Shorter duration of oxygen therapy Decrease of lung damage and reduction in severity of chronic lung disease

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1 Randomized Controlled Trial of Early Compared with Delayed Use of Inhaled Nitric Oxide in Newborns with a Moderate Respiratory Failure and Pulmonary Hypertension A González J Fabres I D Apremont G Urcelay M Avaca C Gandolfi J Kattan Additional Information for Readers Provided by Ikaria This reprint concerns the following uses for INOmax (nitric oxide) for inhalation that are not approved by the Food and Drug Administration. Shorter duration of oxygen therapy Decrease of lung damage and reduction in severity of chronic lung disease Reprinted from Journal of Perinatology, Advance Online Publication 2009

2 Journal of Perinatology (2009), 1 5 r 2009 Nature Publishing Group All rights reserved /09 $32 ORIGINAL ARTICLE Randomized controlled trial of early compared with delayed use of inhaled nitric oxide in newborns with a moderate respiratory failure and pulmonary hypertension A González 1,4, J Fabres 1,4, I D Apremont 2,4, G Urcelay 3,4, M Avaca 1, C Gandolfi 2 and J Kattan 1,4 1 Division of Neonatology, Hospital Clıńico Universidad Cato lica, Santiago, Chile; 2 Newborn ICU, Hospital Dr So tero del Rıó, Santiago, Chile; 3 Division of Pediatric Cardiology, Hospital Clıńico Universidad Cato lica, Santiago, Chile and 4 Departamento de Pediatrıá, Escuela de Medicina, Pontificia Universidad Cato lica de Chile, Santiago, Chile Objective: To evaluate whether early treatment with inhaled nitric oxide (ino) will prevent newborns with moderate respiratory failure from developing severe hypoxemic respiratory failure (oxygenation index (OI)X40). Study Design: A total of 56 newborns with moderate respiratory failure (OI between 10 and 30) were randomized before 48 h after birth to early treatment with 20 p.p.m. of ino (Early ino group, n ¼ 28) or conventional mechanical ventilation with FiO (Control group, n ¼ 28). Infants received ino and/or high-frequency oscillatory ventilation (HFOV) if they developed an OI>40. Result: 7 of 28 early ino patients (25%) compared to 17 of 28 control patients (61%) developed an OI>40 (P<0.05). In the Early ino group mean OI significantly decreased from 22 (baseline) to 19 at 4 h (P<0.05) and remained lower over time: 19 (12 h), 18 (24 h) and 16 at 48 h. In contrast, OI increased in the Control group and remained significantly higher than the Early ino group during the first 48 h of study: 22 (baseline), 29, 35, 32 and 23 at 4, 12, 24 and 48 h, respectively (P<0.01). Of 17, 6 control patients who developed an OI>40 were successfully treated with ino. Nine of the remaining eleven control patients and six of seven Early ino patients who had an OI>40 despite use of ino responded with the addition of HFOV. One patient of the Early ino group and two of the Control group died. Median (range) duration of oxygen therapy was significantly shorter in the Early ino group: 11.5 (5 to 90) days compared to 18 (6 to 142) days of the Control group (P<0.03). Conclusion: Early use of ino in newborns with moderate respiratory failure improves oxygenation and decreases the probability of developing severe hypoxemic respiratory failure. Journal of Perinatology advance online publication, 5 November 2009; doi: /jp Correspondence: Dr A González, Unidad de Neonatología, Departamento de Pediatría, Facultad de Medicina, Pontificia Universidad Católica de Chile, Marcoleta 367, Santiago, Chile. alvgonza@med.puc.cl Received 13 March 2009; revised 14 September 2009; accepted 16 September 2009 Keywords: inhaled nitric oxide; newborn; hypoxic respiratory failure Introduction Persistent pulmonary hypertension of the newborn (PPHN) is a serious condition that may accompany respiratory diseases such as meconium aspiration syndrome, pneumonia, respiratory distress syndrome and congenital diaphragmatic hernia. 1,2 Despite the variety of causes, similar physiopathologic changes characterize this syndrome: a persistently raised pulmonary vascular resistance after birth, which leads to severe hypoxemia due to extrapulmonary shunting. This disease can progressively get worse, due to the fact that sustained hypoxia may injure pulmonary vasculature inducing remodeling and making it unresponsive to vasodilators. 3,4 The sustained hypoxemia and the high oxygen concentrations, and elevated ventilator pressures/volumes needed to treat these infants, may lead to further lung injury and myocardial dysfunction. This sequence of events may result in death, an occurrence made more common in countries that do not have access to extracorporeal membrane oxygenation (ECMO), where mortality of this syndrome is between 10 and 40%. 1,2,5,6 Inhaled nitric oxide (ino) has been shown to be an effective treatment for PPHN Several randomized clinical trials have shown that ino significantly improves oxygenation and decreases the need of ECMO or incidence of death in near-term infants with hypoxic respiratory failure However, their response to ino is not ideal and ranges between 50 and 60%. This may be due to the fact that in most studies, infants were critically ill, either fulfilling ECMO criteria (oxygenation index (OI) X 40) or had severe lung disease and vascular damage, and were unresponsive to therapy. It has been also shown that ino may attenuate lung inflammation and pulmonary vascular remodeling in experimental lung-injured models. 14,15 On the basis this information, we hypothesized that early treatment with ino in newborns with

