Clinical trial NCT01431313
Conditions: Pulmonary Hypertension Heart Failure, Diastolic
Keywords: pulmonary hypertension heart failure
Sponsor: Schmidhofer, Mark, MD
Start date: 2012-06-01; Completion date: 2017-10-01
Enrollment: 48 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This is a single-center, open label phase II study to evaluate the effect of inhaled nitrite delivered in a dose escalation manner on the change in pulmonary vascular resistance (PVR) in subjects with pulmonary hypertension undergoing right heart catheterization.

A total of 50 subjects with a confirmed diagnosis of pulmonary hypertension and meet all inclusion/exclusion criteria will be enrolled in the study which will entail a single right heart catheterization and nebulized nitrite dose of 45mg with one subsequent dosage of 90 mg.

Detailed description:
Screening Visit:Initial screening evaluations including physical examination, medical history, and clinical laboratory assessments will be conducted to determine study eligibilities during a routine clinic visit at the UPMC HVI, CLC or inpatient at UPMC Presbyterian. Subjects who meet the inclusion criteria and none of the exclusion criteria will be entered into the study.

Day 1: This study visit will occur on the same day subjects are scheduled for their clinically indicated right heart catheterization or who volunteer for a research right heart catheterization for this specific study. Subjects on oral background PAH therapy (ETRA or PDE5I) will be instructed to hold their regimen on the day of the study visit.

Subjects will receive nebulized AIR001 doses escalated based upon safety and tolerability. The dose of inhaled nitrite will be delivered via electronic nebulizer. During the study right heart/pulmonary artery hemodynamics will be measured as well as noninvasive systemic blood pressure monitoring. Subjects will be tested for the changes in pulmonary vascular resistance (PVR) using standard clinical protocol hemodynamic recordings of right atrial, right ventricular, and pulmonary artery pressures, in addition to cardiac output at time zero,

3 Day phone follow up

30 Day follow up visit: All subjects enrolled in the study will be followed for 30 days (+/- 5 day window) after completion of the study treatment. A physical exam and clinical labs will be obtained at this visit.

Arms: arm 1: Sodium Nitrite Inhalation Solution (AIR001) 45mg dosage with one subsequent escalation dosage of 90mg based on tolerability.
Interventions: intervention 1: Inhaled Nitrite
Intervention descriptions: intervention 1: Each patient will receive a starting dose of 45 mg of inhaled nitrite, with one planned subsequent planned dose of 90 mg of inhaled nitrite

Protocol documents:
Prot_SAP_000.pdf: 
 
 
 
 
 
 
 
A Dose Escalation Study to Evaluate the Effect of Inhaled Nitrite (AIR001) on 
Cardiopulmonary Hemodynamics in Subjects with Pulmonary Hypertension 
 
 
 
 
 
 
 
 
 
 
 
Marc Simon, MD 
Principal Investigator 
 
 
 
 
 
 
 
 
University of Pittsburgh  
School of Medicine 
Division of Cardiology 
 
 
 
 
 
Version 3.0 
October 12, 2017 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
_________________________________________________________________________________________________________ 
_________________________________________________________________________________________________________ 
CONFIDENTIAL                                                           Page 1 of 51                                                               Protocol v3.0_10/12/2017                                  
 
TABLE OF CONTENTS 
 
PROTOCOL SYNOPSIS………………………………………………………………………………...3 
 
 
1. 
STUDY OBJECTIVE, SPECIFIC AIMS, BACKGROUND AND SIGNIFICANCE ............... 10 
 
1.1 
OBJECTIVE................................................................................................................ 10 
1.2 
SPECIFIC AIMS ......................................................................................................... 10 
1.3 
BACKGROUND .......................................................................................................... 10 
1.4 
SIGNIFICANCE .......................................................................................................... 15 
 
 
2. 
RESEARCH DESIGN AND METHODS ............................................................................. 16 
 
2.1 
CLASSIFICATION AND METHODOLOGICAL DESIGN ............................................. 16 
2.2 
DETAILED DESCRIPTION OF RESEARCH ACTIVITIES .......................................... 16 
2.3 
STUDY TREATMENT ................................................................................................. 21 
2.4 
STUDY DESIGN SCHEMATIC ................................................................................... 22 
2.5 
DURATION OF FOLLOW-UP ..................................................................................... 22 
2.6 
SAFETY MONITORING .............................................................................................. 22 
2.7 
STUDY INVESTIGATIONAL THERAPY SUPPLIES ................................................... 23 
2.8 
STUDY INVESTIGATIONAL THERAPY STORAGE ................................................... 23 
2.9 
STUDY INVESTIGATIONAL THERAPY ACCOUNTABILITY ..................................... 24 
2.10 STUDY PROCEDURES ............................................................................................. 24 
2.11 SCHEDULE OF ACTIVITIES ...................................................................................... 27 
2.12 DESCRIPTION OF STUDY PROCEDURES .............................................................. 28 
2.13 ENDPOINTS ............................................................................................................... 30 
2.14 WITHDRAWAL CRITERIA .......................................................................................... 31 
2.15 STATISTICAL ANALYSIS ........................................................................................... 32 
 
 
3. 
HUMAN SUBJECTS ......................................................................................................... 33 
 
3.1 
SUBJECT POPULATION............................................................................................ 33 
3.2 
INCLUSION CRITERIA............................................................................................... 33 
3.3 
EXCLUSION CRITERIA.............................................................................................. 34 
 
 
4. 
RECRUITMENT AND INFORMED CONSENT PROCEDURES ........................................ 35 
4.1 
RECRUITMENT METHODS ....................................................................................... 35 
4.2 
INFORMED CONSENT PROCEDURES ..................................................................... 35 
 
 
5. 
POTENTIAL RISKS AND BENEFITS ............................................................................... 36 
5.1 
POTENTIAL RISKS .................................................................................................... 36 
5.2 
ALTERNATIVE TREATMENTS .................................................................................. 39 
5.3 
BENEFITS .................................................................................................................. 40 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
_________________________________________________________________________________________________________ 
_________________________________________________________________________________________________________ 
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5.4 
RISK MANAGEMENT ................................................................................................. 40 
5.5 
DATA SAFETY MONITORING PLAN.......................................................................... 40 
 
 
6. 
STUDY ADMINSTRATION ................................................................................................ 45 
6.1 
QUALITY CONTROL AND QUALITY ASSURANCE .................................................. 45 
6.2 
DATA HANDLING AND RECORD-KEEPING.............................................................. 46 
6.3 
ETHICS ...................................................................................................................... 47 
 
 
7. 
COSTS AND PAYMENTS ................................................................................................. 48 
7.1 
COSTS ....................................................................................................................... 48 
7.2 
PAYMENTS ................................................................................................................ 48 
 
 
8. 
QUALIFICATIONS AND SOURCE OF SUPPORT ............................................................ 49 
8.1 
QUALIFICATIONS OF INVESTIGATORS ................................................................... 49 
8.2 
SOURCE OF SUPPORT............................................................................................. 49 
 
 
9. 
REFERENCES .................................................................................................................. 50 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
_________________________________________________________________________________________________________ 
_________________________________________________________________________________________________________ 
CONFIDENTIAL                                                           Page 3 of 51                                                               Protocol v3.0_10/12/2017                                  
 
PROTOCOL SYNOPSIS 
 
Protocol Title: 
A Dose Escalation Study to Evaluate the Effect of Inhaled Nitrite 
on 
Cardiopulmonary 
Hemodynamics 
in 
Subjects 
with 
Pulmonary Hypertension 
 
Protocol Number: 
PRO 11080686 
 
NCT Number: 
NCT01431313 
 
Version # and Date: 
Version 3.0/October 12, 2017 
 
Clinical Phase: 
 
Phase II clinical investigation 
 
Investigational Drug: 
Sodium Nitrite Inhalation Solution (AIR001 Inhalation Solution) 
 
Trial Site: 
Single-Center Trial 
 
Sponsor: 
Mark Schmidhofer, MD 
Associate Professor of Medicine 
University of Pittsburgh 
Heart and Vascular Institute 
 
Investigator: 
 
Marc Simon, MD, MS, FACC 
Associate Professor of Medicine 
University of Pittsburgh 
UPMC Heart and Vascular Institute 
 
Sub-Investigators: 
Mark T. Gladwin, MD            
Michael Risbano, MD 
 
Allison Morris, MD 
Frank Sciurba, MD 
Dennis McNamara, MD 
Study Monitor: 
Mark Schmidhofer, MD 
Associate Professor of Medicine 
University of Pittsburgh 
Heart and Vascular Institute 
 
Research Facilities: 
University of Pittsburgh Medical Center 
200 Lothrop Street 
Pittsburgh, PA. 15213 
 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
_________________________________________________________________________________________________________ 
_________________________________________________________________________________________________________ 
CONFIDENTIAL                                                           Page 4 of 51                                                               Protocol v3.0_10/12/2017                                  
 
Clinical Laboratories: 
University of Pittsburgh Medical Center 
200 Lothrop Street 
Pittsburgh, PA. 15213 
 
Manufacturer: 
Aires Pharmaceuticals, Inc. 
 
Study Rationale: 
Inhaled nitric oxide has been demonstrated to improve 
pulmonary hemodynamics in subjects with pulmonary arterial 
hypertension (PAH).   
 
We hypothesize that inhaled nitrite provides a therapeutic 
approach for patients with PAH for the following reasons: 
1. Inhaled nitrite therapy provides a potent, effective treatment 
that will vasodilate the pulmonary circulation (reduce 
pulmonary vascular resistance), reduce mean pulmonary 
artery pressure, and increase cardiac output. 
 
2. This study will provide valuable efficacy and safety insights 
into the interactions between NO and nitrite, both direct NO 
generators, with or without background PH therapy, 
particularly phosphodiesterase type-5 inhibitors (PDE-5I), 
on pulmonary hemodynamics and right heart function. 
 
3. This study will also provide novel assessments of right 
ventricular global myocardial function. 
 
4. The results obtained from this study will also lay the 
groundwork for future large-scale clinical trials that are 
needed to investigate the efficacy of inhaled nitrite and/or its 
long-term effects on pulmonary hemodynamics in patients 
with pulmonary hypertension.   
 
Study Objectives: 
The main objective of this study is to investigate the effect of 
inhaled nitrite delivered in a dose escalation manner in patients  
that may be on background pulmonary hypertension therapy; 
endothelin receptor antagonist (ETRA), PDE-5I, or prostacyclin 
analog on cardiopulmonary hemodynamics in subjects with 
pulmonary hypertension.  
 
Study Hypothesis: 
• We hypothesize that inhaled nitrite treatment will improve 
clinical parameters of pulmonary vascular and right heart 
function. Our primary endpoint will be the change in 
pulmonary vascular resistance (PVR) measured by right 
heart catheterization. 
 
• We hypothesize that inhaled nitrite treatment will improve 
global right ventricular function. 
 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
_________________________________________________________________________________________________________ 
_________________________________________________________________________________________________________ 
CONFIDENTIAL                                                           Page 5 of 51                                                               Protocol v3.0_10/12/2017                                  
 
• We hypothesize that inhaled nitrite may be delivered safely, 
based on stable mean arterial blood pressure with minimal 
dose limiting symptoms, in patients on background PAH 
therapy, particularly PDE-5I (sildenafil, tadalafil). 
 
Study Aims: 
Specific Aims: 
 
1. To determine the efficacy of inhaled nitrite, on the 
change in pulmonary vascular resistance measured by 
right heart catheterization. 
 
2. To 
explore 
the 
effect 
of 
inhaled 
nitrite 
on 
echocardiographic 
and 
hemodynamic 
parameters 
measured: 
SVR, 
RV 
systolic 
(dP/dtmax/IP, 
PWRmax/EDV, RV EF, TAPSE), RV diastolic function 
(dP/dtmin, Tau), Pulmonary vascular impedance / Wave 
Intensity. 
 
3. To evaluate the safety of inhaled nitrite on systemic 
blood pressure in patients on background therapy for 
PAH. 
 
4. To identify the pharmacokinetic relationships of nitrite 
measures of drug exposure and plasma cGMP, using 
blood sampling. 
   
5. To identify the changes in mitochondrial oxygen 
consumption, using analysis of platelets. 
 
6. To compare effects of inhaled nitrite on pulmonary 
vascular resistance with inhaled NO gas at 40 ppm. 
 
    
This is a single-center, open label phase II study to evaluate the 
effect of inhaled nitrite delivered in a dose escalation manner on 
the change in pulmonary vascular resistance (PVR) in subjects 
with pulmonary arterial hypertension undergoing right heart 
catheterization. 
 
A total of 50 subjects with a confirmed diagnosis of pulmonary 
hypertension and meeting all inclusion/exclusion criteria will be 
enrolled in the study which will entail a single right heart 
catheterization. The study population consisted of subjects with 
WHO group I PAH (n=20), WHO group II PH (n=20) and 
subjects with WHO group III PH (n=10) will receive the dose 
escalation paradigm: 
 
• Each subject will receive a starting dose of 45 mg inhaled 
nitrite (AIR001 Inhalation Solution), followed by a second dose 
of 90 mg inhaled nitrite, based on safety and tolerability. 
 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
_________________________________________________________________________________________________________ 
_________________________________________________________________________________________________________ 
CONFIDENTIAL                                                           Page 6 of 51                                                               Protocol v3.0_10/12/2017                                  
 
The study visit will occur on the same day subjects are 
scheduled for their clinically indicated right heart catheterization 
or who volunteer for a research right heart catheterization for 
this specific study.  Subjects on oral background PAH therapy 
(ETRA or PDE-5I) will be instructed to hold their regular PAH 
regimen on the day of the study visit.   
 
Baseline 
pulmonary 
artery 
hemodynamic 
assessment, 
echocardiogram, micromanometry measured simultaneous 
pressure and flow velocity signals will be performed prior to 
inhaled nitrite treatment.  Responses to 40 ppm inhaled NO will 
be measured before nitrite nebulization via a non-rebreather 
face mask to assess baseline, pre-AIR001 vasodilator 
responsiveness. 
 
Subjects will receive a starting dose of 45 mg (placed in the 
nebulizer cup) of inhaled nitrite (AIR001), with one subsequent 
planned dose of 90 mg of inhaled nitrite, based on safety and 
tolerability, approximately 60 minutes after the first dose. During 
the study, right heart/pulmonary artery hemodynamics will be 
measured continuously, and cardiac output measured at 15 
minute intervals, as well as noninvasive systemic blood 
pressure monitoring. Subjects will be tested for the changes in 
hemodynamics and calculated pulmonary vascular resistance 
(PVR) with inhaled nitrite.  
 
Subjects will be monitored closely for changes in blood pressure 
during the study.  .The presence of severe systemic hypotension 
defined as systolic blood pressure >30 mm Hg drop from pre-
dose baseline AND symptomatic or requiring fluid replacement 
or other therapy will lead to a discontinuation of the study 
treatment, and the next higher dose of inhaled nitrite will not be 
administered.   Subjects will continue to be monitored closely.  
 
Oxygen 
saturation 
measured 
by 
pulse 
oximeter 
and 
methemoglobin levels monitored by co-oximetry prior to dose 
administration and through 2 hours post-dose will be monitored 
closely. 
 
Blood samples may be collected for Cyclic Guanosine 
Monophosphate (cGMP) concentration determination, 
pharmacokinetics (PK) and platelet mitochondrial analysis at 
pre dose and the end of the each peak dose of nitrite (approx.. 
15 minutes post inhaled dose).  PK and cGMP measurements 
will be made on mixed venous blood using PCW pullback 
samples and venous blood will be collected for platelet 
mitochondrial analysis.  
 
Subjects will be monitored carefully for occurrences of adverse 
events, laboratory test abnormalities, and changes in vital signs.  
 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
_________________________________________________________________________________________________________ 
_________________________________________________________________________________________________________ 
CONFIDENTIAL                                                           Page 7 of 51                                                               Protocol v3.0_10/12/2017                                  
 
Following the study treatment, patients will be followed as an 
outpatient on Day 30 (+/- 5 day window). An additional follow-up 
assessment by telephone will occur on Day 3.  
 
