ࡱ> =/2      !"#$%&'()*+,-. _ bjbj 4f Au,\\,,,,,@@@8x|.@/4dddd ex|Wl)++++++,bW,É e eÉÉW ,,ddt" É,d,d( É) ǭWШR[aW<k (W Wz,ѯÉÉÉWW ÉÉÉÉÉÉÉÉÉÉÉÉ\ :   Targeted Therapies In Pulmonary Arterial Hypertension David Montani1,2,3, MD, PhD, Marie-Camille Chaumais1,2,3,4, PharmD, PhD, Christophe Guignabert, PhD1,2,3, Sven Gunther, MD1,2,3, Barbara Girerd, PhD1,2,3, Xavier Jas, MD, Vincent Algalarrondo, MD, PhD, Laura C. Price5, MD, PhD, Olivier Sitbon1,2,3, MD, PhD, Grald Simonneau1,2,3, MD, Marc Humbert 1,2,3, MD, PhD. 1 Universit Paris-Sud, Facult de Mdecine, Kremlin-Bictre, France. 2 AP-HP, Centre de Rfrence de lHypertension Pulmonaire Svre, Service de Pneumologie et Ranimation Respiratoire, DHU Thorax Innovation, Hpital de Bictre, Le Kremlin-Bictre, France. 3 INSERM UMR 999, LabEx LERMIT, Centre Chirurgical Marie Lannelongue, Le Plessis Robinson, France. 4 AP-HP, Service de Pharmacie, DHU Thorax Innovation, Hpital Antoine Bclre, Clamart, France 5 National Heart and Lung Institute, Imperial College London, Royal Brompton Hospital, Dovehouse Street, London SW3 6LY, UK Corresponding author: Marc Humbert, MD, PhD Centre de Rfrence de lHypertension Pulmonaire Svre, Service de Pneumologie Hpital de Bictre, Assistance Publique Hpitaux de Paris, Universit Paris-Sud, 78, rue du Gnral Leclerc 94270 Le Kremlin-Bictre, France. Tel: (33) 1 45 21 79 72; Fax: (33) 1 45 21 79 71; e-mail:  HYPERLINK "mailto:marc.humbert@bct.aphp.fr" marc.humbert@bct.aphp.fr CONTENTS 1. Introduction 2. Molecular basis of PAH and molecular targets 2.1 Nitric oxide (NO) pathway 2.2 Prostacyclin (PGI2) pathway 2.3 Endothelin (ET-1) pathway 2.4 Receptor- (RTKs) and non-receptor tyrosine kinase (NRTKs) 2.5The RhoA/Rho kinase (ROCK) pathway 2.6 Inflammation & Autoimmunity 2.7 Cellular metabolism 2.8 Oxidative stress (OS) 2.9 Genetic Predisposition 3. Conventional therapy 4. Approved Specific PAH therapies 4.1 Calcium channels blockers 4.2 Currently available PAH therapies target endothelial dysfunction 4.2.1 Therapies targeting PGI2 pathway: epoprostenol and derivatives 4.2.2 Therapies targeting ET-1 pathway: Endothelin receptor antagonists 4.2.3 Therapies targeting NO pathway: phosphodiesterases type 5 inhibitors 4.3 Optimization of route administration and pharmakocinetic 4.3.1 New formulation of epoprostenol Oral 4.3.2 Tissue targeting ET-1 receptor antagonist: macitentan 4.3.3 Soluble guanylate cyclase stimulator: riociguat 4.3.4 Oral PGI2 receptor agonists: selexipag 5. NEW THERAPEUTIC TARGETS 5.1 Tyrosine kinase inhibitors 5.1.1 Efficacy of kinase inhibitors in experimental models of PH 5.1.2 Evaluation of kinase inhibitors in human PAH 5.1.3 Cardiotoxicity of kinase inhibitors 5.1.4 PH induced by dasatinib 5.2 Other 5.2.1 Vascular intestinal peptide (VIP) 5.2.2 Statin 5.2.3 Rho Kinases inhibitors 5.2.4 Progenitor cells 6. ERS/ESC guidelines for the treatment of pulmonary hypertension ABBREVIATIONS ACVRL1: activin A receptor type II-like kinase-1 BMPR2: bone morphogenetic protein receptor type 2 cAMP: cyclic adenosine monophosphate cGMP: cyclic guanosine monophosphate CO: cardiac output CYP: cytochrome P450 EC: endothelial cell EGF: epidermal growth factor ENG: endoglin ET: endothelin ERA: endothelin receptor antagonists FGF2: fibroblast growth factor 2 iPAH: idiopathic PAH IPr: PGI2 receptor Kv: voltage-gated potassium channels LVEF: left ventricular ejection fraction MCT: monocrotaline mPAP: mean pulmonary arterial pressure NYHA: New York Heart Association OS: oxidative stress PH: pulmonary hypertension PAH: pulmonary arterial hypertension PASMC: pulmonary arteries smooth muscle cells PDE-5: phosphodiesterase type 5 PDGF: platelet-derived growth factor Ppcw: pulmonary capillary wedge pressure PGI2: prostacycline PVR: pulmonary vascular resistance RTK: receptor tyrosine kinase ROCK: RhoA/Rho kinase ROS: reactive oxygen species sGc: soluble gyanylyl cyclase SOD: superoxyde dismutase SMC: smooth muscle cell TGF: transforming growth factor TKI: tyrosine kinase inhibitor TXA2: thromboxane VIP: vasoactive intestinal polypeptide VPAC: vasoactive intestinal polypeptide receptor ABSTRACT Pulmonary arterial hypertension (PAH) is a rare disorder characterized by progressive obliteration of small pulmonary arteries that leads to elevated pulmonary arterial pressure and right heart failure. During the last decades, the better understanding of the pathophysiology of the disease has resulted in the development of effective therapies targeting endothelial dysfunction (epoprostenol and derivatives, endothelin receptor antagonists and phosphodiesterase type 5 inhibitors). These drugs allow clinical, functional and hemodynamic improvement, but to date, no cure exists for PAH and prognosis remains poor. Recently, several additional pathways have been suggested to be involved in the pathogenesis of PAH, and may represent innovative therapies. In this Review, we reviewed conventional therapy, the pharmacological agents currently available for the treatment of PAH and the benefit/risk ratio of potential future therapies. Key words: endothelial dysfunction, pulmonary hypertension, prostacyclin, endothelin receptor antagonist, phoshodiesterases type 5 inhibitors, kinase inhibitors. 1. Introduction Pulmonary hypertension (PH) is defined as an increase in mean pulmonary arterial pressure (mPAP) e" 25 mmHg at rest as assessed by right heart catheterization  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_72" \o "Galie, 2009 #1" Galie, Hoeper et al. 2009). According to pulmonary capillary wedge pressure (Ppcw), pulmonary vascular resistance (PVR) and cardiac output (CO), different haemodynamic definitions of PH exist as follows. Pre-capillary PH includes the clinical groups 1, 3, 4 and 5 while post-capillary PH includes the clinical group 2 (Table 1) ADDIN EN.CITE Oudiz20072(Oudiz 2007)2217Oudiz, R. J.David Geffen School of Medicine at UCLA, CA 90502-2006, USA. oudiz@humc.eduPulmonary hypertension associated with left-sided heart diseaseClin Chest MedClinics in chest medicineClin Chest Med233-41, x2812007/03/07Antihypertensive Agents/therapeutic useComorbidityExercise TestHeart Diseases/*epidemiology/surgeryHeart TransplantationHumansHypertension, Pulmonary/classification/diagnosis/*epidemiology/physiopathologyPrognosisVentricular Function, Left2007Mar0272-5231 (Print) 0272-5231 (Linking)17338938Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/1733893810.1016/j.ccm.2006.12.001eng( HYPERLINK \l "_ENREF_184" \o "Oudiz, 2007 #2" Oudiz 2007). PAH or group 1 PH comprises PAH due to many different etiologies, despite which patients with PAH share clinical features and pathological changes to the pulmonary circulation including endothelial dysfunction, endothelial and smooth muscle cell (SMC) proliferation, vasoconstriction and in situ thrombosis. In addition, sub-groups of PAH share similarities in term of management  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_227" \o "Simonneau, 2009 #3" Simonneau, Robbins et al. 2009). However, despite many new available therapies over the last two decades, PAH remains an incurable disease process, which if not interrupted, subsequently leads to right heart failure and death  ADDIN EN.CITE Chin20084(Chin and Rubin 2008)4417Chin, K. M.Rubin, L. J.Department of Internal Medicine, Division of Pulmonary and Critical Care, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA. Kelly.Chin@utsouthwestern.eduPulmonary arterial hypertensionJ Am Coll CardiolJournal of the American College of CardiologyJ Am Coll Cardiol1527-3851162008/04/19AlgorithmsAntihypertensive Agents/*therapeutic useDisease ProgressionHumansHypertension, Pulmonary/classification/complications/*drug therapyPrognosisPulmonary Artery/*pathologyRisk Factors2008Apr 221558-3597 (Electronic) 0735-1097 (Linking)18420094Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/1842009410.1016/j.jacc.2008.01.024eng( HYPERLINK \l "_ENREF_26" \o "Chin, 2008 #4" Chin and Rubin 2008). 2. MOLECULAR BASIS OF PAH AND MOLECULAR TARGETS The imbalance in the production of endothelium-derived vasodilator and constrictor factors is a pivotal element in the development and progression of the disease (Figure 1). Over the past two decades, this observation has led to the development of all current approved specific therapies for PAH. 2.1 Nitric oxide (NO) signaling pathway and Phosphodiesterases (PDEs) NO is a gaseous lipophilic free radical generated by three distinct isoforms of nitric oxide synthases (NOS): neuronal (nNOS), inducible (iNOS) and endothelial NOS (eNOS). NO dilates blood vessels, inhibits leukocyte adhesion, platelet aggregation, thrombus formation, and vascular proliferation and modulates many other physiological processes including cell metabolism and intracellular signaling pathways  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_150" \o "Mayer, 1997 #182" Mayer and Hemmens 1997;  HYPERLINK \l "_ENREF_143" \o "Lincoln, 1998 #194" Lincoln, Dey et al. 1998). Reduced NO bioavailability has been reported to be associated with the development of many different vascular diseases including PAH. There are different molecular mechanisms explaining the decreased NO bioavailability in PAH including increased levels of endogenous competitive inhibitor of eNOS (such as asymmetric dimethylarginine or ADMA), eNOS uncoupling, decreased L-arginine levels, increased NO scavenging by haemoglobin and reactive oxygen species (ROS)  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_234" \o "Steinhorn, 2008 #451" Steinhorn 2008;  HYPERLINK \l "_ENREF_276" \o "Zuckerbraun, 2011 #450" Zuckerbraun, George et al. 2011). It has been well documented that exogenous and endogenous NO inhibit vascular SMC proliferation and migration  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_214" \o "Sarkar, 1996 #210" Sarkar, Meinberg et al. 1996;  HYPERLINK \l "_ENREF_277" \o "Zuckerbraun, 2007 #215" Zuckerbraun, Stoyanovsky et al. 2007;  HYPERLINK \l "_ENREF_250" \o "Tsihlis, 2011 #214" Tsihlis, Oustwani et al. 2011). Most of the effects of NO, on smooth muscle cells, platelets and cardiac myocytes, are mediated through its activation of soluble guanylate cyclase and amplifying the production of cyclic guanosine monophosphate (cGMP)  ADDIN EN.CITE Rybalkin2003174(Rybalkin, Yan et al. 2003)17417417Rybalkin, S. D.Yan, C.Bornfeldt, K. E.Beavo, J. A.Department of Pharmacology, University of Washington, Seattle, Wash 98195, USA.Cyclic GMP phosphodiesterases and regulation of smooth muscle functionCirc ResCirculation researchCirc Res280-919342003/08/233',5'-Cyclic-GMP Phosphodiesterases/genetics/*metabolismAnimalsCell Division/physiologyCyclic GMP/metabolismEnzyme ActivationHumansIsoenzymes/genetics/metabolismMultigene Family/geneticsMuscle, Smooth, Vascular/cytology/*physiology2003Aug 221524-4571 (Electronic) 0009-7330 (Linking)12933699Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/1293369910.1161/01.RES.0000087541.15600.2Beng( HYPERLINK \l "_ENREF_207" \o "Rybalkin, 2003 #174" Rybalkin, Yan et al. 2003) (Figure 1). Phosphodiesterases (PDEs) are a superfamily of enzymes that inactivate cAMP and cGMP and have different tissue distributions. Among all PDEs, PDE-5 is abundantly expressed in lung tissue and is upregulated in PAH, contributing to endothelial dysfunction by inactivating cGMP. In addition, a PDE-1C upregulation in hyperproliferative pulmonary arteries SMC (PASMCs) has been noted in patients with PAH  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_217" \o "Schermuly, 2007 #394" Schermuly, Pullamsetti et al. 2007). PDE-5 inhibitors facilitates the antiproliferative and vasodilating effects of endogenous NO and represent the rationale for the use of PDE inhibitors in PAH  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_163" \o "Moncada, 1993 #7" Moncada and Higgs 1993;  HYPERLINK \l "_ENREF_194" \o "Rabe, 1994 #10" Rabe, Tenor et al. 1994;  HYPERLINK \l "_ENREF_83" \o "Giordano, 2001 #11" Giordano, De Stefano et al. 2001;  HYPERLINK \l "_ENREF_157" \o "Michelakis, 2002 #8" Michelakis, Tymchak et al. 2002;  HYPERLINK \l "_ENREF_33" \o "Corbin, 2005 #646" Corbin, Beasley et al. 2005;  HYPERLINK \l "_ENREF_242" \o "Tantini, 2005 #9" Tantini, Manes et al. 2005;  HYPERLINK \l "_ENREF_263" \o "Wharton, 2005 #5" Wharton, Strange et al. 2005). 2.2 Prostacyclin (PGI2) signaling pathway With its very short half-life (t), the major active metabolite of arachidonic acid (AA) PGI2 is a critical endogenous regulator of vascular homeostasis. PGI2 is produced in vascular endothelial cells (ECs) and acts on neighbouring vascular SMCs as well as circulating platelets and cells  ADDIN EN.CITE Vane2003217(Vane and Corin 2003)21721717Vane, J.Corin, R. E.The William Harvey Research Institute, Charterhouse Square, London, UK.Prostacyclin: a vascular mediatorEur J Vasc Endovasc SurgEuropean journal of vascular and endovascular surgery : the official journal of the European Society for Vascular SurgeryEur J Vasc Endovasc Surg571-82662003/11/07AnimalsEpoprostenol/chemistry/*pharmacology/*therapeutic useHumansMicePeripheral Vascular Diseases/*drug therapyPlatelet Aggregation Inhibitors/*pharmacology/*therapeutic use2003Dec1078-5884 (Print) 1078-5884 (Linking)14603414Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/1460341410.1016/S1078eng( HYPERLINK \l "_ENREF_255" \o "Vane, 2003 #217" Vane and Corin 2003). PGI2 is an agonist of adenylate cyclase and a potent vasodilator with antithrombotic properties  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_90" \o "Gryglewski, 1976 #15" Gryglewski, Bunting et al. 1976;  HYPERLINK \l "_ENREF_255" \o "Vane, 2003 #217" Vane and Corin 2003). An imbalance in AA metabolism with decreased PGI2 and increased thromboxane (TXA2) urinary metabolites have been demonstrated in idiopathic PAH (iPAH) patients  ADDIN EN.CITE Christman199216(Christman, McPherson et al. 1992)161617Christman, B. W.McPherson, C. D.Newman, J. H.King, G. A.Bernard, G. R.Groves, B. M.Loyd, J. E.Center for Lung Research, Vanderbilt University School of Medicine, Nashville, TN.An imbalance between the excretion of thromboxane and prostacyclin metabolites in pulmonary hypertensionN Engl J MedThe New England journal of medicineN Engl J Med70-532721992/07/096-Ketoprostaglandin F1 alpha/analogs & derivatives/urineAdultEpoprostenol/*urineFemaleHumansHypertension, Pulmonary/*urineLung Diseases, Obstructive/urineMaleRadioimmunoassayThromboxane A2/*urineThromboxane B2/analogs & derivatives/urine1992Jul 90028-4793 (Print) 0028-4793 (Linking)1603138Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.http://www.ncbi.nlm.nih.gov/pubmed/160313810.1056/NEJM199207093270202eng( HYPERLINK \l "_ENREF_27" \o "Christman, 1992 #16" Christman, McPherson et al. 1992). In addition, expression of the key enzyme for PGI2 synthesis PGI2 synthase (PGIS) is reduced in pulmonary arteries of PAH patients  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_253" \o "Tuder, 1999 #17" Tuder, Cool et al. 1999) (Figure 1). The actions of PGI2 are mediated by binding to cell surface PGI2 receptors (IPr) or by binding to nuclear peroxisome proliferator-activated receptors (PPARs)  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_142" \o "Lim, 2002 #339" Lim and Dey 2002;  HYPERLINK \l "_ENREF_255" \o "Vane, 2003 #217" Vane and Corin 2003;  HYPERLINK \l "_ENREF_266" \o "Wise, 2003 #216" Wise 2003;  HYPERLINK \l "_ENREF_52" \o "Falcetti, 2007 #340" Falcetti, Flavell et al. 2007;  HYPERLINK \l "_ENREF_159" \o "Midgett, 2011 #268" Midgett, Stitham et al. 2011;  HYPERLINK \l "_ENREF_235" \o "Stitham, 2011 #271" Stitham, Midgett et al. 2011). Binding of PGI2 to IPr induces cell-specific signaling that lead to elevation of intracellular cAMP through Gs-protein coupling to adenylate cyclase  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_97" \o "Hashimoto, 1990 #393" Hashimoto, Negishi et al. 1990;  HYPERLINK \l "_ENREF_31" \o "Coleman, 1994 #392" Coleman, Smith et al. 1994;  HYPERLINK \l "_ENREF_22" \o "Chaumais, 2010 #82" Chaumais, Jobard et al. 2010). Administration of PGI2 or its analogues in experimental pulmonary hypertension leads to a decrease in pulmonary arterial pressure and pulmonary vascular resistance  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_138" \o "Leffler, 1979 #396" Leffler and Hessler 1979;  HYPERLINK \l "_ENREF_5" \o "Archer, 1986 #397" Archer, Chesler et al. 1986;  HYPERLINK \l "_ENREF_270" \o "Yuki, 1994 #398" Yuki, Sato et al. 1994;  HYPERLINK \l "_ENREF_162" \o "Miyata, 1996 #399" Miyata, Ueno et al. 1996;  HYPERLINK \l "_ENREF_149" \o "Max, 1999 #395" Max, Kuhlen et al. 1999). 2.3 Endothelin (ET-1) signaling pathway ET-1 is a 21 amino acid peptide that is produced by the vascular endothelium from a 39 amino acid precursor, big ET-1, through the actions of an ET-1 converting enzyme (ECE) found on the EC membrane. In addition to ECs, PASMCs  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_146" \o "Markewitz, 2001 #19" Markewitz, Farrukh et al. 2001) and lung fibroblasts  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_222" \o "Shi-Wen, 2004 #20" Shi-Wen, Chen et al. 2004) can produce small amount of ET-1. The ET-1 biosynthesis is stimulated by different stimuli: including hypoxia, growth factors, cytokines, shear stress, thrombin and angiotensin II. ET-1 acts through two receptors, ET-A and ET-B. Both of these receptors are coupled to a Gq-protein and the formation of IP3. Increased inositol triphosphate (IP3) causes calcium release by the sarcoplasmic reticulum, which causes smooth muscle contraction. In addition to be a potent vasoconstrictor, ET-1 stimulates PASMC proliferation  ADDIN EN.CITE Giaid199322(Giaid, Yanagisawa et al. 1993)222217Giaid, A.Yanagisawa, M.Langleben, D.Michel, R. P.Levy, R.Shennib, H.Kimura, S.Masaki, T.Duguid, W. P.Stewart, D. J.Department of Pathology, McGill University, Montreal, Canada.Expression of endothelin-1 in the lungs of patients with pulmonary hypertensionN Engl J MedThe New England journal of medicineN Engl J Med1732-9328241993/06/17AdultEndothelins/*biosynthesisEndothelium, Vascular/metabolismFemaleHumansHypertension, Pulmonary/*metabolism/pathology/physiopathologyIn Situ HybridizationLung/blood supply/*metabolismMaleMiddle AgedRNA, Messenger/analysisVascular Resistance1993Jun 170028-4793 (Print) 0028-4793 (Linking)8497283Research Support, Non-U.S. Gov'thttp://www.ncbi.nlm.nih.gov/pubmed/849728310.1056/NEJM199306173282402eng( HYPERLINK \l "_ENREF_82" \o "Giaid, 1993 #22" Giaid, Yanagisawa et al. 1993). Binding of ET-1 to ETA and ETB receptors on PASMCs promotes vasoconstriction, whereas activation of ETB receptors on EC causes vasodilatation through increased PGI2 and NO levels  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_99" \o "Hirata, 1993 #23" Hirata, Emori et al. 1993;  HYPERLINK \l "_ENREF_220" \o "Seo, 1994 #24" Seo, Oemar et al. 1994) (Figure 1). Several studies in animal models document that both selective and non selective ET receptors antagonists (ERAs) prevent or attenuate pulmonary hypertension by vasodilatation, decrease of vascular remodelling and right heart hypertrophy  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_23" \o "Chen, 1995 #400" Chen, Chen et al. 1995;  HYPERLINK \l "_ENREF_24" \o "Chen, 1997 #401" Chen, Chen et al. 1997;  HYPERLINK \l "_ENREF_124" \o "Kim, 2000 #402" Kim, Yung et al. 2000;  HYPERLINK \l "_ENREF_246" \o "Tilton, 2000 #403" Tilton, Munsch et al. 2000). 