Inhaled Nitric Oxide Versus Aerosolized Iloprost in
`Secondary Pulmonary Hypertension in Children With
`Congenital Heart Disease
`Vasodilator Capacity and Cellular Mechanisms
`
`Peter C. Rimensberger, MD; Isabelle Spahr-Schopfer, MD; Michel Berner, MD; Edgar Jaeggi, MD;
`Afksendiyos Kalangos, PhD, MD; Beat Friedli, MD; Maurice Beghetti, MD
`
`Background—Inhaled nitric oxide (iNO) has been used to assess the vasodilator capacity of the pulmonary vascular bed
`in children with congenital heart disease and elevated pulmonary vascular resistance. Inhaled iloprost is a pulmonary
`vasodilator for the long-term treatment of pulmonary hypertension (PHT). Because these 2 vasodilators act through
`different pathways (release of cGMP or cAMP, respectively), we compared the pulmonary vasodilator capacity of each.
`Methods and Results—A total of 15 children with congenital heart disease and PHT who had elevated pulmonary vascular
`resistance (preoperative, n⫽10; immediately postoperative, n⫽5) were first given 20 ppm of iNO for 10 minutes; then,
`after baseline values were reached again, they were given aerosolized iloprost at 25 ng 䡠 kg⫺1 䡠 min⫺1 for another 10
`minutes. Finally, iNO and iloprost were given simultaneously for 10 minutes. With iNO, the pulmonary vascular
`resistance and systemic vascular resistance ratio decreased from 0.48⫾0.38 to 0.27⫾0.16 (P⬍0.001). Similarly, iloprost
`decreased the ratio from 0.49⫾0.38 to 0.26⫾0.11 (P⬍0.05). The combination had no additional effect on the resistance
`ratio. Plasma cGMP increased from 17.6⫾11.9 to 34.7⫾21.4 nmol/L during iNO (P⬍0.01), and plasma cAMP
`increased from 55.7⫾22.9 to 65.1⫾21.2 nmol/L during iloprost inhalation (P⬍0.05).
`Conclusions—In children with PHT and congenital heart disease, both iNO and aerosolized iloprost are equally effective
`in selectively lowering pulmonary vascular resistance through an increase in cGMP or cAMP, respectively. However,
`the combination of both vasodilators failed to prove more potent than either substance alone. Aerosolized iloprost might
`be an alternative to iNO for early testing of vascular reactivity and for the postoperative treatment of acute PHT.
`(Circulation. 2001;103:544-548.)
`
`Key Words: hypertension, pulmonary 䡲 heart defects, congenital 䡲 vasodilatation 䡲 nitric oxide 䡲 prostaglandins
`
`The assessment of pulmonary vascular reactivity plays an
`
`important role in the management of patients with
`pulmonary hypertension (PHT) and congenital heart disease
`(CHD). We and others have shown that the response to early
`testing with inhaled nitric oxide (iNO) seems to depend on
`the degree of established vascular disease and, thus, may be
`helpful in selecting patients for operation.1– 4 iNO mediates
`vasodilatation through the stimulation of soluble guanylate
`cyclase to produce cGMP. Prostacyclin, an unstable arachi-
`donic acid metabolite, is another endothelial-derived vasodi-
`lator that increases the concentration of cAMP in vascular
`smooth muscle cells.5,6 Recently, inhaled prostacyclin (PGI2)
`was proven to act as a selective pulmonary vasodilator, and it
`has been proposed as an alternative to iNO for the treatment
`of adult respiratory distress syndrome7 and after repair of
`CHD.8 It has been demonstrated that
`iloprost, a stable
`
`prostacyclin analog, is as potent in its selective vasodilator
`effect as PGI2.9,10
`We compared the hemodynamic effects and changes in
`plasma cGMP and cAMP levels induced by iNO and iloprost
`alone or in combination in patients with PHT and CHD
`preoperatively or postoperatively.
`
`Methods
`
`Study Population
`Approval by the institutional ethics committee and written informed
`consent were obtained before recruiting 15 children (mean age,
`5.4⫾4.3 years) with PHT and CHD to participate in the study.
`Among the 15 children, 10 were studied in the catheterization
`laboratory preoperatively (group 1) and 5 were studied in the
`intensive care unit immediately after surgical repair (group 2). PHT
`was defined as a mean pulmonary pressure ⬎30 mm Hg and an
`
`Received May 2, 2000; revision received September 14, 2000; accepted September 19, 2000.