Planned Sample Size: 
50 subjects 
 
Duration of Treatment: 
One day 
 
Inclusion Criteria: 
WHO Group I PAH (n = 20) 
 Diagnosis of RHC confirmed WHO Group I PAH  in any of the 
following 3 categories: 
• 
Idiopathic, primary or familial pulmonary arterial  
hypertension (IPAH, PPH, or FPAH)  
                       OR 
• 
PAH associated with one of the following 
connective tissue diseases (CTD): 
 
              i. Systemic sclerosis (scleroderma) 
 
             ii. Limited scleroderma 
 
             iii. Mixed connective tissue disease 
 
             iv. Systemic lupus erythematosus 
 
             v. Overlap syndrome;   
                              OR 
• 
PAH associated with:   
                     i. 
Human 
immunodeficiency 
virus 
(HIV)  
infection; 
                     ii. Simple, congenital systemic-to-pulmonary  
shunts at   least one year post-surgical repair. 
                    iii. Exposure to legal drugs, chemicals and 
toxins, such as fenfluramine, derivatives, other anorexigens, 
toxic rapeseed oil or L-tryptophan.  Subjects with PAH 
associated with illegal drug use, such as methamphetamine, are 
excluded. 
 
Stable PH for at least 3 months if on disease specific therapy. 
 
WHO Group II PH (=20) 
Diagnosis of RHC confirmed WHO Group II PH by: 
• 
Pulmonary capillary wedge pressure (PWCP)  > 
15 
AND 
• 
Transpulmonary Gradient (TPG) > 12 
 
WHO Group III PH (n = 10) 
    Has WHO functional class II-IV symptoms 
Had the diagnosis of PH confirmed by a cardiac 
catheterization with the following values: 
o Mean pulmonary artery pressure (mPAP) ≥ 25 mm Hg 
(at rest) 
o PCWP or left ventricular-end diastolic pressure £15 mm 
Hg (if diagnosed with PAH) 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
_________________________________________________________________________________________________________ 
_________________________________________________________________________________________________________ 
CONFIDENTIAL                                                           Page 8 of 51                                                               Protocol v3.0_10/12/2017                                  
 
o Pulmonary Vascular Resistance (PVR) ≥ 3 mm 
Hg/L/min or ≥ 240 dynes*sec/cm5 
 
 
Both WHO Group I PAH, WHO Group II and III PH  
1) Age 18 and older; 
2) Able to participate in right heart catheterization 
3) Evidence of a personally signed and dated informed consent 
document indicating that the subject has been informed of all 
pertinent aspects of the study 
4) Subjects who are willing and able to comply with scheduled 
visits, treatment plan, laboratory tests, and other study 
procedures 
Exclusion Criteria: 
• Age less than 18 years; 
• Baseline systemic hypotension, defined as MAP less than 50 
mmHg; 
• Required intravenous inotropes within 30 days prior to study 
participation; 
• Has uncontrolled systemic hypertension as evidenced by 
sitting systolic blood pressure >160 mm Hg or sitting diastolic 
blood pressure >100 mm Hg at screening; 
• Has a history of portal hypertension or chronic liver disease, 
including hepatitis B and/or hepatitis C (with evidence of 
recent infection and/or active virus replication) defined as 
moderate to severe hepatic impairment (Child-Pugh Class B-
C); 
• Has chronic renal insufficiency as defined by serum 
creatinine >2.5 mg/dL at screening or requires dialysis 
support; 
• Has a hemoglobin concentration <9 g/dL at Screening; 
• History of atrial septostomy within 6 months prior to Day 1 
visit 
• Repaired or unrepaired congenital heart disease (CHD); 
• Pericardial constriction; 
• Confirmed diagnosis of restrictive or constrictive 
cardiomyopathy; 
• Left ventricular ejection fraction (EF) <40% by multiple gated 
acquisition scan (MUGA), angiography or 
echocardiography;  
• Symptomatic coronary disease with demonstrable ischemia; 
• Other severe acute or chronic medical or laboratory 
abnormality that may increase the risk associated with study 
participation or investigational product administration or may 
interfere with the interpretation of study results and, in the 
judgment of the investigator, would make the subject 
inappropriate for entry into this study; 
• Has a psychiatric, addictive or other disorder that 
compromises the ability to give informed consent for 
participating in this study.  This includes subjects with a 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
_________________________________________________________________________________________________________ 
_________________________________________________________________________________________________________ 
CONFIDENTIAL                                                           Page 9 of 51                                                               Protocol v3.0_10/12/2017                                  
 
recent history of abusing alcohol or illicit drugs 30 days prior 
to study screening Day 0 and for the duration of the study; 
• Poorly controlled asthma defined by active wheezing and/or 
cough with FEV1 < 70% predicted, responsive to inhaled BD 
(>15% increase in FEV1 with BD); 
• Investigators, study staff or their immediate families; 
• Clinically significant intercurrent illness (including lower 
respiratory tract infection) or clinically significant surgery 
within 4 weeks before the administration of study drug; 
• Personal or family history of congenital or acquired 
methemoglobinemia; 
• Personal  history of RBC CYP B5 reductase deficiency; 
• Known or suspected hypersensitivity or allergic reaction to 
sodium nitrite; 
• Personal history of glucose-6-phosphate dehydrogenase 
G6PD) deficiency or any contraindication to receiving 
methylene blue; 
• If female, is pregnant or breast feeding, or has a positive urine 
or blood pregnancy test result predose; 
• Receipt of an investigational product or device, or 
participation in a drug research study within a period of 15 
days (or 5 half-lives of the drug, whichever is longer) before 
the first dose of study drug; 
• Blood loss or blood donation >550 mL within 90 days or 
plasma donation >500 mL within 14 days before 
administration of study drug; 
• RHC < 2 weeks from treatment visit unless clinically indicated 
 
 
Study Endpoints: 
Primary endpoint: 
The primary outcome measures for this study is change in 
pulmonary vascular resistance measured by right heart 
catheterization from time zero compared with peak effect within 
60 minutes post completion of nebulized dose of nitrite.  
 
Secondary endpoints: 
The secondary endpoints measure: 
• 
Time to maximum PVR decrease; 
• 
Repeated measures ANOVA (RM-ANOVA) for change in 
PVR calculated from the start of inhalation and at times 15, 
30, 45 and 60 minutes post end of nebulization. 
• 
Change 
in 
mean 
pulmonary 
artery 
pressure, 
transpulmonary gradient and cardiac output (CO)/cardiac 
index (CI),  
• 
Change in systemic blood pressure,  
• 
Change in SVR,  RV systolic (dP/dtmax/IP, PWRmax/EDV, 
RV EF, TAPSE),  RV diastolic function (dP/dtmin, Tau),  


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
_________________________________________________________________________________________________________ 
_________________________________________________________________________________________________________ 
CONFIDENTIAL                                                           Page 10 of 51                                                               Protocol 
v3.0_10/12/2017                                      
 
• 
Change in pulmonary vascular impedance / Wave 
Intensity,   
• 
Change in plasma nitrite concentrations in mixed venous 
blood at specified time points, 
• 
Change in pulmonary artery occlusion (capillary) pullback 
nitrite and plasma cGMP at baseline compared with peak 
nitrite concentration. 
• 
Change in mitochondrial oxygen consumption compared to 
baseline after each dose of nitrite. 
 
 
1.   OBJECTIVE, SPECIFIC AIMS, BACKGROUND, AND SIGNIFICANCE 
 
1.1 OBJECTIVE 
 
The main objective of this study is to investigate the effect of inhaled nitrite delivered in a dose 
escalation manner in patients on background pulmonary hypertension therapy (ETRA, PDE5I, 
or prostacyclin analog) on cardiopulmonary hemodynamics in subjects with pulmonary 
hypertension.  
 
1.2 SPECIFIC AIMS 
 
Hypothesis:  
 
• We hypothesize that inhaled nitrite treatment will reduce pulmonary vascular resistance. Our 
primary endpoint will be the change in pulmonary vascular resistance (PVR) measured by 
right heart catheterization. 
 
• We hypothesize that inhaled nitrite treatment will improve cellular and global right ventricular 
signaling and function. 
 
• We hypothesize that nitrite will be safe and well-tolerated in patients on background PAH 
therapy (PDE-5I and ETRA) 
 
Specific Aims: 
 
1) To determine the efficacy of inhaled nitrite, on the change in pulmonary vascular resistance 
measured by right heart catheterization. 
 
2) To explore the effect of inhaled nitrite on echocardiographic and hemodynamic parameters 
measured: SVR, RV systolic (dP/dtmax/IP, PWRmax/EDV, RV EF, TAPSE), RV diastolic 
function (dP/dtmin, Tau), Pulmonary vascular impedance / Wave Intensity 
 
3) To evaluate the safety of inhaled nitrite on systemic blood pressure in patients on 
background therapy for PAH. 
 
4) To identify the pharmacokinetic relationships of nitrite measures of drug exposure and 
plasma cGMP, using blood sampling. 
 
5) To identify the changes in mitochondrial oxygen consumption, using analysis of platelets  


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
_________________________________________________________________________________________________________ 
_________________________________________________________________________________________________________ 
CONFIDENTIAL                                                           Page 11 of 51                                                               Protocol 
v3.0_10/12/2017                                      
 
 
6) To compare effects of inhaled nitrite on pulmonary vascular resistance with inhaled NO gas 
at 40 ppm. 
 
 
1.3 BACKGROUND 
 
1.3.1. 
Pulmonary Arterial Hypertension  
Pulmonary arterial hypertension (PAH) is a relatively rare and devastating illness 
characterized by high morbidity and mortality rates.1 The 1993 American College of Chest 
Physicians consensus statement reports that the incidence ranges from one to two cases 
per million.2 PAH shows a female bias with F:M ratio of 2.3:1. The median age at diagnosis 
is 36. In the NIH registry, the diagnosis of pulmonary hypertension required a mean 
pulmonary artery pressure greater than 25 mm Hg at rest or greater than 30 mm Hg with 
exercise. PAH may occur in either sporadic or familial forms. Six percent of subjects in the 
NIH registry had a first-order relative with PAH. Familial PPH is inherited as an autosomal 
dominant disorder with incomplete penetrance, and has been mapped to a locus designated 
PPH1 on chromosome 2q33 that produces loss of function mutations in BMPR2.3 Bone 
morphogenetic proteins, members of the transforming growth factor-β (TGF-β) superfamily 
of signaling molecules, regulate cell differentiation and proliferation, apoptosis, 
morphogenesis and cell fate determination. Identified defects of this receptor are predicted 
to disrupt ligand binding, kinase activity, and heteromeric dimer formation – cellular 
processes that may ultimately be important in the maintenance of blood vessel integrity.  
 
Familial and sporadic PAH appear to be phenotypically identical. The median survival for all 
subjects in the NIH registry was 2.8 years; the one, three, and five year survivals were 68%, 
48%, and 34%, respectively.1 With the advent of epoprostenol therapy, the one, three, and 
five year survivals for PAH subjects have improved to 87%, 63%, and 54%, respectively.4 
Despite these improvements in outcome, mortality remains unacceptably high. The lack of a 
routine screening test for PAH, and the fact that early symptoms are nonspecific ensures 
that subjects typically present with advanced disease. 
 
Pulmonary hypertension is characterized by high pulmonary artery pressures and a high 
resistance to blood flow. The development of clinical PPA may be contingent on a genetic 
susceptibility (“hit” one) combined with an inciting injurious event(s) to the pulmonary artery 
EC (“hit” two) that renders it dysfunctional. The inciting event(s) probably cause a state of 
recurrent or chronic pulmonary vascular injury and repair, resulting in an excessive and 
adverse vascular remodeling process with associated alterations in gene expression. 
Adverse vascular remodeling and EC dysfunction results in excessive growth factor 
expression5, decreased eNOS expression and NO production6, vascular smooth muscle 
dysfunction, in situ thrombosis7 and a deficiency in endogenous prostacyclin synthesis8, 9  
 
1.3.2. 
Investigational Drug 
The active ingredient in AIR001 Inhalation Solution is sodium nitrite in a phosphate-buffered, 
pH adjusted solution for nebulization.  Pre-clinical data suggest that under the hypoxic, 
acidemic conditions present in the pulmonary hypertensive lung, inhaled nitrite results in the 
sustained release of NO which will act as an acute pulmonary vasodilator.  In addition, 
because decreased levels of NO have been shown to stimulate vascular remodeling, the 
increased NO resulting from nitrite inhalation is postulated to attenuate or reverse the 
remodeling process, resulting in sustained pulmonary hemodynamic improvement in 
subjects with PAH. 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
_________________________________________________________________________________________________________ 
_________________________________________________________________________________________________________ 
CONFIDENTIAL                                                           Page 12 of 51                                                               Protocol 
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1.3.3. 
Inhaled NO for Pulmonary Hypertension 
Nitric oxide released from the endothelium as a gas or attached to transport molecules 
activates soluble guanylyl cyclase in smooth muscle after binding to its heme group, 
resulting in increased cyclic GMP.  Cyclic GMP activates GMP-dependent kinases that 
decrease intracellular calcium concentration in smooth muscle, producing relaxation.  Nitric 
oxide also inhibits synthesis of a potent constrictor peptide, endothelin-1, an action that may 
contribute to vasodilation.  Inhaled NO may prove efficacious in patients with sickle cell 
disease and secondary pulmonary hypertension due to its ability to selectively dilate the 
pulmonary vasculature.10, 11  Nitric oxide diffuses to ventilated alveoli and dilates the blood 
vessels subserving these gas exchange units but is then inactivated by hemoglobin, 
preventing systemic hypotension.  This results in a selective reduction in pulmonary artery 
pressures. 
 
Inhaled nitric oxide (NO) is a potent acute vasodilator and results in improved 
hemodynamics in the 15% of PAH subjects who demonstrate vasoreactivity.  However, the 
usefulness of NO for chronic therapy is limited by the need for continuous inhalation.  In 
subjects who demonstrate vasoreactivity during an acute challenge with inhaled NO or a 
prostanoid challenge, therapy with calcium channel blockers has been demonstrated to 
result in improved symptoms and survival.  In subjects who do no not demonstrate an acute 
vasodilator response, available therapies for PAH include prostanoids, ETRA, and PDE-5I. 
Inhaled NO is now one of the currently used drugs for vasodilator challenges in the cardiac 
catheterization laboratory and therefore will be used as a comparator for nitrite responses in 
the current study. 
 
Inhaled NO will be delivered using FDA approved doses of 40 ppm for approximately 5-15 
minutes as used routinely in the catheterization laboratory for evaluation of pulmonary 
vascular responses in patients with PAH.  At doses up to 80 ppm (40 ppm will be used in 
this study) inhaled nitric oxide rarely produces adverse effects.  Buildup of NO-O2 reaction 
products in tubing such as NO2, a molecule that can precipitate bronchospasm and 
pulmonary edema, can occur if the NO delivery system is not purged daily.  The system will 
be purged prior to use, and inhaled levels of NO2 maintained well below 1 ppm. The 
INOvent NO delivery system that will be used in this study continuously monitors inhaled NO 
and NO2.  NO will only be used as clinically indicated using standard methodologies to 
evaluate pulmonary arterial vasodilatory capacity. 
 
1.3.4 
Effects of nitrate and nitrite on mitochondria and metabolism.  
Mitochondria are critical for oxidizing fuels (i.e., glucose, fatty acids, amino acids) for cellular 
energy production.  Accumulating evidence suggests that mitochondria dysfunction, 
characterized by decreased mitochondrial number, altered mitochondrial ROS generation or 
decreased substrate metabolism is associated with insulin resistance within skeletal 
muscle.  A human in vivo study by Larsen et al recently reported that skeletal muscle 
mitochondria harvested after dietary nitrate supplementation displayed improved 
mitochondrial efficiency. These findings suggest that dietary nitrate enhances the efficiency 
of energy generation through direct effects on muscle mitochondria and may have 
implications in the metabolic syndrome where mitochondrial dysfunction may play a central 
role. 
 
