion-Mboussa A, Viktrup L. Tadalafil administered once daily for lower urinary tract symptoms secondary to benign prostatic hyperplasia: a dose finding study. J Urol. 2008 Oct; 180(4):1228-34. 111. Dmochowski R, Roehrborn C, Klise S, Xu L, Kaminetsky J, Kraus S. Urodynamic effects of once daily tadalafil in men with lower urinary tract symptoms secondary to clinical benign prostatic hyperplasia: a

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randomized, placebo controlled 12-week clinical trial. J Urol. 2010 Mar; 183(3):1092-7. 112. Porst H, Kim ED, Casabé AR, Mirone V, Secrest RJ, Xu L, Sundin DP, Viktrup L; LVHJ study team. Efficacy and safety of tadalafil once daily in the treatment of men with lower urinary tract symptoms suggestive of benign prostatic hyperplasia: results of an international randomized, double-blind, placebo-controlled trial. Eur Urol. 2011 Nov; 60(5):1105-13. 113. Oelke M, Bachmann A, Descazeaud A, Emberton M, Gravas S, Michel MC, N'dow J, Nordling J, de la Rosette JJ; European Association of Urology. EAU guidelines on the treatment and follow-up of non-neurogenic male lower urinary tract symptoms including benign prostatic obstruction. Eur Urol. 2013 Jul; 64(1):118-40. 114. Bechara A, Romano S, Casabé A, Haime S, Dedola P, Hernández C, Rey H. Comparative efficacy assessment of tamsulosin vs. tamsulosin plus tadalafil in the treatment of LUTS/BPH. Pilot study. J Sex Med. 2008 Sep; 5(9):2170-8. 115. Regadas RP, Reges R, Cerqueira JB, Sucupira DG, Josino IR, Nogueira EA, Jamacaru FV, de Moraes MO, Silva LF. Urodynamic effects of the combination of tamsulosin and daily tadalafil in men with lower urinary tract symptoms secondary to benign prostatic hyperplasia: a randomized, placebo-controlled clinical trial. Int Urol Nephrol. 2013 Feb; 45(1):39-43. 116. Casabé A, Roehrborn CG, Da Pozzo LF, Zepeda S, Henderson RJ, Sorsaburu S, Henneges C, Wong DG, Viktrup L. Efficacy and safety of the coadministration of tadalafil once daily with finasteride for 6 months in men with lower urinary tract symptoms and prostatic enlargement secondary to benign prostatic hyperplasia. J Urol. 2014 Mar; 191(3):727-33.

Modulating cGMP levels as therapeutic drug targets …Modulating cGMP levels as therapeutic drug...

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