Am J Cardiovasc Drugs DOI 10.1007/s40256-015-0125-4

LEADING ARTICLE

Novel Targets of Drug Treatment for Pulmonary Hypertension Jian Hu1 • Qinzi Xu1 • Charles McTiernan1 • Yen-Chun Lai1 David Osei-Hwedieh1 • Mark Gladwin1,2

•

Ó Springer International Publishing Switzerland 2015

Abstract Biomedical advances over the last decade have identified the central role of proliferative pulmonary arterial smooth muscle cells (PASMCs) in the development of pulmonary hypertension (PH). Furthermore, promoters of proliferation and apoptosis resistance in PASMCs and endothelial cells, such as aberrant signal pathways involving growth factors, G protein-coupled receptors, kinases, and microRNAs, have also been described. As a result of these discoveries, PH is currently divided into subgroups based on the underlying pathology, which allows focused and targeted treatment of the condition. The defining features of PH, which subsequently lead to vascular wall remodeling, are dysregulated proliferation of PASMCs, local inflammation, and apoptosis-resistant endothelial cells. Efforts to assess the relative contributions of these factors have generated several promising targets. This review discusses recent novel targets of therapies for PH that have been developed as a result of these advances, which are now in pre-clinical and clinical trials (e.g., imatinib [phase III]; nilotinib, AT-877ER, rituximab, tacrolimus, paroxetine, sertraline, fluoxetine, bardoxolone methyl [phase II]; and sorafenib, FK506, aviptadil, endothelial progenitor cells (EPCs) [phase I]). While substantial progress has been made in recent years in targeting key molecular pathways, PH still remains without a cure, and these novel therapies provide an important conceptual framework

& Jian Hu [email protected] 1

Pittsburgh Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, E1226 BST 200 Lothrop Street, Pittsburgh, PA 15213, USA

2

Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Pittsburgh, USA

of categorizing patients on the basis of molecular phenotype(s) for effective treatment of the disease.

Key Points Several novel strategies have emerged for pulmonary hypertension in recent years. Pre-clinical and clinical trials have been performed to evaluate the novel strategies. The following drugs, acting on novel targets, are currently being evaluated in clinical trials: imatinib (phase III); nilotinib, AT-877ER, rituximab, tacrolimus, paroxetine, sertraline, fluoxetine, bardoxolone methyl (phase II); and sorafenib, FK506, aviptadil, endothelial progenitor cells (EPCs) (phase I).

1 Introduction Pulmonary hypertension (PH) is a progressive vascular disease caused by vasoconstriction and structural remodeling of arterioles that result in the reduction of vessel elasticity and inner diameter. These changes increase the resting mean pulmonary arterial pressure (PAP) (C25 mmHg) and can lead to dyspnea, fatigue, cough, chest pain, palpitations, peripheral edema, syncope, right heart failure, and death [1, 2]. In the US population, the age-standardized mortality rate for PH has significantly increased from 5.5 per 100,000 population in 2001 to 6.5 per 100,000 in 2010. In 2010, women showed significantly higher mortality rates than

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men, and non-Hispanic blacks showed significantly higher mortality rates than non-Hispanic whites. Aged population cohorts (75–84 and C85 years of age) show the highest PH mortality (47.9/100,000 and 108.7/100,000, respectively, 2010 data) [3]. Mechanisms for the development of PH are complex and may be idiopathic, genetic, or secondary to left heart disease, lung diseases, and/or chronic thromboembolism. Five subgroups of PH have been defined on the basis of the etiology [1], and current research progress on the novel therapeutic targets is focused on group 1 PH [pulmonary arterial hypertension (PAH)]. PAH is characterized by a progressive increase in pulmonary vascular resistance (PVR) caused by pulmonary artery remodeling and vaso-occlusion. The most important mechanism for PAH development is endothelial dysfunction and/or injury, which leads to imbalanced production of endogenous vasodilators, such as prostacyclin and nitric oxide, and vasoconstrictors, such as endothelin and serotonin. Without treatment, the survival rates of PAH at 1 year, 3, and 5 years are 68, 48, and 34 %, respectively [4]. Contemporary drugs used to treat PAH target three pathways: the cyclic guanosine monophosphate signaling pathway (e.g., sildenafil and tadalafil), prostacyclin signaling pathway (e.g., epoprostenol and iloprost), and endothelin signaling pathway (e.g., bosentan and ambrisentan). More recently, new drugs targeting these major pathways (e.g., riociguat, selexipag, and macitentan) have been shown to be effective in the treatment of PAH and have been approved by US Food and Drug Administration (FDA). Although current treatments appear to relieve symptoms, improve exercise capacity, and prevent hospitalizations, only limited evidence for disease reversal has been observed. In recent years, several novel strategies have emerged for PH treatment, and this article focuses on new and promising therapeutic approaches.

