Bone Marrow Mesenchymal Stem Cell Derived Extracellular Vesicle Infusion for the Treatment of Respiratory Failure from COVID-19: A Randomized Placebo Controlled Dosing Clinical Trial

Abstract

Background:
Bone marrow mesenchymal stem cell (BM-MSC)-derived extracellular vesicles (EVs), ExoFloTM, convey the immunomodulatory and regenerative properties of intact BM-MSC. This study aimed to determine the safety and efficacy of ExoFlo as treatment for moderate-to-severe Acute Respiratory Distress Syndrome (ARDS) in patients with severe COVID-19.

Research question:
Does two doses of ExoFlo safely reduce mortality in COVID-19 associated moderate to severe ARDS as compared to placebo?

Study design and methods:
A prospective phase 2 multicenter, double-blind, randomized, placebo-controlled dosing trial was conducted at five sites across the US with infusions of placebo, 10 mL of ExoFlo, or 15 mL of ExoFlo on Day 1 and 4. Patients (102) with COVID-19 associated moderate-to-severe ARDS were enrolled and randomized. Adverse events were documented throughout. The primary outcome measure was all-cause 60-day mortality rate. Secondary outcomes included time to death (overall mortality), the incidence of treatment emergent serious adverse events, proportion of discharged patients at 7, 30, and 60 days, time to hospital discharge, and ventilation free days.

Results:
No treatment-related adverse events were reported. Mortality (60-day) in the Intention-to-Treat (ITT) population was reduced in ExoFlo-15 compared to Placebo (not significant, Chi-square p=0.1343). For the post-hoc subgroup analyses, 60-day mortality was decreased in ExoFlo-15 compared to Placebo (Relative Risk=0.385; 95% confidence interval [CI]=0.159,0.931; p=0.0340; N=50). In ExoFlo-15 a Relative Risk of 0.423 (CI=0.173,1.032; p=0.0588; N=24) was determined for participants aged 18-65 with moderate to severe ARDS. Ventilation-free days (VFDs) improved in ExoFlo-15 (p=0.0455; N=50) for all participants aged 18-65.

Interpretation:
ExoFlo (15 mL dose) is safe in patients with severe or critical COVID-19 respiratory failure. In participants aged 18 to 65, the risk reduction in 60-day mortality was further improved from all aged subjects in the ITT population after two doses of 15 mL of ExoFlo as compared to placebo.

Full text

Journal Pre-proof
Bone Marrow Mesenchymal Stem Cell Derived Extracellular Vesicle Infusion for the
Treatment of Respiratory Failure from COVID-19: A Randomized Placebo Controlled
Dosing Clinical Trial
Amy L. Lightner, MD, Vikram Sengupta, MD, Sascha Qian, MD, John T. Ransom,
PhD, Sam Suzuki, MS MBA, David J. Park, MD, Timothy I. Melson, MD, Brian P.
Williams, MD, James J. Walsh, MD, Mustafa Awili, MD
PII: S0012-3692(23)00926-1
DOI: https://doi.org/10.1016/j.chest.2023.06.024
Reference: CHEST 5725
To appear in: CHEST
Received Date: 22 February 2023
Revised Date: 5 June 2023
Accepted Date: 15 June 2023
Please cite this article as: Lightner AL, Sengupta V, Qian S, Ransom JT, Suzuki S, Park DJ, Melson TI,
Williams BP, Walsh JJ, Awili M, Bone Marrow Mesenchymal Stem Cell Derived Extracellular Vesicle
Infusion for the Treatment of Respiratory Failure from COVID-19: A Randomized Placebo Controlled
Dosing Clinical Trial, CHEST (2023), doi: https://doi.org/10.1016/j.chest.2023.06.024.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
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during the production process, errors may be discovered which could affect the content, and all legal
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Copyright © 2023 Published by Elsevier Inc under license from the American College of Chest
Physicians.

1
Bone Marrow Mesenchymal Stem Cell Derived Extracellular Vesicle Infusion for the Treatment of Respiratory Failure
from COVID-19: A Randomized Placebo Controlled Dosing Clinical Trial
Amy L. Lightner MD1, Vikram Sengupta MD1, Sascha Qian MD1, John T. Ransom PhD1, Sam Suzuki MS MBA1, David J. Park
MD2, Timothy I. Melson MD3, Brian P. Williams MD4, James J. Walsh MD5, Mustafa Awili MD6
1Direct Biologics, LLC, 5301 Southwest Pkwy, Austin TX 78745
2St. Joseph Hospital Heritage, Fullerton, CA
3Helen Keller Hospital, Sheffield, AL
4Covenant Health, Lubbock, TX
5Donald Guthrie Foundation, Sayre, PA
6PRX Research, Mesquite, TX
Corresponding author:
Amy L. Lightner, M.D.
Chief Medical Officer
Direct Biologics, LLC
5301 Southwest Pkwy
Austin TX 78745
Phone: 617-901-9915
E-mail: ALightner@directbiologics.com
Funding: Funded by Direct Biologics, LLC
Trial Registration: ClinicalTrials.gov Identifier: NCT04493242.
Abstract word count: 297
Manuscript word count: 3197
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ABSTRACT
1
Background: Bone marrow mesenchymal stem cell (BM-MSC)-derived extracellular vesicles (EVs), ExoFloTM, convey the
2
immunomodulatory and regenerative properties of intact BM-MSC. This study aimed to determine the safety and
3
efficacy of ExoFlo as treatment for moderate-to-severe Acute Respiratory Distress Syndrome (ARDS) in patients with
4
severe COVID-19.
5
Research Question: Does two doses of ExoFlo safely reduce mortality in COVID-19 associated moderate to severe
6
ARDS as compared to placebo?
7
Study Design and Methods: A prospective phase 2 multicenter, double-blind, randomized, placebo-controlled dosing
8
trial was conducted at five sites across the US with infusions of placebo, 10 mL of ExoFlo, or 15 mL of ExoFlo on Day 1 and
9
4. Patients (102) with COVID-19 associated moderate-to-severe ARDS were enrolled and randomized. Adverse events
10
were documented throughout. The primary outcome measure was all-cause 60-day mortality rate. Secondary outcomes
11
included time to death (overall mortality), the incidence of treatment emergent serious adverse events, proportion of
12
discharged patients at 7, 30, and 60 days, time to hospital discharge, and ventilation free days.
13
Results: No treatment-related adverse events were reported. Mortality (60-day) in the Intention-to-Treat (ITT)
14
population was reduced in ExoFlo-15 compared to Placebo (not significant, Chi-square p=0.1343). For the post-hoc
15
subgroup analyses, 60-day mortality was decreased in ExoFlo-15 compared to Placebo (Relative Risk=0.385; 95%
16
confidence interval [CI]=0.159,0.931; p=0.0340; N=50). In ExoFlo-15 a Relative Risk of 0.423 (CI=0.173,1.032; p=0.0588;
17
N=24) was determined for participants aged 18-65 with moderate to severe ARDS. Ventilation-free days (VFDs) improved
18
in ExoFlo-15 (p=0.0455; N=50) for all participants aged 18-65.
19
Interpretation: ExoFlo (15 mL dose) is safe in patients with severe or critical COVID-19 respiratory failure. In participants
20
aged 18 to 65, the risk reduction in 60-day mortality was further improved from all aged subjects in the ITT population
21
after two doses of 15 mL of ExoFlo as compared to placebo.
22
23
TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT04493242.
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KEY WORDS: Extracellular vesicle, bone marrow mesenchymal stem cell, COVID-19, safety, efficacy
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ABBREVIATIONS: ARDS, Acute respiratory distress syndrome; BM-MSC, Bone marrow mesenchymal stem cell; CGMP,
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Current Good Manufacturing Practice; CMC, Chemistry, Manufactuing and Controls; EV, Extracellular vesicle; PEEP,
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Positive end expiratory pressure; SAE, Serious adverse event; SOFA, Sequential organ failure assessment; TEAE,
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Treatment emergent adverse event
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Optimal management of acute respiratory distress syndrome (ARDS) morbidity remains critical. ARDS develops in 33%-
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42% of hospitalized patients with COVID-19 and in 61-81% of patients admitted to the intensive care unit (ICU). COVID-
36
19 ARDS patients demonstrate similar pathologic changes of diffuse alveolar damage as classic ARDS.1,2 Pooled mortality
37
estimates of ARDS cases in COVID-19 patients showed similar mortality to non-COVID-19 ARDS patients.3
38
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Bone marrow mesenchymal stem cells (BM-MSC) show promise for the treatment of ARDS. The phase I START trial
40
monitored outcomes for 60 days following a single IV administration to patients with moderate-to-severe ARDS; no SAEs
41
were observed following infusion of allogeneic BM-MSC.4 Transplantation of healthy donor BM-MSC into patients with
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COVID-19 pulmonary disease improved functional outcomes without any observed adverse effects, and serum level
43
changes in TNF-α and IL-10 suggest BM-MSC may inhibit cytokine storm.5 MSCs from other tissue sources also exhibit
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efficacy.6 Yet, the challenges of cryodamage, fresh product distribution, cell product heterogeneity, immunogenicity,
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thrombotic events, and scalability make BM-MSC technology impractical for global delivery.4,7,8
46
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ExoFloTM is an extracellular vesicle (EV) product manufactured per CGMP regulations from a single donor BM-MSC
48
culture that conveys the immunomodulatory and regenerative properties of BM-MSC without cellular therapy
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limitations.9-12 Extensive characterization of ExoFlo EVs reveals an absence of immunogenic surface epitopes that would
50
cause acute immune reactions. The BM-MSC used to manufacture ExoFlo are fully characterized to meet the ISCT
51
definition of possessing trilineage differentiation capability (bone, adipose and cartilage), and to be positive for the
52
surface markers CD90 and CD166 but negative for CD45. The cells are evaluated by, and have a master file on record,
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with the FDA that includes information about the chemistry, manufacturing and controls (CMC) requirements for an
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approved Phase II IND clinical study. ExoFlo’s efficacy and safety potential was evidenced by an investigator-initiated
55
safety study treating COVID-19-associated ARDS patients.13 These findings combine with the acellular nature,
56
homogeneity, and scalability of ExoFlo to increase its potential as a practical therapeutic for respiratory failure from
57
COVID-19.13,14
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To further evaluate the safety and efficacy of ExoFlo for the treatment of hospitalized patients with respiratory failure
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from severe or critical COVID-19 a randomized, controlled trial, Extracellular Vesicle Infusion Treatment for COVID-19
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(EXIT COVID-19), was conducted. We hypothesized ExoFlo would be safe in the treatment of severe and critical COVID-19
62
patients and compared the safety and efficacy of two doses of ExoFlo to Placebo.
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Methods
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Study design and participants
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A prospective, multi-center, phase 2, randomized, double-blind, placebo-controlled trial was conducted. Enrollment for
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EXIT COVID-19 began September 24, 2020 and completed May 22, 2021. Five clinical trial sites in the United States
69
actively participated in patient recruitment and enrollment. Patients with severe or critical COVID-19 as defined by a
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SpO2 <94% on room air at sea level, partial pressure of arterial oxygen to fraction of inspired oxygen (PaO2/FiO2) <300
71
mm Hg and a respiratory rate >30 breaths/min, or lung infiltrates >50% were included (see e-Table 1, Inclusion Criteria).
72
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The trial protocol was approved by the institutional review board (IRB) at each site (or a centralized IRB as applicable)
74
and overseen by a data and safety monitoring board (DSMB) that was fully independent of both study sponsor and
75
director. Written informed consent (or consent by other IRB-approved process) was obtained from each patient or
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patient’s legally authorized representative if the patient was unable to provide consent.
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Randomization and masking
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See Figure 1 for CONSORT diagram of patient screening and enrollment, and e-Appendix 1 for full description of the
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clinical trial protocol. Patients (102) were randomized 1:1:1 by the clinical trial sites to 15 mL ExoFlo, 10 mL ExoFlo, or
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Placebo arms on Day 1. ExoFlo is colorless when thawed so only treatment masking was required to maintain blinding.
82
Unblinded pharmacists prepared interventions that were delivered to the blinded nursing staff who delivered the
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infusion. Pharmacists are trained on blinding principles, sign a Delegation of Authority Log, and do not intermingle with
84
practitioners or patients and their family.
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Procedures
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Each lot of ExoFlo meets stringent release specifications, including proteomic, mRNA and miRNA
88
characterization. Additionally, the size and quantity of EVs and the presence of an exosome specific tetraspanin profile
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for CD9, CD63 and CD81 are confirmed. Identity assays are combined with validated potency assays to demonstrate the
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mechanism of action is functional.
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Dosing of ExoFlo was calculated based on (1) the 24-patient preliminary COVID-19 ExoFlo pilot study;13 (2) the phase I
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START trial using IV administration of BM-MSC for ARDS, which demonstrated safety at up to 5 million cell/Kg and a
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ceiling dose of 10 million cell/Kg;7 (3) observation of approximately 2,000 extracellular vesicles secreted per cell; and (4)
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lab analysis indicating 60-80 billion EV/mL. Extrapolation from the START trial MSC ceiling dose indicates an IV ExoFlo
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ceiling dose of 17.5 mL/70 Kg adult, and 15 mL and 10 mL of IV ExoFlo were determined as reasonable high and low
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dosing arms providing 1.2 and 0.9 trillion EV particles per dose, respectively.
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All enrolled patients received a 100 mL intravenous infusion over 60 minutes on Day 1. Treatment arms were: 100 mL
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normal saline (NS, Placebo), 10 mL ExoFlo mixed with 90 mL NS (ExoFlo-10), and 15 mL ExoFlo with 85 mL NS (ExoFlo-15).
101
A repeat of the same study treatment occurred on Day 4 if the patient had not recovered (SpO2 ≥93% on room air or
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PaO2/FiO2 ≥300 mmHg). All patients were followed for 60 days, or until hospital discharge or death. Regardless of the
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allocated treatment arm, patients were offered standard supportive care according to hospital guidelines. There was no
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major numerical difference between ExoFlo-15 and placebo arms for 3 types of prior and concomitant medications
105
(Remdesivir, Plasma, Dexamethasone), and >75% and 100% of both groups received prior and concomitant
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glucocorticoids, respectively, per recent guidelines.15 No statistical testing was provided due to a small sample size per
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arm and no pre-defined limits for the test of equivalence. Although all means and percentages between ExFlo 15ml and
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Placebo arms in e-Table 2 were not significant (P>0.1) by a superiority test, we could not make a conclusive statement on
109
equivalence between ExoFlo 15ml and placebo arms. Other experimental treatment or off-label use of marketed
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medications were prohibited. Patients were assessed daily from Day 1 to Day 60 during hospitalization. All serious
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adverse events (SAEs) and grade 3 or 4 adverse events (AEs) representing increased severity from Day 1, and any grade 2
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or higher suspected drug-related hypersensitivity reactions were recorded.
