Prostacyclin Analogue Treprostinil Research Articles

Pharmacology and Emerging Therapies for Group 3 Pulmonary Hypertension Due to Chronic Lung Disease.

Pulmonary hypertension (PH) frequently complicates chronic lung disease and is associated with high morbidity and poor outcomes. Individuals with interstitial lung disease and chronic obstructive pulmonary disease develop PH due to structural changes associated with the destruction of lung parenchyma and vasculature with concurrent vasoconstriction and pulmonary vascular remodeling similar to what is observed in idiopathic pulmonary arterial hypertension (PAH). Treatment for PH due to chronic lung disease is largely supportive and therapies specific to PAH have had minimal success in this population with exception of the recently FDA-approved inhaled prostacyclin analogue treprostinil. Given the significant disease burden of PH due to chronic lung diseases and its associated mortality, a great need exists for improved understanding of molecular mechanisms leading to vascular remodeling in this population. This review will discuss the current understanding of pathophysiology and emerging therapeutic targets and potential pharmaceuticals.

Cutaneous iontophoresis of vasoactive medications in patients with scleroderma-associated pulmonary arterial hypertension.

It remains unknown whether the cutaneous microvascular responses are different between patients with scleroderma-associated pulmonary arterial hypertension (SSc-PAH) and SSc without pulmonary hypertension (PH). We included 59 patients with SSc between March 2013 and September 2019. We divided patients into 4groups: (a) no PH by right heart catheterization (RHC) (n=8), (b) no PH by noninvasive screening tests (n=16), (c) treatment naïve PAH (n=16), and (d) PAH under treatment (n=19). Microvascular studies using laser Doppler flowmetry (LDF) were done immediately after RHC or at the time of an outpatient clinic visit (group b). The median (IQR) age was 59 (54-68) years, and 90% were females. The responses to local thermal stimulation and postocclusive reactive hyperemia, acetylcholine, and sodium nitroprusside iontophoresis were similar among groups. The microvascular response to treprostinil was more pronounced in SSc patients without PH by screening tests (% change: 340 (214-781)) compared with SSc-PAH (naïve+treatment) (Perfusion Units (PU) % change: 153 (94-255) % [p=.01]). The response to A-350619 (a soluble guanylate cyclase (sGC) activator) was significantly higher in patients with SSc without PH by screening tests (PU % change: 168 (46-1,296)) than those with SSc-PAH (PU % change: 22 (15-57) % [p=.006]). The % change in PU with A350619 was directly associated with cardiac index and stroke volume index (R: 0.36, p=.03 and 0.39, p=.02, respectively). Patients with SSc-PAH have a lower cutaneous microvascular response to a prostacyclin analog treprostinil and the sGC activator A-350619 when compared with patients with SSc and no evidence of PH on screening tests, presumably due to a peripheral reduction in prostacyclin receptor expression and nitric oxide bioavailability.

Improvement of diabetic wound healing using prostacyclin analoges encapsulated in lipid nanoparticles

Diabetes is associated with impairment of cutaneous wound healing and can lead to lower limb amputation. The cutaneous microcirculation in diabetes is impaired due to alterations of the nitric oxide and prostacyclin pathways that can be therapeutic targets. Topical application of lipid nanoparticles can increase drug delivery through improved stability and controlled delivery compared to classical formulations. We tested the efficacy of topical delivery of the prostacyclin analogue treprostinil and the IP receptor agonist ACT-333679 (active metabolite of selexipag), encapsulated in lipid nanoparticles, on wound healing in a diabetic mice model. We used 80 nm cationic (C80) or neutral (N80) lipid nanoparticles. Cationic emulsions (EM) were also formulated. For the wound healing study, 12-week-old males db/db mice were used. Two 8 mm 2 diameter ulcers were made on the back of each animal and protected using a transparent, occlusive dressing. Each treatment was evaluated on 12 mice with one ulcer treated with the treatment and the contralateral ulcer treated with the vehicle. Wounds were treated daily over 24 days and evaluated daily using planimetry (ImageJ, NIH). Area under the curve (AUC) of the wound surface (expressed as a % of day 0 size) was calculated and compared between treatments and their vehicles. Encapsulation of ACT-333679 was stable in C80 and N80 for 6 months at 4 °C. Encapsulation of ACT-333679 was stable for 3 months at 4 °C. Encapsulation of Treprostinil was stable for 3 months in C80 while encapsulation was not stable in EM (1 month stability). Both Treprostinil and ACT-333679 increased the rate of wound healing in the db/db mice model. There was no major difference between lipid nanoparticles and emulsions. Stable lipid nanoparticles loaded with IP receptors agonists can be formulated in a topical gel, are stable over time and result in an improved wound healing in a diabetic mouse model.