`From the Pediatric and Neonatal Critical Care Division (P.C.R., M. Berner) and the Pediatric Cardiology Unit (E.J., B.F., M. Beghetti), Hôpital des
`Enfants, and the Department of Pediatrics, the Pediatric Anesthesiology Unit, Department of Anesthesiology (I.S.-S.), and the Clinic for Cardiovascular
`Surgery (A.K.), University Hospital of Geneva, Switzerland.
`Correspondence to Maurice Beghetti, MD, Pediatric Cardiology Unit, Hôpital des Enfants, Department of Pediatrics, University Hospital of Geneva,
`6 Rue Willy-Donze, CH-1211 Geneva 14, Switzerland. E-mail Maurice.Beghetti@hcuge.ch
`© 2001 American Heart Association, Inc.
`Circulation is available at http://www.circulationaha.org
`
`544
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`
`545
`
`TABLE 1. Baseline Hemodynamics of All Patients
`
`Patient
`
`Diagnosis
`
`Age,
`y-mo
`
`Weight,
`kg
`
`Mean
`SAP
`
`Mean
`PAP
`
`Qp/Qs
`
`PVR,
`U 䡠 m2
`
`Rp/Rs
`
`Preoperative
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`Postoperative
`11
`12
`13
`14
`15
`
`2.9
`1.6
`1.8
`1.8
`2.4
`1.7
`2.9
`2.6
`2.5
`0.54
`
`䡠 䡠 䡠
`
`䡠 䡠 䡠
`
`䡠 䡠 䡠
`
`䡠 䡠 䡠
`
`5.45
`9.7
`5.9
`2.6
`7.4
`2.9
`3
`4.14
`6.35
`18.9
`
`0.31
`0.56
`0.35
`0.27
`0.36
`0.27
`0.21
`0.38
`0.4
`1.8
`
`5.4
`5.7
`2.9
`9.7
`6.3
`
`0.47
`0.47
`0.45
`0.52
`0.3
`
`VSD
`AVSD
`TGV corrected, VSD*
`TGV corrected, VSD*
`VSD
`VSD, PDA
`PDA, coarctation
`VSD, PDA
`ASD, PDA
`ASD, VSD
`
`5–7
`1–9
`8–6
`4–0
`5–2
`2–11
`6–5
`11–10
`14
`1–1
`
`17
`8
`16
`12
`15
`8
`10
`17
`27
`8
`
`78
`63
`64
`64
`87
`65
`85
`70
`85
`47
`
`69
`58
`45
`40
`83
`45
`61
`40
`71
`46
`
`VSD closure
`VSD closure
`AVSD repair
`VSD, PDA closure
`VSD closure
`
`4–0
`1–8
`1
`1–11
`11–11
`
`11
`6
`5
`7
`17
`
`62
`53
`51
`71
`86
`
`56
`34
`29
`34
`31
`
`䡠 䡠 䡠
`VSD indicates ventricular septal defect; ASD, atrial septal defect; AVSD, atrioventricular septal defect; TGV,
`transposition of the great vessels; PDA, patent ductus arteriosus; SAP, systemic arterial pressure; and PAP, pulmonary
`arterial pressure.
`*Post-Senning procedure with VSD left open.
`
`indexed pulmonary vascular resistance ⬎2.5 Wood U 䡠 m2. Baseline
`demographic and hemodynamic data are given in Table 1.
`
`Study Design
`The following protocol was used in all patients: (1) baseline
`measurements, (2) administration of 20 ppm iNO for 10 minutes,
`(3) return to baseline for 10 minutes, (4) administration of 25
`ng 䡠 kg⫺1 䡠 min⫺1 iloprost for 10 minutes, and (5) administration of
`iNO and iloprost for 10 minutes. Normoventilation (pH 7.38 to 7.42)
`and inspired oxygen concentration (21% in preoperative patients and
`40% in postoperative patients) were kept constant throughout the
`protocol to avoid confounding factors. At the end of each step,
`hemodynamic changes were recorded, arterial blood gases were
`measured to ensure stable pH conditions, and blood samples were
`drawn from the left atrium or systemic artery for measurements of
`cGMP and cAMP.
