Prolonged Hypoxic Breathing in Healthy Volunteers: a Safety Study

The primary endpoint of this research study is to prove safety in healthy subjects breathing
humidified hypoxic inspiratory gas mixture for 5 days.

The secondary endpoint of our study is to describe the physiological and biochemical changes
during the 5-day hypoxic period and the 2 days after return to normoxia.

The specific aims are as follows:

AIM 1: To provide a safe, controlled setting for prolonged exposure of monitored healthy
adults to a normobaric, low-oxygen environment for five days.

AIM 2: To carry out a seven-days long pilot safety-study in twelve healthy young subjects by
inducing hypoxia with a humidified normobaric gas mixture containing oxygen (O2) content as
low as 11% to reach a peripheral oxygen saturation (SpO2) between 80%-85%.

Screening will consist of a physical examination, 12-lead electrocardiogram, cardiac
echocardiography, sickle cell screening test, and current (within 12 weeks of clearance exam
with no intervening medical treatment) routine blood and urine analyses.

Women using oral, subdermal or injectable contraceptives, and those using other means of
birth control may participate. A urine pregnancy test will be conducted as part of the
screening process for study participation no more than 7 days before starting the study and
it will be repeated on the day of enrollment. The test result will be read by a female staff
member who will keep the result confidential. If a woman declines to have a pregnancy test,
she will not be able to participate.

Recruitment will be done in three ways:

1. Broadcast MGH: Research Studies In Need of Volunteers

2. Enrollment in the Research Study Volunteer Program (RSVP) for Health of Massachusetts
General Hospital (MGH) and Brigham Women Hospital (BWH)

3. Advertising at the Partners clinical trials Potential study subjects will be able to
contact study investigators by phone or email to verify eligibility according to the
inclusion/exclusion criteria and to set up the first visit, at which point the study
will be explained in detail and they will be asked to provide informed consent. Subjects
may contact the study investigators at any time to withdraw from the study.
Confidentiality will be preserved to the fullest extent.

The study will be explained to each volunteer in detail. Signing of an Informed Consent Form
will be requested for participation in the study. Volunteers will sign the informed consent
at the beginning of the study. The Principal Investigator (PI) and/or Co-PIs of the study
will be available to answer any questions the volunteer may have. Consent can be withdrawn at
any time. The investigators will access personal Medical Information (PMI) for study
purposes. There is no randomization in this interventional physiological study. All patients
will receive the same procedures in the same order.

Biostatistical analysis:

A de-identified code will be assigned to the patient and registered on a dedicated enrollment
log. The baseline study volunteer data (vital signs and biochemical measurements) will be
collected from the MGH electronic medical chart (EPIC). Prospective collection of patient
data will include specific data sheet. Data will be expressed as means ± standard deviation
(SD) or median/interquartile range (IQR) as appropriate.

Sample size: this research-project is aimed to study 12 healthy subjects for the entire 7-day
study period. To account for possible dropouts and missing data, up to 18 healthy volunteers
will be enrolled.

Sample size calculation: This is an intra-hospital safety trial with the purpose of testing
methods to re-create a safe hypoxic environment for possible future studies in patients with
mitochondrial dysfunction. While 2 or 3 subjects would likely be sufficient to test our
methods to reliably delivery humidified nitrogen (N2) and (O2) in predetermined
concentrations, this method will be tested in 12 healthy subjects to reproduce our methods
and obtain feedback from the volunteers. To account for possible dropouts, up to 18 healthy
volunteers will be enrolled.

Risk and discomforts

1. Complications of surgical and non-surgical procedures, etc. Participants in the study
will undergo 3 procedures during the study: hypoxic gas administration, phlebotomy, and
echocardiography.

- Facemask.

1. Claustrophobia is one of the exclusion criteria. If a subject develops
claustrophobia, he/she may leave the trial at any point.

2. The most common side effects of facemask are pressure related, such as
redness, irritation over the mask's borders. To decrease possible skin
irritation and abrasions, subjects will be using a tent during the night and
high flow nasal cannula during the day for breakfast, lunch and dinner.
Additionally they will be able to choose whether to use high-flow nasal
cannula or the facemask during the day at any time as an alternative to the
facemask.

- Tent. Risks associated with use of tents include unintended hypoxia, hypercarbia,
and claustrophobia. These tents are made at the MGH shop and routinely used on the
burn unit to maintain elevated temperature (80-90 °F) and humidification in
critically ill patients. Our subjects will be continuously monitored for hypoxia
and clinical condition to limit risk of unintended hypoxia or hypercarbia.
Claustrophobia will be handled as described previously.