2.4 Receptor- (RTKs) and Non-Receptor Tyrosine Kinase (NRTKs) RTKs have critical functions in several cellular processes including regulating cell proliferation, migration, metabolic changes, differentiation and survival. They are transmembrane glycoproteins that act as receptors for growth factors, neurotrophic factors, and other extracellular signaling molecules. Upon ligand binding, intracellular signal transduction pathways are activated within the cell by phosphorylating tyrosine residues on the receptors themselves (autophosphorylation) and on downstream effector molecules. The NRTKs are integral components of the signaling cascades triggered by RTKs and by other cell surface receptors such as G protein-coupled receptors and receptors of the immune system. The NRTKs are divided into ten families: Src, Abl, Jak, Ack, Csk, Fak, Fes, Frk, Tec, and Syk  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_15" \o "Blume-Jensen, 2001 #407" Blume-Jensen and Hunter 2001). In contrast to RTKs, the NRTKs lack the extracellular ligand-binding domain and transmembrane spanning region and are located in the cytoplasm or anchored to cell membrane through amino terminal modification such as myristoylation or palmitoylation. However, NRTKs possess domain that mediate protein-protein, protein-lipid and/ or protein-DNA interactions, in which the most common found protein-protein interaction domains are the Src homology 2 (SH2) and SH3 domain. In PAH, special attention has been devoted to the pathogenic role of platelet-derived growth factor (PDGF)-, epidermal growth factor (EGF)-, fibroblast growth factor (FGF)- and c-kit-receptors. Indeed, altered expression and/ or increased activity of these four RTK signaling pathways and their contribution to the excessive proliferation and migration of SMCs and ECs have been demonstrated in human and experimental models of PH (Figure 2). In addition, their inhibition by specific inhibitors (TKIs) have been shown to exert beneficial effects in rodent models  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_156" \o "Merklinger, 2005 #69" Merklinger, Jones et al. 2005;  HYPERLINK \l "_ENREF_215" \o "Schermuly, 2005 #59" Schermuly, Dony et al. 2005;  HYPERLINK \l "_ENREF_190" \o "Perros, 2008 #60" Perros, Montani et al. 2008;  HYPERLINK \l "_ENREF_112" \o "Izikki, 2009 #180" Izikki, Guignabert et al. 2009;  HYPERLINK \l "_ENREF_252" \o "Tu, 2011 #371" Tu, Dewachter et al. 2011;  HYPERLINK \l "_ENREF_251" \o "Tu, 2012 #366" Tu, De Man et al. 2012). c-Src is abundant and plays a critical role in the integrity and function of the pulmonary vascular bed  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_181" \o "Oda, 1999 #410" Oda, Renaux et al. 1999). Several studies have obtained evidence that Src and its signaling is abnormally activated in PAH, thereby indirectly promote cell growth and migration  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_34" \o "Courboulin, 2011 #411" Courboulin, Paulin et al. 2011;  HYPERLINK \l "_ENREF_40" \o "de Man, 2012 #426" de Man, Tu et al. 2012;  HYPERLINK \l "_ENREF_251" \o "Tu, 2012 #366" Tu, De Man et al. 2012). 2.5 The RhoA/Rho kinase (ROCK) pathway The small GTPase RhoA and its downstream effectors, ROCK1 and ROCK2, regulate many essential cellular processes such as contraction, cell migration, proliferation, survival, and differentiation. RhoA, which belongs to the Rho subfamily, responds to cell surface receptors for various growth factors, cytokines, adhesion molecules, and G-protein-coupled receptors by cycling between an inactive GDP-bound and an active GTP-bound conformation. The activation of RhoA subsequently activates ROCKs, which is known to phosphorylate several substrates, including myosin phosphatase target subunit 1 (MYPT-1), phosphatase and tensin homolog on chromosome 10 (PTEN), myosin light chain, ezrin/radixin/moesin (ERM) family, adducin, and LIM-kinases. Since the RhoA/ROCK pathway plays a crucial in vasoconstriction and vascular remodeling and because it is activated by upstream key molecules for pulmonary vascular remodeling in PH such as PDGF, ET-1, serotonin, angiotensin II, several studies have been undertaken to evaluate its pathogenic role in the disease. Guilluy and collaborators  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_92" \o "Guilluy, 2009 #376" Guilluy, Eddahibi et al. 2009) have reported a twofold increase in Rho kinase and RhoA activity in lungs from patients with idiopathic PAH as compared to control patients. Consistent with this observation, Do and colleagues  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_45" \o "Do e, 2009 #785" Do e, Fukumoto et al. 2009) have noted close relationships between the activity of ROCK and the disease duration. In addition, beneficial effects of Rho-kinase inhibition have been observed in different cardiovascular disorders  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_223" \o "Shimokawa, 2007 #746" Shimokawa and Rashid 2007) as well as in different animal models of PH i.e. in the monocrotaline-induced PH  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_1" \o "Abe, 2004 #750" Abe, Shimokawa et al. 2004;  HYPERLINK \l "_ENREF_171" \o "Nagaoka, 2005 #777" Nagaoka, Fagan et al. 2005;  HYPERLINK \l "_ENREF_244" \o "Tawara, 2007 #753" Tawara, Fukumoto et al. 2007;  HYPERLINK \l "_ENREF_169" \o "Mouchaers, 2010 #786" Mouchaers, Schalij et al. 2010), in the chronic hypoxia-induced PH  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_51" \o "Fagan, 2004 #764" Fagan, Oka et al. 2004;  HYPERLINK \l "_ENREF_93" \o "Guilluy, 2005 #758" Guilluy, Sauzeau et al. 2005;  HYPERLINK \l "_ENREF_2" \o "Abe, 2006 #756" Abe, Tawara et al. 2006), and in the high flow-induced PH  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_140" \o "Li, 2007 #773" Li, Xia et al. 2007). 2.6 Inflammation & Autoimmunity Although the exact pathophysiology remains unknown, there is increasing evidence to suggest an important role for inflammation in the onset and progression of human and experimental PH  ADDIN EN.CITE Perros201126(Perros, Montani et al. 2011)262617Perros, F.Montani, D.Dorfmuller, P.Huertas, A.Chaumais, M. C.Cohen-Kaminsky, S.Humbert, M.Universite Paris-Sud, Faculte de medecine, Le Kremlin-Bicetre, France. frederic.perros@gmail.com[Novel immunopathological approaches to pulmonary arterial hypertension]Presse MedPresse Med1S3-1340 Suppl 12011/05/04HumansHypertension, Pulmonary/drug therapy/*immunologyInflammation/complications/drug therapy2011AprNouvelles approches immunopathologiques de l'hypertension arterielle pulmonaire.0755-4982 (Print) 0755-4982 (Linking)21536178Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/2153617810.1016/S0755-4982(11)70002-1fre( HYPERLINK \l "_ENREF_191" \o "Perros, 2011 #26" Perros, Montani et al. 2011). Circulating levels of several key cytokines and chemokines (such as interleukin (IL)-6, IL-1(, TNF( and MCP-1) are elevated in iPAH patients and may correlate with a worse clinical outcome  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_107" \o "Humbert, 1995 #27" Humbert, Monti et al. 1995;  HYPERLINK \l "_ENREF_10" \o "Balabanian, 2002 #28" Balabanian, Foussat et al. 2002;  HYPERLINK \l "_ENREF_212" \o "Sanchez, 2007 #29" Sanchez, Marcos et al. 2007;  HYPERLINK \l "_ENREF_232" \o "Soon, 2010 #454" Soon, Holmes et al. 2010). The pulmonary vascular lesions observed in lungs of iPAH patients are also sites of intense chemokine production associated with inflammatory cell recruitment and accumulation  ADDIN EN.CITE Dorfmuller200330(Dorfmuller, Perros et al. 2003)303017Dorfmuller, P.Perros, F.Balabanian, K.Humbert, M.Centre for Pulmonary Vascular Diseases UPRES EA 2705, Dept of Respiratory and Intensive Care Medicine, South Paris Cytokine Institute, Antoine Beclere Hospital, Assistance Publique-Hopitaux de Paris, South Paris University, Clamart, France.Inflammation in pulmonary arterial hypertensionEur Respir JThe European respiratory journal : official journal of the European Society for Clinical Respiratory PhysiologyEur Respir J358-632222003/09/04AnimalsAutoimmunityDisease Models, AnimalHumansHypertension, Pulmonary/drug therapy/immunology/*physiopathologyInflammation/physiopathology2003Aug0903-1936 (Print) 0903-1936 (Linking)12952274Research Support, Non-U.S. Gov't Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/12952274eng( HYPERLINK \l "_ENREF_47" \o "Dorfmuller, 2003 #30" Dorfmuller, Perros et al. 2003). Furthermore, a recent study has reported presence of pulmonary lymphoid neogenesis, revealing chronic inflammation in iPAH  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_189" \o "Perros, 2012 #925" Perros, Dorfmuller et al. 2012). In addition, circulating anti-EC and anti-fibroblast autoantibodies have been reported in 10 to 40 % of iPAH patients  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_241" \o "Tamby, 2005 #31" Tamby, Chanseaud et al. 2005;  HYPERLINK \l "_ENREF_245" \o "Terrier, 2008 #32" Terrier, Tamby et al. 2008), suggesting a possible role of autoimmunity in the pathogenesis of PAH pulmonary vascular lesions. Consistent with this notion, a recent study has reported excessive production and release of the cytokine-like hormone leptin in PAH that act as a key modulator of the hyporesponsiveness of regulatory T cells (Treg)  ADDIN EN.CITE Huertas2012429(Huertas, Tu et al. 2012)42942917Huertas, A.Tu, L.Gambaryan, N.Girerd, B.Perros, F.Montani, D.Fabre, D.Fadel, E.Eddahibi, S.Cohen-Kaminsky, S.Guignabert, C.Humbert, M.Faculte de Medecine, Kremlin-Bicetre, F-94276.Leptin and regulatory T lymphocytes in idiopathic pulmonary arterial hypertensionEur Respir JThe European respiratory journal : official journal of the European Society for Clinical Respiratory PhysiologyEur Respir J2012/03/012012Feb 231399-3003 (Electronic) 0903-1936 (Linking)22362850http://www.ncbi.nlm.nih.gov/pubmed/2236285010.1183/09031936.00159911Eng( HYPERLINK \l "_ENREF_105" \o "Huertas, 2012 #429" Huertas, Tu et al. 2012). The importance of inflammatory mechanisms in PAH pathophysiology has also been highlighted by the kinetics of inflammatory patterns in standard experimental models, such as monocrotaline (MCT)-induced and hypoxia-induced PAH in rats. In these models, it has been clearly shown that inflammation precedes vascular remodeling and PAH. It has also been demonstrated, particularly in MCT-induced PH, that immunosuppressive therapies prevent PH development and totally or partially reverse PH lesions  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_259" \o "Voelkel, 1994 #34" Voelkel, Tuder et al. 1994;  HYPERLINK \l "_ENREF_111" \o "Ikeda, 2002 #35" Ikeda, Yonemitsu et al. 2002;  HYPERLINK \l "_ENREF_193" \o "Price, 2011 #937" Price, Montani et al. 2011). Finally, immune mechanisms are obviously implicated in the etiology of PAH associated with autoimmune diseases or with HIV infection  ADDIN EN.CITE Dorfmuller200330(Dorfmuller, Perros et al. 2003)303017Dorfmuller, P.Perros, F.Balabanian, K.Humbert, M.Centre for Pulmonary Vascular Diseases UPRES EA 2705, Dept of Respiratory and Intensive Care Medicine, South Paris Cytokine Institute, Antoine Beclere Hospital, Assistance Publique-Hopitaux de Paris, South Paris University, Clamart, France.Inflammation in pulmonary arterial hypertensionEur Respir JThe European respiratory journal : official journal of the European Society for Clinical Respiratory PhysiologyEur Respir J358-632222003/09/04AnimalsAutoimmunityDisease Models, AnimalHumansHypertension, Pulmonary/drug therapy/immunology/*physiopathologyInflammation/physiopathology2003Aug0903-1936 (Print) 0903-1936 (Linking)12952274Research Support, Non-U.S. Gov't Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/12952274eng( HYPERLINK \l "_ENREF_47" \o "Dorfmuller, 2003 #30" Dorfmuller, Perros et al. 2003), in which PH develops in a clear inflammatory context. In some cases, immunosuppressive or anti-inflammatory treatments significantly improve hemodynamic and clinical parameters  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_114" \o "Jais, 2008 #36" Jais, Launay et al. 2008), highlighting the role of immune mechanisms in PAH pathogenesis or progression. 2.7 Cellular metabolism The Warburg effect is defined as an increased dependence on glycolysis for ATP synthesis, even in the presence of abundant oxygen. The Warburg effect has been found in a wide spectrum of human cancers as well as in PAH  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_198" \o "Rehman, 2010 #471" Rehman and Archer 2010). Restitution of oxidative metabolism with the use of dichloroacetate has been shown to be efficient in several animal models of PH  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_158" \o "Michelakis, 2002 #458" Michelakis, McMurtry et al. 2002;  HYPERLINK \l "_ENREF_155" \o "McMurtry, 2004 #457" McMurtry, Bonnet et al. 2004;  HYPERLINK \l "_ENREF_91" \o "Guignabert, 2009 #374" Guignabert, Tu et al. 2009). Similarly, inhibition of fatty acid oxidation also prevents this metabolic shift and limits the proliferative and anti-apoptotic cell phenotype observed in PH  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_236" \o "Sutendra, 2010 #459" Sutendra, Bonnet et al. 2010). 2.8 Oxidative stress (OS) Free radicals are defined as molecules having an unpaired electron in the outer orbit. They are generally unstable and very reactive. The pathogenic role of reactive oxygen (or ROS: such as superoxide (O2- .), hydroperoxyl (HO2 .), peroxyl (RO2 .), alkoxyl (RO .), hydroxyl (OH- .), and hydroperoxyl (HO2 .) radicals) and nitrogen species (or NOS: such as nitric oxide (NO .), nitrogen dioxide (NO2 .), nitrogen trioxide (N2O3), and peroxynitrite (ONOO")) in PH has been reviewed recently by Voelkel NF and colleagues  ADDIN EN.CITE Voelkel2012578(Voelkel, Bogaard et al. 2012)57857817Voelkel, N. F.Bogaard, H. J.Al Husseini, A. A.Farkas, L.Gomez-Arroyo, J.Natarajan, R.VCU Medical Center, Internal Medicine , 1220 East Broad Street, 6th floor, Room 6052, Richmond, Virginia, United States, 23298-0456, 804-628-9614, 804-628-0325; nvoelkel@mcvh-vcu.edu.Antioxidants for the treatment of patients with severe angioproliferative pulmonary hypertension?Antioxid Redox SignalAntioxidants & redox signalingAntioxid Redox Signal2012/08/092012Aug 71557-7716 (Electronic) 1523-0864 (Linking)22870869http://www.ncbi.nlm.nih.gov/pubmed/2287086910.1089/ars.2012.4828Eng( HYPERLINK \l "_ENREF_258" \o "Voelkel, 2012 #578" Voelkel, Bogaard et al. 2012) and Tabima TM and colleagues  ADDIN EN.CITE Tabima2012729(Tabima, Frizzell et al. 2012)72972917Tabima, D. M.Frizzell, S.Gladwin, M. T.Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA 15213, USA.Reactive oxygen and nitrogen species in pulmonary hypertensionFree Radic Biol MedFree radical biology & medicineFree Radic Biol MedFree radical biology & medicineFree Radic Biol MedFree radical biology & medicine1970-865292012/03/102012May 11873-4596 (Electronic) 0891-5849 (Linking)22401856Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov'thttp://www.ncbi.nlm.nih.gov/pubmed/2240185610.1016/j.freeradbiomed.2012.02.041eng( HYPERLINK \l "_ENREF_239" \o "Tabima, 2012 #729" Tabima, Frizzell et al. 2012). Indeed, it is well recognized that OS contributes to the development and/or progression of many systemic vascular disease including PAH modulating inflammation, energy metabolism, cellular differentiation, proliferation and apoptosis. Excessive OS appears following an imbalance between antioxidant and oxidant molecules or enzymes, leading to overproduction of ROS and NOS. Indeed, eNOS uncoupling, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, xanthine oxidoreductase (XOR) and mitochondria have been identified as the main contributors of this imbalance in PH. In by 2001, Geraci and al., showed an increase in pulmonary expression of oxidant genes in PAH patients  ADDIN EN.CITE Geraci2001520(Geraci, Moore et al. 2001)52052017Geraci, M. W.Moore, M.Gesell, T.Yeager, M. E.Alger, L.Golpon, H.Gao, B.Loyd, J. E.Tuder, R. M.Voelkel, N. F.Division of Pulmonary Sciences and Critical Care Medicine, Pulmonary Hypertension Center, Department of Pathology, University of Colorado Health Sciences Center, Denver, Colorado, USA.Gene expression patterns in the lungs of patients with primary pulmonary hypertension: a gene microarray analysisCirc ResCirculation researchCirc Res555-628862001/04/03AdultFemale*Gene Expression ProfilingHumansHypertension, Pulmonary/*geneticsLung/*metabolism/pathologyMaleMiddle AgedOligonucleotide Array Sequence AnalysisRNA/genetics/metabolism2001Mar 301524-4571 (Electronic) 0009-7330 (Linking)11282888Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.http://www.ncbi.nlm.nih.gov/pubmed/11282888eng( HYPERLINK \l "_ENREF_77" \o "Geraci, 2001 #520" Geraci, Moore et al. 2001), urinary isoprostanes levels were also increased in PAH patients  ADDIN EN.CITE Cracowski2001843(Cracowski, Souvignet et al. 2001)84384317Cracowski, J. L.Souvignet, C.Quirin, N.Grosbois, X.Bayle, F.Stanke-Labesque, F.Vialtel, P.Bessard, G.Laboratory of Pharmacology, Faculte de Medecine de Grenoble, La Tronche, France. Jean-Luc.Cracowski@ujf-grenoble.frUrinary F2-isoprostanes formation in kidney transplantationClin TransplantClinical transplantationClin TransplantClinical transplantationClin TransplantClinical transplantation58-621512001/02/13AdultCase-Control StudiesEnzyme-Linked Immunosorbent AssayFemaleHumans*Kidney Transplantation*Lipid PeroxidationMaleMiddle AgedOxidative StressPostoperative PeriodProstaglandins F/*urineRegression Analysis2001Feb0902-0063 (Print) 0902-0063 (Linking)11168317http://www.ncbi.nlm.nih.gov/pubmed/11168317eng( HYPERLINK \l "_ENREF_37" \o "Cracowski, 2001 #843" Cracowski, Souvignet et al. 2001), which correlated with worse survival  ADDIN EN.CITE Cracowski2012590(Cracowski, Degano et al. 2012)59059017Cracowski, J. L.Degano, B.Chabot, F.Labarere, J.Schwedhelm, E.Monneret, D.Iuliano, L.Schwebel, C.Chaouat, A.Reynaud-Gaubert, M.Faure, P.Maas, R.Renversez, J. C.Cracowski, C.Sitbon, O.Yaici, A.Simonneau, G.Humbert, M.1Clinical Pharmacology Department, INSERM CIC3, University Hospital; Grenoble, France.Independent association of urinary F2-isoprostanes with survival in pulmonary arterial hypertensionChestChestChestChest2012/03/132012Mar 81931-3543 (Electronic) 0012-3692 (Linking)22406961http://www.ncbi.nlm.nih.gov/pubmed/2240696110.1378/chest.11-1267Eng( HYPERLINK \l "_ENREF_36" \o "Cracowski, 2012 #590" Cracowski, Degano et al. 2012). Studies that followed have confirmed the presence of OS in situ in lungs of PAH patients with oxidative stress markers (8-hydroxyguanosine and nitrotyrosine) and NOX-4, an isoform of NADPH-oxidase in the media and adventitial fibroblasts of diseased vessels  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_17" \o "Bowers, 2004 #531" Bowers, Cool et al. 2004;  HYPERLINK \l "_ENREF_160" \o "Mittal, 2007 #534" Mittal, Roth et al. 2007;  HYPERLINK \l "_ENREF_141" \o "Li, 2008 #625" Li, Tabar et al. 2008). A recent study reported that the transfection of a BMPR2 mutation to human PASMCs increased production of OS in vascular human SMCs, and in mice BMPR2-mutated transgenic mice, a significant increase in isoprostanes was seen in lung tissue with a specific location of vascular bed, with mitochondria being a likely source  ADDIN EN.CITE Lane2011545(Lane, Talati et al. 2011)54554517Lane, K. L.Talati, M.Austin, E.Hemnes, A. R.Johnson, J. A.Fessel, J. P.Blackwell, T.Mernaugh, R. L.Robinson, L.Fike, C.Roberts, L. J., 2ndWest, J.Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee.Oxidative injury is a common consequence of BMPR2 mutationsPulm CircPulmonary circulationPulm CircPulmonary circulationPulm CircPulmonary circulation72-83112011/09/1020112045-8940 (Electronic)21904662http://www.ncbi.nlm.nih.gov/pubmed/21904662316717410.4103/2045-8932.78107Eng( HYPERLINK \l "_ENREF_133" \o "Lane, 2011 #545" Lane, Talati et al. 2011). Similar results were seen in mice with ALK-1 mutations with the presence of OS associated with decoupling eNOS  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_115" \o "Jerkic, 2011 #546" Jerkic, Kabir et al. 2011). Many experimental models of PH have emphasized the role of OS in pulmonary artery lesions. In MCT-induced PH in rats, levels of 8-isoprostanes and NOX-4 were increased, in association with a decrease of eNOS  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_148" \o "Mathew, 2002 #552" Mathew, Yuan et al. 2002;  HYPERLINK \l "_ENREF_53" \o "Farahmand, 2004 #549" Farahmand, Hill et al. 2004;  HYPERLINK \l "_ENREF_118" \o "Kamezaki, 2008 #550" Kamezaki, Tasaki et al. 2008;  HYPERLINK \l "_ENREF_38" \o "Csiszar, 2009 #45" Csiszar, Labinskyy et al. 2009;  HYPERLINK \l "_ENREF_221" \o "Seta, 2011 #559" Seta, Rahmani et al. 2011). In the rat MCT/pneumonectomy model, elevation of OS and specifically NOX-4 was also reported, and correlated with inflammation and vascular remodeling  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_46" \o "Dorfmuller, 2011 #678" Dorfmuller, Chaumais et al. 2011). Moreover, development of right heart failure in experimental PH was also associated with increased OS production in the right ventricle  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_197" \o "Redout, 2007 #573" Redout, Wagner et al. 2007). Treatment with antioxidants including superoxide dismutase (SOD), EUK-134 (a SOD and catalase mimetic) and resveratrol improved PH in MCT experimental models  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_53" \o "Farahmand, 2004 #549" Farahmand, Hill et al. 2004;  HYPERLINK \l "_ENREF_38" \o "Csiszar, 2009 #45" Csiszar, Labinskyy et al. 2009;  HYPERLINK \l "_ENREF_196" \o "Redout, 2010 #576" Redout, van der Toorn et al. 2010). In mice exposed to chronic hypoxia, an increase in OS was also noted with an overproduction of superoxide anions in pulmonary arteries; treatment with the antioxidant N-acetylcysteine significantly improved PH  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_104" \o "Hoshikawa, 2001 #40" Hoshikawa, Ono et al. 2001). In addition, the antioxidant edorstein in rats exposed to chronic hypoxia  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_254" \o "Uzun, 2006 #577" Uzun, Balbay et al. 2006). More recently, in the Sugen/chronic hypoxia model, treatment with protadim (an inducer of antioxidant enzymes such as heme-oxygenase-1 and SOD) protected rats from developing right ventricular failure  ADDIN EN.CITE Voelkel2012578(Voelkel, Bogaard et al. 2012)57857817Voelkel, N. F.Bogaard, H. J.Al Husseini, A. A.Farkas, L.Gomez-Arroyo, J.Natarajan, R.VCU Medical Center, Internal Medicine , 1220 East Broad Street, 6th floor, Room 6052, Richmond, Virginia, United States, 23298-0456, 804-628-9614, 804-628-0325; nvoelkel@mcvh-vcu.edu.Antioxidants for the treatment of patients with severe angioproliferative pulmonary hypertension?Antioxid Redox SignalAntioxidants & redox signalingAntioxid Redox Signal2012/08/092012Aug 71557-7716 (Electronic) 1523-0864 (Linking)22870869http://www.ncbi.nlm.nih.gov/pubmed/2287086910.1089/ars.2012.4828Eng( HYPERLINK \l "_ENREF_258" \o "Voelkel, 2012 #578" Voelkel, Bogaard et al. 2012). 