2 Novel Targets of Drug Treatment 2.1 Receptor Tyrosine Kinase Inhibitors Receptor tyrosine kinases (RTKs) are a class of high-affinity cell surface receptors for a variety of growth factors, hormones, and cytokine ligands. Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), and c-kit-receptors play pathogenic roles in the development of PH [5, 6], as these RTK ligands increase proliferation and migration of smooth muscle cells (SMCs) and endothelial cells (ECs) in lungs. Several tyrosine kinase inhibitors (TKIs) are currently under development. Imatinib (GleevecÒ) inhibits PDGF, c-kit, and c-Abl receptors. The use of imatinib has been shown to inhibit proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) in rodents and

humans, and to exert pro-apoptotic effects in isolated PASMCs [7, 8]. Imatinib has also been reported to prevent right ventricular and pulmonary vascular remodeling and to improve pulmonary hemodynamics in animal models [5, 9]. After several reported cases of improved exercise capacity and hemodynamics of PH patients given imatinib treatment [10–14], the compound was evaluated for safety, tolerability, and efficacy in 59 PH patients in a doubleblinded, placebo-controlled multicenter, phase II trial [15]. This clinical trial has shown that imatinib decreases PVR and increases cardiac output, but has no significant improvement in the 6-min walk distance (6MWD). However, subgroup analysis revealed that patients with severely improved PVR showed a significant increase in 6MWD and hemodynamics after receiving imatinib. In a phase III trial of 202 patients with severe PH, patients receiving imatinib showed a significant improvement in 6MWD, decreased mean PAP and PVR, and increased cardiac output [16]. Nilotinib is another TKI that shows greater potency and selectivity in inhibition of the BCR-ABL tyrosine kinase than does imatinib. Nilotinib has been demonstrated to prevent angioproliferation in a rodent model of PH with systemic sclerosis [17]. However, a phase II clinical trial conducted in 23 patients was terminated because of serious adverse effects including cardiopulmonary disorders (e.g., pulseless electrical activity, right ventricular dysfunction, acute respiratory failure, and PAH worsening) and hepatobiliary disorders [e.g., cholecystitis and cholelithiasis (NCT01179737)] [18]. Sorafenib is a multikinase inhibitor targeting PDGF receptor (PDGFR), VEGF receptor (VEGFR), c-kit, and Raf. In a mouse PH model, it has been shown to prevent hemodynamic changes and attenuate PH-associated vascular remodeling by abolishing the upregulation of the mitogen-activated protein kinase (MAPK) cascade [19]. In a clinical trial of 22 patients with PH, sorafenib treatment generated a modest improvement in the exercise capacity without any alteration in cardiac function [20]. It was well tolerated at a 200-mg twice-daily dose, with the most common adverse effects being moderate skin reaction and alopecia. However, there are reports showing that some other agents in the family of TKIs induce PH. For example, treatment of dasatinib in patients with leukemia induced PH [21–23]. Sugen5416, another protein kinase inhibitor, also induces PH in rodents in combination with hypoxia exposure [24]. In summary, although some TKIs offer promising new treatments for PH, more studies are needed to understand how to use them with minimal adverse effects and maximal efficacy. 2.2 Rho-Kinase Inhibitor RhoA is a small monomeric G-protein that belongs to Ras homologous (Rho) family. RhoA and its downstream substrate Rho-kinase (ROCK) play significant roles in the