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Outcomes
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The primary endpoint was improvement in the mortality rate within 60 days from randomization. Secondary endpoints
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included time to death, 2) incidence of treatment-emergent serious adverse events, 3) proportion of discharged patients,
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4) time to hospital discharge at 7, 30, and 60 days from randomization, and 5) ventilation free days. Exploratory outcome
118
measurements included viremia, serum acute phase reactants, immune cell subset counts, Sequential Organ Failure
119
Assessment (SOFA) scores, and Quality of Life (EQ-5D-5L) scores.
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Statistical analysis
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Calculation of the sample size was based upon 60-day binomial mortality rates of 32% for ExoFlo-15 referring to the
123
Expanded Access preliminary data and publication of 43% for placebo.16 Sixty-eight patients in the ITT analysis set
124
generated approximately 38% power based on a type I error rate of 0.2 (80% CI) to reject the null hypothesis with the
125
underlying assumption of 60-day mortality rates.
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The study was designed to assess safety at two doses of ExoFlo towards nominating a safe and effective dose of ExoFlo
127
for the treatment of respiratory failure from COVID-19, and to understand trends in morbidity and mortality for future
128
phase 3 hypotheses and study design. Analysis of the primary outcome of 60-day binominal mortality rate was planned
129
and tested by a Chi-square test as a primary method. Predefined subgroup analyses were performed in patients who met
130
criteria for moderate to severe ARDS and/or post-hoc subgroup of aged ≥18 to <65 to investigate primary and secondary
131
endpoints in this disease-specific cohort.
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Role of the funding source
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The study was conducted in accordance with ethical principles as denoted in the International Council for Harmonization
135
(ICH) E6 requirements. The role of the funding source and sponsor of the trial was protocol development including study
136
design, analysis and interpretation of the data, writing the manuscript, and decision for submission of the manuscript.
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Results
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Trial participants
140
Thirty-four subjects were randomized per treatment arm. There were no significant demographic or clinical differences
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in the treatment arms based on age, gender, race, body mass index (BMI), respiratory rate, intubation prior to
142
enrollment, time from first diagnosis of COVID-19 to time of first treatment dose, total SOFA score, PaO2/FiO2 ratio, and
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prior therapy for COVID-19 (e-Table 2).
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Subjects in the three arms were comparable with respect to the number of doses received, reason for not receiving the
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second infusion, and completion of all 60 days of the study. Of the patients who received two doses, 27 of 34 subjects
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(79.4%) randomized to ExoFlo-15, 29 of 34 subjects (85.3%) to ExoFlo-10 and 27 of 34 subjects (79.4%) to Placebo.
148
149
Safety
150
The Safety Analysis Set (Table 1) consisted of all 68 enrolled subjects who received any dose of ExoFlo. No AEs or SAEs
151
caused a pause in patient recruitment or clinical trial discontinuation. No infusion reaction or AEs were observed in any
152
cohort within the first 72 hours. No AEs were attributed by the investigators to administration of ExoFlo, and there was no
153
apparent difference across the three study arms of the percentage of subjects with AEs or the distribution of types of AE.
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155
AEs included worsening hypoxic respiratory failure requiring intubation (N=4), expiration (N=4), acute renal failure (N=3),
156
and pulmonary embolism (N=1). All events occurred more than 72 hours following treatment and were evaluated by an
157
independent DSMB to be reasonably attributable to COVID-19 disease progression or a temporally correlated provoking
158
stimulus. Both Treatment-Emergent Adverse Events (TEAEs) and serious TEAEs of grade 3 or 4 occurred with comparable
159
frequency between ExoFlo-15 and placebo, as did TEAEs of any grade. The frequency of serious TEAEs of any grade in
160
ExoFlo-15 was less than that of Placebo and ExoFlo-10. The only treatment related TEAE (grade 2 hypotension) occurred
161
in the Placebo arm. No serious treatment related TEAEs occurred in any of the three arms. TEAEs that led to death occurred
162
in 47.1% of the subjects in Placebo, 38.2% in ExoFlo-10, and 29.4% in ExoFlo-15. For all clinical laboratory parameters, the
163
mean values for the three groups were comparable at baseline and there were no apparent major differences across the
164
three groups in changes from baseline (not shown).
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Efficacy
167
Intention-to-Treat (ITT) Population Analysis
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The overall mortality rate among all subjects was 61%. The 60-day mortality was numerically lower in the ITT ExoFlo groups
169
compared with the Placebo group (Table 2). Although alpha significance level of 0.2 was suboptimal and may not indicate
170
true statistical significance, the study rejected the null hypothesis for the primary endpoint (p=0.1343) for ExoFlo-15. For
171
all other analyses including ExoFlo-10 and secondary endpoints, subgroup analyses were not pre-defined with a properly
172
adjusted type I error rate, and p-values were calculated for a descriptive purpose only. No multiplicity adjustment applies
173
to subgroup analyses.
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The overall mortality (Kaplan-Meier (KM), Figure 2) was improved at all timepoints for ExoFlo-15 compared with ExoFlo-
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10, which was superior at all timepoints than Placebo. The overall mortality comparison between Placebo and ExoFlo-15
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was measured by the KM curves and a hazard ratio with 95% Confidence Intervals (CI) using a Cox regression model and
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tested using a log-rank test. No arm reached median overall mortality with a 60-day follow-up. Although statistical
179
significance was not achieved for the log-rank test (p=0.1820) or the hazard ratio (HR=0.59; 95% CI=[0.27, 1.30]), the KM
180
curves suggest an increasing reduction in the mortality risk over time in ExoFlo-15 compared to placebo. The relative
181
difference in mortality rates across the three groups increased with time from randomization; ExoFlo-15 was 3% better
182
than Placebo at Day 15, 9% better at Day 30, and 18% better at Day 60. Similar trends, although of lesser magnitude, were
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observed in ExoFlo-10 vs Placebo. Mortality rates for ExoFlo-15 and ExoFlo-10 diverged by 60 days, and the mortality rate
184
for ExoFlo-10 at 60 days was similar to that of Placebo.
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The percentage of subjects discharged was highest for ExoFlo-15 (58.8%), followed by ExoFlo-10 (52.9%), and Placebo
187
(50.0%). The median time to hospital discharge was estimated to be 22 days for ExoFlo-15, 29 days for ExoFlo-10 and not
188
reached by Placebo when evaluated with KM curve. The KM curves suggested a decreasing time to discharge from Placebo
189
to ExoFlo-10 to ExoFlo-15, although statistical significance was not achieved for the log-rank test (p=0.5554) or the recovery
190
ratio (HR=1.21; 95% CI=[0.63, 2.31]) as estimated by a Cox regression model when comparing time to hospital discharge
191
between ExoFlo-15 and Placebo.
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In the ITT population, ventilation-free days (VFDs) were highest for ExoFlo-15 (Mean (SD) = 41.3 (25.8)) and similar for
194
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ExoFlo-10 (Mean (SD) = 32.0 (26.2)) and Placebo (Mean (SD) = 33.9 (28.1)). The difference in VFDs between ExoFlo-15 and
195
Placebo failed to reach statistical significance (p=0.303, Wilcoxon rank sum test), but encouraging trends in several
196
endpoints emerged in analyses of subpopulations of the ITT population that were not pre-defined (e-Tables 3-5).
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Subpopulations
199
Important post-hoc sub analyses were in the patients aged 18-65 with respiratory failure or moderate to severe ARDS.
200
Those with respiratory failure had a 60-day mortality of 50% in the Placebo and 19.2% in the ExoFlo-15, representing
201
absolute risk reduction of 30.8% and Relative Risk of 0.385 (95%CI=0.159,0.931, p=0.0340, Table 3). For this age group
202
who met modified Berlin criteria for moderate to severe ARDS, the 60-day mortality was 72.7% in the Placebo and 30.8%
203
in ExoFlo-15, yielding Absolute Risk reduction of 41.9% and a Relative Risk of 0.423 (95%CI=0.173,1.032, p=0.0588),
204
indicating a trend towards improvement (Table 3).
205
206
For the 18-65 year age group the number of VFDs in ExoFlo-15 (47.6 days) was improved (p=0.0455, Wilcoxon rank-sum
207
test) compared to Placebo (30.3 days, e-Table 4). A dose response effect trend was observed for VFDs in both Moderate
208
and Severe ARDS in this age group (e-Table 5): Moderate ARDS – 47.0 (ExoFlo-15), 25.3 (ExoFlo-10), 13.3 (Placebo); Severe
209
ARDS – 34.6 (ExoFlo-15), 26.5 (ExoFlo-10), 19.6 Placebo).
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Discussion
212
This prospective, double-blind, randomized, placebo-controlled phase 2 trial is the first trial to show BM-MSC EVs are
213
safe and exhibit potential for efficacy based on post-hoc subgroup analyses in the treatment of severe or critical COVID-
214
19. A critical finding of this study was the safety profile of ExoFlo. There was a lack of adverse or serious adverse events
215
related to ExoFlo at either 10 mL or 15 mL treatment doses. Given the severity of illness in this patient population, the
216
overwhelming safety profile is highly encouraging for regulatory path in severely impaired COVID-19 patients.
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Fortunately, there were no differences in the safety profile at either dose despite the difference in efficacy trends
218
observed between ExoFlo-10 compared to ExoFlo-15, and no adverse events were related to investigational product. The
219
rate of TEAEs and SAEs of any severity grade did not increase beyond Placebo with either dose of ExoFlo. The number of
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patients in treatment arms who died were lower with treatment relative to Placebo. In fact, overall mortality trended
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lowest in ExoFlo-15 and improved with increasing time from randomization. This safety profile is superior to the known
222
side effects attributed to dexamethasone, remdesivir and IL-6 antagonists.17,18
223
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All-cause 60-day mortality in the ITT population was 29.4% with ExoFlo-15 and 47.1% with placebo. Although not
225
statistically significant, our findings are consistent with the findings of the initial investigator-initiated trial and expanded
226
access program (NCT04657458) wherein two treatments of ExoFlo-15 resulted in a mortality reduction among patients
227
hospitalized with severe or critical COVID-19. Additional secondary endpoints here supporting the benefit of ExoFlo-15
228
included confirmation of overall mortality by the KM curves, shorter time to hospital discharge, increased ventilation free
229
days, and biomarker trends. Dose-response trends were observed in the ITT population for 60-day mortality rate, overall
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mortality (KM), median time to discharge (KM) and VFDs. In the age 18-65 patient subgroup with moderate or severe
231
ARDS, the VFDs showed a dose-response trend with ExoFlo-15 > ExoFlo-10 > Placebo. Although these metrics did not
232
reach statistical significance these results will inform the subsequent Phase 3 trial design. While this study was not
233
adequately powered for a mortality benefit between treatment arms, a larger mortality risk reduction was identified in
234
subjects aged 18-65 experiencing respiratory failure due to COVID-19, and a similar trend toward risk reduction was seen
235
in this age group with moderate to severe ARDS.
236
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In the subgroup of patients aged 18-65 who met modified Berlin criteria for moderate to severe ARDS, mortality was
238
30.8% for ExoFlo-15 (N=11) as compared to 72.7% (N=13) in the placebo group, demonstrating a 60-day mortality
239
absolute risk reduction of 41.9% and a Relative Risk of 0.423 (95%CI=0.173,1.032). Some of this difference may be due to
240
significant co-morbidities in the aging population, age as the known independent prognostic factor that affects the >65
241
age group overwhelmingly to decrease treatment effects on mortality, or small sample size and a Type I error. Another
242
reason for such a mortality benefit in the ARDS cohort is that ExoFlo may have a more substantial impact in patients both
243
nearing intubation and those intubated at the time of treatment, demonstrating the value of ExoFlo in a critically ill
244
patient population. Importantly, this suggests that ExoFlo could be beneficial in pre-ARDS patients. Larger sample sizes
245
are needed to confirm a significant difference in mortality, as the treatment arm size was too small to adequately power
246
this question.
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While this is the first completed prospective, randomized, placebo controlled trial of an extracellular vesicle (EV) product
249
for the treatment of respiratory failure from COVID-19, several other randomized clinical trials have been conducted with
250
anti-viral and immunomodulatory therapeutics for the treatment of COVID-19.15,19-29 Those with documented effects on
251
mortality include remdesivir (anti-viral), and dexamethasone and IL-6 antagonists (immunomodulatory). The trial herein
252
is the first to show an EV product with potential mortality benefit, that, in phase 3, may be superior to the
253
aforementioned clinical trial results.
254
255
While weaknesses of our study include that insufficient power was proposed for the primary endpoint and indications of
256
efficacy may arise from the small sample size and post-hoc subgroup analyses, preliminary efficacy inferences may be
257
drawn from trends in the endpoint data to guide generation of hypotheses for the future phase 3 trial design.
258
Suggestions of efficacy were observed, particularly in subjects receiving the higher dose of ExoFlo; overall mortality,
259
VFDs, and days to discharge all trended in favor of the higher dose of ExoFlo versus placebo. In addition, these trends
260
seemed to be improved in a younger patient cohort with ARDS.
261
262
Interpretation
263
Based on preliminary results demonstrated from ExoFlo, the FDA issued a regenerative medicine advanced therapeutic
264
(RMAT) designation and also authorized proceeding with a phase 3 clinical trial that is currently underway to confirm the
265
results described herein. Given the limited approved therapeutics with proven mortality benefit, expedient results of our
266
phase 3 will be critical to the ongoing treatment of ARDS patients. Evidence of significant efficacy against respiratory
267
failure from COVID-19 disease by ExoFlo would represent a significant advancement in efforts to reduce morbidity and
268
mortality caused by SARS-CoV-2.
269
270
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ACKNOWLEDGEMENTS:
271
Guarantor statement: The first author is responsible for the content of this manuscript.
