The Protocol of Evaluation of Safety and Efficiency of Method of Exosome Inhalation in SARS-CoV-2 Associated Two-Sided Pneumonia
Phase: PHASE1
Conditions: Covid19, SARS-CoV-2 PNEUMONIA, COVID-19
Sponsor: State-Financed Health Facility "Samara Regional Medical Center Dinasty"

 
 
 
«Approved» 
Director «MC Dinasty» 
Tyumina O.V  
06 July 2020                  
 
IRB/IEC APPROVED 
№ 002 form 06 July 2020 
 
IND 06072020 
Protocol ID COVID-19 EXO 
Protocol Nr NCT04491240 
 
 
 
PROTOCOL AND STATISTICAL PLAN 
EVALUATION OF SAFETY AND EFFICIENCY OF METHOD OF 
EXOSOME INHALATION IN SARS-COV-2 ASSOCIATED PNEUMONIA  
(COVID-19EXO) 
(COVID-19EXO ver.1 from 06.07.2020)

 
 
Protocol and statistical plan COVID-19 EXO 
 
Pages 2 form 8 
History of changes 
Rev 1. 
• 
Primary document 
 
 

 
 
Protocol and statistical plan COVID-19 EXO 
 
Pages 3 form 8 
Pricipal investigator 
State-Financed Health Facility “Samara Regional Medical Center Dinasty”.  
 
Contact: 
Stanislav Volchkov, MD, PhD +78469564455   quality@cordbank.ru 
Contact: 
Pavel Ovchinnikov, MD +78469564455 
Principal Investigator: Olga Tiumina, PhD, MD 
Principal Investigator: Stanislav Volchkov, PhD, MD 
Principal Investigator: Elena Strebkova, PhD, MD 
Principal Investigator: Dmitriy Konstantinov, PhD, MD 
 
Sponsor/Collaborators 
Sponsor: State-Financed Health Facility “Samara Regional Medical Center 
Dinasty” 
Responsible Party: Sponsor 
 
Collaborators: Clinics of the Federal State Budgetary Educational Institution SSMU 
Samara Regional Clinical Hospital V.D. Seredavin 
 
 
AIM 
Evaluation of the safety and efficacy of the method of inhalation administration of exosomes in bilateral 
pneumonia caused by the new coronavirus infection SARS-CoV-2. 
 
GOALS 
1. 
Develop an algorithm for introducing exosomes in case of pneumonia caused by COVID-19. 
2. 
Formulate patient selection criteria for this technique. 
3. 
To analyze the safety and efficiency of exosomal administration in case of pneumonia caused by 
COVID-19. 
 
Study Description 
Brief Summary: Coronavirus is an acute viral disease with prevailing upper respiratory tract 
infections caused by the RNA-containing virus of the genus Betacoronavirus of 
the Coronaviridae family. Most patients with severe COVID-19 develop 
pneumonia in the first week of the disease. As the infection progresses, the 
infiltration increases, and the affected areas increases. Excessive and 
uncontrolled immune system response with rapidly developing fatal cytokine 
storm plays the main role in the pathogenesis of acute respiratory distress 
syndrome (ARDS) due to SARS‑CoV‑2 infection. 
According to available data, exosomes can regulate inflammation and 
regenerative processes due to the change in the concentration of anti-
inflammatory cytokines and switch the immune cell to regenerative secretome. 
Inhalation of exosomes may reduce inflammation and damage to the lung tissue 
and stimulate the regenerative processes. 
This protocol has been developed based on the literature, information about the 
ongoing tests NCT04276987 (A Pilot Clinical Study on Inhalation of 
Mesenchymal Stem Cells Exosomes Treating Severe Novel Coronavirus 
Pneumonia) and NCT04384445 (Organicell Flow for Patients With COVID-19), 
Patent No 271036826 of 2019. “A method for obtaining and concentrating 
microRNA-containing exosomal multi-potent mesenchymal-stromal cells for use 

 
 