INHALED TREPROSTINIL FOR PATIENTS AT RISK FOR ARDS

SESSION TITLE: Tuesday Abstract Posters SESSION TYPE: Original Investigation Posters PRESENTED ON: 10/22/2019 01:00 PM - 02:00 PM PURPOSE: Inhaled prostacyclin analogs have been utilized to improve oxygenation in severe refractory hypoxic respiratory failure. Administration of inhaled prostacyclins in patients with pneumonia or early acute lung injury not requiring positive pressure ventilation has not been studied. The prostacyclin analogue treprostinil may have anti-inflammatory effects, as well as improve oxygenation, potentially preventing or delaying onset or severity of acute respiratory distress syndrome (ARDS). We sought to determine the feasibility and safety of administration of inhaled treprostinil, as well as the effect on oxygenation, in a group at risk for developing ARDS. METHODS: This was a single center, double-blind, placebo-controlled, randomized trial for feasibility and safety (NCT02370095). Patients with hypoxemia due to pneumonia or signs of low pressure pulmonary edema were randomized to receive inhaled treprostinil or placebo (2:1 ratio respectively) via the TD-100 nebulizer device. Inclusion criteria were a unilateral or bilateral infiltrate on chest imaging and a ≥ 4 liter per minute supplemental oxygen requirement. Study drug was initiated at 6 breaths every four hours and titrated up to 12 breaths over 16 hours. Patients were maintained on study drug or placebo for 7 days, and then tapered off over a period of 4 days. If oxygenation improved rapidly, patients could be titrated off sooner. Study drug was stopped sooner if positive pressure ventilation was required. The primary outcome measure was change in daily SaO2/FiO2 (S/F) ratios. Adverse events were assessed for relatedness to study drug. RESULTS: Fourteen patients were enrolled over a period of 31 months. Baseline characteristics of the treprostinil and placebo cohorts were not significantly different with respect to age, gender, race, APACHE score or LIPS score. The baseline mean S/F ratio in the inhaled treprostinil group was 194 vs 212 in the placebo group (p = 0.62). Average time on study drug was not significantly different between the two groups. Four patients required positive pressure ventilation in the treprostinil group vs one in the placebo group. Utilizing a daily pre-dose S/F value (collected during and after treatment was discontinued) and adjusting for pre-treatment baseline S/F ratio, there was no significant treatment effect (p=0.07). CONCLUSIONS: Inhaled treprostinil administration is feasible in patients at risk for ARDS. Treatment with inhaled treprostinil was not associated with improvement in the S/F ratio relative to placebo. Adverse events associated with study drug were not severe nor unexpected based on the known adverse effect profile of inhaled treprostinil. CLINICAL IMPLICATIONS: Inhaled treprostinil does not appear to be harmful in patients with hypoxia who are at risk for ARDS, however the clinical benefit is unclear at this time in the absence of larger studies to explore this question further. DISCLOSURES: No relevant relationships by Wayne Anderson, source=Web Response No relevant relationships by Thomas Bice, source=Web Response Research Grant relationship with United Pharmaceuticals Please note: $5001 - $20000 Added 03/15/2019 by Shannon Carson, source=Web Response, value=Grant/Research Support Investigator relationship with Biomarck Pharmaceuticals Please note: $20001 - $100000 Added 03/15/2019 by Shannon Carson, source=Web Response, value=Grant/Research Support No relevant relationships by Agathe Ceppe, source=Web Response Consultant relationship with United Therapeutics Please note: $5001 - $20000 Added 02/14/2019 by Hubert Ford, source=Web Response, value=Travel also Consultant relationship with Actelion Please note: $1001 - $5000 Added 03/14/2019 by Hubert Ford, source=Web Response, value=Honoraria Consultant relationship with Liquidia Please note: $1-$1000 Added 03/14/2019 by Hubert Ford, source=Web Response, value=Honoraria Research Grant relationship with United Therapeutics Please note: $5001 - $20000 Added 03/14/2019 by Hubert Ford, source=Web Response, value=Grant/Research Support No relevant relationships by Joyce Lanier, source=Web Response No relevant relationships by Blair Wendlandt, source=Web Response