Developing Drugs for Pulmonary Hypertension

pathogenesis of PH, mediating vascular remodeling and vasoconstriction [25–28]. Long-term inhibition of ROCK with fasudil, a selective ROCK inhibitor, improved pathological parameters of PH and pulmonary artery remodeling in monocrotaline- or hypoxia-induced rodent models of PH [29–31]. Fasudil was the first ROCK inhibitor approved in 1995 for the treatment of subarachnoid hemorrhage–induced brain vessel vasospasms [32, 33]. Clinical trials have shown that intravenous administration of fasudil has acute beneficial vasodilatory effects on the pulmonary circulation in PH patients [34, 35]. Inhalation of fasudil was shown to be as effective as nitric oxide inhalation in PH patients [36]. It was also reported that fasudil reduced pulmonary artery pressure in patients with high-altitude PH in a randomized, double-blind study [37]. AT-877ER (fasudil hydrochloride) is a specific ROCK inhibitor with an extended-release formulation. It has been used in a double-blind, placebo-controlled clinical trial to treat PH in Japan. Although there was no significant difference observed in 6MWD between the AT-877ER group and placebo group, the mid-term results showed improved pulmonary hemodynamics and cardiac index in the AT877ER–treated group [38, 39]. 2.3 Vasoactive Intestinal Peptide Vasoactive intestinal peptide (VIP) is a neuropeptide hormone that induces pulmonary vasodilation, inhibits vascular SMC proliferation and platelet aggregation, and shows antiinflammatory properties [40–46]. Mediated by G-protein coupled receptors (VPAC1, VPAC2, and PAC1), VIP can stimulate the adenylate cyclase signaling pathway, activate phospholipase-D, tyrosine kinases, and RhoA-GTPAses, and increase intracellular calcium via calcium channel activation. Downregulation of VIP has been proposed as one of the defining features of PH on the basis of the observation of pulmonary vascular remodeling and hypertension in VIPdeficient mice and a reversal of the pathology with VIP replacement therapy [47]. Lungs from VIP knockout mice showed increased expression of gene transcripts associated with vasoconstriction/proliferation and inflammation, and decreased expression of gene transcripts associated with vasodilation/anti-proliferation. This gene expression profile, however, was reversed to that seen in wild-type mice by VIP treatment [48]. Interestingly, serum and pulmonary levels of VIP are decreased in PH patients, but the expression levels of VIP receptors are increased in PASMCs isolated from PH patients [49]. A clinical trial of 20 PH patients receiving a single inhalation dose of 100 lg aviptadil, an analog of VIP, showed minimal side effects and yielded an acute and mild, but statistically significant, decrease of pulmonary arterial pressure with a significant increase in stroke volume and mixed venous oxygen saturation [50]. A total of six patients

in this trial have been reported to have a reduction in PVR of [20 %. Additionally, a combination of VIP and endothelin receptor antagonist, bosentan, has been tested in pre-clinical PH models. This combination therapy was more effective than either drug alone, perhaps because of the synergistic effects of both drugs on suppression of the endothelin-endothelin (ET-ET) receptor pathway [51]. 2.4 Selective Serotonin Reuptake Inhibitors Upregulation of serotonin released from the pulmonary endothelium is associated with the development of PH in animal models, likely through its effects on pulmonary vasoconstriction and SMC proliferation [52–61]. Selective serotonin reuptake inhibitors (SSRIs) can attenuate and even reverse the development of PH induced by chronic hypoxia or monocrotaline in animal models [62–67]. Several clinical studies have suggested that SSRIs may be associated with a decreased development of PH and mortality in PH patients [68, 69]. However, one phase II clinical trial failed to show the beneficial effects of SSRIs (paroxetine, sertraline, or fluoxetine) in the treatment of PH, while a separate trial showed that the use of SSRIs correlated to even higher mortality and a greater risk of clinical worsening [70, 71]. Because these trials may have failed to appropriately match all clinical variables of the treated and control patient populations, further studies are necessary to conclusively determine whether SSRIs are an appropriate therapeutic category for PH. 2.5 Endothelial Progenitor Cells Since PH is associated with loss of or damage to the pulmonary vascular endothelium, it has been theorized that the delivery of additional endothelial progenitor cells (EPCs) may provide a therapeutic benefit. The main sources of EPCs are bone marrow and peripheral blood [72–78]. EPC population is most commonly characterized through expression of CD34, CD133, and kinase domain receptor (KDR) [79–81]. Some studies have suggested that patients with PH had reduced peripheral EPCs [82–86]; however, the relationship between the number of peripheral EPCs and PH remains controversial [87–90]. EPCs derived from hypoxia-derived PH mice were found to have altered functions such as decreased migratory response to stromal cell-derived factor-1a (SDF-1a), adhesion to fibronectin, and incorporation into the vascular network [88]. It has been demonstrated that treatment with autologous or umbilical cord blood-derived EPC can prevent and reverse PH in small and large animal models, with improved hemodynamics and amelioration in the medial thickness of the small pulmonary arteries [78, 90, 91]. Studies in animal models of PH have shown that EPCs transfected with genes