3 ino in newborns with moderate respiratory failure A González et al 2 moderate respiratory failure (OI between 10 and 30) and pulmonary hypertension would improve oxygenation and attenuate the development of severe hypoxemic respiratory failure (OI > 40). Methods This was a prospective, randomized controlled and unblinded trial performed in two neonatal intensive care units in Santiago, Chile between March 1997 and December This study was approved by the investigational review board of the Pontificia Universidad Católica de Chile School of Medicine and by the ethics committees of both hospitals. A written informed consent was obtained from the parents of all infants at the time of enrollment. Study population Near-term infants (X35 weeks gestation) with birth weights >2000 g and p48 h old were eligible for enrollment. We included 56 infants requiring mechanical ventilation with moderate hypoxemic respiratory failure with an OI between 10 and 30. This was calculated on two consecutive measurements of post-ductal arterial blood gases and after ventilatory support had been adjusted to achieve a PCO 2 < 60 mm Hg and ph > All infants had a previous echocardiogram, which was negative for cardiac anomalies and had evidence of pulmonary hypertension. This was defined as a tricuspid insufficiency jet with an estimated systolic pulmonary artery pressure X2/3 of systemic systolic arterial blood pressure and/or evidence of right-to-left shunting through the foramen ovale or ductus arteriosus. Exclusion criteria included the presence of a lifethreatening congenital anomaly, suspected or confirmed chromosomal abnormality, congenital heart disease, congenital diaphragmatic hernia and other forms of lung hypoplasia syndromes. Protocol Infants were randomized using sequenced sealed envelopes into two study groups: (1) Early ino group, which received ino with conventional mechanical ventilation, and (2) Control group, which received conventional mechanical ventilation with 1.0 FiO 2. Patients in both groups were kept on conventional mechanical ventilation and settings were adjusted to keep PaO 2 between 60 and 100 mm Hg and PaCO 2 between 35 and 50 mm Hg. Inotropic drugs were administered to keep mean systemic arterial blood pressures X50 mm Hg. Sodium bicarbonate was given if necessary to keep the arterial ph between 7.35 and 7.5. Inhaled nitric oxide was started at 20 p.p.m. in the Early ino group and ventilatory settings were not changed during the first 30 min unless acute deterioration occurred. This initial dose was kept constant for the first 4 h. Attempts were made to reduce ino dose in a stepwise manner by 5 p.p.m. every 2 to 4 h until 5 p.p.m. was reached. If condition of the patient deteriorated with the reduction of dose, the dose was increased to obtain the minimal dose necessary to keep oxygen saturations X88%. The new dose was maintained for at least 24 h. Attempts were made to discontinue ino, if the patient had been stable on an ino dose p5 p.p.m. and with an OI < 10 for 24 h. If this was not possible, ino was continued for an additional 24 h. Treatment failure was defined as a worsening in respiratory status during first 48 h of treatment based on an increase in the OI to >40. Patients in the Control group who reached an OI > 40 were treated with ino. Infants in both groups who persisted with an OI >40 despite ino were treated with high-frequency oscillatory ventilation (HFOV) and ino. ECMO treatment was not available in Chile during the study period. Inhaled nitric oxide was delivered using a 990 p.p.m. sealed tank (AGA Chile SA, Santiago, Chile/INO Therapeutics; Port Allen, LA, USA) and ino and NO 2 levels were continuously measured by electrochemical cell analyzers (PAC II Analyzer; Draeger, Lübeck, Germany and NOxBox I; Bedfont, Rochester, England, UK). An indwelling arterial line was placed in all patients to monitor systemic arterial blood pressure and draw serial arterial blood gases at baseline, 4, 12 and 24 h and at least every 12 h thereafter while the patient was receiving ino. Methemoglobin levels were also measured at baseline and every 24 h while the patient was on ino. All these variables along with the clinical data of patients including pregnancy history and delivery, birth weight, age, and the presence of other complications of the neonatal period such as intraventricular hemorrhage, pneumothorax, chronic lung disease (defined as oxygen dependence for 28 or more days and an abnormal chest X-ray), were recorded on standardized forms and saved in a computer database. Statistical analyses Sample size was estimated based on previous data. 6 Assuming that 60% of Control group patients would develop an OI X 40, we calculated that approximately 54 patients would need to be enrolled to provide the study with 80% power to detect a reduction in treatment failure to 33% in the Early ino group, with a type I error of We conducted the analysis according to the intentionto-treat principle. Clinical and demographic characteristics were compared using the Student s t-test and analysis of variance for continuous variables with normal distribution, and Mann Whitney test for independent samples for those with a not normal distribution. Categorical variables were compared using w 2 -test and if the expected number of observations was less than 5, Fisher s exact test was used instead. A P-value p0.05 was considered significant. Results A total of 56 newborns were enrolled in this study, 28 in each group. As seen in Table 1, characteristics of infants did not differ Journal of Perinatology

4 ino in newborns with moderate respiratory failure A González et al 3 Table 1 Patient characteristics Early ino (n ¼ 28) Controls (n ¼ 28) P-value Birth weight (g) 3225± ±475 NS Gestational age (weeks) 38± ±1.7 NS 5 0 Apgar, median (range) 8 (3 9) 8 (2 9) NS Gender (Male/Female) 15/13 16/12 NS Age at enrollment (h) 25±14 26±12 NS Inborns, n (%) 6 (22) 6 (22) NS Inotrope use, n (%) 21 (75) 20 (71) NS Oxygenation Index * * Early NOi * Control * Primary diagnosis (n) Meconium aspiration 8 7 Pneumonia/sepsis 7 9 NS RDS/HMD 11 9 Idiopathic/other 2 3 Abbreviations: HMD, hyaline membrane disease; ino, inhaled nitric oxide; RDS, respiratory distress syndrome. Table 2 Respiratory variables at enrollment Early ino (n ¼ 28) Controls (n ¼ 28) P-value FiO ± ±0.08 NS Mean airway pressure (cm H 2 O) 14.2± ±3.5 NS Oxygenation index 22.2± ±5.3 NS PaO 2 (mm Hg) 61±14 64±25 NS PaCO 2 (mm Hg) 39±8 40±10 NS ph 7.4± ±0.1 NS Surfactant use, n (%) 19 (68) 20 (71) NS PIP (cm H 2 O) 30.3±6 31.7±7 NS PEEP (cm H 2 O) 4.3± ±1.2 NS Ventilator rate (breaths/min) 59±10 57±9 NS Abbreviations: ino, inhaled nitric oxide; NS, not