2.9 Genetic Predispositions The transforming growth factor (TGF) family can be divided into two groups: 1) the bone morphogenetic proteins (BMPs) and certain growth and differentiation factors, which act through Smad 1, 5 and 8; 2) the TGF(s, activins, nodal and myostatin, which act through Smad 2 and 3. TGF- signaling is initiated by the binding of TGF- to its serine and threonine kinase receptors, the type II (TRII) and type I (TRI) receptors on the cell membrane. Several exonic mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene  ADDIN EN.CITE Newman200849(Newman, Phillips et al. 2008)494917Newman, J. H.Phillips, J. A., 3rdLoyd, J. E.Division of Pulmonary and Critical Care Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2650, USA. john.newman@vanderbilt.eduNarrative review: the enigma of pulmonary arterial hypertension: new insights from genetic studiesAnn Intern MedAnnals of internal medicineAnn Intern Med278-8314842008/02/20Activin Receptors, Type II/drug effects/geneticsBone Morphogenetic Protein Receptors, Type II/drug effects/geneticsHumansHypertension, Pulmonary/drug therapy/*geneticsMutation2008Feb 191539-3704 (Electronic) 0003-4819 (Linking)18283205Research Support, N.I.H., Extramural Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/18283205148/4/278 [pii]eng( HYPERLINK \l "_ENREF_174" \o "Newman, 2008 #49" Newman, Phillips et al. 2008), in the activin A receptor type II-like kinase-1 (ACVRL1)  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_84" \o "Girerd, 2010 #50" Girerd, Montani et al.) and to a lesser extent in the endoglin (ENG) gene have been reported in patients with heritable (70%) and idiopathic (3.5 to 40%) PAH, or in PAH associated with appetite suppressants, or in PAH and hereditary hemorrhagic telangiectasia with co-existent PAH  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_41" \o "Deng, 2000 #52" Deng, Morse et al. 2000;  HYPERLINK \l "_ENREF_132" \o "Lane, 2000 #54" Lane, Machado et al. 2000;  HYPERLINK \l "_ENREF_248" \o "Trembath, 2001 #51" Trembath, Thomson et al. 2001;  HYPERLINK \l "_ENREF_106" \o "Humbert, 2002 #53" Humbert, Deng et al. 2002). BMPR2 gene mutations confer a reduction in BMPR-II signaling activity. However BMPR-II expression is also substantially reduced in patients with various form of PH without a mutation  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_7" \o "Atkinson, 2002 #856" Atkinson, Stewart et al. 2002), as well as in experimental animal models  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_240" \o "Takahashi, 2006 #982" Takahashi, Goto et al. 2006;  HYPERLINK \l "_ENREF_200" \o "Reynolds, 2012 #992" Reynolds, Holmes et al. 2012). PAH patients carrying a BMPR2 mutation present approximately 10 years earlier the disease than non-carriers and have a more severe hemodynamic compromise at diagnosis  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_238" \o "Sztrymf, 2008 #55" Sztrymf, Coulet et al. 2008). ACVRL1 mutation carriers are also significantly younger at PAH diagnosis, as compared with BMPR2 mutation carriers and non-carriers, suggesting a more rapid disease evolution  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_84" \o "Girerd, 2010 #50" Girerd, Montani et al. 2010). These patients are also less likely to respond to vasodilator therapy  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_203" \o "Rosenzweig, 2008 #56" Rosenzweig, Morse et al. 2008). No differences in survival and time to death or lung transplantation were found in male and female PAH patients carrying a BMPR2 mutation  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_85" \o "Girerd, 2010 #998" Girerd, Montani et al. 2010). 3. Conventional therapy An appropriate level of physical activity is required for patients with PAH avoiding breathlessness, which can precipitate PH worsening and extreme effort limitation  ADDIN EN.CITE Rubin199770(Rubin 1997)707017Rubin, L. J.Departments of Medicine and Physiology, University of Maryland School of Medicine, Baltimore 21201, USA.Primary pulmonary hypertensionN Engl J MedThe New England journal of medicineN Engl J Med111-733621997/01/09AlgorithmsAnticoagulants/therapeutic useHumans*Hypertension, Pulmonary/diagnosis/etiology/therapyIncidenceLung TransplantationVasodilator Agents/therapeutic use1997Jan 90028-4793 (Print) 0028-4793 (Linking)8988890Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/898889010.1056/NEJM199701093360207eng( HYPERLINK \l "_ENREF_204" \o "Rubin, 1997 #70" Rubin 1997). In addition, situations associated with hypobaric hypoxia should be avoided, such as high altitude travel. The prevention of infections, which may provoke deteriorations in PH, is important, thus routine immunizations against influenza and pneumococcal infections are advised  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_166" \o "Montani, 2005 #71" Montani, Sitbon et al. 2005;  HYPERLINK \l "_ENREF_72" \o "Galie, 2009 #1" Galie, Hoeper et al. 2009). Diuretics in PAH decrease right ventricular overload and improve symptoms. Careful dose adjustment is needed on the basis of clinical, echocardiographic and hemodynamic findings  ADDIN EN.CITE Montani200572(Montani, Jais et al. 2005)727217Montani, D.Jais, X.Sitbon, O.Capron, F.Simonneau, G.Humbert, M.Centre des Maladies Vasculaires Pulmonaires, UPRES EA2705, Service de Pneumologie et Reanimation respiratoire, Hopital Antoine-Beclere, Universite Paris-Sud, Assistance Publique, Hopitaux de Paris, Clamart, France.[Pulmonary arterial hypertension]Rev Mal RespirRevue des maladies respiratoiresRev Mal Respir651-662242005/11/19Antihypertensive Agents/therapeutic useDrug Therapy, CombinationElectrocardiographyEpoprostenol/therapeutic useHumans*Hypertension, Pulmonary/classification/diagnosis/drug therapyPhosphodiesterase Inhibitors/therapeutic useSulfonamides/therapeutic useTreatment Outcome2005SepHypertension arterielle pulmonaire.0761-8425 (Print) 0761-8425 (Linking)16294183Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/16294183MDOI-RMR-09-2005-22-4-0761-8425-101019-200530049 [pii]fre( HYPERLINK \l "_ENREF_165" \o "Montani, 2005 #72" Montani, Jais et al. 2005). Although there is no controlled trial examining the effect of anticoagulation in PAH, three open label studies suggest a survival benefit with oral anticoagulants  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_67" \o "Fuster, 1984 #73" Fuster, Steele et al. 1984;  HYPERLINK \l "_ENREF_202" \o "Rich, 1992 #74" Rich, Kaufmann et al. 1992;  HYPERLINK \l "_ENREF_60" \o "Frank, 1997 #75" Frank, Mlczoch et al. 1997). This benefit is partially attributable to a decrease of in situ thrombosis in PAH patients characterized by right-heart failure and immobility. Vitamin K antagonists are therefore prescribed for PAH patients (INR: 1.5 to 2.5), unless there is a contraindication  ADDIN EN.CITE Montani200572(Montani, Jais et al. 2005)727217Montani, D.Jais, X.Sitbon, O.Capron, F.Simonneau, G.Humbert, M.Centre des Maladies Vasculaires Pulmonaires, UPRES EA2705, Service de Pneumologie et Reanimation respiratoire, Hopital Antoine-Beclere, Universite Paris-Sud, Assistance Publique, Hopitaux de Paris, Clamart, France.[Pulmonary arterial hypertension]Rev Mal RespirRevue des maladies respiratoiresRev Mal Respir651-662242005/11/19Antihypertensive Agents/therapeutic useDrug Therapy, CombinationElectrocardiographyEpoprostenol/therapeutic useHumans*Hypertension, Pulmonary/classification/diagnosis/drug therapyPhosphodiesterase Inhibitors/therapeutic useSulfonamides/therapeutic useTreatment Outcome2005SepHypertension arterielle pulmonaire.0761-8425 (Print) 0761-8425 (Linking)16294183Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/16294183MDOI-RMR-09-2005-22-4-0761-8425-101019-200530049 [pii]fre( HYPERLINK \l "_ENREF_165" \o "Montani, 2005 #72" Montani, Jais et al. 2005). In PAH patients with severe hypoxia (PaO2 < 60mmHg), oxygen therapy (for at 15 hours a day) can improve quality of life by improving dyspnea and exercise capacity  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_165" \o "Montani, 2005 #72" Montani, Jais et al. 2005;  HYPERLINK \l "_ENREF_12" \o "Barst, 2009 #76" Barst, Gibbs et al. 2009), although no controlled trials have been performed. Pregnancy is associated with 30% to 50% mortality in patients with PAH and is a formal contraindication in pulmonary hypertension  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_16" \o "Bonnin, 2005 #77" Bonnin, Mercier et al. 2005). A safe and effective method of contraception is always advised in PAH women of childbearing age. However the best contraception is not yet specified  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_12" \o "Barst, 2009 #76" Barst, Gibbs et al. 2009;  HYPERLINK \l "_ENREF_58" \o "Frachon, 2010 #78" Frachon, Gaudin et al.). 4. Approved Specific PAH therapies 4.1 Calcium channels blockers (CCBs) CCBs decrease calcium influx into SMCs of arterial wall and myocardium cells by inhibition of L-type voltage-dependant calcium channels. All CCBs are peripheral arterial dilators leading to vasodilatation. Historically, many CCBs treatments were proposed in PAH management. However, only a small proportion of patients respond to this treatment. Therefore, vasoreactivity testing is performed at the time of PAH diagnosis, for example using inhaled NO, to identify patients who may benefit from long-term therapy with CCB. A response to vasoreactivity testing is defined as a fall in mPAP by a least 10 mmHg to reach an absolute level of < 40 mmHg, with stable or increased cardiac output  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_71" \o "Galie, 2009 #79" Galie, Hoeper et al. 2009). Sustained benefit with CCBs is defined by the improvement of dyspnea to NYHA class II or less in concert with sustained hemodynamic improvement after a year of treatment  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_76" \o "Galie, 2004 #53" Galie, Torbicki et al. 2004;  HYPERLINK \l "_ENREF_230" \o "Sitbon, 2005 #211" Sitbon, Humbert et al. 2005). The current approach is to initiate CCBs if the patient has a positive vasodilator test, adequate systemic blood pressure (above 90 mmHg) and a stable dyspnea in NYHA class I-IV prior to initiation of therapy  ADDIN EN.CITE Barst200976(Barst, Gibbs et al. 2009)767617Barst, R. J.Gibbs, J. S.Ghofrani, H. A.Hoeper, M. M.McLaughlin, V. V.Rubin, L. J.Sitbon, O.Tapson, V. F.Galie, N.Columbia University, New York, New York 10583, USA. robyn.barst@gmail.comUpdated evidence-based treatment algorithm in pulmonary arterial hypertensionJ Am Coll CardiolJournal of the American College of CardiologyJ Am Coll CardiolS78-84541 Suppl2009/07/09AlgorithmsAntihypertensive Agents/administration & dosageCalcium Channel Blockers/administration & dosageEpoprostenol/administration & dosageEvidence-Based MedicineHumansHypertension, Pulmonary/*drug therapyInfusions, IntravenousRandomized Controlled Trials as TopicTreatment Outcome2009Jun 301558-3597 (Electronic) 0735-1097 (Linking)19555861Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/1955586110.1016/j.jacc.2009.04.017eng( HYPERLINK \l "_ENREF_12" \o "Barst, 2009 #76" Barst, Gibbs et al. 2009). The agents commonly recommended are long-acting nifedipine, diltiazem or amlodipine  ADDIN EN.CITE Barst200976(Barst, Gibbs et al. 2009)767617Barst, R. J.Gibbs, J. S.Ghofrani, H. A.Hoeper, M. M.McLaughlin, V. V.Rubin, L. J.Sitbon, O.Tapson, V. F.Galie, N.Columbia University, New York, New York 10583, USA. robyn.barst@gmail.comUpdated evidence-based treatment algorithm in pulmonary arterial hypertensionJ Am Coll CardiolJournal of the American College of CardiologyJ Am Coll CardiolS78-84541 Suppl2009/07/09AlgorithmsAntihypertensive Agents/administration & dosageCalcium Channel Blockers/administration & dosageEpoprostenol/administration & dosageEvidence-Based MedicineHumansHypertension, Pulmonary/*drug therapyInfusions, IntravenousRandomized Controlled Trials as TopicTreatment Outcome2009Jun 301558-3597 (Electronic) 0735-1097 (Linking)19555861Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/1955586110.1016/j.jacc.2009.04.017eng( HYPERLINK \l "_ENREF_12" \o "Barst, 2009 #76" Barst, Gibbs et al. 2009). The choice between nifedipine and diltiazem is guided by heart rate at rest: diltiazem is only used if the value is > 80/min  ADDIN EN.CITE Rich199274(Rich, Kaufmann et al. 1992)747417Rich, S.Kaufmann, E.Levy, P. S.Department of Medicine, University of Illinois College of Medicine, Chicago 60680.The effect of high doses of calcium-channel blockers on survival in primary pulmonary hypertensionN Engl J MedThe New England journal of medicineN Engl J Med76-8132721992/07/09AdultBlood Pressure/drug effectsCalcium Channel Blockers/*administration & dosage/pharmacologyDiltiazem/pharmacologyHumansHypertension, Pulmonary/drug therapy/*mortalityNifedipine/pharmacologyProspective StudiesPulmonary Artery/drug effectsSurvival RateVascular Resistance/drug effectsWarfarin/therapeutic use1992Jul 90028-4793 (Print) 0028-4793 (Linking)1603139Clinical Trialhttp://www.ncbi.nlm.nih.gov/pubmed/160313910.1056/NEJM199207093270203eng( HYPERLINK \l "_ENREF_202" \o "Rich, 1992 #74" Rich, Kaufmann et al. 1992). Due to its prominent negative inotropic effect, verapamil is not recommended  ADDIN EN.CITE Packer1984212(Packer, Medina et al. 1984)21221217Packer, M.Medina, N.Yushak, M.Wiener, I.Detrimental effects of verapamil in patients with primary pulmonary hypertensionBr Heart JBritish heart journalBr Heart JBritish heart journalBr Heart JBritish heart journal106-115211984/07/01AdultDepression, ChemicalFemaleHeart Ventricles/physiopathologyHemodynamics/drug effectsHumansHypertension, Pulmonary/*drug therapy/physiopathologyMaleMiddle AgedTime FactorsVerapamil/*adverse effects/pharmacology/therapeutic use1984Jul0007-0769 (Print) 0007-0769 (Linking)6743418Research Support, U.S. Gov't, P.H.S.http://www.ncbi.nlm.nih.gov/pubmed/6743418481593eng( HYPERLINK \l "_ENREF_186" \o "Packer, 1984 #212" Packer, Medina et al. 1984). 4.2 Currently available PAH therapies target endothelial dysfunction For many years PAH treatment was extremely limited and the prognosis was poor, with a mean survival time of less than 3 years after diagnosis  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_39" \o "D'Alonzo, 1991 #609" D'Alonzo, Barst et al. 1991;  HYPERLINK \l "_ENREF_8" \o "Badesch, 2009 #80" Badesch, Champion et al. 2009). The introduction of current therapies over the last quarter of the century has significantly improved PAH management and patients survival  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_109" \o "Humbert, 2010 #615" Humbert, Sitbon et al. 2010). However, PAH remains a progressive disease with unacceptably high mortality, and no long-term exists  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_72" \o "Galie, 2009 #1" Galie, Hoeper et al. 2009;  HYPERLINK \l "_ENREF_227" \o "Simonneau, 2009 #3" Simonneau, Robbins et al. 2009). All current therapeutic options predominantly act to restore the imbalance in vascular tone of PAH patients. 4.2.1 Therapies targeting prostacyclin (PGI2) pathway: Epoprostenol and PGI2 derivatives Epoprostenol Therapeutic use of prostaglandin has a convincing rationale in PAH patients. Epoprostenol is the synthetic form (sodium salt) of PGI2, and was the first PGI2 analogue approved by the FDA in 1995. Epoprostenol binds to the IP receptor (IPr) and has two major pharmacologic actions in PAH: direct vasodilation of pulmonary and systemic arterial vascular bed, and inhibition of platelet aggregation  ADDIN EN.CITE Friedman199781(Friedman, Mears et al. 1997)818117Friedman, R.Mears, J. G.Barst, R. J.Department of Pediatrics, Columbia University College of Physicians & Surgeons, New York, NY 10032, USA.Continuous infusion of prostacyclin normalizes plasma markers of endothelial cell injury and platelet aggregation in primary pulmonary hypertensionCirculationCirculationCirculationCirculation2782-49691997/12/31AdolescentAdultChildChild, PreschoolEndothelium, Vascular/*drug effects/physiologyEpoprostenol/*pharmacology/therapeutic useHumansHypertension, Pulmonary/*blood/drug therapyMiddle AgedPlatelet Aggregation/*drug effectsVasodilator Agents/*pharmacology1997Nov 40009-7322 (Print) 0009-7322 (Linking)9386137Research Support, U.S. Gov't, P.H.S.http://www.ncbi.nlm.nih.gov/pubmed/9386137eng( HYPERLINK \l "_ENREF_64" \o "Friedman, 1997 #81" Friedman, Mears et al. 1997). Like native PGI2, the structure of the epoprostenol molecule is unstable in solution, prone to rapid degradation. Therefore, epoprostenol must be delivered by continuous intravenous route through a permanent indwelling central venous catheter via a portable infusion pump system. Treatment with epoprostenol therefore has limitations based on its pharmacological characteristics. Complications of chronic intravenous therapy with epoprostenol include catheter associated venous thrombosis and line-related infections (which range from exit site reactions, tunnel infections and cellulitis, to bacteremia or sepsis)  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_14" \o "Barst, 1996 #83" Barst, Rubin et al. 1996;  HYPERLINK \l "_ENREF_9" \o "Badesch, 2000 #84" Badesch, Tapson et al. 2000;  HYPERLINK \l "_ENREF_117" \o "Kallen, 2008 #85" Kallen, Lederman et al. 2008). Other serious drug-related complications include mild ascites and thrombocytopenia  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_206" \o "Rubin, 1990 #87" Rubin, Mendoza et al. 1990;  HYPERLINK \l "_ENREF_25" \o "Chin, 2009 #86" Chin, Channick et al. 2009). Thrombocytopenia was reported in 34 % of patients treated with epoprostenol compared with 15 % of patients receiving specific oral therapy for PAH  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_25" \o "Chin, 2009 #86" Chin, Channick et al. 2009). Moreover, interruption of epoprostenol delivery, either as a result of pump malfunction or catheter obstruction or damage, can result in potentially fatal rebound pulmonary hypertension. Common side effects of epoprostenol therapy include flushing, headache, jaw pain, diarrhea, nausea, photosensitivity, erythroderma, anxiety and musculoskeletal aches and pain, predominantly involving legs and feet  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_14" \o "Barst, 1996 #83" Barst, Rubin et al. 1996;  HYPERLINK \l "_ENREF_145" \o "Magnani, 1996 #91" Magnani and Galie 1996;  HYPERLINK \l "_ENREF_4" \o "Ahearn, 2002 #90" Ahearn, Selim et al. 2002;  HYPERLINK \l "_ENREF_154" \o "McLaughlin, 2002 #89" McLaughlin, Shillington et al. 2002;  HYPERLINK \l "_ENREF_231" \o "Sitbon, 2002 #88" Sitbon, Humbert et al. 2002). Metabolism perturbations as hyperglycemia or hyperthyroidism are also reported  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_237" \o "Szczeklik, 1980 #92" Szczeklik, Pieton et al. 1980;  HYPERLINK \l "_ENREF_206" \o "Rubin, 1990 #87" Rubin, Mendoza et al. 1990). These side effects tend to be dose-dependent and they often respond to a cautious dose reduction. To date, epoprostenol remains the only PAH therapy to have been associated with a mortality benefit in a randomized clinical trial and, on this basis, was granted regulatory approval for treatment of patients with NYHA functional class III or IV symptoms. Trepostinil Other prostanoids are more chemically stable in solution and their plasma t is much longer than epoprostenol. Treprostinil, a tricyclic benzidine analogue of epoprostenol, is chemically stable at room temperature (for up to 72 h) and at neutral pH. These characteristics allow administration of the compound by the intravenous, subcutaneous, inhaled or oral routes. The intravenous and subcutaneous forms of treprostinil are considered to be bioequivalent at steady state at reference dose of 10 ng/kg/min  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_130" \o "Laliberte, 2004 #85" Laliberte, Arneson et al. 2004). At steady-state, the elimination t of treprostinil is 4.4 and 4.6 h following intravenous and subcutaneous administration, respectively  ADDIN EN.CITE Mubarak2010999(Mubarak 