During ischemia nitrite is reduced to NO and N2O3 by different nitrite reductase enzyme 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
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systems12, 13.  Mitochondrial NO and S-nitrosothiols formed from nitrite dynamically and 
reversibly inhibit complex I during reperfusion, which limits ROS formation from complex I 
and III 14, 15. This ultimately prevents the opening of the mitochondrial permeability transition 
pore and the release of cytochrome c. It has recently been shown that the site of nitrosation 
is on Cys 39 of the ND3 subunit of complex I 15. A number of enzymes are required to 
convert nitrite into NO during organ ischemia. For example, in the heart, deoxygenated 
myoglobin acts as a functional nitrite reductase16 Nitrite-dependent NO formation is 
significantly decreased in myoglobin deficient hearts17 and nitrite administration reduces 
myocardial infarction with abrogated effects in the myoglobin knockout mice18  These 
studies by the Shiva and Gladwin laboratories have demonstrated that nitrite is a potent 
cytoprotective agent to mitochondria after ischemia and reoxygenation by inhibiting 
mitochondrial complex I activity, thereby blocking electron transfer and blocking the 
formation of superoxide during reperfusion. Further, nitrite has been shown to induce the 
expression of PGC1a as well as the mitochondrial biogenesis pathway in other cell types. 
These data suggest that nitrite inhibits mitochondrial respiration and attenuates 
mitochondrial ROS generation. Preliminary data from our research team suggest that nitrite 
specifically inhibits platelet mitochondrial basal respiration and enhances maximal 
respiratory capacity in vivo, effects which potentially decrease mitochondrial ROS 
generation and enhance substrate utilization in the platelet thereby attenuating platelet 
activation 19. 
 
Measurement of platelet mitochondrial respiration and dysfunction from a standard blood 
draw. We have now developed new methodologies to measure mitochondrial function and 
respiration in isolated platelets 19.  Considering emerging data on the role of nitrite and NO in 
regulating mitochondrial function, we plan to obtain fresh platelets to utilize with extracellular 
flux analysis (XF analysis (XF24, Seahorse Biosciences, Billerica, MA), a high throughput 
methodology to evaluate mitochondrial bioenergetics. We aim to measure platelet cell 
mitochondrial function before and after nitrite inhalation studies. Further, platelet 
mitochondrial bioenergetics will be measured along with thrombotic activation in patients 
with pulmonary hypertension. 
1.3.4. 
Effects of Inhaled Nitrite and Interaction between Inhaled Nitrite and 
Background Therapies for PAH 
Nitric oxide and nitrite.  
NO produced from eNOS is subject to rapid inactivation reactions with hemoglobin that 
greatly limit its lifetime in blood. Recent studies also indicate that NO formed from eNOS is 
can be oxidized to nitrite by oxygen or plasma ceruloplasmin.12 Nitrite transport in blood 
provides an endocrine form of NO that can be shuttled from the lungs to the periphery, while 
limiting the exposure of authentic NO to the scavenging red cell environment. Then during 
the rapid hemoglobin deoxygenation from artery-to-vein, nitrite is reduced back to NO via 
reactions with hemoglobin, myoglobin, and other heme containing enzymes, such as 
xanthine oxidoreductase. Once formed, nitrite is a potent vasodilator, potentially regulating 
basal blood flow and hypoxic vasodilation 14, 20-25 Nitrite limits ischemia/reperfusion-induced 
apoptosis and cytotoxicity in heart, liver and brain26-28.  Additionally, nitrite limits hypoxic 
pulmonary vasodilation in a deoxyhemoglobin- and pH-dependent fashion29.  Given the 
central role of NO in vasoregulation, the association of decreased NO bioavailability in PAH 
and recently emerging signaling actions of nitrite, a strong foundation of knowledge supports 
that inhaled nitrite can reduce pulmonary pressures in patients with PAH. 
 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
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PRO11080686 
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Inhaled nitrite for hypoxia-induced pulmonary hypertension 
in a new born sheep model.  
The above supports that inhaled nitrite could potentially 
exert the same actions as inhaled NO, by locally generating 
NO and promoting pulmonary vasodilation. Nitrite therapy 
has advantages over inhaled NO: ease of administration 
(intermittent use with a standard nebulizer), better 
availability, and lower cost.  Pre-clinical data from a new-
born sheep model of hypoxemia-induced fetal pulmonary 
hypertension indicate that a single nebulized dose of 
sodium nitrite produced the same vasodilatory effect as 20 
ppm inhaled NO (Figure 1). From this, we hypothesize that 
(a) low-dose, nebulized nitrite can reduce pulmonary artery 
pressures and pulmonary vascular resistance in patients 
with PAH. 
 
Nitrite reverses experimental mouse hypoxia-induced PAH.   
Chronic exposure of mice to hypoxia (10% O2) results in 
pulmonary vascular remodeling, characterized by increased 
muscularization, increased right ventricular pressures (RVP) 
and right ventricular hypertrophy. Treatment with low 
concentrations of nebulized nitrite (dosing and delivery as 
described in experimental plan), either once or three times 
per week, significantly prevented the development of PAH, 
as defined by all outcome measurements (Figure 2, A-C).  
Additionally, nitrite treatment 2 weeks into the hypoxic 
exposure, after the establishment of PAH, halted the 
progression of PAH and reversed increases in right 
ventricular pressures. Thus, inhaled nebulized nitrite 
inhibited both the development of hypoxia-induced PAH and 
reversed established PAH.  
 
Nitrite reverses monocrotaline-induced PAH.   
To determine whether the effects of nitrite therapy on PAH were specific to chronic hypoxia, 
we examined an additional model of experimental PAH, sodium monocrotaline (MCT)-
induced PAH. Similar to its effects in the hypoxia model of PAH, nitrite reduced 
monocrotaline-induced PAH (Figure 3 A-D).  Nitrite treatment significantly reversed 
monocrotaline-induced right ventricular hypertrophy and protected against increases in right 
ventricular pressures.   
 
 
 
 
 
 
 
 
 
 
 
Figure 1. Vasodilatory Effects of
Nitrite During Hypoxia-Induced 
Pulmonary Hypertension 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
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1.3.5 
Rationale for Dose of Nitrite 
The low doses of nitrite used in these treatment regimens –  less than 45 mg or 13% of the 
dose used in the emergency treatment of cyanide poisoning – do not produce 
methemoglobin levels greater than 3% and have not been associated with clinically 
significant hypotension.  Aires Pharmaceuticals, who licensed intellectual property pertaining 
to inhaled nitrite from the NIH, has completed a Phase 1a single dose escalation study 
designed to define the maximum tolerated dose of inhaled sodium nitrite delivered to normal 
volunteers following electronic nebulization.  These data, presented in abstract form at the 
2009 ATS meetings, show that at the highest dose (176 mg) a significant decrease in 
systemic blood pressure and increase in heart rate was found.  At the maximal tolerated 
dose (MTD);(125 mg) there was an increase in HR (+24 +/-5 bpm) without significant 
changes in systolic (-3 +/- 2 mmHg) or diastolic (-3 +/- 2 mmHg) blood pressure.  
Bioconversion of nitrite was demonstrated by an increase in exhaled NO.  Methemoglobin 
levels increased at the highest doses administered, but remained less than 3.5% in all 
subjects.  Pharmacokinetic modeling demonstrated dose-proportional increases in peak and 
AUC plasma nitrite.   
 
Note that in 80 volunteers in phase I studies at the NIH methemoglobin levels never rose 
higher than 3%.  If levels of methemoglobin rise above approximately 30% of total 
hemoglobin, a subject may appear cyanotic and experience dyspnea, due to the reduced 
oxygen carrying capacity of hemoglobin (methemoglobin cannot bind oxygen).  Levels 
above 50% can cause seizures, hypotension, coma and death.   
 
The effects of the interaction between FDA approved oral therapies for PAH (ETRA and 
PDE-5I) and inhaled nitrite are unknown. It is possible that the combination of the drugs 
 
Fig.2. Nitrite prevents and reverses hypoxia (10% 
O2) induced pulmonary hypertension. Nitrite 
treatments were 1X per week, 3X per week, and 
when administered after PAH development in week 
3 and 4.    
 
 
 
 
!
Fig. 
3. 
Nitrite 
reverses 
monocrotaline–induced 
pulmonary hypertension in rats with treatment 3X per 
week.  A. Reduction in RV mass. B. Reduction in RV mass 
to body mass.  C. Reduction in RV pressure.  D. a) control, 
b) vehicle treated rat with PAH showing hypertrophied 
right ventricle (RV), c) nitrite treated with normal RV. 
 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
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could lower systemic blood pressure to potentially clinically significant levels.  This is of 
particular concern for the PD5 inhibitors which will increase the levels of cGMP for any given 
dose of NO or nitrite.  For this reason the doses have been reduced significantly below the 
MTD of 125 mg and a dose escalation plan will be followed with clear stopping rules.  The 
patient will be carefully monitored with a pulmonary artery catheter during drug 
administration and intravenous fluids and vasopressors will be immediately available for the 
treatment of symptomatic hypotension. In addition we propose a dose escalation regimen to 
ensure careful drug dose titration. 
 
1.4   
SIGNIFICANCE 
Inhaled nitric oxide has been demonstrated to improve pulmonary hemodynamics acutely in 
subjects with pulmonary hypertension.  It has been demonstrated to lower pulmonary arterial 
pressure acutely in preclinical models of pulmonary hypertension, putatively through a 
mechanism of sustained NO release.  Repeat dosing of inhaled sodium nitrite has also been 
demonstrated to result in sustained improvement in pulmonary hemodynamics and in 
pulmonary vasculopathy in animal models of pulmonary hypertension. 
We hypothesize that inhaled nitrite provides an innovative therapeutic approach for patients     
with PAH for the following reasons: 
1) Inhaled nitrite therapy provides a potent, effective treatment that will vasodilate the pulmonary 
circulation (reduce pulmonary vascular resistance), reduce mean pulmonary artery pressure, 
and increase cardiac output. 
 
2) This study will provide valuable efficacy and safety insights into the interactions between NO 
and nitrite, both direct NO generators, with or without background PH therapy, particularly 
PDE5I, on pulmonary hemodynamics and right heart function. 
 
3) This study will also provide novel assessments of right ventricular global myocardial function. 
 
4) The results obtained from this study will also lay the groundwork for future large-scale clinical 
trials that are needed to investigate the efficacy of inhaled nitrite and/or its long-term effects 
on pulmonary hemodynamics in patients with pulmonary hypertension.   
 
2.   STUDY DESIGN AND METHODS 
 
2.1 CLASSIFICATION AND METHODOLOGICAL DESIGN 
This is a single-center, open label phase II study to evaluate the effect of inhaled nitrite 
delivered in a dose escalation manner on the change in pulmonary vascular resistance (PVR) in 
subjects with pulmonary arterial hypertension undergoing right heart catheterization. 
 
 
2.2 STUDY DESIGN AND METHODS 
 
2.2.1. Study Design 
A total of 50 subjects with a confirmed diagnosis of pulmonary hypertension and meeting all 
inclusion/exclusion criteria will be enrolled in the study which will entail a single right heart 
catheterization. The study population consisted of subjects with WHO group I PAH (n=20), 
WHO group II (n=20) and subjects with WHO group III PH (n=10) will receive the following 
dose escalation paradigm: Each subject will receive a starting dose of 45 mg inhaled nitrite 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
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PRO11080686 
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(AIR001 Inhalation Solution), with one subsequent planned dose escalation of 90 mg inhaled 
nitrite, based on pulmonary vascular resistance response and safety and tolerability. 
 
Screening (Day 0): 
The potential study subjects are followed on a routine basis in the UPMC Heart and Vascular 
Institute (HVI), Comprehensive Lung Center (CLC) or an inpatient at UPMC Presbyterian, are 
well known or referred to the study investigator. Initial screening evaluations including physical 
examination, medical history, and clinical laboratory assessments will be conducted to 
determine study eligibilities during a routine clinic visit at the UPMC HVI, CLC or inpatient at 
UPMC Presbyterian. Subjects who meet the inclusion criteria and none of the exclusion 
criteria will be entered into the study.  
 
Experimental Procedures (Day 1): 
The study visit will occur on the same day subjects are scheduled for their clinically indicated 
right heart catheterization or who volunteer for a research right heart catheterization for this 
specific study. Subjects on oral background PAH therapy (ETRA or PDE5I) will be instructed 
to hold their regimen on the day of the study visit.   
 
Subjects will be evaluated for additional medical history, physical examination, vital signs and 
pulse oximetry since screening. Laboratory testing will be repeated if baseline laboratory 
values are clinically significant and warrant repeat testing or inadvertently not collected at the 
screen visit.  An echocardiogram with documentation of TRV for estimation of right ventricular 
systolic pressure and assessment of LV systolic and diastolic function will be performed pre 
and post nitrite inhalation.   
 
Right heart catheterization is performed routinely as a clinical standard of care procedure for 
diagnostic purposes in this disease population. The standard right heart catheterization is 
performed with a balloon tipped, flow-directed pulmonary artery catheter (Swan-Ganz 
catheter), inserted through a sheath in the internal jugular vein. As per standard clinical 
protocol, hemodynamic recordings of right atrial, right ventricular, and pulmonary artery 
pressures, in addition to cardiac output, are made at baseline, and then repeated following 
pulmonary vasodilator challenge with inhaled nitric oxide (iNO, which is part of the standard 
clinical right heart catheterization when assessing pulmonary hypertension).  Responses to 
40 ppm iNO will be measured before nitrite nebulization to assess vasodilator responsiveness.  
As a part of this protocol the subjects may undergo an additional hemodynamic measurement 
using a micromanometer pressure catheter that may be inserted via the Swann-Ganz catheter 
distal port.  Measurements with micromanometer and/or Swann-Ganz catheter will be made 
with simultaneous flow velocity from transthoracic echocardiographic doppler ultrasound at 
baseline just prior to nebulized AIR001 administration and at the time of the final 
hemodynamic assessment (60 minutes after second dose). This portion of the study that 
involves the micromanometer instrumentation is expected to add an additional 5-10 minutes 
onto the procedure. 
 
Subjects will receive nebulized AIR001 doses escalated based upon safety and tolerability.  
The dose of inhaled nitrite will be delivered via electronic nebulizer. During the study right 
heart/pulmonary artery hemodynamics will be measured as well as noninvasive systemic 
blood pressure monitoring. Subjects will be tested for the changes in pulmonary vascular 
resistance (PVR) using standard clinical protocol hemodynamic recordings of right atrial, right 
ventricular, and pulmonary artery pressures, in addition to cardiac output at time zero, and 
approximate timelines 15, 30, 45 and 60 minutes after completion of each nebulization dose. 
For details, see sections 2.10.2 and 2.12 below. 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
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PRO11080686 
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Subjects will be monitored closely for changes in blood pressure during the study.  A decrease in 
systolic blood > 30 mm Hg drop from pre-dose baseline AND symptomatic or requiring fluid 
replacement or other therapy will lead to a discontinuation of the study treatment, and the next 
higher dose of inhaled nitrite will not be administered.  Subjects will continue to be monitored 
closely and treated as needed. 
 
Oxygen saturation measured by pulse oximeter and methemoglobin levels monitored by co-
oximetry prior to dose administration and through 2 hours post-last dose will be monitored 
closely.  Venous methemoglobin levels may be assessed periodically at investigator discretion.    
Due to circumstances unknown, co-oximetry may not read MetHgb level, in the event, venous 
metHgb may be collected at approx. time intervals 30 and 45 minutes for each inhaled dose. 
 
Blood samples may be collected for Cyclic Guanosine Monophosphate (cGMP) concentration 
determination, pharmacokinetics (PK) and platelet mitochondrial analysis at pre dose and the 
end of the peak dose of nitrite may be collected.  PK and cGMP measurements will be made on 
mixed venous blood using PCW pullback samples and venous blood will be collected for platelet 
mitochondrial analysis 
 
Subjects will be monitored carefully for adverse events, laboratory test abnormalities, and 
changes in vital signs.  Adverse experiences will be evaluated according to criteria outlined in 
the NCI Common Terminology Criteria for Adverse Events (CTCAE), version 4.0.   
 