significant; PEEP, positive end expiratory pressure; PIP, peak inspiratory pressure. Results are mean±s.d., unless specified. between the groups in terms of birth weight, age at randomization, condition at birth and the underlying respiratory diseases associated with their respiratory failure. The baseline ventilatory and hemodynamic conditions were also similar between the Early ino and Control groups at the time of enrollment (Table 2). The infants required moderately high levels of conventional ventilatory support, with FiO 2 ranging between 0.75 and 1. Acute changes in oxygenation Infants assigned to the Early ino group had a significant increase in PaO 2 from baseline, which led to a significant decrease in their OI: (mean±s.d.) OI decreased from 22.2±4.3 at baseline to 19.0±7.2 at 4 h (P<0.05), which continued decreasing over time (Figure 1). In contrast, OI increased in the Control group and remained significantly higher than the Early ino group during the first 48 h of study (P<0.01) Time (h) *: p<0.01 Figure 1 Change in oxygenation index over time in the two study groups. Points represent mean±s.e.m. The oxygenation indexes (OIs) were significantly higher after baseline in the Control group (P<0.01). Table 3 Respiratory outcomes Early ino (n ¼ 28) Controls (n ¼ 28) Treatment failure Seven of 28 infants (25%) receiving early ino and 17 of 28 infants (61%) on conventional therapy developed an OI >40 treatment failure (P<0.05). Of 17, 6 control patients who developed an OI>40 were successfully treated with ino subsequently. Nine of the remaining eleven control patients and six of seven Early ino patients who had an OI > 40 despite ino responded with the addition of HFOV. One patient of the Early ino group and two of the Control group died. Table 3 shows the respiratory outcomes of both groups: duration of mechanical ventilation was not significantly different, whereas duration of oxygen therapy was significantly lower among infants of the Early ino group (P<0.03). This was confirmed with the survival analysis for the need of oxygen therapy, which was significantly higher over time in Control group (Figure 2). Patients treated with ino did not have elevated blood levels of methemoglobin or high levels of NO 2 in the ventilatory circuit. Also, there were no differences between the groups in the incidence of other neonatal complications such as bleeding and/or coagulation disorders, hypotension or infections. 48 P-value Treatment failure (OI X40), n (%) 7 (25) 17 (61) <0.05 Deaths (n) 1 2 NS Mech. ventilation days, median (range) 6 (3 28) 8 (4 37) NS Oxygen therapy days, median (range) 11.5 (5 90) 18 (6 142) <0.03 Chronic lung disease, n (%) 4/27 (15) 7/26 (27) NS Abbreviations: ino, inhaled nitric oxide; NS, not significant; OI, oxygenation index. Journal of Perinatology

5 ino in newborns with moderate respiratory failure A González et al Early ino Group 0.8 Control Group Days after enrollment p < 0.03 Figure 2 Survival plot of probability of oxygen requirement over time in study groups. As observed, infants of the Early ino (inhaled nitric oxide) group spent significantly less time with oxygen therapy compared with those of the Control group (P<0.03). P of Oxygen Requriement Discussion The present study shows that early treatment with ino in neonates with moderate hypoxemic respiratory failure increases oxygenation and decreases the probability of developing severe PPHN/respiratory failure (OI>40). The observed improvement in oxygenation with ino therapy is consistent with the results of several randomized controlled studies The novel aspect of this trial is the early use of ino in the disease s course in centers without ECMO availability, preventing the development of severe respiratory failure in most treated infants. This reduction in PPHN severity may decrease mortality and respiratory sequelae in patients with respiratory failure in places where ECMO is not available. As the observed mortality was low in both groups, unfortunately this study was not powered to confirm this point. Until now, most published reports have not shown a decrease in mortality with ino, possibly this can be explained because ECMO was used in infants when ino failed. Along with their oxygenation improvement, infants who received early ino required less time with oxygen supplementation and therefore their oxygen exposure was reduced. It is possible that this strategy of early ino use may decrease lung damage and the severity of chronic lung disease in infants with moderate respiratory failure. Most studies have not shown a clear benefit of ino in long-term respiratory outcomes. In the majority of these studies, treated infants were sicker, enrolled later in the disease course and all of them had ECMO as a rescue therapy when ino failed. In this study however, ECMO was not available and therefore those infants whose oxygenation did not improve remained either on conventional or HFOV with high settings and elevated oxygen concentrations for longer periods of time. There are few studies that have evaluated a relatively early ino use strategy. Davidson et al. 16 did not find clear differences in the length of oxygen supplementation or mechanical ventilation; however the studied infants seem sicker and ECMO was available. The Franco-Belgium Collaborative NO Trial Group 17 showed a decrease in length of mechanical ventilation and a lower proportion of infants requiring oxygen at 28 days when ino was used in near-term infants younger than 7 days with moderate respiratory failure (OI of 15 to 40). Sadiq et al. 18 compared ino treatment with standard treatment in near-term infants with moderate PPHN (alveolar-arterial oxygen gradient, (AaDO 2 )of 500 to 599 torr), they found that ino treatment improved PaO 2, reduced the amount of ventilatory support and prevented progression to severe PPHN defined as AaDO 2 > 600 torr. Infants of the Control group also received ino if their AaDO 2 was >600, there were no differences in need for ECMO or incidence of death. More recently, Konduri et al. 19 conducted a large randomized controlled multicenter trial in