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of adrenomedullin [92] or endothelial nitric oxide synthase (eNOS) [93] caused greater improvement in PH than EPCs alone. The combination of EPCs and sildenafil therapy also has synergistic effects when compared with a single treatment with EPCs [94]. EPCs may benefit the prevention of PH by vascular endothelial repair [95–97] and providing protective paracrine effects [98–101]. Several clinical studies have shown that EPC treatment is safe, feasible, and can improve exercise capacity and hemodynamics [42, 44, 76, 77, 102]. There are two main types of EPCs; one is colony-forming unit-Hill (CFU-Hill) cells, and the other one is endothelial colony-forming cells (ECFCs). It has been reported that ECFCs can form functional vessels in vivo, while CFU-Hill cells cannot [103]. There is evidence showing that CFU-Hill cells are hematopoietic cells which may never become long-term intimal ECs in vivo [103, 104]. Studies showed that the ECFCs could prevent PH in rats treated with bleomycin [105], but no reports have suggested CFU-Hill cells in preventing or improving PH. While more clinical studies are needed to confirm the safety, adverse effects, and efficacy of EPC treatment, it is a novel approach for the future treatment of PH (Table 1). 2.6 Inflammation and Immunity As perivascular inflammation is observed in patients and animals with PH, several studies have assessed the role of

inflammation and immunity in the pathogenesis of PH. Perivascular inflammatory infiltration is a complex interplay between T- and B-lymphocytes, macrophages, dendritic cells, and mast cells [106]. It was found that inflammation precedes vascular remodeling in animal PH models, which suggests that altered immunity is the cause rather than a consequence of vascular disease [107]. In addition, the circulating levels of cytokines and chemokines such as interleukin-1b (IL-1b), IL-6, IL-8, monocyte chemoattractant protein 1 (MCP1), fractalkine, CCL5/ RANTES, and tumor necrosis factors-a (TNF-a) are increased. These cytokines may lead to pulmonary vascular remodeling by mediating or controlling proliferation, migration and differentiation of pulmonary vascular cells [108]. In pre-clinical studies, targeting of several immune mediators or processes, such as CD20, IL-1, transforming growth factor-b (TGF-b), nuclear factor of activated T cells (NFAT), tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), 5-lipoxygenase/5-lipoxygenase-activating protein (5-LO/FLAP), Leukotriene B4 (LTB4), and purine synthesis, has been shown to be effective in preventing or even reversing PH [108]. Immunosuppressive agents such as dexamethasone, mycophenolate mofetil, cyclosporine, and etanercept have also been shown to improve PH in animal models [109]. The highly selective elastase inhibitor elafin has been demonstrated to attenuate fully developed PAH in an animal model, with a significant

Table 1 Summary of the action of the drugs Class RTKI

Agent

Mechanism of action

Imatinib

Inhibition of PDGF, c-kit, and c-Abl receptors

Nilotinib

Inhibition of BCR-ABL tyrosine kinase

Sorafenib

Inhibition of PDGFR, VEGFR, c-kit, and Raf

Rho-kinase inhibitor

AT-877ER (fasudil hydrochloride)

Inhibition of Rho-kinase

VIP

Aviptadil

Inducing pulmonary vasodilation, inhibiting vascular SMCs proliferation, platelet aggregation

SSRI

Paroxetine, sertraline, fluoxetine

Inhibition of serotonin reuptake

EPCs Inflammation and immunity

Repairing pulmonary vascular endothelium Elafin

Inhibition of elastase

Bardoxolone methyl

Inducing Nrf2 and suppressing NF-jB activation

Tacrolimus

Inhibition of the NFAT family

Rituximab

Antibody of CD20 on B cells

CCR5 Antagonist

Maraviroc

Inhibition of CCR5, protective effect on vascular lesions

Wnt

Mesd (blocker of Wnt ligands)