Protocol and statistical plan COVID-19 EXO 
 
Pages 4 form 8 
in cosmetic and pharmaceutical products to stimulate regenerative processes 
and slow down aging. 
Detailed 
Description: 
COVID-19 is an infectious disease caused by the most recently discovered 
coronavirus. This new virus and disease were unknown before the outbreak 
began in Wuhan, China, in December 2019. COVID-19 is now a pandemic 
affecting many countries worldwide. Globally, as of 1:09 pm CEST, 27 July 2020, 
there have been 16 096 741 confirmed cases of COVID-19, including 646 384 
deaths, reported to WHO. 
The main and rapidly achievable target of SARS-CoV-2 is lung type II alveolar 
cells (AT2), which determines the development of diffuse alveolar damage. In the 
pathogenesis of ARDS due to COVID-19, the main role is played by an over-
response of the immune system with rapidly developing severe life-threatening 
cytokine release syndrome (cytokine storm). Cytokine release syndrome 
threatens the emergence and progression of ARDS. The key components of the 
pathogenesis of ARDS also include disruption of cell cytotoxicity mechanisms, 
excessive activation of cytotoxic lymphocytes and macrophages with a massive 
release of proinflammatory cytokines (FNO-α, IL-1, IL-2, IL-6, IL-8, IL-10), 
granulocytic colony-stimulating factor, monocytic chemoattractive protein 1), and 
inflammatory markers (CRP, serum ferritin), infiltration of internal organs and 
tissues by activated T-lymphocytes and macrophages, resulting in a 
hyperinflammatory reaction. Such severe lesions can lead to death or severe 
lung damage, including long rehabilitation after discharge. 
Experimental studies have demonstrated that mesenchymal stem cells (MSCs) 
may significantly reduce lung inflammation and pathological impairment resulting 
from different types of lung injury. Many researchers connect the anti-
inflammatory effect of MSC with their secretome which includes MSC derived 
exosomes. It is highly likely that MSC exosomes have the same therapeutic 
effect on inoculation pneumonia as MSCs themselves. Moreover, exosomes 
show a strong effect of regenerative stimulation on different wounds so the 
regenerative effect can be extended on patients with COVID-19 pneumonia. 
The purpose of this protocol is to explore the safety and efficiency of aerosol 
inhalation of the exosomes in the treatment of severe patients hospitalized with 
novel coronavirus pneumonia (NCP). 
 
Conditions 
Conditions: Covid19 
SARS-CoV-2 PNEUMONIA 
COVID-19 
 
Keywords: Covid-19 
SARS-CoV-2 
exosomes 
MSC 
 
Study Design 
Study Type: Interventional 
Primary 
Purpose: 
Other 
Study Phase: Phase 1/Phase 2 
Interventional 
Study Model: 
Parallel Assignment 
The trial has three groups, each with 10 subjects (n=30). All eligible study subjects 
will be randomized, double-blinded, to either the two treatment groups or placebo 

 
 
Protocol and statistical plan COVID-19 EXO 
 
Pages 5 form 8 
group. 
Number of 
Arms: 
3 
Masking: Double (Participant, Care Provider) 
Two main groups will be provided with exosomes in a specially provided solution, the 
third group (control) will receive the same solution without exosomes. Due to 
exosomes are nanoparticles and requires special methods and devices to be 
detected the hospital staff and patients have no way to check which group receives 
exosomes. 
Allocation: Randomized 
Enrollment: 30 [Actual] 
Arms and Interventions 
Arms 
Assigned Interventions 
Experimental: EXO-1 
Participants (n=10) in this group will 
receive standard therapy and 
exosomes of the first type. 
 
Procedure/Surgery: EXO 1 inhalation 
Twice a day during 10 days inhalation of 3 ml special 
solution contained 0.5-2x10^10 of nanoparticles 
(exosomes) of the first type. 
 
Experimental: EXO-2 
Participants (n=10) in this group will 
receive standard therapy and 
exosomes of the second type. 
 
