Inhaled nitric oxide: Current clinical concepts

Accepted Manuscript Inhaled Nitric Oxide: Current Clinical Concepts Pavan Bhatraju, MD, Fellow in Pulmonary and Critical Care Medicine, Jack Crawford, PhD, MD, Assistant Professor, Program Director Pediatric Anesthesiology Fellowship, Michael Hall, MD, Assistant Professor, John D. Lang, Jr., MD, Associate Professor, Clinical Director of Operative and Perioperative Operations PII:

S1089-8603(15)30013-6

DOI:

10.1016/j.niox.2015.08.007

Reference:

YNIOX 1513

To appear in:

Nitric Oxide

Received Date: 9 April 2015 Revised Date:

31 July 2015

Accepted Date: 26 August 2015

Please cite this article as: P. Bhatraju, J. Crawford, M. Hall, J.D. Lang Jr., Inhaled Nitric Oxide: Current Clinical Concepts, Nitric Oxide (2015), doi: 10.1016/j.niox.2015.08.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Inhaled Nitric Oxide: Current Clinical Concepts Pavan Bhatraju, MD Fellow in Pulmonary and Critical Care Medicine Department of Medicine University of Washington Medical Center Seattle, WA

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Authors:

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Jack Crawford, PhD, MD Assistant Professor Program Director Pediatric Anesthesiology Fellowship Division of Cardiothoracic Anesthesiology | Pediatric Cardiac Section Department of Anesthesiology The University of Alabama at Birmingham Birmingham, AL

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Michael Hall, MD Assistant Professor Division of Cardiothoracic Anesthesiology Department of Anesthesiology and Pain Medicine University of Washington Medical Center Seattle, WA

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Corresponding Author: John D. Lang, Jr, MD Associate Professor Clinical Director of Operative and Perioperative Operations Department of Anesthesiology and Pain Medicine University of Washington Medical Center 1959 NE Pacific St., Suite EE-201 Seattle, WA 98195-6540 Phone # 206.498.8013 Email: [email protected]

The manuscript has is not funded by any granting agency or industry sponsor. We have no conflicts of interest to declare.

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ABSTRACT

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Nitric oxide (NO) has come far way since being discovered serendipitously to relax vascular smooth muscle. Initially, administered to animals to reduce pulmonary artery pressures and improve oxygenation. It now enjoys FDA approval for administration to newborns with pulmonary hypertension but is used common place for other critical cardiopulmonary ailments. While never quite living up to expectations, newer applications show greater promise as a therapy especially in the area of ischemiareperfusion. The following will give a clinical overview of inhaled nitric oxide as a gas, as applied to the pediatric patient population, and to those adults suffering with cardiopulmonary and hematologic disease. Lastly, due to more recent discoveries, the effects of how NO may be used to treat disorders sucha as ischemia-reperfusion, will also be reviewed.

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1. INTRODUCTION

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Nitric Oxide (NO) is an odorless, colorless gas first identified by Joseph Priestley in 1774. At that time, it was described to be toxic and highly reactive. Following this initial description, NO was largely thought of as a pollutant and discussion was generally limited to its toxic potential and industrial uses. It would not be until several centuries later that its role in biologic systems was suggested and subsequently proven

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In 1987, Palmer and colleagues published work in Nature characterizing endothelium-derived relaxing factor (EDRF) as NO. [1; 2; 3] This work demonstrated NO’s critical role in biologic systems. Specifically, these investigators revealed that NO was released from vascular endothelium and played a crucial role in mediating smooth muscle vasorelaxation. Subsequently, numerous investigations have provided additional insights into the complexities and ubiquitous nature of NO. However, much remains to be learned.

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Briefly, NO is a generated in vivo from the amino acid L-arginine in the presence nitric oxide synthase (NOS). NO exerts its effects as a vasorelaxant via activation of guanylate cyclase (sGC), which enhances cyclic guanosine monophosphate (cGMP), ultimately resulting in smooth muscle relaxation. However, its role in biologic systems extends way beyond vasomotor control. In fact, NO plays a dual role both as a pro- and anti-inflammatory mediator. Moreover, NO down regulates leukocyte responses, decreases platelet aggregation, facilitates neurotransmission, augments bronchodilation, and attenuates inflammatory responses from such perturbations as ischemia and reperfusion. [4; 5; 6; 7] As its functions have been further elucidated, NO has attained a therapeutic role in a variety of disorders. Following Food and Drug Administration (FDA) approval in 1999 for a select group of neonatal patients, commercial availability of delivery systems and increased understanding of its role in disease states assisted in expanding the variety of applications it now enjoys.[6; 8] Inhaled NO’s utility in the modulation of pulmonary vasomotor tone is proven, and deemed part of standard clinical practice for pulmonary hypertension, especially in the pediatric population. Other disorders where inhaled NO has been used therapeutically include cardiac and lung transplantation, and acute respiratory distress syndrome (ARDS). Fittingly, continued work has focused on extending its therapeutic application in a host of other disorders such as acute myocardial infarction, sickle cell disease and solid organ 2

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transplantation. The information that follows presents a summary of NO biochemistry, potential NO toxicities, and will then expand on current clinical disorders where inhaled NO has demonstrated clinical efficacy or where there is emerging evidence of clinical promise. 1.1 NO BIOCHEMISTRY

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NO is a highly reactive molecule with other free radical species and possesses an extremely short halflife.[2] NO is produced endogenously or delivered exogenously where it can react with a variety of cellular targets resulting in vasorelaxation, enhanced neuronal transmission, reduced apoptosis, inhibition of neutrophil aggregation and adhesion, and modulate vascular smooth muscle proliferation.[9] NO synthesis is dependent on the enzyme nitric oxide synthase (NOS). This complex enzyme system generates NO from the terminal nitrogen atom of L-arginine in the presence of NADPH and dioxygen. NOS binds flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), heme, tetrahydrobiopterin (BH4) and calmodulin from L-arginine and oxygen by a family of three NO synthases (NOS), all of which are expressed in numerous cell types. The generation of NO leads to several actions that promotes smooth muscle relaxation. First, activation of guanylate cyclase raises the level of intracellular cGMP which in turn inhibits the entry of calcium into the cell thereby inducing smooth muscle relaxation. Secondly, activation of K+ channels leads to cellular hyperpolarization and relaxation. Finally, stimulation of cGMP-dependent protein kinase activates of myosin light chain phosphatase leading to dephosphorylation of myosin light chains resulting in smooth muscle relaxation. NOS proteins are related but encoded by distinct genes. Three distinct isoforms have been described. Neuronal NOS (NOS I), is produced in central and peripheral nerves and is pivotal in neuronal transmission and cell to cell communication within the central nervous system; Inducible NOS (NOS II), is exactly that, NOS that is induced by an inflammatory stimulus such as a microbe.[10] Unlike the other types of NOS (I and III), NOS II is generally not considered constitutive and is independent of calcium regulation. While NOS II is expressed by immune cells such as neutrophils and macrophages, it is also present in other cell lines including hepatocytes. Endothelial NOS (NOS III), is constitutively expressed by endothelial cells and is critical for the regulation of vascular function, more specifically vasorelaxation. Classically, the ability of NO to elicit vasorelaxation is due to its ability to increase intracellular levels of cyclic guanosine monophosphate (cGMP) through the activation of soluble guanylate cyclase (sGC). cGMP–dependent protein kinases in turn decrease the sensitivity of myosin to calcium-induced contraction and lower intracellular calcium by activation of calcium-sensitive potassium channels and inhibit the release of calcium from the sarcoplasmic reticulum. 1.2 FATE OF INHALED NO Chemical Reactivity