The prostacyclin analogue treprostinil in the treatment of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare but life-threatening disease that progresses rapidly and is currently without a cure. Pharmacological treatments aim to slow down disease progression and to reduce symptoms by targeting the prostacyclin, the endothelin or the nitric oxide pathway. Drugs targeting the prostacyclin pathway have been shown to be favourable for PAH patients by causing vasodilatative, anti-proliferative as well as anti-inflammatory effects, but tend to be underused, partially due to adverse effects and difficulties associated with their intravenous administration. Treprostinil, a stable prostacyclin analogue, was FDA-approved in 2002 to improve exercise capacity in PAH patients and is available in intravenous, subcutaneous, inhaled and oral form. The four different possible ways of administration, a long half-life and its stability at room temperature give treprostinil an advantage over epoprostenol, iloprost and selexipag, the three other FDA-approved drugs targeting the prostacyclin pathway. In clinical trials, treprostinil improved exercise capacity, quality of life (QOL), functional class and clinical status. While the different forms of treprostinil lead to specific complications, its general adverse effects are dizziness, nausea, pain in the jaw and extremities, diarrhoea, flushing and headache. This MiniReview will assess the benefits and drawbacks of treprostinil in the treatment of PAH by examining its specific mechanism of action and pharmacological properties, such as pharmacokinetics, pharmacodynamics, adverse effects and interactions. In addition, we will analyse and discuss results from different clinical trials, comparing treprostinil's four different forms to each other as well as to other drugs targeting the prostacyclin pathway.

Treprostinil reduces endothelial damage in murine sinusoidal obstruction syndrome

Sinusoidal obstruction syndrome (SOS) is a major complication after hematopoietic stem cell transplantation and belongs to a group of diseases increasingly identified as transplant-related systemic endothelial disease. Administration of defibrotide affords some protection against SOS, but the effect is modest. Hence, there is unmet medical need justifying the preclinical search for alternative approaches. Prostaglandins exert protective actions on endothelial cells of various vascular beds. Here, we explored the therapeutic potential of the prostacyclin analog treprostinil to prevent SOS. Treprostinil acts via stimulation of IP, EP2, and EP4 receptors, which we detected in murine liver sinusoidal endothelial cells (LSECs). Busulfan-induced cell death was reduced when pretreated with treprostinil in vitro. In a murine in vivo model of SOS, concomitantly administered treprostinil caused lower liver weight-to-body weight ratios indicating liver protection. Histopathological changes were scored to assess damage to liver sinusoidal endothelial cells, to hepatocytes, and to the incipient fibrotic reaction. Treprostinil indeed reduced sinusoidal endothelial cell injury, but this did not translate into reduced liver cell necrosis or fibrosis. In summary, our observations provide evidence for a beneficial effect of treprostinil on damage to LSECs but unexpectedly treprostinil was revealed as a double-edged sword in SOS.Key messagesMurine liver sinusoidal endothelial cells (LSECs) express prostanoid receptors.Treprostinil reduces busulfan-induced cell death in vitro.Treprostinil lowers liver weight-to-body weight ratios in mice.Treprostinil positively affects LSECs in mice but not hepatic necrosis/fibrosis.

Inhibition of Adrenergic and Non-Adrenergic Smooth Muscle Contraction in the Human Prostate by the Phosphodiesterase 10-Selective Inhibitor TC-E 5005.

The phosphodiesterase (PDE) 5 inhibitor tadalafil is available for treatment of male lower urinary tract symptoms (LUTS), while the role of other PDE isoforms for prostate smooth muscle tone is still unknown. Here, we examined effects of the PDE10-selective inhibitor TC-E 5005 on smooth muscle contraction in human prostate tissue. Prostate samples were obtained from patients undergoing radical prostatectomy. Expression of PDE10 was addressed by RT-PCR, Western blot, and fluorescence staining with different markers. Effects of TC-E 5005 and tadalafil on contraction, and relaxation of prostate strips were studied via organ bath. PDE10A was detectable by RT-PCR, Western blot, and fluorescence staining in prostate tissues. Colocalization with markers suggested expression of PDE10A in smooth muscle cells and catecholaminergic nerves. Norepinephrine, the α1 -adrenergic agonist phenylephrine, the thromboxane A2 analogue U46619, and endothelins 1-3 induced concentration-dependent contractions of prostate strips, while electric field stimulation (EFS) induced frequence-dependent contractions. Application of TC-E 5005 (500 nM) caused significant inhibition of norepinephrine-, phenylephrine-, and endothelin-3-induced contractions. Inhibition of EFS-induced contractions by TC-E 5005 ranged around 50%, resembling inhibition of EFS-induced contractions by tadalafil (10 μM). The prostacyclin analog treprostinil and the nitric oxide donor DEA NONOate induced relaxations of precontracted prostate strips, which were significantly amplified by TCE 5005. The PDE10-selective inhibitor TC-E 5005 inhibits adrenergic and neurogenic smooth muscle contractions in the human prostate. TC-E 5005 inhibits neurogenic contractions with similar efficacy than tadalafil, so that urodynamic effects in vivo appear possible. Prostate 76:1364-1374, 2016. © 2016 Wiley Periodicals, Inc.