- Nasal cannula and non-invasive high flow humidified gas. All participants will wear
non-invasive high flow humidified gas for part of study period. Unlike masks, high
flow nasal cannula is well tolerated in hospitalized patients and warm humidified
air alleviates discomfort of the high flow. In order to avoid abrasions, we will
inspect subjects' nasal mucosa in the morning and evening.

- Intravenous (IV) punctures and phlebotomy. Risks related to phlebotomy include
bruising, hematoma formation, cellulitis, superficial thrombosis, bleeding and
phlebitis. An IV nurse or an anesthesiologist will perform phlebotomy to minimize
subject discomfort.

- Trans-thoracic echocardiography. Echocardiography will be performed on daily basis:
Echocardiographic assessment of pulmonary artery pressure and cardiac output is a
noninvasive and painless maneuver. It is commonly used in clinical practice, has
been used in the field under hypoxic conditions, and is safe for the study
subjects. If the echocardiographic study reveals any unexpected cardiac disease the
subjects will be informed and also their primary care physician (PCP) will be
informed. If the subject does not have a PCP, we will provide information about
choosing a PCP.

In presence of an abnormal physical exam and/or additional abnormal laboratory results
(including a positive pregnancy test), subjects will be informed.

2. Drug side effects and toxicities

A gas mixture enriched with nitrogen will be tested. It is expected that subjects may
suffer to a certain degree of acute mountain sickness. Symptoms may include: headache,
dizziness, poor sleep, poor appetite and fatigue that are related to hypoxia. These
symptoms are well described in mountain climbers and will not require interruption of
the study, unless the subject requests exiting the study. Furthermore, in this proposed
model of normobaric hypoxia, the risk of acute mountain sickness symptoms has been
reported to be lower than hypobaric hypoxia (simulated altitude). The following risks
might also occur:

- Risks Associated with Hypoxia: Severe hypoxemia (SpO2 ≤ 70%), especially with
decreased respiratory rate can ensue. Since the fraction of inspired of oxygen can
be quickly increased, there is no risk of developing prolonged hypoxia events. If
SpO2 ≤ 70% decreases for more than one continuous minute, nitrogen administration
will be stopped and the fraction of inspired of oxygen will be increased to 50%.
The study will be stopped. Symptoms of hypoxia (light headedness, dizziness)
typically resolve immediately upon return to a normal range of SpO2. If hypoxia
symptoms are present, subjects will exit the study only after all symptoms of
light-headedness or dizziness are resolved, and headache or nausea is absent.

- Acute mountain sickness can very rarely progress to high altitude pulmonary edema
(HAPE) or high altitude cerebral edema (HACE) and would be case reportable were
either to occur in normobaria at the limited hypoxic challenge we will be
employing. Though HAPE is very unlikely to occur, we will provide daily monitoring
by echocardiography, clinical exam (auscultation for rales and unusual dyspnea at
rest solicited), and pulse oximetry. If HAPE does occur, the study will stop and
the subject treated with oxygen until resolution of symptoms.

- HACE is also a very unlikely potential risk in this study. If a subject develops
any evidence of ataxia or altered mental status, they will be immediately withdrawn
from the study. FiO2 will be increased to 50%, dexamethasone 8 mg IV will be
administered, and head computed tomography (CT) will be performed to rule out other
much more common sources (e.g. spontaneous subarachnoid hemorrhage) of these
complaints. If no CT abnormalities are noted, a brain magnetic resonance imaging
(MRI) will be obtained to assess for cerebral edema and characteristic T2 weighted
signal change in the corpus callosum consistent with HAPE..

Any study subject who experiences side effects sufficient to prompt premature
termination of the study will not be allowed to continue in the protocol. He or she will
be monitored until vital signs normalize and transthoracic echocardiography (TTE) shows
normal right heart function.

3. Device complications/malfunctions Nitrogen generator: If the nitrogen generator is
malfunctioning, air will be enriched with nitrogen from nitrogen-tanks. The manufacturer
will be contacted (Higher Peak LLC, Winchester, Massachusetts (MA) 01890) in order to
deliver a new nitrogen generator.