Inhaled NO will react avidly with other free radical species, various amino acids and transition metals due to its unpaired electrons. Atmospheric concentrations vary between 10-50 parts per billion up to 1.5 parts per million (ppm). Inhaled NO is generally administered clinically in a range of 1-80 ppm depending on the clinical end-point being targeted. Due to NO being delivered with varying concentrations of oxygen, the potential exists for the formation of nitrogen dioxide (NO2), an oxidant 3

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that can be injury inducing to the lung if its accumulation is significant. [8] In the setting of inflammation in which inhaled NO is commonly administered, reactions are expected with other free radical species such as superoxide yielding peroxynitrite or reacting with tyrosine residues of proteins which can alter cellular function.[11] Nitric oxide will also avidly bind with hemoglobin via reactions with iron from nitrosylhemoglobin. NO will also form methemoglobin, which should be monitored at scheduled intervals throughout its administration. Excessive met-hemoglobin accumulation can be appropriately treated by reducing or discontinuing NO therapy. Numerous other molecules can bind NO including albumin and thiol groups. Binding of NO to thiols forms S-nitrosothiol groups with can serve as downstream extra-pulmonary NO donors. High concentrations of NO (up to 80 ppm) are associated with increased concentrations of nitrite which is also may serve as substrate for downstream NO generation.

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Classic Physiological Actions with NO Inhalation

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As NO is delivered via inhalation, it will be preferentially directed to ventilated alveolar capillary units. The consequences of this will be selective pulmonary vasorelaxation resulting in reduced pulmonary artery pressures, pulmonary vascular resistance and right ventricular afterload. In other pathologic situations such as with the acute respiratory distress syndrome (ARDS), varying improvements in systemic arterial oxygen tensions are likely. Interestingly, who and why patients respond or not, and to what degree this occurs is complex and not fully understood, but conditions leading to increases in NOS II production are associated with decreased responsiveness compared to healthy volunteers.[12] Brisk withdrawal of inhaled NO when administered at clinically significant concentrations can lead to withdrawal as observed by significant “rebound” pulmonary hypertension that can promote the formation of right heart dysfunction. Withdrawal is thought to result from significant reductions in NOS III via down regulation from the exogenously delivered NO and elevated endothelin-1 (ET-1), a potent vasoconstrictor. Risk of rebound hypertension can be minimized by gradual weaning of the inhaled NO. Most recently, agents such as sildenafil, a phosphodiesterase-5 inhibitor that can prolong the effects of cGMP, have been observed to facilitate and prevent rebound hypertension. [3]

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1.4 SYSTEMIC EFFECTS AND TOXICITY OF INHALED NO

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Despite its short half-life and selectivity to the pulmonary circulation, inhaled NO possesses the potential to produce undesirable systemic side effects. Fortunately, most clinical trials have revealed relatively few untoward systemic effects or significant toxicities. Toxicity

In the presence of high concentrations of inspired O2, NO is oxidized to nitrogen dioxide (NO2). NO2 reacts with the alveolar lining fluid yielding nitric acid. Once dissolved in the alveolar lining fluid, NO reacts with superoxide, leading to the formation of peroxynitrite (OONO), a potentially potent oxidant that possesses the potential to decompose into the injury producing, hydroxyl anion.[2] In addition, peroxynitrite may react with tyrosine residues on proteins leading to the production of tyrosine nitrated proteins (nitrotyrosine). Nitrotyrosine residues have the potential to be cytotoxic and have been implicated in impaired surfactant production. Nitrotyrosine residues can be quantitated are considered a marker of oxidative stress. The rate at which NO is oxidized to NO2 depends on the square of NO 4

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concentration and concentration of oxygen to which it is exposed. In order to protect against NO2 toxicity, inhaled NO should be administered with the lowest possible O2 concentration. NO and NO2 concentrations should be monitored and exhaled gases scavenged. [13] The Occupational Health and Safety Administrations (OSHA) permissible exposure limit to NO for employees in the workplace is 25 ppm averaged over an 8 hour work shift. NO – Hemoglobin Interactions

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NO reacts with oxyhemoglobin to yield methemoglobin (MetHb). MetHb is not an effective oxygen carrier and may lead to systemic hypoxemia when significant concentrations accrue. MetHb shifts the oxyhemoglobin curve to the left thus preventing unloading of oxygen from hemoglobin. At concentrations of 0-15% patients are generally asymptomatic, at 15-20% concentrations cyanosis develops, at 20-25% weakness develops progressing to acidosis, and lastly coma occurs when concentrations attain 50-70%. Concentrations > 70% are fatal. MetHb levels should be methodically monitored by co-oximetry during administration of inhaled NO and appropriate reductions in inhaled NO concentrations made according to MetHb concentrations. 1.5 EARLY CLINICAL USE

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2. PEDIATRICS

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The discovery of NO and its action of vasorelaxation resulted in a relatively fast translation to a clinically applied therapeutic agent. Since NO is short-lived and exists in a gaseous form, it quickly made its way to the bedside as an inhaled agent for patients with cardiopulmonary derangements. While a number of compounds were known to induce pulmonary artery vasorelaxation, some via NO generating pathways, virtually all exerted this effect systemically and led to unwanted reductions in critical perfusion pressures and counteracted important compensatory actions such as hypoxic pulmonary vasoconstriction. In 1991, Frostell et al demonstrated the clinical efficacy of inhaled NO in lambs suffering from pulmonary hypertension.[14] As the group had hypothesized, the inhalation of NO predictably decreased pulmonary artery pressures without altering systemic arterial pressures. This series of investigations provided significant evidence of NO’s therapeutic potential and catalyzed other clinical investigations accelerating its routine use as inhaled therapy.

2.1 PERSISTENT PULMONARY HYPERTENSION OF THE NEWBORN (PPHN) The initial FDA approval for the first clinical application of inhaled NO released in 1999 was and remains: “The safety and efficacy of nitric oxide for inhalation has been demonstrated in term and near term neonates with hypoxic respiratory failure associated with evidence of pulmonary hypertension…” Following the approval much hope and excitement led to clinical trials to try and prove iNO would serve to prevent the pathologic increase in pulmonary vascular resistance associated with PPHN and more importantly decrease the incidence and mortality associated with the development of bronchopulmonary dysplasia.