`Children in group 1 were premedicated with midazolam (Dormi-
`cum; 0.5 mg/kg PO; maximum dose 15 mg). For heart catheteriza-
`tion, the children were intubated and sedated using the following
`protocol: 10 ␮g/kg atropine as a single dose; 10 ␮g/kg alfentanil
`(Rapifen) as a single dose; 0.5 mg/kg atracurium as a single dose to
`facilitate intubation; and 2.5 to 3.5 mg/kg propofol (Disoprivan) as a
`bolus, followed by 10 to 15 mg 䡠 kg⫺1 䡠 h⫺1 propofol as a continuous
`infusion.
`Children in group 2, who were already intubated and ventilated,
`were sedated by a continuous infusion of morphine (40 to 80
`␮g 䡠 kg⫺1 䡠 h⫺1) and midazolam (0.2 mg 䡠 kg⫺1 䡠 h⫺1), as it is standard
`practice in our unit in the early postoperative period. Normoventi-
`lation was assured during the test protocol for both groups.
`
`Drug Administration
`Medical-grade quality NO at 300 ppm in nitrogen was administered
`through a separate flowmeter into the inspiratory limb of the
`ventilator circuit, where it was volumetrically diluted to 20 ppm and
`monitored from a sampling port in the inspiratory limb close to the
`endotracheal tube connector. Peak NO and NO2 concentrations were
`
`continuously measured with an electrochemical device during NO
`administration (PrinterNOx, Micro Medical Ltd).
`Iloprost (Ilomedin, Schering AG, Schlieren, Switzerland) was
`prepared from a vial of 50 ␮g/0.5 mL and diluted to obtain a
`theoretical alveolar deposition of 25 ng 䡠 kg⫺1 䡠 min⫺1. Dilution was
`calculated according the following formula: aerosol concentration/
`mL⫽(dose ⫻ body weight [kg])/nebulization rate (mL/min) of the
`nebulizer. For nebulization, we used the Respirgard II (Marquest
`Medical Products) nebulizer connected to the inspiratory limb of the
`ventilator or to a modified Jackson-Rees system for bag ventilation,
`as described previously.11 This jet nebulizer shows the following
`characteristics: mass median aerodynamic diameter, 2.1 ␮m; mass
`distribution, 80% of droplets were ⬍6 ␮m, allowing a high intrapul-
`monary deposition in small children; and nebulization rate, ⬇0.15
`mL/min at a flow rate of 6 L/min.12 Iloprost was continuously
`aerosolized at a flow rate of 6 L/min.
`
`Hemodynamic Assessment
`Catheters were inserted femorally and placed in the superior vena
`cava, the pulmonary artery, the left atrium, and the aorta (or a
`peripheral systemic artery) to allow simultaneous measurements of
`pressure and blood gases and to avoid time-consuming and con-
`founding measurements due to catheter manipulations. For patients
`with an atrial septal defect or a patent foramen ovale, a catheter was
`placed in a pulmonary vein instead of the left atrium. In the absence
`of an interatrial communication, pulmonary arterial wedge pressure
`was measured instead of left atrial pressure.
`Intravascular pressures were measured with fluid-filled transduc-
`ers, and oxygen content was calculated from hemoglobin concentra-
`tions and oxygen saturations. Blood gases were measured from
`systemic arterial blood at each level. Cardiac index and indexed
`pulmonary and systemic blood flow (Qp and Qs, respectively)
`calculations, based on the Fick principle, were obtained from
`assumed oxygen consumption. Systemic and pulmonary vascular
`resistances were calculated with standard formulas and indexed to
`body surface area. The pulmonary-to-systemic vascular resistance
`ratio (Rp/Rs) was then calculated. Oxygen consumption was as-
`sumed constant throughout the study and was not measured during
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`drug administration, because the calculation of Rp/Rs avoids the
`influence of oxygen consumption changes. Hemodynamic parame-
`ters were measured at baseline and at the end of each period of drug
`exposure.
`
`cGMP and cAMP Assays
`Blood samples were collected in an EDTA vacutainer (Becton
`Dickinson Vacutainer systems) and immediately placed on ice.
`Plasma was obtained by centrifugation at 1600g for 10 minutes at
`4°C and then stored at ⫺70°C until analysis. cGMP and cAMP levels
`were determined in batches using a commercial radioimmunoassay
`kit (Immunotech catalog #1118 and 1117). The cGMP assay had a
`measurement range of 0.1 to 100 nmol/L and a sensitivity of 10
`pmol/L. The cAMP assay had a measurement range of 5 to 50
`nmol/L, and the sensitivity of the assay was 0.2 nmol/L. cGMP and
`cAMP levels are expressed in nmol/L.