4. Psychosocial (non-medical) risks Psychosocial risks include prolonged indoor exposure
and perceived isolation from a subject's daily routine, family and friends. Subjects
will have ready access to their personal communication devices (e.g. smart phone for
voice/ social media use) during times when they are not actively participating in
testing procedures. Subjects may be visited in person on the study floor. We do not
anticipate any other psychosocial risks to the study subjects from participation in this
protocol. Strict confidentiality will be maintained by the research team at all times,
including keeping all data in a secure, locked cabinet with limited access. All
specimens will be coded after they are obtained and the code key kept in a locked
cabinet. All electronic data will be stored in a Partners encrypted laptop. Samples
given to parties outside of MGH for analysis will be coded to maintain confidentiality.

Monitoring and Quality Insurance:

Due to the small size of the study, no independent monitoring is deemed necessary. The PI
himself will be responsible for the monitoring of the study.

The PI and co-Is will be responsible for the monitoring of the study. Dr. Lorenzo Berra is
the Principal Investigator and he is an Anesthesiologist and Intensivist at MGH and Assistant
Professor at Harvard Medical School.

Stopping rules

The principal investigator and co-investigators will perform the review and decision
regarding altering or stopping the protocol. Mild or moderate adverse events will be
presented in progress reports at continuing reviews. Protocol exit criteria will be:

- Desaturation: SpO2<70% for more than 1 minute not immediately responsive to oxygen
therapy.

- Chest pain with evidence of ischemia on electrocardiography (EKG) (e.g., T-wave
inversions, ST segment changes).

- Signs and symptoms of acute pulmonary edema:

- Rapidly progressive, severe shortness of breath at rest..

- Severe dyspnea, or a feeling of suffocating or drowning despite return to normoxia.

- Wheezing or gasping accompanied with anxiety, restlessness or a sense of
apprehension.

- A cough that produces frothy sputum that may be tinged with blood

- Signs and symptoms of acute cerebral edema:

- acute ataxia or altered mental status

- any focal neurologic deficit.

- Increase in systolic pulmonary artery pressure of 15 mmHg from baseline (first
trans-echocardiography in ambient air)

- Syncope.

- Febrile illness (> 100.4 on two contiguous assays 1 hour apart)

- Subject may voluntarily withdraw from the study at any time.

Inclusion criteria

- Have a photo identification (ID)

- Male or female individuals age between 18 and 40 years old

- BMI between 19 and 24.9 kg/m2

- Having capacity to consent to the study Exclusion criteria

- Evidence of any physical, mental, and/or medical conditions that would make the
proposed studies relatively more hazardous

- Prior high altitude pulmonary edema (HAPE) or high-altitude cerebral edema (HACE)
diagnosis

- Born at altitudes greater than 2,100 m (~7,000 ft)

- Systemic disease with or without any functional limitation; including

- controlled hypertension

- controlled diabetes without systemic effects

- Pregnancy determined by urine pregnancy test, detecting presence of human chorionic
gonadotropin (hCG), or less than six weeks postpartum

- Women who are not willing to receive urine pregnancy tests

- Active smoking. Volunteers may be enrolled if they quit smoking for more than 1 year.

- Excess alcohol use: more than ½ L/day of wine consumption or equivalent

- Any current medication use except oral contraceptives.

- Living in areas that are more than 1,200 m (~4,000 feet), or have traveled to areas
that are more than 1,200 m for more than four days within the last 2 months

- Tobacco chewers

- Abnormal hemoglobin or hematocrit levels or presence of hemoglobin S

- Evidence of apnea or other sleeping disorders

- Evidence of asthma

- Lower respiratory infection within the last 30 days

- If applicable, unwilling to refrain from using energy drinks or other caffeinated
beverages for 7 days prior to and during the study

- If applicable, unwilling to refrain from use of all over-the-counter oral medications,
herbal remedies, and nutritional supplements for 7 days prior to and during the study

- Not willing to have blood drawn from an arm vein each test day of the study

- Claustrophobia (inability to wear a facemask) or other active psychiatric conditions
or not willingness to cooperate with the investigators and the other medical team

- Currently enrolled in another research study

- Facial abnormalities that would preclude proper use of a face mask

Pregnancy Prevention/Testing: Women using oral, subdermal or injectable contraceptives, and
those using other means of birth control may participate. A urine pregnancy test will be
conducted as part of the screening process for study participation no more than 7 days
before starting the study. The test result will be read by a female staff member who will
keep the result confidential. If a woman declines to have a pregnancy test, she will not be
able to participate.