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In utero, the lung develops within a hypoxic environment created by the fetal circulation. The developing alveoli are perfused with approximately 20% of the fetal cardiac output and the pulmonary vasculature exhibits increased resistance as a component of normal fetal development. With delivery the lung is exposed to mechanical expansion and increasing oxygen concentration with the onset of respiration. These factors serve to dramatically decrease pulmonary vascular resistance (PVR) over the ensuing first few hours of life and ultimately drop to near adult levels within the first six weeks of life.[15] Failure to achieve this decrement in resistance within the early post-natal period results in persistent pulmonary hypertension. Although multiple etiologies exist for pulmonary hypertension within the neonate this review is primarily focused on clinical applications of inhaled NO in PPHN. PPHN may occur as the result of meconium aspiration syndrome, pulmonary hypoplasia, or abnormal pulmonary vascular remodeling also known as idiopathic PPHN. [16]

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PPHN remains a significant problem occurring in up to 1-2 per 1000 live births with a high mortality of 10-20%. [17; 18] Typically within hours of delivery the baby with PPHN will exhibit cyanosis often to a greater degree from a post ductal compared to a preductal sample due to right to left shunting at the level of the ductus. The ensuing workup will evaluate a broad differential including meconium aspiration, sepsis, or congenital heart disease. Standard therapy is focused on maintaining adequate hemodynamics and tissue oxygen delivery while correcting contributing factors that increase PVR. Treatments to control PVR include oxygen supplementation, and avoidance of hypothermia, agitation and acidosis. Frequently the cyanosis is refractory to treatment and escalation in therapy often results involving mechanical ventilation, oxygen therapy and sedation. Although, not uniformly accepted, alkalinisation, remains practiced with special care to avoid iatrogenic lung injury. Further deterioration in hemodynamics may require inotropic support of the right heart, high frequency oscillatory ventilation, and potentially ECMO. Nitric oxide having recently been discovered as the molecule previously described as “endothelium derived relaxing factor (EDRF)” was being developed to serve a therapeutic role in this disease process and human applications were trialed in the early 1990’s.

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A few pre-clinical studies and case series first described the positive effects of iNO on PPHN and the ability of iNO to improve hemodynamics and oxygenation.[14; 19; 20] These results were followed with multi-center trials that have helped shape our treatment guidelines for iNO.[6; 21; 22] These trials have been compiled in a relatively recent review by the Cochrane Library which concluded that inhaled NO provided a clear benefit with regard to ventilation/perfusion mismatching and oxygenation. In addition inhaled NO therapy in term and near term neonates with PPHN decreases the need for ECMO with few side effects. Despite short term gains in these clinical parameters, no mortality benefit has been reported in these trials and as outlined below the long term prevention of bronchopulmonary dysplasia is less clear. [21; 22; 23] Current recommendations suggest inhaled NO can be started in term on near term neonates when the oxygen index (OI = mean airway pressure × FiO2 × 100/PaO2) exceeds 25 or when the PaO2 is <100mmHg when the patient is receiving 100% FiO2. Of note neither these recommendations nor the inclusion criteria of some of the major trials (Neonatal Inhaled Nitric Oxide Study, NINOS) include the language of the initial FDA application with regards to evidence of pulmonary hypertension. In these patients, inhaled NO has shown a clear benefit in improving oxygenation and decreasing the need for ECMO.[23] 6

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Dosing of inhaled NO however remains a more complex question as one of the early randomized controlled trials, the NINOS trial, reported that the most effective dose of inhaled NO was 20ppm. However, within this study 6% of the patients who did not respond to a dose of 20 ppm did exhibit a response to 80 ppm.[6] Given the small size of the population requiring high doses of inhaled NO, the increased risk of met-hemoglobinemia, and multiple reports displaying equivalent responses to doses as small as 5 ppm, the Cochrane review best addresses the question of dose by concluding that current dosing regimens are likely above the response threshold. Therefore, many centers initiate inhaled NO at a dose of 20 ppm and wean as tolerated.[23] 2.2 BRONCHO-PULMONARY DYSPLASIA (BPD)

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The definition of broncho-pulmonary dysplasia or chronic lung disease of infancy recently underwent a revision and is now described as arrested lung growth with reduced alveolarization and dysmorphic vasculature.[24] As alluded to in the previous section the development of BPD was hoped to be the prime target for inhaled NO therapy due to the high morbidity of BPD in neonates suffering from PPHN. Early animal studies showed that inhaled NO therapy increased angiogenesis and alveolarisation in animal models of disease.[25; 26; 27; 28] However as outlined below the ability of inhaled NO to prevent the progression of pulmonary disease in the neonate to BPD have been difficult to realize in clinical trials.

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Clinical trials to search for prevention or improvement of BPD in infants with varying degrees of respiratory distress have been inconclusive. Inhaled NO was not associated with any positive endpoints in one study investigating infants with severe respiratory compromise but was affective in less severely compromised infants.[29; 30] Further studies have suggested inhaled NO therapy may reduce the incidence of acute brain injury while other studies have been unable to reproduce this association.[31; 32] In light of these inconclusive findings and the continued application of inhaled NO in these settings the National Institutes of Health produced a Consensus and State of the Science Statement regarding application of inhaled NO in infants at risk for developing BPD.

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The NIH consensus statement outlined that available evidence does not support the use of inhaled NO in early-routine, early-rescue, or later rescue regimens in preterm infants requiring respiratory support.[33] The NIH went on to acknowledge the existence of certain, although rare, clinical scenarios such as pulmonary hypertension in which inhaled NO may be of benefit but that will require further study. Additionally an update to the Cochrane review discussed above was published in 2010 in response to the conclusion of the EUNO study.[34] EUNO investigated the relationship between a low dose inhaled NO therapy and lung vascularization and development.[35] After consideration of the EUNO trial the Cochrane database updated the conclusions of the review in 2010 to reflect that: “there are no clear indications for inhaled NO in preterm infants.” Taken together these consensus statements highlight that inhaled NO has failed to demonstrate the therapeutic potential to limit the progression of pulmonary disease of the newborn and development of bronchopulmonary dysplasia. Certainly, further work is needed to determine the cause of the gap between benefits measured in preclinical animal studies and trials in humans. As of publication of this review there are currently four trials ongoing to

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gain further insight into the application of iNO in combination with various dosing regimens or adjuvant therapies. 2.3 TOXICITY IN NEWBORNS

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As described in previous sections of this review the toxicity of inhaled NO is due in part to its chemical reaction with oxygen resulting in the formation of more toxic intermediates such as nitrogen dioxide. Additionally, NO reacts with oxyhemoglobin within the red cell resulting in oxidation of the hemoglobin to form met-hemoglobin (metHb). MetHb is unable to reversibly bind and transport oxygen. Although inhaled NO maintains a large therapeutic window in adults due to the reductive capacity of metHb reductase within the red cell it is still important to monitor the patient for development of methemoglobinemia when receiving inhaled NO therapy.

2.4 CONGENITAL CARDIAC DISEASE

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Within the pediatric patient, and especially in the neonate, the risk for injury is greater due to two important differences. First, the presence of fetal hemoglobin presents a target for inhaled NO that is more readily oxidized to metHb. This is compounded by the fact that RBC’s within pre-term infants express significantly less metHb reductase.[36] Therefore, utilization of inhaled NO in neonates involves minimizing dose and close monitoring for the development of met-hemoglobinemia.

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Although application of inhaled NO therapy in patients undergoing repair of congenital cardiac lesions has been less extensively studied it has likely been more extensively employed as therapy. One of the earliest clinical studies of inhaled NO by Wessel et al in 1992 investigated the etiology of postoperative pulmonary hypertension in pediatric patients undergoing cardiac surgery.[37] This work supported the concept of pulmonary vascular endothelial dysfunction as evidenced by a decreased response to acetylcholine post bypass. This injury was accompanied by an enhanced response to inhaled NO compared to pre-bypass conditions. Interestingly this study led to other small trials investigating the application of inhaled NO in refractory pulmonary hypertension in children after cardiac surgery which suggested a potentially lifesaving role for inhaled NO and the need for more rigorous randomized studies.[38; 39]

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One of the largest studies to date investigated 124 patients undergoing an atrio-ventricular septal defect or unrestrictive ventricular septal defect repair with evidence of increased pulmonary artery pressure or blood flow pre-operatively. Patients were randomized to receive 10 ppm iNO or placebo (nitrogen) postsurgery until extubation. Patients receiving inhaled NO had 30% fewer pulmonary hypertensive crises and shorter median time to extubation readiness.[39] However, the study was underpowered to address endpoints such as mortality which is a common and understandable difficulty of developing randomized trials addressing mortality in congenital cardiac patients. This point is further emphasized by a Cochrane database review performed in 2008 which despite limitations of methodological variability, lack of adequate sample size, and differences in patient populations, drew the conclusion that inhaled NO made no difference in the outcomes evaluated.[40] However, the limitations listed make drawing any conclusions from the Cochrane review difficult to interpret.