272
Author contributions: ALL: protocol design, interpretation of data, drafting and finalizing manuscript; JTR: protocol
273
design, interpretation of data, editing manuscript; SS: protocol design, interpretation of data, editing manuscript;
274
SS: protocol design, interpretation of data, editing manuscript; VS: protocol design, interpretation of data, editing
275
manuscript; DJP: enrollment of patients, protocol design, editing manuscript; TIM: enrollment of patients, protocol
276
design, editing manuscript; BPW: enrollment of patients, protocol design, editing manuscript ; JJW: enrollment of
277
patients, protocol design, editing manuscript ; MA: enrollment of patients, protocol design, editing manuscript
278
Names of collaborators: none
279
280
Financial/nonfinancial disclosures: Amy L. Lightner: CMO at Direct Biologics, John Ransom: PhD at Direct Biologics,
281
Sascha Qian: prior associate CMO at Direct Biologics, Vikram Sengupta: prior CMO at Direct Biologics
282
283
Role of the sponsors: Sponsors funded the study, interpreted the data and drafted the manuscript.
284
TAKE HOME POINTS:
285
Study Question: Does two doses of ExoFlo safely reduce mortality in severe COVID-19 moderate to severe ARDS as
286
compared to placebo?
287
288
Results: No AEs or SAEs were related to investigational product. For participants aged 18-65 with respiratory failure, 60-
289
day mortality was significantly decreased in ExoFlo-15 compared to Placebo (Relative Risk or 0.385, 95%CI=0.159,0.931,
290
p=0.0340).
291
292
Interpretation: Two doses of ExoFlo safely and significantly reduces mortality in patients aged 18-65 with respiratory
293
failure due to critical or severe COVID-19.
294
295
296
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297
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2020;14(11):1149-1163. doi:10.1080/17476348.2020.1804365
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5. Leng Z, Zhu R, Hou W, et al. Transplantation of ACE2(-) Mesenchymal Stem Cells Improves the Outcome of
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12. Tang Y, Zhou Y, Li HJ. Advances in mesenchymal stem cell exosomes: a review. Stem cell research & therapy. Jan
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19. Beigel JH, Tomashek KM, Dodd LE, et al. Remdesivir for the Treatment of Covid-19 - Final Report. N Engl J Med.
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20. Bramante CT, Huling JD, Tignanelli CJ, et al. Randomized Trial of Metformin, Ivermectin, and Fluvoxamine for
Covid-19. N Engl J Med. Aug 18 2022;387(7):599-610. doi:10.1056/NEJMoa2201662
21. Gordon AC, Mouncey PR, Al-Beidh F, et al. Interleukin-6 Receptor Antagonists in Critically Ill Patients with Covid-
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open-label, platform trial. Lancet. May 1 2021;397(10285):1637-1645. doi:10.1016/s0140-6736(21)00676-0
23. Higgins AM, Berry LR, Lorenzi E, et al. Long-term (180-Day) Outcomes in Critically Ill Patients With COVID-19 in
the REMAP-CAP Randomized Clinical Trial. Jama. Jan 3 2023;329(1):39-51. doi:10.1001/jama.2022.23257
24. Li G, Hilgenfeld R, Whitley R, De Clercq E. Therapeutic strategies for COVID-19: progress and lessons learned.
Nature reviews Drug discovery. Apr 19 2023:1-27. doi:10.1038/s41573-023-00672-y
25. Reis G, Silva E, Silva DCM, et al. Effect of Early Treatment with Ivermectin among Patients with Covid-19. N Engl J
Med. May 5 2022;386(18):1721-1731. doi:10.1056/NEJMoa2115869
26. Shankar-Hari M, Vale CL, Godolphin PJ, et al. Association Between Administration of IL-6 Antagonists and
Mortality Among Patients Hospitalized for COVID-19: A Meta-analysis. Jama. Aug 10 2021;326(6):499-518.
doi:10.1001/jama.2021.11330
27. Spinner CD, Gottlieb RL, Criner GJ, et al. Effect of Remdesivir vs Standard Care on Clinical Status at 11 Days in
Patients With Moderate COVID-19: A Randomized Clinical Trial. Jama. Sep 15 2020;324(11):1048-1057.
doi:10.1001/jama.2020.16349
28. Wang Y, Zhang D, Du G, et al. Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-
controlled, multicentre trial. Lancet. May 16 2020;395(10236):1569-1578. doi:10.1016/s0140-6736(20)31022-9
29. Yu SY, Koh DH, Choi M, et al. Clinical efficacy and safety of interleukin-6 receptor antagonists (tocilizumab and
sarilumab) in patients with COVID-19: a systematic review and meta-analysis. Emerg Microbes Infect. Dec
2022;11(1):1154-1165. doi:10.1080/22221751.2022.2059405
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Table 1. Overall Summary of Safety Events (Safety Analysis Set)
Safety Parameter
ExoFlo-15
(N=34)
ExoFlo-10
(N=34)
Placebo
(N=34)
ExoFlo Total
(N=68)
Any TEAEs [1]
Any Grade (%)
24 (70.6)
26 (76.5)
23 (67.6)
50 (73.5)
Grade 3 or 4 (%)
5 (14.7)
9 (26.5)
5 (14.7)
14 (20.6)
Serious TEAEs [1]
Any Grade (%)
10 (29.4)
18 (52.9)
16 (47.1)
28 (41.2)
Grade 3 or 4 (%)
3 (8.8)
7 (20.6)
3 (8.8)
10 (14.7)
Study Treatment-Related TEAEs (%)
0
0
1 (2.9)
0
Study Treatment-Related Serious TEAEs (%)
0
0
0
0
TEAEs That Led to Dose Interruption (%)
1 (2.9)
0
0
1 (1.5)
TEAEs That Led to Missing Dose or Discontinued the
Treatment Early (%)
0
0
1 (2.9)
0
TEAEs That Led to Death (%)
10 (29.4)
13 (38.2)
16 (47.1)
23 (33.8)
TEAE = Treatment-Emergent Adverse Events, are defined as any adverse event that started between the first dose date
and 30 days post the last dose date, inclusively.
[1] Toxicity grades of adverse events are evaluated based on criteria of NCI-CTCAE v5.0. Each subject is counted once to
the worst grade at subject-level.
Note: Related = Possibly Related, or Probably Related.
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Table 2. Summary of Efficacy (ITT Analysis Set)
Study Endpoints
Statistics
ExoFlo-15
(N=34)
ExoFlo-10
(N=34)
Placebo
(N=34)
Subjects Who Discharged Within 60
Days
n (%)
20 (58.8)
18 (52.9)
17 (50.0)
Subjects Who Discharged Within 30
Days
n (%)
19 (55.9)
17 (50.0)
17 (50.0)
Subjects Who Discharged Within 7 Days
n (%)
11 (32.4)
9 (26.5)
11 (32.4)
Median Time to Discharge (KM) [1]
n
34
34
34
Median
22.0 days
29.0 days
NR
95% CI
(6.0, NE)
(9.0, NE)
(7.0, NE)
Subjects Who Died Within 30 Days
n (%)
9 (26.5)
10 (29.4)
12 (35.3)
Subjects Who Died Within 60 Days
n (%)
10 (29.4)
14 (41.2)
16 (47.1)
80% CI
(19.1, 41.6)
(29.6, 53.6)
(35.0, 59.4)
95% CI
(15.1, 47.5)
(24.6, 59.3)
(29.8, 64.9)
ExoFlo-15 vs Placebo
P-value [2]
0.1343
Median Time to Death (KM)
Median
NR
NR
NR
Mortality Rate at 15 Days (KM)
%
21.2
22.2
24.2
Mortality Rate at 30 Days (KM)
%
27.3
32.3
36.3
Mortality Rate at 60 Days (KM)
%
30.4
46.6
48.4
95% CI
(17.7, 49.2)
(30.6, 65.8)
(33.1, 66.4)
P/F Ratio Increase from Baseline to Day
7 (mmHg) [3]
n
17
18
18
Mean (SD)
55.5 (86.37)
42.9 (53.39)
48.9 (78.38)
95% CI
(18.9, 92.1)
(21.0, 64.8)
(16.8, 81.0)
Min, Max
0, 311
0, 176
0, 303.16
#Ventilation-Free Days (within 60 Days)
n
34
34
34
Mean (SD)
41.3 (25.78)
32.0 (26.23)
33.9 (28.06)
95% CI
(33.8, 48.7)
(24.4, 39.6)
(25.8, 42.1)
Min, Max
0, 61
0, 61
0, 61
ExoFlo-15 vs Placebo
P-value [4]
0.3030
KM = Kaplan Meier method, NE = Not Evaluable, NR = Not Reached
[1] Subjects who died or discontinued from the study due to a reason other than discharge before reaching 60
days (Day 61) are censored at Day 61.
[2] Chi-square test for 60-day mortality rates. P-value is displayed for a descriptive purpose.
[3] P/F ratio: All treated subjects with baseline and at least one P/F ratio measured at Day 4 or 7. For missing Day 7
data, 380 mmHg was assigned for discharged patients, and no change (0) was assigned to patients with negative
change from the baseline or died before Day 7.
#Ventilation-free days: days when patients are not on mechanical ventilation within 60 days of follow-up.
[4] Wilcoxon rank-sum test. P-value is displayed for a descriptive purpose.
80% CI and 95% CI of Subjects Who Died Within 60 Days are calculated using exact (Clopper-Pearson) method; 95%
CI of P/F Ratio Increase and Ventilation-Free Days are calculated using the student's T distribution.
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18
Table 3. 60-Day Mortality Rate for Patients Aged 18-65 with Respiratory Failure or Moderate to Severe ARDS
INDICATION
PLACEBO
N/N
(%)
EXOFLO 15 ML
N/N
(%)
ABSOLUTE
RISK
REDUCTION
RELATIVE
RISK (RR)
(95% CI)
N
P-VALUE
for RR
Respiratory Failure due to Severe
or Critical COVID-19, Age 18-65
12/24
(50.0)
5/26
(19.2)
30.8%
0.385
(0.159, 0.931)
50
0.0340
Moderate to Severe ARDS
Subgroup (CPAP, BiPAP, MV), All
Ages
11/17
(64.7)
6/16
(37.5)
27.2%
0.580
(0.281, 1.195)
33
0.1394
Moderate to Severe ARDS
Subgroup (CPAP, BiPAP, MV), Age
18-65
8/11
(72.7)
4/13
(30.8)
41.9%
0.423
(0.173, 1.032)
24
0.0588
Absolute Risk Reduction = (60-Day Mortality Placebo) – (60-Day Mortality ExoFlo)
Relative Risk = 60-Day mortality rate in ExoFlo / 60-day mortality rate in Placebo
Moderate to Severe ARDS defined per the modified Berlin definition where moderate ARDS is to be 100 mmHg < P/F ratio ≤
200 mmHg, and severe ARDS is to be P/F ratio ≤ 100 mmHg.
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19
Figure Legends:
Figure 1. CONSORT diagram for study enrollment, allocation of treatment arm and follow-up.
Figure 2. Mortality (ITT population).
Time to death was compared between the IP 15 mL and Placebo using a log-rank test. Median time to death was
estimated by the KM method. The hazard ratio of IP 15 mL to Placebo was estimated using a Cox regression model with a
95% Confidence Interval (CI).
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Figure 1. CONSORT diagram for study enrollment, allocation of treatment arm and follow-up.
121 Assessed for eligibility
19 Screen Failed
7 Not meeting inclusion criteria
12 Not meeting exclusion criteria
102 Randomized
34 Allocated to ExoFlo-15
27 Received 2 scheduled infusions
5 Did not receive 2nd scheduled
infusion due to discharge
1 Did not receive 2nd scheduled
infusion due to death
1 Did not receive 2nd scheduled
infusion due to unknown reason or
not reaching Day 4
34 Allocated to Placebo
27 Received 2 scheduled infusions
4 Did not receive 2nd scheduled
infusion due to discharge
2 Did not receive 2nd scheduled
infusion due to death
1 Did not receive 2nd scheduled
infusion due to unknown reason or
not reaching Day 4
34 Allocated to ExoFlo-10
29 Received 2 scheduled infusions
4 Did not receive 2nd scheduled
infusion due to discharge
1 Did not receive 2nd scheduled
infusion due to death
0 Did not receive 2nd scheduled
infusion due to unknown reason
or not reaching Day 4
10 Discontinued due to death
1 Discontinued due to
withdrawal by subject
1 Discontinued due to other
1 Lost to follow-up
14 Discontinued due to death
0 Discontinued due to
withdrawal by subject
1 Discontinued due to other
1 Lost to follow-up
16 Discontinued due to death
0 Discontinued due to
withdrawal by subject
0 Discontinued due to other
0 Lost to follow-up
34 Analysed
0 Excluded from analysis
34 Analysed
0 Excluded from analysis
34 Analysed
0 Excluded from analysis
Allocation
Follow-Up
Analysis
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Figure 2. Mortality (ITT population).
Time to death was compared between the IP 15 mL and Placebo using a log-rank test. Median time to death was
estimated by the KM method. The hazard ratio of IP 15 mL to Placebo was estimated using a Cox regression model
with a 95% Confidence Interval (CI).
NR = Not Reached
Time to Death is the interval in days from randomization to subject's death. The interval is censored to study
discontinuation or completion if the subject is alive.
[1] p-value is from the log-rank test.
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Conflict of interest: CMO at Direct Biologics, Vikram Sengupta: prior CMO at Direct Biologics, Sascha Sengupta:
prior associate CMO at Direct Biologics, John Ransom: PhD at Direct Biologics, Sam Suzuki: statistician at Direct
Biologics
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REQUEST FOR REGENERATIVE MEDICINE ADVANCED THERAPY (RMAT)
DESIGNATION
Investigational New Drug (IND) Number:
21669
Study Investigational Product:
Date & Format:
Bone Marrow Mesenchymal Stem Cell (bmMSC)-Derived
Extracellular Vesicles (via Intravenous Administration)
8 February 2021| Cover Letter for Protocol Amendment
Based on the following criteria from Section 3033 of the 21st Century Cures Act, the study
investigational product (IP), ExoFlo™, meets eligibility for regenerative medicine advanced
therapy (RMAT) designation:
A.
As a biologic product derived from human bone marrow mesenchymal stem cells, ExoFlo is
an extracellular vesicle isolate product (EVIP) and a regenerative medicine therapy.
B.
ExoFlo is intended to treat, modify and reverse COVID-19 associated moderate-to-severe
Acute Respiratory Distress Syndrome (ARDS), which is a life-threatening disease.
C.
Preliminary clinical evidence indicates that ExoFlo has the potential to address unmet
medical needs for such disease or condition.