`
`Statistical Analysis
`The data were computerized and analyzed using the GraphPad Prism
`Software package. Mean, median, SD, and lower and upper 95%
`confidence intervals were calculated for all parameters. Results are
`expressed as mean⫾SD. Analyses were performed for all patients
`and separately for groups 1 and 2. For each parameter, repeated
`measures ANOVA for normally distributed data or the Friedman’s
`test for non-normally distributed data were used, with post hoc
`corrections as appropriate, for all pairwise multiple comparisons
`(Tukey’s or Dunn multiple comparison test, respectively). P⬍0.05
`was considered significant.
`
`Results
`Patient demographics and hemodynamic data are given in
`Table 1. Inhaled NO caused a selective pulmonary vasodila-
`tation in 14 of 15 patients and an overall decrease in Rp/Rs
`from 0.48⫾0.38 (baseline 1) to 0.27⫾0.16 (P⬍0.001). Sim-
`ilarly, aerosolized iloprost resulted in a selective pulmonary
`vasodilatation in the same 14 patients, and Rp/Rs decreased
`from 0.49⫾0.38 (baseline 2) to 0.26⫾0.11 (P⬍0.05). The
`combination of both drugs did not result in an additional
`decrease of Rp/Rs (0.26⫾0.11 to 0.24⫹0.09).
`One patient (patient 3 in Table 1), who had a ventricular
`septal defect after a previous palliative atrial switch proce-
`dure (Senning) for transposition of the great arteries, did not
`respond at all. One patient (patient 10 in Table 1), who had a
`ventricular septal defect and an atrial septal defect, presented
`with suprasystemic pulmonary arterial pressures and a right-
`to-left shunt (Qp/Qs, 0.54; Rp/Rs, 1.8) before testing. The
`intracardiac shunt diminished under iNO (Qp/Qs, 0.95) and
`inversed under aerosolized iloprost (Qp/Qs, 1.51) due to a
`decrease of Rp/Rs to 0.73 and 0.55, respectively. Rp/Rs
`decreased further to 0.43 when both substances were com-
`bined (Figure 1). When this patient was excluded from
`statistical analysis because of his very high initial pulmonary
`resistance, thus allowing for the testing of a more homoge-
`nous group, results still remained significant. The Rp/Rs
`decreased from 0.39⫾0.11 (baseline 1)
`to 0.24⫾0.10
`(P⬍0.001) under iNO and from 0.39⫾0.11 (baseline 2) to
`0.24⫾0.08 (P⬍0.001) under aerosolized iloprost. The com-
`bination of both drugs did not further decrease Rp/Rs
`(0.24⫾0.08 to 0.22⫾0.08; Figure 2).
`Plasma cGMP levels (Table 2) increased by 97%, from
`17.6⫾11.9 nmol/L (baseline 1) to 34.7⫾21.4 nmol/L, during
`iNO (P⬍0.01) and normalized to baseline values after iNO
`was discontinued. During combined iNO and iloprost inha-
`
`Figure 1. Changes in Rp/Rs (F, solid line) and intracardiac
`shunt (Qp/Qs; ‚, dotted line) in patient 10, showing inversion of
`shunt with iNO and under iloprost aerosolization.
`
`lation, cGMP increased from 17.4⫾14.1 nmol/L (on iloprost
`alone) to 36.1⫾15.2 nmol/L (P⬍0.001). Plasma cAMP levels
`(Table 2) remained stable under iNO (baseline, 55.7⫾22.9
`nmol/L; during iNO, 54.0⫾21.8 nmol/L). During the 10-
`minute administration of iloprost, cAMP increased by 20%,
`from 54.0⫾21.8 to 65.1⫾21.2 nmol/L (P⬍0.05). It remained
`elevated, at 66.0⫾19.85 nmol/L, during the combined admin-
`istration of iNO and iloprost (P⬍0.05 versus baseline and
`iNO). Subgroup analyses of preoperative or postoperative
`patients showed no differences in response when compared
`with all patients.