Effects of chronic treprostinil treatment on experimental right heart hypertrophy and failure.

Right heart function is an important predictor of morbidity and mortality in pulmonary arterial hypertension and many CHD. We investigated whether treatment with the prostacyclin analogue treprostinil could prevent pressure overload-induced right ventricular hypertrophy and failure. Male Wistar rats were randomised to severe pulmonary trunk banding with a 0.5-mm banding clip (n=41), moderate pulmonary trunk banding with a 0.6-mm banding clip (n=36), or sham procedure (n=10). The banded rats were randomised to 6 weeks of treatment with a moderate dose of treprostinil (300 ng/kg/minute), a high dose of treprostinil (900 ng/kg/minute), or vehicle. Pulmonary trunk banding effectively induced hypertrophy, dilatation, and decreased right ventricular function. The severely banded animals presented with decompensated heart failure with extracardial manifestations. Treatment with treprostinil neither reduced right ventricular hypertrophy nor improved right ventricular function. In the pulmonary trunk banding model of pressure overload-induced right ventricular hypertrophy and failure, moderate- and high-dose treatment with treprostinil did not improve right ventricular function neither in compensated nor in decompensated right heart failure.

Abstract 18488: Metabolic Modulation of the Diseased Human Lung; A Translational, Novel ex vivo Lung Perfusion Model

Introduction: Pulmonary arterial hypertension (PAH) and idiopathic pulmonary fibrosis (IPF) are deadly diseases that often require lung transplantation. Preclinical evidence suggests that mitochondrial suppression underlies the pro-proliferative and anti-apoptotic phenotype of pulmonary artery smooth muscle cells and pulmonary fibroblasts, respectively. Two therapies to improve mitochondrial function are a) dichloroacetate (DCA), an activator of pyruvate dehydrogenase (PDH), the gate-keeping enzyme for glucose oxidation, and b) 4-phenylbutyrate (4-PBA), an inhibitor of endoplasmic reticulum stress, a response that suppresses mitochondrial function during cellular stress. Although these drugs can be studied in vitro or in animals, their metabolic effects on the whole human lung may only by studied through ex vivo lung perfusion (EVLP). Methods: We used a custom-made EVLP system to study explanted, diseased human lungs of patients undergoing lung transplant for PAH (n=2), or IPF (n=4). The bronchi were connected to a ventilator, while a pump-driven system perfused the pulmonary artery (PA) with modified Krebs-Henseleit buffer or STEEN solution™ in recirculation. Lung biopsies were collected to measure mitochondrial respiration (using a Seahorse analyzer) and PDH activity at baseline, and after drug intervention with DCA or 4-PBA for 1 hour. Results: In the PAH lungs, hypoxic pulmonary vasoconstriction, a validated marker of lung health, was absent. However administrating the prostacyclin analogue treprostinil reduced PA pressure by 31.6% (at a level of flow corresponding to the patient’s cardiac output), confirming abnormal but present vasoreactivity in the model. DCA caused a 61.1% increase in oxygen consumption, and a 34-fold increase in PDH activity. In IPF lungs, PBA increased oxygen consumption by 62.0%. Conclusions: EVLP is a feasible model to study the direct effects of drugs on diseased human lungs. Since before-after biopsies are not possible in clinical trials, this is a valuable model to facilitate translation of preclinical therapies to clinical trials. Our results show, for the first time in human lungs, that the metabolic remodeling of the PAH and IPF can be reversed by clinically available small molecules like DCA and 4-PBA.