1. UNMET NEED: A systematic prospective observational analysis published in The Lancet
in November 2020 suggests that the lung injury in COVID-19 associated ARDS is similar
to that of classic ARDS. The overall 28-day mortality was high (36%). Furthermore, the
latest statistics demonstrate that the current U.S. healthcare system is unable to
accommodate all patients who should be admitted with COVID-19 associated ARDS: As
of January 13th, 2021, 95% of intensive care hospital beds are occupied nationwide. More
than a third of Americans live in areas where hospitals are running critically short of
intensive care beds. Intensive care units are already at full capacity in states like
California and Texas—a scenario which will only worsen in the upcoming months.
According to the CDC, the number of new deaths in the next 4 weeks will increase with
the total number of deaths estimated around 479,000 to 514,000 by February 20th, 2021—the
primary cause of death is COVID-19 associated ARDS.
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Bone Marrow Mesenchymal Stem Cell Derived Extracellular Vesicles
Protocol Number: DB-EF-PHASEII-001
Direct Biologics, LLC
2
2. CLINICAL EVIDENCE FOR ADDRESSING UNMET NEED: In an open-label
investigator-initiated study in April 2020, 17 out of 24 patients with COVID-19 moderate-
to-severe ARDS were able to reverse their profound hypoxia following a single infusion
of the investigational product; patients were discharged from the hospital with median
time to discharge of 5.6 days (whereas median to recovery for Remdesivir is 10 days).
The mortality rate of 17% was lower compared to retrospective institutional control of
approximately 30% for the same period. To date, 42 patients have been randomized in
EXIT COVID-19, the Phase II double blinded, placebo-controlled, randomized controlled
trial and 7 patients have received the investigational product through single patient
Expanded Access for Compassionate Use without any adverse reactions and 5 out of 7
patients were able to recover.
3. THERAPEUTIC SPECIFICITY FOR UNMET NEED: Potential therapeutic mechanisms
of the investigational Product may be specific for COVID-19 associated ARDS and
include the following:
▪ The reduction of inflammation as demonstrated by decrease in acute phase reactants
(CRP, Ferritin, D-dimer) and the reconstitution of adaptive immunity as demonstrated
by increase in CD4+, CD8+ lymphocytes.
▪ In Vitro study show ExoFlo has moderate capacity (approximately 39.9%) to directly
inhibit SARS-CoV-2 activity.
▪ In Vivo acute lung injury murine study showed that following treatment with ExoFlo,
cytokine storm activities were reduced as demonstrated by the inhibition of GM-CSF,
M-CSF, as well as CXCL-9.
▪ Independent molecular characterization identified 27 miRNA as potential inhibitors
of the ACE-2 related protein network; 7 miRNA as inhibitors of serine protease 2
(TMPRSS2)—a transmembrane protease that is required for Spike (S) protein
priming and SARS-CoV-2 entry into a host cell; 5 miRNA as inhibitors of IL-6 and 2
miRNA that target tumor necrosis factor alpha (TNF-a)—consistent with the clinical
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Bone Marrow Mesenchymal Stem Cell Derived Extracellular Vesicles
Protocol Number: DB-EF-PHASEII-001
Direct Biologics, LLC
3
observation and in vivo observation that ExoFlo may down-regulate the cytokine
storm.
Bone Marrow Mesenchymal Stem Cell Derived Extracellular Vesicles Infusion Treatment for COVID-19
Associated Acute Respiratory Distress Syndrome (ARDS): A Phase II Clinical Trial
Protocol Number:
DB-EF-PHASEII-001
Investigational New Drug (IND) Number:
21669
Study Phase:
II
Study Type:
Double-Blinded, Placebo-controlled, Randomized Controlled Trial
Study Investigational Product:
Bone Marrow Mesenchymal Stem Cell (bmMSC)-Derived
Extracellular Vesicles (via Intravenous Administration)
Administrative Amendment:
Date & Document Version:
6
8 February 2021
|
7.0
Administrative Amendment:
Date & Document Version:
5
31 December 2020
|
6.0
Administrative Amendment:
Date & Document Version:
4
2 November 2020
|
5.0
Administrative Amendment:
Date & Document Version:
3
7 October 2020
|
4.0
Administrative Amendment:
Date & Document Version
2
31 August 2020
|
3.0
Administrative Amendment:
Date & Document Version
1
7 August 2020
|
2.0
Original Protocol:
Date & Document Version
-
24 July 2020
|
1.0
Sponsor:
Direct Biologics, LLC
13492 Research Blvd, Ste 120-758
Austin, TX 78750
CONFIDENTIAL
This protocol is provided to you as an Investigator, potential Investigator, or consultant for review by you, your staff, and
Ethics Committee/Institutional Review Board (EC/IRB). The information contained in this document is regarded as
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Bone Marrow Mesenchymal Stem Cell Derived Extracellular Vesicles
Protocol Number: DB-EF-PHASEII-001
Direct Biologics, LLC
4
confidential and, except to the extent necessary to obtain informed consent, may not be disclosed to another party unless such
disclosure is required by law or regulations. Persons to whom the information is disclosed must be informed that the
information is confidential and may not be further disclosed by them.
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Bone Marrow Mesenchymal Stem Cell Derived Extracellular Vesicles
Protocol Number: DB-EF-PHASEII-001
Direct Biologics, LLC
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PROTOCOL AUTHORIZATION
Signature of Direct Biologics Chief Medical Officer
Vik Sengupta, MD
Chief Medical Officer
Direct Biologics, LLC
Printed Name of Direct Biologics Chief Medical Officer
Date Signed (mm/dd/yy)
Signature of Direct Biologics Chief Scientific Officer
Timothy Moseley, PhD
Chief Scientific Officer
Direct Biologics, LLC
Printed Name of Direct Biologics Chief Scientific Officer
Date Signed (mm/dd/yy)
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Bone Marrow Mesenchymal Stem Cell Derived Extracellular Vesicles
Protocol Number: DB-EF-PHASEII-001
Direct Biologics, LLC
6
INVESTIGATOR’S AGREEMENT
Title: Bone Marrow Mesenchymal Stem Cell Derived Extracellular Vesicles Infusion Treatment
for COVID-19 Associated ARDS: A Phase II Clinical Trial
Protocol Number: DB-EF-PHASEII-001
Signature of Investigator
dd/mm/yy
Printed Name of Investigator and Title
Site Number(s):
By my signature, I agree to supervise and oversee the conduct of this study and to ensure its
conduct is in compliance with the protocol, informed consent, IRB/EC procedures,
instructions from Direct Biologics representatives, the Declaration of Helsinki, International
Conference on Harmonisation (ICH) Good Clinical Practices (GCP) guidelines, and the
applicable parts of the United States (US) Code of Federal Regulations (CFR) and local
regulations governing the conduct of clinical studies.
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Bone Marrow Mesenchymal Stem Cell Derived Extracellular Vesicles
Protocol Number: DB-EF-PHASEII-001
Direct Biologics, LLC
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TABLE OF CONTENTS
PROTOCOL AUTHORIZATION ............................................................................................................................ 5
INVESTIGATOR’S AGREEMENT ......................................................................................................................... 6
LIST OF IN-TEXT TABLES ................................................................................................................................... 11
STATEMENT OF COMPLIANCE ......................................................................................................................... 12
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS ......................................................................... 13
1.
PROTOCOL SUMMARY ........................................................................................................................... 16
1.1
Synopsis ................................................................................................................................................ 16
1.2
Schema ................................................................................................................................................. 18
1.3
Schedule of Activities* ......................................................................................................................... 19
2.
INTRODUCTION ........................................................................................................................................ 22
2.1
Study Rationale ..................................................................................................................................... 22
2.2
Background ........................................................................................................................................... 24
2.2.1
The Status Quo in Healthcare ............................................................................................ 24
2.2.2
Rationale for Exploring New Treatment ........................................................................... 25
2.2.3
COVID-19: Pathophysiology ............................................................................................ 26
2.2.4
A Potential Treatment for COVID-19 Associated ARDS ................................................. 28
2.3
Risk/Benefit Assessment ...................................................................................................................... 33
2.3.1
Known Potential Risks ...................................................................................................... 33
2.3.2
Known Potential Efficacy ................................................................................................. 34
2.3.3
Assessment of Potential Risks and Benefits ...................................................................... 34
3.
OBJECTIVES AND ENDPOINTS ............................................................................................................. 35
4.
STUDY DESIGN .......................................................................................................................................... 37
4.1
Overall Design ...................................................................................................................................... 37
4.2
Scientific Rationale for Study Design ................................................................................................... 37
4.3
Justification for Dose ............................................................................................................................ 38
4.3.1
Rationale for Dosing ......................................................................................................... 38
4.3.2
Rationale for Dosing in Special Populations ..................................................................... 38
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4.3.3
Rationale for Redosing ...................................................................................................... 39
4.4
End of Study Definition ........................................................................................................................ 39
5.
STUDY POPULATION ............................................................................................................................... 40
5.1
Inclusion Criteria .................................................................................................................................. 40
5.2
Exclusion Criteria ................................................................................................................................. 41
5.3
Screen Failures ...................................................................................................................................... 42
5.4
Strategies for Recruitment and Retention ............................................................................................. 42
6.
STUDY INTERVENTION .......................................................................................................................... 43
6.1
Study Intervention(s) Administration .................................................................................................... 43
6.1.1
Study Intervention Description .......................................................................................... 43
6.1.2
Dosing and Administration ................................................................................................ 43
6.1.3
Monitoring of study intervention ....................................................................................... 43
6.1.4
Detailed Treatment Plan in the Event of an Infusion Reaction .......................................... 43
6.2
Preparation/Handling/Storage/Accountability ....................................................................................... 45
6.2.1
Acquisition and accountability .......................................................................................... 45
6.2.2
Formulation, Appearance, Packaging, and Labeling ......................................................... 45
6.2.3
Product Storage and Stability ............................................................................................ 46
6.2.4
Study Treatment Preparation ............................................................................................. 46
6.3
Measures to Minimize Bias: Randomization and Blinding ................................................................... 47
6.4
Staggering Protocol............................................................................................................................... 48
6.5
Study Intervention Compliance ............................................................................................................. 49
6.6
Concomitant Medications ..................................................................................................................... 49
6.7
Rescue Medications for infusion reaction ............................................................................................. 49
7.
STUDY INTERVENTION & PARTICIPANT DISCONTINUATION ................................................... 51
7.1
Discontinuation of Study Intervention .................................................................................................. 51
7.1.1
Clinical Criteria* for Slowing or Suspension of Study Intervention ................................. 51
7.1.2
Clinical Criteria for Not Receiving the Next Study Intervention ....................................... 51
7.1.3
Stopping Rules: ................................................................................................................. 52
7.1.4
Discontinuation of the Study Intervention ......................................................................... 54
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7.2
Participant Discontinuation/Withdrawal from the Study ...................................................................... 54
7.3
Lost to Follow-Up ................................................................................................................................. 54
8.
STUDY ASSESSMENTS AND PROCEDURES ....................................................................................... 56
8.1
Study Procedures By Visit .................................................................................................................... 56
8.2
Efficacy Assessments ............................................................................................................................ 59
8.3
Safety and Data Safety Monitoring Board ............................................................................................ 59
8.4
Adverse Events and Serious Adverse Events ........................................................................................ 59
8.4.1
Definition of Adverse Events ............................................................................................ 59
8.4.2
Definition of Serious Adverse Events ............................................................................... 60
8.4.3
Follow-Up Reporting ........................................................................................................ 61
8.4.4
Definition of Adverse Events of Special Interest (AESI) .................................................. 62
8.4.5
Classification of Severity of Adverse Events and Relationship to Study Treatment ......... 62
8.4.6
Expected Adverse Reactions ............................................................................................. 64
8.4.7
Time Period and Frequency for Event Assessment and Follow-up ................................... 64
8.4.8
Adverse Event Reporting .................................................................................................. 65
8.4.9
Serious Adverse Event Reporting...................................................................................... 65
8.5
Unanticipated Problems ........................................................................................................................ 65
9.
STATISTICAL CONSIDERATIONS ........................................................................................................ 67
9.1
Statistical Hypotheses ........................................................................................................................... 67
9.2
Sample Size Determination ................................................................................................................... 68
9.3
Analysis Population .............................................................................................................................. 69
9.4
Statistical Analyses ............................................................................................................................... 69
9.4.1
Analysis of the Safety Endpoint ........................................................................................ 69
9.4.2
Analysis of the Primary and Secondary Efficacy as well as Exploratory Endpoints ......... 69
9.4.3
AE Analysis ...................................................................................................................... 70
9.4.4
Baseline and Disposition Summaries ................................................................................ 70
9.4.5
Interim Analysis ................................................................................................................ 70
9.4.6
Planned Early Unblinding Analysis .................................................................................. 71
9.4.7
Sub-Group Analyses ......................................................................................................... 71
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10.
REGULATORY ETHICAL AND STUDY OVERSIGHT CONSIDERATIONS ................................... 72
10.1
Informed Consent Process .................................................................................................................... 72
10.2
Consent Procedures & Documentation ................................................................................................. 72
10.3
Study Discontinuation and Closure ....................................................................................................... 72
10.4
Confidentiality and Privacy .................................................................................................................. 73
10.5
Data Storage ......................................................................................................................................... 74
10.6
DSMB Safety Oversight ....................................................................................................................... 74
10.7
Clinical Monitoring ............................................................................................................................... 74
10.8
Quality Assurance and Quality Control ................................................................................................ 75
10.9
Protocol Deviation ................................................................................................................................ 75
10.10
Public Access ........................................................................................................................................ 76
10.11
Conflict of Interest Policy ..................................................................................................................... 76
10.12
Protocol Amendment History ............................................................................................................... 76
11.
APPENDICES .............................................................................................................................................. 81
11.1
Imputing PaO2 from SpO2 and Estimating FiO2 ..................................................................................................................................
81
11.2
Sample EQ-5D-5L and Scoring ............................................................................................................ 84
12.
REFERENCES ............................................................................................................................................. 87
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LIST OF IN-TEXT TABLES
Table 1. Schedule of Activities .................................................................................................................. 19
Table 2: Classes of Factors Contained in the IP for this Study ................................................................... 30
Table 3: Study Objectives, Endpoints, and Justification for Endpoints ....................................................... 35
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STATEMENT OF COMPLIANCE
The trial will be conducted in accordance with ICH GCP and applicable United States CFR. The
Principal Investigator (PI) will assure that no deviation from, or changes to the protocol will take
place without prior agreement from the IND, funding agency and documented approval from the
central IRB, except where necessary to eliminate an immediate hazard(s) to the trial participants.