2010)99999917Mubarak, K. K.Division of Pulmonary, Critical Care and Sleep Medicine, University of Florida, 1600 SW Archer Road, M452A, Gainesville, FL 32610, USA. mubarak@ufl.eduA review of prostaglandin analogs in the management of patients with pulmonary arterial hypertensionRespir MedRespiratory medicineRespir Med9-2110412009/08/18Dose-Response Relationship, DrugEpoprostenol/therapeutic useHumansHypertension, Pulmonary/*drug therapyProstaglandins, Synthetic/*therapeutic useTreatment Outcome2010Jan1532-3064 (Electronic) 0954-6111 (Linking)19683911Research Support, Non-U.S. Gov't Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/1968391110.1016/j.rmed.2009.07.015eng( HYPERLINK \l "_ENREF_170" \o "Mubarak, 2010 #999" Mubarak 2010). Intravenous treprostinil provides similar beneficial hemodynamic results to epoprostenol, with an acute decrease in pulmonary vascular resistance (22% with epoprostenol and 20% reduction with treprostinil) despite a higher dose for intravenous treprostinil  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_153" \o "McLaughlin, 2003 #96" McLaughlin, Gaine et al. 2003;  HYPERLINK \l "_ENREF_185" \o "Oudiz, 2009 #95" Oudiz and Farber 2009). Approval of subcutaneous treprostinil was granted in 2002 in the USA for patients with NYHA functional class IIIV symptoms. In Europe, approval was granted in 2005, but only for PAH with NYHA class III symptoms. The major limitation of this formulation is infusion site pain, which occurs in the majority of patients. A recent study reported that if up-titration beyond 6-months is tolerated, effective doses of treprostinil are reached and outcomes of patients are good with a survival rate of 57% at 9 years  ADDIN EN.CITE Sadushi-Kolici2012631(Sadushi-Kolici, Skoro-Sajer et al. 2012)63163117Sadushi-Kolici, R.Skoro-Sajer, N.Zimmer, D.Bonderman, D.Schemper, M.Klepetko, W.Glatz, J.Jakowitsch, J.Lang, I. M.Department of Internal Medicine II, Division of Cardiology, Medical University of Vienna, Vienna, Austria.Long-term treatment, tolerability, and survival with sub-cutaneous treprostinil for severe pulmonary hypertensionJ Heart Lung TransplantThe Journal of heart and lung transplantation : the official publication of the International Society for Heart TransplantationJ Heart Lung Transplant735-433172012/04/072012Jul1557-3117 (Electronic) 1053-2498 (Linking)22480725Research Support, Non-U.S. Gov'thttp://www.ncbi.nlm.nih.gov/pubmed/2248072510.1016/j.healun.2012.02.025eng( HYPERLINK \l "_ENREF_208" \o "Sadushi-Kolici, 2012 #631" Sadushi-Kolici, Skoro-Sajer et al. 2012). Intravenous treprostinil is only licensed in USA with the advantage of less frequent need for drug reservoir replacement (every 48h compared with every 12-24h for epoprostenol)  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_130" \o "Laliberte, 2004 #85" Laliberte, Arneson et al. 2004). Switching between epoprostenol and treprostinil is feasible in cases of clinical worsening or intolerance to epoprostenol  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_88" \o "Gomberg-Maitland, 2005 #628" Gomberg-Maitland, Tapson et al. 2005;  HYPERLINK \l "_ENREF_199" \o "Reisbig, 2005 #629" Reisbig, Coffman et al. 2005). Inhaled treprostinil was approved by the FDA in 2009 for PAH patients with NYHA functional class III symptoms, after beneficial results obtained in the TRIUMPH-1 trial regarding 6-MWD and quality of life, despite no improvements in time to clinical worsening, Borg dyspnea scores or NYHA functional class  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_152" \o "McLaughlin, 2010 #630" McLaughlin, Benza et al. 2010). The efficacy and safety of oral treprostinil with concomitant ERA and/or iPDE5 was studied in the FREEDOM-C study. Additional oral treatment with treprostinil did not significantly improved the primary end point of 6minute walk distance after 16 weeks, although patients able to achieve higher treatment doses showed greatest improvement in exercise capacity  ADDIN EN.CITE Tapson2012632(Tapson, Torres et al. 2012)63263217Tapson, V. F.Torres, F.Kermeen, F.Keogh, A. M.Allen, R. P.Frantz, R. P.Badesch, D. B.Frost, A. E.Shapiro, S. M.Laliberte, K.Sigman, J.Arneson, C.Galie, N.1Division of Pulmonary, Allergy, and Critical Care Medicine, Duke University Medical Center, Durham, NC;Oral Treprostinil for the Treatment of Pulmonary Arterial Hypertension in Patients on Background Endothelin Receptor Antagonist and/or Phosphodiesterase Type 5 Inhibitor Therapy (The FREEDOM-C Study): A Randomized Controlled TrialChestChestChestChest2012/05/262012May 241931-3543 (Electronic) 0012-3692 (Linking)22628490http://www.ncbi.nlm.nih.gov/pubmed/2262849010.1378/chest.11-2212Eng( HYPERLINK \l "_ENREF_243" \o "Tapson, 2012 #632" Tapson, Torres et al. 2012). Iloprost Iloprost is a carbacyclin analog of PGI2 available for intravenous and inhaled administration  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_103" \o "Hoeper, 2000 #97" Hoeper, Schwarze et al. 2000). Inhaled iloprost rapidly enters into the systemic circulation, with peak concentrations immediately observed after cessation of the inhalation. Iloprost has a short serum t (20-25 min) and it undergoes -oxidation to an inactive metabolite  ADDIN EN.CITE <EndNote><Cite><Author>Krause</Author><Year>1986</Year><RecNum>98</RecNum><DisplayText>(Krause and Krais 1986)989817Krause, W.Krais, T.Pharmacokinetics and pharmacodynamics of the prostacyclin analogue iloprost in manEur J Clin PharmacolEuropean journal of clinical pharmacologyEur J Clin Pharmacol61-83011986/01/01Administration, OralAdultBiological AvailabilityBlood Chemical AnalysisBlood Pressure/drug effectsCardiovascular Agents/adverse effects/metabolism/*pharmacologyCyclic AMP/bloodElectrocardiographyEpoprostenol/adverse effects/metabolism/*pharmacologyHalf-LifeHeart Rate/drug effectsHumansIloprostInfusions, ParenteralKineticsMalePlatelet Aggregation/drug effectsRespiration/drug effects19860031-6970 (Print) 0031-6970 (Linking)2423342http://www.ncbi.nlm.nih.gov/pubmed/2423342eng( HYPERLINK \l "_ENREF_126" \o "Krause, 1986 #98" Krause and Krais 1986), therefore doses are administered 6 to 9 times a day. In the AIR study, 17% of inhaled iloprost-treated patients reached the combined primary end point of improvement in functional class at 12 weeks, compared with 5% of placebo-treated patients, and 10% increase in 6MWD in the absence of clinical deterioration or death  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_182" \o "Olschewski, 2002 #634" Olschewski, Simonneau et al. 2002). Following the AIR study, the European authorities and the FDA granted regulatory approval for inhaled iloprost in 2003 and in late 2004, respectively  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_182" \o "Olschewski, 2002 #634" Olschewski, Simonneau et al. 2002). Data from non-randomized studies suggested that intravenous iloprost has an efficacy profile similar to that of intravenous epoprostenol, with a similar improvement in hemodynamics and in exercise tolerance in PAH patients  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_98" \o "Higenbottam, 1998 #99" Higenbottam, Butt et al. 1998;  HYPERLINK \l "_ENREF_183" \o "Opitz, 2003 #719" Opitz, Wensel et al. 2003). Intravenous iloprost can be considered for patients with advanced PAH, although it has not been formally tested in a randomized, placebo-controlled trial. Moreover, a retrospective analysis of 79 patients with PAH treated with intravenous iloprost (introduced primarily because of clinical worsening despite initial inhaled iloprost) revealed a high rate of treatment failure and poor survival  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_101" \o "Hoeper, 2009 #720" Hoeper, Gall et al. 2009). New Zealand remains the only country to have granted regulatory approval for intravenous iloprost. Due to their similar biological profile, intravenous treprostinil and iloprost have similar side effects as epoprostenol. Dose limiting adverse events are similar for epoprostenol and intravenous treprostinil including headache, nausea, chest pain, jaw pain and restlessness  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_153" \o "McLaughlin, 2003 #96" McLaughlin, Gaine et al. 2003). The safety profiles of epoprostenol and intravenous treprostinil during short-term therapy appear comparable. Similarly, intravenous iloprost is well tolerated with headaches, diarrhea and abdominal pain  ADDIN EN.CITE Oudiz200995(Oudiz and Farber 2009)959517Oudiz, R. J.Farber, H. W.Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA 90502, USA. oudiz@humc.eduDosing considerations in the use of intravenous prostanoids in pulmonary arterial hypertension: an experience-based reviewAm Heart JAmerican heart journalAm Heart J625-3515742009/04/01Dose-Response Relationship, DrugHumansHypertension, Pulmonary/*drug therapy/physiopathologyInjections, IntravenousProstaglandins/*administration & dosage/therapeutic usePulmonary Wedge Pressure/drug effectsTreatment Outcome2009Apr1097-6744 (Electronic) 0002-8703 (Linking)19332188Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/1933218810.1016/j.ahj.2008.10.029eng( HYPERLINK \l "_ENREF_185" \o "Oudiz, 2009 #95" Oudiz and Farber 2009). Beraprost Oral beraprost is rapidly absorbed during fasting, peak concentration is reached after 30 min and the elimination t is obtained 35-40 min after oral administration  ADDIN EN.CITE Galie2002100(Galie, Manes et al. 2002)10010017Galie, N.Manes, A.Branzi, A.n.galie@bo.nettuno.itThe new clinical trials on pharmacological treatment in pulmonary arterial hypertensionEur Respir JThe European respiratory journal : official journal of the European Society for Clinical Respiratory PhysiologyEur Respir J1037-492042002/11/05Dose-Response Relationship, DrugDrug Administration ScheduleEpoprostenol/*administration & dosage/*analogs & derivativesFemaleHumansHypertension, Pulmonary/diagnosis/*drug therapy/mortalityIloprost/*administration & dosageMalePrognosisPyridines/*administration & dosageRandomized Controlled Trials as TopicSeverity of Illness IndexSurvival RateTreatment OutcomeVasodilator Agents/*administration & dosage2002Oct0903-1936 (Print) 0903-1936 (Linking)12412701Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/12412701eng( HYPERLINK \l "_ENREF_74" \o "Galie, 2002 #100" Galie, Manes et al. 2002). However, the clinical improvement in exercise capacity persists only up to 3 -to- 6 months. In addition, there is no hemodynamic benefit compared to PGI2 analogues, whereas the side effects are similar. Therefore, beraprost is neither approved in Europe nor in the US  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_73" \o "Galie, 2002 #101" Galie, Humbert et al. 2002;  HYPERLINK \l "_ENREF_13" \o "Barst, 2003 #102" Barst, McGoon et al. 2003). 4.2.2 Therapies targeting endothelin pathway: endothelin receptor antagonists (ERAs) Dual ERAs Bosentan is a non-peptide pyrimidine derivative that competitively antagonizes the binding of ET-1 to both ETA and ETB receptor subtypes, and irreversibly blocks their activities  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_29" \o "Clozel, 1994 #638" Clozel, Breu et al. 1994). Bosentan decreases pulmonary and systemic vascular resistances and increases CO without increasing heart rate. In addition to its hemodynamic effects, bosentan has been reported to significantly improve pulmonary fibrosis, and to inhibit cell proliferation of PASMCs and inflammatory response in pulmonary tissue to injection of ET-1 in guinea-pig and mouse lung  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_56" \o "Filep, 1995 #858" Filep, Fournier et al. 1995;  HYPERLINK \l "_ENREF_30" \o "Clozel, 2000 #640" Clozel and Roux 2000;  HYPERLINK \l "_ENREF_94" \o "Guimaraes, 2000 #861" Guimaraes, Da-Silva et al. 2000). Bosentan has also been reported to substantially improve the primary endpoint 6MWD, dyspnea score and delayed time to clinical worsening in the clinical trial BREATHE1  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_205" \o "Rubin, 2002 #104" Rubin, Badesch et al. 2002). Following its approval by the US Food and Drug administration (FDA) in 2001, bosentan is now available in many parts of the world. For the time being, the authorities have approved the drug only for patients with PAH of functional class III (Europe) or III/IV (USA and Canada) of the NYHA. Bosentan is associated with reversible and in most cases asymptomatic elevation of transaminases (Benedict et al., 2007; Hoeper, 2005), possibly as a consequence of the cellular accumulation of bile acids due to impaired canalicular excretion  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_54" \o "Fattinger, 2001 #695" Fattinger, Funk et al. 2001). In order to obtain further safety data, European authorities required the introduction of a post-marketing surveillance system. Within 30 months, this system has assembled data from 4,994 patients, representing 79% of those exposed to bosentan in Europe during that time period  ADDIN EN.CITE Humbert2007105(Humbert, Segal et al. 2007)10510517Humbert, M.Segal, E. S.Kiely, D. G.Carlsen, J.Schwierin, B.Hoeper, M. M.Dept of Respiratory Medicine, Antoine Beclere Hospital, Clamart, France. marc.humbert@abc.ap-hop-paris.frResults of European post-marketing surveillance of bosentan in pulmonary hypertensionEur Respir JThe European respiratory journal : official journal of the European Society for Clinical Respiratory PhysiologyEur Respir J338-443022007/05/17Adverse Drug Reaction Reporting SystemsAntihypertensive Agents/*adverse effectsEuropeHumansHypertension, Pulmonary/*drug therapyInternet*Product Surveillance, PostmarketingProspective StudiesSulfonamides/*adverse effects2007Aug0903-1936 (Print) 0903-1936 (Linking)17504794http://www.ncbi.nlm.nih.gov/pubmed/1750479410.1183/09031936.00138706eng( HYPERLINK \l "_ENREF_108" \o "Humbert, 2007 #105" Humbert, Segal et al. 2007). The reported annual rate of aminotransferase level elevation was 10.1%, and 3.2% of patients had to discontinue the drug for this reason. Aminotransferase level elevation was reversible in all cases, and there was no permanent liver injury. Monthly monitoring of liver transaminases is mandatory in patients treated with bosentan. Anaemia is another biologic side effect that could appear during bosentan treatment. Due to its vasodilator properties, bosentan induces potential headache and oedema. Teratogenic effects were also reported with bosentan, thus pregnancy is a formally contraindication  ADDIN EN.CITE Dhillon2009106(Dhillon and Keating 2009)10610617Dhillon, S.Keating, G. M.Adis, a Wolters Kluwer Business, Auckland, New Zealand. demail@adis.co.nzBosentan: a review of its use in the management of mildly symptomatic pulmonary arterial hypertensionAm J Cardiovasc DrugsAmerican journal of cardiovascular drugs : drugs, devices, and other interventionsAm J Cardiovasc Drugs331-50952009/10/02AdolescentAdultAntihypertensive Agents/administration & dosage/adverseeffects/pharmacokinetics/*therapeutic useDrug InteractionsHumansHypertension, Pulmonary/*drug therapyMulticenter Studies as TopicRandomized Controlled Trials as TopicReceptor, Endothelin A/drug effectsSulfonamides/administration & dosage/adverseeffects/pharmacokinetics/*therapeutic use20091175-3277 (Print) 1175-3277 (Linking)19791841Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/1979184110.2165/11202270-000000000-00000eng( HYPERLINK \l "_ENREF_43" \o "Dhillon, 2009 #106" Dhillon and Keating 2009). However, contraception only with hormonal strategy, whatever its administration route, is not reliable due to powerful enzymatic induction of bosentan on CYP2C9 and CYAP3A4. Generally, multiple drug interactions with bosentan are reported due to its property of enzymatic induction such as cyclosporin A and glyburide (contraindicating) or simvastatin, lopinavir/ritonavir, and rifampicin (dose adjustments requiring)  ADDIN EN.CITE Venitz2011721(Venitz, Zack et al. 2011)72172117Venitz, J.Zack, J.Gillies, H.Allard, M.Regnault, J.Dufton, C.Clinical Pharmacokinetics and Drug-Drug Interactions of Endothelin Receptor Antagonists in Pulmonary Arterial HypertensionJ Clin PharmacolJournal of clinical pharmacologyJ Clin PharmacolJournal of clinical pharmacologyJ Clin PharmacolJournal of clinical pharmacology2011/12/302011Dec 281552-4604 (Electronic) 0091-2700 (Linking)22205719http://www.ncbi.nlm.nih.gov/pubmed/2220571910.1177/0091270011423662Eng( HYPERLINK \l "_ENREF_256" \o "Venitz, 2011 #721" Venitz, Zack et al. 2011). Selective ET-A receptors antagonists Selective ET-A receptor inhibition may provide benefits by preserving vasodilator and clearance functions specific to ET-B receptors, while preventing vasoconstriction and cellular proliferation mediated by ET-A receptors  ADDIN EN.CITE Wilkins2004107(Wilkins 2004)10710717Wilkins, M. R.Selective or nonselective endothelin receptor blockade in pulmonary arterial hypertensionAm J Respir Crit Care MedAmerican journal of respiratory and critical care medicineAm J Respir Crit Care Med433-416942004/02/10Antihypertensive Agents/therapeutic useExercise TestHumansHypertension, Pulmonary/diagnosis/*drug therapyIsoxazoles/*therapeutic useReceptors, Endothelin/*antagonists & inhibitorsSulfonamides/therapeutic useThiophenes/*therapeutic use2004Feb 151073-449X (Print) 1073-449X (Linking)14766656Comment Editorialhttp://www.ncbi.nlm.nih.gov/pubmed/1476665610.1164/rccm.2312017eng( HYPERLINK \l "_ENREF_264" \o "Wilkins, 2004 #107" Wilkins 2004). Ambrisentan is an orally active highly selective ET-A receptor antagonist approved for treatment of PAH patients in the ARIES study  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_75" \o "Galie, 2008 #108" Galie, Olschewski et al. 2008) although patients with certain subclasses of PAH (including portopulmonary hypertension (PoPH) and congenital pulmonary-to-systemic shunts) were excluded. Of note, patients with PoPH have also demonstrated beneficial outcomes  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_21" \o "Cartin-Ceba, 2011 #109" Cartin-Ceba, Swanson et al. 2011). Ambrisentan has low potential for drug-drug interactions and requires only once daily administration compared to bosentan  ADDIN EN.CITE Frampton110(Frampton)11011017Frampton, J. E.Adis, a Wolters Kluwer Business, Auckland, New Zealand.AmbrisentanAm J Cardiovasc DrugsAmerican journal of cardiovascular drugs : drugs, devices, and other interventionsAm J Cardiovasc Drugs215-261142011/06/01Antihypertensive Agents/adverseeffects/pharmacokinetics/*pharmacology/therapeutic useClinical Trials as TopicHumansHypertension, Pulmonary/*drug therapyPhenylpropionates/adverse effects/pharmacokinetics/*pharmacology/therapeutic usePyridazines/adverse effects/pharmacokinetics/*pharmacology/therapeutic useTreatment Outcome2011Aug 11179-187X (Electronic) 1175-3277 (Linking)21623643Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/2162364310.2165/11207340-000000000-00000eng( HYPERLINK \l "_ENREF_59" \o "Frampton, 2011 #110" Frampton). Furthermore, ambrisentan has not been shown to increase the risk of liver dysfunction over placebo  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_151" \o "McGoon, 2009 #111" McGoon, Frost et al. 2009). However, there is a higher incidence of peripheral edema observed in patients with PAH treated with selective ET-A receptor antagonists, such as ambrisentan, compared with dual ERAs  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_249" \o "Trow, 2009 #112" Trow and Taichman 2009) which is paradoxical as ETB receptors are known to promote natriuresis and diuresis. One possible explanation for different rates of edema with selective versus dual ERAs may reflect differences in affinities to the ETA receptor  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_249" \o "Trow, 2009 #112" Trow and Taichman 2009). Another potential explanation may involve the renin/angiotensin system (RAS). Selective ETA receptor blockade during early congestive heart failure caused sustained sodium retention by activating the RAS, resulting in edema  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_219" \o "Schirger, 2004 #724" Schirger, Chen et al. 2004). Finally, a study in rats compared bosentan with the ERA sitaxsentan, and suggested that ERA-induced fluid retention was occurring by activation of the vasopressin system via secondary stimulation by endothelin of the uninhibited ETB receptors  ADDIN EN.CITE Vercauteren20121001(Vercauteren, Strasser et al. 2012)1001100117Vercauteren, M.Strasser, D.Vezzali, EStalder, A.Iglarz, M.Hess, P.Clozel, M.Vasopressin is involved in endothelin receptor antagonist-induced fluid retention in rat. Differential effect of selective ETA and dual ETA/ETB receptor antagonists ERSERS2012( HYPERLINK \l "_ENREF_257" \o "Vercauteren, 2012 #1001" Vercauteren, Strasser et al. 2012). A final consideration is the occurrence of pulmonary