Conditions: Primary Graft Dysfunction
Keywords: Lung transplant Pulmonary Graft Dysfunction Sodium Nitrite
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 3
Healthy volunteers: no

Brief summary:
This is a Phase 2 observational nonrandomized pilot investigation to evaluate the safety and efficacy of Sodium Nitrite administration for the reduction of Primary Graft Dysfunction (PGD) in patients undergoing lung transplant. The study will enroll 8 subjects, undergoing lung transplant at the University of Pittsburgh Medical Center (UPMC).

Detailed description:
While increasing numbers of patients with advanced lung disease are candidates for lung transplantation, the short- and long-term outcomes are severely compromised by graft dysfunction, primarily in the form of organ rejection. The earliest manifestation of lung allograft dysfunction, termed primary graft dysfunction (PGD), represents a form of ischemia-reperfusion acute lung injury, and occurs in its severest form (Grade 3) in from 10 to 35% of lung transplant recipients 1-6. PGD is the primary cause of early morbidity and mortality after transplantation and is strongly associated with the late development of chronic lung rejection or Bronchiolitis Obliterans Syndrome (BOS. Early graft dysfunction contributes significantly to the suboptimal outcomes of lung transplantation and to the failure of lung transplant recipients to achieve five-year survival rates comparable to patients who receive other solid organs such as the heart and liver. The risk of PGD further limits the time that lungs can be stored ex-vivo, therefore restricting the pool of available donors. A critical advance in the prevention of both early and late lung allograft dysfunction will occur if PGD can be successfully prevented or minimized.

In this study, the investigators propose to test the hypothesis that administration of Sodium Nitrite to donor lungs and lung transplant recipients at the time of transplantation will be safe and will reduce the incidence of grades 2 and 3 PGD, thereby improving clinical outcomes with minimal toxicity.

Sodium Nitrite will be obtained from a commercial preparation (Hope Pharmaceuticals) and the UPMC Pharmacy will prepare the formulations, which will be infused at three time points. First it will be infused into the preservation solution bag at the time of organ procurement from the donor, then to the allograft at the time of transplantation, and finally as a direct infusion into the organ recipient.

The investigators plan to enroll total of 8 subjects undergoing lung transplantation for this Phase 2 observational non-randomized pilot investigation to evaluate the safety, efficacy, and pharmacokinetics of Sodium Nitrite administration when administered to the procured lung and lung transplant recipient, for the prevention of Primary Graft Dysfunction (PGD). It is anticipated that positive results from this trial lead to a larger clinical investigation of Sodium Nitrite administration directed at producing a reduction in PGD and perhaps secondary obliterative bronchiolitis; and will potentially allow for extended organ storage, extended use of more marginal organs, and more effective use of Donation after Cardiac Death (DCD) organs which undergo combination of warm and cold ischemia for organ procurement.

Arms:
arm 1: Sodium Nitrite will be administered at three time points:

At the time of organ procurement, a pre-prepared syringe of sodium nitrite will be added to each of the 2.8 liter bags of Perfadex solution to flush the donor lungs.

At the time of transplant just prior to reperfusion of lungs, the donor lungs are flushed with a cold pneumoplegia solution after the bronchial (1st) anastomosis and with warm pneumoplegia solution after the portal vein (3rd, last) anastomosis. The drug will be added to pneumoplegia solution just prior to both the flushes.

Sodium Nitrite will be delivered intravenously to the recipient immediately prior to lung reperfusion as a single infusion at rate of 4 mL/min for the first 30 min, followed by 2.2 mL/min for the next 60 min.

Interventions: intervention 1: Sodium Nitrite
Intervention descriptions: intervention 1: Same as the details in Arm Description.

Protocol document:
Prot_SAP_000.pdf: 
Clinical Study protocol  
 
  March 3, 2016, Version 1.9 
 
 
 
1 
 
 
 
 
Clinical Protocol 
 
 
 
 
Sodium Nitrite Administration at the Time of Lung Organ 
Procurement and Transplantation to Minimize the Risk of 
Pulmonary Graft Dysfunction 
 
 
 
 
 
 
 
 
 
 
March 3, 2016 
 
 
 
 
 
 


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CONTENTS 
 
1 
ABSTRACT .......................................................................................................................... 6 
2 
OBJECTIVE, SPECIFIC AIMS AND ENDPOINTS ............................................................. 7 
2.1 
OBJECTIVE ........................................................................................................................ 7 
2.2 
HYPOTHESIS ..................................................................................................................... 7 
2.3 
SPECIFIC AIMS .................................................................................................................. 8 
2.4 
ENDPOINTS ....................................................................................................................... 8 
3 
BACKGROUND AND RATIONALE .................................................................................... 9 
4 
SIGNIFICANCE ................................................................................................................. 14 
5 
STUDY DESIGN AND METHODS .................................................................................... 15 
5.1 
OVERVIEW ...................................................................................................................... 15 
5.2 
STUDY TIME LINE ............................................................................................................ 15 
5.3 
SCREENING PROCEDURES ............................................................................................... 15 
5.4 
STUDY DRUG PREPARATION ............................................................................................ 16 
5.5 
STUDY DRUG INTERVENTION ............................................................................................ 17 
5.6 
DOSING ADJUSTMENTS AND/OR DISCONTINUATION DUE TO ADVERSE EVENTS ................... 18 
5.7 
STUDY PROCEDURES ...................................................................................................... 19 
5.7.1 RANDOMIZATION AND BLINDING .................................................................................... 19 
5.7.2 STUDY MONITORING .................................................................................................... 19 
5.7.3 OPTIONAL END POINTS TO BE OBTAINED FROM ADMISSION TO ICU DISCHARGE BASED                                
ON STANDARD CLINICAL MONITORING ........................................................................... 21 
5.8 
SAFETY ASSESSMENT ...................................................................................................... 23 
5.9 
EFFICACY ASSESSMENT ................................................................................................... 23 
6 
STATISTICAL CONSIDERATION ..................................................................................... 23 
7 
HUMAN SUBJECTS .......................................................................................................... 23 
7.1 
SUBJECT POPULATION ..................................................................................................... 23 
7.1.1 HUMAN SUBJECTS INVOLVEMENT AND CHARACTERISTICS ............................................. 23 
7.1.2 DONOR EXCLUSION CRITERIA ...................................................................................... 24 
7.1.3 RECIPIENT INCLUSION CRITERIA ................................................................................... 24 
7.1.4 RECIPIENT EXCLUSION CRITERIA .................................................................................. 24 
7.2 
RECRUITMENT AND INFORMED CONSENT PROCEDURES .................................................... 25 
8 
POTENTIAL RISKS AND BENEFITS ............................................................................... 25 


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8.1 
RISK OF EXPERIMENTAL INTERVENTION ............................................................................ 25 
9 
PROTECTION AGAINST PATIENT RISK ........................................................................ 26 
9.1 
PROTECTION AGAINST GENERAL PATIENT RISK ................................................................ 26 
9.2 
PROTECTION AGAINST PATIENT RISKS RELATED TO THE STUDY DRUG ............................... 27 
10 
DATA AND SAFETY MONITORING PLAN ...................................................................... 28 
10.1 
DATA AND SAFETY MONITORING BOARD ........................................................................... 28 
10.2 
DATA AND SAFETY MONITORING PLAN .............................................................................. 28 
10.3 
PARAMETERS TO BE MONITORED ..................................................................................... 29 
10.4 
FREQUENCY OF MONITORING ........................................................................................... 29 
10.5 
RECORDING/REPORTING REQUIREMENTS ......................................................................... 30 
10.5.1 ELICITING ADVERSE EVENT INFORMATION .................................................................... 30 
10.5.2 RECORDING REQUIREMENT.......................................................................................... 30 
10.6 
REPORTING OF ADVERSE EVENTS .................................................................................... 31 
10.6.1 REPORTING OF ADVERSE EVENTS TO THE FDA ............................................................ 32 
10.7 
REPORTING ADVERSE EVENTS TO THE RESPONSIBLE IRB ................................ 33 
11 
COSTS AND PAYMENTS ................................................................................................. 34 
12 
POTENTIAL BENEFITS OF THE PROPOSED RESEARCH TO THE SUBJECTS  
 
AND OTHERS.................................................................................................................... 34 
13 
IMPORTANCE OF THE KNOWLEDGE TO BE GAINED ................................................. 34 
14 
QUALIFICATIONS OF THE INVESTIGATORS ................................................................ 34 
15 
REFERENCES ................................................................................................................... 37 
 
 


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PROTOCOL TITLE: 
Sodium nitrite administration at the time of lung 
organ procurement and transplantation to 
minimize the risk of pulmonary graft dysfunction. 
 