All personnel involved in the conduct of this study have completed Human Subjects Protection
and ICH GCP Training. The protocol, informed consent form(s), recruitment materials, and all
participant materials will be submitted to the central IRB for review and approval. Approval of
both the protocol and the consent form must be obtained before any participant is enrolled. Any
amendment to the protocol will require review and approval by the central IRB before the changes
are implemented to the study. All changes to the consent form will be central IRB approved; a
determination will be made regarding whether a new consent needs to be obtained from
participants who provided consent, using a previously approved consent form.
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LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS
ABG
Arterial Blood Gas
ACE2
Angiotensin-converting Enzyme 2
ACLS
Advanced Cardiac Life Saving
AE
Adverse Events
AESI
Adverse Event of Special Interest
ALI
Acute Lung Injury
ANC
Absolute Neutrophil Count
ANOVA
Analysis of Variance
ARB
Angiotensin Receptor Blocker
BMI
Body Mass Index
bmMSCs
Bone marrow mesenchymal stem cells
BMP
Basic Metabolic Profile
CBC
Complete Blood Count
CBER
Center for Biologics Evaluation and Research
CD
Cluster of Differentiation, i.e., CD4+ T cell
CFR
Code of Federal Regulations
cGMP
Current Good Manufacturing Practice
COVID-19
Coronavirus Disease 2019
CRO
Contract Research Organization
CRP
C-Reactive Protein
CRF
Case Report Form
CT
Computerized Tomography
CXR
Chest X-ray
DNA
Deoxyribonucleic Acid
DOB
Date of Birth
DSMB
Data Safety Monitoring Board
EC
Ethics Committee
ECG or EKG
Electrocardiogram
ECMO
Extracorporeal Membrane Oxygenation
eCRF
Electronic Case Report Form
EMR
Electronic Medical Record
EQ-5D-5L
EuroQol-5D, a widely validated metric for quality of life; the 5 five dimensions
(5D) include mobility, self-care, usual activities, pain/discomfort, and
anxiety/depression
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EV
Extracellular Vesicles
EVIP
Extracellular Vesicle Isolate Product
FM
Face mask
FDA
Food and Drug Administration
FiO2
Fraction of Inspired Oxygen
GvHD
Graft versus Host Disease
HF O2
High Flow Oxygen
HFOV
High Frequency Oscillatory Ventilation
HIV
Human Immunodeficiency Virus
HR
Heart Rate
ICH GCP
International Conference on Harmonisation Good Clinical Practice
IgM
Immunoglobulin M
IL
Interleukin, i.e., IL-6
IND
Investigational New Drug
IP
Investigational Product
IRB
Institutional Review Board
IRT
Interactive Response Technology
IV
Intravenous
LFTs
Liver Function Tests
LPM
Liters Per Minute
MAP
Mean Arterial Pressure
MAR
Medical Administration Record
microRNA or miRNA
Microcoding Ribonucleic Acid
MOP
Manual of Procedures
MRN
Medical Record Number
mRNA
Messenger RNA
MV
Mechanical Ventilation
NIH
National Institutes of Health
NK cells
Natural Killer cells
NRB
Nonrebreather
PaO2/FiO2
Partial Pressure of Arterial Oxygen to Fraction of Inspired Oxygen Ratio
PCR
Polymerase Chain Reaction
PEEP
Positive End Expiratory Pressure
PI
Principal Investigator
PT/INR
Prothrombin Time / International Normalized Ratio
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PTSD
Posttraumatic Stress Disorder
PTT
Partial Thromboplastin Time
QC
Quality Control
QOL
Quality of Life
RCT
Randomized Control Trial
RNA
Ribonucleic Acid
RR
Respiratory Rate
RT-PCR
Reverse Transcriptase Polymerase Chain Reaction
SAE
Serious Adverse Event
SARS-CoV-2
Severe Acute Respiratory Syndrome Coronavirus 2
SOFA score
Sequential Organ Failure Assessment score
SpO2
Peripheral Capillary Oxygen Saturation, commonly also referred to as Oxygen
Saturation
SUSAR
Suspected Unexpected Serious Adverse Event
T Cells
Thymus Cells, also known as T Lymphocytes
TNF
Tumor Necrosis Factor, i.e., TNF-α
UP
Unanticipated Problems
VFD
Ventilator-free Day
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1. PROTOCOL SUMMARY
1.1 Synopsis
Title:
Bone Marrow Mesenchymal Stem Cell Derived Extracellular Vesicles Infusion
Treatment for COVID-19 Associated Acute Respiratory Distress Syndrome
(ARDS): A Phase II Clinical Trial
Study
Description:
ExoFloTM Infusion Treatment for COVID-19 Associated ARDS (EXIT
COVID-19), a multicenter, double-blinded, placebo-controlled, randomized
control trial to evaluate the efficacy and safety in COVID-19 associated
moderate to severe ARDS.
Objectives:
To evaluate the safety and efficacy of intravenous (IV) administration of bone
marrow mesenchymal stem cell derived extracellular vesicles (EVs), ExoFlo,
versus placebo as treatment for COVID-19 associated moderate-to-severe
Acute Respiratory Distress Syndrome (ARDS).
Endpoints:
Primary Endpoint:
1)
Improvement in partial pressure of arterial oxygen to fraction of inspired
oxygen (PaO2/FiO2) ratio from pre-infusion baseline (Day 0) to Day 7.
PaO2 may be calculated from arterial blood gas (ABG) or imputed from the
SpO2 daily (See Appendix 11.1).
Note: Day 0 is designated as Day of Screening and Day 1 as Day of the First
Study Intervention. Patients may be screened and treated within the same 24
hours—in this scenario, Day 1 will be synonymous with Day 0 & the pre-
infusion value used will be from Day 1.
Secondary Endpoints:
2)
Time to recovery as defined by the number of days from the first study
treatment until return of oxygenation saturation (SpO2) ≥ 93% on room air
(or PaO2/FiO2 ≥ 300 mmHg). If patient has chronic lung disease, recovery
is defined as pre-COVID-19 SpO2 and O2 support.
3)
Incidence of serious adverse events.
4)
All-cause mortality.
Exploratory Endpoints:
5)
Viremia: qualitative serum severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) ribonucleic acid (RNA) load on Days 0 or 1, 15, 29, and 61.
6)
Acute phase reactants: C-reactive protein (CRP), D-dimer, Ferritin,
interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) on Days 1, 4, 7,
10, 15, 29. CRP, D-dimer, & Ferritin also on Day 0.
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7)
Immune cell counts: Absolute neutrophil count (ANC); CD3+, CD4+,
CD8+ thymus cells also known as T lymphocytes (T cells); natural killer
(NK) cells on Days 1, 4, 7, 10, 15, 29.
8)
Sequential Organ Failure Assessment (SOFA) Score on Days 1, 15, 29 for
patients who are still hospitalized.
9)
Quality of life (QOL) assessment for patients who are discharged. EQ-5D-
5L on Days 29 and 61. See Appendix 11.2 for sample EQ-5D-5L, which
includes dimensions of mobility, self-care, usual activities, pain/discomfort,
and anxiety/depression.
Study
Population:
Up to 120 adult male and female patients between 18 and 85 years of age
hospitalized with COVID-19 associated ARDS.
Phase:
Phase II
Site Number:
2-15
Description of
Intervention:
Patients will be randomized to one of the following:
Treatment Arm 1: PLACEBO Normal saline 100 mL
Treatment Arm 2: IP (Exoflo) 10ml dose in Normal saline 90 mL, which is
approximately 800 billion EVs (Lot# P-441-1901-E5, P-441-2004-C5).
Treatment Arm 3: IP (Exoflo) 15ml dose in Normal saline 85 mL, which is
approximately 1.2 trillion EVs (Lot# P-441-1901-E5, P-441-2004-C5).
The intervention will be infused over 60 minutes on Day 1 and repeated on Day
4 provided that the patient has not recovered as defined by SpO2 ≥ 93% on RA
or PaO2/FiO2 ≥ 300 mmHg. If patient has chronic lung disease, recovery is
defined as baseline SpO2 and O2 support.
Allocation of
Intervention:
Patients will be randomized 1:1:1 to each of the three treatment arms initially.
The Interactive Response Technology will notify the DSMB such that a safety
and interim analysis can be held following day 7 of the 60th patient randomized.
Duration:
Each patient will be enrolled in the study for an estimated 60 days.
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Perform basic evaluation, respiratory evaluation, standard labs, acute phase
reactants, flow cytometry labs, SOFA score, adverse event review per section 1.3.
Repeat the same treatment received on day =1, provided that the patient has not
recovered as defined by SpO2 ≥ 93% on RA (or P/F ratio ≥ 300 mmHg) or pre-
COVID-19 O2 support & SpO2 if patient has chronic lung disease.
Perform basic evaluation, respiratory evaluation (includes SARS-CoV-2 RT PCR & O2
support recording), standard labs (BMP, CBC differential, LFTs, PT/INR), acute phase
reactants, flow cytometry labs, SOFA score, microbiology & rule out labs, adverse
event review per section 1.3 Schedule of Activities.
Administer the randomly assigned treatment.
Perform basic evaluation, respiratory evaluation, standard labs, acute phase
reactants, flow cytometry labs, SOFA score, adverse event review per section 1.3 if
still hospitalized.
Randomize
Arm 1
100 mL NS + SOC
N=40*
Arm 2
10 mL IP & 90 mL
NS + SOC N=40*
N Up To 120: Obtain written informed consent. Screen potential participants by
inclusion and exclusion criteria; obtain history and lab values.
1.2 Schema
Prior to
Enrollment
Arm 3
15 mL IP & 85 mL
NS + SOC N=40*
Day = 1
Day = 4
Day = 7, 10, 15, 29
Day = 15, 29, 61
Abbreviations: AE=adverse event; BMP=basic metabolic profile; CBC=complete blood count; EQ-5D-5L=quality of life
assessment; IP=investigational product; IV=intravenous; LFT-liver function test; NK cells=natural killer cells;
NS=normal saline; P/F ratio = partial pressure of arterial oxygen to fraction of inspired oxygen ratio; PT/INR=
prothrombin time/international normalized ratio; RNA=ribonucleic acid; SOC=standard of care; SOFA=sequential
organ failure assessment score; SARS-CoV-2=severe acute respiratory syndrome coronavirus 2.
Perform basic evaluation, respiratory evaluation, standard labs, acute phase
reactants, SOFA score per section 1.3 if still hospitalized. Follow-up call with EQ-5D-
5L if patient is discharged. Interim adverse event review is performed.
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1.3 Schedule of Activities*
NOTE: If eligibility criteria are met, patients may receive the first study intervention on the
same day as screening, such that Day 1 is synonymous with Day 0 and the pre-infusion value
from Day 1 may be used in place of the value from Day 0. See table footnotes A-J for answers
to other common site questions.
Table 1. Schedule of Activities.
Screen
Day
PROCEDURES
0 or 1
1
4
7
10
15
29
61
Visit
0
1
2
3
4
5
6
7
BASIC EVALUATION
Informed consent
X
Inclusion/Exclusion
X
X
Demographics
X
Medical history
X
Days of Illness Before Admission
XA
Concomitant Meds
X
X
XB
XB
XB
XB
XB
XB
Vital SignsC
X
X
XB
XB
XB
XB
XB
XB
Height & WeightI
XI
Physical examination
X
X
XB
XB
XB
XB
XB
XB
Glasgow Coma Score
X
X
XB
XB
XB
XB
XB
XB
Pregnancy TestD
XD
XD
EKGI
XI
PRN AS INDICATED; N/A AFTER D/C
Randomization
X
Administer IV Study Intervention
X
XE
RESPIRATORY EVAL
SARS-CoV-2 RT-PCRF
X
XJ
XB
XB
XB
Record Prone Posn (Y/N, Freq)
X
XJ
XB
XB
XB
XB
XB
XB
PaO2/FiO2 ratio
X
CALCULATE DAILY UNTIL D/C; SEE SECTION 11.1
Record O2 Support
X
DAILY UNTIL D/C; NOTE NC (LPM), FM (LPM), NRB, BiPAP (FiO2),
HFNC O2 (FiO2), MV (FiO2, PEEP), HFOV (FiO2).
CXR or CT chest
X
PRN AS INDICATED; N/A AFTER D/C
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STANDARD LABS
BMP
X
XJ
XB
XB
XB
XB
XB
XB
CBC with differential
X
XJ
XB
XB
XB
XB
XB
XB
LFTS (including Bilirubin)
X
XJ
XB
XB
XB
XB
XB
XB
PT/INR
X
XJ
XB
XB
XB
XB
XB
XB
PTT
X
XJ
XB
XB
XB
XB
XB
XB
ACUTE-PHASE REACTANTS
CRP
X
XJ
XB
XB
XB
XB
XB
D-dimer
X
XJ
XB
XB
XB
XB
XB
Ferritin
X
XJ
XB
XB
XB
XB
XB
Il-6
XJ
XB
XB
XB
XB
XB
TNF-α
XJ
XB
XB
XB
XB
XB
FLOW CYTOMETRY
T-lymphocyte panel (stain CD3+,
CD4+, CD8+)
XJ
XB
XB
XB
XB
XB
NK cells count (CD3- CD56+ subset
of lymphocyte gate)
XJ
XB
XB
XB
XB
XB
METRIC
SOFA Score
XJ
XG
XG
EQ-5D-5L (Call Survey)
XH
XH
MICROBIOLOGY
UrinalysisI
XI
PRN AS INDICATED; N/A AFTER D/C
Urine cultureI
XI
PRN AS INDICATED; N/A AFTER D/C
Blood culture x 2I
XI
PRN AS INDICATED; N/A AFTER D/C
Sputum cultureI
XI
PRN AS INDICATED; N/A AFTER D/C
RULE OUT TESTS
Mycoplasma IgMI
XI
QuantiFERON GoldI
XI
Legionella AgI
XI
Strep. Pneumoniae AgI
XI
Influenza A/B PCRI
XI
Adverse Events Review
X
DAILY UNTIL D/C; CALL F/U ON DAY 15, 29, 61
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Abbreviations: Ag=antigen; ANC=absolute neutrophil count; BiPAP=bilevel positive airway pressure; BMP=basic
metabolic profile; CRP=C-reactive protein; CBC=complete blood count; char=characteristics; CT=computed
tomography; CXR=chest x-ray; D/C=discharge; EKG=electrocardiogram; EQ-5D-5L=5-dimentional quality of life
assessment; FiO2=fraction of inspired oxygen; FM=face mask; Freq=frequency; HF NC O2=high flow nasal cannula
oxygen support; HFOV=high frequency oscillatory ventilation; IgM=immunoglobulin M; IP=investigational
product; IV=intravenous; LFT-liver function test; LPM=liters per minute; Meds=medications; MV=mechanical
ventilation; N/A=not applicable; NC=nasal cannula; NK cells=natural killer cells; NRB=nonrebreather;
PCR=polymerase chain reaction; PEEP=positive end expiratory pressure; POS=positioning; PT/INR= prothrombin
time/international normalized ratio; PTT=partial prothrombin time; Quant=quantitative; RT-PCR=reverse
transcriptase polymerase chain reaction; SARS-CoV-2=severe acute respiratory syndrome coronavirus 2;
SOFA=sequential organ failure assessment score; T cells=thymus cells also known as T lymphocytes; TNF-
α=tumor necrosis factor alpha.