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Beyond post-operative pulmonary hypertensive crises, congenital cardiac surgery involving palliative conversion to cavo-pulmonary circulation such as the Glenn procedure or Fontan presents a unique setting for the application of iNO. Patients who have undergone these procedures depend on minimization of the trans-pulmonary pressure gradient between the venous supply of pulmonary blood flow and atria of the heart in order to facilitate adequate cardiac preload. Therefore, these patients are evaluated very closely pre-operatively for the existence of elevated pulmonary vascular resistance and the potential reversibility of this resistance. Within the catheterization lab oxygen and iNO are employed to identify patients that may be amenable to other therapies such as oral phosphodiesterase inhibitors for medical optimization. Additionally, this data is used to identify referral candidates for surgical repair or transplant procedures.[41]

3.0 ADULTS

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Once these patients progress on to operative repair further studies have been done investigating the postoperative course of patients receiving either partial (bidirectional Glenn) or total cavo-pulmonary (Fontan) anastomosis. The variable contribution of mixed venous saturation via either inferior cava blood return (Glenn) or flow across a fenestration (Fontan) to the systemic arterial saturation can be measured postoperatively to determine the effect of therapies to alter pulmonary vascular resistance. Taken together these studies demonstrate the ability of inhaled NO at doses from 1.5-20ppm to decrease PVR and improve systemic saturations in patients with elevated trans-pulmonary gradients after cavo-pulmonary anastomosis.[42; 43] However, beyond these case series supporting a test application of iNO in patients with elevated pulmonary vascular resistance after cardiac surgery as a component of goal directed therapy, there are currently no adequate clinical trials to inform us of the mortality benefit of iNO after congenital cardiac surgery. Certainly future multi-center trials are likely required to obtain the power sufficient to more clearly define the role of inhaled NO after cardiac surgery in children.

3.1 Acute Respiratory Distress Syndrome (ARDS)

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The utilization of inhaled NO to treat patients suffering from ARDS dates back to the early 1990s.[44] Numerous therapies have been tested to reduce hypoxemia in ARDS and NO was the new kid on the block. However, over the last two decades the potential therapeutic role of inhaled NO remains unclear. Results of studies thus far have been only moderately successful. This review will discuss the physiologic basis, address the literature evaluating the clinical indications and describe future therapeutic areas of interest for patients with ARDS. Before evaluating the role of NO it is important to emphasize that the history of inhaled NO with ARDS has paralleled a time period of significant changes to the management of patients with ARDS. Even the definition of ARDS has undergone several iterations over this time period. In 1994, the AmericanEuropean Consensus Conference (AECC) provided the initial definition of ARDS as acute onset of hypoxemia (arterial partial pressure of oxygen to fraction of inspired oxygen [PaO2/FIO2] ≤ 200 mm Hg) with bilateral infiltrates on frontal chest radiograph, and no evidence of left atrial hypertension. Also, a new entity with less severe hypoxemia but similar criteria was defined, acute lung injury.[45] However 9

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this initial definition led to some ambiguities that required an updated definition of ARDS proposed by the ‘ARDS Definition Task Force’ termed the Berlin criteria. The updated definition specified “acute” as < 1 week, bilateral opacities as not fully explained by effusions, lobar collapse or pulmonary nodules, origin of edema as respiratory failure not fully explained by cardiac failure or fluid overload and Pa02/FI02 ratio as measured with a minimum of 5 mmHg of positive end expiratory pressure (PEEP).[46] The initial publication delivering inhaled NO to patients suffering from ARDS was published in 1993 but did not provide a specific definition of ARDS and even the acronym was different. Instead of “A” in ARDS standing for “acute” it stood for “adult”. This led to patients being enrolled in the 1993 study 14 days after being ventilated. While this limitation of a changing definition is worth understanding, practically all the literature reviewed in this article used the AECC definition and was published prior to the Berlin definition.

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Another limitation of the literature is to understand the change in management of patients with ARDS over the last two decades. In the early 1990s the standard of care included limiting peak pressures to less than 40 mmHg, utilizing 10-15 cm H20 of PEEP, proning patients and employing veno-venous extracorporeal membrane oxygenation if necessary.[47] This compares to present day practice protocols that employ volume and pressure-limited ventilation, places emphasis on fluid limitation , employs PEEP and relies on minimizing harmful therapies. [48; 49; 50; 51] Many of the advancements in ARDS paralleled the studies iNO and require effort to review the literature in the time period of the standard of care in which it was published.

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ARDS is a heterogeneous entity that leads to pathologic diffuse alveolar damage of lung units (Figure 1). [52] The affected alveoli are not able to maintain effective ventilation and perfusion matching thus results in hypoxemia. One of the benefits of inhaled NO is that, unlike intravenous therapies, it selectively targets lung units with varying degrees of ventilation. As inhaled NO diffuses across the alveolar membrane, it avidly binds hemoglobin, which may limit its systemic signs. This property allows for inhaled NO to positively modulate the pulmonary circulation. The inhalation of NO selectively works on the area of the lung that maintains ventilation and dilates the associated pulmonary vessels to improve the matching of ventilation and perfusion.[53] The lung unit in ARDS that maintains ventilation will be the same lung unit whose vasculature will dilate and preferentially receive more of the systemic blood flow. The lung units with impaired ventilation will receive a lower ratio of perfusion and provide a lower amount of the body’s total arterial oxygenation.[14; 54] Initial studies of patients with ARDS compared systemic vasodilators (prostacyclin) with inhaled NO and discovered that systemic therapies worsened oxygenation due to counteracting the body’s hypoxic pulmonary vasoconstriction (HPV). Inhaled NO, instead, preserved HPV and augmented the partial pressure of oxygen due to the aforementioned mechanism.[44] Why is inhaled NO thought to improve clinical outcomes for patients with ARDS? Conventional wisdom was that an increase in PaO2 resulting from inhaled NO would allow for a decrease in FiO2 and in turn lead to less detrimental effects from reactive oxygen species, a major contributor of inflammatorymediate injury.[55; 56] Furthermore, decreases in pulmonary artery pressures may improve right ventricular function and reductions in pulmonary capillary wedge pressure may diminish pulmonary edema formation. In addition, benefit may have been gained via an anti-inflammatory and anti10

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thrombotic property of inhaled NO which may have benefits for patients suffering from ARDS. [57; 58; 59; 60]