near-term infants who were <14 days old with moderate respiratory failure (OI of 15 to 25). They compared early use of ino with a Control group that received simulated initiation of ino. Infants received standard ino if they developed an OI X25. Although the Early ino group had significantly better initial oxygenation and Control group progressed to standard ino and developed an OI >40 more often, the incidence of death, ECMO use and other respiratory outcomes was not different. Although this study evaluated a population similar to our study, controls had a lower threshold to receive standard ino (OI >25) and ECMO was used as a back-up therapy. In addition, the mean OI at entry was approximately 20, which was close to the threshold to receive standard ino. In fact, a significantly larger proportion of the control infants (54%) received standard ino, and therefore there was not a clear separation between the Early and the Control groups that can explain the lack of a clear treatment effect. A subgroup analysis of the 176 infants enrolled at an OI of 15 to 20 in that study showed a trend for lower incidence of ECMO use/mortality (10% with Early ino vs 18% in the Control group, P ¼ 0.12). 19,20 Also, the incidence of ECMO use in the whole study (11%) was lower than that observed in previous large clinical trials with ino (approximately 40%). Therefore, similar to our results, these data indicate that the earlier use of ino may be associated with further reductions in short-term respiratory morbidity. There are several recently published studies that evaluated ino treatment in premature infants. Although different strategies were evaluated and results are conflicting, it seems that early routine use in mildly sick ventilated preterm infants may improve their survival without bronchopulmonary dysplasia This study also supports a possible beneficial effect of early ino therapy in decreasing lung damage. There are a number of factors that limit the impact of our trial. First, this trial was not blind because we had limited numbers of personnel and equipment to have independent teams masked to the assigned therapy or in charge of the ino administration and monitoring. Second, this study took more than 5 years to be Journal of Perinatology

6 ino in newborns with moderate respiratory failure A González et al 5 completed, because of the difficulty in early recruitment of these patients and also because we had a limited and interrupted supply of nitric oxide during the study period. After the design and onset of this study, it became known that the use of HFOV improves the oxygenation response to ino in newborns with hypoxic respiratory failure due to parenchymal lung disease. 25 However, because we had a limited availability of HFOV during the study period, this mode of ventilation was added to ino only in infants who persist with severe respiratory failure (OI>40) despite the use of ino. Despite these limitations, we believe that the observed results in improved oxygenation and mitigation of the development of severe PPHN/respiratory failure (OI >40) with early ino use are important, and support a strategy that may ameliorate the development of lung injury. Further studies are needed to confirm the impact of this strategy of ino use in long-term respiratory outcomes. Conflict of interest The authors declare no conflict of interest. Acknowledgments We thank Alessandra Gederlini for her help with the statistical analyses. We also thank Margarita Bidegain and Ronald Goldberg for their critical review of the paper. This study was funded in part by project DIPUC 96/01E, Pontificia Universidad Católica de Chile and AGA Chile SA. References 1 Morin FC, Stenmark KR. Persistent pulmonary hypertension of the newborn. Am J Respir Crit Care Med 1995; 151: Fox WW, Duara S. Persistent pulmonary hypertension in the neonate: diagnosis and management. J Pediatr 1983; 103: Durmowicz AG, Stenmark KR. Mechanisms of structural remodeling in chronic hypoxic pulmonary hypertension. Pediatr Rev 1999; 20: Aschner JL, Fike CD. New developments in the pathogenesis and management of neonatal pulmonary hypertension In: Bancalari E, Polin R (eds). The Newborn Lung: Neonatology Questions and Controversies. Sauders Elsevier Inc.: Philadelphia, PA: 2008: p Walsh-Sukys MC, Tyson JE, Wright LL, Bauer CR, Korones SB, Stevenson DK et al. Persistent pulmonary hypertension of the newborn in the era before nitric oxide: practice variations and outcomes. Pediatrics 2000; 105: González A, Fabres J, Cifuentes J, Kattan J, Estay A, Tapia JL et al. Tratamiento de la hipertensión Pulmonar persistente severa neonatal con oxido nitrico inhalatorio. Libro de Resu menes XI Congreso Latinoamericano de Pediatrıá 1998; Frostell C, Fratacci M, Wain JC, Jones R, Zapol WN. Inhaled nitric oxide: a selective pulmonary vasodilator reversing hypoxic pulmonary vasoconstriction. Circulation 1991; 83: Berger J, Gibson R, Redding G, Standaert TA, Clarke NR, Truog WE. Effect of inhaled nitric oxide during group B streptococcal sepsis in piglets. Am Rev Respir Dis 1993; 147: Kinsella J, Neish S, Shaffer E, Abman S. Low-dose inhalational nitric oxide in persistent pulmonary hypertension of the newborn. Lancet 1992; 340: Roberts J, Polaner D, Lang P, Zapol WM. Inhaled nitric oxide in persistent pulmonary hypertension of the newborn. Lancet 1992; 340: The Neonatal Inhaled Nitric Oxide Study Group. Inhaled nitric oxide in full-term infants with hypoxic respiratory failure. N Engl J Med 1997; 336: Roberts JD, Fineman JR, Morin FC, Shaul PW, Rimar S, Schreiber MD et al. Inhaled nitric oxide and persistent pulmonary hypertension of the newborn. N Engl J Med 1997; 336: Clark R, Kueser T, Walker MW, Southgate WM, Huckaby JL, Perez JA et al. Low-dose nitric oxide therapy for persistent pulmonary hypertension of the newborn. N Engl J Med 2000; 342: Kinsella J, Parker TA, Galan H, Sheridan BC, Halbower AC, Abman SH et al. Effects of inhaled nitric oxide on pulmonary edema and lung neutrophil accumulation in severe experimental hyaline membrane disease. Pediatr Res 1997; 41: Roberts JD, Chiche JD, Weimann