A: The number of COVID-19 symptomatic days prior to the current admission is recorded along with the number
of days from admission to first study administration.
B: Patients will be monitored for 72 hours as inpatients following each study intervention. Recovery will be defined
in the study as SpO2 reaching ≥ 93% on RA (or P/F ≥ 300 mm Hg) or pre-COVID-19 SpO2 or O2 support if patient
has chronic lung disease.
•
Following the first treatment on Day 1, the patient may be discharged on Day 4 or later.
•
Following the second treatment on Day 4, the patient may be discharged on Day 7 or later.
•
If a patient received the first but not the second treatment, he/she may be discharged prior to Day 7.
•
Following patient’s discharge, no labs will be drawn. Follow-up via phone-call only on Days 29 and 61.
C: During the infusion, measurements +/- 2 minutes of specified times for q5min vitals and +/- 5 minutes of
specified times for q15min vitals are considered permissible; following the infusion, vital sign measurements
are permissible +/-15 minutes of the specified times. Temperature is required only 5 minutes prior and q15min
during the course of the infusion.
D: Serum pregnancy test (in women of childbearing potential) will be obtained on Day 0. Urine dipstick obtained
on Day 1 at 4 hours prior to the first study treatment if >24 hours passed since the serum pregnancy test.
E: Not all patients who recovered will receive a repeat study intervention. Patients who recovered by Day 4 do
not receive a repeat study intervention.
F: Qualitative SARS-CoV-2 RT PCR will be used for Day 0 or 1 (prior to the first dose), 15, 29, 61. If there is a
shortage of tests, documentation of positive test within 14 days prior to admission suffices for Day 0 or 1.
G:
SOFA Score will be administered only if patient is still hospitalized.
H: ED-5D-5L score will be administered over the phone only if patient is discharged. Note: This is not a meaningful
measure for patients who are still hospitalized due to common use of IV sedatives or PO antipsychotics for
delirium. A baseline prior to study infusion is not obtained because the patient is hypoxic, and the hospital
staff needs to prioritize diagnostics and treatment rather than time-consuming quality of life metric.
I: The Screen column refers to tests obtained prior to the first study treatment, usually obtained on Day 0, i.e., 24
hours prior to the first study treatment. However, height & weight, EKG, microbiology, and rule out tests do
not have to be repeated on Day 0 if obtained once following the current hospital admission.
J: All lab tests including SARS-CoV-2 viremia, standard tests, acute phase reactants, flow cytometry will be
obtained around 1-2 hours prior to the first study treatment on Day 1. On Day 1, SOFA score will be calculated
on the morning values prior to the study treatment.
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2. INTRODUCTION
2.1 Study Rationale
The objective of this clinical trial is to evaluate the safety and efficacy of ExoFlo, a bone-marrow
mesenchymal stem cell (bmMSC) derived extracellular vesicles product, via IV infusion, for the
treatment for ARDS in patients with severe COVID-19. Containing a panoply of chemokines,
messenger RNA (mRNA), and noncoding RNA (microRNA or miRNA) secreted from bmMSCs,
extracellular vesicles are the essential paracrine mediators of bmMSC function—which is not
necessarily engraftment or differentiation at the target tissue, but rather cell-to-cell
communication.[1,2] Extracellular vesicles retain the potent anti-inflammatory effects of bmMSCs,
but are acellular and nonimmunogenic, containing no nucleus or deoxyribonucleic acid (DNA).[3,4]
And at 1/1000 the size of an MSC, extracellular vesicles pass easily through capillaries, potentially
rendering safer IV dosing and redosing compared to allogeneic stem cells.
Treatment with bmMSCs has already shown promise in the treatment of COVID-19 related ARDS,
other sepsis-based ARDS, and other hyperinflammatory disease states.[4-11] These findings taken
together with the role of extracellular vesicles as the primary therapeutic mediators of bmMSC
function, and the ongoing absence of any proven therapy for COVID-19, prompted our physician-
investigators to conduct the first clinical trial worldwide on therapeutic use of bmMSC-derived
extracellular vesicles — in an open label phase I study, 24 patients with moderate to severe ARDS
in setting of severe COVID-19 were enrolled at the height of the pandemic and received a single
IV 15 mL dose of IP.[12] All safety endpoints were met with no immediate infusion-related adverse
reactions in addition to no treatment-attributable adverse events (AEs). Furthermore, following a
single infusion of IP, 17 out of 24 patients demonstrated a profound reversal of their initial hypoxia,
correlating with rapid reduction in supplemental oxygen and median time to recovery of 5.6
days.[12]
Exploratory endpoints in our preliminary clinical trial revealed statistically significant
improvements in acute phase reactants, absolute neutrophil counts (ANC), and T-lymphocyte
subsets following treatment with the IP—suggesting that the therapeutic mechanisms of action for
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ARDS may be related to the reduction of inflammation and the reconstitution of adaptive
immunity.[12] Independent molecular characterization of the IP have since indicated other
promising therapeutic actions, which may be specific for COVID-19 associated ARDS. Notably,
we have identified many highly expressed and unique chemokines in IP including 27 miRNA,
which are potential inhibitors of the ACE-2 related protein network; 7 miRNA, which are
inhibitors of serine protease 2 (TMPRSS2)—a transmembrane protease that is required for Spike
(S) protein priming and SARS-CoV-2 entry into a host cell; 5 miRNA that inhibit IL-6, a regulator
of the acute phase response that can lead to the severe systemic inflammatory response known as
“cytokine storm” and activation of the coagulation cascade when present at an excessive level; and
2 miRNA that target tumor necrosis factor alpha (TNF-), a proinflammatory cytokine, that also
plays a prominent role in mediating cytokine storm.
As the COVID-19 pandemic has continued, with substantial concern for resurgence in the US,
these findings evince the need for further and more rigorous study of extracellular vesicle therapy
in the way of a double-blinded, randomized controlled trial (RCT). However, to date, there has
been no randomized study on the safety and efficacy of bone-marrow derived extracellular vesicles
for the treatment of severe COVID-19 nor for the treatment of ARDS.
This study should be of national and global interest as timely treatment with MSC extracellular
vesicles can potentially turn the tide of the devastating disease process of COVID-19, not to
mention its catastrophic and evolving economic consequences. Thus far, purported treatments for
COVID-19 associated viral pneumonia and ARDS, involving single-target agents such as
hydroxychloroquine and ritonavir/lopinavir, have shown mixed results.[13-17] A phase III study of
remdesivir reached statistical significance with median time to recovery (11 vs 17 days in treatment
vs placebo) but not reduction in mortality rate.[18] Our preliminary clinical study using the IP as
treatment for COVID-19 showed a superior median time to recovery of 5.6 days and was performed
in a population of patients with baseline demographics and clinical characteristics, which
portended poorer outcomes. Eighty-three percent (83%) of the patients had type II diabetes; all
patients had pre-enrollment PaO2/FiO2 ratio consistent with moderate (46%) or severe ARDS
(54%).[12]
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Due to the potent immunomodulatory properties, the IP may have the therapeutic potential of
reducing mortality rate in COVID-19 related ARDS as well as other sepsis-associated ARDS,
especially when used as part of an early goal directed therapy. To date, there is still no disease-
altering treatment for ARDS that has been shown to reduce mortality rate.[19-23] The lack of
significant therapeutic advancement has been acknowledged by National Institutes of Health (NIH)
ARDS Clinical Trials Network, which has since declared the necessity of prioritizing prevention
and early treatment of ARDS.[24,25] If the success of early goal directed therapy in sepsis is any
indication, recognizing ARDS as a continuum rather than fixating on the positive pressure
mechanical ventilation (MV) criteria of the Berlin definition may be the essential first step in
finally changing mortality outcomes in patients with ARDS.[24,26] Thus, one distinguishing feature
of this study will be the inclusion of non-intubated patients with ARDS as defined by all other
criteria of the 2012 Berlin definition—a purposeful identification, which has been adopted in other
key studies in critical care, in order to identify at risk patients and facilitate timely treatment.[27-34]
2.2 Background
2.2.1 The Status Quo in Healthcare
Since December 2019, COVID-19, the disease caused by the SARS-CoV-2 strain of the
coronavirus, has expanded into a global pandemic.[35-38] Due in part to its population density, New
York City and its immediate vicinity, became the initial epicenter of COVID-19 in the US.[39,40]
Common symptoms of COVID-19 include fever, non-productive cough, nasal congestion, and
fatigue. A significant portion of the population may not exhibit pronounced symptoms during the
first 10-20 days of infection, while symptomatic patients may present to a healthcare facility with
reduced oxygen saturation (SpO2), ABG revealing hypoxia, and chest X-ray revealing bilateral
infiltrates.[37,41,42] The remaining labs may be significant for leukocytosis, lymphopenia, elevated
acute phase reactants, and biomarkers of end-organ damage such as renal and myocardial
injury.[43,44] In patients with underlying health conditions, an estimated 30% of the infected
population will require hospitalization—once admitted, roughly half of this population will require
MV in an intensive care unit. Early observations cites mortality rates as high as 88-94% following
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intubation, indicating MV as a significant predictor of mortality at rates which exceeded even those
observed in other-sepsis associated ARDS.[37-40,45] Early observations also cite surprisingly high
mortality rates of 68% and higher in patients requiring noninvasive oxygen support, which
indicates the importance of timely intervention..[40,46-48]
Due to the explosion of cases in the first wave, concerns regarding resource limitations, and
emerging understanding of how best to treat COVID-19, major urban hospitals have developed
increasing thresholds for hospital admission as well as MV.[49,50] Prior to the pandemic, patients
presenting with acute onset of fever, shortness of breath, and hypoxia, meeting criteria for
moderate to severe ARDS, would typically be intubated. However, these patients are now first
maintained with noninvasive supplemental O2 and other optimization measures like intermittent
proning, with endotracheal intubation delayed for as long as possible. This group of patients holds
significant interest for this study, as an appropriately timed intervention could substantially reduce
progression to complete hypoxic respiratory failure requiring MV, a critical event associated with
high morbidity, mortality, and healthcare expenditure.
The paradigm shifts in the treatment algorithm for COVID-19 induced respiratory failure has
evolved in response both due to the perceived shortage in resources in addition to the increasing
controversy surrounding the current understanding of COVID-19 pulmonary disease.[51] There is
a growing consensus among physicians that COVID-19 pulmonary disease is poorly understood,
and MV may pose more harm than benefit.
2.2.2 Rationale for Exploring New Treatment
Common practices for COVID-19 thus far included: (1) empiric antibiotics for presumptive
bacterial coinfection, (2) initiation of potential disease altering medications such as antivirals, (3)
daily intermittent proning of both intubated and non-intubated patients, (4) addition of steroids in
patient who met criteria for moderate to severe ARDS, and (5) the addition of empiric
anticoagulation in hypercoagulable patients.[46,52,53] Strictly speaking, while all therapies for
COVID-19 are considered experimental therapies, many practices have already been adopted by
hospitals systems across the US as a basic regimen for severe COVID-19.
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Trials for experimental single target agents, including antivirals, antibiotics, and biologics, like
remdesivir, hydroxychloroquine, and tocilizumab, respectively, have yielded mixed outcomes with
some associated with significant mortality and morbidity.[13,14,16-18] For example, remdesivir has
been associated with significant hepatotoxicity that requires discontinuation in some patients and
Actemra® (tocilizumab) has been associated with significant thrombocytopenia, gastrointestinal
perforation, and leukopenia.[16,54] Other options for prevention and treatment include vaccination
and convalescent plasma—both of which may pose the risk of antibody- dependent
enhancement—a cascade of events whereby non-neutralizing antibodies bind to the newly
infecting virus and enhance the viral invasion of host cells.[55-58] Vaccination and convalescent
plasma also notably require relatively stable viral epitopes for efficacy—this is a problematic
requirement for treatment efficacy since much like the RNA virus HIV, SARS-CoV-2 directly
suppresses host T-cell function.[59,60] Clinically, this has been borne out with frequent presentations
of multi-organ failure secondary to immunodeficiency even in previously healthy individuals; use
of antiretroviral may show initial efficacy but only transiently.
The Food and Drug Administration (FDA) has yet to approve for official use a COVID-19 potent
medication that can account for the mutability of SARS-CoV-2 coronavirus as well as enhance
both innate and adaptive immunity.[61-64] The mortality rates for critically ill patients with COVID-
19 range 50-94% in some hospitals, vastly exceeding the well-studied mortality rate of 27-45%
reported for mild, moderate, and severe ARDS of the past.[23,39,40] Clearly, the continued
exploration of treatments for severe COVID-19 is greatly needed.
2.2.3 COVID-19: Pathophysiology
The pathophysiology of COVID-19 has been studied with intense interest while the exact
mechanism remains elusive. The prevailing theory is that the virus gains entry into host cells
through cross-linking of the SARS-CoV-2 spike protein (S protein) with angiotensin-converting
enzyme 2 (ACE2) receptor on host cells, thereby leading to the fusion of the viral and host cell
membrane.[65] Following entry into the host cell, the viral RNA is translated into protein in the host
cell’s cytoplasm and then assembled into more viral copies (viral envelope containing genomic
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RNA) with subsequent secretion and further invasion of host cells.[66] The normal role of ACE2 is
the inactivation of angiotensin II. Early studies show that ACE2 is downregulated in response to
SARS-CoV2, rendering it unavailable to play its physiologic role, and thereby leading to the
accumulation of Angiotensin II. In addition to acting as a profound vasoconstrictor, Angiotensin
II, also elicits increased synthesis and elaboration of pro-inflammatory, pro-fibrotic, pro-
thrombotic, and pro-oxidative factors, thereby causing local injury to the tissue.[67] Especially
considering its ubiquitous expression in lung, heart and kidney tissues, the accumulation of
Angiotensin II may be central to the hyper-inflammatory state, referred to as cytokine storm.[63,64]
This state observed in some cases of severe COVID-19 is believed to be amplified and sustained
by two evolving parallel processes: (1) the activation of macrophages and other antigen presenting
cells (APC), which then alert lymphocytes to the presence of the virus; (2) viral RNA replication
within host cells, which activates synthesis of proinflammatory factors.[68] Both pathways lead to
immune dysregulation and hyperinflammation. Indeed, immunological studies from Wuhan,
China, revealed lymphopenia in patients with severe cases of COVID-19, specifically among
CD3+, CD4+ and CD8+ thymus cells also known as T lymphocytes (T cells), as well as regulatory
T cells, the depletion of which likely facilitates ongoing immune evasion by the virus.[43]
Following findings of high mortality among intubated patients with COVID-19, many intensivists
have expressed doubt that the initial presentations were entirely consistent with classic ARDS.