IDENTIFYING WORDS: 
 
Lung transplant, Pulmonary graft dysfunction 
DATE: 
September 29, 2011 
VERSION NUMBER: 
1.9 
STUDY INVESTIGATORS  
 
Principal Investigators: 
Matt Morrell, MD 
 
Co-Investigators: 
 
Norihisa Shigemura, MD 
 
Jonathan D'Cunha, MD, PhD 
Mark T. Gladwin, MD     
Joseph Pilewski, MD  
Maria Crespo, MD 
John Mc Dyer, MD 
Patricia George, MD 
 
Bruce Johnson, MD 
 
Matthew Pipeling, MD 
Michael Donahoe, MD 
Yingze Zhang, Ph.D 
Diana Zaldonis, MPH, BSN 
 
Jeremiah Hayanga MD 
Silpa Kilaru MD 
Elizabeth Lendermon MD 
Simeng Wang 


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Synopsis 
Protocol Title:  
Sodium nitrite administration at the time of lung organ procurement and 
transplantation to minimize the risk of pulmonary graft dysfunction 
Study Site:  University of Pittsburgh Medical Center  
Phase of Development:  Phase  A  
Objectives:   
Primary Objective(s):  
 
To evaluate the safety of Sodium Nitrite infusion into the procured lungs and 
lung transplant recipient as reflected in blood mean arterial pressure and 
methemoglobin levels. 
Secondary Objectives:   
 
To evaluate the efficacy of Sodium Nitrite infusion into the procured lungs 
and the    lung transplant recipient in the prevention of grade 2-3 Primary 
Graft Dysfunction.   
 
To evaluate the efficacy of Sodium Nitrite infusion into the procured lungs 
and the lung transplant recipient in the prevention of delayed allograft 
complications including the incidence of acute rejection and BOS 
 
To identify the pharmacokinetic relationship of Sodium Nitrite and 
Ernest Chan MD 
Nicholas Hess 
 
Collaborators: 
 
Susan Stuart, RN, MPM 


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metabolism of sodium nitrite in lung transplant recipients using blood 
sampling measures before and after drug exposure (will measure levels of 
plasma and whole blood nitrite, plasma nitrate, red cell iron-nitrosyl-
hemoglobin, S-nitroso-hemoglobin and methemoglobin). 
Statement of Primary Efficacy Hypothesis and Primary Analysis:  
The administration of Sodium Nitrite at the time of organ procurement and 
transplantation will be safe and significantly reduce the rate of Primary Graft 
Dysfunction in patients undergoing lung transplantation. 
Planned Sample Size: A total of 8 subjects will be enrolled into the protocol.  
Recruitment is expected to continue for six months. 
Study Design: Phase IIA observational non-randomized cohort. 
Diagnosis and Key Patient Selection Criteria:  Males or females greater than 18 
years of age or older undergoing lung transplantation. 
Treatments:  Sodium Nitrite infusion to the donor lungs and the lung transplant 
recipient. 
 
1 Abstract 
While increasing numbers of patients with advanced lung disease are candidates for lung 
transplantation, the short- and long-term outcomes are severely compromised by graft 
dysfunction, primarily in the form of organ rejection.  The earliest manifestation of lung allograft 
dysfunction, termed primary graft dysfunction (PGD), represents a form of ischemia-reperfusion 
acute lung injury, and occurs in its severest form (Grade 3) in from 10 to 35% of lung transplant 
recipients 1-6.  PGD is the primary cause of early morbidity and mortality after transplantation 
and is strongly associated with the late development of chronic lung rejection or Bronchiolitis 
Obliterans Syndrome (BOS) 7-9. Early graft dysfunction contributes significantly to the 
suboptimal outcomes of lung transplantation and to the failure of lung transplant recipients to 
achieve five-year survival rates comparable to patients who receive other solid organs such as 
the heart and liver. The risk of PGD further limits the time that lungs can be stored ex-vivo, 
therefore restricting the pool of available donors. A critical advance in the prevention of both 
early and late lung allograft dysfunction will occur if PGD can be successfully prevented or 
minimized.  
 


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In this study, we propose to test the hypothesis that administration of Sodium Nitrite to donor 
lungs and lung transplant recipients at the time of transplantation will be safe and will reduce the 
incidence of grades 2 and 3 PGD, thereby improving clinical outcomes with minimal toxicity. 
 
Sodium Nitrite will be obtained from a commercial preparation (Hope Pharmaceuticals, Sodium 
Nitrite Injection USP (30mg/mL) NDC Number 60267-079-02) and the UPMC Pharmacy will 
prepare the formulations, which will be infused at three time points. First it will be infused into 
the preservation solution bag at the time of organ procurement from the donor, then to the 
allograft at the time of transplantation, and finally as a direct infusion into the organ recipient. 
 
We plan to enroll total of 8 subjects undergoing lung transplantation for this Phase IIA 
observational non-randomized pilot investigation to evaluate the safety, efficacy, and 
pharmacokinetics of Sodium Nitrite administration when administered to the procured lung and 
lung transplant recipient, for the prevention of Primary Graft Dysfunction (PGD). It is anticipated 
that positive results from this trial lead to a larger clinical investigation of Sodium Nitrite 
administration directed at producing a reduction in PGD and perhaps secondary obliterative 
bronchiolitis; and will potentially allow for extended organ storage, extended use of more 
marginal organs, and more effective use of Donation after Cardiac Death (DCD) organs which 
undergo combination of warm and cold ischemia for organ procurement. 
 
2 Objective, Specific Aims and Endpoints 
 
2.1 Objective 
 
The objective of this Phase IIA observational non-randomized pilot investigation is to evaluate 
the safety, efficacy, and pharmacokinetics of Sodium Nitrite administration when administered to 
the procured lung and lung transplant recipient, for the prevention of Primary Graft Dysfunction 
(PGD). 
2.2 Hypothesis  
Administration of Sodium Nitrite to donor lungs and lung transplant recipients at the time of 
transplantation will be safe and will reduce the incidence of grades 2 and 3 PGD, thereby 
improving clinical outcomes with minimal toxicity. 


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2.3 Specific Aims 
The specific aims of this study are: 
Primary Objective(s):  
 
To evaluate the safety of Sodium Nitrite infusion into the procured lungs and the lung 
transplant recipient as reflected in blood mean arterial pressure and methemoglobin 
levels. 
 
Secondary Objectives:   
 
To evaluate the efficacy of Sodium Nitrite infusion into the procured lungs and the lung 
transplant recipient in the prevention of grade 2-3 primary graft dysfunction.   
 
To evaluate the efficacy of Sodium Nitrite infusion into the procured lungs and the lung 
transplant recipient in the prevention of delayed allograft complications including the 
incidence of acute rejection and BOS. 
 
To identify the pharmacokinetic relationship of Sodium Nitrite and metabolism of sodium 
nitrite in lung transplant recipients using blood sampling measures before and after 
drug exposure (levels of plasma and whole blood nitrite, plasma nitrate, red cell iron-
nitrosyl-hemoglobin, S-nitroso-hemoglobin and methemoglobin will be measured). 
2.4    Endpoints 
Primary Endpoints Analysis  
 
The primary endpoint for the safety of Sodium Nitrite administration will be assessed by 
measurement of methemoglobin levels and mean arterial pressure during the nitrite 
infusion in the transplant recipient. 
 
The primary endpoint for the efficacy of Sodium Nitrite infusion will be the incidence of 
grades 2 and 3 PGD using the International Society for Heart and lung transplant 
(ISHLT) criteria for PGD based upon the worst value obtained over the initial 72 hours 
post lung reperfusion (T0-72). 
 
Secondary Endpoint Analysis  
 
Secondary endpoints for efficacy will include AUC curves for the oxygenation index 
during the initial 72 hours post transplantation, ventilator free days, intensive care free 
days and hospital days. 
 
The secondary endpoints for the prevention of Bronchiolitis Obliterans(BO) will be 
captured for up to 12-months following lung transplantation and include clinically 


Cli ni c al St u d y pr ot o c ol  
 
  M ar c h 3, 2 0 1 6, V er si o n 1. 9 
 
 
 
9  
 
i n di c at e d s pir o m etri c a n d l u n g v ol u m e a s s e s s m e nt s of l u n g f u n cti o n p erf or m e d a s a n 
i n di c at or  of  Br o n c hi oliti s  O blit er a n s  S y n dr o m e  ( B O S),  a n d  e vi d e n c e  of  p at h ol o gi c al  
r ej e cti o n b y s ur v eill a n c e tr a n s br o n c hi al l u n g bi o p si e s. 
 
