Effect of Nitric Oxide in Cardiac Surgery Patients With Endothelial Dysfunction.

I. SUBJECT ENROLLMENT

1. Methods of enrollment, including procedures for patient registration. Patients will be
screened in the "Cardiac surgery pre-operative clinic", Massachusetts General Hospital
(MGH) Cox building floor 6 (before surgery). If the patient is eligible and after
obtaining the consent of the primary cardiac surgeon, the patient will be approached for
consent to participate in this clinical trial. Consent will be obtained exclusively from
the patient, and not from a surrogate, because cardiac surgery is usually a scheduled
procedure. The obtaining of the consent directly from the patient is preferred, since
there will be the opportunity of interaction with the patient when he/she is fully
awake. After consent is obtained, the patient will be randomized to the study group. A
de-identified code will be assigned to the patient and registered on a dedicated
enrollment log.

2. Procedures for obtaining informed consent (including timing of consent process).
Patients will be screened in the "Cardiac surgery pre-operative clinic", MGH Cox
building floor 6 (before surgery). If a patient is eligible for the study and after
obtaining the consent of the primary cardiac surgeon, the patient will be approached to
consent to participate in the clinical trial. There is no need for surrogate consent for
this trial, as explained above. Signing the Informed Consent Form will be requested for
participation in the study. Personal Medical Information (PMI) will be accessed by the
investigator(s) only for study purposes. Patients who choose not to participate in this
study will receive standard care according to the procedures of the ICU, without any
repercussion.

3. Treatment assignment and randomization (if applicable). To ensure a robust and unbiased
approach, randomization should account for demographic characteristics of the patients
(i.e.: age, sex) and glomerular filtration rate (GFR). Only patients who are cared for
by experienced anesthesiologists and cardiac surgeons (more than 5 years of staff
experience) will participate in this study. The patients will be randomized to receive
either NO (study drug) or N2 (placebo) alone. In order to account for an elevated
baseline pulmonary artery pressure, patients with pre-operative mean PAP ≥30 mmHg will
be allocated equally to the two groups during the stratified randomization process. The
random allocation sequence will be created using a computerized random generation
program. The randomization will be in blocks of 10 patients. The intervention will
consist of administering the test gas via the cardiopulmonary bypass (CPB) machine and
after CPB via the inspiratory limb of the anesthetic or ventilator circuit, and
thereafter via the mechanical ventilator in the Intensive Care Unit (ICU). When patients
are extubated they will breathe test gas via a facemask or nasal prongs. Test gas
administration will commence at the onset of CPB and last for 24 hours. At the end of 24
hours, inhaled NO (iNO) will be weaned and discontinued while carefully monitoring
hemodynamics for a period of 2-4 hours. Local guidelines for NO gas discontinuation will
be followed. Using commercially available tanks of Nitric Oxide (Airgas Inc, Radnor
Township, Pennsylvania) or volumetrically-calibrated flowmeters, pure N2 (placebo) or
850 ppm NO gas in N2 will be mixed with pure oxygen (O2) or air to obtain the desired
concentration of O2 and, in the NO treatment group, a final concentration of 80 ppm NO.
For safety, nitric oxide, nitrogen dioxide (NO2), O2 and methemoglobin (Met-Hb) levels
will be continuously monitored by the personnel unblinded to the study. Patients in the
placebo (N2) group will receive nitrogen test gas during the same 24 hour-period. The
inspired oxygen levels will be maintained at the usual levels required for routine
post-operative care. No changes to the usual and customary standards of care for any
intraoperative or postoperative treatment will be made during the study period.

4. Blinding procedure for Nitric Oxide and Nitrogen delivery. A double-blind study will be
performed to avoid potential patient and investigator bias, the study is blinded to:
participants, perfusionists, surgeons, anesthesiologists, ICU physicians and the nursing
staff. The test gas tank and the gas delivery system in the operating room (OR) and at
the bedside will be masked. The respiratory therapist in the ICU and a member of the
study staff will be unblinded and will prepare the appropriate test gas tanks and NO/N2
meters. Blood Met-Hb levels and NO/NO2 concentrations are safety concerns that the
respiratory therapist and one member of the study staff will monitor and regulate (NO2
levels will be maintained below 5 ppm, and Met-Hb below 5%, if necessary by reducing NO
concentration to 40 ppm or less according to MGH guidelines NO therapy).