These reservations originated from the observation that hypoxemia in a subset of patients with
severe COVID-19 is often out of proportion to their relatively normal work of breathing and lung
compliance.[51] Some physician researchers hypothesize that this uncoupling of hypoxemia and
lung mechanics may be due to polymorphisms of SARS-CoV-2 pathogenicity that causes distinct
phenotypes. Alternatively, some have suggested that distinct phenotypes may be separated into
early versus stages.
Ultimately, irrespective of the presenting phenotype, hospitalized patients with persistent COVID-
19 associated ARDS develop lung-mechanics and pathology similar to other sepsis- associated
ARDS. These patients accumulate inflammatory infiltrates in the lung parenchyma and airspaces,
oxidative stress from high FiO2 and mechanical microtrauma from positive pressure
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MV—all factors leading to diffuse alveolar damage, which is the most common histologic pattern
identified in patients with ARDS.[69] Pathologic examinations of patients with COVID-19
associated ARDS in Wuhan, China reveal alveolar epithelial injury, reactive hyperplasia of type
II pneumocytes, hyaline membrane formation, and fibroblastic plugs in the air-spaces, all of which
are indicative of how COVID-19 pulmonary disease can rapidly devolve into a fatal state of
minimal gas exchange.[70]
2.2.4 A Potential Treatment for COVID-19 Associated ARDS
As a regenerative medical therapy, bone-marrow derived extracellular vesicles can potentially shift
the tide of the devastating disease process caused by COVID-19. Extracellular vesicles are
naturally occurring vesicles produced by most eukaryotic cells and are the primary mode of
intercellular paracrine signaling.[2,3,71-73] Exosomes, one of the primary types of extracellular
vesicles, are typically 30-150 nanometers in diameter and approximately 1/1000th size of a cell.
These signals are not cells and contain no nucleus or DNA. The population of extracellular vesicles
secreted by bone marrow-derived mesenchymal stem cells have been studied extensively in
preclinical studies of inflammation and are notable for their ability to downregulate inflammation
and upregulate repair.[1-3,74-81]
IV administration of bmMSCs-derived exosomes has already shown safety and potential efficacy
in investigational studies in patients with ARDS. In 2015, the phase I START trial enrolled 9
patients with moderate-to-severe ARDS and monitored outcomes for 60 days following a single
dose of IV administration; no SAEs were observed in the 6 hours following the infusion nor in the
weeks following the one dose infusion of allogeneic bmMSCs, including up to 10 million
MSCs/kg.[7] In a recent pivotal study in Wuhan, China in March 2020, transplantation of bmMSCs
from healthy donors into seven patients with COVID-19 pulmonary disease improved functional
outcomes without any observed adverse effects including infusion-related or allergic reactions
within two hours after treatment nor delayed hypersensitivity or secondary infections.[5] Within 2
days of the treatment, 6 out of 7 patients recovered; within 3 days, 3 out of 7 patients were
discharged
from
the
hospital.
Exploratory
endpoints
were
significant
for
decreased
pro-
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inflammatory cytokine TNF-α and for increased anti-inflammatory IL-10—suggesting that one
therapeutic value of bmMSCs in patients with COVID-19 may be cytokine storm inhibition.[5]
Given that the therapeutic properties of bmMSCs is primarily due to mediation of cell-to-cell
communication via secretion of soluble factors and extracellular vesicles, rather than actual
engraftment and differentiation into target tissue, the treatment potential of extracellular vesicles
purified from bmMSCs hold particular clinical interest during the COVID-19 pandemic.
Containing a panoply of chemokines, mRNA, microRNA, exosomes and other extracellular
vesicles promote the synthesis of regenerative, tissue protective, antimicrobial, and anti-
inflammatory proteins and silence the expression of pro-inflammatory genes that fuel the cytokine
storm.[1] These signals also regulate immune dysregulation at an epigenetic level by influencing
histone methylation and acetylation. And since extracellular vesicles are the primary therapeutic
effector of MSCs, the question arises as to why US physicians would accept the risks of allogeneic
stem cell transplant, such as graft versus host disease (GvHD), when the same therapeutic effects
can be derived from a significantly more standardized product and administered at much lower
risk to the patient.
The IP is a bone marrow derived extracellular vesicle product notable for its high production
standards, purity, and potency. Extensive characterization of the extracellular vesicles contained
in the IP has revealed an absence of immunogenic surface epitopes that make it highly unlikely to
cause acute immune reactions, and there are no known product reactions or reported adverse
reactions to date. The IP exerts a powerful anti-inflammatory effect as well as promotes
regeneration of tissues damaged by inflammation. Each mL of IP, from the lots to be used in this
study (P-441-1901-E5 and P-441-2004-C5), contains approximately 80 billion extracellular
vesicles, and each mL is comprised of over 2000 different anti-inflammatory cytokines, anti-
oxidative stress, pro-regeneration, tumor suppressor, and antimicrobial agents--the potential for
changing the course of ARDS in patients with COVID-19 is therefore compelling (see Table 2 for
a sample overview).
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Table 2: Classes of Factors Contained in the IP for this Study
Class
Factor
Description
Function
Angiogenesis
& Wound
Healing
uPAR
CD87
Wound and Tissue Healing
VEGF
Vascular endothelial growth
New blood vessel formation
Thrombomodulin
CD141, thrombin cofactor
Anti-clotting factor, wound remodeling
CD97
G protein-coupled receptor
Promotes angiogenesis attracts endothelial
Chemotaxis,
Cell Migration
IGFBP2
Insulin growth factor binding
Regulates IGF levels, Supports TIMPs and
TSLP
Thymic Stromal Lymphoprotein
T-Cell Differentiation and Recruitment
NCAM
Neuronal Cell Adhesion
Cell Adhesion to Neurons, NK Cells
NUP85
Nucleoporin85
Monocyte migration; mRNA transport &
Immune
Modulation
MIF
Macrophage inhibitory Factor
Regulates macrophages-Anti-inflammatory
TNF-𝑎 RI
Tumor necrosis factor- 𝑎 receptor
Negative regulation of TNF-a- Anti-
IL1-R6
Interleukin 1 Receptor 6
Cytokine, chemokine and antimicrobial
PF4
Platelet factor 4
Antimicrobial activity
Tumor
suppressor
IGFBP-4
Insulin growth factor binding
Anti-tumorigenic In vivo & in vitro; binds
bIG-H3
TGFB Induced protein
ECM protein induced by TGFB to inhibit
Serpin F1
Secreted multifunctional protein
Anti-tumorogenic, anti-angiogenic,
DKK3
Dickkopf-related protein 3
Wnt signaling, tumor suppressor
ECM
Development
&
Remodeling
Cathepsin B
Catabolic Protease
Collagen matrix Remodeling
TIMP-1
Collagenase Inhibitor
Regulates Collagen Remodeling
TIMP-2
Collagenase Inhibitor
Regulates Collagen Remodeling
FAP-A
Fibroblast activation protein,
Regulation ECM integrity, collagen content
Regenerative
Semaphorin 6c
Signal regulator of tissue
Nervous system development, cartilage r-
IGF2
Insulin-like Growth Factor 2
Reproductive and brain tissue regeneration
FGF-16
Fibrolast Growth Factor 16
Cardiomyocte proliferation, heart tissue
Molecular characterization of over 2000 specific chemokines, mRNA, and microRNA in the IP
reveal the therapeutic potential for ARDS and COVID-19 due to their associations with increasing
bacterial clearance, restoring lung protein permeability, increasing alveolar fluid clearance,
stabilizing the capillary alveolar barrier, and reprogramming immune cells toward anti-
inflammatory phenotype. This observation is supported by multiple preclinical studies on the role
of MSC exosomes in acute lung injury and ARDS models revealing that (1) these signals can
suppress the secretion of TNF-α and other pro-inflammatory cytokines, (2) promote the secretion
of anti-inflammatory cytokines, including IL-10, (3) repair human lung microvascular endothelial
cells by increasing expression of Ang-1, (4) induction of anti-inflammatory M2 phenotypes in
macrophages, (5) promote regulatory T cell proliferation by increasing expression of TGFβ1 and
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reducing circulating levels of IL-6, and (6) enhance alveolar and lung edema fluid
clearance.[2,74,77,80]
Clinically, the safety and efficacy potential of the IP has already been explored in our preliminary
open-label single-arm prospective study. During April 2020, at the height of the pandemic,
24 patients with severe COVID-19 received a single dose IV IP for moderate to severe ARDS.[12]
Following a single dose of IV IP, 17 out of 24 patients showed profound improvement in
oxygenation, as measured by the PaO2/FiO2 ratio, within 48-72 hours. The median time to
discharge was 5.6 days. Exploratory endpoints were significant were reduced acute phase reactants
include CRP, D-dimer, and Ferritin in addition to reduced neutrophil count and improved CD3+,
CD4+, and CD8+ T cells.
In addition to the availability of preliminary clinical safety and efficacy data, the IP (bone marrow-
derived extracellular vesicles) manufactured by Direct Biologics, should be considered for use due
the following reasons pertaining to safety, efficacy, reproducibility, and overall quality.
(1)
The IP has undergone current good manufacturing processes (cGMP): the IP is a bmMSC
derived extracellular vesicle product produced under cGMP standards. The bmMSCs and
media both have master files on record at the FDA. The IP meets donor and manufacturing
safety profiles, has lot-specific tissue traceability and a comprehensive labeling including
detailed instructions for use.
(2)
Safety profile in clinical use: There are no documented AEs associated with the use of this
product. Our preliminary Institutional Review Board (IRB) approved clinical study showed
no adverse effects within 24 hours of IV administration and no suspected unexpected
serious adverse reaction (SUSAR) or treatment-attributable SAE within 14 days following
IV administration of the IP to 24 patients with severe COVID-19.
(3)
No allogeneic DNA exposure: In contrast to MSC transplantation treatment with
extracellular vesicles does not involve the introduction of foreign DNA, thereby
eliminating the complexities and variability involved in human-to-human MSC
transplantation.
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(4)
Size advantage: It has been demonstrated that due to their size, nearly 100% of MSCs
administered by an IV route become lodged in and occlude pulmonary capillaries. In
contrast, extracellular vesicles (30-150 nanometers) are roughly 1000x smaller than MSCs
and easily pass through capillaries, thereby eliminating the risk of pulmonary vascular
occlusion in the setting of COVID-19 pulmonary disease. An additional benefit conferred
by the smaller size of these vesicles, as in the case of the IP, they can be delivered in highly
concentrated doses, is that multiple doses can be given without increasing the risk for
vascular occlusion.
(5)
Extensive characterization: Extracellular vesicles derived from bmMSCs are the most
extensively studied in the peer-reviewed literature and have been shown to be safer and
possess more favorable signaling factors from a clinical standpoint. In addition, the
molecular characterization of the IP is readily available.
(6)
Lack of immunogenicity: Extracellular vesicles are not known to express surface epitopes
that would be recognized by the immune system as foreign and can be easily redosed for
efficacy whereas a cellular biologic has a much higher risk of eliciting an immune reaction,
GvHD, or other reactions which could be amplified with redosing. Additionally, the use of
xeno-free medium in the IP’s manufacturing process further reduces the likelihood of an
adverse immune response. To date, there are no known reactions to infused or injected IP.
(7)
Standardization & scalability: As in the case of the IP, which is produced under rigorous
conditions described below, extracellular vesicle products can be produced with a high
level of quality and consistency that most closely approximates a pharmaceutical grade
product. In contrast, perinatal exosomes have a high level of batch-to-batch variability and
contamination rate related to the use of multiple donors, the absence of external FDA
validation processes, the use of immunogenic bovine serum in cellular cultivation
processes, and the heterogeneity of exosomes obtained from a source where maternal and
fetal exosomes are inevitably co-mingled.
(8)
Product stability and ease of storage: While extracellular vesicles require storage ≤ -40°C,
short-term storage up to 14 days at 2-8°C has been shown not to degrade the particle
distribution size of the IP, indicating its stability. Storage of these vesicles does not require
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preservatives nor a full facility, which is necessary, for example, for the storage of live
cells in the context of bone marrow or stem cell transplantation.
(9)
Scalability: the IP, the bone marrow derived extracellular vesicles product used in this
study has been uniformly tested for quality. These signals originate from a single human
donor and are abundantly available due to proprietary production technology. In contrast,
bmMSCs and perinatal exosomes must be harvested from different human donors after a
meticulous vetting process, posing challenges to uniform quality control, and immediate
availability. Furthermore, the use of extracellular vesicles precludes the need for a
transplant facility to store living cells, which increases the number of facilities that can
administer therapy.
(10)
Lack of carcinogenicity: bmMSCs and their signals have been extensively characterized
without evidence of oncogene expression. The safety and standardization of bone marrow
derived extracellular vesicles is superior compared to MSCs in addition to amniotic
exosomes, also known as amniosomes, which is a tissue product of inherently high
variability when considering each lot is harvested from a different donor.
2.3 Risk/Benefit Assessment
2.3.1 Known Potential Risks
There are no known immediate or long-range potential risks based on preclinical studies of bone
marrow derived extracellular vesicles. Specifically regarding ExoFlo, there are no known reported
immediate or long-range potential risks following intra-articular treatment for osteoarthritis based
on a preliminary clinical study. There are also no known immediate risks (≤72 hours) based on our
preliminary open-label safety study on 24 patients with severe COVID-19 following a single
15 mL infusion of IP.[12] The patients were followed for 14 days following treatment. There were
no immediate infusion-related adverse reactions within 4 hours or SAEs which were attributable
to the IP following independent Data Safety Monitoring Board (DSMB) review. Currently, there
are no known long-range potential risks of IV administration of the IP as treatment for severe
COVID-19.