J, Steudel W, Zapol WM, Bloch KD. Nitric oxide inhalation decreases pulmonary artery remodeling in the injured lungs of rat pups. Circ Res 2000; 87: Davidson D, Barefield ES, Kattwinkel J, Dudell G, Darmask M, Straube R et al. Inhaled nitric oxide for the early treatment of persistent pulmonary hypertension of the term newborn: a randomized, double-masked, placebo-controlled, dose-response, multicenter study. Pediatrics 1998; 101: The Franco-Belgium Collaborative Group. Early compared with delayed inhaled nitric oxide in moderately hypoxemic neonates with respiratory failure: a randomized controlled trial. Lancet 1999; 354: Sadiq HF, Mantych G, Benawra RS, Devaskar UP, Hocker JR. Inhaled nitric oxide in the treatment of moderate persistent pulmonary hypertension of the newborn: a randomized controlled, multicenter trial. J Perinatol 2003; 23: Konduri GG, Solimano A, Sokol GM, Singer J, Ehrenkranz RA, Singhal N et al. A randomized trial of early versus standard inhaled nitric oxide therapy in term and near term newborn infants with hypoxic respiratory failure. Pediatrics 2004; 113: Konduri GG. New approaches for persistent pulmonary hypertension of newborn. Clin Perinatol 2004; 31: Schreiber M, Gin-Mestan K, Marks J, Huo D, Lee G, Srisuparp P. Inhaled nitric oxide in premature infants with respiratory distress syndrome. N Engl J Med 2003; 349: Kinsella JP, Cutter GR, Walsh WF, Gerstmann DR, Bose CL, Hart C et al. Early inhaled nitric oxide therapy in premature newborns with respiratory failure. N Engl J Med 2006; 355: Ballard RA, Truog WE, Cnaan A, Martin RJ, Ballard PL, Merrill JD et al. Inhaled nitric oxide in preterm infants undergoing mechanical ventilation. N Engl J Med 2006; 355(4): Barrignton K, Finer N. Inhaled nitric oxide for respiratory failure in preterm infants. Cochrane Database Syst Rev 2009, Issue 1. Art no CD DOI / CD pub Kinsella J, Truog W, Walsh W, Goldberg RN, Bancalari E, Mayock DE et al. Randomized, multicenter trial of inhaled nitric oxide and high frequency oscillatory ventilation in severe persistent pulmonary hypertension of the newborn. J Pediatr 1997; 131: Journal of Perinatology

7 INOmax (nitric oxide) for inhalation HIGHLIGHTS OF PRESCRIBING INFORMATION These highlights do not include all the information needed to use INOmax safely and effectively. See full prescribing information for INOmax. INOmax (nitric oxide) for inhalation Initial U.S. Approval: 1999 RECENT MAJOR CHANGES Warnings and Precautions, Heart Failure (5.4) 8/2009 INDICATIONS AND USAGE INOmax is a vasodilator, which, in conjunction with ventilatory support and other appropriate agents, is indicated for the treatment of term and near-term (>34 weeks gestation) neonates with hypoxic respiratory failure associated with clinical or echocardiographic evidence of pulmonary hypertension, where it improves oxygenation and reduces the need for extracorporeal membrane oxygenation (1.1). Monitor for PaO 2, methemoglobin, and inspired NO 2 during INOmax administration (1.1). Utilize additional therapies to maximize oxygen delivery (1.1). DOSAGE AND ADMINISTRATION Dosage: The recommended dose of INOmax is 20 ppm, maintained for up to 14 days or until the underlying oxygen desaturation has resolved (2.1). Administration: INOmax must be delivered via a system which does not cause generation of excessive inhaled nitrogen dioxide (2.2). Do not discontinue INOmax abruptly (2.2). DOSAGE FORMS AND STRENGTHS INOmax (nitric oxide) is a gas available in 100 ppm and 800 ppm concentrations. CONTRAINDICATIONS Neonates known to be dependent on right-to-left shunting of blood (4). WARNINGS AND PRECAUTIONS Rebound: Abrupt discontinuation of INOmax may lead to worsening oxygenation and increasing pulmonary artery pressure (5.1). Methemoglobinemia: Methemoglobin increases with the dose of nitric oxide; following discontinuation or reduction of nitric oxide, methemoglobin levels return to baseline over a period of hours (5.2). Elevated NO 2 Levels: NO 2 levels should be monitored (5.3). Heart Failure: In patients with pre-existing left ventricular dysfunction, inhaled nitric oxide may increase pulmonary capillary wedge pressure leading to pulmonary edema (5.4). ADVERSE REACTIONS Methemoglobinemia and elevated NO 2 levels are dose dependent adverse events. Worsening oxygenation and increasing pulmonary artery pressure occur if INOmax is discontinued abruptly. Other adverse reactions that occurred in more than 5% of patients receiving INOmax in the CINRGI study were: thrombocytopenia, hypokalemia, bilirubinemia, atelectasis, and hypotension (6). To report SUSPECTED ADVERSE REACTIONS, contact INO Therapeutics at and or FDA at FDA-1088 or DRUG INTERACTIONS Nitric oxide donor agents: Nitric oxide donor compounds, such as prilocaine, sodium nitroprusside, and nitroglycerin, when administered as oral, parenteral, or topical formulations, may have an additive effect with INOmax on the risk of developing methemoglobinemia (7). Revised: August 2009 FULL PRESCRIBING INFORMATION: CONTENTS* 1. Indications and Usage 1.1 Treatment of Hypoxic Respiratory Failure 2. Dosage and administration 2.1 Dosage 2.2 Administration 3. Dosage Forms and Strengths 4. Contraindications 5. Warnings and Precautions 5.1 Rebound 5.2 Methemoglobinemia 5.3 Elevated NO 2 Levels 5.4 Heart Failure 6. Adverse Reactions 6.1 Clinical Trials Experience 6.2 Post-Marketing Experience 7. Drug Interactions 8. Use in Specific Populations 8.1 Pregnancy 8.2 Labor and Delivery 8.3 Nursing Mothers 8.4 Pediatric Use 8.5 Geriatric Use 10. Overdosage 11. Description 12. Clinical Pharmacology 12.1 Mechanism of Action 12.2 Pharmacodynamics 12.3 Pharmacokinetics 12.4 Pharmacokinetics: Uptake and Distribution 12.5 Pharmacokinetics: Metabolism 12.6 Pharmacokinetics: Elimination 13. Nonclinical Toxicology 13.1 Carcinogenesis, Mutagenesis, Impairment of Fertility 14. Clinical Studies 14.1 Treatment of Hypoxic Respiratory Failure (HRF) 14.2 Ineffective in Adult Respiratory Distress Syndrome (ARDS) 16. How Supplied/Storage and Handling *Sections or subsections omitted from the full prescribing information are not listed.