3  
B a c k gr o u n d a n d R ati o n al e 
W hil e  i n cr e a si n g  n u m b er s  of  p ati e nt s  wit h  a d v a n c e d  l u n g  di s e a s e  ar e  c a n di d at e s  f or  l u n g  
tr a n s pl a nt ati o n,  t h e  s h ort-  a n d  l o n g-t er m  o ut c o m e s  ar e  s e v er el y  c o m pr o mi s e d  b y  gr aft  
d y sf u n cti o n, pri m aril y i n t h e f or m of or g a n r ej e cti o n.  T h e e arli e st m a nif e st ati o n of l u n g all o gr aft 
d y sf u n cti o n, t er m e d pri m ar y gr aft d y sf u n cti o n ( P G D), r e pr e s e nt s a f or m of i s c h e mi a-r e p erf u si o n 
a c ut e l u n g i nj ur y, a n d o c c ur s i n it s s e v er e st f or m ( Gr a d e 3) r a n gi n g fr o m 1 0 t o 3 5 % of l u n g 
tr a n s pl a nt  r e ci pi e nt s  1- 6.   P G D  i s  t h e  pri m ar y  c a u s e  of  e arl y  m or bi dit y  a n d  m ort alit y  aft er  
tr a n s pl a nt ati o n a n d i s str o n gl y a s s o ci at e d wit h t h e l at e d e v el o p m e nt of c hr o ni c l u n g r ej e cti o n or 
br o n c hi oliti s o blit er a n s s y n dr o m e ( B O S) 7- 9. E arl y gr aft d y sf u n cti o n c o ntri b ut e s si g nifi c a ntl y t o 
t h e s u b o pti m al o ut c o m e s of l u n g tr a n s pl a nt ati o n a n d t o t h e f ail ur e of l u n g tr a n s pl a nt r e ci pi e nt s 
t o a c hi e v e fi v e- y e ar s ur vi v al r at e s c o m p ar a bl e t o p ati e nt s w h o r e c ei v e ot h er s oli d or g a n s s u c h 
a s t h e h e art a n d li v er. T h e ri s k of P G D f urt h er li mi t s t h e ti m e t h at l u n g s c a n b e st or e d e x- vi v o, 
t h er ef or e r e stri cti n g t h e p o ol of a v ail a bl e d o n or s. A criti c al a d v a n c e i n t h e pr e v e nti o n of b ot h 
e arl y  a n d  l at e  l u n g  all o gr aft  d y sf u n cti o n  will  o c c ur  if  P G D  c a n  b e  s u c c e s sf ull y  pr e v e nt e d  or  
mi ni mi z e d. 
 
T h e I nt er n ati o n al S o ci et y f or H e art a n d L u n g Tr a n s pl a nt (I S H L T) g ui d eli n e s h a v e pr o vi d e d a 
st a n d ar di z e d d efi niti o n f or P G D b a s e d o n t h e P a O 2/ Fi O 2 ( P/ F) r ati o a n d t h e pr e s e n c e of c h e st 
r a di o gr a p h  i nfiltr at e s  a s s e s s e d  wit hi n  t h e  fir st  7 2  h o ur s  f oll o wi n g  tr a n s pl a nt ati o n  1 0 .   T h e  
fr e q u e n c y of P G D h a s v ari e d i n p u bli s h e d r e p ort s wit hi n t h e r a n g e of 7 0- 8 0 % a s o utli n e d i n 
T a bl e 1.   
 
T a bl e 1: Fr e q u e n c y of R e p ort e d P G D b y Gr a d e a n d D efi niti o n U si n g a C o m p o n e nt of 
I S H L T Crit eri a 
R ef er e n c e  
Ti m e ( N)  
D efi niti o n  
P G D 0 ( %) P G D 1 ( %) P G D 2 ( %) P G D 3 ( %)
Pr e k k er
 2 0 0 5 ( 5) 
1 9 9 2- 2 0 0 4 ( 4 0 2) T ( 0- 4 8) 
 
3 8 % 
2 8 % 
3 4 % 
B o b a dill a 2 0 0 8 ( 1) 
1 9 9 9- 2 0 0 8 ( 3 3) 
T 7 2 
 
 
 
3 0 % 
D a u d * 2 0 0 7 ( 7) 
1 9 9 8- 2 0 0 4 ( 3 3 4) T 0 
1 9 % 
3 9 % 
2 1 % 
2 1 % 


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Table 1: Frequency of Reported PGD by Grade and Definition Using a Component of 
ISHLT Criteria 
Reference 
Time (N) 
Definition 
PGD 0 (%) PGD 1 (%) PGD 2 (%) PGD 3 (%)
Huang * 2008 (3) 
1998-2004 (334) T48 
17% 
65% 
8% 
9% 
Christie 2009 (11) 
2003-2007 (317) T24 
 
 
 
20% 
Pittsburgh, 
unpublished 
2003-2004  (76) T (0-72) 
21% 
34% 
20% 
25% 
 
+ Grouped 0-1 into Group 1.  Did not include radiographic scoring component 
*  Identical cohort analyzed at different time points 
 
 
PGD grade 3 has been associated with a 90-day mortality of 16.2% in comparison to a mortality 
of 9.5% for subjects with PGD Grades 0-1. However, the prognostic significance of PGD 
extends well beyond the immediate post-operative period.  Grade 3 PGD has been associated 
with a more rapid decline in post-operative pulmonary function and the late development of 
bronchiolitis obliterans syndrome (BOS) 7. These data suggest that new approaches to reduce 
PGD are fundamental to improving the morbidity and mortality in lung transplant recipients.  
 
 
 
4.3.1 Complex Interaction of Donor and Recipient Risk Factors Contributing to Primary Graft 
Dysfunction 
 
Using the standardized definition for PGD, numerous donor, recipient, and treatment variables 
have been identified as potential clinical risk factors for the development of PGD 11. These 
reported associations remain limited by the lack of multicenter, prospective, observational 
studies using consistent procurement techniques.  Despite this limitation, both single center and 
registry reviews have suggested that variables such as donor age 6, prolonged ischemia time 12, 
recipient pulmonary hypertension 6,13,14, recipient diagnosis 15, and donor requirement for blood 
transfusion 16, may all be independent risk factors for PGD. 
 
4.3.2 Rationale for the Investigation of Nitrite Therapy in PGD 
 


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Figure 
1: 
Inhibition 
of 
mitochondrial 
ROS 
and 
cytochrome C release by nitrite during anoxia and 
reoxygenation. A) nitrite inhibits complex I activity; B) 
decreases ROS generation; C) decreases permeability 
pore opening (measured as mitochondrial swelling; and 
D) Western blot for cytochrome c remaining in the 
mitochondria.  Shiva et al. J. Ex. Med 2008. 
Sodium Nitrite has been shown to limit ischemia-reperfusion injury and myocardial infarction. 
Nitrite is a potent cytoprotective agent, with its optimal dose (48 nmol - which raises murine 
plasma nitrite concentration to 10 µM) decreasing infarct size by 50-70% in the heart, liver and 
brain of respective animal models 17-20.  This 
cytoprotective 
effect 
of 
nitrite 
after 
ischemia/reperfusion (IR) has now been 
observed by a number of groups and in a 
number of organs/models.   Although this 
cytoprotection 
has 
been 
shown 
to 
be 
dependent on the formation of nitric oxide 
(NO), it is independent of endothelial NO 
synthase. 
 
Recent 
data 
have 
also 
demonstrated 
that 
dietary 
nitrite 
supplementation can increase plasma and 
tissue levels of nitrite, and these increased 
levels can protect against IR injury in the 
heart and liver 
21. The mechanism of 
bioactivation of nitrite in the ischemic organ is 
the subject of intense current study, with 
groups proposing a central role for xanthine 
oxidoreductase 
22, mitochondrial enzymes 
23,24, NO synthase 
27, as well as non-
enzymatic acidic disproportionation 26-28.   
 