Procedure/Surgery: EXO 2 inhalation 
Twice a day during 10 days inhalation of 3 ml special 
solution contained 0.5-2x10^10 of nanoparticles 
(exosomes) of the second type. 
 
Placebo Comparator: Placebo 
Participants (n=10) in this group will 
receive standard therapy and 
inhalation placebo solution. 
Procedure/Surgery: Placebo inhalation 
Twice a day during 10 days inhalation of 3 ml special 
solution free of nanoparticles (exosomes). 
 
 
Eligibility 
Minimum Age: 18 Years 
Maximum 
Age: 
65 Years 
Sex: All 
Gender 
Based: 
No 
Accepts 
Healthy 
Volunteers: 
No 
Criteria: Inclusion Criteria: 
•  Ability to understand the study objectives and risks and provide signed and dated 
informed consent; 
•  Confirmed COVID-19 infection (by PCR or antibody test); 
•  Pneumonia requiring hospitalization, and oxygen saturation of <94% indoors or a 
need for auxiliary oxygen. The confirmed volume of lung damage by CT: not less 
than 30% and not more than 80%; 
•  ability to proceed with inhalation by self; 
  
Exclusion Criteria: 
•  Severe respiratory failure at the time of screening due to COVID-19 pneumonia; 

 
 
Protocol and statistical plan COVID-19 EXO 
 
Pages 6 form 8 
•  Known to undergo medical resuscitation for 14 days before randomization; 
•  Any serious medical condition or deviation of the clinical laboratory parameter 
that, in the opinion of the researcher, prevents safe participation and completion of 
the study by the participant Confirmed uncontrolled active bacterial, fungal, viral or 
other infection (other than SARS-CoV-2). 
•  According to the researcher, the progression to death is inevitable and will occur 
within the next 24 hours, regardless of the therapy. 
•  The life expectancy of fewer than 28 days, taking into account a medical condition 
already existing that cannot be corrected, e.g. participants with the following 
conditions or suspicions: polyorganic insufficiency, poorly controlled neoplasms, 
terminal stage heart disease, cardiopulmonary cardiac arrest that required 
cardiopulmonary resuscitation, or electrical activity not accompanied by a pulse, or 
asystole within the last 30 days, terminal stage liver disease, terminal stage liver 
disease, or liver disease; 
•  Pregnancy or breastfeeding; 
•  Liver function failure (Class C for Child-Pugh), detected within 24 hours at 
screening (local laboratory); 
•  Absolute neutrophil count (ANC) <500 cells/µL at screening (local laboratory); 
•  Platelet count <50000 cells/µL at screening (based on laboratory data); 
•  Creatinine level ≥ 1.5 from the upper limit; 
•  Uncontrolled or untreated arrhythmia with clinical manifestations, myocardial 
infarction within the last 6 weeks or congestive heart failure (NYHA Degrees 3 or 4); 
•  Respiratory failure in the last 6 months or home use of oxygen in severe chronic 
respiratory disease (COPD); 
•  Quadriplegia; 
•  Primary immunodeficiency, tuberculosis, progressive multifocal 
leukoencephalopathy, aspergillosis or other invasive mold/fungal infection in 
anamnesis, or internal or bone marrow transplantation for 6 months before 
randomization; 
•  Known infection with hepatitis B or C viruses requiring therapy; 
  
 
Research methods and STATISTICAL CONSIDERATIONS 
Title  
Purpose 
Method 
Frequency 
Statistics 
Number of 
participants with 
minor and 
serious side 
effects 
Safety 
assessment 
of the 
procedure. 
Collection of 
information about 
unwanted reactions 
to inhalation, for 
example, 
1) temperature 
increase,  
2) allergic reaction 
in the form of 
swelling Quincke, 
rash, seizures, etc.; 
3) bronchospasm 
for inhalation 
After each 
procedure 
Numeric data, calculated 
as a percent from whole 
patients in the group. 
Biochemistry of 
blood 
Evaluation of 
procedure 
efficiency 
C-reactive protein, 
LDH 
At the 
beginning of 
inhalation (day 
1) and on next 
day of last 
inhalation (day 
11) 
The difference in each 
group between baseline 
and day 11 will be 
measured by T-test. The 
data will be presented as a 
Mean with full range (min-
max). The difference will 
be calculated as Mean 