8 FULL PRESCRIBING INFORMATION 1 Indications and Usage 1.1 Treatment of Hypoxic Respiratory Failure INOmax is a vasodilator, which, in conjunction with ventilatory support and other appropriate agents, is indicated for the treatment of term and near-term (>34 weeks) neonates with hypoxic respiratory failure associated with clinical or echocardiographic evidence of pulmonary hypertension, where it improves oxygenation and reduces the need for extracorporeal membrane oxygenation. Utilize additional therapies to maximize oxygen delivery. In patients with collapsed alveoli, additional therapies might include surfactant and highfrequency oscillatory ventilation. The safety and effectiveness of inhaled nitric oxide have been established in a population receiving other therapies for hypoxic respiratory failure, including vasodilators, intravenous fluids, bicarbonate therapy, and mechanical ventilation. Different dose regimens for nitric oxide were used in the clinical studies [see Clinical Studies (14)]. Monitor for PaO 2, methemoglobin, and inspired NO 2 during INOmax administration. 2 Dosage and administration 2.1 Dosage Term and near-term neonates with hypoxic respiratory failure The recommended dose of INOmax is 20 ppm. Treatment should be maintained up to 14 days or until the underlying oxygen desaturation has resolved and the neonate is ready to be weaned from INOmax therapy. An initial dose of 20 ppm was used in the NINOS and CINRGI trials. In CINRGI, patients whose oxygenation improved with 20 ppm were dosereduced to 5 ppm as tolerated at the end of 4 hours of treatment. In the NINOS trial, patients whose oxygenation failed to improve on 20 ppm could be increased to 80 ppm, but those patients did not then improve on the higher dose. As the risk of methemoglobinemia and elevated NO 2 levels increases significantly when INOmax is administered at doses >20 ppm, doses above this level ordinarily should not be used. 2.2 Administration The nitric oxide delivery systems used in the clinical trials provided operator-determined concentrations of nitric oxide in the breathing gas, and the concentration was constant throughout the respiratory cycle. INOmax must be delivered through a system with these characteristics and which does not cause generation of excessive inhaled nitrogen dioxide. The INOvent system and other systems meeting these criteria were used in the clinical trials. In the ventilated neonate, precise monitoring of inspired nitric oxide and NO 2 should be instituted, using a properly calibrated analysis device with alarms. The system should be calibrated using a precisely defined calibration mixture of nitric oxide and nitrogen dioxide, such as INOcal. Sample gas for analysis should be drawn before the Y-piece, proximal to the patient. Oxygen levels should also be measured. In the event of a system failure or a wall-outlet power failure, a backup battery power supply and reserve nitric oxide delivery system should be available. Do not discontinue INOmax abruptly, as it may result in an increase in pulmonary artery pressure (PAP) and/or worsening of blood oxygenation (PaO 2 ). Deterioration in oxygenation and elevation in PAP may also occur in children with no apparent response to INOmax. Discontinue/wean cautiously. 3 Dosage Forms and Strengths Nitric oxide is a gas available in 100 ppm and 800 ppm concentrations. 4 Contraindications INOmax is contraindicated in the treatment of neonates known to be dependent on right-to-left shunting of blood. 5 Warnings and Precautions 5.1 Rebound Abrupt discontinuation of INOmax may lead to worsening oxygenation and increasing pulmonary artery pressure. 5.2 Methemoglobinemia Methemoglobinemia increases with the dose of nitric oxide. In clinical trials, maximum methemoglobin levels usually were reached approximately 8 hours after initiation of inhalation, although methemoglobin levels have peaked as late as 40 hours following initiation of INOmax therapy. In one study, 13 of 37 (35%) of neonates treated with INOmax 80 ppm had methemoglobin levels exceeding 7%. Following discontinuation or reduction of nitric oxide, the methemoglobin levels returned to baseline over a period of hours. 5.3 Elevated NO 2 Levels In one study, NO 2 levels were <0.5 ppm when neonates were treated with placebo, 5 ppm, and 20 ppm nitric oxide over the first 48 hours. The 80 ppm group had a mean peak NO 2 level of 2.6 ppm. 5.4 Heart Failure Patients who had pre-existing left ventricular dysfunction treated with inhaled nitric oxide, even for short durations, experienced serious adverse events (e.g., pulmonary edema). 6 Adverse Reactions Because clinical trials are conducted under widely varying conditions, adverse reaction rates observed in the clinical trials of a drug cannot be directly compared to rates in the clinical trials of another drug and may not reflect the rates observed in practice. The adverse reaction information from the clinical studies does, however, provide a basis for identifying the adverse events that appear to be related to drug use and for approximating rates. 6.1 Clinical Trials Experience Controlled studies have included 325 patients on INOmax doses of 5 to 80 ppm and 251 patients on placebo. Total mortality in the pooled trials was 11% on placebo and 9% on INOmax, a result adequate to exclude INOmax mortality being more than 40% worse than placebo. In both the NINOS and CINRGI studies, the duration of hospitalization was similar in INOmax and placebo-treated groups. From all controlled studies, at least 6 months of follow-up is available for 278 patients who received INOmax and 212 patients who received placebo. Among these patients, there was no evidence of an adverse effect of treatment on the need for rehospitalization, special medical services, pulmonary disease, or neurological sequelae. In the NINOS study, treatment groups were similar with respect to the incidence and severity of intracranial hemorrhage, Grade IV hemorrhage, periventricular leukomalacia, cerebral infarction, seizures requiring anticonvulsant therapy, pulmonary hemorrhage, or gastrointestinal hemorrhage. The table below shows adverse reactions that occurred in at least 5% of patients receiving INOmax in the CINRGI study with event rates >5% and greater than placebo event rates. None of the differences in these adverse reactions were statistically significant when inhaled nitric oxide patients were compared to patients receiving placebo. Table 1: Adverse Reactions in the CINRGI Study Adverse Event Placebo (n=89) Inhaled NO (n=97) Hypotension 9 (10%) 13 (13%) Withdrawal 9 (10%) 12 (12%) Atelectasis 8 (9%) 9 (9%) Hematuria 5 (6%) 8 (8%) Hyperglycemia 6 (7%) 8 (8%) Sepsis 2 (2%) 7 (7%) Infection 3 (3%) 6 (6%) Stridor 3 (3%) 5 (5%) Cellulitis 0 (0%) 5 (5%) 6.2 Post-Marketing Experience The following adverse reactions have been identified during postapproval use of INOmax. Because these reactions are reported voluntarily from a population of uncertain size, it is not always possible to estimate their frequency reliably or to establish a causal relationship to drug exposure. The listing is alphabetical: dose errors associated with the delivery system; headaches associated with environmental exposure of INOmax in hospital staff; hypotension associated with acute withdrawal of the drug; hypoxemia associated with acute withdrawal of the drug; pulmonary edema in patients with CREST syndrome.