The mechanism of nitrite cytoprotective effect involves the inhibition of mitochondrial respiration 
and decreased ROS at reperfusion. Nitrite appears to be mitochondrially-targeted and able to 
modulate mitochondrial reactive oxygen species (ROS) generation 29,30. We have demonstrated 
that nitrite dose dependently S-nitrosates and inhibits mitochondrial complex I in vivo models of 
IR as well as in vitro models of anoxia/reoxygenation (Figure 1).  
This post-translational modification decreases electron transfer through complex I, which 
effectively decreases ROS generation upon reperfusion and prevents protein oxidation and loss 
of function 30. Similar to other complex I inhibitors, the nitrite dependent S-nitrosation of complex 
I reduces reperfusion reactive oxygen species formation and oxidative damage to the 
mitochondria (indicated by a reduction in aconitase oxidation). In addition, nitrite prevents 


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opening of the mitochondrial permeability transition pore and the release of cytochrome c from 
the mitochondria in response to calcium, an effect similar to that of cyclosporine on 
mitochondrial function 
 
 
4.3.3 NO And Nitrite Regulate Cellular Apoptosis 
 
The mitochondrial apoptotic pathway is initiated by opening of the mitochondrial permeability 
transition pore and cytochrome c of mitochondrial ROS and cytochrome C release in a process 
inhibited by nitrite (Figure 1) and regulated by the pro- and anti-apoptotic proteins bax and bcl-2.  
This pathway drives the activation of caspase 9 and assembly of the apoptosome, which leads 
to the eventual activation of caspase 3 31. This pathway plays a central role in the cytotoxicity of 
ischemia and reperfusion, as demonstrated by studies in which inhibiting caspase 9 32 or 
overexpressing the anti-apoptotic protein bcl-2 33, significantly decreases infarct size in 
myocardial infarction models.  
 
At low concentrations, NO regulates several steps in the apoptotic pathway including inhibiting 
the release of cytochrome C 34 and the activation of caspases 9 and 3 35, and maintaining levels 
of the anti-apoptotic protein bcl-2 36.   
 
 
 
Consistent with these data, nitrite inhibits caspase 
3 activation (Figure 2).  
 
 
 
 
 
 
 
4.3.4 Sodium Nitrite Therapy as the Most Promising Pilot Trial to Model in The Treatment 
Of PGD  
 
Figure 2: Caspase 3 activity in wild type 
mice after I/R with or without nitrite 
treatment. 


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Accumulating evidence suggests that the ubiquitous anion, nitrite (NO2-), is a physiological 
signaling molecule, with roles in intravascular endocrine nitric oxide (NO) transport, hypoxic 
vasodilatation, and cellular cytoprotection 37-40.  
 
Numerous pre-clinical studies in mice, rats, dogs, sheep and primates have shown that nitrite 
has both vasodilatory and tissue protective effects in models of ischemia-reperfusion injury to 
the central nervous system, liver, kidney, heart and lung 41-47.  Additionally, recent pre-clinical 
studies, as outlined below, now indicate that nitrite has potent effects at reducing primary graft 
dysfunction and late rejection after lung and heart transplantation 39,40,47.  Most importantly, 
phase I-II studies of nitrite infusion, at doses much higher than the 40 mg dose planned for this 
trial, have been completed in more than 80 human volunteers and patients with sickle cell 
disease with no observed toxicity 37,38,40.  In collaborative studies at the University of Pittsburgh 
with the Department of Surgery and the Starzl Transplantation Institute, we have evaluated 
Sodium Nitrite in rat models of lung transplantation and heart transplantation.  These studies 
show that nitrite not only potently inhibits early ischemia and reperfusion injury but also prevents 
down-stream allograph rejection. 
 
Heart Transplantation Models: In these studies, fully allogeneic heterotopic heart transplantation 
was performed in Lewis (LEW) to Brown Norway (BN) rats. Animals were all given tacrolimus 
therapy, and either a regular diet or a low nitrite/nitrate chow validated in Dr. Gladwin’s 
laboratory or the low chow but with water containing nitrite (50 mg/L) for 120 days, beginning 7 
days before transplant.  Serum nitrite/nitrate levels were significantly higher in animals given 
nitrite water for 30 days, and lower in the animals fed with low nitrite/nitrate diet than those in 
animals who received standard diet. Supplementation of drinking water with nitrite enhanced 
heart graft survival to a median of >120 days from 49.5 days in animals fed a standard diet. In 
contrast, the low nitrite/nitrate diet led to nitrite and nitrate insufficiency and resulted in 
significantly earlier rejection of allografts (30.5 days). These data demonstrate that nitrite 
supplementation is significantly effective in preventing development of heart allograft rejection in 
a relevant pre-clinical model 47. 


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Figure 3: Increase in oxygenation 
after lung transplantation (LTx) in 
mice receiving nitrite in both the lung 
preservation solution (LPD) and IV at 
the time of reperfusion. 
 
Lung transplantation models: In these studies, 
orthotropic left lung transplantation (LTx) was 
performed syngeneic rats. Grafts were preserved 
with or without Sodium Nitrite (10 M) in the cold 
storage   
 solution at 4°C for 6 hours.  
 Groups of recipients also received Sodium Nitrite at 
a    
 dose of 480 nmoles intravenously 5 minutes before  
 graft reperfusion to target a plasma concentration of  
10 μM. Graft function, as measured by the pulmonary 
vein PaO2 while breathing 100% oxygen and 
inflammatory lung injury were assessed 2 hours after 
transplantation. 
 
We found that oxygenation was significantly higher in 
animals 
receiving 
Sodium 
Nitrite 
in 
both 
the 
preservation solution and given to the recipient 
compared to those in the control group (Figure 3; 
234±53 vs 99±18 mm Hg; P<0.05). In contrast, nitrate 
did not alter gas exchange.  
 
The increase of graft mRNA for IL-6, IL-1, TNF-α, iNOS, and COX-2 in the control group was 
markedly inhibited in nitrite group. Lung myeloperoxidase activity, lung wet to dry weights and 
histological evidence of edema and cellular inflammation were all significantly reduced with 
nitrite treatment.  
 
4 Significance 
 
It is anticipated that positive results in this trial will lead to a larger clinical investigation of 
Sodium Nitrite administration directed at producing a reduction in PGD, and perhaps secondary 
obliterative bronchiolitis; and will potentially allow for extended organ storage, extended use of 


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more marginal organs, and more effective use of Donation after Cardiac Death (DCD) organs 
which undergo combination warm and cold ischemia for organ procurement. 
 
5 Study Design and Methods 
5.1 Overview  
This is a Phase IIA observational nonrandomized pilot investigation to evaluate the safety and 
efficacy of Sodium Nitrite administration for the reduction of PGD in patients undergoing lung 
transplant. The study will enroll 8 subjects, undergoing lung transplant at the University of 
Pittsburgh Medical Center (UPMC). 
 
5.2 Study Time Line 
We propose to enroll eight patients over a 6-month interval. 
 
5.3 Screening Procedures 
Patients with end stage lung disease who are deemed eligible for placement on the lung 
transplant waiting list will be eligible for participation and written informed consent will be 
obtained at the time of their listing for lung transplantation. The medical records of the lung 
transplant patients will be reviewed for eligibility criteria by the PI and other Co- Investigators 
who by virtue of their role as a clinician already have the knowledge of and access to the 
patients’ identifiable medical information. Subjects will be approached for consent after the 
decision has been reached to place the subject on the waiting list. After enrollment and prior to 
transplantation, blood will be drawn for baseline studies that include complete blood count 
(CBC), chemistry profile; and for storage to allow for subsequent exploratory PBMC gene 
expression profiling and proteomics which would  include evaluation of IL6, IL, TNF-a, iNOS 
and COX-2. 
 
 


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5.4 Study Drug Preparation 
The Sodium Nitrite will be obtained from a commercially available, FDA-approved preparation 
(Hope Pharmaceuticals, Sodium Nitrite Injection USP (30mg/mL) NDC Number 60267-079-02) 
and the UPMC Pharmacy will prepare the formulations for this clinical study. 
To minimize waste of study drug, the UPMC Pharmacy will prepare Sodium Nitrite as two stock 
solutions in syringes.  The first stock solution will be a 12 mL (0.16mg/mL) stock solution to be 
used in the Perfadex preservation solution and the pneumoplegia solutions.  The second stock 
solution will be 1.33 mL (30 mg/mL) stock solution to be used in the preparation of a 40 mg 
Sodium Nitrite infusion is normal saline.   The stock solutions in syringe will be prepared from 
Hope Pharmaceuticals Sodium Nitrite Injection, USP.    The stock solutions is syringe will be 
frozen for long term storage to be used as needed as enrollment progresses. 
 
A specifically labeled frozen syringe of prepared stock Sodium Nitrite solution (12 mL, 0.16 
mg/mL)  will be available for the addition to the 2.8 L Perfadex bag.  Once thawed, the addition 
of the 12 mL contents of the syringe will achieve a final concentration of 0.69 mg/L of solution 
(i.e., 10 uMolar or 1.93 mg for each 2.8 liter bag. Thawed syringes that are not used will be 
discarded.   
  An initial 1l bag of Perfadex administered to the donor organ will not include the addition of 
Sodium Nitrite.  
 