Following the study treatment, subjects will be followed as an outpatient on Day 30 (+/- 5 day 
window). An additional follow-up assessment by telephone will occur on Day 3.  
 
2.2 STUDY TREATMENT 
 
Following initial standard clinical measurements for right heart catheterization, 40 ppm inhaled 
NO gas will be administered for approximately 5-15 minutes through a facemask, continuously 
monitoring subject vasoreactivity.  This is considered part of the clinical right heart 
catheterization procedure for pulmonary hypertension. Repeat hemodynamic measurements will 
be performed upon completion of inhaled NO.  Pre-dose baseline PK and methemoglobin levels 
may be collected.  The inhaled nitrite will be delivered per protocol below. 
 
Following completion of all baseline assessments, each subject will receive a starting dose of 
inhaled nitrite, with one subsequent planned dose escalation of inhaled nitrite, based on safety 
and tolerability. Subjects will be monitored carefully during and after the study treatment.  
 
The study will be conducted at UPMC Presbyterian Hospital, 3rd FL Catheterization Laboratory. 
Baseline vasoreactivity will be recorded after Inhaled NO at 40 ppm for approximately 5-15 
minutes. Subjects will receive a starting dose of AIR001 (45 mg), with one subsequent planned 
dose escalation to 90 mg based upon safety and tolerability. The dose of AIR001 will be delivered 
by an electronic nebulizer system that is portable, highly efficient utilizing continuously vibrating 
mesh aerosol generation technology that allows a high percentage respirable dose delivery, 
minimal loss of drug to the environment between inhalations, and a reproducible droplet size 
distribution for optimal delivery of drugs to the distal pulmonary tree. 
 
During, and at approximate timelines of 15, 30, 45 and 60 minutes post-inhalation for each dose, 
right heart/pulmonary artery hemodynamics will be measured as well as noninvasive systemic 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
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blood pressure monitoring.  Subjects will be monitored closely during the study. Dose limiting 
toxicity (DLT) is defined in Table 1.   
A dose level will also be considered intolerable if, in the judgment of the Investigator, the type, 
frequency, or severity of AEs becomes unacceptable. If an intolerable dose is identified, no further 
doses at that or higher doses will be administered. 
 
Table 1  
Dose Limiting Toxicity Criteria 
Sign or Symptom 
Moderate 
Severe1 
Bronchospasm/ wheezing 
Symptomatic but not requiring therapy 
Symptomatic and requiring therapy 
SBP2 
Systolic > 20 mm Hg drop from pre-
dose baseline AND symptomatic, or 
SBP <80 mm Hg AND symptomatic 
Systolic > 30  mm Hg drop from pre-dose 
baseline  AND symptomatic or requiring fluid 
replacement or other therapy  
Dyspnea 
Causes more discomfort (than a mild, 
easily tolerated AE) and interrupts the 
subject’s usual daily activities  
Incapacitating and causes considerable 
interference with the subject’s usual daily 
activities 
Cough 
Causes more discomfort (than a mild, 
easily tolerated AE) and interrupts the 
subject’s usual daily activities 
Incapacitating and causes considerable 
interference with the subject’s usual daily 
activities 
Hypoxia3 
SaO2 < 5% from baseline AND 
symptomatic  
SaO2 ≤10% baseline AND symptomatic 
Venous or percutaneous 
methemoglobin4,5 
>5% and <7% 
≥7% 
Other drug-related signs or 
symptoms6 
Causes more discomfort (than a mild, 
easily tolerated AE) and interrupts the 
subject’s usual daily activities 
OR 
CTCAE v.46 Grade 2 toxicity 
Incapacitating and causes considerable 
interference with the subject’s usual daily 
activities 
OR 
CTCAE v.46 Grade 3 or 4 toxicity 
AE = adverse event; CTCAE = Common Toxicity Criteria for Adverse Events; DLT = dose limiting toxicity;   
SBP = systolic blood pressure; SaO2 = oxygen saturation (hemoglobin);  
 
1 
Severe DLT seen in any subject which is deemed to be study drug-related will preclude further dosing with AIR001 
for that subject. 
2      Moderate or Severe SBP without symptoms will be continuously monitored with BP measurements every 5 minutes.  
Symptomatic refers to dizziness or fainting.  Discontinuation of further dosing will be based upon follow up blood 
pressure measurements, subject symptoms and physician-investigator discretion (see Stopping Rules). 
3 
Moderate or Severe SaO2 without symptoms will be continuously monitored and recorded every 5 minutes.  As 
most clinically feasible; in patients with difficulty measuring SaO2 at baseline, most usually those with scleroderma 
but not limited to this condition, the follow up will not be based upon repeated SaO2 measurements but on clinical 
decision of the operating physician that will be documented as such. 
4 
Elevated percutaneous methemoglobin levels measured by pulse co-oximeter will be continuously monitored 
and confirmed by venous methemoglobin level.  Follow up will be based upon the clinical decision of the 
operating physician and will be documented as such. 
5 
A single occurrence of venous methemoglobin levels >5% in an individual subject will result in no further dosing 
for that individual subject for the remainder of the study period 
6 
Common Terminology Criteria for Adverse Events v. 4.0 (CTCAE). CTCAE criteria will be applied if considered 
related to study drug and confirmed on repeat testing. 
 
 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
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Baseline for the calculation of supine blood pressure changes is defined as the supine 
measurement taken prior to AIR001 dosing. 
 
The presence of severe hypotension will be defined by a decrease in systolic blood pressure 
>30  mm Hg from pre-dose baseline AND symptomatic or requiring fluid replacement or other 
therapy will lead to a discontinuation of the study treatment, and the next higher dose of inhaled 
nitrite will not be administered to that subject. Subjects will continue to be closely monitored. 
Oxygen saturation will be measured by pulse oximeter and methemoglobin levels monitored by 
continuous co-oximetry prior to dose administration and 2 hours post last-dose will be monitored 
closely. 
 
Blood samples for PK analysis, Cyclic Guanosine Monophosphate (cGMP) concentration 
determination, and platelet mitochondrial analysis may be collected at pre-dose and at the end of 
nebulization at the presumed peak nitrite concentration for each tolerated dosage time points; 
approximately 15 minutes post inhalation.  Blood samples collected for measurements will be 
made on mixed venous blood using PCW pullback samples at baseline (pre-dose) and after each 
nitrite dose. Platelets will be isolated by differential centrifugation. Measurements of platelet 
mitochondrial activity will be determined at baseline (pre-dose) and after each nitrite dose.   
 
Subjects will be monitored carefully for adverse events, laboratory test abnormalities, and 
changes in vital signs.  Adverse experiences will be evaluated according to criteria outlined in the 
NCI Common Terminology Criteria for Adverse Events (CTCAE), version 4.0.   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
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2.3. 
STUDY DESIGN SCHEMATIC 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Day 0 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Day 1 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Day 3 
 
 
 
 
Day 30 
Nitrite Dose 1 
(nebulized) 
NIBP 
RA PA, PCWP, CO/CI 
(approx.15, 30, 45, 60 min 
after neb) 
PK, cGMP, platelet 
mitochondrial samples 
MetHgb measurements 
Baseline Values 
Echo/ NIBP  
Hemodynamic Measurements: RA, RV, PA, PCWP, 
CO/CI, Micromanometry Pressure and Doppler Velocity 
iNO value: measurements during inhalation 
Pre dose PK, cGMP & platelet mitochondrial samples 
Pre dose MetHgb measurement 
Nitrite Dose 2 
(nebulized) 
NIBP 
RA, PA, PCWP, CO/CI 
(approx.15, 30, 45, 60 min 
after neb) 
PK, cGMP, platelet 
mitochondrial samples 
MetHgb measurements  
DISCO
treat
DO NO
with n
 
Con
mo
Inpatient or Outpatient Eligibility 
 
Symptomatic 
Systemic 
hypotension,  
desaturation 
and/or adverse 
changes in 
pulmonary 
hemodynamics 
Yes 
 
STOP
No
Final Measurement 
Micromanometry pressure and Doppler velocity measurements 
RV measurements 
Echo  
SpO2 and MetHgb measurements 
Follow-Up Assessment by Telephone 
Follow-Up Assessment - Outpatient Clinic 
Proceed to 2nd dose? 
No 
Symptomatic 
Systemic 
hypotension,  
desaturation 
and/or adverse 
changes in 
pulmonary 
hemodynamics 
 
Yes 
Yes
No


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
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All subjects enrolled in the study will be followed for 30 days (+/- 5 day window) after completion 
of the study treatment. Subjects removed from study for unacceptable adverse events will be 
followed until resolution or stabilization of the adverse event. 
 
2.5. SAFETY MONITORING 
 
Prior studies suggested that hypotension and methemoglobinemia may occur with the use of 
sodium nitrite. The study treatment of inhaled nitrite will be used safely with careful monitoring 
and prompt discontinuation of the study treatment in response to systemic hypotension with 
definitions of dose limiting toxicity as outlined in Table 1. 
 
All subjects will be studied in UPMC Presbyterian Hospital Catheterization Laboratory with an 
investigator and nurse present at all times, and with continuous monitoring of vital signs and 
oxygen saturation. Subjects will also be monitored closely for methemoglobin levels during the 
study treatment.  
 
 
Following study drug treatment, subjects will be evaluated as an outpatient at the clinic on Day 
30 (+/- 5 day window). In addition, an interval telephone assessment will occur on Day 3. Subjects 
will be followed for evidence of acute or delayed adverse effects from the study treatment and to 
assess their clinical status. All adverse events experienced by subjects will be collected from the 
time of dosing, through the study and until the final study visit. Subjects continuing to experience 
toxicity at the off study visit may be contacted for additional assessments until the toxicity has 
resolved or is deemed irreversible. If possible, symptoms should be managed symptomatically. 
In the case of toxicity, appropriate medical treatment should be used. 
 
In addition to these formal evaluations, subjects will be encouraged to immediately contact the 
study investigator and/or the study coordinator with questions, concerns, or to report new 
symptoms that occur during their study participation. If appropriate, based upon the evaluation, 
medical treatments will be provided to subjects, including appropriate referral to physicians or 
other services at the UPMC.  
 
2.6. STOPPING RULES 
 
In addition to the dose limiting toxicity as outlined in Table 1 above, study treatment will be 
discontinued if any of the following occurs: 
 
• 
A decrease in systolic BP > 40 mm Hg drop from pre-dose baseline; 
• 
Desaturation > 10% from baseline; 
• 
A single occurrence of venous methemoglobin level >5%; 
• 
Severe DLT criteria met and symptomatic or requiring fluids or treatment; 
• 
Any serious adverse event thought to be possibly related to the study treatment.   
 
 
2.7. DRUG SUPPLIES 
 
 
2.7.1. Formulation and Packaging 
Formulation: 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
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The active ingredient of the Sodium Nitrite Inhalation Solution (AIR001 Inhalation Solution) is 
sodium nitrite in a phosphate-buffered, pH adjusted solution for nebulization. The Sodium 
Nitrite Inhalation Solution is intended to be prepared prior to inhalational delivery to patients 
via electronic nebulization.   
Packaging: 
All vial formulations are labeled, packaged and distributed by Aires Pharmaceuticals, Inc. 
Aires Pharmaceuticals, Inc. will provide drug supplies to the investigational site. 
 
2.7.2. Availability 
AIR001 is an investigational drug and will be obtained from Aires Pharmaceuticals, Inc, San 
Diego, California. AIR001 will be supplied free-of-charge to investigators under a 
Collaborative Agreement between Aires Pharmaceuticals, Inc. and the University of 
Pittsburgh. 
 
2.7.3. Preparing and Dispensing 
The study drug will only be diluted and dispensed by the research pharmacist to the required 
dose concentrations. 
 
The drug product will be delivered to subjects utilizing a nebulizer system supplied by Aires 
that is portable, highly efficient and that utilizes continuously vibrating mesh aerosol 
generation technology that allows a high percentage respirable dose delivery, minimal loss of 
drug to the environment between inhalations, and a reproducible droplet size distribution for 
optimal delivery of drugs to the distal pulmonary tree.  
 
2.7.4. Drug Administration 
The route of administration is by nebulization.   It is anticipated that each nebulization will take 
approximately 10 – 15 minutes to deliver the dose. All doses specified in the protocol are the 
amounts of AIR001 placed into the nebulizer cup.    
 
 
2.8 DRUG STORAGE 
 
The investigational drug will be kept in its original packaging and stored in a locked, secure area 
at controlled room temperature (20 – 25º C) with the investigation drug services (IDS).  The 
temperature of the storage area must be monitored to ensure compliance with required 
temperatures. A temperature log will be maintained to make certain that the drug supplies are 
stored at the correct temperature at all times maintained by IDS. Access to and administration of 
the investigational drug will be limited to the study investigators and authorized research staff. 
Study drugs may only be dispensed to subjects enrolled in this study.  
 
2.9. DRUG ACCOUNTABILITY 
 
The study investigators or the study coordinator will document the amount of study drugs 
dispensed and/or administered to subjects. The study drugs accountability records will be 
maintained throughout the course of the clinical trial by IDS.  Any discrepancies in drug supplies 
will be noted and explained. 
 
2.10 STUDY PROCEDURES 
 
2.10.1. Screening  


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
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Screening evaluations to determine eligibility will take place prior to receiving study treatment. 
The potential study subjects are followed on a routine basis in the UPMC Heart and Vascular 
(HVI), Comprehensive Lung Center (CLC) or an inpatient at UPMC Presbyterian and are well 
known or referred to the study investigators.  During routine clinical visit, patients who express 
interest in participation will undergo the screening assessment to determine that all 
inclusion/exclusion criteria are met prior to receiving the study treatment.  
 
If the patient has had any of the tests done within the 6 months prior to the screening period, 
they will not be repeated for study purposes. The results collected from these tests and 
procedure will serve as a baseline for comparison of the subject’s overall condition following 
study treatment. This visit may take up to 2 hours to complete. 
 
Screening  
 
• 
Medical history review and demographics 
• 
Complete history and physical examination to include vital signs, blood pressure, body 
weight, and height. 
• 
Assessment of oxygen saturation 
• 
Baseline clinical laboratory evaluations including complete blood count with differential,  
platetlets, electrolytes, glucose, BUN, serum creatinine, liver function tests, PT/PTT, nt-
ProBNP.. 
• 
Urine pregnancy test for female of childbearing potential. 
• 
Informed, written consent may be obtained after subject meets eligibility criteria or on day 
of Study visit (Day 1) 
 
2.10.2. Experimental procedures (Day 1) 
Subjects who meet the inclusion criteria and none of the exclusion criteria will receive the 
study treatment of inhaled nitrite. The study will take place at UPMC Presbyterian Hospital, 
3rd FL Catheterization Laboratory, and may take up to 6 hours to complete.  
 