II. STUDY PROCEDURES a. Study visits and parameters to be measured (e.g., laboratory tests,
x-rays, and other testing). SCREENING visit: Screening will take place in the "Cardiac
surgery pre-operative clinic", MGH Cox building floor 6 (before surgery). Patients will be
screened if they require prolonged CPB (>90 minutes on CPB, i.e., valve replacement ±
coronary artery bypass grafting (CABG)) and if the primary cardiac surgeon of the patient
agrees on enrolling the patient in the study.

Screening consists of:

- Review eligibility entry criteria of the study: inclusion/exclusion criteria will be
reviewed.

- Review the research protocol: This is a protected time for the subject to ask questions
and become familiar with all aspects of the study protocol.

- Administration of a brief questionnaire to determine endothelial function with the
subject verbal assent on the day of pre-surgical evaluation. The questionnaire evaluates
medical history, integrating clinical and laboratory data of screened patients. In males
> 40 year old and females > 50 year old, endothelial dysfunction has been shown to be
associated with at least one the following criteria: Previous coronary artery bypass
graft or PTCA (+ stent); History or presence of intermittent claudication; Critical limb
ischemia, or peripheral vascular disease with the Exception of vasculitis; History of
transient ischemic attack and/or ischemic stroke; Diagnosis of diabetes (IDDM or NIDDM)
requiring oral hypoglycemic agents or insulin; Hypercholesterolemia (total cholesterol >
200 mg/dl or LDL > 160 mg/dl) treated with statins, ion- exchange resins or other oral
agents; BMI > 40; Hypertension (SBP 140 ≥ mmHg) treated with antihypertensive drugs;
Active smoking ≥ 10 pack - years

- Consent form: The subject will be asked to sign the consent form.

Laboratory tests will be reviewed by the same physician, and if inclusion/exclusion criteria
are met, subjects will be enrolled.

RANDOMIZATION. Patients will be randomly allocated to one of the test gas study groups
(inhaled 80 parts per million (ppm) nitric oxide in nitrogen) or the placebo group (inhaled
N2). The intervention will consist of giving the test gas both via the CPB machine and after
CPB via the anesthetic circuit, and thereafter via the mechanical ventilator and/or with face
mask/nasal prongs in the ICU/ward. Test gas administration will commence at the onset of CPB
and last for 24 hours. At the end of 24 hours, iNO will be weaned and discontinued while
carefully monitoring hemodynamics for a period of 2-4 hours. Local guidelines for iNO
discontinuation will be adopted. Using an Inovent (Ikaria Inc, N.J., USA) or
volumetrically-calibrated flowmeters, pure nitrogen (placebo) or 850 ppm NO gas in N2 is
mixed with pure O2 or air to obtain a final concentration of 80 ppm NO. During CPB the test
gas is delivered through the extracorporeal oxygenator, after CPB the NO is delivered through
the inspiratory limb of the anesthetic or ventilator circuit. NO, NO2 and O2 and
methemoglobin levels are monitored by an unblinded observer. Patients in the placebo group
will receive nitrogen test gas during the same 24 hour-period. When patients are extubated
they will breathe test gas via a face mask or nasal prongs. The inspired oxygen levels will
be maintained at the usual levels required for routine post-operative care. The test gas tank
in the OR and at the bedside will be covered and blinded from the clinicians treating the
patient. Only the respiratory therapist in the ICU and a member of the study staff will be
unblinded and will prepare the appropriate test gas tanks and NO/N2 meters. No changes to the
usual and customary standards of care for any intraoperative or postoperative treatments will
be made during the study period.

III. MONITORING AND QUALITY ASSURANCE

1. Independent monitoring of source data. Informed consent forms, case report forms, and
data will be reviewed by the principal investigator following enrollment of every 5
subjects and an independent data and safety monitor following enrollment of every 50
subjects to the protocol to ensure safety of the study subjects. The safety data that
will be reviewed includes: patient-, nurse-, research-assistant, or
investigator-reported adverse events such as reason for early termination of the
protocol, or adverse reaction to NO. Other data to be reviewed includes appropriate
handling and processing of blood samples and maintenance of patient confidentiality
throughout the study.