Organ Implantation Prior To Reperfusion 
A specifically labeled frozen syringe of prepared stock Sodium Nitrite solution (12 mL, 0.16 
mg/mL) will be available to the OR at the time of lung transplantation.   Once thawed, the 
Sodium Nitrite will be added to the pneumoplegia solution to achieve a final concentration of 
0.69 mg/L (10 uMolar),  Thawed syringes that are not used will be discarded. The pneumoplegia 
solution (see Appendix C) is prepared by the UPMC Pharmacy and the Sodium Nitrite will be 
added immediately prior to the infusion of the pneumoplegia solution into the donor lung. 
 
 
Organ Reperfusion to Recipient 
A specifically labeled frozen syringe of prepared stock Sodium Nitrite solution (1.33 mL, 30 
mg/,L) will used by the UPMC pharmacy to prepare a 40 mg Sodium Nitrite in a 250 ML infusion 


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bag of normal saline (i.e., 0.16 mg/mL)  The entire volume of this bag will be used to administer 
to the patient at the time of Organ Perfusion. 
 
5.5 Study Drug Intervention 
Sodium Nitrite will be administered to the donated lung at the time of procurement and to the 
lung transplant recipient at the time of reperfusion.  
 
Organ Procurement from the Donor  
Consistent with current practice, the initial liter of antegrade flush of the donor lung with 
Perfadex includes the addition of prostaglandin and nitroglycerin to a 1L bag of the preservation 
solution.  This initial liter of solution WILL NOT include the addition of Sodium Nitrite.  
Subsequent to this initial liter, any additional preservation solution (Perfadex) will be provided in 
2.8 Liter bags only and will include the addition of Sodium Nitrite for injection to achieve a final 
concentration of 0.69 mg/mL (10 uMolar).  Other additives to the preservation solution will 
include THAM and calcium chloride consistent with local practice.  The Sodium Nitrite will be 
added  prior to the infusion of the preservation solution.  The actual volume of solution used for 
both antegrade (PA to PV) and retrograde (PV to PA) flush will be determined by the surgical 
team directing the procurement and recorded for the purpose of this study.   A member of the 
study team will be present for monitoring of this procedure. 
 
Organ Implantation prior to Reperfusion 
At the time of transplant, the first donor lung will be flushed with a cold pneumoplegia solution 
after the bronchial (1st) anastomosis. The pneumoplegia solution used clinically at UPMC is a 
50% Dextrose/autologous blood preparation, which includes Nitroglycerine, Verapamil and 
Heparin (refer to Appendix C for description of UPMC pneumoplegia solution); and the Sodium 
Nitrite will be added just prior to use.   A second flush of pneumoplegia solution will occur after 
the PV (3rd, last) anastomosis.  The identical pneumoplegia solution as outlined above will be 
used for the second flush, but at a temperature range of 34- 38 oC. The same process of 
flushing with pneumoplegia solution will be followed for the second donor lung.  
 
 
To the recipient at the time of reperfusion: 


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Sodium Nitrite will be delivered intravenously to the recipient immediately prior to the first lung 
reperfusion as a single infusion of  Infuse at 4 mL/min for the first 30 min, followed by 2.2 
mL/min for the next 60 min.  
  
5.6 Dosing Adjustments and/or Discontinuation due to Adverse Events 
The Sodium Nitrite infusion will only be initiated if the patient’s mean arterial pressure (MAP) > 
50 mm Hg.  The patient’s blood pressure may be supported by vasoactive medications and/or 
volume resuscitation at the time of study drug infusion as directed by the patient’s clinical 
involved anesthesiologist. 
 
If the patient meets criteria for initiation of the study medication, the infusion will be started at 
the prescribed rate and continue as outlined above.  The pre-infusion MAP will be established 
by the treating anesthesiologist.  The study medication will continue unless the patient’s MAP 
falls > 20% compared to the recorded baseline at the start of the study drug infusion.  A fall in 
MAP > 20% will result in the study drug being stopped for 10 minutes and the patient’s MAP   
will be continuously monitored.   
 
If the MAP returns to within 10% of the MAP baseline determined prior to study drug infusion, 
the infusion may be restarted after a 10 minute interval.  This transient fall in MAP  with rapid 
reversal will be attributed to the study drug.  If the MAP  falls again to > 20% from the MAP 
baseline, the cycle may be repeated for a maximum of two intervals.  The patient’s MAP must 
return to within 10% of the baseline before any restart of the study medication.  
If the MAP fails to return to within 10% of the MAP baseline determined prior to study drug 
infusion, the infusion will be suspended pending hemodynamic resuscitation as directed by the 
staff anesthesiologist.   A more prolonged period of hypotension suggests the effects of organ 
reperfusion rather than study medication but the infusion will be held to allow stabilization of the 
patient.  The study drug infusion will be restarted once the patient’s MAP returns to within 10% 
of the baseline value.   
The study medication may be infused up to 1 hour following the organ reperfusion if 
hemodynamic stability is delayed. 
 


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5.7 Study Procedures  
5.7.1 Randomization and blinding  
This study will be a non-randomized pilot observational study. 
5.7.2 Study monitoring  
1) Required end points during study drug infusion 
 
Mean Arterial Pressure:  
The recipient’s mean arterial pressure (MAP) will be monitored at 1 minute intervals 
during study drug infusion followed by 15 minute intervals for 1 hour.  
 
Nitrite and metabolite levels:  
Blood samples will be obtained prior to the infusion; at 5, 30, and 60 minutes during the 
infusion; and at 5, 10, 30, 60, 90, 150 after the infusion to measure plasma and whole 
blood nitrite, plasma nitrate, red cell iron-nitrosyl-hemoglobin, S-nitroso-hemoglobin and 
methemoglobin concentrations. However, no sample will be sent for methemoglobin 
concentration prior to drug infusion.  Blood samples will also be obtained every 24 hours 
for two days for future exploratory biomarker/proteomic studies  which would  include 
evaluation of IL6, IL, TNF-a, iNOS and COX-2. Nitrite levels will be measured by 
established methodologies (ozone-based reductive chemiluminescence) routinely 
performed in Dr. Gladwin’s NO Metabolomics Core facility. 
2) Required end points post study drug infusion to hospital discharge 
 
Following administration of the Sodium Nitrite, the study subjects will continue to receive 
usual care for their condition consistent with local transplant treatment protocols.  The 
study will not introduce any modification to the normal care algorithms.  Day 0 (D0) will 
be defined as the day of transplantation and reperfusion.  Subsequent study days will be 
numbered sequentially.  The following parameters will be required for collection 
prospectively.  They will be obtained specifically for the study protocol if not requested 
by the treating physician.  If requested by the treating physician the parameters will be 
recorded from the medical record.  Specific parameters required to be monitored 
following lung transplantation/reperfusion will include: 


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 
Arterial blood gas measurements from D0 to D3  
The Pa02/FiO2 ratio and oxygenation index will be calculated based upon an arterial 
blood gas at D0 (within 6 hours of reperfusion), and D1, D2, and D3. The initial 
measurement (D0) will be obtained on a FiO2 of 1.0 and PEEP of 5 while on mechanical 
ventilation.  Subsequent measurements will be obtained on the prescribed ventilator 
settings.  Ventilator parameters will be obtained with each blood gas determination. 
There will be no arterial blood gas measurement on days patient is extubated.  
 
Chest radiography from D0 to D3   
Plain film portable chest radiography will be obtained on D0 (within 6 hours of 
reperfusion), and D1, D2, and D3.   All chest radiography will be referred to a radiology 
core for independent review and classification independent of the review required for 
clinical documentation. 
 
The primary end point of PGD will be scored using the following criteria: 
 
 
 
 
 
 
 
 
 
 
 
 
The absence of pulmonary infiltrates on plain film radiograph will be assigned Grade 0 
independent of the PaO2/FiO2 ratio. All extubated patients will be graded as 0 or 1 based 
upon the interpretation of the chest radiograph.  The need for extracorporeal support 
and/or inhaled nitric oxide will be documented and automatically assigned a Grade III 
classification. 
 
Grade 
PaO2/FiO2 
Radiograph 
0 
>300 
Absent 
1 
>300 
Present 
2 
200-300 
Present 
3 
<200 
Present 


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The diagnosis of Primary Graft Dysfunction (PGD) requires the exclusion of alternative 
conditions, which might produce a similar radiographic and physiologic pattern including 
hyperacute rejection, cardiogenic pulmonary edema, venous anastomotic obstruction, 
and allograft infection. Exclusion of these conditions will be made by a PGD grading 
committee composed of transplant physicians not involved in the patient’s care at the 
time of transplant until hospital discharge.  Medical records will be reviewed within six to 
eight weeks of transplantation by the committee to classify PGD. 
 