Pre-Dose: Baseline 
• 
Physical examination to include vital signs, blood pressure, weight and oxygen saturation 
• 
Laboratory testing  that clinically warrants repeating testing or inadvertently not collected 
for screening may be obtained prior to research procedures 
• 
Echocardiogram 
• 
Non-invasive blood pressure measurement 
• 
Right Heart Catheterization (RHC)-RA, RV, PA, pulmonary capillary wedge pressure 
(PCWP), CO/CI  
• 
Inhaled NO testing at 40 ppm if PCWP < 25 with hemodynamic response recorded (RA, 
PA, PCWP, CO/CI) 
• 
Micromanometry to record high fidelity pressure signals with simultaneous flow velocity 
from transthoracic echocardiographic ultrasound for the measurement of pulmonary 
arterial input impedance. Micromanometer pressure catheter is inserted via the Swann-
Ganz catheter distal port.  
• 
Repeat 2nd baseline hemodynamic measurement: RA, RV, PA, PCWP. CO/CI 
• 
Pre-dose baseline PK, cGMP and platelet mitochondrial samples 
• 
Pre-dose methemoglobin measurment 
 
AIR001 Dose 1 45 mg Nebulized  
 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
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• 
Non-invasive blood pressure measurement 
• 
PK, cGMP and platelet mitochondrial samples (approx. 15 minutes after nebulization) 
• 
RA, PA, PCWP, CO/CI (approx.15, 30, 45, 60 minutes after nebulization) 
• 
AE assessment 
• 
Methemoglobin (MetHgb) measurements (approx. 5,15, 30, 60 minutes after nebulization) 
 
 
 AIR001 Dose 2 90mg Nebulized 
 
• 
Non-invasive blood pressure measurement  
• 
PK, cGMP and platelet mitochondrial samples (approx. 15 minutes after nebulization) 
• 
RA, PA, PCWP, CO/CI (approx. 15, 30, 45, 60 minutes after nebulization) 
• 
AE assessment 
• 
MetHgb measurements (approx. 5,15, 30, 60 minutes after nebulization) 
 
Final Measurements:  
 
• 
Includes 60 minute hemodynamic measurements (RA, PA, PCWP, CO/CI) from last 
tolerated AIR001 dose 
• 
RV measurement 
• 
AE assessment 
• 
Micromanometry to record high fidelity pressure signals with simultaneous flow velocity 
from transthoracic echocardiographic ultrasound for the measurement of pulmonary 
arterial input impedance. Micromanometer pressure catheter is inserted via the Swann-
Ganz catheter distal port. 
• 
Echocardiogram 
• 
SpO2 and MetHgb measurements at approx. 120 minutes post last tolerable dose 
 
PK Sampling for Nitrite/Nitrate: 
 
• 
Blood samples for PK analysis may be collected at pre-dose and at the end of 
nebulization at the presumed peak nitrite concentration for each tolerated dosage time 
points; approximately 15 minutes post inhalation. 
 
Methemoglobin (metHgb) levels: 
 
• 
MetHgb levels will be recorded via co-oximetry at the following approximate time points: 
pre-dose, 5, 15, 30, 60 minutes after each dose of AIR001 and at 120 minutes post last 
dose of AIR001.  Venous methemoglobin levels will be assessed as clinically indicated at 
the discretion of the investigator.  Due to circumstances unknown, co-oximetry may not 
pick up methemoglobin readings.  In those instances, venous methemoglobin levels will 
be collected at approximate 30 and 45 minute post inhalation time points. 
 
2.10.3. Follow-up monitoring (Day 3)- Telephone Assessment 
The interval telephone assessment should be completed as close to the scheduled visit date 
as possible. However, a variance of 2 days will be allowed for the follow-up monitoring to 
facilitate scheduling or to account for weekends or holidays. 
 
Subjects will be contacted by the study investigator and/or the study coordinator for a follow-
up assessment by telephone to assess for symptoms potentially related to adverse effects of 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
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study drug, review of medication changes, and interval medical of surgical histories including 
emergency or physician office visits. This phone assessment may last about 30 minutes. 
 
• 
Interval history assessment 
• 
AE assessment 
 
2.10.4. Follow-up monitoring (Day 30 +/- 5 day window)- Outpatient Clinic Assessment 
Outpatient visits should be completed as close to the scheduled visit dates as possible. 
However, a variance of 5 days will be allowed for the follow-up monitoring to facilitate 
scheduling or to account for weekends or holidays. This visit will take place at the UPMC HVI 
and CLC outpatient clinic, and may take up to 2 hours to complete. 
 
• 
Physical examination to include vital signs, blood pressure and oxygen saturation 
• 
Clinical laboratory evaluations 
• 
AE assessment 
 
In addition to these formal evaluations, subjects will be encouraged to immediately contact 
the study coordinator and/or the investigators with questions, concerns, or to report new 
symptoms that occur during their study participation. If appropriate, based upon the 
evaluation, medical treatments will be provided to subjects, including appropriate referral to 
physicians or other services at the UPMC.  
 
Any clinically significant adverse event, laboratory test or physical examination observed 
during final assessments will be followed as medically appropriate until resolved or explained. 
 
 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
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2.11. SCHEDULE OF ACTIVITIES (STUDY ASSESSMENT TABLE) 
The table below summarizes the protocol procedures that will be performed at screening, during 
the study treatment, and at follow-up.   
Study Phase 
Screening 
Pre-
Treatment 
Treatment 
Post-Treatment 
Follow-Up Monitoring 
Visit Type 
Outpatient 
Clinic 
Outpatient Cath Lab 
Phone 
Assessment 
Outpatient 
Clinic 
Days on Intervention             
Day 0 
Day 1 
Day 3 
Day 30 
Study Procedures 
Baseline 
Dose 1 
Dose 2 
Final 
Measurement 
Informed Consent 
x 
 
 
 
 
 
 
Medical History and 
Demographics 
x 
 
 
 
 
 
 
I/E Criteria 
x 
 
 
 
 
 
 
Physical Exam 
x 
x 
 
 
 
 
x 
Vital Signs 
x 
x 
 
 
 
 
x 
Oxygen Saturation 
x 
x 
x 
x 
x 
 
x 
Laboratory Tests*** 
x 
x 
 
 
 
 
x 
Urine Pregnancy Test ∆ 
x 
x 
 
 
 
 
 
Non-invasive blood pressure 
measurement  
 
x 
x 
x 
 
 
 
Right Heart Catheterization 
(RHC):  RA, RV, PA, PCWP, 
CO/CI 
(RV measurements at 
baseline,  pre-dose and 
60minute interval post last 
dose AIR001) 
 
x 
x 
x 
x 
 
 
Micromanometry 
 
x 
 
 
x 
 
 
Echocardiogram 
 
x 
 
 
x 
 
 
Inhaled NO at 40 ppm if 
PCWP < 25 
 
x 
 
 
 
 
 
PK Sampling* 
 
x 
x 
x 
 
 
 
metHgb Level§ 
 
x 
x 
x 
x 
 
 
Plasma cGMP 
 
x 
x 
x 
 
 
 
Platelet  Mitochondrial 
Analysis 
 
x 
x 
x 
 
 
 
AE Assessment 
x 
x 
x 
x 
x 
x 
x 
Interval History Assessment 
 
 
 
 
 
x 
 
∆   Urine pregnancy test will be performed only on female of childbearing potential. 
*** Clinical laboratory tests may be repeated on study visit 1 if screen lab results deemed clinically significant and warrant repeat testing or inadvertently 
not collected at screen visit. 
*    PK Sampling for Nitrite: Blood samples will be obtained at pre-dose and at the end of nebulization at the presumed peak nitrite concentration for   
each tolerated dosage time points; 15 minutes post inhalation. 
§   Methemoglobin (metHgb) levels:will be measured at the following time points: pre-dose, 5, 15, 30 and 60 minutes each dose of AIR001 and at 120 
minutes post last dose of AIR001.  
 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
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2.12. DESCRIPTION OF STUDY PROCEDURES 
 
2.12.1. Right Heart Catheterization and Simultaneous Pressure and Doppler Flow  
Velocity Measurement 
The right heart catheterization is performed as a clinical standard of care procedure, routinely 
performed for diagnostic purposes in this disease population. The standard right heart 
catheterization is performed with a balloon tipped, flow-directed pulmonary artery catheter 
(Swan-Ganz catheter), inserted through a sheath in the internal jugular vein.  As per standard 
clinical protocol, hemodynamic recordings of right atrial, right ventricular, and pulmonary 
artery pressures, in addition to cardiac output, are made at baseline, and then repeated 
following pulmonary vasodilator challenge with inhaled nitric oxide (iNO, which is part of the 
standard clinical right heart catheterization when assessing pulmonary hypertension).  
As a part of this protocol the subjects may undergo an additional hemodynamic measurement 
using a micromanometer pressure catheter that will be inserted via the Swann-Ganz catheter 
distal port. The micromanometer catheter will record high fidelity pressure signals for the 
measurement of pulmonary arterial input impedance. Measurements with micromanometer 
will be made with simultaneous flow velocity from transthoracic echocardiographic ultrasound 
at baseline just prior to nebulized AIR001 administration and at the time of the final 
hemodynamic assessment (60 minutes after second dose). This portion of the study that 
involves the micromanometer instrumentation is expected to add an additional 5-10 minutes 
onto the approximate 4 hour procedure. 
Cardiac Output (CO) 
Cardiac output is the amount of blood pumped through the left ventricle of the heart in a time 
interval of one minute.  There are a number of ways to calculate CO.  Thermodilution and/or 
Fick cardiac index are 2 means which will be used for measuring CO for this protocol.  The 
CO will be the average of the recorded CO collected.    
Cardiac Index (CI) 
Cardiac index is a measurement that relates the CO from the left ventricle in one minute to 
body surface area (BSA) therefore determining cardiac function to size of the individual.  CI is 
calculated by CO/BSA. 
 
 
2.12.2. Pulmonary Capillary Wedge Pressure 
Pulmonary capillary wedge pressure (PCWP) provides an indirect estimate of left atrial 
pressure (LAP). PCWP is measured by the balloon-tipped, multi-lumen catheter (Swan-Ganz 
catheter) while it is in a branch of the pulmonary artery. Just behind the tip of the catheter is 
a small balloon that can be inflated with air (~1 cc). The catheter has one opening (port) at 
the tip (distal to the balloon) and a second port several centimeters proximal to the balloon. 
These ports are connected to pressure transducers. When properly positioned in a branch of 
the pulmonary artery, the distal port measures pulmonary artery pressure and the proximal 
port measures right atrial pressure. The balloon is then inflated, which occludes the branch of 
the pulmonary artery. When this occurs, the pressure in the distal port rapidly falls, and after 
several seconds, reaches a stable lower value that is very similar to left atrial pressure 
(normally about 8-10 mmHg). The balloon is then deflated and pulmonary artery pressure can 
again be recorded. The same catheter can be used to measure cardiac output.  Blood samples 
will be withdrawn from the tip of the wedged catheter at baseline (pre-dose), and at 15 minutes 
after each dose of inhaled nitrite (45 mg dose and 90mg). 
 
2.12.3  Echocardiography 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
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Transthoracic echocardiograms will be completed to assess RV function by measures such 
as TAPSE as well as to assess the Doppler waveform of the pulmonary artery.  The echoes 
will be completed pre-dose and after final dose.  This is a noninvasive test. 
 
2.12.4. Percutaneous Oxygen Saturation and Methemoglobin 
The device (Masimo Rainbow SET® CO-Oximeter) selected for use in this clinical trial has 
been validated to provide accurate determinations of both SaO2 and methemoglobin levels 
under conditions of hypoxia and methemoglobinemia. Continuous SaO2 and 
methemoglobin monitoring will occur prior to study drug administration until 2 hours post-
dose. Measurements will be recorded pre-dose, approx. 5, 15, 30 and 60 minutes after each 
dose of AIR001 and approximately 2 hours post last dose of AIR001. 
 
2.12.5. Pharmacokinetic (PK) Studies 
Pharmacokinetic studies will be performed on all subjects enrolled to measure the 
concentration of inhaled nitrite and its metabolites in blood. The evaluation of the relationship 
beteween dosage and blood concentrations, and the relationship between blood 
concentrations and pharmacologic effects will provide valuable mechanistic information about 
the effects of inhaled nitrite on cardiopulmonary hemodynamics in Subjects with Pulmonary 
Hypertension. 
 
To determine the pharmacokinetic profiles of the inhaled nitrite, blood samples will be 
collected at pre-nitrite dosing and at the end of nebulization at the presumed peak nitrite 
concentration for each tolerated dosage time points; approximately 15 minutes post 
inhalation.  Vital signs, blood pressure and pulse oximetry will be measured and monitored 
closely at each time points. Samples will be immediately centrifuged and frozen at -20°C until 
the time of analysis.  
 
 
PK samples will be accurately recorded for the collection time. Covariate information, e.g., 
age, body weight, height, disease type, concomitant medications, laboratory values, etc., will 
be obtained at the study visit. This is expected to yield an information-rich data set through 
dense sampling, enabling better characterization of the PK relationship of inhaled nitrite in 
subjects with PAH.  
 
2.12.6. Laboratory Testing 
 
Peripheral blood samples will be evaluated for baseline and follow-up comparison purposes 
which include complete blood count with differential, platelets, electrolytes, glucose, BUN, 
serum creatinine, liver function tests, PT/PTT, and nt-ProBNP.  Urine pregnancy test may be 
performed only on female of child-bearing potential on Day 0 and Day 1.  
 
Screening lab samples may be collected and analyzed at UPMC laboratories or at outlying 
facility of subject’s choice.  Lab results will be added to research chart for comparison.  All 30-
day samples will be analyzed at UPMC Presbyterian laboratory with the exception of the 
ntPro-BNP which may be processed and analyzed through the Pulmonary, Allergy and Critical 
Care Medicine (PACCM) research lab.  
 
Baseline laboratory results within 6 months of screen visit may be used as baseline 
comparison and will not be repeated for research purposes.  Blood samples may be 
collected on study visit 1 if baseline screening results are deemed clinically significant and 
warrant repeat testing or inadvertently not collected at screen visit. 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
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2.12.7. Additional Laboratory Testing 
Blood samples may be collected for Cyclic Guanosine Monophosphate (cGMP) 
concentration determination and platelet mitochondrial will be collected at the end of 
nebulization at the at pre dose and the end of the peak dose of nitrite may be collected.  
cGMP measurements will be made on mixed venous blood using PCW pullback samples 
and venous blood will be collected for platelet mitochondrial analysis. These de-identified 
blood samples will be sent to the research lab on the 12th floor BST for processing.   
 
2.12.8. Specimen collection and management 
Specimen Collection / Documentation 
At each sampling time, one mL of blood will be withdrawn and discarded to assure that the 
solution used to maintain catheter patency does not dilute the sample.  Blood samples will be 
accurately recorded for the actual sample collection time. Each research PK sample will be 
labeled with subject’s unique identifier, sample date, scheduled sample collection time, and 
actual sample collection time.  Covariate information, e.g., age, body weight, height, disease 
type, concomitant medications, laboratory values, etc., will be obtained at each time point. 
This is expected to yield an information-rich data set through dense sampling, enabling better 
characterization of the biomarkers in this patient population.  
 
Specimen Handling and Labeling (De-Identification) 
Specimens collected will be properly labeled. All research biological specimens and all 
records associated with the samples will be labeled only with a unique code that contains no 
personal identifiers. The information linking these code numbers to the corresponding 
subject’s identity will be kept in a secure location in the investigator’s office, and will not be 
available to staff managing samples at the research laboratories.  
 
Immediately upon receipt of the biological specimens, all attempts will be made to process, 
isolate, collect, and store the specimens. The code number and date on which the specimen 
is frozen, all other information about the specimen, and subsequent processing will be entered 
on the specimen processing worksheet. 
 
Specimen Management and Storage 
Specimens in excess of immediate assay requirements may be stored indefinitely in a locked 
freezer under the control of the principal investigator. 
 
The blood samples will be stored after appropriate coding to remove patient identifiers. The 
coding information linking patient identifiers to the stored samples will be maintained in a 
locked, secure area that will be accessible only to the study investigator. Subjects may request 
to have their samples destroyed at any time. These samples will be destroyed immediately 
upon receipt of the subjects’ written request to do so. Identification of which samples to destroy 
will be available from the coding information linking patient identifiers to the stored samples 
as described earlier in this paragraph.  
 
Restrictions to Direct Access of Specimens 
Specimens will be kept in the responsible study investigators’ laboratories indefinitely and will 
be under the control of the principal investigator.  Investigators or other personnel not involved 
with the management or operations of the study are not permitted direct access to the 
specimens.  
 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
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2.13. 
ENDPOINTS 
 
2.13.1. Primary Endpoints 
The primary outcome measures for this study is change in pulmonary vascular resistance 
(PVR) measured by right heart catheterization from time zero compared with peak effect 
within 60 minutes post completion of nebulized dose of nitrite. 
 