2. Safety monitoring (e.g., Data Safety Monitoring Board, etc.). An interim analysis by a
Data Safety management Board (DSMB) comprised of 3 members is planned at reaching 50% of
the study population (125 patients enrolled). The study will only be stopped if the
interim analysis detects a significant increase of mortality, acute kidney injury (AKI),
need for renal replacement therapy (RRT), myocardial infarction (MI), post-operative
hemorrhage, other significant morbidity in the NO test gas group.

Members of the Data Safety Management Board (DSMB) consist of an anesthesiologist with
clinical expertise in NO, a cardiologist and a nephrologist.

3. Outcomes monitoring. Plasma creatinine will be measured daily until day 7 after CPB or
until discharge from the hospital if this occurs earlier. Urine output will be assessed
until day 7 after CPB or until discharge from the hospital if this occurs earlier.
Follow up will be completed at 1 year after surgery as described in "V. Study
procedure".

4. Adverse event reporting guidelines. The principal investigator will review any adverse
events immediately following their occurrence and report them to the Institutional
Review Board (IRB) in accordance with Partners Investigator guidelines. Specifically,
for this study, serious adverse events will be reported by phone, fax, or email within
24 hours to the Human Research Committee (HRC), followed by a full written report within
10 business/14 calendar days. Mild or moderate adverse events will be presented in
progress reports at continuing reviews.

Stopping rules. The review and decision regarding altering or stopping the protocol will be
performed by the principal investigator together with the DSMB. Mild or moderate adverse
events will be presented in progress reports at continuing reviews. Protocol exit criteria
will be:

1. Acute worsening hypotension: decrease in mean blood pressure of > 20 mmHg not
attributable to other causes, i.e., hypovolemia, hemorrhage, sepsis, acute heart
failure.

2. Sudden worsening of hypoxemia: decrease of peripheral oxygen saturation (SatO2) < 70% at
100% of inspiratory oxygen fraction (FiO2) not attributable to other causes, i.e.,
pulmonary edema, acute respiratory distress syndrome (ARDS), pulmonary embolism.

3. An increase in NO2 levels > 5 ppm from baseline or any life-threatening symptom
potentially attributed to NO administration by the physician investigator.

IV. BIOSTATISTICAL ANALYSIS

a. Specific data variables being collected for the study

Preoperative collection of patient data from the MGH electronic medical chart (EPIC) will
include:

- Patient demographics

- Past and current medical and surgery history and therapy

- Co-morbidities

- Pertinent cardiopulmonary test results: i.e., echocardiography, cardiac catheterization,
pulmonary function tests, and chest imaging).

Prospective collection of patient data will include:

- ICU and length of hospital stay

- Duration of mechanical ventilation

- Hospital course

- Intrahospital mortality and mortality at 6 weeks, 90 days and 1 year after cardiac
surgery.

- Activity of daily living at and postoperative health related quality of life (PROMIS
questionnaire for global health) at 6 weeks, 90 days and 1 year after surgery.

- Nonfatal stroke before surgery and at 6 weeks after surgery

- Perioperative and non-perioperative nonfatal myocardial infarction, as defined by the
third universal definition of MI released in 2012 by the European Society of Cardiology
(ESC)/American College of Cardiology Foundation (ACCF)/American Heart Association
(AHA)/World Heart Federation (WHF)

- Renal failure requiring dialysis at any time during hospitalization and at 6 weeks, 90
days and 1 year after cardiac surgery.

- Acute Kidney Injury as difened by KDIGO criteria.

- Organ dysfunction assessed daily in ICU by using Sequential organ failure assessment
(SOFA).

- Delirium assessed daily in the first 7 days after surgery by using the confusion
assessment method for intensive care unit (CAM-ICU).

- Cardiovascular support: vasopressors and inotropic agents requirement
(Vasoactive-inotropic score), need for balloon pump or ventricular-assist device.

- Post-operative infections, cardiac arrhytmias and non cardiac complications.

Samples to be collected:

1. Blood samples will be collected before surgery, at the end of surgery, each day until
day 7 after the cardiac procedure or until hospital discharge if before day 7, and at 6
weeks. The samples will be tested for: creatinine (after surgery, 24 hours after the end
of CPB, 48 hours after the end of CPB, POD3, POD4, POD5, POD6, POD7 and 6 weeks) and
biomarkers for kidney injury (immediately before CPB, 15 minutes after the end of CPB,
24 hours after the end of CPB, 48 hours after the end of CPB and at 6 weeks). Urine
samples will be collected for urinary biomarkers of kidney injury immediately before
CPB, 15 minutes after the end of CPB, 24 hours after the end of CPB, 48 hours after the
end of CPB and at 6 weeks.