Sequential Organ Failure Assessment Score (SOFA):  
The SOFA score is a recognized ICU scoring system to define the extent of the patient’s 
organ failure.  The scoring system has an individual component score for each of 6 
organ systems including respiratory, cardiovascular, hepatic, coagulation, renal and 
neurological systems 58. The SOFA Score will be obtained daily at D1, D2, D3, and D4.  
 
Ventilator Free Days (VFD): Defined as the number of days off mechanical ventilationat 
8am through D30 post transplantation. 
 
ICU Free Days (IFD): Defined as the number of days outside the ICU through  D30 post 
transplantation. 
 
Hospital Days: Defined as the total number of acute and long term acute (LTAC) hospital 
days during the index transplant admission. 
 
Outcome at 90 days: Patient clinical status at D90 post transplantation including living or 
deceased, and location. To confirm clinical status of patient on Day 90 post transplant as 
living or deceased and if living current location , a follow up call with made to the patient 
or the family n day 90 +/- 7 days.  
 
5.7.3 Optional end points to be obtained from admission to ICU 
discharge based on standard clinical monitoring 
(1)  Arterial blood gas measurements from D4 to ICU discharge 
The Pa02/FiO2 ratio and oxygenation index will be calculated and the worst value for each 
individual day recorded for analysis.  Corresponding ventilator parameters will be recorded. 


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(2) Plain film portable chest radiography from D4 to ICU discharge 
All chest radiography will be referred to a radiology core for independent review and 
classification independent of the review required for clinical documentation. 
(3) Requirement for extracorporeal support and days 
Any day beyond D0 to ICU discharge in which the patient receives extracorporeal 
support will be counted. 
(4) Requirement for inhaled NO support and days  
Any day beyond D0 to ICU discharge in which the patient receives inhaled NO 
support will be counted. 
 
Results of Echocardiography -specifically the left ventricular function and estimated 
pulmonary arterial pressure (PA systolic, PA diastolic, and mean) to evaluate cardiac 
and pulmonary arterial function.  
 
D0 to ICU discharge 
- ventilator settings 
-vital signs 
- 24 hour fluid balance 
- hemodynamic indices including MAP, PA pressures, central  venous pressures, cardiac     
index. 
3) Additional end points to one year post hospital discharge obtained only if needed for 
patient’s standard care 
 
Results of pulmonary function tests - specifically the FVC, FEV1, and FEV1/FVC ratio. 
 
Lung Biopsy Results – specifically the diagnosis of acute cellular rejection and grade, 
bronchiolitis obliterans (BO), and treatment. 
 
Complications 
– 
including 
major 
opportunistic 
infections 
and 
subsequent 
hospitalizations. 
 


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5.8 Safety Assessment  
The primary safety assessment will be based upon measured methemoglobin levels and 
hemodynamic indices during the Sodium Nitrite infusion.  Because of the exploratory nature of 
this trial, all potential Adverse Events will be recorded for review. 
 
5.9 Efficacy Assessment  
The primary efficacy assessment will be the incidence of PGD Grade 3 or greater in comparison 
to age and disease-matched historical controls for the UPMC Lung Transplantation program.  
 
6 Statistical Consideration 
Statistical analysis for the protocol will be primarily exploratory (i.e., hypothesis generating), and 
no firm conclusion will be drawn based upon a confirmation oriented statistical analysis. The 
results will be displayed as mean±standard deviation for continuous variables, or frequency 
(percentage), for discrete variables. For variables with skewed distributions, both nonparametric 
analysis, and parametric analysis using logarithmically transformed data will be performed. The 
continuous efficacy variables will be analyzed by analysis of variance (ANOVA) models, which 
will contain terms of treatment dose, disease type, and treatment-by-disease type interaction. 
The treatment-by-disease type interaction will be assessed at significance level 0.10. If the 
treatment-by-disease type interaction is not statistically significant, the interaction effect will be 
removed from the model. The treatment differences will be assessed by pair-wise comparison 
between the study population and historical controls, using least-square means from the 
ANOVA model. If baseline value of each continuous efficacy variable is to be adjusted, data will 
be analyzed by analysis of covariance (ANCOVA). 
 
7 Human subjects 
7.1 Subject Population 
7.1.1 Human subjects involvement and characteristics 
Subjects of both genders and all ethnic backgrounds will be considered for inclusion in this 
study.   Patients with end stage lung disease who are deemed eligible for placement on the lung 


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transplant list will be enrolled. All volunteer subjects must be at least 18 years of age and have 
provided informed, written consent for participation in this study.  Based on the population of 
patients referred for lung transplantation, we expect that approximately 50% of eligible subjects 
will be women and 10% will be non-Caucasian. Children under 18 years of age will not be 
considered for inclusion in this study because children are not eligible for lung transplant at the 
adult lung transplant programs involved in this protocol (children are referred to a pediatric lung 
transplant program).  If the results of this study demonstrate beneficial effects of Sodium Nitrite 
administration for lung transplant recipients, future studies will attempt to incorporate children 
through collaboration with pediatric lung transplant centers. 
7.1.2 Donor exclusion criteria 
 
Age > 55 years.  
 
Mechanical ventilation > 5 days prior to procurement 
 
Significant chest trauma or lung contusion 
 
Smoking history > 20 pack-year 
 
PaO2/FiO2 (O2 challenge) < 300 
 
Donor radiograph with 2 quadrant infiltrates 
 
Donor that are determined single lung donors prior to transplant will be excluded 
7.1.3 Recipient inclusion criteria 
 
Subjects undergoing lung transplantation.  
 
 Subjects in the age range of 18-70 years Ability to understand and provide consent.  
Proxy consent will not be accepted. 
7.1.4 Recipient exclusion criteria 
 
Recipient age > 70 years. 
 
Recipient history of pulmonary hypertension (idiopathic PAH, or secondary PAH with 
mean PA > 30 mm Hg) 
 
Recipient history of abnormal cardiac function defined as  prior CABG or LVEF < 45 %)  
 
 Recipient history of open thoracotomy/prior pleurodesis as exclusion criteria.  However 
patients who have had limited VATS procedures for biopsies would NOT be excluded 
from the study.Recipient history of  cirrhosis 
 
Recipient history of mechanical ventilation or extracorporeal support pre-operatively 


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 
Recipient pre-operative hypotension defined by a systolic blood pressure less than 90 
mm Hg not responsive to intravenous fluids or requirement for vasoactive medications 
 
Recipient preoperative history of renal insufficiency, dialysis or estimated glomerular 
filtration rate <30 ml/min/1.73 m2 BSA 
 
Patients undergoing retransplantation 
 
Recipient history of significant coronary artery disease that is flow limiting and unable to 
be corrected by further percutaneous coronary artery interventions. 
 
7.2 Recruitment and Informed Consent Procedures   
Only patients with end stage lung disease deemed eligible for placement on the lung transplant 
list will be enrolled. All volunteer subjects must be at least 18 years of age and have provided 
informed, written consent for participation in this study at the time of listing for lung 
transplantation.  The medical and surgical team will review the participation of the patient in this 
study at the time a decision is made for listing.  Patients deemed eligible for participation will be 
approached by one of the study physicians for informed consent. 
 
8 Potential Risks and Benefits 
8.1 Risk of Experimental Intervention 
Sodium Nitrite:  Sodium nitrite has been used commercially as a food preservative, an anti-
corrosive agent, a coloring agent, and an anti-anginal agent, with additional uses in laxatives, 
burn ointments, and liniments.  Amyl nitrite has been inhaled or ingested as an euphoric 
stimulant.  Nitrite has also been found as a contaminant in well water.   Literature searches 
generated case reports of nausea, vomiting, abdominal pain, dizziness, headache, flushing, 
cyanosis, tachypnea, dyspnea, hypotension and death attributed to excess nitrite (high-dose) 
exposure from these sources as a consequence of methemoglobinemia due to oxidation of 
heme-iron in oxyhemoglobin.  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.  Note that methemoglobin levels have 
never risen higher than 3% at the currently used doses (30 mg) in 80 volunteers in phase I 
studies at the NIH. 