2.13.2. Secondary Endpoints 
The secondary endpoints measure: 
• 
Time to maximum PVR decrease; 
 
• 
Repeated measures ANOVA (RM-ANOVA) for change in PVR calculated from the start 
of inhalation and at times 15, 30, 45 and 60 minutes post end of nebulization. 
• 
Change in mean pulmonary artery pressure, transpulmonary gradient and cardiac output 
(CO)/cardiac index (CI), 
• 
Change in systemic blood pressure,  
• 
Change in SVR,  RV systolic (dP/dtmax/IP, PWRmax/EDV, RV EF, TAPSE),  RV diastolic 
function (dP/dtmin, Tau),  
• 
Changes in pulmonary vascular impedance / Wave Intensity,   
• 
Change in plasma nitrite concentrations, cGMP, and platelet mitochondrial activity in 
mixed venous blood at specified time-points, 
Change in pulmonary artery occlusion (capillary) pullback nitrite and plasma cGMP at baseline 
compared with both  nitrite doses. 
 
2.14. 
SUBJECT WITHDRAWAL  
 
A subject may voluntarily discontinue participation in this study at any time. The investigator may 
also, at his or her discretion, discontinue a subject from this study at any time. Every effort should 
be made by the investigator to keep the subject in the study.   
 
Subjects may be withdrawn from the study prior to completion if any of the following criteria are 
observed: 
• 
Intercurrent illness or an unexpected fatal or life-threatening adverse event, which requires 
discontinuation of study treatment 
• 
Pregnancy 
• 
Subject reached protocol-defined stopping criteria 
• 
Request by the subject to withdraw from the study 
• 
Protocol violations 
• 
Persistent non-compliance 
• 
Lost to follow-up 
• 
Investigator discretion 
• 
Study closed/terminated 
 
2.14.1. Dropouts and withdrawals 
To be considered complete, a subject must complete all study visits as specified in the 
protocol without violations of the protocol so significant as to obscure the response to study 
treatment. 
 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
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PRO11080686 
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Subjects who fail to complete all study required visits will not be considered complete and 
may not enroll at a later date and will not be replaced. A record will be kept of all subjects who 
fail to complete all study visits and their primary reasons for discontinuation. 
 
In the event of subject withdrawal, subjects will be encouraged to continue all follow-up visits 
for safety monitoring or to continue follow up as directed by their personal primary physicians 
,unless the subject withdraws consent at any time (without having to justify the decision). All 
available data from subjects who discontinued during the study, for whatever reason, will be 
included in the safety analysis. 
 
2.15 STATISTICAL ANALYSIS 
 
2.15.1 Sample Size and Power 
Limited data are available on expected vasodilatory responses to nitrite in patients with Group 
I PAH or Group III PH.  We reviewed data from studies examining the PVR response to inhaled 
NO, fasudil, iloprost, and treprostinil30-32, which reported percent decreases in PVR after 
therapy ranging from 5-25%.  Assuming a distribution of PVR among PAH patients similar to 
that reported by Voswinckel, et al (911 +/- 102 dyn∙sec∙cm-5), we estimate using the paired t-
test that we will need 14 participants to achieve 80% power to detect a statistically significant 
10% decrease in PVR at a significance level of 0.05. At a projected enrollment of 
approximately 20 patients with group I PAH for our primary outcome measure, we anticipate 
more than sufficient power to detect significant acute vasodilatory effects of nitrite. 
 
2.15.2 Statistical Analysis 
General Approach 
The primary efficacy outcome variable is pulmonary vascular resistance (PVR), measured by 
right heart catheterization. T-test will be performed to determine difference of mean values for 
outcome variables at baseline and highest tolerated nitrite.  The secondary endpoints will be 
analyzed with repeated measures ANOVA (RM-ANOVA) to compare the overall 
cardiopulmonary hemodynamic effects and the PK endpoints (eg: AUC) for all doses of nitrite. 
 
Safety Analysis 
Adverse events (AEs) will be grouped by body system. AEs occurring pre-treatment, during 
active treatment and post-treatment will be summarized separately. The number and 
percentage of subject experiencing at least one AE of any type, AEs within each body system 
and AEs within each preferred term will be tabulated and listed. Separate summaries will be 
provided for all AEs, AEs by maximum severity, drug related AEs, severe adverse events 
(SAEs), and for AEs leading to withdrawal. 
 
Pharmacokinetic Analysis 
PK endpoints will be summarized using descriptive statistics. The actual sampling times will 
be used in calculating PK parameters using non-compartmental methods. The PK 
endpoints namely, AUC, Incremental recovery, t½, Cl, AUClast, Cmax will be analyzed using a 
multiplicative linear mixed effect models. 
 
Handling of missing data: 
Every effort will be made to collect complete data on each study day. With respect to safety 
evaluation, it is not planned to impute missing data. 
 
3.   HUMAN SUBJECTS 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
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PRO11080686 
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3.1 SUBJECT POPULATION 
 
The racial, gender and ethnic characteristics of the proposed subject population in this research 
protocol shall reflect the demographics of the population of Pittsburgh and the surrounding area. 
We shall attempt to recruit subjects in proportion to these demographics. No exclusion criteria 
shall be based on race, ethnicity or gender. 
 
Every effort will be made to keep subjects in the study until they complete all study procedures. 
3.1.1 Inclusion of Women and Minority 
Both men and women of all races and ethnic groups are eligible for this trial. Women who 
meet the inclusion criteria, and have none of the exclusion criteria, will be enrolled without 
restriction as dictated by the study protocols.  Because of the use of a study medication, 
woman of child bearing potential must meet specialized inclusion/exclusion criteria to 
minimize this risk.   
 
 
3.1.2 Inclusion of Children 
Children under the age of 18 will not be recruited for this protocol because of the need to 
explore potential adverse effects of the study treatment more fully in adults. . 
 
3.2 INCLUSION CRITERIA 
 
Potential study subjects must satisfy the following criteria to be enrolled in the study 
 
WHO Group I PAH (n = 20) 
 Diagnosis of RHC confirmed WHO Group I PAH in any of the following categories: 
• 
Idiopathic, primary or familial pulmonary arterial hypertension (IPAH, PPH, or 
FPAH)  
                       OR 
• 
PAH associated with one of the following connective tissue diseases: 
 
              i. Systemic sclerosis (scleroderma) 
 
             ii. Limited scleroderma 
 
             iii. Mixed connective tissue disease 
 
             iv. Systemic lupus erythematosus 
 
             v. Overlap syndrome;   
                              OR 
• 
PAH associated with:   
                     i. Human immunodeficiency virus (HIV)   infection; 
                     ii. Simple, congenital systemic-to-pulmonary shunts at least one year post-  
                         surgical repair. 
                    iii. Exposure to legal drugs, chemicals and toxins, such as fenfluramine, 
                         derivatives, other anorexigens, toxic rapeseed oil or L-tryptophan.  
                        Subjects with PAH associated with illegal drug use, such as 
                        methamphetamine excluded. 
 
Stable PAH for at least 3 months if on therapy 
 
WHO Group II (n=20) 
• 
Pulmonary capillary wedge pressure (PWCP)  > 15 
AND 
 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
PRO11080686 
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• 
Transpulmonary Gradient (TPG) > 12 
 
WHO Group III PH (n = 10) 
Has WHO functional class II-IV symptoms 
 
Had the diagnosis of PH confirmed by a cardiac catheterization with the following values: 
• 
Mean pulmonary artery pressure (mPAP) ≥ 25 mm Hg (at rest); 
• 
Pulmonary capillary wedge pressure (PCWP) or left ventricular-end diastolic pressure  
£15 mm Hg (if diagnosed with PAH); and 
• 
Pulmonary vascular resistance (PVR) ≥ 3 mm Hg/L/min or ≥ 240 dynes*sec/cm5; 
 
WHO Group I, II and III  
     Age 18 and older; 
     Able and willing to participate in right heart catheterization;. 
Evidence of a personally signed and dated informed consent document indicating that the 
subject has been informed of all pertinent aspects of the study; 
Subjects who are willing and able to comply with scheduled visits, treatment plan, laboratory 
tests, and other study procedures; 
 
3.3 EXCLUSION CRITERIA 
 
Subjects meeting any of the exclusion criteria at baseline will be excluded from participating in 
study. 
 
• Age less than 18 years; 
• Baseline systemic hypotension, defined as MAP less than 50 mmHg; 
• Required intravenous inotropes within 30 days prior to study participation; 
• Has uncontrolled systemic hypertension as evidenced by sitting systolic blood pressure 
>160 mm Hg or sitting diastolic blood pressure >100 mm Hg at Screening; 
• Has a history of portal hypertension or chronic liver disease, including hepatitis B and/or 
hepatitis C (with evidence of recent infection and/or active virus replication) defined as 
moderate to severe hepatic impairment (Child-Pugh Class B-C); 
• Has chronic renal insufficiency as defined by serum creatinine >2.5 mg/dL at Screening or 
requires dialytic support; 
• Has a hemoglobin concentration <9 g/dL at Screening; 
• History of atrial septostomy within 6 months prior to Day 1 visit; 
• Repaired or unrepaired congenital heart disease (CHD); 
• Pericardial constriction; 
• Confirmed diagnosis of restrictive or constrictive cardiomyopathy; 
• Left ventricular ejection fraction <40% by multiple gated acquisition scan (MUGA), angiography 
or echocardiography; 
• Symptomatic coronary disease with demonstrable ischemia; 
• Other severe acute or chronic medical or laboratory abnormality that may increase the risk 
associated with study participation or investigational product administration or may interfere 
with the interpretation of study results and, in the judgment of the investigator, would make the 
subject inappropriate for entry into this study; 
• Has a psychiatric, addictive or other disorder that compromises the ability to give informed 
consent for participating in this study.  This includes subjects with a recent history of abusing 
alcohol or illicit drugs 30 days prior to study screening Day 0and for the duration of the study; 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
PI: Marc Simon, MD 
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• Poorly controlled asthma defined by active wheezing and/or cough with FEV1 < 70% predicted, 
responsive to inhaled BD (>15% increase in FEV1 with BD); 
• Investigators, study staff or their immediate families; 
• Clinically significant intercurrent illness (including lower respiratory tract infection) or clinically 
significant surgery within 4 weeks before the administration of study drug; 
• Personal or family history of congenital or acquired methemoglobinemia; 
• Personal or family history of RBC CYP B5 reductase deficiency; 
• Known or suspected hypersensitivity or allergic reaction to sodium nitrite; 
• Personal history of glucose-6-phosphate dehydrogenase (G6PD) deficiency or any 
contraindication to receiving methylene blue; 
• If female, is pregnant or breast feeding, or has a positive pregnancy test result predose; 
• Receipt of an investigational product or device, or participation in a drug research study within 
a period of 15 days (or 5 half-lives of the drug, whichever is longer) before the first dose of 
study drug; 
• Blood loss or blood donation >550 mL within 90 days or plasma donation >500 mL within 14 
days before administration of study drug; 
• 
RHC < 2 weeks from treatment visit unless clinically indicated 
4.   RECRUITMENT AND INFORMED CONSENT PROCEDURES 
 
4.1 RECRUIMENT METHODS 
 
The potential study subjects will be recruited from the UPMC Heart and Vascular (HVI) and 
Comprehensive Lung Center (CLC) or inpatients at UPMC Presbyterian Hospital. Potential 
subjects will be first identified or referred to the investigator who may also be the primary care or 
treating physician.  The investigator who may already have knowledge of and access to subjects' 
information may review the subjects’ records to identify potential research subjects for the study. 
After identifying potentially eligible subjects, the investigator will then approach these subjects to 
discuss the research opportunity.  
 
To minimize the possibility that subjects will feel obligated to participate, investigators will 
reinforce with their subjects that participation is voluntary, that they do not have to participate, and 
the decision not to participate will not affect their care, now or in the future. The investigator will 
also allow subjects to make further inquiries if they are interested. 
 
 
4.2 INFORMED CONSENT PROCEDURES 
 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in 
the study and continuing throughout the individual’s study participation.  Extensive discussion of 
risks and possible benefits of participation in this study will be provided to the subjects and their 
families.  Consent forms describing in detail the study procedures and risks are given to the 
subject and written documentation of informed consent is required prior to enrolling in the study.  
Consent forms will be IRB approved and the subject will be asked to read and review the 
document. Upon reviewing the document, the investigator will explain the research study to the 
subject and answer any questions that may arise.  The subjects will sign the informed consent 
document prior to being enrolled in the study.  The subjects should have the opportunity to discuss 
the study with their surrogates or think about it prior to agreeing to participate.  The subjects may 
withdraw consent at any time throughout the course of the study.  A copy of the informed consent 
document will be given to the subjects for their records.  The rights and welfare of the subjects 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
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will be protected by emphasizing to them that the quality of their medical care will not be adversely 
affected if they decline to participate in this study. 
 
Prior to performing any of the research study procedures or interventions, subjects must provide 
informed consent. The investigator will verbally explain the study to the potential subject in a 
language understandable to subjects, providing all pertinent information (purpose, procedures, 
risks, benefits, alternatives to participation, etc.), and will allow potential subjects ample 
opportunity to ask questions to elicit a better understanding of the study. Following this verbal 
explanation, potential subjects will be provided with a local IRB approved consent form and will 
be asked to read and review the document. Upon reviewing the document, the investigator will 
provide adequate opportunity for the subject to consider all options, answer any additional 
questions the potential subject may have. Every effort will be made to ensure that subjects have 
comprehended the study information prior to obtaining subject's voluntary agreement to 
participate. 
 
In addition, older potential participants whose competency to consent is in question will be tested 
for sufficient comprehension and recall of the information presented. Prospective subjects who 
do not remember the important facts about participation in the research study after repeated 
testing will not be included in the study. The investigators will also assess whether a participant 
understands experimental procedures over time, including assessment throughout the full 
duration of participation in the study. 
 
The subjects may withdraw consent at any time throughout the course of the study.  A copy of 
the informed consent document will be given to the subjects for their records.  The rights and 
welfare of the subjects will be protected by emphasizing to them that the quality of their medical 
care will not be adversely affected if they decline to participate in this study. 
 
5.   POTENTIAL RISKS AND BENEFITS 
 
5.1 POTENTIAL RISKS 
 
As with any experimental procedure, there may be adverse events or side effects that are 
currently unknown, and certain of these unknown risks could be permanent, severe or life 
threatening.  Every attempt will be taken to minimize these risks.   
 
Risks of Sodium Nitrite Inhalation Solution: 
The first-in-man clinical study of AIR001 Inhalation Solution, Study AIR001-CS01, was a dose 
escalation study designed to identify the dose limiting toxicity, the maximum tolerated dose, and 
the pharmacokinetics of AIR001 administered.  The most important safety finding was a decline 
in systolic and diastolic blood pressure at the highest dose tested, 176 mg.  Dose-dependent 
asymptomatic increases in heart rate were noted at all doses, although at doses up to and 
including the maximum tolerated dose of 125 mg, the heart rate changes were well tolerated.  
Increase in heart rate was not always associated with a change in blood pressure.  Significant 
levels of methemoglobin, or changes in pulmonary function, were not observed. Methemoglobin 
levels increased at the highest doses administered, but remained less than 3.5% in all subjects.   
 
As with any drug product administered via inhalation, it is possible that susceptible individuals, 
such as those with unrecognized asthma, will develop bronchospasm upon exposure to inhaled 
sodium nitrite solution.  There were no changes in pulmonary function noted in AIR001-CS01, 
which excluded subjects with asthma or abnormal baseline pulmonary function tests. 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
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PRO11080686 
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In Study AIR001-CS02, inhaled nitrite was generally well tolerated under hypoxic conditions at 
doses up to and including 113 mg.  Additionally, a sustained reduction in hypoxia-induced 
pulmonary hypertension was demonstrated, suggesting that safely delivered doses of AIR001 
Inhalation Solution B result in clinically significant vasodilatation, presumably through a 
mechanism of intrapulmonary reduction of nitrite to NO. 
 
In this study, the significant treatment related adverse event was a decrease in blood pressure.  
The magnitude of the change in blood pressure from the hypoxic baseline was less than from the 
normoxic baseline, suggesting that hypoxia itself has an effect on BP. 
 