Competitively blocking the binding of Wnt ligands to LRP5/6

miRNAs

MRX34 Miravirsen

Mimic of miRNA tumor suppressor miR-34 Inhibitor of miRNA-122

CCR5 C-C motif chemokine receptor 5, EPC endothelial progenitor cell, LRP5/6 low-density lipoprotein receptor-related protein 5/6, miRNA microRNA, NFAT nuclear factor of activated T cells, NF-kB nuclear factor kappa-light-chain-enhancer of activated B cells, Nrf2 nuclear factor erythroid 2–related factor 2, PDGF platelet-derived growth factor, PDGFR PDGF receptor, RTKI receptor tyrosine kinase inhibitor, SMC smooth muscle cell, SSRI selective serotonin reuptake inhibitor, VEGFR VEGF receptor, VIP vasoactive intestinal peptide

Developing Drugs for Pulmonary Hypertension

improvement of the vascular pathology, parameters of pulmonary hemodynamics, and right ventricular function [110]. In fact, elafin is an FDA-approved orphan drug for the treatment of PH. Bardoxolone methyl (RTA402) is an orally available semi-synthetic triterpenoid that induces nuclear factor erythroid 2–related factor 2 (Nrf2) and suppresses nuclear factor kappa-light-chain-enhancer of activated B cells (NF-jB) activation. Bardoxolone methyl can suppress activation of proinflammatory mediators, enhance endothelial nitric oxide (NO) bioavailability, improve metabolic dysfunction, inhibit vascular proliferation, and prevent maladaptive remodeling. Bardoxolone methyl also targets multiple cell types relevant to PH, including ECs, SMCs, and macrophages. A phase II clinical trial is currently underway to evaluate its efficacy as a therapeutic for PH (NCT02036970) [111]. Recently, an inhibitor of the NFAT family, FK506 (Tacrolimus), was reported to reverse severe PH in the SUGEN/hypoxia model by restoring bone morphogenetic protein type 2 receptor (BMPR2) signaling [112], and its safety and efficacy are being evaluated in a clinical trial (NCT01647945) [113]. Rituximab is a chimeric monoclonal antibody recognizing CD20 on B cells and is currently in use in a National Institute of Allergy and Infectious Diseases (NIAID)-sponsored trial (NCT01086540) for the treatment of systemic sclerosis-associated PH [114]. However, no results have been reported yet for the clinical trials utilizing bardoxolone methyl, FK506, or rituximab for the treatment of PH. The effectiveness and safety of numerous immune targets still need to be evaluated, and the immunotherapies may have to be adjusted on a patient-bypatient basis according to different subtypes of PH characterized by distinct inflammatory profiles. 2.7 G-Protein-Coupled Receptor CCR5 CCR5 is strongly expressed in principal cell types implicated in PH progression, including ECs, SMCs, T cells, and macrophages [115–122]. It is considered a therapeutic target in human immunodeficiency virus (HIV) infection because it is a co-receptor for HIV cell entry [120, 122]. The CCR5 pathway also plays an important role in atherogenesis, and studies have suggested that inhibition of the CCR5 pathway has a protective effect on vascular lesions [117, 122]. Pulmonary expression of CCR5 is increased in mice subjected to hypoxia-induced PH, as well as in the explanted lungs from patients with idiopathic PH. Notably, CCR5 expression is more marked in areas characterized by greater severity of vascular medial hypertrophy. The activation of CCR5 leads to the proliferation of cultured human PASMCs, while a human CCR5 antagonist developed as an HIV therapeutic, maraviroc, inhibits this proliferation [115]. It is reported that CCR5-deficient mice developed less severe PH than wild-type mice after