edema associated with circulating ET-1 via activation of the ET-B receptor that can be prevented by ET-B blockade  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_32" \o "Comellas, 2009 #113" Comellas, Briva et al. 2009). Together these results, at least regarding peripheral and pulmonary edema, propose the safer profile of bosentan. Sitaxsentan was another selective ET-A receptor antagonist available in Europe, Australia and Canada  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_48" \o "Dupuis, 2008 #103" Dupuis and Hoeper 2008). However, it was taken off the market after two cases of fatal liver toxicity  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_135" \o "Lavelle, 2009 #644" Lavelle, Sugrue et al. 2009;  HYPERLINK \l "_ENREF_3" \o "Affsaps, 2010 #114" Affsaps 2010;  HYPERLINK \l "_ENREF_137" \o "Lee, 2011 #642" Lee, Kirkham et al. 2011). After this market withdrawal a study assessing the efficacy and safety of lower dosages of sitaxsentan in PAH was conducted  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_213" \o "Sandoval, 2012 #115" Sandoval, Torbicki et al.). In this study PAH patients mostly in WHO functional class II and receiving sitaxsentan 100 mg retained or improved their WHO functional class status and none experienced a clinical worsening event. There was no significant effect of sitaxsentan 100 mg on 6MWD; sitaxsentan 50 mg appeared subtherapeutic. Sitaxsentan was generally well tolerated. The 100-mg dose of sitaxsentan was associated with a low incidence of liver enzyme abnormalities (liver transaminases >3 _ ULN) that was similar to that observed with placebo treatment  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_213" \o "Sandoval, 2012 #115" Sandoval, Torbicki et al.). This study may have been relevant if no other ERA was available on the market for PAH treatment. However, bosentan and ambrisentan are available and proved their efficacy and safety in PAH. 4.2.3 Therapies targeting NO pathway: phosphodiesterase (PDE) type 5 inhibitors When compared with expression of PDE-5 in other tissues such as the penile corpus cavernosum and the myocardium, the expression and activity of PDE-5 is considerably higher in lung and in pulmonary vascular smooth muscle cells  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_33" \o "Corbin, 2005 #646" Corbin, Beasley et al. 2005). Therefore, the three PDE-5 inhibitors initially approved for the treatment of erectile dysfunction (sildenafil, tadalafil, and vardenafil) were evaluated in PAH. Among them, sildenafil and tadalafil are currently approved for the treatment of PAH in NYHA functional class II and III. Sildenafil, tadalafil, and vardenafil cause significant pulmonary vasodilatation in patients with maximum effects observed after 60, 75 to 90 minutes, and 40 to 45 minutes, respectively  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_81" \o "Ghofrani, 2004 #648" Ghofrani, Voswinckel et al. 2004). Sildenafil and vardenafil have very similar molecular structures, derived from cGMP, whereas tadalafil has a different chemical structure  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_164" \o "Montani, 2009 #660" Montani, Chaumais et al. 2009). These structural differences are reflected in the pharmacokinetic properties and selectivity for the PDE isoenzyme  ADDIN EN.CITE Wright2006116(Wright 2006)11611617Wright, P. J.Belmont Surgery, Belmont, Durham, UK. patwright@doctors.org.ukComparison of phosphodiesterase type 5 (PDE5) inhibitorsInt J Clin PractInternational journal of clinical practiceInt J Clin Pract967-756082006/06/20Administration, OralCost-Benefit AnalysisDouble-Blind MethodErectile Dysfunction/*drug therapyHumansMaleMulticenter Studies as TopicPatient SatisfactionPhosphodiesterase Inhibitors/chemistry/pharmacokinetics/*therapeutic useRandomized Controlled Trials as TopicTreatment Failure2006Aug1368-5031 (Print) 1368-5031 (Linking)16780568Comparative Study Research Support, Non-U.S. Gov't Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/1678056810.1111/j.1742-1241.2006.01049.xeng( HYPERLINK \l "_ENREF_268" \o "Wright, 2006 #116" Wright 2006). Both Sildenafil and vardenafil have a terminal half-life of approximately 4 hours, and tadalafil has a half-life of 17.5 hours. Due to its half-life, tadalafil is administered once per day compared to sildenafil, which have to be taken thrice per day. All three PDE-5 inhibitors are eliminated by hepatic metabolism  ADDIN EN.CITE Wright2006116(Wright 2006)11611617Wright, P. J.Belmont Surgery, Belmont, Durham, UK. patwright@doctors.org.ukComparison of phosphodiesterase type 5 (PDE5) inhibitorsInt J Clin PractInternational journal of clinical practiceInt J Clin Pract967-756082006/06/20Administration, OralCost-Benefit AnalysisDouble-Blind MethodErectile Dysfunction/*drug therapyHumansMaleMulticenter Studies as TopicPatient SatisfactionPhosphodiesterase Inhibitors/chemistry/pharmacokinetics/*therapeutic useRandomized Controlled Trials as TopicTreatment Failure2006Aug1368-5031 (Print) 1368-5031 (Linking)16780568Comparative Study Research Support, Non-U.S. Gov't Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/1678056810.1111/j.1742-1241.2006.01049.xeng( HYPERLINK \l "_ENREF_268" \o "Wright, 2006 #116" Wright 2006). Sildenafil is predominantly metabolized by CYP3A4 into a N-desmethyl metabolite that also has some PDE-5 activity. This metabolite is thought to account for approximately one fifth of the drugs activity. As bosentan induces CYP3A4, this leads to a pharmacokinetic interaction whereby sildenafil plasma levels are reduced and bosentan plasma levels are increased if the two drugs are co-administered  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_20" \o "Burgess, 2008 #664" Burgess, Hoogkamer et al. 2008). However, clinical studies reported no significant transaminases elevation as well as no clinical inefficiency of sildenafil when both drugs are coadministered  ADDIN EN.CITE Humbert2007105(Humbert, Segal et al. 2007)10510517Humbert, M.Segal, E. S.Kiely, D. G.Carlsen, J.Schwierin, B.Hoeper, M. M.Dept of Respiratory Medicine, Antoine Beclere Hospital, Clamart, France. marc.humbert@abc.ap-hop-paris.frResults of European post-marketing surveillance of bosentan in pulmonary hypertensionEur Respir JThe European respiratory journal : official journal of the European Society for Clinical Respiratory PhysiologyEur Respir J338-443022007/05/17Adverse Drug Reaction Reporting SystemsAntihypertensive Agents/*adverse effectsEuropeHumansHypertension, Pulmonary/*drug therapyInternet*Product Surveillance, PostmarketingProspective StudiesSulfonamides/*adverse effects2007Aug0903-1936 (Print) 0903-1936 (Linking)17504794http://www.ncbi.nlm.nih.gov/pubmed/1750479410.1183/09031936.00138706eng( HYPERLINK \l "_ENREF_108" \o "Humbert, 2007 #105" Humbert, Segal et al. 2007). Vardenafil also has an active metabolite that accounts for approximately 7% of total pharmacological activity. The activity of tadalafil is solely through the parent drug  ADDIN EN.CITE Wright2006116(Wright 2006)11611617Wright, P. J.Belmont Surgery, Belmont, Durham, UK. patwright@doctors.org.ukComparison of phosphodiesterase type 5 (PDE5) inhibitorsInt J Clin PractInternational journal of clinical practiceInt J Clin Pract967-756082006/06/20Administration, OralCost-Benefit AnalysisDouble-Blind MethodErectile Dysfunction/*drug therapyHumansMaleMulticenter Studies as TopicPatient SatisfactionPhosphodiesterase Inhibitors/chemistry/pharmacokinetics/*therapeutic useRandomized Controlled Trials as TopicTreatment Failure2006Aug1368-5031 (Print) 1368-5031 (Linking)16780568Comparative Study Research Support, Non-U.S. Gov't Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/1678056810.1111/j.1742-1241.2006.01049.xeng( HYPERLINK \l "_ENREF_268" \o "Wright, 2006 #116" Wright 2006). The adverse event profiles of the different PDE-5 inhibitors are generally similar. PDE-5 inhibitor class-specific side effects include headache, flushing, nasal congestion, digestive disorders (mainly dyspepsia and nausea), and myalgia, which are a reflection of their vasodilatory effects  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_268" \o "Wright, 2006 #116" Wright 2006;  HYPERLINK \l "_ENREF_228" \o "Simonneau, 2008 #119" Simonneau, Rubin et al. 2008;  HYPERLINK \l "_ENREF_69" \o "Galie, 2009 #118" Galie, Brundage et al. 2009;  HYPERLINK \l "_ENREF_116" \o "Jing, 2009 #117" Jing, Jiang et al. 2009). Most adverse events were transient and reported as mild or moderate and are dose-dependent. Sildenafil inhibits also PDE analogs retinal PDE6 and could therefore alter the vision  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_70" \o "Galie, 2005 #667" Galie, Ghofrani et al. 2005). In controlled trials, sildenafil and tadalafil monotherapy have been shown to improve clinical status, exercise capacity and hemodynamic parameters. The pivotal study of sildenafil given at 20 mg, 40 mg, or 80 mg three times a day versus placebo in more than 200 patients suffering from PAH demonstrated a significant increase in 6MWD, improvement in WHO functional class and decrease in mean PAP after 12 weeks  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_70" \o "Galie, 2005 #667" Galie, Ghofrani et al. 2005). Although sildenafil approval in Europe is limited to 20 mg three times daily, no data is currently available on the long-term efficacy of this lower dosage and uptitration beyond this dosage (mainly 40 mg to 80 mg three times a day) may be needed in clinical practice. In combination therapy to bosentan or epoprostenol, sildenafil improved clinical status, 6MWD, time to clinical worsening and quality of life  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_100" \o "Hoeper, 2004 #674" Hoeper, Faulenbach et al. 2004;  HYPERLINK \l "_ENREF_228" \o "Simonneau, 2008 #119" Simonneau, Rubin et al. 2008). Efficacy and safety of tadalafil was studied in The PHIRST (Pulmonary Arterial Hypertension and ReSponse to Tadalafil) trial double blind, placebo-controlled study followed by a long-term extension phase  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_69" \o "Galie, 2009 #118" Galie, Brundage et al. 2009). In this 16-week study, 405 PAH patients either treatment-nave or receiving bosentan, were randomly assigned to receive placebo or tadalafil (2.5, 10, 20 or 40 mg once daily). Tadalafil increased the 6MWD in a dose-dependent manner but only the highest dose (40 mg) reached a level of significance as well in time to clinical worsening and quality of life  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_69" \o "Galie, 2009 #118" Galie, Brundage et al. 2009). In clinical practice, tadalafil is given at 40 mg (2x20mg) once a day. All of these current therapeutic options mainly act at restoring an imbalance in vascular tone by prostanoid replacement, ET-1 antagonism or PDE-5 inhibition. However, it is now well recognized that all these therapeutic agents exhibit also antiproliferative  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_262" \o "Wharton, 2000 #120" Wharton, Davie et al. 2000;  HYPERLINK \l "_ENREF_28" \o "Clapp, 2002 #121" Clapp, Finney et al. 2002;  HYPERLINK \l "_ENREF_242" \o "Tantini, 2005 #9" Tantini, Manes et al. 2005;  HYPERLINK \l "_ENREF_263" \o "Wharton, 2005 #5" Wharton, Strange et al. 2005) and immunomodulatory effects  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_195" \o "Raychaudhuri, 2002 #128" Raychaudhuri, Malur et al. 2002;  HYPERLINK \l "_ENREF_247" \o "Toward, 2004 #123" Toward, Smith et al. 2004;  HYPERLINK \l "_ENREF_113" \o "Jaffar, 2007 #126" Jaffar, Ferrini et al. 2007;  HYPERLINK \l "_ENREF_274" \o "Zhou, 2007 #127" Zhou, Hashimoto et al. 2007;  HYPERLINK \l "_ENREF_121" \o "Karavolias, 2010 #1003" Karavolias, Georgiadou et al. 2010), and that they act against oxidative stress  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_55" \o "Ferrari, 1989 #133" Ferrari, Cargnoni et al. 1989;  HYPERLINK \l "_ENREF_224" \o "Shinmura, 2005 #129" Shinmura, Tamaki et al. 2005;  HYPERLINK \l "_ENREF_49" \o "Elgebaly, 2007 #131" Elgebaly, Portik-Dobos et al. 2007;  HYPERLINK \l "_ENREF_95" \o "Gur, 2008 #130" Gur, Sikka et al. 2008;  HYPERLINK \l "_ENREF_225" \o "Shukla, 2009 #132" Shukla, Rossoni et al. 2009). Unfortunately, at this time there is no cure for PAH. Mastering of chemistry leads to the development of new promising drugs in term of kinetic and side effects, increasing our chances of having a new, better-tolerated and more powerful therapeutic tools. In addition, our improved understanding of additional pathways in this condition will presumably lead to the identification of novel molecular targets and the development of novel therapeutic strategies in the near future. 4.3 Optimization of route administration and pharmacokinetic improvements 4.3.1 Novel formulation of epoprostenol A novel formulation of epoprostenol with a greater chemical stability is currently under investigation (EPITOME-1). It is reconstituted with sterile water for injection or sodium chloride 0.9% injection  ADDIN EN.CITE Lambert2012716(Lambert, Bandilla et al. 2012)71671617Lambert, O.Bandilla, D.Iyer, R.Witchey-Lakshmanan, L.Palepu, N.Actelion Pharmaceuticals Ltd, Allschwil, Switzerland. olivier.lambert@actelion.comStability and microbiological properties of a new formulation of epoprostenol sodium when reconstituted and dilutedDrug Des Devel TherDrug design, development and therapyDrug Des Devel TherDrug design, development and therapyDrug Des Devel TherDrug design, development and therapy61-7062012/05/09Chemistry, PharmaceuticalDrug StabilityDrug StorageEpoprostenol/*chemistry/*pharmacologyHemolysis/drug effectsHydrogen-Ion ConcentrationPreservatives, Pharmaceutical/*pharmacologyTemperature20121177-8881 (Electronic) 1177-8881 (Linking)22563237Research Support, Non-U.S. Gov'thttp://www.ncbi.nlm.nih.gov/pubmed/22563237334010710.2147/DDDT.S29916eng( HYPERLINK \l "_ENREF_131" \o "Lambert, 2012 #716" Lambert, Bandilla et al. 2012). The reconstituted solution has a pH of > 11. This new formulation is stable for 24 h at room temperature when diluted at concentrations of 15,000 ng/ml compared to 12 h for the first formulation, reducing the frequency of use of the drug delivery pump system  ADDIN EN.CITE Witchey-Lakshmanan L2010146(Witchey-Lakshmanan L, Palepu N et al. 2010)14614617Witchey-Lakshmanan L, Palepu N,Teja BB.A novel formulation of epoprostenol with antimicrobial properties, administered under ambient conditions, for treatment of patients with pulmonary arterial hypertension.AJRCCMAJRCCM181A33642010( HYPERLINK \l "_ENREF_267" \o "Witchey-Lakshmanan L, 2010 #146" Witchey-Lakshmanan L, Palepu N et al. 2010). If this new formulation has the same efficacy in PAH to the previous epoprostenol, the quality of life of patients would be likely to improve. In United States, the FDA has approved the novel formulation of epoprostenol Veletri (Actelion Pharmaceuticals Ltd, Allschwil, Switzerland) in 2008. 4.3.2 Oral PGI2 receptor (IPr) agonists: selexipag Clinical efficacy and tolerability of current PGI2 analogs may be compromised by concomitant activation of other prostanoid receptors. Therefore, the selectivity of PGI2 replacement therapies for the IPr has emerged as an important consideration in the management of PAH. For example, vasorelaxant effect of iloprost and beraprost is attenuated in pulmonary artery from rats with monocrotaline (MCT)-induced pulmonary hypertension (Kuwano et al, 2008) via a mechanism that involves co-activation of the contractile EP3 receptor. A promising therapeutic approach under investigation is the use of a non-prostanoid agonist to directly activate the IPr. Selexipag is a first-in-class orally active prodrug metabolized to the highly selective IPr agonist ACT-333679 (previously known as MRE-269), which has a half-life of over 6 h  ADDIN EN.CITE Asaki2007727(Asaki, Hamamoto et al. 2007)72772717Asaki, T.Hamamoto, T.Sugiyama, Y.Kuwano, K.Kuwabara, K.Discovery Research Laboratories, Nippon Shinyaku Co Ltd, Kisshoin, Kyoto, Japan. t.asaki@po.nippon-shinyaku.co.jpStructure-activity studies on diphenylpyrazine derivatives: a novel class of prostacyclin receptor agonistsBioorg Med ChemBioorganic & medicinal chemistryBioorg Med ChemBioorganic & medicinal chemistryBioorg Med ChemBioorganic & medicinal chemistry6692-70415212007/09/04HumansPyrazines/chemical synthesis/*chemistry/*pharmacologyReceptors, Epoprostenol/*agonistsStructure-Activity Relationship2007Nov 10968-0896 (Print) 0968-0896 (Linking)17764960http://www.ncbi.nlm.nih.gov/pubmed/1776496010.1016/j.bmc.2007.08.010eng( HYPERLINK \l "_ENREF_6" \o "Asaki, 2007 #727" Asaki, Hamamoto et al. 2007). With its high selectivity for the IPr over other prostanoid receptors (at least 130-fold selectivity), selexipag can be distinguished from beraprost or iloprost currently used in the management of PAH  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_128" \o "Kuwano, 2007 #135" Kuwano, Hashino et al. 2007). With no affinity for the prostaglandin E receptor 3 (EP3), selexipag exerts similar vasodilatory activity on both large and small pulmonary arterial branches  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_129" \o "Kuwano, 2008 #134" Kuwano, Hashino et al. 2008) and its relaxant efficacy is not modified under conditions associated with PAH, whereas relaxation to treprostinil may be limited in the presence of mediators of disease such as ET-1  ADDIN EN.CITE Morrison2012220(Morrison, Studer et al. 2012)22022017Morrison, K.Studer, R.Ernst, R.Haag, F.Kauser, K.Clozel, M.Actelion Pharmaceuticals Ltd.Differential effects of selexipag and prostacyclin analogs in rat pulmonary arteryJ Pharmacol Exp TherThe Journal of pharmacology and experimental therapeuticsJ Pharmacol Exp Ther2012/08/252012Aug 231521-0103 (Electronic) 0022-3565 (Linking)22918043http://www.ncbi.nlm.nih.gov/pubmed/2291804310.1124/jpet.112.197152Eng( HYPERLINK \l "_ENREF_168" \o "Morrison, 2012 #220" Morrison, Studer et al. 2012). Preclinical study results showed that twice-daily administration of selexipag attenuates right ventricular hypertrophy, improves pulmonary hemodynamics, and significantly increases survival in MCT-treated rats  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_129" \o "Kuwano, 2008 #134" Kuwano, Hashino et al. 2008). In a microdosing study using 100 g of selexipag in healthy, white, male volunteers, headache was the most commonly reported adverse event  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_128" \o "Kuwano, 2007 #135" Kuwano, Hashino et al. 2007). A 2010 report from a phaseII study, involving 43 patients with PAH, showed that treatment with selexipag for 17 weeks conferred significant improvements in PVR (-30.3% versus placebo) values compared with placebo  ADDIN EN.CITE Simonneau2012680(Simonneau, Torbicki et al. 2012)68068017Simonneau, G.Torbicki, A.Hoeper, M. M.Delcroix, M.Karlocai, K.Galie, N.Degano, B.Bonderman, D.Kurzyna, M.Efficace, M.Giorgino, R.Lang, I. M.Dept of Pneumology, Clamart, France.Selexipag, an oral, selective IP receptor agonist for the treatment of pulmonary arterial hypertensionEur Respir JThe European respiratory journal : official journal of the European Society for Clinical Respiratory PhysiologyEur Respir J2012/03/012012Feb 231399-3003 (Electronic) 0903-1936 (Linking)22362844http://www.ncbi.nlm.nih.gov/pubmed/2236284410.1183/09031936.00137511Eng( HYPERLINK \l "_ENREF_229" \o "Simonneau, 2012 #680" Simonneau, Torbicki et al. 2012). A numerical improvement in 6MWD was also observed (+24.7 m and +0.4 m in the selexipag and placebo groups, respectively). Treatment with selexipag was well tolerated by most patients in this study. Adverse events were consistent with the known side effect profile of IPr agonism such as headache, pain in extremity, pain in jaw, nausea, and diarrhea. These side effects decreased over time in patients treated with selexipag  ADDIN EN.CITE Simonneau2012680(Simonneau, Torbicki et al. 2012)68068017Simonneau, G.Torbicki, A.Hoeper, M. M.Delcroix, M.Karlocai, K.Galie, N.Degano, B.Bonderman, D.Kurzyna, M.Efficace, M.Giorgino, R.Lang, I. M.Dept of Pneumology, Clamart, France.Selexipag, an oral, selective IP receptor agonist for the treatment of pulmonary arterial hypertensionEur Respir JThe European respiratory journal : official journal of the European Society for Clinical Respiratory PhysiologyEur Respir J2012/03/012012Feb 231399-3003 (Electronic) 0903-1936 (Linking)22362844http://www.ncbi.nlm.nih.gov/pubmed/2236284410.1183/09031936.00137511Eng( HYPERLINK \l "_ENREF_229" \o "Simonneau, 2012 #680" Simonneau, Torbicki et al. 2012). It is likely that they were related to the rapid up-titration to maximal tolerated dosage, which was relatively aggressive. Gastric side-effects including cramping, nausea, vomiting, disruption of gastric contractility and development of emesis could be related to activation of gastric smooth muscle via stimulation of EP3 receptors  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_167" \o "Morrison, 2010 #308" Morrison, Ernst et al. 2010)  ADDIN EN.CITE Pal2007688(Pal, Brasseur et al. 2007)68868817Pal, A.Brasseur, J. G.Abrahamsson, B.Department of Mechanical and Bio Engineering, The Pennsylvania State University, 205 Reber Building, University Park, PA 16802, USA.A stomach road or "Magenstrasse" for gastric emptyingJ BiomechJournal of biomechanicsJ BiomechJournal of biomechanicsJ BiomechJournal of biomechanics1202-104062006/08/29Biological Clocks/physiologyComputer SimulationGastric Emptying/*physiologyHumans*Models, BiologicalMuscle, Smooth/*physiologyPeristalsis/*physiologyStomach/*physiology20070021-9290 (Print) 0021-9290 (Linking)16934271Research Support, Non-U.S. Gov'thttp://www.ncbi.nlm.nih.gov/pubmed/1693427110.1016/j.jbiomech.2006.06.006eng( HYPERLINK \l "_ENREF_187" \o "Pal, 2007 #688" Pal, Brasseur et al. 2007)  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_119" \o "Kan, 2002 #692" Kan, Jones et al. 2002;  HYPERLINK \l "_ENREF_57" \o "Forrest, 2009 #689" Forrest, Hennig et al. 2009). In the rat stomach, selexipag does not evoke gastric contraction, nor disrupts gastric function  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_167" \o "Morrison, 2010 #308" Morrison, Ernst et al. 2010). A role for EP3 receptors has also been invoked in the development of peripheral pain reported in patients receiving treatment with PGI2 analogs (Minami et al, 2001; Southall and Vasko, 2001). Thus, the development of a selective IPr agonist that is devoid of off-target effects may provide improved efficacy and tolerability in patients with PAH. A phaseIII randomized trial GRIPHON  ADDIN EN.CITE NIH2011136(NIH 2011)13613646NIHClinicalTrials.gov. ACT��������������������������������������������������������������������������������� 2�9�3�9�8�7� �i�n� �p�u�l�m�o�n�a�r�y� �a�r�t�e�r�i�a�l� �h�y�p�e�r�t�e�n�s�i�o�n� �[�o�n�l�i�n�e�]�,�<�/�t�i�t�l�e�>�<�/�t�i�t�l�e�s�>�<�d�a�t�e�s�>�<�y�e�a�r�>�2�0�1�1�<�/�y�e�a�r�>�<�/�d�a�t�e�s�>�<�u�r�l�s�>�<�r�e�l�a�t�e�d�-�u�r�l�s�>�<�u�r�l�>�h�t�t�p�:�/�/�c�l�i�n�i�c�a�l�t�r�i�a�l�s�.