Risks of Nitric Oxide Inhalation: 
The inhalation of NO is usually well tolerated without side effects. 
Infrequent risks: decrease pulmonary vascular tone, but because of its short biological half-life 
will be metabolized or inactivated before reaching the systemic circulation.  Other risks: Massive 
overdoses of nitric oxide are fatal (from 500-1000ppm). 
 
Risks of Sildenafil/Tadalafil (PDE-5 Inhibitor): 
These medications are commonly used and FDA approved to treat Pulmonary Hypertension.  
These medications may lower blood pressure significantly if taken together with some other 
medications like nitroglycerin.  AIR001 may cause similar effects in this study in combination with 
PDE-5I. 
Subjects will be closely monitored throughout the entire procedure and should blood pressure 
drop significantly with the first inhaled dose of AIR001, study participant will not receive the next 
higher dose. 
 
Risks of Blood Drawing: 
The amount of blood to be drawn over the course of this research study could be a maximum of 
5 tablespoons. To minimize the risks of blood tests, a licensed technician or registered nurse will 
draw your blood.   
Common risks include temporary discomfort, bruising which may last for several days, redness, 
swelling, lower hemoglobin level. Infrequent risks include a subject may feel lightheaded or faint 
when blood is drawn. This is usually due to nervousness and is not usually serious. Rare risks 
include infection, and bleeding.  
 
Risks of Echocardiography: 
This is a noninvasive procedure. There is no known risk associated with this procedure. 
 
Risks of Right heart Catheterization: 
Common risks: pain at the needle entry site and slight risk of bleeding around the site. bruising at 
site, lightheadedness or dizziness during the needle stick. 
Infrequent risks: puncturing the lung which would require a chest tube insertion, irregular 
heartbeats which usually stop when the long tube is removed from the heart. 
Very rare complications include cardiac arrhythmias, cardiac tamponade, low blood pressure, 
infection, or embolism caused by blood clots at the tip of the catheter. 
 
Risks of the Use of Micromanometer Catheter during Catheterization: 
The micromanometer pressure and flow catheter are FDA approved, and have been used for 
clinical/investigative indications for over 25 years. Moreover, these catheters do not pose any 
inherent risk to patients. The catheter will be placed through the clinical Swann-Ganz catheter 
that is within the preexisting jugular venous sheath. This obviates the need for an additional 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
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venipuncture. This catheter has successfully been used without complications by the study 
Principal Investigator for the past 5+ years.  
  
Risks of Medications used during Catheterization: 
The medications used for sedation are relatively brief in duration and should wear off within 
several hours.  The side effects are listed below for each drug. 
 
1 % Lidocaine: will be used to numb the area prior to the insertion of the cardiac catheter into      
either your neck or groin vein. A common side effect is slight burning at the site which dissipates 
quickly.  
 
Fentanyl: Common side effects include temporary light-headedness, dizziness, nausea, 
vomiting or sweating.  
 
Midazolam: Common side effect includes drowsiness. Infrequent side effects are nausea,    
vomiting. Breathing problems are rare. 
 
Allergic reactions (e.g., hives, itching, etc.) to lidocaine, fentanyl, or midazolam are rare. 
 
Risks of Chest X-Ray during the Catheterization 
A chest x-ray may be done, if clinically indicated, on participants that a neck vein is used for the 
catheterization.  A chest X-ray performed for the purpose of this research study involves exposure 
to radiation. Each chest X-ray will result in a radiation dose of approximately 0.03 rem to the chest 
with minimal exposure of other body areas. For comparison, radiation workers are permitted, by 
Federal regulation, to receive a maximum annual radiation dose of 20 rems to the most sensitive 
organs of their body. There is no minimum amount of radiation exposure that is recognized as 
being totally free of the risk of causing genetic mutations (abnormal cells) or cancer. However, 
the risk associated with the amount of radiation exposure received from participation in this study 
is considered to be low and comparable to everyday risks.  
 
Reproductive Risks:   
It is not known if the study drug can affect an unborn baby. Therefore, subjects should not become 
pregnant or father a baby while on this study.  If subjects are physically able to father a baby, 
subjects must use an effective method of birth control while on this study.  If subjects become 
aware that they or their sexual partner is pregnant during the course of their participation in this 
research study, subjects must contact, as soon as possible, the study investigator.  
 
Risks of Breach of Confidentiality:  
Participation in this research study does involve the potential risks of a breach of confidentiality 
of the medical record information and associated privacy of the participants.  This research study 
will result in identifiable information that will be placed into the subject’s medical records held at 
the University of Pittsburgh Medical Center. The nature of the identifiable information resulting 
from participation in this research study that will be recorded in the medical record includes 
laboratory test results. This potential for breach of confidentiality could impact future insurability, 
employability, or have other personal consequences for the subjects. 
 
5.2. PROTECTIONS AGAINST RISK 
 
Protection Against Patient Risks Related to the Study Drug 
The study has been designed with a focus on protecting subjects against risk from the medication 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
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including: 
• 
Specific exclusion criteria to provide a stable population of subjects and non-enrollment of 
subjects with significant co-morbidities that might place them at excess risk (see exclusion 
criteria above). 
• 
Continuous monitoring by the DSMB. 
• 
Involvement by trained staff / investigators with experience in the administration of inhaled 
nitrite. 
• 
Specific holding criteria related to study adverse events. 
• 
Frequent monitoring the cardiopulmonary hemodynamics for over the duration of the 
study. 
 
Protection Against General Risks of Study Procedures 
All research interventions/activities will be conducted in private patient care areas. The collection 
of sensitive information about subjects is limited to the amount necessary to achieve the aims of 
the research, so that no unneeded sensitive information is being collected. 
 
All demographic and clinical information about the subject will be stored on an electronic 
password-guarded study database under the supervision of the investigator for this protocol. The 
electronic database has not been validated to be in full compliance with the FDA regulations at 
21 CFR Part 11.  Use of this electronic database is, however, felt to be acceptable due to the 
limited scope of this research study and the extent of data that will be collected. The data will be 
stored anonymously with a subject number. Information linking subject identifiers with the coded 
subject number will be stored under password protection on computers in locked areas, with 
access only to the database manager. Maintaining records in locked files in locked offices will 
protect confidentiality of subjects.  Access to the database will be limited to the data manager and 
staff under the supervision of the PIs.  
 
To prevent excessive blood sampling, a single withdrawal of blood for a combination of clinically 
indicated and this research study will not exceed 5% of the circulating blood volume, and the 
cumulative withdrawal over 1 month will not exceed 10% of the circulating blood volume. In 
addition, careful attention will be made to cardiovascular status and hemoglobin evaluation. 
 
Specimens will be stripped of subject identifiers and stored securely according to a similar coding 
protocol as described above. These specimens will be stored safely in the custody of the Principal 
Investigator responsible for the individual assays. These Investigators will limit future access to 
any remaining sample to only those investigators with prior IRB approval for their studies. 
 
All staff involved in this study are properly credentialed and instructed in the areas of testing, 
confidentiality, and safety. To minimize the risks associated with the study procedures and/or 
collection of specimens, trained staff or experienced investigators will perform the study 
procedures.  
   
5.3 ALTERNATIVE TREATMENTS 
 
If subjects choose not to participate in this study, they are to continue their medical care under 
the direction of their primary physicians. 
 
5.4 POTENTIAL BENEFITS 
 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
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There will be no direct benefit to the subjects participating in this study, but the society at large 
may benefit from the increased knowledge gained from this study that will lead to new treatment 
for individuals diagnosed with PAH in the future. 
 
5.5 DATA SAFETY MONITORING PLAN 
      
5.5.1 Data Safety Monitoring Board 
A Data and Safety Monitoring Board (iDSMB) independent of the study investigators will 
monitor this clinical trial for additional measure of subject protection. The iDSMB consists of 
clinicians completely independent of the investigators who have no financial, scientific, or 
other conflict of interest with the trial. 
 
The iDSMB will conduct interim monitoring of accumulating data from research activities to 
assure the continue safety of human subjects, relevance and appropriateness of the study, 
and the integrity of research data. 
 
5.5.2 Data Safety Monitoring Plan 
Assuring patient safety is an essential component of this protocol.  The study Principal 
Investigator has primary responsibility for the oversight of the data and safety monitoring.  The 
study investigators will evaluate all adverse events.  All subjects who have AEs, whether 
considered associated with the use of the study medication or not, must be monitored to 
determine the outcome.  The clinical course of the AE will be followed up according to 
accepted standards of medical practice, even after the end of the period of observation, until 
a satisfactory explanation is found or the Principal Investigator considers it medically justifiable 
to terminate follow-up. 
 
All untoward medical occurrences observed in subjects receiving the study drug will be 
recorded on the participants’ adverse event worksheets by the study coordinator under the 
supervision of the principal investigator. The worksheets will then be reviewed for 
completeness and internal consistency. In addition to internal safeguards built into a 
computerized system, external safeguards will be put in place to ensure that access to the 
computerized system and to the data is restricted to authorized personnel. Training conducted 
by qualified individuals on a continuing basis will be provided to individuals in the specific 
operations with regard to computerized systems that they are to perform during the course of 
the study.  
 
*Stopping Rule: 
 
For safety reasons, we propose to discontinue this study treatment if any of the following occurs: 
 
• 
A decrease in systolic BP > 40 mm Hg drop from pre-dose baseline;  
• 
Desaturation > 10% from baseline; 
• 
A single occurrence of venous methemoglobin level >5%; 
• 
Severe DLT criteria met and symptomatic or requiring fluids or treatment; 
• 
Any serious adverse event thought to be possibly related to the study treatment. 
  
The Sponsor and Investigator will prepare a detailed written summary of serious, unexpected, 
and treatment related adverse events, and will compare, and contrast the event with prior events. 
The detailed written summary will be provided to the DSMB and the IRB.  
 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
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In addition, the DSMB Report addressed the following information will be submitted to the IRB 
at the time of continuing review annually or more often as required: 
 
• A list of the research personnel who participated in the data and safety monitoring. 
 
• The frequency of monitoring that took place during the renewal intervals and/or the dates 
that data and safety monitoring was conducted. 
 
• A summary of cumulative data related to unanticipated problems (including adverse 
events) including a determination of causality and whether the risk to benefit assessment 
has changed. 
 
• If appropriate, a summary of pertinent scientific literature reports, therapeutic 
developments, or results of related studies that may have an impact on the safety of study 
participants or the ethics of the research study. 
 
• A summary of the outcome of reviews conducted to ensure subject privacy and research 
data confidentiality. 
 
• Final conclusions regarding changes to the anticipated benefit-to-risk assessment of the 
study participation and final recommendations related to continuing, changing, or 
terminating the study. 
 
5.5.3 Parameters to be Monitored 
The following progress will be monitored throughout the course of the research to ensure the 
safety of subjects as well as the integrity and confidentiality of their data. 
 
• 
An evaluation of the progress of the research study, including subject recruitment and 
retention, and an assessment of the timeliness and quality of the data. 
 
• 
A review of collected data (including adverse events, unanticipated problems, and subject 
withdrawals) to determine whether there is a change to the anticipated benefit-to-risk 
assessment of study participation and whether the study should continue as originally 
designed, should be changed, or should be terminated. 
 
• 
An assessment of external factors or relevant information (eg. Pertinent scientific literature 
reports or therapeutic development, results of related studies) that may have an impact 
on the safety and study participants or the ethics of the research study. 
 
• 
A review of study procedures designed to protect the privacy of the research subjects and 
the confidentiality of their research data. 
 
5.5.4 Frequency of Monitoring 
The Investigator will review subject safety data as it generated. The Investigator and the 
research staff will meet on a quarterly interval to re-evaluate study goals, subject recruitment, 
data coding and retention, documentation and identification of adverse events, complaints 
and confidentiality of subjects. There will be an evaluation of the progress of the research 
study, including assessments of data quality, time lines, participant recruitment, accrual, and 
retention. The Investigator will also review the outcome and adverse event data to determine 
whether there is any change to the anticipated benefit-to-risk ratio of study participation and 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
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whether the study should continue as originally designed or should it be re-evaluated and 
changed. 
  
The iDSMB is expected to meet at least two times a year at the call of the Chairperson to 
review the progression of the study including patient enrollment, protocol compliance, and 
adverse event reports. An emergency meeting of the iDSMB may be called at any time by the 
Chair should participant safety questions or other unanticipated problems arise. 
 
5.5.5. 
Adverse Event Reporting: 
The study investigators will be responsible for detecting, documenting and reporting events 
that meet the following definition of an adverse event. 
 
5.5.5.1. 
Adverse event definitions 
 
Adverse event. Any untoward medical occurrence in a clinical study; regardless of the causal 
relationship of the event with the investigational drug or study treatment(s). 
 
Associated with the use of the investigational drug or study treatment(s).  There is a 
reasonable possibility that the adverse event may have been caused by the investigational 
drug or study treatment(s). 
 
Disability.  A substantial disruption of a person’s ability to conduct normal life functions. 
 
Life-threatening adverse event.  Any adverse event that places the patient or subject, in the 
view of the investigator, at immediate risk of death from the event as it occurred (i.e., does not 
include an adverse event that, had it actually occurred in a more severe form, might have 
caused death). 
 
Serious adverse event. Any adverse event occurring at any dose that results in any of the 
following outcomes: death, a life-threatening adverse event, inpatient hospitalization or 
prolongation of existing hospitalization, a persistent or significant disability/incapacity, or a 
congenital anomaly/birth defect. 
 
• 
Hospitalization shall include any initial admission (even if less than 24 hours) to a 
healthcare facility as a result of a precipitating clinical adverse event; to include transfer 
within the hospital to an intensive care unit.  Hospitalization or prolongation of 
hospitalization in the absence of a precipitating, clinical adverse event (e.g., for a 
preexisting condition not associated with a new adverse event or with a worsening of the 
preexisting condition; admission for a protocol-specified procedure) is not, in itself, a 
serious adverse event.   
 
Unexpected adverse event.  Any adverse event, the frequency, specificity or severity of which 
is not consistent with the risk information described in the clinical protocol(s) or elsewhere in 
the current IND application, as amended. 
 
5.5.5.2. Recording/Reporting requirements  
Eliciting adverse event information 
Clinical study subjects will be routinely questioned about adverse events at study visits. 
 
Recording requirements 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
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All observed or volunteered adverse drug events (serious or non-serious) and abnormal test 
findings, regardless of treatment group or suspected causal relationship to the investigational 
drug or study treatment(s) will be recorded in the subjects’ case histories.  For all adverse 
events, sufficient information will be pursued and/or obtained so as to permit 1) an adequate 
determination of the outcome of the event (i.e., whether the event should be classified as a 
serious adverse event) and; 2)  an assessment of the casual relationship between the adverse 
event and the investigational drug or study treatment(s).    
 
Adverse events or abnormal test findings felt to be associated with the investigational drug or 
study treatment(s) will be followed until the event (or its sequelae) or the abnormal test finding 
resolves or stabilizes at a level acceptable to the Investigator. 
 
Abnormal test findings 
An abnormal test finding will be classified as an adverse event if one or more of the following 
criteria are met: 
 
• 
The test finding is accompanied by clinical symptoms. 
 
• 
The test finding necessitates additional diagnostic evaluation(s) or medical/surgical 
intervention; including significant additional concomitant drug treatment or other therapy  
 
Note: simply repeating a test finding, in the absence of any of the other listed 
 
criteria, does not constitute an adverse event.   
• 
The test finding leads to a change in study dosing or discontinuation of subject 
participation in the clinical study 
• 
The test finding is considered an adverse event by the investigator-sponsor of the IND 
application 
 
 
Causality and severity assessment 
The investigator-sponsor of the IND application will promptly review documented adverse 
events and abnormal test findings to determine 1) if the abnormal test finding should be 
classified as an adverse event; 2) if there is a reasonable possibility that the adverse event 
was caused by the investigational drug or study treatment(s); and 3) if the adverse event 
meets the criteria for a serious adverse event. 
 