hypoxia exposure, and maraviroc treatment markedly attenuated hypoxia-induced PH development (distal pulmonary artery muscularization) in CCR5 knock-in mice [122]. In addition to the preventive effect, maraviroc can also partially reverse PH in chronically hypoxic mice [122]. Although no clinical trials evaluating the effects of CCR5 inhibitors on PH have been reported, it may have therapeutic potential for the treatment of PH. 2.8 Wnt Signaling Wnt signaling has been linked to more than 20 clinical diseases. Its effects in regulating angiogenesis and cell growth have prompted researchers to hypothesize that it can play a role in the treatment of PH in which none of the current therapies can promote angiogenesis or reverse medial thickening. Both the Wnt/b-catenin (Wnt/bC) pathway and Wnt/planar cell polarity (Wnt/PCP) pathway have been shown to be involved in the development of PH [123–127]. Wnt7a and Wnt5 downregulate b-catenin and have antiproliferative effects on PASMC cultured under hypoxic conditions [127, 128]. In the lung tissue of PH patients, it was found that the expression of PCP mediators are upregulated, suggesting a pathophysiological role of the Wnt/PCP pathway [129]. Low-density lipoprotein receptorrelated protein 5/6 (LRP5/6) are Wnt co-receptors that bind to Wnt ligands and mediate canonical Wnt/bC signaling, while Mesd is a cellular-encoded chaperone protein for LRP5/6 that competitively blocks the binding of Wnt ligands to LRP5/6 [130]. Systemic delivery of Mesd protein attenuated PH and pulmonary vascular remodeling in a rodent model of hyperoxia-induced PH [131]. ICG001, a novel b-catenin inhibitor, has been shown to increase alveolarization and reduce pulmonary vascular remodeling and PH in the rodent model of hyperoxia-induced PH. ICG001 could also decrease PASMC proliferation and the expression of extracellular matrix remodeling molecules under hyperoxic conditions [132]. Gene expression array analyses of different cell types from PH patients revealed that molecular lesions associated with PH were present in all cell types, and the Wnt signaling pathway was a common molecular defect in both heritable and idiopathic PH [133]. However, due to a wide range of cellular functions and molecular targets of Wnt signaling, extensive screening of Wnt modulators will be required to find therapeutic candidates for PH that have minimal adverse effects and toxicity with maximal efficacy. 2.9 MicroRNAs MicroRNAs (miRNAs) are a category of small noncoding RNAs, which regulate an array of proteins that affect many biological processes including cell differentiation, survival,

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Fig. 1 Drugs currently in discovery, development, and/or approved for the treatment of pulmonary hypertension. miRNA microRNA

and proliferation [134]. As many miRNAs are associated with cancer development and have a potential as anti-cancer agents, and because there are many overlapping pathways (e.g., BMPR2, Src/STAT3/Pim1, and hypoxia) in both cancer and PH [135, 136], the evaluation of miRNAs in PH treatment remains an attractive idea. Some miRNAs are thought to be modulators of signaling in pathogenic protein expression, which leads to pulmonary vascular remodeling, PH development, right ventricular (RV) hypertrophy, and RV failure [137, 138]. There are two strategies for miRNAbased therapies: miRNAs mimetics and miRNA antagonists. Mimetics of miR-204, miR-424, miR-503, and let-7f are reported to ameliorate or reverse PH in animal models [139– 141]. While miRNA antagonism may have more side effects than miRNA mimetics, several studies have demonstrated that specific miRNA antagonists, such as anti-miR-17, can provide a beneficial therapy for PH [142]. Two drugs in this category are being evaluated in clinical trials for other diseases. MRX34, a mimic of miRNA tumor suppressor miR34, is being evaluated for treatment of liver cancer by intravenous (IV) delivery [143]. Miravirsen, an inhibitor of miRNA-122, is in clinical trials for the treatment of hepatitis C virus infection, via subcutaneous administration [144]. Currently, there are no clinical trials for miRNA-based therapy for PH. However, further investigation may lead to the development of novel and effective therapeutic strategies.

3 Summary PH is a disease with complex and diverse underlying mechanisms and a poor prognosis. In recent years, significant progress has been made toward understanding the pathophysiology and biochemistry of PH development, which has led to considerable advances in the search for new treatments. Alongside the refinement of existing treatment strategies, many new molecular and cellular pathways and targets involved in PH have been explored in the development of new drugs and therapies (Fig. 1). Although there are new therapies in pre-clinical or earlystage clinical trials, most of the clinical trials conducted are short term. More studies need to be conducted to assess the safety and efficacy of such novel therapeutics before such promising approaches can be added to our armamentarium of treatments for PH. Acknowledgments We thank Dr. Sergei Snovida for helpful comments on the manuscript and Elfy Chiang for the graphic assistance. Compliance with ethical standards There are no conflicts of interests to declare for Jian Hu, Qinzi Xu, Charles McTiernan, YenChun Lai, and David Osei-Hwedieh. Mark Gladwin receives National Institutes of Health (NIH) grant funding, and is a consultant for Bayer for sickle cell disease. He is also the co-inventor of an NIH patent for the use of nitrite for cardiovascular indications. He also receives royalties as co-author of a textbook of medical students. No financial assistance was received for preparing this manuscript.

Developing Drugs for Pulmonary Hypertension

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