2. Statistical methods. The primary aim of this study is to determine whether
supplementation with NO decreases the incidence of AKI in patients with signs and
symptoms suggesting endothelial dysfunction. A total of 250 cardiac-surgery patients
will be enrolled, requiring more than 90 minutes of CPB. The two arms will be compared
for homogeneity of baseline characteristics (age, weight, sex, and GFR), intraoperative
course (CPB time, amount of blood transfusions) and postoperative data (post-operative
transfusions, adverse events directly related to surgery procedures). To examine
differences between the two groups, we will use an unpaired Student's t-test for
continuous variables with a normal distribution, a Mann-Whitney U test for continuous
variables that are not normally distributed and a Fisher's exact test for dichotomous
variables. Continuous variables will be described as mean±standard deviation (SD) when
normally distributed, median with interquartiles (IQR) when not normally distributed, or
count (%) if a dichotomous variable. The change in the laboratory markers over time and
between the two groups will be tested with a repeated measures ANOVA. The incidence of
AKI and postoperative adverse events after prolonged CPB will be described in terms of
relative risk with significance level and confidence intervals for the two groups. Based
on the occurrence of missing data, the strategy to manage them will be decided. However,
our aim would be not to use any kind of imputation.

3. Power analysis. The sample size of this trial was calculated based on the primary
endpoint: the reduction of AKI incidence. Despite we do not have track incidence, AKI is
very common in our MGH patients after open-heart surgery, and it is reasonable to assume
that AKI incidence is similar to those recent reports from US and European studies at
major academic centers CPB (AKI incidence has been reported to be between 50 to 60%). In
our prior trial on NO in cardiac surgery, the sample size was calculated to find a 30%
reduction in the incidence of AKI (n=212 in that trial). We estimate a greater (35%)
reduction in the incidence of AKI because we anticipate that this population with signs
and symptoms of endothelial dysfunction will be more affected by the beneficial
therapeutic properties of NO. Thus, in the NO group the incidence of AKI is expected to
decrease from 55% to 35.75%. The sample size needed to detect a difference, assuming an
alpha error of 0.05 (two-sided test) and a study power of 0.8, is 114 patients per
group. In order to account for possible dropouts, we increased our sample size by 10%.
We will enroll 250 patients, 125 in the NO group and 125 patients in the control (N2)
group.

Inclusion Criteria:

1. Provide written informed consent

2. Age ≥ 18 years of age

3. Elective cardiac or aortic surgery with CPB>90 minutes

4. Stable pre-operative renal function without evidence of plasma creatinine level
increase of ≥ 0.3 mg/dL over the prior 3 months and without renal replacement therapy
(RRT).

5. Clinical evidence of endothelial dysfunction assessed by a specifically designed
questionnaire.

Exclusion Criteria:

1. eGFR less than 30 ml/min/1.73 m2

2. Emergent cardiac surgery.

3. Life expectancy < 1 year at the time of enrollment.

4. Hemodynamic instability as defined by a systolic blood pressure <90 mmHg.

5. Mean pulmonary artery pressure ≥ 40 mm Hg and PVR > 4 Wood Units.

6. Left ventricular ejection fraction < 30% by echocardiography obtained within three
months of enrollment

7. Administration of one or more Packed Red Blood Cells (RBCs) transfusion in the week
prior to enrollment.

8. X-ray contrast infusion less than 48 hours before surgery.

9. Evidence of intravascular or extravascular hemolysis from any other origin:

i. Intravascular: Intrinsic RBCs defects leading to hemolytic anemia (eg, enzyme
deficiencies, hemoglobinopathies, membrane defects). Extrinsic: liver disease,
hypersplenism, infections (eg, bartonella, babesia, malaria), treatment with oxidizing
exogenous agents (eg, dapsone, nitrites, aniline dyes), exposure to other hemolytic
agents (eg, lead, snake and spider bites), lymphocyte leukemia, autoimmune hemolytic
disorders.

ii. Extravascular: Infection (eg, clostridial sepsis, severe malaria), paroxysmal cold
hemoglobinuria, cold agglutinin disease, paroxysmal nocturnal hemoglobinuria, iv
infusion of Rho(D) immune globulin, iv infusion of hypotonic solutions.