If the investigator-sponsor’s final determination of causality is “unknown and of questionable 
relationship to the investigational drug or study treatment(s)”, the adverse event will be 
classified as associated with the use of the investigational drug or study treatment(s) for 
reporting purposes.  If the investigator-sponsor’s final determination of causality is “unknown 
but not related to the investigational drug or study treatment(s)”, this determination and the 
rationale for the determination will be documented in the respective subject’s case history.  
 
5.5.6. 
Reporting of adverse events 
 
5.5.6.1. 
Reporting of adverse events to the FDA 
Written IND Safety Reports 
The investigator-sponsor will submit a written IND Safety Report (i.e., completed FDA Form 
3500 A) to the responsible new drug review division of the FDA for any observed or 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
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volunteered adverse event that is determined to be 1) associated with the investigational drug 
or study treatment(s); 2) serious; and 3) unexpected.  Each IND Safety Report will be 
prominently labeled, “IND Safety Report”, and a copy will be provided to all participating sub-
investigators.   
 
Written IND Safety Reports will be submitted to the FDA as soon as possible and, in no event, 
later than 15 calendar days following the Sponsor’s receipt of the respective adverse event 
information. 
 
For each written IND Safety Report, the investigator-sponsor and Aires Pharmaceuticals, Inc. 
chief medical officer or designee  will identify all previously submitted IND Safety Reports that 
addressed a similar adverse event experience and will provide an analysis of the significance 
of newly reported adverse event in light of the previous, similar report(s). 
 
Follow-up information to an IND Safety Report will be submitted to the applicable review 
division of the FDA as soon as the relevant information is available.  If the results of the 
sponsor-investigator’s follow-up investigation show that an adverse event that was initially 
determined to not require a written IND Safety Report does, in fact, meet the requirements for 
reporting; the investigator-sponsor will submit a written IND Safety Report as soon as 
possible, but in no event later than 15 calendar days, after the determination was made. 
 
Telephoned IND Safety Reports 
In addition to the subsequent submission of a written IND Safety Report (i.e., completed FDA 
Form 3500A), the investigator-sponsor will notify the responsible review division of the FDA 
by telephone or facsimile transmission of any observed or volunteered adverse event that is 
1) associated with the use of the investigational drug or study treatment(s); 2 fatal or life-
threatening; and 3) unexpected. 
 
The telephone or facsimile transmission of applicable IND Safety Reports will be made as 
soon as possible but in no event later than 7 calendar days after the investigator-sponsor’s 
initial receipt of the respective human adverse event information. 
 
5.5.7. 
Reporting adverse events to the responsible IRB 
 
In accordance with applicable policies of the University of Pittsburgh Institutional Review 
Board (IRB), the Investigator will report, to the IRB, any observed or volunteered adverse 
event that is determined to be 1) associated with the investigational drug or study treatment(s); 
2) serious; and 3) unexpected.  Adverse event reports will be submitted to the IRB in 
accordance with the respective IRB procedures.  
 
Applicable adverse events will be reported to the IRB as soon as possible and, in no event, 
later than 10 calendar days following the Investigator’s receipt of the respective information.  
Adverse events which are 1) associated with the investigational drug or study treatment(s); 2) 
fatal or life-threatening; and 3) unexpected will be reported to the IRB within 24 hours of the 
Investigator’s receipt of the respective information.  
 
Follow-up information to reported adverse event will be submitted to the IRB as soon as the 
relevant information is available.  If the results of the sponsor-investigator’s follow-up 
investigation show that an adverse event that was initially determined to not require reporting 
to the IRB does, in fact, meet the requirements for reporting; the Investigator will report the 


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adverse event to the IRB as soon as possible, but in no event later than 10 calendar days, 
after the determination was made. 
 
6 
STUDY ADMINSTRATION 
 
 
6.1. 
QUALITY CONTROL AND QUALITY ASSURANCE 
 
Independent monitoring of the clinical study for protocol and GCP compliance will be conducted 
periodically (i.e., at a minimum of annually) by qualified staff of the Education and Compliance 
Office – Human Subject Research, Research Conduct and Compliance Office, University of 
Pittsburgh. 
 
The investigator-sponsor and the University of Pittsburgh and University of Pittsburgh Medical 
Center will permit direct access of the study monitors and appropriate regulatory authorities to the 
study data and to the corresponding source data and documents to verify the accuracy of this 
data. 
 
 
6.2. 
DATA HANDLING AND RECORD-KEEPING 
 
6.2.1. Data recording 
Study worksheets will be completed for each subject enrolled into the clinical study.  The 
Investigator will review, sign and date completed worksheets; the Investigator’s signature 
serving as attestation of the Investigator’s responsibility for ensuring that all clinical and 
laboratory data are complete, accurate and authentic.   
 
Appropriate coded identifications (i.e. Subject ID number) will be used. Every effort will be 
made to collect complete data for each study visit. Causes of missing data will be fully 
documented. With respect to safety evaluation, it is not planned to impute missing data. 
 
6.2.2. Record maintenance and retention   
The Sponsor and Investigator will maintain records in accordance with Good Clinical Practice 
guidelines; to include: 
 
• 
FDA correspondence related to the IND and clinical protocol, including copies of 
submitted Safety Reports and Annual Reports  
• 
IRB correspondence (including approval notifications) related to the clinical protocol; 
including copies of adverse event reports and annual or interim reports 
• 
Current and past versions of the IRB-approved clinical protocol and corresponding 
IRB-approved consent form(s) and, if applicable, subject recruitment advertisements 
• 
Signed FDA Form 1572 Statements of Investigator (i.e., for the Sponsor and all 
identified sub-investigators) 
• 
Financial disclosure information (Investigator-sponsor and clinical protocol sub-
investigators) 
• 
Curriculum vitae (Sponsor and clinical protocol sub-investigators) 
• 
Certificates of required training (e.g., human subject protections, Good Clinical 
Practice, etc.) for Sponsor and listed sub-investigators 
• 
Listing of printed names/signatures of Investigator-sponsor and listed sub-
investigators 


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• 
Normal value(s)/range(s) for medical/laboratory/technical procedures or tests included 
in the clinical protocol 
• 
Laboratory certification information 
• 
Instructions for on-site preparation and handling of the investigational drug(s), study 
treatment(s), and other study-related materials (i.e., if not addressed in the clinical 
protocol) 
• 
Decoding procedures for blinded trials (incorporate only if applicable) 
• 
Master randomization list (incorporate only if applicable) 
• 
Signed informed consent forms 
• 
Completed worksheets; signed and dated by Investigator 
• 
Source Documents or certified copies of Source Documents 
• 
Monitoring visit reports  
• 
Copies of Sponsor communications to the Investigator and copies of Investigator 
communications to sub-investigators  
• 
Subject screening and enrollment logs 
• 
Subject identification code list 
• 
Investigational drug accountability records, including documentation of drug disposal. 
• 
Retained biological specimen log 
• 
Interim data analysis report(s)  
• 
Final clinical study report 
 
Subject-specific data and will be coded and the subject identification code list will be stored 
so as to protect the subjects’ confidentiality.  Subject names or other directly identifiable 
information will not appear on any reports, publications, or other disclosures of clinical 
study outcomes. 
 
The Investigator-sponsor and Aires Pharmaceuticals, Inc.  will retain the specified 
records and reports for up to 2 years after the marketing application is approved for the 
investigational drug; or, if a marketing application is not submitted or approved for the 
investigational drug, until 2 years after investigations under the IND have been 
discontinued and the FDA so notified. 
 
 
6.3. 
ETHICS  
 
Institutional Review Board (IRB) Approval 
The Investigator will obtain, from the University of Pittsburgh Institutional Review Board (IRB), 
prospective approval of the clinical protocol and corresponding informed consent form(s); 
modifications to the clinical protocol and corresponding informed consent forms, and brochures 
(i.e., directed at potential research subjects and clinical faculty/staff) for study recruitment.    
 
The only circumstance in which a deviation from the current IRB-approved clinical 
protocol/consent form(s) may be initiated in the absence of prospective IRB approval is to 
eliminate an apparent immediate hazard to the research subject(s).  In such circumstances, the 
Investigator will promptly notify the University of Pittsburgh IRB of the deviation.  
 
The University of Pittsburgh IRB operates in compliance with FDA regulations at 21 CFR Parts 
50 and 21 CFR 56, and in conformance with applicable International Conference on 
Harmonization (ICH) Guidelines on Good Clinical Practice (CGP).  
 


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In the event that the University of Pittsburgh IRB requires, as a condition of approval, substantial 
changes to a clinical protocol submitted under an FDA-accepted IND application, or in the event 
of the Investigator’s decision to modify the previously accepted clinical protocol: 
 
The Investigator will submit (i.e., in advance of implementing the change) a Protocol Amendment 
to the IND describing any change to this Phase 1 clinical protocol that significantly affects the 
safety of the subjects.  For changes that do not affect critical safety assessments, the revisions 
to the clinical protocol will be addressed in the Annual Report to the IND.  
 
 
Ethical and scientific conduct of the clinical study  
The clinical study will be conducted in accordance with the current IRB-approved clinical protocol; 
ICH Guidelines on GCP; and relevant policies, requirements, and regulations of the University of 
Pittsburgh IRB, University of Pittsburgh and UPMC, Commonwealth of Pennsylvania, and 
applicable federal agencies. 
 
The Investigator will make certain that appropriate processes and procedures are in place to 
ensure that ongoing questions and concerns of enrolled subjects are adequately addressed and 
that the subjects are informed of any new information that may affect their decision to continue 
participation in the clinical study.  In the event of substantial changes to the clinical study or the 
risk-to-benefit ratio of study participation, the Investigator will obtain the informed consent of 
enrolled subjects for continued participation in the clinical study. 
 
7.   COSTS AND PAYMENTS 
 
7.1 COSTS 
 
The study is jointly supported by the NIH Program Project Grant (PPG; grant number: 
P01HL103455) and by Aires Pharmaceuticals, Inc. The current proposal is the human core 
component of this PPG. The NIH funding covers the majority of the study procedures as well as 
the study infrastructure. Study drugs and research testing will be supported by Aires 
Pharmaceuticals, Inc. Medication such as ETRA or PDE-5I, routine lab tests, and any procedures 
not related to this research study are considered routine medical care and will be billed to the 
subjects’ health insurance company. Subjects will be responsible for paying any deductibles, co-
payments or co-insurance that are a normal part of their health insurance plan. Subjects who do 
not have health insurance will be responsible for these costs.   
 
 
7.2 PAYMENTS 
 
Subjects will be compensated $250.00 after completion of right heart catheterization and study 
treatment at the end of Day 1 study visit, and $50.00 after completion of the Day 30 outpatient 
clinic follow-up study visit. The total compensation for participation in all aspects of the clinical 
study will be $ 300.00.  Parking validations will be given for study visit 1 and Day 30. 


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8.   QUALIFICATIONS AND SOURCES OF SUPPORT 
 
 
8.1 QUALIFICATIONS OF THE INVESTIGATORS 
 
Sponsor: 
Mark Schmidhofer, MD is an Associate Professor of Medicine, at the University of Pittsburgh, 
Heart and Vascular Institute.  Dr. Schmidhofer is the Director of the Coronary Intensive Care Unit 
and Director of Quality Improvement, Division of Cardiology.  He has served as coauthor on 
several publications.  Dr. Schmidhofer will work closely with Dr. Simon 
 in maintaining the necessary regulatory documents for this established IND.   
 
Investigator: 
Marc Simon, MD, is an Associate Professor of Medicine in the Heart Failure and Cardiac 
Transplantation Section at the University of Pittsburgh. His clinical specialty is advance heart 
failure and pulmonary hypertension. His research has largely focused on adaptation and failure 
of the right ventricle in pulmonary hypertension. Dr. Simon has been involved in many research 
projects. He will play a large role in the accrual of patients who are seen at the UPMC Heart and 
Vascular Institute on a regular basis. 
 
Sub-Investigators: 
Mark T. Gladwin, MD, is a Professor of Medicine, Director of the Hemostasis & Vascular Biology 
Research Institute and Chief of the Division of Pulmonary, Allergy, and Critical Care Medicine at 
the University of Pittsburgh.  Dr. Gladwin has established himself as one of leading international 
authorities on nitrite anion biology and its application to treat a variety of cardiopulmonary 
diseases.  He is currently the PI on two, large multi-center clinical trials to test the efficacy of nitric 
oxide and sildenafil in subjects with sickle cell disease and pulmonary hypertension.  He has 
served as a principal or associate investigator on more than 25 human subjects protocols and 
holds seven FDA INDs for the use of investigational therapeutic medications, including nitrite, 
carbon monoxide, L-NMMA, and sildenafil.   
 
Dennis McNamara, MD is a Professor of Medicine at the University of Pittsburgh in the Heart 
and Vascular Institute.  He is the Director of the Heart Failure/Transplantation Section at the 
University of Pittsburgh Medical Center.  Dr. McNamara is recognized as a national leader in 
pharmacogenetic research in heart failure, and is the National Principal Investigator of an NHLBI 
multi-center genetics outcomes study. 
 
Alison Morris, MD, is an Associate Professor of Medicine, Division of Pulmonary, Allergy, and 
Critical Care Medicine.  She is the PI of the NIH R01 to study the pathogenesis of HIV-associated 
emphysema.  Dr. Morris has also been the PI on a K23, a California Tobacco-disease Related 
Program grant, and PI or co-investigator on numerous research projects examining lung diseases 
in various populations.  
 
Michael Risbano, MD, MA is an Assistant Professor in the Department of Medicine, Division of 
Pulmonary, Allergy, and Critical Care Medicine.  Dr. Risbano’s clinical interests are medications 
primarily focused on the diagnosis, management and treatment of pulmonary hypertension. He 
is an attending physician in the Comprehensive Pulmonary Hypertension Program. Dr. 
Risbano’s past research has focused on the discovery and implementation of biomarkers for the 
early diagnosis of pulmonary hypertension in patients with scleroderma (SSc-PAH). He is 


Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
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interested in the hemodynamic evaluation of subjects with pulmonary hypertension and 
correlation of hemodynamic values with biomarker levels. Dr. Risbano also has a research 
interest in the hemodynamic responses to vasoactive medications. 
 
Frank Sciurba, MD, is an Associate Professor of Medicine at the University of Pittsburgh, 
Pulmonary, Allergy and Critical Care Medicine division and Director of The Emphysema 
Research Center and Pulmonary Function Exercise Physiology Laboratory. He is an active 
member of the American College of Chest Physicians, and serves on the Pulmonary 
Physiology, Function & Rehabilitation Network Steering Committee. He is a member of the 
American Thoracic Society and has served on the Ad Hoc Committee on Standards for Clinical 
Exercise Testing and currently serves on the Corporate Relations Committee and Clinical 
Problems Program committee and has chaired the ATS International Conference post graduate 
course in COPD for four consecutive years. 
 
 
8.2 SOURCE OF SUPPORT 
 
National Heart, Lung, and Blood Institute and Aires Pharmaceuticals, Inc. 
 
9.   REFERENCES 
 
1. 
D'Alonzo GE, Barst RJ, Ayres SM, Bergofsky EH, Brundage BH, Detre KM, Fishman 
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Rubin LJ. Primary pulmonary hypertension. Chest. 1993;104:236-250 
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JE. An imbalance between the excretion of thromboxane and prostacyclin metabolites in 
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Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
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10. 
Cockrill BA. The use of nitric oxide in primary pulmonary hypertension. Respiratory 
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Tejero J, Gladwin MT. The globin superfamily: Functions in nitric oxide formation and 
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Shiva S, Huang Z, Grubina R, Sun J, Ringwood LA, MacArthur PH, Xu X, Murphy E, 
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SF, Wang X, Kevil CG, Gladwin MT, Lefer DJ. Cytoprotective effects of nitrite during 
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Study Short Title: Inhaled Nitrite in Pulmonary Hypertension                                                                  
 
  
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23. 
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with severe pulmonary hypertension. Heart. 2005;91:391-392 
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