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Rossaint at el published the seminal trial in 1993 comparing inhaled NO to intravenous prostacyclin to improve oxygenation and hemodynamic parameters in patients with ARDS. The trial included 10 patients that had been intubated for 14 days prior to enrollment. Concentrations of inhaled NO administered ranged from 5-20 ppm. Inhaled NO administration led to a significant reduction in pulmonary-artery pressures, increase in PaO2/FiO2 and improved pulmonary gas exchange. In contrast, intravenously administered vasodilators led to an increase in intrapulmonary shunting resulting in reductions in PaO2. These observations served as a springboard for further investigations of inhaled NO’s efficacy in the clinical setting of critical disease.[44]

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A larger study published by the same group assessing longer-term outcomes with the use of inhaled NO was undertaken. They retrospectively studied thirty patients from 1991-1992 that presented to their tertiary referral center for care due to ARDS and who required treatment with inhaled NO. Prior to administration of inhaled NO the patients on average had been mechanically ventilated for 20 days. The average dose of medication administered was 11.5 +/- 1.4 ppm for more than 48 hours. Similar to previous studies they found an increase in PaO2/FIO2 from 98 +/- 8 mm Hg to 132 +/- 12 mm Hg (p<0.001) and a reduction of pulmonary arterial pressures (PAP) from 35 +/- 2 mmHg to 31 +/- 2 mmHg (p<0.001). [61; 62; 63] Also, when the inhaled NO was temporarily discontinued arterial oxygenation would fall and the PAP would rise to pre-treatment values. Tachyphylaxis was not observed with prolonged administration. They also did not find an improvement in survival with administration of inhaled NO.[47] While the results were not as compelling as predicted, the study was a retrospective evaluation and so encouraged researchers to develop a prospective multicenter randomized trial to evaluate the long-term effects of inhaled NO.

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A common clinical observation was that similar patients with ARDS responded to inhaled NO while other patients did not.[64; 65] Manktelow et al sought to understand if patients who initially respond favorably to inhaled NO were the same patients that would continue to respond after 48 hours of therapy. They completed a retrospective study of 92 patients with ARDS and were administered inhaled NO. They discovered that almost two thirds of patients responded to inhaled NO and of those patients that responded the PaO2/FIO2 remained significantly higher (120 +/- 46 vs 89 +/-32 mmHg) and the pulmonary artery pressures were lower.[12] Also consistent with previous studies is that clinically significant met-hemoglobinemia was a rare occurrence when administration of inhaled NO concentrations were < 40 ppm. [44; 47; 61; 65] Another intriguing finding was that patients with septic shock were less likely to respond favorably. This later finding will be readdressed as this influenced study inclusion in subsequent trials, restricting inclusion to patients with non-sepsis cases of ARDS.[66] One of the first multi-center, randomized prospective trials evaluating inhaled NO in ARDS patients was completed by Dellinger et al.[67] (Table 1) This trial included 30 centers across the United States, a total of 177 patients and the patients were followed for 28 days. All patients met the AECC definition of ARDS and were enrolled in the study if onset of disease was 72 hours prior to randomization. Patients were excluded if they had a non-pulmonary cause of ARDS. Randomization included placebo or one of 11

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five different concentrations of NO (1.25, 5, 20, 40, 80 ppm). Patients were maintained on the treatment gas until the end of the study (day 28) or until adequate oxygenation was achieved. The standard of care for all patients with ARDS included maintaining PEEP to optimize compliance (usually 8 to 12 cm H2O) and to decrease inspiratory plateau pressure to
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Subsequently, a European multicenter randomized controlled trial was published that had confirmed Dellinger et al. conclusions.[67; 69] In this study, 180 patients were randomized to either inhaled NO versus placebo with the primary outcome being frequency of reversal of acute lung injury and the secondary outcome being patient mortality. Conclusions from this study supported a previous unblinded small trial as well as Dellinger’s et al RCT that no treatment difference existed between the intervention group and placebo.[70] One important point of note was that therapy for ARDS other than inhaled NO administration was not standardized and so questions arose as to the groups truly being similar. Also of note is that this was the first trial to demonstrate that inhaled NO treated patients required more renal replacement therapy than the placebo group, an observation that has not been a consistent finding.

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The most recent RCT to evaluate the efficacy of inhaled NO was published in 2004.[71] This study was based on the previous observation that patients had a significant improvement in ventilation-free survival at 28 days who received 5 ppm of inhaled NO (Figure 1). [67] Interestingly, patients were enrolled between 1996 and 1999 but the study was not published until 2004. This is noteworthy because the standard of care for both arms of the study changed significantly during the time interval. At the time this study was completed the goals of mechanical ventilation included limiting plateau pressure to < 35 cm H2O. However there was no limitation or guidance with respect to tidal volume, which we now know contributes to lung injury. The administration of neuromuscular blocker or fluid management was not standardized as was the use of adjunctive therapies such as prone positioning or extracorporeal membrane oxygenation. Consequently, another seminal trial provided support for a volume and pressure limited strategy of lung ventilation and research showed that limited tidal volumes even in patients at an optimal plateau pressure was still beneficial.[71] Further, our knowledge as to the benefits of PEEP all resulted in a reduction in ARDS mortality. With this information serving as a background this study found that inhaled NO administered at 5 ppm had no effect on patient ventilator time or mortality. Several meta-analyses have concluded inhaled NO has not influenced mortality in

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ARDS patients. Inhaled NO may lead to variable improvements in oxygenation and possibly predispose patients to renal dysfunction. [72; 73; 74]

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Interestingly, Dellinger et al. analyzed the 2004 Taylor et al. study to assess for long-term pulmonary function benefits from inhaled NO in survivors.[75] They found that ARDS patients who survived after treatment and had received low-dose inhaled NO had significantly better pulmonary function values at 6 months compared to the placebo-treated patients. The mechanism to explain this finding is unknown and premorbid pulmonary function tests were unavailable. However this observation does make us ponder inhaled NO’s potential on other important clinical outcomes other than mortality.

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Why inhaled NO has not decreased mortality seems multifactorial. In the Lundin et al trial there was a signal for increased renal failure in patients who received inhaled NO and in the Taylor et al trial patients receiving inhaled NO demonstrated an increased incidence of nosocomial infections.[67; 70; 76] Both of these unintended consequences may have negated a benefit of inhaled NO. It also is important to note that while studies have consistently shown an increase in PaO2 when patients received inhaled NO, studies have also shown that hypoxemia is rarely the cause of death in patients with ARDS. Only 10-15% of patients die of refractory hypoxemia with ARDS while a majority of patients die of multiple organ failure.[76]

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We now know that lower tidal volumes are beneficial for patients. Lower tidal volumes may also lead to worsened short-term oxygenation. Interestingly, of all possible outcomes the positive outcome most consistently demonstrated from inhaled NO in patients suffering from ARDS is that inhaled NO improves oxygenation.[77] One may argue that if a trial was completed with the standard of care being lung protective ventilation then supplemental inhaled NO may assist to achieve even lower tidal volumes in patients unable to achieve their targets due to worsening oxygenation. It would appear that inhaled NO should not be routinely used in the treatment for patients with ARDS. It seems reasonable to consider inhaled NO in patients with refractory hypoxemia knowing that it may not improve long-term outcomes but may serve as bridge of sorts to other therapies or to allow for therapeutic benefit to be had from other therapies. Could inhaled NO improve the patient’s oxygenation to allow the patient to continue on lung protective ventilation at a goal tidal volume and possibly lead to improved outcomes?