Assessing the agonist profiles of the prostacyclin analogues treprostinil and naxaprostene, particularly their DP1 activity

In this study, the inhibitory profiles of the prostacyclin analogues treprostinil and naxaprostene on several isolated smooth muscle preparations have been investigated. Treprostinil was an agonist for prostanoid DP1, EP2 and IP receptors, but not EP4 receptors; its DP1 potency was only 3–4 times less than PGD2 itself. Naxaprostene was much more selective for IP receptors and tended towards partial agonism. Treprostinil is a 13,14-dihydro analogue and the role of conformation around C12–15 in controlling agonist specificity is debated; the synthesis of new analogues is proposed and possible clinical usage discussed. In terms of selective prostanoid antagonists employed, BW-A868C/MK-0524 (DP1), ACA-23 (EP2) and GW-627368 (EP4) were found fit for purpose. However, the IP antagonist RO-1138452 was compromised by α1 and α2-adrenoceptor-mediated contractile activity on rat tail artery and anti-muscarinic activity on mouse trachea. There is a need for IP receptor antagonists with better selectivity and higher affinity.

New Oral Drugs for Pulmonary Arterial Hypertension: Macitentan, Riociguat, and Treprostinil

In 2013, the US Food and Drug Administration (FDA) approved 3 new oral drugs for the treatment of pulmonary arterial hypertension (PAH; World Health Organization [WHO] Group 1 pulmonary hypertension [PH]). These include the endothelin receptor antagonist macitentan, the soluble guanylate cyclase stimulator riociguat, and the prostacyclin analogue treprostinil. In addition, riociguat was approved for the treatment of patients with inoperable or postsurgery recurrent or persistent chronic thromboembolic PH (CTEPH; WHO Group 4 PH). The approval of these drugs has several important clinical implications: first, in a disease where many of the currently available treatments are complicated by significant side effects and/or complex administration regimens, the availability of new oral drugs clearly represents a valuable addition to the armamentarium. Second, the macitentan study was the first long-term, event-driven trial to be published in the PAH field, making the results more robust and paving the way for improved clinical trial design in the future. Third, riociguat is the first FDA-approved medical treatment regimen for selected CTEPH patients, thus providing a critical treatment option for patients with inoperable or recurrent/persistent CTEPH. Lastly, the approval of oral treprostinil made this drug the first oral prostacyclin analogue to be available in the United States. In this article, the authors will discuss the mechanisms of action of macitentan, riociguat, and oral treprostinil; review the landmark trials that led to the FDA approval of these drugs, and discuss their clinical use.

Differential actions of the prostacyclin analogues treprostinil and iloprost and the selexipag metabolite, MRE-269 (ACT-333679) in rat small pulmonary arteries and veins

The prostacyclin (IP) receptor agonists, treprostinil, iloprost and the selexipag metabolite, MRE-269 (ACT-333679) were evaluated in rat distal pulmonary blood vessels. Small pulmonary arteries and veins were pre-contracted with the thromboxane mimetic, U46619 (25 and 100nM, respectively), and relaxation determined with and without IP receptor antagonists, RO1138452 and RO3244794. In arteries, treprostinil was a more potent vasorelaxant than iloprost, while the efficacy of iloprost was greater. In pulmonary arteries, treprostinil-induced relaxation was essentially abolished by both IP antagonists (1μM), while responses to iloprost were partially inhibited. Both treprostinil and iloprost were equipotent, prominently relaxing pulmonary veins with responses being similarly and partially sensitive to IP antagonists. In contrast, RO1138452 failed to inhibit relaxations to MRE-269 in either pulmonary arteries or veins, suggesting no involvement of typical IP receptors. Thus, rat pulmonary tissues cannot be considered appropriate to assess classical IP receptors using the proposed highly selective non-prostanoid agonist MRE-269, contrasting with the IP receptor agonism profile of prostacyclin analogues, iloprost and treprostinil.

Efficacy and Safety of Oral Treprostinil Monotherapy for the Treatment of Pulmonary Arterial Hypertension