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Nitrite in the form of sodium nitrite for parenteral administration is currently available and 
approved by the FDA for use in the emergency treatment of cyanide poisoning.  Sodium Nitrite 
administration for this clinical indication at the labeled dosage of 300 mg  causes 
methemoglobinemia, a desirable effect in the treatment of cyanide poisoning, as methemoglobin 
binds to cyanide, thus protecting cellular mitochondria.  Sodium nitrite (4mg/kg, approximately 
300 mg) was infused intravenously over 10 minutes in eight healthy volunteer subjects. 
Methemoglobin levels in venous blood samples rose from 0.02% at baseline to 5.7% of total 
hemoglobin following infusion of sodium nitrite, with no subject experiencing adverse events. 
The sponsor of this IND application, Dr. Gladwin, has previously held an IND for sodium nitrite 
(IND # 70,411) for cardiovascular applications.  The cardiovascular IND involved the 
administration of Sodium Nitrite to 69 normal volunteers in 4 phase I-II clinical trials without 
observed adverse effects.  We have also treated 11 subjects with sickle cell disease on this IND 
without observed adverse effects.  The low doses of nitrite used in these investigational 
treatment regimens –  approximately 40 mg or  13% of the dose (300mg) 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.  There have been no 
adverse events noted in the 80 treated normal human volunteers and patients with sickle cell 
disease.   While the safety of nitrite in lung transplant recipients has not been established, the 
short plasma half-life (T1/2 = 42 mins 49) and dose chosen for this study minimize the risk of 
subjects experiencing the known effects of nitrite, specifically methemoglobinemia and 
hypotension.  
 
9 Protection against Patient Risk 
9.1 Protection against General Patient Risk 
All research interventions/activities will be conducted in private room/areas. The collection of 
sensitive information about subjects is limited to the amount necessary to achieve the aims of 
the research. 
 
All demographic and clinical information including the required and optional end points about the 
subject will be collected on hard copy case report forms used for data entry into a locked 
database.  The hard copy casebooks will be maintained in the data center under the supervision 


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of Drs. Gladwin and Donahoe. The data will be stripped of individual identifiers and 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. Research Data are maintained on a password-protected 
computer that is accessible only to the data manager and his staff. All staff will sign 
confidentiality statements. Access to the database will be limited to the data manager and staff 
under the supervision of Drs. Gladwin and Donahoe.  Blood specimens will be stripped of 
subject identifiers and stored according to a similar coding protocol as described above. These 
specimens will be stored safely in the custody of the sub-investigators responsible for the 
individual assays. These sub-investigators will limit future access to any remaining samples to 
only those investigators with prior IRB approval for their studies. For the PK assays these will be 
stored by Dr. Gladwin’s laboratory.   
 
All staff involved in this study are properly credentialed and instructed in the areas of testing, 
confidentiality, and safety.  
 
The Investigator will retain the data for up to 2 years following termination of this IND 
application. The investigator may continue to use and disclose subjects’ identifiable information 
for the purpose of this study for a minimum of seven  years after final reporting or publication of 
the study.  If the subject and/or legal representative decide to withdraw or be withdrawn from 
study participation, they may request that the study data and samples be destroyed. 
 
9.2 Protection against Patient Risks Related to the Study Drug 
The following components have been incorporated to protect the study subjects from risk 
related to the study drug: 
 
Small sample size consistent with the exploratory nature of the trial. 
 
Specific exclusion criteria to provide a stable population of patients listed for single or 
double lung transplant, and non-enrollment of patients listed for retransplantation with 
significant co-morbidities that might place them at excess risk (see exclusion criteria 
above). 
 
Continuous monitoring by an independent data and safety monitoring board. 


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 
Involvement by expert staff with a broad range of expertise in the administration of 
Sodium Nitrite, and the pre and postoperative care of patients undergoing lung 
transplantation. 
 
Frequent monitoring for adverse effects of the study drug will be conducted during and 
following its administration. 
10 Data and Safety Monitoring Plan 
10.1 Data and Safety Monitoring Board 
An Independent Data and Safety Monitoring Board (IDSMB) will be established; to be 
comprised of individuals who are not involved with this study protocol.  The DSMB will oversee 
patient safety in this clinical trial. The DSMB will be comprised of members including senior 
experts in pulmonary medicine, clinical research, clinical trial design, biostatistics, psychosocial 
issues relating to chronic disease, and research ethics. The Data and Safety Monitoring Board 
will conduct interim mentoring 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. 
 
10.2 Data and Safety Monitoring Plan  
Assuring patient safety is an essential component of this protocol.  The study investigators have 
primary responsibility for subject safety monitoring.  The investigators will evaluate all adverse 
events.  The study coordinators must view patient records for possible adverse events until 72 
hours after the last dose of study drug. All untoward medical occurrences observed in subjects 
receiving the study drug will be recorded on the participants’ adverse event case report forms 
(CRF) by the study coordinator under the supervision of the clinical investigator. The CRF will 
then be reviewed for completeness and internal consistency. Subsequently, the CRFs will be 
recorded on an electronic password-guarded study database.  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.  
 


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The Clinical Investigator will work with the reporting sub-investigators to prepare a detailed 
written summary of serious, unexpected, and study drug-related adverse events, and will 
compare, and contrast the event with prior events. The detailed written summary will be 
provided to the IDSMB and also to the sponsor of the IND, Dr Gladwin. Adverse events will be 
reported. Adverse events will be reported to IRB as per guidelines. 
 
10.3 Parameters to be Monitored 
The Clinical Investigator will evaluate any changes in laboratory values and physical signs, and 
will determine if the change is clinically important, and different from what is expected in the 
clinical course of patients following a lung transplant.  Expected events for lung transplant 
patients are untoward clinical occurrences that are perceived by the clinical investigator to occur 
with reasonable frequency in the day to day care of patients with a lung transplant, treated in the 
hospital setting. Examples of adverse events that are expected in the course of lung transplant 
care include infection, rejection, and organ failures.  Such events will not be considered 
reportable adverse events unless the event is considered by the clinical investigator to be 
associated with the study drug or experimental procedures, or is unexpectedly severe or 
infrequent for a patient undergoing lung transplantation.  
 
 An event will be considered to be study drug- or experimental procedure-related if the event 
follows a reasonable, temporal sequence from the study drug/procedure, and could readily have 
been produced by the study drug/procedure.  An event will be considered to be unexpected for 
the study drug if it is not identified as a potential adverse event in this clinical protocol, or is 
unexpectedly severe, or occurs more frequent than events described herein.   The most 
common adverse events expected for this intervention would be hypotension and 
methemoglobinemia.  These events will be reported to the Sponsor of the IND, Dr Gladwin 
within 5 days of the event.  
 
10.4 Frequency of Monitoring 
The Clinical Investigator, sub-investigators, and the research staff will meet will meet 
every 4 months to re-evaluate study goals, subject recruitment, data coding and 
retention, documentation and identification of adverse events, complaints and 
confidentiality of subjects. However, the meeting may be held more frequently, if 


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necessary, for example depending on the subject enrollment. 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 Clinical 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 whether the study should 
continue as originally designed or should it be re-evaluated and changed. Summary of 
all adverse events and subject safety data will be prepared by the Clinical Investigator 
and submitted to the sponsor of the IND, Dr Gladwin. Dr Gladwin will review subject 
safety data as it generated to submit to the FDA, if needed. 
 
The IDSMB will meet at least yearly and more often as necessary with trial. The IDSMB will also 
meet prior to initiation of the study. 
The DSMP and IDSMB reports will be submitted to the IRB at the time of renewal. 
 
10.5 Recording/Reporting Requirements  
 
10.5.1 
Eliciting adverse event information 
Clinical study subjects will be routinely questioned about adverse events at study visits. 
10.5.2 
Recording requirements 
All observed or volunteered adverse drug events (serious or non-serious) and abnormal test 
findings, regardless of 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/and study treatment will be followed until the event (or its sequel), or the abnormal test finding 
resolves or stabilizes at a level acceptable to the investigator-sponsor. 


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1) 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 sponsor of the IND 
application. 
 
 
2) Causality and severity assessment 
The clinical investigator 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 clinical investigator’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. It will be promptly reported to the sponsor within 24 hours of notification of 
the event.  If the investigator’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.  
 
10.6 Reporting of Adverse Events   
10.6.1  Reporting of Adverse event to the Sponsor  
All adverse events that are unexpected, fatal or life threatening and associated with the use of 
the study drug will be reported by the Clinical investigator to the sponsor of the IND application 


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within 24 hours of notification of event either by telephone or secure email. Investigator will 
provide the sponsor of the IND with the detailed report upon complete investigation of the 
adverse event within 5 days of being notified of the event. 
 
Any adverse event unexpected, serious but not life threatening and associated with the use of 
the drug will also be reported to the sponsor within 5-7 days of notification of the event.   
 