9 7 Drug Interactions No formal drug-interaction studies have been performed, and a clinically significant interaction with other medications used in the treatment of hypoxic respiratory failure cannot be excluded based on the available data. INOmax has been administered with tolazoline, dopamine, dobutamine, steroids, surfactant, and high-frequency ventilation. Although there are no study data to evaluate the possibility, nitric oxide donor compounds, including sodium nitroprusside and nitroglycerin, may have an additive effect with INOmax on the risk of developing methemoglobinemia. An association between prilocaine and an increased risk of methemoglobinemia, particularly in infants, has specifically been described in a literature case report. This risk is present whether the drugs are administered as oral, parenteral, or topical formulations. 8 Use in Specific Populations 8.1 Pregnancy Pregnancy Category C Animal reproduction studies have not been conducted with INOmax. It is not known if INOmax can cause fetal harm when administered to a pregnant woman or can affect reproductive capacity. INOmax is not intended for adults. 8.2 Labor and Delivery The effect of INOmax on labor and delivery in humans is unknown. 8.3 Nursing Mothers Nitric oxide is not indicated for use in the adult population, including nursing mothers. It is not known whether nitric oxide is excreted in human milk. 8.4 Pediatric Use Nitric oxide for inhalation has been studied in a neonatal population (up to 14 days of age). No information about its effectiveness in other age populations is available. 8.5 Geriatric Use Nitric oxide is not indicated for use in the adult population. 10 Overdosage Overdosage with INOmax will be manifest by elevations in methemoglobin and pulmonary toxicities associated with inspired NO 2. Elevated NO 2 may cause acute lung injury. Elevations in methemoglobinemia reduce the oxygen delivery capacity of the circulation. In clinical studies, NO 2 levels >3 ppm or methemoglobin levels >7% were treated by reducing the dose of, or discontinuing, INOmax. Methemoglobinemia that does not resolve after reduction or discontinuation of therapy can be treated with intravenous vitamin C, intravenous methylene blue, or blood transfusion, based upon the clinical situation. 11 Description INOmax (nitric oxide gas) is a drug administered by inhalation. Nitric oxide, the active substance in INOmax, is a pulmonary vasodilator. INOmax is a gaseous blend of nitric oxide and nitrogen (0.08% and 99.92%, respectively for 800 ppm; 0.01% and 99.99%, respectively for 100 ppm). INOmax is supplied in aluminum cylinders as a compressed gas under high pressure (2000 pounds per square inch gauge [psig]). The structural formula of nitric oxide (NO) is shown below: 12 Clinical Pharmacology 12.1 Mechanism of Action Nitric oxide is a compound produced by many cells of the body. It relaxes vascular smooth muscle by binding to the heme moiety of cytosolic guanylate cyclase, activating guanylate cyclase and increasing intracellular levels of cyclic guanosine 3,5 -monophosphate, which then leads to vasodilation. When inhaled, nitric oxide selectively dilates the pulmonary vasculature, and because of efficient scavenging by hemoglobin, has minimal effect on the systemic vasculature. INOmax appears to increase the partial pressure of arterial oxygen (PaO 2 ) by dilating pulmonary vessels in better ventilated areas of the lung, redistributing pulmonary blood flow away from lung regions with low ventilation/perfusion (V/Q) ratios toward regions with normal ratios Pharmacodynamics Effects on Pulmonary Vascular Tone in PPHN Persistent pulmonary hypertension of the newborn (PPHN) occurs as a primary developmental defect or as a condition secondary to other diseases such as meconium aspiration syndrome (MAS), pneumonia, sepsis, hyaline membrane disease, congenital diaphragmatic hernia (CDH), and pulmonary hypoplasia. In these states, pulmonary vascular resistance (PVR) is high, which results in hypoxemia secondary to right-to-left shunting of blood through the patent ductus arteriosus and foramen ovale. In neonates with PPHN, INOmax improves oxygenation (as indicated by significant increases in PaO 2 ) Pharmacokinetics The pharmacokinetics of nitric oxide has been studied in adults Pharmacokinetics: Uptake and Distribution Nitric oxide is absorbed systemically after inhalation. Most of it traverses the pulmonary capillary bed where it combines with hemoglobin that is 60% to 100% oxygen-saturated. At this level of oxygen saturation, nitric oxide combines predominantly with oxyhemoglobin to produce methemoglobin and nitrate. At low oxygen saturation, nitric oxide can combine with deoxyhemoglobin to transiently form nitrosylhemoglobin, which is converted to nitrogen oxides and methemoglobin upon exposure to oxygen. Within the pulmonary system, nitric oxide can combine with oxygen and water to produce nitrogen dioxide and nitrite, respectively, which interact with oxyhemoglobin to produce methemoglobin and nitrate. Thus, the end products of nitric oxide that enter the systemic circulation are predominantly methemoglobin and nitrate Pharmacokinetics: Metabolism Methemoglobin disposition has been investigated as a function of time and nitric oxide exposure concentration in neonates with respiratory failure. The methemoglobin (MetHb) concentration-time profiles during the first 12 hours of exposure to 0, 5, 20, and 80 ppm INOmax are shown in Figure 1. Figure 1: Methemoglobin Concentration Time Profiles Neonates Inhaling 0, 5, 20 or 80 ppm INOmax % Methemoglobin Hours of INOmax Administration INOmax 0 ppm (n = 41) INOmax 5 ppm (n = 41) INOmax 20 ppm (n = 36) INOmax 80 ppm (n = 37) Methemoglobin concentrations increased during the first 8 hours of nitric oxide exposure. The mean methemoglobin level remained below 1% in the placebo group and in the 5 ppm and 20 ppm INOmax groups, but reached approximately 5% in the 80 ppm INOmax group. Methemoglobin levels >7% were attained only in patients receiving 80 ppm, where they comprised 35% of the group. The average time to reach peak methemoglobin was 10 ± 9 (SD) hours (median, 8 hours) in these 13 patients, but one patient did not exceed 7% until 40 hours Pharmacokinetics: Elimination Nitrate has been identified as the predominant nitric oxide metabolite excreted in the urine, accounting for >70% of the nitric oxide dose inhaled. Nitrate is cleared from the plasma by the kidney at rates approaching the rate of glomerular filtration. 13 Nonclinical Toxicology 13.1 Carcinogenesis, Mutagenesis, Impairment of Fertility No evidence of a carcinogenic effect was apparent, at inhalation exposures up to the recommended dose (20 ppm), in rats for 20 hr/day for up to two years. Higher exposures have not been investigated.