Background— Pulmonary arterial hypertension (PAH) is a progressive, fatal disease with no cure. Parenteral and inhaled prostacyclin analogue therapies are effective for the treatment of PAH, but complicated administration requirements can limit the use of these therapies in patients with less severe disease. This study was designed to evaluate the safety and efficacy of the oral prostacyclin analogue treprostinil diolamine as initial treatment for de novo PAH. Methods and Results— Three hundred forty-nine patients (intent-to-treat population) not receiving endothelin receptor antagonist or phosphodiesterase type-5 inhibitor background therapy were randomized (treprostinil, n=233; placebo, n=116). The primary analysis population (modified intent-to-treat) included 228 patients (treprostinil, n=151; placebo, n=77) with access to 0.25-mg treprostinil tablets at randomization. The primary end point was change from baseline in 6-minute walk distance at week 12. Secondary end points included Borg dyspnea index, clinical worsening, and symptoms of PAH. The week 12 treatment effect for 6-minute walk distance (modified intent-to-treat population) was 23.0 m ( P =0.0125). For the intent-to-treat population, 6-minute walk distance improvements were observed at peak (26.0 m; P =0.0001) and trough (17.0 m; P =0.0025) plasma study drug concentrations. Other than an improvement in the combined 6-minute walk distance/Borg dyspnea score, there were no significant changes in secondary end points. Oral treprostinil therapy was generally well tolerated; the most common adverse events (intent-to-treat) were headache (69%), nausea (39%), diarrhea (37%), and pain in jaw (25%). Conclusions— Oral treprostinil improves exercise capacity in PAH patients not receiving other treatment. Oral treprostinil could provide a convenient, first-line prostacyclin treatment option for PAH patients not requiring more intensive therapy. Clinical Trial Registration:— URL: http://www.clinicaltrials.gov . Unique identifier: NCT00325403 .

Cathodal Iontophoresis of Treprostinil Induces a Sustained Increase in Cutaneous Blood Flux in Healthy Volunteers

Prostacyclin analogues are the most effective drugs to treat sclerodermic digital ulcers, but their systemic use is limited by their frequent side effects. The authors tested whether the prostacyclin analogues treprostinil and iloprost, delivered by cutaneous iontophoresis, induce sustained vasodilatation. Twenty healthy volunteers received treprostinil by cathodal iontophoresis on the forearm. Skin blood flux was quantified using laser Doppler imaging. Treprostinil 250 µM induced an increase in cutaneous vascular conductance AUC(80) (min) (31 897 ± 24 390 %BL·min) compared with NaCl 0.9% (P < .005), treprostinil 2.5 µM (P < .005), and treprostinil 25 µM (P < .005). This was confirmed when flux was recorded for up to 10 hours. Systemic and skin tolerance of treprostinil were good. Iloprost was stopped early because of local toxicity. Treprostinil cathodal iontophoresis induces a sustained increase in cutaneous blood flow with good local tolerance. This could be investigated as a new local therapy for digital ulcers in scleroderma.

Inhaled treprostinil: a therapeutic review

Pulmonary arterial hypertension (PAH) is a life-threatening disease which, if untreated, leads to right ventricular failure and often death. Several effective therapies are now available for PAH, including endothelin receptor antagonists, phosphodiesterase-5 inhibitors, and prostacyclin analogs. The prostacyclin analog treprostinil has proven efficacious when delivered by subcutaneous or intravenous infusion, and most recently by inhalation. Inhaled treprostinil has been shown to be 64%–72% bioavailable in healthy volunteers. Pilot clinical studies have elucidated the acute hemodynamic effects and relative pulmonary selectivity of this agent, as well as established target dosing in PAH and nonoperable chronic thromboembolic PAH. Likewise, chronically administered inhaled treprostinil resulted in clinical and hemodynamic improvement. Both pilot studies confirmed a satisfactory safety profile in patients with PAH. The pivotal Phase III trial, TRIUMPH-I, demonstrated the efficacy and safety of inhaled treprostinil (target dose of 54 μg four times daily) in PAH patients added to background therapies of bosentan or sildenafil, as assessed by improvements in the primary endpoint, peak six-minute walk distance (median placebo-corrected treatment effect of 20 m), as well as select secondary endpoints. Inhaled treprostinil is approved by the US Food and Drug Administration for patients with World Health Organization Group I PAH to improve exercise ability. Studies establishing effectiveness included predominately patients with New York Heart Association functional class III symptoms and etiologies of idiopathic or heritable PAH (56%) or PAH associated with connective tissue diseases (33%).

Treprostinil Inhibits the Adhesion and Differentiation of Fibrocytes via the Cyclic Adenosine Monophosphate–Dependent and Ras-Proximate Protein–Dependent Inactivation of Extracellular Regulated Kinase