`
`Discussion
`In our study population, inhaled NO and aerosolized iloprost,
`at
`the concentration or doses used, were proven to be
`equipotent and equally selective in lowering pulmonary
`vascular resistance through an increase in either cGMP or
`cAMP, respectively. We observed no changes in systemic
`vascular resistance or in systemic arterial pressures during
`iloprost inhalation such as other investigators have reported,13
`although we delivered slightly higher doses of 25
`ng 䡠 kg⫺1 䡠 min⫺1 over a longer time (ie, over a total of 20
`minutes when combining both substances) than those authors
`did. This may be explained by the use of a nebulizer, different
`characteristics of the aerosol spray, and the different intrapul-
`monary deposition characteristics between children and
`adults and between intubated and nonintubated patients.
`
`Figure 2. Individual responses from all patients in Rp/Rs to iNO
`and aerosolized iloprost (with exception of patient 10).
`Responders are indicated by F and nonresponders by 䡩.
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`Comparison of Inhaled Pulmonary Vasodilators
`
`547
`
`TABLE 2.
`
`Levels of cGMP and cAMP in Plasma Samples
`
`extracellular matrix, or plexiform obstructive vascular lesions
`may be responsible for the absent reaction. Although we did
`not see a synergistic effect of the combination of both
`vasodilators when statistical analysis were performed, there
`were a few patients who showed a further decrease in Rp/Rs
`when both substances were combined. One patient with both
`ventricular and atrial septal defects and a right-to-left shunt
`(Qp/Qs, 0.54) showed an impressive response to both iNO
`and iloprost alone, with inversion of the intracardiac shunt on
`aerosolized iloprost, and further improvement of Rp/Rs when
`both substances were combined (Figure 1). This observation
`suggests that there might be a subgroup of patients who will
`respond to a combined treatment better than to a single
`treatment.18 Whether this is of clinical or prognostic impor-
`tance remains to be evaluated.
`iNO increased cGMP and iloprost increased cAMP (Table
`2). Both responses were highly selective, indicating different
`independent mediator pathways for the 2 vasodilators. Al-
`though initially different messenger pathways will be acti-
`vated,
`the final pathway leading to smooth muscle cell
`relaxation is probably the same for both substances (ie, a
`decrease in the concentration of free intracellular cytosolic
`Ca2⫹).20,21 This might explain the lack of a synergistic effect
`when combining both pulmonary vasodilators. We found no
`evidence for an iloprost-induced endothelial cell release of
`NO, which would again stimulate soluble guanylate cyclase
`to produce cGMP, as has been postulated to be the case for
`PGI2.14,22
`PGI2 has been proposed as an alternative to iNO after the
`repair of CHD.8 The nebulization of PGI2 or iloprost might
`have some advantages over the inhalation of NO, such as its
`lack of toxic reactions23,24 (which require monitoring NO2
`and methemoglobin formation during NO inhalation) and its
`easy administration by conventional nebulizers compared
`with the more complicated delivery systems required for NO.
`Furthermore, possibly life-threatening rebound phenomenons
`have been described with iNO25,26 but not with aerosolized
`PGI2 or iloprost withdrawal. These advantages might favor
`the use of iloprost for the treatment of PHT; however,
`because of the lack of well-documented studies with prosta-
`cyclin or prostacyclin analogs,
`it might be too early to
`recommend the use of iloprost
`instead of iNO for the
`postoperative management of patients with PHT and CHD.
`The use of iNO has been shown to be helpful for the
`assessment of pulmonary vascular reactivity in selecting
`patients for surgery.1– 4 In this setting, the aerosolization of
`prostacyclin might offer a real alternative to iNO. However,
`as with any nebulizer device, some uncertainty will exist
`regarding the amount of prostacyclin effectively delivered
`into the alveolar space. This leads to a potential risk to falsely
`classify some patients as nonresponders. The problem may be
`more present in children, in whom aerosol deposition shows
`more variability and is less well studied than in adults.27,28
`Our study has several
`limitations. First,
`iloprost was
`always given after iNO because of its longer half-life; iloprost
`has a documented hemodynamic effect of ⬇1 to 2 hours,9,13
`which would have excluded a 2-group study protocol with a
`randomized drug allocation. Such a protocol would have
`required a longer testing protocol, which we thought was not
`
`Baseline
`18⫾12
`cGMP, nmol/L
`56⫾23
`cAMP, nmol/L
`Values are mean ⫾ SD.
`*P⬍ 0.01 vs baseline; † P⬍ 0.05 vs baseline or iNO (repeated measures
`ANOVA).