10 Nitric oxide has demonstrated genotoxicity in Salmonella (Ames Test), human lymphocytes, and after in vivo exposure in rats. There are no animal or human studies to evaluate nitric oxide for effects on fertility. 14 Clinical Studies 14.1 Treatment of Hypoxic Respiratory Failure (HRF) The efficacy of INOmax has been investigated in term and near-term newborns with hypoxic respiratory failure resulting from a variety of etiologies. Inhalation of INOmax reduces the oxygenation index (OI= mean airway pressure in cm H 2 O fraction of inspired oxygen concentration [FiO 2 ] 100 divided by systemic arterial concentration in mm Hg [PaO 2 ]) and increases PaO 2 [see Clinical Pharmacology (12.1)]. NINOS Study The Neonatal Inhaled Nitric Oxide Study (NINOS) group conducted a double-blind, randomized, placebo-controlled, multicenter trial in 235 neonates with hypoxic respiratory failure. The objective of the study was to determine whether inhaled nitric oxide would reduce the occurrence of death and/or initiation of extracorporeal membrane oxygenation (ECMO) in a prospectively defined cohort of term or near-term neonates with hypoxic respiratory failure unresponsive to conventional therapy. Hypoxic respiratory failure was caused by meconium aspiration syndrome (MAS; 49%), pneumonia/sepsis (21%), idiopathic primary pulmonary hypertension of the newborn (PPHN; 17%), or respiratory distress syndrome (RDS; 11%). Infants 14 days of age (mean, 1.7 days) with a mean PaO 2 of 46 mm Hg and a mean oxygenation index (OI) of 43 cm H 2 O / mm Hg were initially randomized to receive 100% O 2 with (n=114) or without (n=121) 20 ppm nitric oxide for up to 14 days. Response to study drug was defined as a change from baseline in PaO 2 30 minutes after starting treatment (full response = >20 mm Hg, partial = mm Hg, no response = <10 mm Hg). Neonates with a less than full response were evaluated for a response to 80 ppm nitric oxide or control gas. The primary results from the NINOS study are presented in Table 2. Table 2: Summary of Clinical Results from NINOS Study Control (n=121) NO (n=114) P value Death or ECMO*, 77 (64%) 52 (46%) Death 20 (17%) 16 (14%) 0.60 ECMO 66 (55%) 44 (39%) * Extracorporeal membrane oxygenation Death or need for ECMO was the study s primary end point Although the incidence of death by 120 days of age was similar in both groups (NO, 14%; control, 17%), significantly fewer infants in the nitric oxide group required ECMO compared with controls (39% vs. 55%, p = 0.014). The combined incidence of death and/or initiation of ECMO showed a significant advantage for the nitric oxide treated group (46% vs. 64%, p = 0.006). The nitric oxide group also had significantly greater increases in PaO 2 and greater decreases in the OI and the alveolar-arterial oxygen gradient than the control group (p<0.001 for all parameters). Significantly more patients had at least a partial response to the initial administration of study drug in the nitric oxide group (66%) than the control group (26%, p<0.001). Of the 125 infants who did not respond to 20 ppm nitric oxide or control, similar percentages of NOtreated (18%) and control (20%) patients had at least a partial response to 80 ppm nitric oxide for inhalation or control drug, suggesting a lack of additional benefit for the higher dose of nitric oxide. No infant had study drug discontinued for toxicity. Inhaled nitric oxide had no detectable effect on mortality. The adverse events collected in the NINOS trial occurred at similar incidence rates in both treatment groups [see Adverse Reactions (6.1)]. Follow-up exams were performed at months for the infants enrolled in this trial. In the infants with available follow-up, the two treatment groups were similar with respect to their mental, motor, audiologic, or neurologic evaluations. CINRGI Study This study was a double-blind, randomized, placebo-controlled, multicenter trial of 186 term and near-term neonates with pulmonary hypertension and hypoxic respiratory failure. The primary objective of the study was to determine whether INOmax would reduce the receipt of ECMO in these patients. Hypoxic respiratory failure was caused by MAS (35%), idiopathic PPHN (30%), pneumonia/sepsis (24%), or RDS (8%). Patients with a mean PaO 2 of 54 mm Hg and a mean OI of 44 cm H 2 O / mm Hg were randomly assigned to receive either 20 ppm INOmax (n=97) or nitrogen gas (placebo; n=89) in addition to their ventilatory support. Patients who exhibited a PaO 2 >60 mm Hg and a ph < 7.55 were weaned to 5 ppm INOmax or placebo. The primary results from the CINRGI study are presented in Table 3. Table 3: Summary of Clinical Results from CINRGI Study Placebo INOmax P value ECMO*, 51/89 (57%) 30/97 (31%) <0.001 Death 5/89 (6%) 3/97 (3%) 0.48 * Extracorporeal membrane oxygenation ECMO was the primary end point of this study Significantly fewer neonates in the INOmax group required ECMO compared to the control group (31% vs. 57%, p<0.001). While the number of deaths were similar in both groups (INOmax, 3%; placebo, 6%), the combined incidence of death and/or receipt of ECMO was decreased in the INOmax group (33% vs. 58%, p<0.001). In addition, the INOmax group had significantly improved oxygenation as measured by PaO 2, OI, and alveolar-arterial gradient (p<0.001 for all parameters). Of the 97 patients treated with INOmax, 2 (2%) were withdrawn from study drug due to methemoglobin levels >4%. The frequency and number of adverse events reported were similar in the two study groups [see Adverse Reactions (6.1)] Ineffective in Adult Respiratory Distress Syndrome (ARDS) ARDS Study In a randomized, double-blind, parallel, multicenter study, 385 patients with adult respiratory distress syndrome (ARDS) associated with pneumonia (46%), surgery (33%), multiple trauma (26%), aspiration (23%), pulmonary contusion (18%), and other causes, with PaO 2 /FiO 2 <250 mm Hg despite optimal oxygenation and ventilation, received placebo (n=193) or INOmax (n=192), 5 ppm, for 4 hours to 28 days or until weaned because of improvements in oxygenation. Despite acute improvements in oxygenation, there was no effect of INOmax on the primary endpoint of days alive and off ventilator support. These results were consistent with outcome data from a smaller dose ranging study of nitric oxide (1.25 to 80 ppm). INOmax is not indicated for use in ARDS. 16 How Supplied/Storage and Handling INOmax (nitric oxide) is available in the following sizes: Size D Size D Size 88 Size 88 Portable aluminum cylinders containing 353 liters at STP of nitric oxide gas in 800 ppm concentration in nitrogen (delivered volume 344 liters) (NDC ) Portable aluminum cylinders containing 353 liters at STP of nitric oxide gas in 100 ppm concentration in nitrogen (delivered volume 344 liters) (NDC ) Aluminum cylinders containing 1963 liters at STP of nitric oxide gas in 800 ppm concentration in nitrogen (delivered volume 1918 liters) (NDC ) Aluminum cylinders containing 1963 liters at STP of nitric oxide gas in 100 ppm concentration in nitrogen (delivered volume 1918 liters) (NDC ) Store at 25 C (77 F) with excursions permitted between C (59 86 F) [see USP Controlled Room Temperature]. Occupational Exposure The exposure limit set by the Occupational Safety and Health Administration (OSHA) for nitric oxide is 25 ppm, and for NO 2 the limit is 5 ppm. INO Therapeutics 6 Route 173 West Clinton, NJ USA 2009 INO Therapeutics SPC-0303 V:4.0 03/2010 IMK