10.6.2 Reporting of adverse events to the FDA 
2) (1) Written IND Safety Reports 
The 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 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 the clinical investigator and 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 sponsor of this IND 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’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. 
 


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3) Telephoned IND safety reports 
In addition to the subsequent submission of a written IND Safety Report (i.e., completed FDA 
Form 3500A), the sponsor of the IND  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 sponsor’s initial receipt of the 
respective human adverse event information. 
 
10.7 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-
sponsor’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 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-sponsor will report the adverse event 
to the IRB as soon as possible, but in no event later than 10 calendar days, after the 
determination was made. 
 


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11  Costs and Payments 
 At this point, the patients will not be compensated for participation in this study however 
neither the patients nor their insurance provider be charged for the study drug, Sodium 
Nitrite and any procedures done specifically for the research purposes.  
Any procedures done as a part of their routine clinical care will be charged to either the patients 
or their insurance provider. 
 
12  Potential Benefits of the Proposed Research to the Subjects and     
Others 
There are potential direct benefits for the volunteer subjects participating in this study, as the 
pre-clinical data suggests that Sodium Nitrite administration at time of lung transplantation 
reduces the severity of Primary Graft Dysfunction (PGD).    Studies in animal models of lung 
transplantation also suggest improved oxygenation, reduced inflammation and reduced chronic 
rejection.  However, at the present time, it is not known whether these same benefits will also be 
observed in humans. 
 
13 Importance of the Knowledge to be Gained 
This study may help improve the outcomes of patients with end stage lung disease who 
undergo lung transplantation. In addition, the study may advance our understanding of the 
mechanisms involved in primary graft dysfunction and the effects of nitrite on lung function.  The 
individuals and society stand to benefit from findings if nitrite is found to have beneficial effects. 
14 Qualifications of the Investigators 
Principal Investigator 
Matt Morrell, MD: Dr. Morrell is Assistant Professor in the Division of Pulmonary, Allergy, and 
Critical Care Medicine.  He has extensive clinical experience in the postoperative care of 
patients following lung transplantation.   
 
Co-Investigators: 


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Norihisa Shigemura, MD: Dr. Shigemura is Assistant Professor in the Department of Surgery.  
He has extensive clinical experience in the pre, post, and intraoperative care of the lung 
transplant recipient.   
 
Jonathan D'Cunha, MD, PhD: Dr D’Cunha is the Associate Professor of Cardiothoracic 
Surgery and Associate Director, Lung Transplantation. He has extensive clinical experience in 
the pre, post and intraoperative care of Lung transplant recipient. 
 
Mark T. Gladwin MD: Dr Gladwin is the Chief of Pulmonary, Allergy, and Critical Care Medicine 
and Director of the Vascular Medicine Institute at the University of Pittsburgh.  He is an 
internationally recognized authority in the field of sodium nitrite including both the basic science 
and a broad range of clinical applications in cardiovascular disease.  He is a current IND holder 
for the investigation of sodium nitrite in cardiovascular disease.   
 
Joseph Pilewski, MD: Dr. Pilewski is the Medical Chief of the Lung Transplantation Program at 
UPMC.  He has extensive experience in the areas of management of patients both pre and post 
lung transplantation.  He has also been an international leader in the development and 
implementation of complex clinical trials in cystic fibrosis. 
 
Maria Crespo, MD: Dr. Crespo is Assistant Professors in the Division of Pulmonary, Allergy, 
and Critical Care Medicine and is involved in the daily care of the lung transplant patient 
population. Dr. Crespo has been actively involved in clinical research trials. 
 
John McDyer, MD: Dr. McDyer is Associate Professor of Medicine, Division of Pulmonary, 
Allergy, and Critical Care Medicine (PACCM), Department of Medicine, University of Pittsburgh 
School of Medicine. Dr. McDyer also serves as the Director, Lung Transplantation Translational 
Research Program. Dr. McDyer is a transplant pulmonologist and immunologist, and has 
extensive experiences in human and mouse studies related to mechanisms of rejection and 
tolerance in lung transplant, and antiviral host defense. 
 
Patricia George, MD: Dr. George is Assistant Professors in the Division of Pulmonary, Allergy, 
and Critical Care Medicine Dr. George’s clinical and research interests centered on lung 
transplantation and lung disease in immunocompromised hosts. 
 


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Matthew Pipeling, MD: Dr. Pipeling is Assistant Professors in the Division of Pulmonary, 
Allergy, and Critical Care Medicine and is involved in the daily care of the lung transplant patient 
population. 
 
Bruce Johnson, MD: Dr. Johnson is Assistant Professors in the Division of Pulmonary, Allergy, 
and Critical Care Medicine. Dr. Johnson’s practice focuses on the pre and post-transplant care 
of lung transplant recipients, and he is the senior clinician in lung transplantation at UPMC.   
 
Elizabeth Lendermon MD and Silpa Kilaru MD are both Assistant Professors in the Division of 
Pulmonary, Allergy, and Critical Medicine and are involved in the daily care of the lung 
transplant patient population. 
 
Michael Donahoe, MD: Dr. Donahoe is Associate Professor in the Division of Pulmonary, 
Allergy, and Critical Care Medicine.  Dr. Donahoe also serve as the Director of Medical 
Intensive Care Unit (MICU), and has extensive experience in the design and execution of 
numerous NIH-sponsored complex clinical trials, and co-investigator for studies in nutrition 
support in COPD, aging in critical care medicine, and the care of critically ill patients. In addition, 
he has been an active participant in industry sponsored clinical research trials in nutrition 
support in COPD, ARDS, ICU infections and sepsis investigational drug therapy.  
 
Dr. Yingze Zhang is an Associate Research Professor at the Division of Pulmonary, Allergy 
and Critical Care Medicine. She is the Director of the Translational Research Core Laboratory 
(TRCL) at the PACCM division and has been conducting biomedical research for more than 20 
years. The TRCL is dedicated to providing resources and technical support for clinical sample 
processing, sample banking, and high-throughput genetics and biomarker analysis associated 
with translational research of lung and vascular diseases. We have archived and are actively 
banking clinical research samples for multiple NIH-funded studies. The biological samples 
collected from this study will be banked in Translational Research Core Laboratory (TRCL) at 
the University of Pittsburgh under her oversight. 
 
Diana Zaldonis MPH, BSN is a Clinical Research Coordinator of the Department of 
Cardiothoracic Surgery. Ms. Zaldonis is a registered nurse with 29 years of experience in 
cardio-thoracic surgery with the past 22 years in cardio-pulmonary transplantation as a clinical 
and research coordinator. She possesses a master’s degree from the University Of Pittsburgh 


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Graduate School Of Public Health in Epidemiology. Ms. Zaldonis has extensive experience in 
managing and coordinating clinical trials. Her ojor role wolud be to  provide guidance on 
regulatory submssions. She would not be involved with  the consenting the patients.  
 
Jeremiah Hayanga MD is an Assistant Professor of Surgery in the Department of Cardiothoracic 
Surgery.  He is also the Director of Lung Transplant Outcomes Committee in the Throacic 
Transplant Division.  He is actively involved in clinical research and listed as Co-Investigator on 
several research projects within the Department of Cardiothoracic Surgery.   
Ernest Chan MD is a Faculty Research Instructor at the University of Pittsburgh.  He has 
previously worked on research projects in the Department of Cardiothoracic Surgery.   
 
Nicholas Hess and Simeng Wang are both Medical Students at the University of Pittsburgh and 
involved in Lung Transplant Research in the Department of Cardiothoracic Surgery. 
Collaborators 
 
Susan Stuart, RN, MPM: Susan Stuart is the CEO for the Center for Organ Recovery and 
Education (CORE).  She is experienced in the daily operations for organ procurement. 
 
 
15 References 
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Munoz del Rio A, Meyer K, Greenspan DS, et al. Th-17, monokines, collagen type v, and 
primary graft dysfunction in lung transplantation. Am J Respir Crit Care Med 
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2. Christie JD, Van Raemdonck D, de Perrot M, Barr M, Keshavjee S, Arcasoy S, Orens J. 
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31. Oberst A, Bender C, Green DR. Living with death: The evolution of the mitochondrial 
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SF, Wang X, Kevil CG, et al. Cytoprotective effects of nitrite during in vivo ischemia-
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44. Gonzalez FM, Shiva S, Vincent PS, Ringwood LA, Hsu LY, Hon YY, Aletras AH, Cannon 
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49. Dejam A, Hunter CJ, Tremonti C, Puta RM, Hon YY, Grimes G, Partovi K, Pelletier MM, 
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Circulation  2007; 116:1821-1831