`
`Iloprost
`17⫾14
`65⫾21†
`
`iNO⫹Iloprost
`36⫾15*
`66⫾19†
`
`iNO
`35⫾21*
`54⫾22
`
`However, the positive hemodynamic response and the signif-
`icant
`increase in cAMP levels with aerosolized iloprost
`exclude an underdosing of this substance in our study.
`Although a synergistic effect might be theoretically ex-
`pected when combining both substances, because the vaso-
`dilatation is induced by different second messengers, we
`failed to demonstrate such a synergistic effect except in one
`patient. The combination of 2 different vasodilators directly
`applied to the airways has, to our knowledge, never been
`tested clinically, although results from animal studies would
`suggest to do so.14,15 In these animal studies, the combination
`of PGI2 and iNO produced a more enhanced vasodilator effect
`compared with their separate effects, and this was dose-
`dependent. It was suggested that the combination of both
`drugs might be useful in the management of PHT. Also, a
`synergistic effect on pulmonary vasodilatation has been
`shown in clinical studies in children with PHT, in which oral
`beraprost, a PGI2 analog, was combined with iNO.16 –18 All
`these studies showed major differences compared with the
`present study. The results may differ for the following
`reasons. (1) The doses of prostacyclin used by Ikeda et al15 in
`animals were ⬇100-fold greater than those commonly used
`clinically (and those tested to be safe and without the risk of
`overdosing); this must result in a spillover into the systemic
`circulation, with a subsequent decrease of arterial pressure.19
`(2) An oral preparation of a PGI2 analog was used in 3
`studies16 –18; therefore, drug dosing can hardly be compared
`with an application of iloprost by aerosol. Indeed, there are no
`studies in children with CHD and PHT thus far in whom
`prostacyclin or iloprost was given by aerosolization into the
`airways.
`All but 1 of our 15 patients responded to iNO and to
`iloprost. The only nonresponder to either substance even
`failed to respond to the combination, despite a net increase in
`cGMP and cAMP levels (Table 3). This observation suggests
`that mechanisms other than the vascular smooth muscle cell
`concentration and release of the 2 second messengers may
`play an important role in vasodilatation. A defect further
`down the pathway, fixed rigid vessels caused by increased
`
`TABLE 3. Hemodynamics and cGMP and cAMP Levels of
`Patient 3 (Nonresponder)
`
`Mean SAP, mm Hg
`Mean PAP, mm Hg
`Rp/Rs
`cGMP, nmol/L
`cAMP, nmol/L
`
`Baseline
`
`64
`45
`0.35
`13.9
`34
`
`iNO
`
`65
`47
`0.32
`19.6
`35
`
`Iloprost
`
`70
`49
`0.37
`14.1
`56
`
`iNO⫹Iloprost
`67
`46
`0.37
`24.6
`58
`
`SAP indicates systemic arterial pressure; PAP, pulmonary arterial pressure.
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`acceptable for ethical reasons. Furthermore, it would have
`been more difficult to insure stable conditions and a return to
`baseline over such a long period. Second, although we made
`sure of a return back to baseline hemodynamics after iNO
`inhalation before administering iloprost, a potential interac-
`tion between iNO-induced effects and the iloprost-induced
`vasodilator capacities on the endothelial cell cannot be
`excluded. However, the return to baseline of cGMP levels
`after iNO inhalation under iloprost makes this unlikely.
`Finally, dose-response studies were not performed. There-
`fore, we cannot exclude that higher doses of iloprost might
`have resulted in a dose-dependent additional decrease in
`pulmonary arterial resistance. However, this is unlikely on
`the basis of other reports.10,29,30
`In children with PHT and CHD, both iNO and aerosolized
`iloprost are equally effective in selectively lowering pulmo-
`nary vascular resistance through a selective increase in cGMP
`or cAMP, respectively. However, the combination of both
`vasodilators failed to prove more potent than either substance
`alone. Although aerosolized iloprost may be an alternative to
`iNO for early testing of vascular reactivity and for the
`postoperative treatment of acute PHT, it may prove to be
`more useful for the prolonged treatment of PHT.
`
`Acknowledgments
`Supported by Schering AG, Schlieren, Switzerland. The authors
`thank Lazlo Vadas, PhD, Department of Medical Biochemistry,
`University of Geneva, Switzerland, for his assistance and for his
`measurements of cGMP and cAMP levels.
`
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`IKARIA EXHIBIT 2012
`Praxair v. INO Therapeutics
`IPR2015-00522
`
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