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3.2 Post-Cardiac Arrest

The use of inhaled NO is in the early stages of evaluation in patients following cardiac arrest following the demonstration of potential injury protection during ischemia and reperfusion. Previous animal investigations have demonstrated that inhaled NO when administered prior to ischemia-reperfusion reduces vital organ injury via a mechanism based on endothelial protection.[78] Other studies in animal models have supported a protective immunomodulatory role from inhaled NO post-transplant either through inhibition of pulmonary vasoconstriction or limiting peripheral blood polymorphonuclear neutrophil adhesion to vascular endothelium.[79; 80] Further, studies have demonstrated that inhaled NO decreases myocardial infarction size and improves left ventricular function in rats.[81] The beneficial effects of inhaled NO have been confirmed in follow up studies assessing myocardial injury post-cardiac arrest.[82] Kida et al found that breathing inhaled NO within 1 hour after return of spontaneous 13

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circulation improved neurological and myocardial function as well as overall survival at 10 days.[83] In addition injury attenuation, the absence of systemic hypotension from inhaled NO has been viewed as advantageous. These findings are very encouraging and should spawn further research in more sophisticated animal models and ultimately in humans that are post-cardiac arrest.

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3.3 Sickle Cell Disease (SCD) and Acute Chest Syndrome (ACS)

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SCD is a disorder caused by the substitution of a single amino acid (valine for glutamic acid) in the human B-globulin subunit. Patients with this disorder are at a higher risk of vaso-occlusive complications. The sickled hemoglobin when deoxygenated has a higher affinity to polymerize and lead to decreased red cell deformability and distortion of the cell into a crescent sickle shape. These cells have a higher affinity for vaso-occlusion and hemolysis. ACS is an acute pulmonary disease secondary to vaso-oclusion within the pulmonary vascular. Studies define the syndrome as a new pulmonary infiltrate involving at least one complete lung segment consistent with consolidation but excluding atelectasis, with at least one of the following: chest pain, fever, tachypnea, wheezing, sputum production or cough. These were the definitions used for inclusion criteria in two large studies of patients with acute chest syndrome.[84; 85] The etiologies of ACS are variable but the two most common causes are fat embolism and infection. ACS is responsible for one-quarter of mortality in sickle cell disease. [86]

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The proposed mechanisms of inhaled NO in alternative disease processes such as ARDS can also be used as a construct to understand inhaled NO and ACS. Previous reports have found that inhaled NO can improve pulmonary hypertension and ventilation-perfusion matching. Inhaled NO will preferentially vasodilate the pulmonary circulation in continuity with non-diseased alveoli and therefore improves ventilation-perfusion matching. NO will also decrease pulmonary vascular resistance by this same mechanism. Other research supports NO ability to improve hemoglobin’s affinity to oxygen, lessen deoxygenated hemoglobin and possibly decrease polymerization of hemoglobin. NO can decrease the P50 (partial pressure of oxygen at 50% hemoglobin saturation) of hemoglobin S, thus decreasing polymerization.[87; 88] It has also been postulated that NO may have hematologic effects, such as inhibition of platelet aggregation, reducing cellular-adhesion molecule and cytokine expression, but this research has primarily been performed in in animal models and has not been tested in humans with ACS.[89; 90] Unfortunately, inhaled NO has only been used in isolated cases of mechanically ventilated patients with ACS and there are no randomized studies to assess the benefits and risks of this treatment.[91] In the case reports concentrations of inhaled NO varied between 20-80 ppm and in most patients there was a dramatic improvement in oxygenation and drop in pulmonary vascular resistance.[92; 93; 94] Further, met-hemoglobinemia remained below levels. From these case reports, the long term effects of inhaled NO are unknown or if these patients would have improved on their own without inhaled NO. A recent study evaluated the effectiveness of inhaled NO in treating pain crises secondary to vaso-occlusive disease in patients with SCD.[95] The study objective was to determine if inhaled NO administered via face mask could reduce the duration of pain crises in patients with established SCD. Study participants initially received 80 ppm of inhaled NO for 4 hours followed by 40 ppm for another 4 hours. The 14

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concentrations were subsequently decreased to 5 ppm for the duration of treatment protocol.[95] At the time of enrollment no patients were suffering from ACS and there was no difference between the placebo group and the inhaled NO in the freedom from parenteral opioids, pain relief using Visual Analogue Score of 6 or lower or ability to ambulate. While this study did not specifically evaluate patients with ACS it did establish the fact that inhaled NO is safe in patients with SCD. From the current literature it is plausible inhaled NO may yet benefit certain patients with ACS but until further studies are completed, inhaled NO should not be routinely used for treatment of patients with ACS. 3.4 Chronic Obstructive Pulmonary Disease (COPD)

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Patients with COPD may develop hypoxemia from severe ventilation-perfusion defects, elevated pulmonary pressures from vascular destruction and air trapping ultimately leading to cor pulmonle and right ventricular failure. Small studies have sought to evaluate the efficacy of inhaled NO in decreasing pulmonary arterial pressures and/or improvements in arterial oxygenation. Thus far, few studies exist and patient enrollments are small. In the investigations that do exist inhaled NO has failed to improve oxygenation in patients with COPD and may actually worsen it.[96; 97] Measurements of the effects on pulmonary artery pressure and right heart function have not been studied in this specific patient population. 3.5 Extracorporeal Membrane Oxygenation (ECMO)

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ECMO is used in severe cardiac and/or pulmonary failure. It carries a high mortality rate (mostly related to the underlying etiology of the primary disease process for which it is indicated). ECMO provides temporary support for these patients with the hope of recovery or potential bridging to organ transplantation. There is also a high morbidity associated with ECMO including bleeding, thromboembolic events, infection, limb ischemia and vascular injury. [98; 99]

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Inhaled NO has been used in conjunction with ECMO to assist right ventricular function along with potentially improving oxygenation in weaning from ECMO support. Inhaled pulmonary vasodilators selectively dilate ventilated-perfused pulmonary vasculature thereby decreasing pulmonary vascular resistance and improving right ventricular performance. This decrease in pulmonary vascular resistance reduces the stroke work done by the right ventricle. The net improvement in oxygenation and reduction in right ventricle work provides at least a theoretical advantage for improving right ventricular function and oxygenation to allow for weaning from veno-venous and veno-arterial ECMO. There is insufficient data or randomized controlled trials demonstrating efficacy of inhaled NO used in conjunction with ECMO. However, when used for right ventricular (RV) support or for decreasing pulmonary artery pressures in support of RV this drug has utility. In a trial randomizing patients to ECMO versus conventional ventilation with severe respiratory failure (CESAR), inhaled NO was used in a higher percentage of patients on ECMO support.[100] In our experience, we often use inhaled NO as an adjunct for decreasing pulmonary vascular resistance and improving right ventricular performance when weaning from ECMO support but prospective clinical trials have not been conducted probably due to the fact that inhaled NO is in many cases already being used for the primary disease process requiring the implementation to ECMO. 15

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3.6 Cardiopulmonary Bypass

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3.7 Lung and Heart Transplantation

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Cardiopulmonary bypass (CPB) has been shown to have a detrimental effect on lung pulmonary vascular resistance. The underlying mechanism is thought to be related to pulmonary hypo-perfusion or activation of systemic inflammatory response during (CPB).[101; 102] In a murine model with left anterior descending (LAD) coronary artery occlusion, inhaled NO decreased infarct size and improved left ventricular performance without causing hypotension.[81] Studies are limited assessing the effects of inhaled NO in patients undergoing cardiopulmonary bypass. Gianetti and colleagues enrolled 29 consecutive patients for cardiac procedures requiring CPB. Significant decreases in myocardial injury were appreciated when receiving inhaled NO for eight hours during and after CPB .[103] The protective mechanism was not elucidated but postulated to be due to anti-inflammatory modulation. [104] A subsequent randomized controlled study of 58 patients with severe mitral stenosis, pulmonary hypertension and reversible pulmonary vascular resistance showed an increase in cardiac output, a decrease in pulmonary pressures and resistance when inhaled NO and prostacyclin were utilized.[105] In a study of 29 patients with mitral stenosis and severe pulmonary hypertension, shorter ICU stays and fewer vasoactive medications were attributed to the administration of inhaled NO.[106] In children undergoing Tetrology of Fallot repair, if treated with inhaled NO decreased ventilator time, ICU length of stay, lower troponin and BNP levels were observed.[107] Additional positive findings in the inhaled NO treated group were reduced fluid balance, reduced need for diuretics, and overall less complicated ICU course. At the present it appears that select patients undergoing cardiopulmonary bypass benefit demonstrate some benefit when inhaled NO is employed.