�g�o�v�/�c�t�2�/�s�h�o�w�/�N�C�T�0�1�1�0�6�0�1�4�?�t�e�r�m�=�G�R�I�P�H�O�N�&�a�m�p�;�r�a�n�k�=�1�<�/�u�r�l�>�<�/�r�e�l�a�t�e�d�-�u�r�l�s�>�<�/�u�r�l�s�>�<�/�r�e�c�o�r�d�>�<�/�C�i�t�e�>�<�/�E�n�d�N�o�t�e�>��(�� �H�Y�PERLINK \l "_ENREF_177" \o "NIH, 2011 #136" NIH 2011) to examine the effect of selexipag on morbidity and mortality in PAHis underway and will afford more information regarding efficacy and safety of selexipag. If positive results are obtained in this clinical trial, an orally active PGI2 analog will be available for the first time in Europe and in US. 4.3.3 Tissue targeting ET-1 receptors: macitentan Macitentan, also called ACT-064992 [N-[5-(4-bromophenyl)- 6-(2-(5-bromopyrimidin-2-yloxy)ethoxy)-pyrim�i�d�i�n�-�4�-�y�l�]�-�N�2 -� �p�r�o�p�y�l�a�m�i�n�o�s�u�l�f�a�m�i�d�e�]�,� �i�s� �a� �n�o�v�e�l�,� �h�i�g�h�l�y� �p�o�t�e�n�t�,� �t�i�s�s�u�e�-�t�a�r�g�e�t�i�n�g� �b�o�t�h� �E�T�-�1� �r�e�c�e�p�t�o�r�s� �a�n�d� �c�h�a�r�a�c�t�e�r�i�z�e�d� �b�y� �h�i�g�h� �l�i�p�o�p�h�i�l�i�c�i�t�y� �� �A�D�D�I�N� �E�N�.�C�I�T�E� �� �A�D�D�I�N� �E�N�.�C�I�T�E�.�D�A�T�A� �����(�� �H�Y�P�E�R�L�I�N�K� �\�l� �"�_�E�N�R�E�F�_�1�1�0�"� �\�o� �"�I�g�l�a�r�z�,� �2�0�0�8� �#�1�3�7�"� ���I�g�l�a�r�z�,� �B�i�n�k�e�r�t� �e�t� al. 2008). Twenty-six-weeks of multiple oral dosing (10 mg/Kg/day) of macitentan in rats leads to a maximal plasma concentration to 1.5 g/L and a four to five higher level of its major and pharmacology active metabolite ACT-132577. ACT-132577 is formed by oxidative depropylation of macitentan and has a longer half-life than the parent compound (8.4 h versus 2h respectively)  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_110" \o "Iglarz, 2008 #137" Iglarz, Binkert et al. 2008). By remaining active in local tissue environments, macitentan and its metabolite have the advantage of extended functional in vivo half-life targeting ET-1 receptors in tissues. Moreover, macitentan increased binding to receptors than existing ERAs indicating a more potent pharmacological activity in vivo  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_110" \o "Iglarz, 2008 #137" Iglarz, Binkert et al. 2008;  HYPERLINK \l "_ENREF_127" \o "Kummer, 2009 #696" Kummer, Haschke et al. 2009). In rats with pulmonary hypertension, macitentan prevented both the increase of pulmonary pressure and the right ventricular hypertrophy, and it markedly improved survival, without effect on systemic arterial blood pressure (Iglarz 2008). Moreover, macitentan does not inhibit canalicular bile acid transport in rats, which could lead to better liver safety profile than bosentan (Bolly et al., J Med Chem 2012). In a phase II clinical trial, macitentan was slowly absorbed and, at a dose of 300 mg, the t was about 17.5 h  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_226" \o "Sidharta, 2011 #1004" Sidharta, van Giersbergen et al. 2011). Formation of the ACT-132577 active metabolite is slow with maximum plasma concentrations attained at least 30 h after dosing and a t around 65.6 h, in accordance with a once-a-day dosing regimen. Macitentan is well tolerated: Headache, nausea and vomiting were the dose-limiting adverse events  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_226" \o "Sidharta, 2011 #1004" Sidharta, van Giersbergen et al. 2011). During the time of the study, administration of macitentan had no effect on serum total bile salt concentrations, providing some support that, in contrast to other ERAs, macitentan may be devoid of liver toxic effects  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_226" \o "Sidharta, 2011 #1004" Sidharta, van Giersbergen et al. 2011). However, as elevations in liver aminotransferases induced by ERAs are typically seen after repeated dosing and sometimes only after months of treatment  ADDIN EN.CITE <EndNote><Cite><Author>Humbert</Author><Year>2007</Year><RecNum>105</RecNum><DisplayText>(Humbert, Segal et al. 2007)</DisplayText><record><rec-number>105</rec-number><foreign-keys><key app="EN" db-id="tvpz225sued5v9edsfp59fpgdw5xxfrdfrp9">105</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Humbert, M.</author><author>Segal, E. S.</author><author>Kiely, D. G.</author><author>Carlsen, J.</author><author>Schwierin, B.</author><author>Hoeper, M. M.</author></authors></contributors><auth-address>Dept of Respiratory Medicine, Antoine Beclere Hospital, Clamart, France. marc.humbert@abc.ap-hop-paris.fr</auth-address><titles><title>Results of European post-marketing surveillance of bosentan in pulmonary hypertensionEur Respir JThe European respiratory journal : official journal of the European Society for Clinical Respiratory PhysiologyEur Respir J338-443022007/05/17Adverse Drug Reaction Reporting SystemsAntihypertensive Agents/*adverse effectsEuropeHumansHypertension, Pulmonary/*drug therapyInternet*Product Surveillance, PostmarketingProspective StudiesSulfonamides/*adverse effects2007Aug0903-1936 (Print) 0903-1936 (Linking)17504794http://www.ncbi.nlm.nih.gov/pubmed/1750479410.1183/09031936.00138706eng( HYPERLINK \l "_ENREF_108" \o "Humbert, 2007 #105" Humbert, Segal et al. 2007), trials with longer duration of treatment are needed to truly demonstrate a lack of liver injury associated with macitentan which was found in this phase II study. In plasma, only macitentan and the pharmacologically active oxidative depropyl metabolite, ACT-132577, were found whereas in urine two minor metabolites were detected. Another study regarding the metabolic profile of macitentan in humans by radioactivity reported that in addition to macitentan and the ACT-132577 metabolite, a carboxylic acid metabolite ACT-373898 was also identified in plasma  ADDIN EN.CITE Bruderer20121012(Bruderer, Hopfgartner et al. 2012)1012101217Bruderer, S.Hopfgartner, G.Seiberling, M.Wank, J.Sidharta, P. N.Treiber, A.Dingemanse, J.Clinical Pharmacology, Allschwil, Switzerland. Shirin.bruderer@actelion.comAbsorption, distribution, metabolism, and excretion of macitentan, a dual endothelin receptor antagonist, in humansXenobioticaXenobiotica; the fate of foreign compounds in biological systemsXenobioticaXenobiotica; the fate of foreign compounds in biological systemsXenobioticaXenobiotica; the fate of foreign compounds in biological systems901-104292012/03/312012Sep1366-5928 (Electronic) 0049-8254 (Linking)22458347http://www.ncbi.nlm.nih.gov/pubmed/2245834710.3109/00498254.2012.664665eng( HYPERLINK \l "_ENREF_19" \o "Bruderer, 2012 #1012" Bruderer, Hopfgartner et al. 2012). Moreover, in urine, four entities were identified, with the hydrolysis product of ACT-373898 as the most abundant one. In faeces, five entities were identified, macitentan, ACT-132577, ACT-373898, its hydrolysis product (M 323u), and ACT-080803  ADDIN EN.CITE Bruderer20121012(Bruderer, Hopfgartner et al. 2012)1012101217Bruderer, S.Hopfgartner, G.Seiberling, M.Wank, J.Sidharta, P. N.Treiber, A.Dingemanse, J.Clinical Pharmacology, Allschwil, Switzerland. Shirin.bruderer@actelion.comAbsorption, distribution, metabolism, and excretion of macitentan, a dual endothelin receptor antagonist, in humansXenobioticaXenobiotica; the fate of foreign compounds in biological systemsXenobioticaXenobiotica; the fate of foreign compounds in biological systemsXenobioticaXenobiotica; the fate of foreign compounds in biological systems901-104292012/03/312012Sep1366-5928 (Electronic) 0049-8254 (Linking)22458347http://www.ncbi.nlm.nih.gov/pubmed/2245834710.3109/00498254.2012.664665eng( HYPERLINK \l "_ENREF_19" \o "Bruderer, 2012 #1012" Bruderer, Hopfgartner et al. 2012). The urinary excretion is the most important route of elimination of drug-related material than faeces in humans. Concomitant treatment with rifampin, a strong inducer of CYP3A4, reduced significantly the exposure to macitentan and its metabolite ACT-373898 at steady-state but did not affect the exposure to the active metabolite ACT-132577 to a clinically relevant extent  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_18" \o "Bruderer, 2012 #1013" Bruderer, Aanismaa et al. 2012). In the phase III placebo-controlled SERAPHIN trial  ADDIN EN.CITE NIH2010139(NIH 2010)13913946NIHClinicalTrials.gov. Study of ACT����������������������������������������������������������������������������������������������������������������������������� 0�6�4�9�9�2� �o�n� �m�o�r�b�i�d�i�t�y� �a�n�d� �m�o�r�t�a�l�i�t�y� �i�n� �p�a�t�i�e�n�t�s� �w�i�t�h� �s�y�m�p�t�o�m�a�t�i�c� �p�u�l�m�o�n�a�r�y� �a�r�t�e�r�i�a�l� �h�y�p�e�r�t�e�n�s�i�o�n�<�/�t�i�t�l�e�>�<�/�t�i�t�l�e�s�>�<�d�a�t�e�s�>�<�y�e�a�r�>�2�0�1�0�<�/�y�e�a�r�>�<�/�d�a�t�e�s�>�<�u�r�l�s�>�<�r�e�l�a�t�e�d�-�u�r�l�s�>�<�u�r�l�>�h�t�t�p�:�/�/�c�l�i�n�i�c�a�l�t�r�i�a�l�s�.�g�o�v�/�c�t�2�/�s�h�o�w�/�<�/�u�r�l�>�<�u�r�l�>�N�C�T�0�0�6�6�0�1�7�9�?�t�e�r�m�=�S�E�R�A�P�H�I�N�&�a�m�p�;�r�a�n�k�=�2�<�/�u�r�l�></related-urls></urls></record></Cite></EndNote>( HYPERLINK \l "_ENREF_175" \o "NIH, 2010 #139" NIH 2010), the efficacy and safety of macitentan will be tested in approximately 750 patients with PAH. The primary objective in this study is to establish whether treatment with macitentan prolongs time to the first morbidity or mortality event. Results are expected in the end of 2012. 4.3.4 Soluble guanylate cyclase stimulator: riociguat Endothelium-derived NO regulates vascular homeostasis through PASMC relaxation via the activation of the second messenger cGMP. The clinical benefits associated with the PDE5 inhibitor class has led to interest in testing whether other agents that modulate NO signaling might be similarly beneficial in PAH. Riociguat is a first-in-class drug that augments cGMP biosynthesis through direct stimulation of the enzyme soluble guanylate cyclase (sGC) promoting vasodilatation by direct stimulation of sGC in an NOindependent fashion, and by sensitization of sGC to low endogenous NO levels  ADDIN EN.CITE <EndNote><Cite><Author>Ghofrani</Author><Year>2009</Year><RecNum>141</RecNum><DisplayText>(Ghofrani and Grimminger 2009)</DisplayText><record><rec-number>141</rec-number><foreign-keys><key app="EN" db-id="tvpz225sued5v9edsfp59fpgdw5xxfrdfrp9">141</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Ghofrani, H. A.</author><author>Grimminger, F.</author></authors></contributors><auth-address>Dept of Internal Medicine, University Hospital Giessen and Marburg, Germany. ardeschir.ghofrani@innere.med.uni-giessen.de</auth-address><titles><title>Soluble guanylate cyclase stimulation: an emerging option in pulmonary hypertension therapyEur Respir RevEuropean respiratory review : an official journal of the European Respiratory SocietyEur Respir Rev35-41181112009/03/01Enzyme ActivationGuanylate Cyclase/*metabolismHumansHypertension, Pulmonary/*drug therapyPyrazoles/*therapeutic usePyrimidines/*therapeutic useVasodilator Agents/*therapeutic use2009Mar1600-0617 (Electronic) 0905-9180 (Linking)20956121Research Support, Non-U.S. Gov't Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/2095612110.1183/09059180.00011112eng( HYPERLINK \l "_ENREF_79" \o "Ghofrani, 2009 #141" Ghofrani and Grimminger 2009). Since the therapeutic effect of PDE5 inhibitors is dependent on baseline NO expression (levels of which are typically reduced in PAH)  ADDIN EN.CITE Evgenov2006142(Evgenov, Pacher et al. 2006)14214217Evgenov, O. V.Pacher, P.Schmidt, P. M.Hasko, G.Schmidt, H. H.Stasch, J. P.Department of Anesthesia and Critical Care, Massachusetts General Hospital, Harvard Medical School, 55 Fruit Street, CLN 309, Boston, Massachusetts 02114, USA.NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potentialNat Rev Drug DiscovNature reviews. Drug discoveryNat Rev Drug Discov755-68592006/09/07AnimalsDrug DesignEnzyme Activation/drug effectsEnzyme Activators/chemistry/*pharmacology/therapeutic useGuanylate Cyclase/*metabolismHumansMolecular StructureNitric Oxide/*metabolism2006Sep1474-1776 (Print) 1474-1776 (Linking)16955067Research Support, N.I.H., Extramural Research Support, N.I.H., Intramural Research Support, Non-U.S. Gov't Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/16955067222547710.1038/nrd2038eng( HYPERLINK \l "_ENREF_50" \o "Evgenov, 2006 #142" Evgenov, Pacher et al. 2006), treatments that act directly on sGC could potentially have a greater efficacy than PDE5inhibitors. sGC forms  HYPERLINK "http://en.wikipedia.org/wiki/Heterodimers" \o "Heterodimers" heterodimers consisting of a larger alpha-subunit and a smaller hem-binding beta-subunit  ADDIN EN.CITE Zabel1999701(Zabel, Hausler et al. 1999)70170117Zabel, U.Hausler, C.Weeger, M.Schmidt, H. H.Department of Pharmacology and Toxicology, Julius-Maximilians-University, D-97078 Wuerzburg, Germany.Homodimerization of soluble guanylyl cyclase subunits. Dimerization analysis using a glutathione s-transferase affinity tagJ Biol ChemThe Journal of biological chemistryJ Biol Chem18149-52274261999/06/22Affinity Labels/*metabolismAnimalsCells, CulturedChromatography, GelDimerizationElectrophoresis, Polyacrylamide GelGlutathione Transferase/*metabolismGuanylate Cyclase/*metabolismHumansProtein ConformationSolubilitySpodoptera1999Jun 250021-9258 (Print) 0021-9258 (Linking)10373411Research Support, Non-U.S. Gov'thttp://www.ncbi.nlm.nih.gov/pubmed/10373411eng( HYPERLINK \l "_ENREF_271" \o "Zabel, 1999 #701" Zabel, Hausler et al. 1999). Binding of NO to the heme group leads to an approximately 200-fold increase in the conversion of GTP to cGMP  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_63" \o "Friebe, 2003 #704" Friebe and Koesling 2003). First sGC activators such as YC-1 or BAY 41-2272 require the presence of a reduced hem group in order to activate the enzyme and can be blocked by drugs oxidizing the hem-group as the sGC inhibitor ODQ  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_233" \o "Stasch, 2001 #706" Stasch, Becker et al. 2001). Then, a new class of NO-independent sGC-activators has been discovered, notably with the BAY 58-2667, also called cinaciguat (Stasch et al., 2002). Cinaciguat can activate the enzyme without requiring the presence of a reduced hem group and provides synergistic effects when combined with NO donors (evgenov 2006). Due to its effects in lowering systemic blood pressure, cinaciguat have led its development as treatment for acute decompensated heart failure. Further research led to riociguat (BAY 63-2521), which is structurally similar to BAY 41-2272, with improved pharmacokinetic and pharmacological properties  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_161" \o "Mittendorf, 2009 #707" Mittendorf, Weigand et al. 2009). Riociguat directly stimulates sGC activity independent of NO and also acts in synergy with NO to produce anti-aggregatory, anti-proliferative, and vasodilatory effects. An initial study found that oral riociguat attenuated acute hypoxic pulmonary vasoconstriction in mice in a dose-dependent fashion and improved established MCT-induced pulmonary hypertension in rats  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_218" \o "Schermuly, 2008 #143" Schermuly, Stasch et al. 2008), with a greater effect than sildenafil in PH induced by hypoxia and SU5416 in rats  ADDIN EN.CITE Lang2012715(Lang, Kojonazarov et al. 2012)71571517Lang, M.Kojonazarov, B.Tian, X.Kalymbetov, A.Weissmann, N.Grimminger, F.Kretschmer, A.Stasch, J. P.Seeger, W.Ghofrani, H. A.Schermuly, R. T.University of Giessen and Marburg Lung Center, Giessen, Germany.The Soluble Guanylate Cyclase Stimulator Riociguat Ameliorates Pulmonary Hypertension Induced by Hypoxia and SU5416 in RatsPLoS OnePloS onePLoS OnePloS onePLoS OnePloS onee43433782012/08/2320121932-6203 (Electronic) 1932-6203 (Linking)22912874http://www.ncbi.nlm.nih.gov/pubmed/22912874342230610.1371/journal.pone.0043433eng( HYPERLINK \l "_ENREF_134" \o "Lang, 2012 #715" Lang, Kojonazarov et al. 2012). A phase I randomized placebo-controlled study in 58 healthy male subjects were given riociguat orally was designed to test the safety profile,  HYPERLINK "http://en.wikipedia.org/wiki/Pharmacokinetics" \o "Pharmacokinetics" pharmacokinetics and  HYPERLINK "http://en.wikipedia.org/wiki/Pharmacodynamics" \o "Pharmacodynamics" pharmacodynamics of single oral doses of riociguat (0.255mg). Doses of riociguat were increased stepwise, and riociguat was well tolerated up to 2.5mg  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_62" \o "Frey, 2008 #698" Frey, Muck et al. 2008). Plasma concentrations reached maximal levels 0.5 - 1.5 h after administration and declined thereafter with a terminal half-life of 5 - 10 h  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_61" \o "Frey, 2008 #697" Frey, Muck et al. 2008). A proof-of-concept study was conducted to investigate oral riociguat in patients with moderate to severe PH in a two-part, non-randomized, open-label, single center trial  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_89" \o "Grimminger, 2009 #709" Grimminger, Weimann et al. 2009). Riociguat was well tolerated in doses up to 2.5 mg, whereas 5.0 mg caused asymptomatic hypotension in one patient. Therefore the 2.5 mg dose (n = 10) was used in the second part of the trial to demonstrate efficacy. Riociguat significantly reduced mPAP, PVR and systemic vascular resistance and increased cardiac index CI  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_89" \o "Grimminger, 2009 #709" Grimminger, Weimann et al. 2009). Systolic blood pressure was significantly reduced but this effect was not associated with systemic hypotension. Results from a multicenter, open-label, uncontrolled phaseII trial involving 75 patients with PAH (n = 33) and chronic thromboembolic pulmonary hypertension (n = 42) showed that 12weeks of oral riociguat given 3 times daily conferred improvements in symptoms, NYHA functional class, exercise capacity, NTproBNP level, and pulmonary hemodynamics  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_80" \o "Ghofrani, 2010 #144" Ghofrani, Hoeper et al.). Treatment was well tolerated overall up to the highest planned 2.5 mg dose. A decrease in systemic arterial diastolic pressure was the only significant side effect reported, with none associated with symptoms leading to a permanent discontinuation of riociguat  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_80" \o "Ghofrani, 2010 #144" Ghofrani, Hoeper et al. 2010). Riociguat is also under investigation in other form of PH as PH associated with chronic obstructive pulmonary disease, with interstitial lung disease or with left ventricular dysfunction  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_216" \o "Schermuly, 2011 #711" Schermuly, Janssen et al. 2011;  HYPERLINK \l "_ENREF_78" \o "Ghio, 2012 #713" Ghio, Bonderman et al. 2012;  HYPERLINK \l "_ENREF_102" \o "Hoeper, 2012 #712" Hoeper, Halank et al. 2012). An ongoing multicentre randomized, double-blind placebo-controlled phaseIII study of riociguat (PATENT1)  ADDIN EN.CITE NIH2011145(NIH 2011)14514546NIHClinicalTrials.gov. A study to evaluate efficacy and safety of oral BAY632521 in patients with pulmonary arterial hypertension (PAH)2011http://clinicaltrials.gov/ct2/show/NCT00810693?term=BAY632521&rank=1( HYPERLINK \l "_ENREF_176" \o "NIH, 2011 #145" NIH 2011) assessed the effects of riociguat on 462patients with PAH. The primary endpoint is 6MWD; secondary endpoints include changes in PVR, NT-pro-BNP levels, WHO FC, Borg dyspnoea score, time to clinical worsening and quality of life. Results from PATENT-1will be available at the end of 2012. PATENT-2 (NCT00863681) is a non-randomized, open-label, multicenter, long-term extension study involving patients who complete PATENT-1. Another Phase III clinical study, CHEST-1 (NCT00855465), is a multicenter randomized, double blind placebo involves patients with CTEPH that is either inoperable or with persistent or recurrent PH after surgery for CTEPH. The primary and secondary endpoints are similar to those of PATENT-1. As PATENT-2, CHEST-2 (NCT00910429) is a nonrandomized, open-label, multicenter, long-term extension study involving patients who complete CHEST-1. Results from PATENT-1 and CHEST-1 are expected for 2013. 