 
 
Protocol and statistical plan COVID-19 EXO 
 
Pages 7 form 8 
difference with Standard 
Deviation (SD) 
Time to Clinical 
Recovery 
(TTCR) 
Evaluation of 
procedure 
efficiency 
Calculation of days 
in hospital 
Once at the 
end of trial 
The data will be presented 
as a Mean with Standard 
Deviation (SD) 
SpO2 
Concentration 
Changes 
Evaluation of 
procedure 
efficiency 
The concentration 
of SpO2 by Pulse 
oximetry device 
during procedures 
and compared to 
placebo.  
Before and 
after each 
inhalation 
(total 4 
measures per 
day). 10 days. 
The intraday data (4 
measures per day) will be 
calculated as a Median by 
day. The Median by day 
data (10 measures) will be 
processed by T-test 
intragroup and between 
groups. The data in tables 
will be presented as a 
Median (Me) with 25-75 
quartile range.  
 
The results will be statistically processed using parametric and non-parametric statistics methods using 
STATA version 9.0, Statistica for Windows version 6.0 and MS Office Excel 2007. Changes from the start 
of therapy and after will be compared using the T-test, comparing data between the main group and the 
control group using non-parametric method box-plot (data are presented in Me - median format with an 
indication of 25% (q1) and 75% (q3) quartile. 
 
STATISTICAL CONSIDERATIONS  
This study will enroll patients with PCR confirmed COVID according inclusion criteria.  
The trial has three groups, each with 10 subjects (n=30). All eligible study subjects will be randomized, 
double-blinded, to either the two treatment groups or placebo group. Two main groups will be provided with 
exosomes in a specially provided solution, the third group (control) will receive the same solution without 
exosomes. Due to exosomes are nanoparticles and requires special methods and devices to be detected 
the hospital staff and patients have no way to check which group receives exosomes. 
This is interventional, prospective, randomized double-blinded with a control group study. 
The primary outcomes will be assessed at discharge of the clinic and 1 month after.  Duration of study 
participation will be 1 months from the time of discharge from the clinic.  
It is estimated that up to 2-3 research participants will be enrolled each week and that approximately 4 
weeks of accrual will be necessary to enroll 10 participants.  
Study Duration  
Research participants will be followed for safety for 1 months after the discharge from the clinic.  
Demographics and Baseline Characteristics  
Demographics and baseline characteristics will be summarized for all research participants. Characteristics 
to be examined include age, sex. The results will be statistically processed using parametric and non-
parametric statistics methods using STATA version 9.0, Statistica for Windows version 6.0 and MS Office 
Excel 2007. Changes from the start of therapy and after will be compared using the T-test, comparing data 
between the main group and the control group using non-parametric method, box-plot.  
 
POSSIBLE APPLICATION AREA 
    Specialized hospitals. 
 
DATA TO BE OBTAINED DURING IMPLEMENTATION 
- correct selection of criteria for selecting patients for treatment of patients with exosomes; 
- developed algorithm for using the method of administration of exosomes; 

 
 
Protocol and statistical plan COVID-19 EXO 
 
Pages 8 form 8 
-information on safety and efficacy of exosomal administration in case of pneumonia caused by SARS-
CoV-2; 
- appropriateness and effectiveness of this treatment method. To evaluate its impact on the tactics and 
treatment results; 
EXPECTED RESULTS 
It is assumed that the inhalation method of injection of exosomes will speed up the rehabilitation of patients, 
reduce the volume of pulmonary tissue lesions and reduce the time of stay of patients in hospital conditions.