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No large randomized controlled studies exist assessing the efficacy of prophylactic inhaled NO use in orthotopic lung transplant (OLT) patients, however smaller series do exist. Based on these in aggregate, no evidence that the use of prophylactic nitric oxide reduces complications or prevents primary graft dysfunction.[108; 109] Collectively, there is no decreased hospital stay, ventilator times or mortality, however one study (n=14 patients compared to historical controls) displayed a decreased ICU length of stay and trend towards decreased in acute rejection. Moreno et al demonstrated a decreased incidence of primary graft dysfunction and expression of inflammatory mediators (interleukin-6 and interleukin-10) in patients receiving 10 ppm of inhaled NO that was begun at the start of the case and continued for the first 48 hours post-operatively.[110] A prospective study from Columbia University demonstrated inhaled NO was associated with improved oxygenation, need for mechanical ventilation and decreased two-month mortality rate. There was no difference in long-term mortality, however. [111] In summary, inhaled NO is commonly used during and after lung transplantation surgery to assist in enhancing oxygenation and reduced pulmonary artery pressures but larger trials are need to definitively demonstrated improvement of major outcome metrics. As with OLT, inhaled NO is also commonly used as an adjunct in orthotopic heart transplantations (OHT) but there is a paucity of clinical trials specifically addressing the efficacy of inhaled NO in this patient population. In one prospective study use of inhaled NO demonstrated no difference in survival,

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however there was an improvement in right ventricular function and reduction in pulmonary pressures and resistance. [111]

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Right ventricular dysfunction and hypoxemia are commonly encountered difficulties associated with OHT and OLT surgeries providing a niche for inhaled NO both in and out of the operating room. Larger cohort studies or ideally randomized controlled trials are necessary to determine their clinical benefit, but in our clinical practice and that of others it appears the inhaled NO use has largely become the standard of care without the confirmatory data to back it up. 3.8 Left Ventricular Assist Devices

4.0 ISCHEMIA-REPERFUSION INJURY (IRI)

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Left ventricular assist devices (LVADs) have been used in patients with end stage heart failure for several decades. These devices are used as a bridge to transplant or as destination therapy. Right ventricular failure is encountered in 5-39% of post-LVAD implantations. There are many advantages of an LVAD over a total artificial heart or biventricular assist device, which are beyond the scope of this article. In a prospective RCT with assessing the effect of inhaled NO in LVAD placement there was no difference in RV dysfunction, reduction in the need for right ventricular assist device placement, or intubation times. [112]

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IRI is a complex series of events involving intracellular injury and giving rise to inflammatory responses that can cause vital organ injury and failure. [113] Ischemia leads to anoxic injury that results in the loss of adenosine triphospate (ATP) production thus results in a failure of the cell to sustain its homeostatic functions. As a consequence of these intracellular alterations there is an increase in membrane permeability and inability to regulate cytosolic pH and Na+/K+ ATPases. The net effect can be mitochondrial permeability transition and subsequent cellular necrosis, cellular apoptosis (programmed cell death) or necroapoptosis. Reperfusion occurs with the reintroduction of molecular oxygen in conjunction with the reestablishment of blood flow following either warm or cold ischemia. The inflammatory responses triggered are extremely complex but appear to begin with the production of reactive oxygen species such as superoxide radical which serve to attract neutrophils whereby they adhese to the primed cellular endothelium and synthesize cytokines. The balance between NO, a local vasodilator in this circumstance and endothelin-1 (ET-1) is lost resulting in reductions in NO concentrations and increases in ET-1 with the net effect being vasoconstriction and microcirculatory failure due to a low or low flow state. Thus, replenishing NO via therapeutic administration may serve to abrogate reperfusion injury in vulnerable patients. NO-mediated protection in IRI can occur via multiple mechanisms including cytoprotection, anti-inflammatory effects, modulation of mitochondrial respiration, antioxidant effects and maintenance of vasomotor tone within a vulnerable organ. In contrast, NO can also contribute to IRI via formation of secondary reactive nitrogen species including, peroxynitrite. [114] As previously pointed out, traditional thinking has been that inhaled NO crosses the alveolar-capillary membrane and is rendered inactive by rapid reactions with oxy- or deoxy hemoglobin in the red blood cell (RBC). However, seminal studies by Fox-Robichaud et al dismissed this concept, demonstrating that inhaled NO possesses extra-pulmonary bioactivity in the mesenteric vasculature by 17

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preventing neutrophil adhesion in a feline model of IRI.[115] These concepts have been extended to show inhaled NO inhibits myocardial IRI in mice, inhibits myocardial injury in patients undergoing cardiopulmonary bypass, improves forearm blood flow in healthy volunteers and inhibits IR-dependent inflammatory injury in patients undergoing knee surgery. [81; 103; 116] (Nagasaka et al. Brief periods of nitric oxide inhalation protect against myocardial IRI. Anesthesiology 2008). Current How inhaled NO mediates extra-pulmonary effects remains unclear with the general hypothesis being that it forms a relatively stable, NO-containing intermediate in the circulation, which then mediates systemic effects either directly or after being recycled to NO. Recent evidence in feline model of IRI suggest the NOcontaining intermediate may be plasma S-nitrosothiols (e.g. S-nitrosoalbumin), whereas studies in humans and mice indicate nitrite anion as a possible mediator. [11; 117; 118; 119]

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Most recently, administration of inhaled NO was demonstrated to be hepatoprotective when given prior to patients undergoing liver transplantation. [120] Specifically, patients were randomized to receive either 80 ppm of inhaled NO or placebo (nitrogen gas). Inhalation began after the induction of anesthesia and was terminated at the time of wound closure. There were no observed significant accumulations of NO2, nor was the formation of MetHb significant. Surprisingly, platelet transfusion was reduced by 50% in patients receiving inhaled NO. When adjusted for cold ischemia time and gender, inhaled NO significantly decreased hospital length of stay and evaluation of serum transaminases (ALT/AST) and coagulation (PT/PTT) times indicated inhaled NO was associated with the rate at which liver function was restored post-transplantation. Inhaled NO did not significantly effect changes in inflammatory markers in liver tissue 1 hour post-reperfusion, but significantly lowered the occurrence of hepatocellular apoptosis by 75% (Figure 3). Evaluation of circulating NO-metabolites indicated that the most likely candidate transducer of extra-pulmonary effects of inhaled NO was nitrite. While this research is not definitive, this evidence is certainly suggestive that inhaled NO may have a beneficial role in patients undergoing procedures where there is predictable ischemia–reperfusion such as in organ transplantation. In a follow-up study including two large volume transplantation on centers inhaled NO enhanced allograft function indexed by liver function tests and reduced complications at 9-months. Intensive care unit and hospital length of stay were not decreased. Inhaled NO increased concentrations of nitrate, nitrite and nitrosylhemoglobin, with nitrite being postulated as a protective mechanism. Mean costs of iNO were $1,020 per transplant in this study.[121] Another recent set of publications utilizing models of non-heart beating donors and transplanted steatotic livers also support inhaled NO in significantly reducing ALT levels, enhancing microcirculatory perfusion and reducing other injury indices. [122; 123] These studies are consistent with others demonstrating injury mitigating efficacy when inhaled NO is used prior to predictable IRI. 5.0 Nitrite Anion