5. NEW THERAPEUTIC TARGETS 5.1 The tyrosine kinase inhibitors 5.1.1. Efficacy of kinase inhibitors in experimental models of pulmonary hypertension Imatinib It has been demonstrated that imatinib could reverse PAH in animal models (monocrotaline-induced or hypoxic PH)  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_196" \o "Schermuly, 2005 #59" Schermuly, Dony et al. 2005). These authors demonstrated that the development of PAH in rats exposed to monocrotaline is associated with an increase in PDGF receptor expression, and particularly in its activated (phosphorylated) form. In animal model, imatinib was able to restore a normal expression of this receptor. Moreover, a 2-week treatment by imatinib of rats exposed to monocrotaline corrected hemodynamic characteristics, reversed vascular remodelling and significantly improved survival  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_196" \o "Schermuly, 2005 #59" Schermuly, Dony et al. 2005). Sorafenib Sorafenib, a multikinase inhibitor (MKI) targeting c-kit, VEGFR, Raf, has been evaluated in several animal models of pulmonary hypertension  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_114" \o "Klein, 2008 #147" Klein, Schermuly et al. 2008;  HYPERLINK \l "_ENREF_153" \o "Moreno-Vinasco, 2008 #148" Moreno-Vinasco, Gomberg-Maitland et al. 2008). Klein et al demonstrated that sorafenib could reverse vascular remodelling of rats with monocrotaline-induced PH  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_114" \o "Klein, 2008 #147" Klein, Schermuly et al. 2008). Moreover, Klein et al. showed that this improvement was associated with a decrease of proliferation and an increase of apoptosis in PAH lesions. These results were confirmed in two other animal models (a conventional hypoxia-induced PH model and a model of severe PH combining dual VEGFR-1 and -2 inhibition and hypoxia). In these models, sorafenib prevents pulmonary vascular remodelling and the onset of PAH in these animal models. Nilotinib As imatinib, nilotinib inhibits PDGFR, c-kit and c-Abl, with a stronger effect on c-Abl than imatinib. Nilotinib has been approved for the treatment of patients with chronic myeloid leukemia in the context of imatinib resistance or intolerance. Preliminary studies have reported a benefit effect with nilotinib in experimental pulmonary hypertension. Nilotinib is under investigation in human PAH with a phase II clinical trial (ClinicalTrials.gov Identifier NCT01179737). 5.1.2 Evaluation of kinase inhibitors in human PAH Imatinib After highlighting the importance of the PDGF pathway in the pathophysiology of PAH and the possibility to reverse PAH in experimental models, several isolated cases of clinical, functional and hemodynamic have been reported  ADDIN EN.CITE Ghofrani2005149(Ghofrani, Seeger et al. 2005)14914917Ghofrani, H. A.Seeger, W.Grimminger, F.Imatinib for the treatment of pulmonary arterial hypertensionN Engl J MedThe New England journal of medicineN Engl J Med1412-3353132005/09/30Exercise Tolerance/drug effectsHumansHypertension, Pulmonary/*drug therapy/physiopathologyIntracellular Signaling Peptides and Proteins/therapeutic useMaleMiddle AgedPiperazines/*therapeutic usePyrimidines/*therapeutic useReceptors, Platelet-Derived Growth Factor/*antagonists & inhibitorsVascular Resistance/drug effects2005Sep 291533-4406 (Electronic) 0028-4793 (Linking)16192491Case Reportshttp://www.ncbi.nlm.nih.gov/pubmed/1619249110.1056/NEJMc051946eng( HYPERLINK \l "_ENREF_72" \o "Ghofrani, 2005 #149" Ghofrani, Seeger et al. 2005) ADDIN EN.CITE Souza2006150(Souza, Sitbon et al. 2006)15015017Souza, R.Sitbon, O.Parent, F.Simonneau, G.Humbert, M.Long term imatinib treatment in pulmonary arterial hypertensionThoraxThoraxThoraxThorax7366182006/08/01Antihypertensive Agents/*therapeutic useHumansHypertension, Pulmonary/*drug therapyPiperazines/*therapeutic usePyrimidines/*therapeutic use2006Aug0040-6376 (Print) 0040-6376 (Linking)16877696Letter Research Support, Non-U.S. Gov'thttp://www.ncbi.nlm.nih.gov/pubmed/16877696210467910.1136/thx.2006.064097eng( HYPERLINK \l "_ENREF_214" \o "Souza, 2006 #150" Souza, Sitbon et al. 2006). Following these encouraging results, a randomized (imatinib vs placebo), double-blind, 24 weeks Phase II study was realized in 59 PAH patients in NYHA functional class II to IV receiving specific PAH therapies  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_71" \o "Ghofrani, 2010 #151" Ghofrani, Morrell et al.). Change from baseline in the 6MWD after 24 weeks, was the primary endpoint and was not different between the 2 groups (+22m in imatinib group as compared to placebo, p=0.21)  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_71" \o "Ghofrani, 2010 #151" Ghofrani, Morrell et al.). Only secondary endpoints, including cardiac output and pulmonary vascular resistance (PVR), were significantly improved. Post-hoc subgroup analyses suggested a greater improvement in patients with severe hemodynamic impairment. Serious adverse events occurred in 11 imatinib recipients (39%) and 7 placebo recipients (23%). Three deaths occurred in each group. This preliminary study does not allow concluding on the potential benefits of imatinib in PAH, but led to development of a clinical trial phase III (IMPRES) evaluating imatinib in a randomized and double-blind trial of 24-week, in severe PAH patients (PVR> 800 dynes.sec.cm-5) treated with at least two PAH specific treatments. After 24 weeks, it was observed a significantly improvement of the primary endpoint which was the 6MWD (+32m in imatinib group vs placebo) and an improvement of secondary endpoints such as hemodynamic characteristics (PVR and cardiac output). However, it was no observed improvement in NYHA functional class or in time before clinical deterioration. This 24-week randomized trial was followed by a 3-year open-label extension phase including 143 patients, all of them receiving the imatinib treatment. An interim analysis of safety was recently reported (Hoeper, oral presentation, ATS 2012). In this study, 112 patients developed adverse events, mainly nausea (30.8%), vomiting (18.2%), peripheral edema (16.8%), ENT symptoms (15.4%), periorbital edema (15.4%), headache (14.7%) and diarrhea (12.6%). Moreover, 2 cases of subdural hematoma (SDH) were reported in the double-blind period of 6 months (both in the group treated with imatinib) and 7 supplemental cases of SDH were observed in the open-label trial, including one death directly related to this complication. Estimation of the SDH incidence >0.01 SDH /patient-year in patients treated by imatinib. The precise mechanism of SDH in these patients is not elucidated and the high incidence of SDH observed in PAH may be partly favoured by anticoagulation recommended for PAH patients. Netherless, SDH seems to be a specific complication of imatinib and SDH have been reported in other indications, including oncology or pulmonary fibrosis. Indeed, this complication never reported at this level in previous clinical trial evaluated PAH therapies. Sorafenib Sorafenib (MKI) was evaluated in a 16-week, phase Ib, single-center, open-label trial in PAH patients receiving parenteral prostanoids in NYHA functional class I to III. The aim of this study was to evaluate the safety of sorafenib; eleven PAH patients were treated and only 2 patients reached the maximum dose of 400mg twice daily. The most common adverse events were moderate skin reactions on the hands and feet, alopecia and diarrhea (only one adverse event (skin reaction) led to discontinuation of sorafenib)  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_79" \o "Gomberg-Maitland, 2010 #152" Gomberg-Maitland, Maitland et al.). No clinical improvement (6MWD +23 m, p=0.33) were observed and hemodynamic evaluation arise concerns about safety of this drug in PAH patients with a decrease of cardiac output in all the eleven patients (see above)  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_79" \o "Gomberg-Maitland, 2010 #152" Gomberg-Maitland, Maitland et al.). 5.1.3 Cardiotoxicity of kinase inhibitors Although TKI and MKI represent potential innovative therapeuties in the setting of PAH, their use in clinical practice may raise safety concerns. Indeed, experimental data and human reports suggested that most of these molecules may induce cardiotoxicity. Although this risk seems rare in their common use (chronic myeloid leukemia, cancer), the existence of an underlying heart disease appears to be the main risk factor for development of cardiotoxicity. Thus, prevalence of cardiotoxicity of TKI and MKI could be higher when use in PAH patients because of the right heart failure classically associated with PAH. To date, the mechanism of this cardiotoxicity is not completely elucidated. Indeed, each molecule inhibits a large number of targets, and cardiotoxicity is probably not specific to a single target and may be shared by several MKI or TKI. Clinical trials evaluating imatinib as a treatment for chronic myeloid leukemia, have reported a high incidence of peripheral and pulmonary edema (52-68%) and 5 -16% of patients complain of dyspnea  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_27" \o "Cohen, 2002 #153" Cohen, Williams et al. 2002). In 2006, Kerkela et al. reported 10 patients displaying signs of cardiac dysfunction despite a normal cardiac evaluation prior to treatment with imatinib  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_112" \o "Kerkela, 2006 #154" Kerkela, Grazette et al. 2006). These patients presented a decrease in left ventricular ejection fraction (LVEF) associated with mild left ventricular dilation. Transmission electron micrographs of cardiac biopsies showed prominent membrane whorls in the myocytes, in cardiomyocyte mitochondria and in the sarcoplasmic reticulum, and glycogen accumulation. The authors demonstrated that mice treated with high-dose of imatinib may develop similar lesions in cardiomyocyte mitochondria and sarcoplasmic reticulum, associated with a decrease in LVEF  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_112" \o "Kerkela, 2006 #154" Kerkela, Grazette et al. 2006). In addition, the authors demonstrated that the main mechanism of cardiotoxicity of imatinib was associated with mitochondrial toxicity, leading to a decrease in mitochondrial membrane potential, a release of cytochrome c into the cytosol, activation of caspases 3 and 7, and cardiomyocytes apoptosis  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_112" \o "Kerkela, 2006 #154" Kerkela, Grazette et al. 2006). Interestingly, cardiomyocytes transfected with a gene encoding for a c-Abl receptor resistant to imatinib, were at least in part protected of toxicity induced by imatinib, suggesting a possible implication of this tyrosine kinase in the mechanism of imatinib induced cardiotoxicity  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_112" \o "Kerkela, 2006 #154" Kerkela, Grazette et al. 2006). Moreover, Atallah et al. conducted a meta-analysis of patients treated with imatinib in published clinical trials  ADDIN EN.CITE Atallah2007155(Atallah, Durand et al. 2007)15515517Atallah, E.Durand, J. B.Kantarjian, H.Cortes, J.Department of Leukemia, The University of Texas, M. D. Anderson Cancer Center, Houston, TX 77030, USA.Congestive heart failure is a rare event in patients receiving imatinib therapyBloodBloodBloodBlood1233-711042007/04/24AgedAged, 80 and overAntineoplastic Agents/*therapeutic useFemaleHeart Failure/*etiology/mortalityHematologic Neoplasms/complications/*drug therapyHumansIncidenceMaleMiddle AgedPiperazines/*therapeutic useProtein-Tyrosine Kinases/antagonists & inhibitorsPyrimidines/*therapeutic useSurvival Rate2007Aug 150006-4971 (Print) 0006-4971 (Linking)17449798http://www.ncbi.nlm.nih.gov/pubmed/1744979810.1182/blood-2007-01-070144eng( HYPERLINK \l "_ENREF_5" \o "Atallah, 2007 #155" Atallah, Durand et al. 2007). Among 1276 patients enrolled, 22 (1.7%) patients were identified as having symptoms that could be attributed to systolic heart failure, 8 out of them (0.6%) were considered possibly or probably related to imatinib  ADDIN EN.CITE Atallah2007155(Atallah, Durand et al. 2007)15515517Atallah, E.Durand, J. B.Kantarjian, H.Cortes, J.Department of Leukemia, The University of Texas, M. D. Anderson Cancer Center, Houston, TX 77030, USA.Congestive heart failure is a rare event in patients receiving imatinib therapyBloodBloodBloodBlood1233-711042007/04/24AgedAged, 80 and overAntineoplastic Agents/*therapeutic useFemaleHeart Failure/*etiology/mortalityHematologic Neoplasms/complications/*drug therapyHumansIncidenceMaleMiddle AgedPiperazines/*therapeutic useProtein-Tyrosine Kinases/antagonists & inhibitorsPyrimidines/*therapeutic useSurvival Rate2007Aug 150006-4971 (Print) 0006-4971 (Linking)17449798http://www.ncbi.nlm.nih.gov/pubmed/1744979810.1182/blood-2007-01-070144eng( HYPERLINK \l "_ENREF_5" \o "Atallah, 2007 #155" Atallah, Durand et al. 2007). The two factors that seemed to favour the occurrence of cardiotoxicity were age and preexisting cardiovascular disease: congestive heart failure, diabetes, coronary artery disease, hypertension, arrhythmia, cardiomyopathy. This observation underlines the potential risk of imatinib in patients with pre-existing cardiac failure, such as PAH. Sunitinib, a MKI used in metastatic renal-cell carcinoma and gastrointestinal stromal tumours, blocks several kinases, including PDGFR, VEGFR, c-kit, RET, FLT3 but not c-Abl. Chu et al assessed the cardiovascular risk associated with sunitinib in patients with metastatic gastrointestinal stromal tumours enrolled in a phase I/II trial  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_23" \o "Chu, 2007 #156" Chu, Rupnick et al. 2007). These authors showed that 11% of patients treated by sunitinib had cardiovascular events, mainly due to left heart failure (8%)  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_23" \o "Chu, 2007 #156" Chu, Rupnick et al. 2007). As demonstrated for imatinib, the main risk factor of developing cardiotoxicity was the presence of an underlying cardiac pathology  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_23" \o "Chu, 2007 #156" Chu, Rupnick et al. 2007). Echocardiography analysis of 36 patients receiving 4 weeks of sunitinib showed an absolute LVEF reduction >15% or more in seven (19%) patients  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_23" \o "Chu, 2007 #156" Chu, Rupnick et al. 2007). This LVEF decrease was proportional to the cumulative dose of sunitinib  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_23" \o "Chu, 2007 #156" Chu, Rupnick et al. 2007). Endomyocardial biopsies were obtained in two patients showing abnormalities evocative of mitochondrial toxicity. Indeed, these authors confirmed in animal model, that sunitinib may induce mitochondrial damage, release of cytochrome c and apoptosis of cardiomyocytes  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_23" \o "Chu, 2007 #156" Chu, Rupnick et al. 2007). Sorafenib, another MKI used in kidney cancer and hepatocellular carcinoma, share a broadly similar profile of targets with sunitinib (inhibition of PDGFR, c-kit, VEGFR, Raf, FLT3). In an open-label trial evaluated the safety of sorafenib in 12 PAH patients, no beneficial effect was observed of the treatment but a decrease in cardiac output in all patients after 16 weeks of treatment (-0,6 L/min, p<0,01) ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_79" \o "Gomberg-Maitland, 2010 #152" Gomberg-Maitland, Maitland et al.). Thus, while sorafenib has shown efficacy in experimental models of PAH, the benefit/risk ratio of this treatment contraindicated its use in human PAH. These observations support the hypothesis that the expected cardiotoxicity of TKI or MKI is higher in PAH patients than in the common population. Cardiotoxicity of TKI or MKI may be a limitation to the development of these drugs in the management of PAH. 5.1.4 PAH induced by dasatinib Dasatinib is a dual Src/Abl kinase inhibitor displaying a greater inhibition of BCR/ABL kinase than imatinib (>300 times in vitro) [Lombardo, 2004]. Dasatinib was initially proposed as a second-line therapy in chronic myeloid leukemia. Of note, pulmonary complications, and specifically pleural effusions, have been reported more frequently with dasatinib use compared with other TKIs [Bergeron, 2007; Masiello, 2009; Quintas-Cardama, 2007]. Recently, we reported a series of 9 cases of severe precapillary PAH confirmed by right heart catheterization, occurring in patients treated with dasatinib in France [Montani, circ 2012]. Median delay between initiation of dasatinib and PAH diagnosis was 34 months. Clinical, functional, or hemodynamic improvements were usually observed after dasatinib discontinuation. However, no patients demonstrate complete clinical and hemodynamic recovery even after long-term follow-up. Interestingly, all patients had received imatinib prior dasatinib and 6 patients received nilotinib after discontinuation of dasatinib without any recurrence of PAH, suggesting that PAH may be a specific complication of dasatinib. Paradoxically, dasatinib inhibit PDGFR, c-kit and c-Abl, and should have been a potential candidate in PAH treatment. However, dasatinib has a very wide spectrum of targets including Src kinase family, suggesting that one of these targets may be responsible of the PAH occurrence. The lowest estimate of incident PH occurring in patients exposed to dasatinib in France was 0.45% (Montani, Circ 2012). Of note, it has been recently demonstrated that first-line dasatinib may induce significantly higher and faster rates of complete cytogenetic response in newly diagnosed CML in comparison with imatinib [Kantarjian, 2010]. Therefore, this agent is likely to become increasingly prescribed in the future, and the number of cases of dasatinib-induced PAH may increase, particularly with extended duration of therapy. 