Fibrocytes comprise a recently described cell type of blood-derived, fibroblast-like cells that are recruited from the circulation to sites of wound repair, vascular remodeling, or fibrotic tissue remodeling. We recently showed that the stable prostacyclin analogue treprostinil, a clinically approved drug for pulmonary arterial hypertension (PAH), significantly reduced the recruitment of fibrocytes to sites of vascular remodeling in experimental hypoxic pulmonary hypertension. Here we report on the molecular mechanism underlying the inhibitory action of treprostinil on the adhesion and differentiation of human fibrocytes. Human fibrocytes expressed the prostanoid receptors, prostaglandin I (IP) receptors and prostaglandin E subtype receptors (EP2 and EP4). The generation of intracellular cyclic adenosine monophosphate (cAMP) by treprostinil reduced the expression of the integrins CD49 and CD29 when freshly isolated human peripheral blood mononuclear cells were treated with treprostinil. Cell-matrix adhesion was significantly impaired by treatment with treprostinil. We present evidence for a treprostinil/cAMP-induced downstream suppression of extracellular regulated kinase (ERK) that is transmitted via a protein kinase A-independent pathway through Rap proteins, which sequester Ras. The resulting dephosphorylated state of c-Raf limits the activity of ERK. The cell-matrix adhesion assay with the ERK inhibitor further confirmed that the adhesion of fibrocytes was impaired. Thus our data suggest that treprostinil inhibits the adhesion and differentiation of fibrocytes by limiting the activity of ERK via the cAMP-Rap axis.

Abstract 4923: Prostacyclin Analogues Activates Calcium-dependent Potassium (K Ca ) Channels via PPAR β / δ in Pulmonary Artery

Pulmonary hypertension is associated with vasoconstriction, thrombosis and proliferation, and the lack of endogenous prostacyclin. Prostacyclin and its analogues are potent vasodilators, representing an important pillar of modern therapy of this devastating disease. Potassium channels determine the membrane potential of pulmonary artery smooth muscle cells (PASMCs) and are important controllers of their calcium homeostasis and consequently the pulmonary arterial tone. In the present study, we investigated the signaling pathway and effects of the stable prostacyclin analogues treprostinil and iloprost in primary human PASMCs and isolated rat pulmonary artery. We employed the whole-cell patch-clamp technique combined with small interfering (si)RNA and live-cell calcium imaging on human PASMCs and the myograph-mounted isolated intrapulmonary artery model. We found that treprostnil (10 nM) and iloprost (10 nM) hyperpolarized primary human PASMCs (13.1 ± 2.2 mV, p &lt; 0.01 and 15.7 ± 2 mV, p &lt; 0.01 respectively, n = 6 for each group) and activated K Ca (IC 50 = 0.2 ± 0.05 nM and IC 50 = 0.5 ± 0.2 nM respectively, n = 5 for each group) at clinically relevant concentrations. The IP receptor antagonist RO1138452 (10 μ M) and PKA blocker KT5720 (1 mM) abolished these effects. In the siRNA-transfected cells no enhancement of K Ca current was detected. The novel PPAR β / δ antagonist GSK0660 (10 nM), abolished the activation of K Ca current by iloprost or treprostinil, whereas the selective PPAR β / δ agonist GW0742 (10 nM) activated the K Ca current. Calcium imaging measurements supported these data showing that GW0742 reduced the basal intracellular calcium concentration by 9.41±2.99 % (n = 25, p &lt; 0.01) compared to the baseline in human PASMCs. Consequently, GW0742 caused relaxation in U46619 -preconstricted rat intrapulmonary arteries (IC 50 = 9.42 μ M, n = 5). This is the first study implicating K Ca in the prostacyclin analogue- mediated hyperpolarization via PPAR β / δ in human PASMCs. This might represent a novel pathway related to the vasodilating effects of prostanoids.

G020 Properties of pulmonary artery smooth muscle from task-1 knockout mice

In pulmonary artery (PA), compelling evidence have suggested that TWIK-related Acid Sensitive K+ -1 (TASK-1) channel contribute to the background K+ current that support the membrane potential (Em) of the smooth muscle cells. However, due to poor selectivity of its modulators, the role of TASK-1 channel in the functional regulation of tone in pulmonary arteries remains elusive. The purpose of this study was to investigate the properties of PA isolated from mice in which the TASK-1 and TASK-3 genes have been deleted (TASK1/3 knockout, KO), compared with their wild type controls (C57BL/6 mice, WT). Intra-PAs were isolated from adult male WT and KO mice. Vessels were mounted on a wire myograph for isometric tension recordings. PAs were also processed for gene expression studies using RT-PCR. The addition of 10mM KCl, the K+ channel modulators 4-AP (1mM) and levcromakalim (10μm), or the L-type calcium channel blocker nifedipine (1μm), did not elicit any significant contractile response in PAs from either KO (N=3) or WT mice (N=3), suggesting that the Em is not depolarised and that artery is fully dilated in both groups. The contractile response of PA to phenylephrine, serotonin and PGF2α did not show any difference between the two strains. Potency (pEC50) of the prostacyclin analog treprostinil to induce relaxation on preconstricted vessels was significantly higher in KO mice (6.7 ± 0.1, N = 3) than in WT mice (7.2 ± 0.1N = 4). RT-PCR confirmed TASK-1 expression in PA from WT but not KO mice whereas TASK-3 was not expressed in either WT or KO mouse PA. TASK-2, TASK-5, TWIK-2, Kv1.5 and Kv2.1 RNA levels did not show any significant alteration between WT (N = 3) or KO mice (N = 3). The PA isolated from KO mice does not display any resting tone or altered responses to the studied agonists. Surprisingly, response to treprostinil was increased, and TASK-1 absence in PA was not compensated by alteration of other K+ channels expression. The results thus far do not support a major physiological role for TASK-1 in mouse PA.