Inhaled NO is ultimately metabolized via oxidation to anion nitrite and nitrate. A number of sophisticated recent investigations have revealed that nitrite administration results in vasorelaxation and that under certain pathophysiological states, mainly IRI, confers cytoprotection.[124] Under distinct conditions such as hypoxia and ischemia, nitrite can be reduced to NO by deoxyhemoglobin and deoxymyoglobin and probably by other enzymes such as xanthine oxidoreductase, components of the electron transport chain and nitric oxide synthase. Thus, nebulized nitrite may serve as biological 18

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reservoir of sorts for NO with the potential to significantly lessen the costs and delivery times (via delivery system setup) encountered with traditional inhaled NO administration. In fact, inhaled nebulized nitrite in concentrations equivalent to 20 ppm of inhaled NO, rapidly reduced and sustained hypoxia-induced pulmonary hypertension in newborn lambs.[125] In a murine model of lung transplantation, nebulization of 3mg of sodium nitrite to the donor lung prior to procurement resulted in improved oxygenation and lessened lung injury one to two hours following reperfusion.[126] There was an observed increase in cGMP and significant decrease in inflammatory cytokines. Most recently, investigators using a porcine model of hemorrhagic shock, demonstrated that inhaled nitrite (11 mg) resulted in lower lactate production and preserved mitochondrial function compared to controls.[127] It must also be mentioned that in addition to nitrite, that carbon monoxide inhalation was equally efficacious.

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Conclusions: Inhaled NO has demonstrated itself to be a novel and versatile molecule without significant side effects. It has managed to work its way into daily clinical practice as a complimentary therapy for a myriad of cardiopulmonary disorders. While its net impact in these areas has been somewhat less impactful as originally predicted, the recent emergence of data demonstrating organ protection for niche patients such as organ transplantation or cardiopulmonary bypass is meaningful. Currently, the FDA has approved inhaled NO only for use in the pediatric population for hypoxic respiratory failure in the term or near term newborn. However, as stated previously there are multiple areas of new research in selected patients especially when IRI involved. Thus, further focused translational and clinical investigations regarding inhaled NO seem warranted.

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Figure Legends

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Figure 1. Mechanism of action and inaction of inhaled nitric oxide. Panel A shows normal ventilation– perfusion. Hypoxic pulmonary vasoconstriction (Panel B) minimizes ventilation–perfusion mismatching in the presence of abnormal ventilation. Inhaled vasodilators with a short half-life improve oxygenation by increasing blood flow to ventilated lung units (Panel C). If a vasodilator is administered intravenously (Panel D) or if diseases are associated with dysregulated pulmonary vascular tone, such as sepsis and acute lung injury (Panel E), hypoxic pulmonary vasoconstriction is counteracted, leading to worsening oxygenation. Long-term administration of inhaled nitric oxide, with the accumulation of nitric oxide or leakage between lung units associated with collateral ventilation, as may occur in chronic obstructive pulmonary disease (Panel F), may negate the beneficial effects of inhaled nitric oxide on oxygenation.

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Figure 2. Mean PaO2 and positive end-expiratory pressure during the first 7 days of therapy. Data are mean (1.96 standard errors). There was a statistically significant increase in the group means of PaO2 during the initial 24 hours that resolved by 48 hours. Figure 3. Analysis of myocardial injury determined in CPB patients by measurements of the release of total CK (A) and CK-MB (C) and by measurements of the cumulative release (AUC) of total CK (B) and CKMB (D) over 48 hours post surgery. Data shown are mean ± SEM for patients who received NO (lighter blocks) and for controls (darker blocks). *P < .05. CPB – cardiopulmonary bypass. AUC – area under the curve. CK –creatinine kinase. MB – myocardial band.

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Figure 4. Inhaled NO decreases reperfusion-dependent hepatic cell death. (A) Histopathological scoring of hepatic tissue samples before (white bars) and 1 hour after reperfusion (black bars). P values represent significance calculated by paired t-test. (B) Representative H&E-stained sections indicating increased injury in LB2. Original magnification, ×25. The circled area is shown at a higher magnification (×100) in the inset and shows increased PMN infiltration adjacent to the hepatic vein (zone 3). (C) Representative fluorescence micrographs showing changes in TUNEL-positive cells (green); blue staining: DAPI. Original magnification, ×25. (D) Paired changes in TUNEL-positive objects in liver biopsies before (LB1) and 1 hour after reperfusion (LB2). P values represent significance calculated by paired t test. (E) Average reperfusion-dependent increases in TUNEL-positive objects. *P ≤ 0.0005 relative to placebo. LB – liver biopsy. PMN – polymorphonuclear neutrophils.

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Study/Year

Patients (n)

Primary Outcome

Control

Dose of iNO

18 and 36 ppm Improvement in partial pressure of oxygen and reduction of pulmonary pressures 1.25, 5, 20, 40, Length of time on 80 ppm mechanical ventilation

N =10

IV prostacyclin

Dellinger et al. 1998[67]

N =177 ARDS within 72 hrs. Excluded if nonpulmonary cause of ARDS, ie sepsis. N =180 NO responders only included

Placebo

Placebo

1-40 ppm at the lowest effective dose

Reversal of acute lung injury

N =385 ARDS with P:F ratio < 250. Excluded sepsis as cause of ARDS and any nonpulmonary organ failure 92 Survivors from prior RCT

Placebo

5 ppm

Survival without need for mechanical ventilation during the first 28 days

Dellinger et al. 2012 [75]

M AN U

TE D

EP AC C

Taylor et al. 2004[66]

N/A

Secondary Outcome

None

SC

Rossaint et al. 1993 [47]

Lundin et al. 1999[69]

RI PT

Table 1. Studies of Inhaled Nitric Oxide in Patients with Acute Respiratory Distress Syndrome (ARDS)

5 ppm

Pulmonary function tests six months posttreatment

Oxygenation, pulmonary artery pressure and 28 day mortality Development of acute lung injury, mortality Oxygenation and 28 day survival

Conclusion Proof of concept that inhaled NO can selectively affect lung units.

Post-hoc analysis found less time on mechanical ventilation in group administered 5 ppm. Improvement in oxygenation did not change ALI. No mortality benefit. Inhaled NO had no effect on patient ventilator time or mortality at 28 days. Took 5 years after study completed to publish. Significant change in ARDS care in the interim. ARDS patients surviving six months post-treatment had significant better values for pulmonary function.

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Highlights for review