5.2 Other 5.2.1 Vascular intestinal peptide (VIP) Vascular intestinal peptide (VIP) causes PASMC relaxation  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_209" \o "Saga, 1984 #158" Saga and Said 1984;  HYPERLINK \l "_ENREF_173" \o "Nandiwada, 1985 #157" Nandiwada, Kadowitz et al. 1985), neutralizes a variety of pulmonary vasoconstrictors, including endothelin and hypoxia  ADDIN EN.CITE Hamidi159(Hamidi, Lin et al.)15915917Hamidi, S. A.Lin, R. Z.Szema, A. M.Lyubsky, S.Jiang, Y. P.Said, S. I.Department of Medicine, State University of New York at Stony Brook, NY, USA.VIP and endothelin receptor antagonist: an effective combination against experimental pulmonary arterial hypertensionRespir ResRespiratory researchRespir Res141122011/10/28AnimalsDrug Therapy, CombinationHypertension, Pulmonary/*chemically induced/*drug therapy/pathologyMonocrotaline/toxicityRatsRats, Sprague-DawleyReceptors, Endothelin/*antagonists & inhibitors/physiologySulfonamides/*administration & dosageVasoactive Intestinal Peptide/*administration & dosage20111465-993X (Electronic) 1465-9921 (Linking)22029879Comparative Study Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.http://www.ncbi.nlm.nih.gov/pubmed/22029879321009510.1186/1465-9921-12-141eng( HYPERLINK \l "_ENREF_96" \o "Hamidi, 2011 #159" Hamidi, Lin et al.), inhibits airway and PVSMCs proliferation  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_147" \o "Maruno, 1995 #160" Maruno, Absood et al. 1995;  HYPERLINK \l "_ENREF_192" \o "Petkov, 2003 #161" Petkov, Mosgoeller et al. 2003), and has broad anti-inflammatory actions  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_192" \o "Petkov, 2003 #161" Petkov, Mosgoeller et al. 2003). VIP acts by biding to its associated receptors, vasoactive intestinal polypeptide receptor 1 (VPAC1) and VPAC2, downstream activation of the cAMP and cGMP second messenger systems occurs, leading to modulation of vascular tone  ADDIN EN.CITE Couvineau2012162(Couvineau and Laburthe 2012)16216217Couvineau, A.Laburthe, M.Centre de recherche biomedicale Bichat-Beaujon, Faculte de medecine X. Bichat, Paris, France. alain.couvineau@inserm.fr orVPAC receptors: structure, molecular pharmacology and interaction with accessory proteinsBr J PharmacolBritish journal of pharmacologyBr J Pharmacol42-5016612011/09/29AnimalsDrug DesignHumansInflammation/drug therapy/physiopathologyModels, MolecularMolecular Dynamics SimulationNeurodegenerative Diseases/drug therapy/physiopathologyProtein ConformationReceptors, Vasoactive Intestinal Peptide, Type II/*metabolismReceptors, Vasoactive Intestinal Polypeptide, Type I/*metabolismVasoactive Intestinal Peptide/*metabolism2012May1476-5381 (Electronic) 0007-1188 (Linking)21951273Research Support, Non-U.S. Gov't Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/21951273341563610.1111/j.1476-5381.2011.01676.xeng( HYPERLINK \l "_ENREF_35" \o "Couvineau, 2012 #162" Couvineau and Laburthe 2012). VIP was therefore considered a potent new target therapy in PAH management. Experimental studies reported involvement of VIP deficiency in the pathophysiology of PH  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_211" \o "Said, 2007 #163" Said, Hamidi et al. 2007;  HYPERLINK \l "_ENREF_210" \o "Said, 2008 #164" Said 2008). Patients with PAH showed reduced lung and serum VIP levels and upregulated pulmonary artery expression of both VIP receptor subtypes  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_192" \o "Petkov, 2003 #161" Petkov, Mosgoeller et al. 2003), suggesting that targeting VIP might be a useful therapeutic approach. VIP has been studied in clinical trials with conflicting results. Aviptadil improved exercise capacity, dyspnea, and pulmonary hemodynamics among eight treatment-naive patients with IPAH. A subsequent study that evaluated 20 patients with pulmonary hypertension of various etiologies demonstrated that inhalation of 100 g of aviptadil was associated with transient pulmonary vasodilatation, increased stroke volume and mixed venous oxygen saturation  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_139" \o "Leuchte, 2008 #165" Leuchte, Baezner et al. 2008). A modest increase in CO was also observed. However, results from an as-yet unpublished double-blind, placebo-controlled, dose-finding phaseII study involving 56 patients with PAH already treated with endothelin receptor antagonists, PDE5 inhibitors, or both showed no significant effects on exercise capacity or pulmonary hemodynamics after addition of inhaledaviptadil  ADDIN EN.CITE Gali2010166(Gali 2010)16616626N GaliEffects of inhaled aviptadil (vasoactive intestinal peptide) in patients with pulmonary arterial hypertension (PAH)181, B16 2010Am. J. Respir. Crit. Care Med. ( HYPERLINK \l "_ENREF_68" \o "Gali, 2010 #166" Gali 2010). In line with these clinical results, PAH treatment with ViP appears to be compromised at least for the next few years. 5.2.2 Statins Statins are well known inhibitors of endogenous cholesterol synthesis, acting on the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Different statins were shown to improve biomarkers, hemodynamic, pulmonary vascular and right cardiac hypertrophy in animal models of PH with an additive effect in combination with specific therapeutics as tadalafil  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_86" \o "Girgis, 2003 #168" Girgis, Li et al. 2003;  HYPERLINK \l "_ENREF_87" \o "Girgis, 2007 #169" Girgis, Mozammel et al. 2007);  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_179" \o "Nishimura, 2002 #170" Nishimura, Faul et al. 2002;  HYPERLINK \l "_ENREF_180" \o "Nishimura, 2003 #171" Nishimura, Vaszar et al. 2003;  HYPERLINK \l "_ENREF_272" \o "Zhang, 2012 #829" Zhang, Liu et al. 2012). Human studies are limited, with one observational study showing benefits  ADDIN EN.CITE Kao2005832(Kao 2005)83283217Kao, P. N.Pulmonary and Critical Care Medicine, Stanford University Medical Center, Stanford, CA 94305-5236, USA. peterkao@stanford.eduSimvastatin treatment of pulmonary hypertension: an observational case seriesChestChestChestChest1446-5212742005/04/12AdultAgedFemaleHumansHydroxymethylglutaryl-CoA Reductase Inhibitors/*therapeutic useHypertension, Pulmonary/*drug therapyMaleMiddle AgedSimvastatin/*therapeutic use2005Apr0012-3692 (Print) 0012-3692 (Linking)15821229Case Reportshttp://www.ncbi.nlm.nih.gov/pubmed/1582122910.1378/chest.127.4.1446eng( HYPERLINK \l "_ENREF_120" \o "Kao, 2005 #832" Kao 2005), but results from small, short-term randomized trials have been conflicting. A study with pravastatin showed general benefits on both biomarkers and lung physiology  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_136" \o "Lee, 2009 #839" Lee, Chen et al. 2009), whereas the simvastatin as a treatment for PH trial with simvastatin added to PDE-5 inhibitors and endothelin-1 antagonists showed transient improvement in NT-proBNP levels and right ventricular mass at 6 months but not over 1 year  ADDIN EN.CITE Wilkins2010826(Wilkins, Ali et al. 2010)82682617Wilkins, M. R.Ali, O.Bradlow, W.Wharton, J.Taegtmeyer, A.Rhodes, C. J.Ghofrani, H. A.Howard, L.Nihoyannopoulos, P.Mohiaddin, R. H.Gibbs, J. S.The Sipht Study, GroupDepartment of Experimental Medicine & Toxicology, Imperial College London, Hammersmith Hospital, London, United Kingdom.Simvastatin as a Treatment for Pulmonary Hypertension Trial (SiPHT)Am J Respir Crit Care MedAmerican journal of respiratory and critical care medicineAm J Respir Crit Care Med2010/01/302010Jan 281535-4970 (Electronic) 1073-449X (Linking)20110553http://www.ncbi.nlm.nih.gov/pubmed/2011055310.1164/rccm.200911-1699OCEng( HYPERLINK \l "_ENREF_265" \o "Wilkins, 2010 #826" Wilkins, Ali et al. 2010). Similarly, rosuvastatin showed benefits on biomarkers t not on physiological function at 6 months  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_11" \o "Barreto, 2008 #834" Barreto, Maeda et al. 2008). Statins seem to improve biomarkers in human PAH, but as yet studies are too preliminary to indicate whether these effects are clinically relevant  ADDIN EN.CITE Katsiki2011172(Katsiki, Wierzbicki et al. 2011)17217217Katsiki, N.Wierzbicki, A. S.Mikhailidis, D. P.Department of Clinical Biochemistry (Vascular Disease Prevention Clinics), Royal Free Hospital Campus, University College London Medical School, University College London, UK.Pulmonary arterial hypertension and statins: an updateCurr Opin CardiolCurrent opinion in cardiologyCurr Opin Cardiol322-62642011/04/12AnimalsBiological Markers/bloodDisease Models, AnimalDyslipidemias/complications/*drug therapyHumansHydroxymethylglutaryl-CoA Reductase Inhibitors/blood/*pharmacologyHypertension, Pulmonary/*prevention & controlRandomized Controlled Trials as TopicTreatment Outcome2011Jul1531-7080 (Electronic) 0268-4705 (Linking)21478744Research Support, Non-U.S. Gov't Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/2147874410.1097/HCO.0b013e32834659bfeng( HYPERLINK \l "_ENREF_122" \o "Katsiki, 2011 #172" Katsiki, Wierzbicki et al. 2011). Recently, a randomized clinical trial of aspirin and simvastatin for pulmonary hypertension failed  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_123" \o "Kawut, 2011 #173" Kawut, Bagiella et al. 2011). 5.2.3 Rho Kinases inhibitors Rho Kinases inhibitors are a new class of agents which may be beneficial in the treatment of PAH and two different approaches exist to inhibit to RhoA-ROCK axis in PAH: The direct ROCK inhibition with fasudil and aminofurazan derivatives, and an indirect ROCK inhibition with statins. Fasudil (HA-1077) is the main specific ROCK inhibitor  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_172" \o "Nagumo, 2000 #790" Nagumo, Sasaki et al. 2000). In small-caliber arteries exposed to hypoxia, fasudil inhibited the MLC phosphorylation produced by the ROCK activation and produced the relaxation of the PASMCs in a dose-dependent manner  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_261" \o "Wang, 2001 #801" Wang, Jin et al. 2001). Fasudil has also been reported to inhibit in vitro PDGF produced by PASMC from humans  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_144" \o "Liu, 2011 #809" Liu, Ling et al. 2011). In PH animal models, fasudil decreases mPAP, PVR, pulmonary vascular remodeling and right cardiac hypertrophy  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_171" \o "Nagaoka, 2005 #777" Nagaoka, Fagan et al. 2005;  HYPERLINK \l "_ENREF_2" \o "Abe, 2006 #756" Abe, Tawara et al. 2006;  HYPERLINK \l "_ENREF_169" \o "Mouchaers, 2010 #786" Mouchaers, Schalij et al. 2010). In monocrotaline-induced PH in rats, fasudil improved PH to a greater degree than did bosentan and sildenafil, with no synergistic effect when fasudil is combined with bosentan or sildenafil  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_169" \o "Mouchaers, 2010 #786" Mouchaers, Schalij et al. 2010), whereas combination with prostacyclin has been shown a better improvement than fasudil alone  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_244" \o "Tawara, 2007 #753" Tawara, Fukumoto et al. 2007). Fasudil was studied in PAH patients in its intravenous and inhaled form  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_65" \o "Fujita, 2010 #788" Fujita, Fukumoto et al. 2010;  ADDIN EN.CITE  ADDIN EN.CITE.DATA  HYPERLINK \l "_ENREF_66" \o "Fukumoto, 2005 #789" Fukumoto, Matoba et al. 2005). The first clinical trial showed that intravenous fasudil exerts an acute pulmonary vasodilator effects in patients with severe PAH with a decrease of PVR by 17 %  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_66" \o "Fukumoto, 2005 #789" Fukumoto, Matoba et al. 2005). However, despite a decreased PAP and increased cardiac index in these patients, there were no significant differences. A further trial of intravenous fasudil in 8 PAH patients allowed to obtain a decrease of mPAP but induced also and decrease of systemic vascular resistance and systolic arterial pressure  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_201" \o "Rhodes, 2009 #792" Rhodes, Davidson et al. 2009). These systemic effects could be avoided by the inhalation route. In 15 PAH patients, inhalation of fasudil significantly reduced mPAP and tended to decrease pulmonary vascular resistance  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_65" \o "Fujita, 2010 #788" Fujita, Fukumoto et al. 2010). Moreover, fasudil in inhalation form selectively affects lung tissues  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_65" \o "Fujita, 2010 #788" Fujita, Fukumoto et al. 2010). Recently, inhalation of fasudil leads to a decrease of systolic PAP and PVR in 19 patients with high-altitude PH  ADDIN EN.CITE Kojonazarov2012811(Kojonazarov, Myrzaakhmatova et al. 2012)81181117Kojonazarov, B.Myrzaakhmatova, A.Sooronbaev, T.Ishizaki, T.Aldashev, A.Effects of fasudil in patients with high-altitude pulmonary hypertensionEur Respir JThe European respiratory journal : official journal of the European Society for Clinical Respiratory PhysiologyEur Respir J496-83922012/02/031-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/*analogs & derivatives/therapeuticuseAcclimatizationAltitude Sickness/complications/*drug therapyHumansHypertension, Pulmonary/*drug therapy/etiologyTreatment OutcomeVasodilator Agents/*therapeutic use2012Feb1399-3003 (Electronic) 0903-1936 (Linking)22298615Clinical Trial Letter Research Support, Non-U.S. Gov'thttp://www.ncbi.nlm.nih.gov/pubmed/2229861510.1183/09031936.00095211eng( HYPERLINK \l "_ENREF_125" \o "Kojonazarov, 2012 #811" Kojonazarov, Myrzaakhmatova et al. 2012). Another specific ROCK inhibitor, azaindole-1, was recently characterized, with a higher specificity than fasudil. However, despite benefit effects on PAP in PH animal models, azaindole-1 induces also a strong decrease of systemic arterial pressures  ADDIN EN.CITE Pankey2012813(Pankey, Byun et al. 2012)81381317Pankey, E. A.Byun, R. J.Smith, W. B., 2ndBhartiya, M.Bueno, F. R.Badejo, A. M.Stasch, J. P.Murthy, S. N.Nossaman, B. D.Kadowitz, P. J.Department of Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana 70112-2699, USA.The Rho kinase inhibitor azaindole-1 has long-acting vasodilator activity in the pulmonary vascular bed of the intact chest ratCan J Physiol PharmacolCanadian journal of physiology and pharmacologyCan J Physiol PharmacolCanadian journal of physiology and pharmacologyCan J Physiol PharmacolCanadian journal of physiology and pharmacology825-359072012/05/182012Jul1205-7541 (Electronic) 0008-4212 (Linking)22591047Research Support, N.I.H., Extramuralhttp://www.ncbi.nlm.nih.gov/pubmed/2259104710.1139/y2012-061eng( HYPERLINK \l "_ENREF_188" \o "Pankey, 2012 #813" Pankey, Byun et al. 2012). SB-772077-B is a potent aminofurazan derivative which was recently characterized and demonstrated to inhibit the ROCK activity. A treatment with in SB-772077-B rats with monocrotaline-induced PH reported a decrease of PAP but also systemic arterial pressure  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_42" \o "Dhaliwal, 2009 #818" Dhaliwal, Badejo et al. 2009). In addition to its inhibition on HMG-CoA reductase, statins have also been found to inhibit synthesis of isoprenoids such as geranylgeranylpyrophosphate and farnesylpyrophosphate, which are involved in the activation of various signaling molecules such as RhoA by facilitating the mediator interaction with the cell membrane, and its coupling to GTP  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_201" \o "Rhodes, 2009 #792" Rhodes, Davidson et al. 2009). By inhibiting the addition of geranylgeranylpyrophosphate to RhoA, statins inhibit the RhoA-ROCK activation  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_269" \o "Xing, 2007 #821" Xing, Gan et al. 2007). These properties explain in part the benefits observed in PH animal models with statins associated to their antioxidant, anti-inflammatory and antithrombotic actions. 5.2.4 Endothelial progenitor cells Endothelial progenitor cells (EPCs) have the property to proliferate and migrate in response to angiogenic growth factors and subsequently differentiate into mature cells in situ for blood vessel formation. In PAH patients, a deficiency of bone-marrow derived EPCs might contribute to endothelial dysfunction and the progression of the disease  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_44" \o "Diller, 2008 #863" Diller, van Eijl et al. 2008). Introduction of EPCs in the circulation of PAH patients could therefore repair and regenerate blood vessels improving endothelial dysfunction and damaged in pulmonary microvasculature. Administration of EPCs in rats with monocrotaline-induced PH led to prevention of PH with a decrease of RVSP and a restoring of microvasculature structure  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_273" \o "Zhao, 2005 #873" Zhao, Courtman et al. 2005). Pulmonary hypertension and Cell Therapy (PHACeT) is a clinical trial ongoing in Canada in order to assess safety of administrating autologous mononuclear cells transduced with eNOS in idiopathic PAH patients  ADDIN EN.CITE NIH.2010909(NIH. 2010)90990945US NIH.ClinicalTrials.gov. Pulmonary hypertension: assessment of cell therapy (PHACeT) [online],2010http://clinicaltrials.gov/ct2/show/NCT00469027?term=PHACeT&rank=1( HYPERLINK \l "_ENREF_178" \o "NIH., 2010 #909" NIH. 2010). Associated to this safety study, efficacy of EPCs infusion have been reported in adult and children patients with idiopathic PAH with improvement on exercise capacity and pulmonary hemodynamics  ADDIN EN.CITE  ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_260" \o "Wang, 2007 #905" Wang, Zhang et al. 2007;  HYPERLINK \l "_ENREF_275" \o "Zhu, 2008 #908" Zhu, Wang et al. 2008). 6. guidelines for the treatment of pulmonary hypertension AND FUTURE DIRECTIONS In 2009, the Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS) proposed an algorithm for the treatment of pulmonary arterial hypertension. This algorithm is presented in Figure 3. First, this algorithm underlines the importance to test vasoreactivity during right heart catheterization to screen potential long-term responders to CCB. In the absence of acute vasodilator response or absence of long-term response to CCB, specific PAH therapies targeting endothelial dysfunction (epoprostenol and prostacyclin derivatives, endothelin receptor antagonists or PDE5i) are indicated. The choice of the drugs and route of administration depends on the clinical severity based on NYHA functional class. Of note, intravenous epoprostenol is recommended as a first line therapy for all the most severe patients in NYHA functional class IV. All approved drugs (AREs, PDE5i, inhaled iloprost, i.v epoprostenol) may be proposed as first-line therapy for patients in NYHA functional class III. Few data are currently available in patients in NYHA functional class II and ERS/ESC guidelines proposed oral specific PAH therapy (AREs, PDE5i) as first-line therapy. These guidelines proposed sequential combination therapy in absence of adequate response. This combination therapy may include the three classes of drugs currently available, and no specific recommendation on the best strategy of association have been proposed. Preliminary reports suggest that a more aggressive therapeutic strategy with first-line triple combination therapy (prostacyclin, ARE, PDE5i) may be more effective to improve hemodynamics in severe PAH patients. The 5th World Symposium on Pulmonary Hypertension will be held in Nice in 2013 and new algorithm of treatment should be discussed including recent advances in the management of PAH. CONCLUSION @@@@Marc@@@ FIGURES  Figure 1. Pathological pathways in pulmonary arterial hypertension and current and emerging therapeutic target therapies. Pathogenesis of pulmonary arterial hypertension corresponds to an imbalance between excessive vasoconstrictors (endothline-1) and lack of vasodilators (nitric oxide and prostacyclin) leading to vasoconstriction and proliferation. Pulmonary artery smooth muscle cell therapeutic targets are representing as licensed (full line blue boxes) or investigational (dotted line brown boxes) treatment approaches for pulmonary arterial hypertension. Arrows represent receptor stimulation, whereas terminated lines show receptor blockade. Abbreviations: AC, adenylate cyclase; cAMP, cyclic AMP; cGMP, cyclic GMP; ECE1, endothelin converting enzyme 1; eNOS, endothelial nitric oxide synthase ; ET-A, endothelin receptor type A; ET-B, endothelin receptor type B; ETRA, endothelin receptor antagonists; IP, prostaglandin I2; PDE5, phosphodiesterase type 5; PGIS, prostaglandin I synthase; sGC, soluble guanylate cyclase.  Figure 2. Receptor tyrosine kinases involved in pathogenesis of pulmonary arterial hypertension Binding of growth factors to their receptor tyrosine kinase induce proliferation, migration and survival of smooth muscle cells (pink), endothelial cells (blue) and activation of inflammatory cells (purple). These cells act on both leading to a self-maintained phenomenon and vascular remodeling. Abbreviations: SCF: stem cell factor; PDGF: platelet derives growth factor, PDGF-R: PDGF receptor; FGF-2: fibroblast growth factor 2: FGF-R: FGF receptor, EGF: epidermal growth factor; EGF-R: EGF receptor.  Figure 3. Evidence-based treatment algorithm for pulmonary arterial hypertension patients (adapted from  ADDIN EN.CITE  ADDIN EN.CITE.DATA  HYPERLINK \l "_ENREF_72" \o "Galie, 2009 #1" Galie, Hoeper et al. 2009). APAH: associated pulmonary arterial hypertension; BAS: balloon atrial septostomy; CCB: calcium channel blocker; ERA: endothelin receptor antagonist; IPAH: idiopathic pulmonary arterial hypertension; PAH: pulmonary arterial hypertension; PDE-5 I: phosphodiesterase type-5 inhibitor; s.c.: subcutaneously; WHO-FC: World Health Organization functional class. # : to maintain arterial blood O2pressure >8 kPa (60 mmHg). 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Jin, et al. (2001). "Rho-kinase activation is involved in hypoxia-induced pulmonary vasoconstriction." Am J Respir Cell Mol Biol 25(5): 628-635.    !56CHinprwиииУxxbM8(h;Wh$GCJOJQJ^JaJmH sH (h;WhjyCJOJQJ^JaJmH sH +h;WhjyCJH*OJQJ^JaJmH sH +h;WhPALCJH*OJQJ^JaJmH sH (h;WhPALCJOJQJ^JaJmH sH (h;WhPALCJ$OJQJ^JaJmH sH .h;WhN 5CJ$OJQJ\^JaJmH sH .h;WhB5CJ$OJQJ\^JaJmH sH .h;WhPAL5CJ$OJQJ\^JaJmH sH 6q r v 8 3 !$d1$]a$gdf*0m$!$ Hd1$]a$gdf*0m$$d]a$gdf*0m$ $dha$gdf*0m$ $dha$gd}m$ $da$gdf*0m$ $da$gd;Wm$ $da$gdf>m$     ) . 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