Rapid Switch From Intravenous Epoprostenol to Intravenous Treprostinil in Patients With Pulmonary Arterial Hypertension

Intravenous epoprostenol improves exercise capacity and survival in patients with pulmonary arterial hypertension (PAH); however, chemical instability and a short half-life have caused limitations in its use. The chemically stable prostacyclin analogue treprostinil has a longer half-life, and improves hemodynamics and signs/symptoms of PAH. This study investigated the feasibility of transitioning patients with PAH from intravenous epoprostenol to intravenous treprostinil using a rapid switch protocol. Twelve PAH patients were enrolled in a 12 week prospective open label study. Patients were switched from intravenous epoprostenol to intravenous treprostinil (1:1 ng/kg/min) by a direct switch of the medication reservoir from epoprostenol to treprostinil. The dose of treprostinil was adjusted throughout the study to achieve a 2-fold increase of treprostinil compared with the baseline epoprostenol dose. Rapid transition to treprostinil was achieved without serious adverse events and, baseline clinical status was maintained over 12 weeks. The mean baseline epoprostenol dose was 28 +/- 14 ng/kg/min. At week 12, the mean treprostinil dose was 62 +/- 30 ng/kg/min. All patients reported less prostacyclin-related side effects with treprostinil and remained on treprostinil after study completion. Selected patients with PAH can be safely transitioned from intravenous epoprostenol to intravenous treprostinil using a rapid switch protocol.

Efficacy of Long-term Subcutaneous Treprostinil Sodium Therapy in Pulmonary Hypertension

The aim of this long-term multicenter analysis was to investigate whether subcutaneously infused treprostinil could provide sustained improvements of exercise capacity and survival benefits in patients with pulmonary arterial hypertension (PAH) and inoperable chronic thromboembolic pulmonary hypertension (CTEPH). Subcutaneous administration of the prostacyclin analog treprostinil is an effective treatment for PAH that, unlike epoprostenol, does not require the insertion of a permanent central venous catheter. Multicenter retrospective study. Three European university hospitals. Ninety-nine patients with PAH and 23 patients with CTEPH in New York Heart Association (NYHA) classes II-IV were followed up for a mean of 26.2 +/- 17.2 months (+/- SE) [range, 3 to 57 months]. Long-term efficacy was assessed by 6-min walking distance (SMWD), Borg dyspnea score, and NYHA class. Clinical events were monitored to assess survival and event-free survival. At 3 years, significant improvements from baseline were observed in mean SMWD (305 +/- 11 to 445 +/- 12 m, p = 0.0001), Borg dyspnea score (5.7 +/- 0.2 to 4.5 +/- 1, p = 0.0006), and NYHA class (3.20 +/- 0.04 to 2.1 +/- 0.1, p = 0.0001). These changes were observed under a mean dose of subcutaneously infused treprostinil at 40 +/- 2.6 ng/kg/min (range, 16 to 84 ng/kg/min). Subcutaneously infused treprostinil was well tolerated, and local pain at the subcutaneous site accounted for treatment interruption in only 5% of the cases. Survival was 88.6% and 70.6% at 1 year and 3 years, respectively. At the same time points, the event-free survival rates, defined as survival without hospitalization for clinical worsening, transition to IV epoprostenol, and need for combination therapy or atrial septostomy, were 83.2% and 69%, respectively. Long-term subcutaneous therapy with treprostinil appears to continuously improve exercise tolerance and symptoms in patients with PAH and inoperable CTEPH. Moreover, treatment may provide a significant survival benefit.