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2.3.2 Known Potential Efficacy
Based our preliminary safety study, which was also revealing for potential efficacy of the IP,
known potential immediate benefit (≤72 hours) of the IP as IV treatment for severe COVID-19 is
improved oxygenation, as demonstrated by improved PaO2/FiO2 ratio and de-escalation of oxygen
support requirement within 48-72 hours following a single IV infusion; reduced inflammation, as
demonstrated by reduction of D-dimer, Ferritin, and CRP; reconstitution of adaptive immunity, as
demonstrated by improved neutrophilia and lymphopenia.[12]
One major potential long-range benefit (>72 hours) of the IP as IV treatment for severe COVID-
19 is improved survival. Seventy-one percent (71%) of the patients (17/24) recovered and were
discharged from the hospital within a mean of 5.6 days. Estimated 45-55% of the patients with
severe COVID-19 were discharged from the same hospital in the same month following standard
of care only, suggesting treatment with the IP was associated with increased likelihood of recovery.
Other potential long-range benefits include decreased incidence of intubation among non-
intubated patients, in addition to shorter duration of hospitalization.
2.3.3 Assessment of Potential Risks and Benefits
Given that the safety and efficacy of IV administration of the IP for severe COVID-19 has yet to
be proven in an appropriately powered randomized clinical trial (RCT), one primary feature of this
investigational study to maximize the potential benefit to risk ratio is that patients will only be
enrolled only if they are clinically deteriorating despite receiving standard of care for COVID-19
associated moderate-to-severe ARDS and no established alternative treatment is available. The
rationale of the necessity of exposing participants to potential risks is that they are manifesting
clinical deterioration from severe COVID-19, which is a life-threatening disease, despite the best
available and established clinical treatments. Other features of the study design that maximize the
potential benefit to risk ratio is the daily evaluation of AEs, use of stopping rules, and a staggering
protocol.
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3. OBJECTIVES AND ENDPOINTS
Table 3: Study Objectives, Endpoints, and Justification for Endpoints
OBJECTIVES
ENDPOINTS
JUSTIFICATION FOR ENDPOINTS
PRIMARY
The primary
Improvement in Partial
There is a high variation in physician practices and hospital
objective is to
pressure of arterial oxygen
practices, which affect chosen mode of O2 support and rate
evaluate efficacy of
to fraction of inspired
of intubation & ventilator weaning. Focusing on
IP as treatment for
oxygen (PaO2/FiO2) from
dichotomous variables of intubated/non-intubated or
COVID-19 associated
pre-infusion (Day 0)
ordinal scales of oxygen support overinflates the relative
moderate to severe
baseline to Day 7.
measure of true hypoxia. For example, FiO2 of a
ARDS* compared to
nonrebreather (NRB) can reach 0.8-0.9 while high flow
placebo.
oxygen (HF O2) is set at FiO2 of 0.8-1.0—defining HF O2 as
an entire class above NRB is problematic. Therefore, even
when imputing PaO2 from SpO2 (see Appendix 11.1),
PaO2/FiO2 ratio is still a comparatively more meaningful as
a standardized measure of oxygenation.
SECONDARY
The secondary
objective is to
evaluate the safety
and efficacy of IP as
treatment for COVID-
19 associated
moderate to severe
ARDS* compared to
placebo.
Time to recovery as defined
by the number of days from
the first study treatment
until return of oxygenation
saturation (SpO2) ≥ 93% on
room air (or PaO2/FiO2 ≥
300 mmHg).
Incidence of Serious
Adverse Events (SAE)
Patients who received IP + SOC are more likely to recover
faster compared to patients who received Placebo + SOC.
Recovery is defined as days from the first study treatment
until return of oxygen saturation (SpO2) ≥ 93% on room air
or to pre-COVID-19 baseline SpO2 if patient has chronic
lung disease. Recovery may also be defined as PaO2/FiO2
ratio ≥ 300 mmHg.
All adverse events must be reviewed and evaluated by an
independent Data Safety Monitoring Board (DSMB).
All-cause mortality rate.
All-cause mortality is included in most studies of ARDS and
COVID-19.
EXPLORATORY
The explorative
Qualitative SARS-CoV-2
Progression of severe COVID-19 is associated with
objectives are to
RNA level on days = 0 or 1
detectable viral load, rising levels of acute phase reactants,
evaluate whether IV
(prior to first dose), 15, 29,
worsening neutrophilia, depletion of lymphocytes, and
treatment with the IP
61 following the 1st
decreased functioning of natural killer cells (NK cells) in
is associated with
infusion of IP. CRP, D-
addition to concurrent multiorgan failure. Preliminary
significant surrogate
dimer, Ferritin, IL-6, TNF-α;
study on the IP shows acute phase reactants, neutrophilia
markers compared to
ANC; CD3+, CD4+, CD8+ T
and lymphocytopenia improved significantly within 48-72
placebo.
cells; NK cells on days = 1,
hours of the IP. The secondary endpoints will help
4, 7, 10, 15, 29 and 61.
determine whether the treatment mechanism of action is
via reduction of COVID-19 viremia, reduction of
inflammation, and/or enhancement of both adaptive and
innate immunity.
SOFA Score on days = 1, 15,
SOFA score is a common mortality prediction score used in
29.
sepsis research.[84] Individual scores can be useful as a
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Table 3: Study Objectives, Endpoints, and Justification for Endpoints
OBJECTIVES
ENDPOINTS
JUSTIFICATION FOR ENDPOINTS
EQ-5D-5L among patients
discharged from the
hospital on days = 29, 61
measure of organ dysfunction as pulmonary,
cardiovascular, hematologic, hepatic, renal, and neurologic
systems are all affected.
EQ-5D-5L is the widely used metric used in cost-effective
analysis.[82] Post-Intensive Care Syndrome among survivors
is underrecognized; prominent features include cognitive
dysfunction, posttraumatic stress disorder (PTSD)-like
symptoms, and muscle weakness.[85]
Abbreviations: ANC=absolute neutrophil count; BMP=basic metabolic profile; CRP=C-reactive protein;
CBC=complete blood count; CT=computed tomography; CXR=chest x-ray; EKG=electrocardiogram; EQ-5D-5L=5-
dimentional quality of life assessment; FiO2=fraction of inspired oxygen; HFOV=high frequency oscillatory
ventilation; IgM=immunoglobulin M; IP=investigational product; IV=intravenous; LFT-liver function test;
LPM=liters per minute; NK cells=natural killer cells; PCR=polymerase chain reaction; PEEP=positive end expiratory
pressure; PT/INR= prothrombin time/international normalized ratio; PTT=partial prothrombin time; RT-PCR=reverse
transcriptase polymerase chain reaction; SARS-CoV-2=severe acute respiratory syndrome coronavirus 2;
SOFA=sequential organ failure assessment score; Sp02=peripheral capillary oxygen saturation; T cells=thymus cells
also known as T lymphocytes.
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4. STUDY DESIGN
4.1 Overall Design
Phase:
Phase II
Description:
Multicenter, placebo-controlled, dose-ranging, double-blinded, RCT.
Intervention
Model:
All eligible participants will receive standard of care for severe COVID-19 while
being randomized to one of the following three treatment arms—in each arm, a 60-
minute study intervention is administered on Day 1. If the patient has not recovered
by Day 4, the study intervention is repeated on Day 4.
# Research Sites:
2-15; competitive enrollment will be used.
Study
Intervention:
(1) Placebo or IV 100 mL of NS, (2) IV 10 mL of IP mixed with 90 mL of NS, (3)
IV 15 mL of IP mixed with 85 mL of NS. (Lots# P-441-1901-E5, P-441-2004-C5).
Allocation &
Stratification:
An Interactive Response Technology (IRT) system will be used to electronically
randomize subjects in a blinded fashion. Participants will be randomized to three
treatment arms following stratification by research site.
Masking
Description:
Following randomized allocation of participants to each of the 3 treatment arms, the
hospital pharmacists, who are provided the study intervention identity by the IRT,
will independently prepare the appropriate infusion of (1) 100 mL of IV normal
saline, (2) 10 mL of IP mixed with 90 mL of normal saline, or (3) 15 mL of IP
mixed with 85 mL of normal saline for each patient.
4.2 Scientific Rationale for Study Design
The rationale for choosing a double blinded, placebo controlled, RCT is that this is the gold
standard for proving safety and efficacy of the IP compared to standard of care. Randomized block
design further minimizes selection bias. In this case, institutions may vary in terms of clinician
preferences, hospital resources, patient demographics, other characteristics (such as poor versus
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good outpatient follow-up), and severity of illness, in addition to overall effectiveness of ICU care.
Intubation status may be better stronger predictor of clinical deterioration rather than P/F ratio.
Adopting stratified randomization allows the randomization to be performed within each stratum
so that the balance between the treatment arms is as close to equal as possible.
4.3 Justification for Dose
4.3.1 Rationale for Dosing:
Dosing of the IP was calculated based on (1) the 24-patient preliminary COVID-19 clinical case
series with the IP; (2) the START trial, which is a phase I trial of IV administration of bone-marrow
derived stromal cells as treatment for ARDS (NCT01775774), which demonstrated safety of using
IV doses of 1 million cell/Kg, 5 million cell/Kg, in addition to a ceiling dose of 10 million cell/Kg;
(3) observation of approximately 2,000 extracellular vesicles secreted per stromal cell; and (4) lab
analysis of the IP (Lots# P-441-1901-E5 and P-441-2004-C5), which showed that each mL
contained approximately 80 billion extracellular vesicles. For an adult of 70 Kg, extrapolation
from the START trial MSC ceiling dose would yield an IV IP ceiling dose of 17.5 mL. Given a
range of body mass indexes (BMIs) that is typically on the higher end (BMI ≥30) when analyzing
hospitalized patients with severe COVID-19, 15 mL of IV IP was determined as a reasonable
starting dose for one treatment arm while 10 mL of IV IP was determined as a reasonable lower
end dose for a second treatment arm.
* Note: only IP lots # 441-1905-E5 and P-441-2004-C5 will be used in the clinical study.
4.3.2 Rationale for Dosing in Special Populations:
Pediatric population: The safety and efficacy of the IP for adults with COVID-19 have not been
assessed yet via RCT. Current use of the IP for children with severe COVID-19 is not under
consideration.
Pregnancy: The safety and efficacy of the IP for adults with COVID-19 have not been assessed
yet via RCT. Current use of the IP for pregnant patients is not under consideration. Of note, in the
proof-of-concept study, the IP was infused safely in one post-partum patient. Further investigation,
including any risk to the fetus, is required before use of the IP as treatment for severe COVID-19
in pregnant patients.
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Renal impairment: It is currently not known if dosage adjustment is needed in patients with renal
impairment. Preclinical studies of pharmacokinetics of exosomes reveal that clearance is via the
reticuloendothelial system, specifically the mononuclear phagocyte system (i.e., monocytes and
macrophages) in the liver and the spleen, rather than renal clearance, suggesting that dose
adjustment in this study for renal impairment is most likely unnecessary.[86, 87]
Hepatic impairment: It is not currently known if dosage adjustment is needed in patients with
hepatic impairment, especially if clearance of the IP can also be accomplished by monocytes and
macrophages localized to the lungs, spleen, and lymph nodes. Clearance of the IP likely also
depends on the total dose as well as the biodistribution to sites of injury.[88]
4.3.3 Rationale for Redosing:
Pharmacokinetics studies of the IP in patients with severe COVID-19 are ill advised as it would
require not only fluorescent tagging of surface proteins of the IP, but also serial MRI analysis of
the biodistribution of immunofluorescence. Sending a patient with severe COVID-19 for serial
MRI scans is neither practical nor safe, especially considering the progressing hypoxemia in many
of these patients. Therefore, our previous proof-of-concept study focused on exploratory endpoints
in order to observe a time-dependent effect—in this study, 23 out of 24 patients showed a favorable
trend in biomarkers within 48-72 hours, with the peak of the favorable biomarkers and PaO2/FiO2
occurring closer to 72 hours following the first infusion of the IP. Seventeen (17) out of the 23
initial responders had a sustained response following a single dose of the IP whereas the remaining
6 appeared to have “lost the effect” after 72-96 hours, suggesting that a second dose at day = 4
may have been helpful in improving clinical outcomes. Therefore, our RCT study will involve a
second dose on day 4 for patients who have not recovered when the second dose is due, where
recovery is defined by return of oxygenation saturation (SpO2) ≥ 93% on room air (or PaO2/FiO2
≥ 300 mmHg). If patient has chronic lung disease, recovery is defined as pre-COVID-19 SpO2
and O2 support.
4.4 End of Study Definition
A subject is considered to have completed the study if he or she has completed all the phases of
the study shown the Schedule of Activities (Section 1.3) or if the subject has expired.
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5. STUDY POPULATION
5.1 Inclusion Criteria
Eligibility for study enrollment includes meeting all of the following criteria:
1. Provision of signed and dated informed consent form (either by the individual or by the
individual’s healthcare proxy).
2. Stated willingness to comply with all study procedures and availability for the duration of
the study
3. Male or female aged 18-85.
4. COVID-19 positive as defined by positive Reverse Transcriptase Polymerase Chain
Reaction (RT-PCR) SARS-CoV-2.
5. Moderate to severe ARDS as defined by modified Berlin definition, * which includes
timing within 1 week of known clinical insult or new or worsening respiratory symptoms;
bilateral opacities not fully explained by effusions, or lung collapse; respiratory failure not
fully explained by cardiac failure or fluid overload; PaO2/FiO2 ≤ 200 mm Hg.
6. Hypoxia requiring noninvasive oxygen support such as Nasal Cannula (NC),
Nonrebreather (NRB), Bilevel Positive Airway Pressure (BIPAP), Continuous Positive
Airway Pressure (CPAP), high flow nasal cannula oxygen (HFNC O2) or mechanical
ventilation (MV) despite initiating standard of care.
7. If the candidate is either a male or female of reproductive potential, he or she must agree
to use of double barrier method of highly effective birth control contraception such as
condoms with oral contraceptive pill or choose to remain abstinent if already practicing
abstinence during the screening period. The required duration of usage of double barrier
method OR maintenance of abstinence must include the time from the beginning of the
screening period until 90 days following the last dose of the study treatment.
*Modified Berlin definition used in this study is the full Berlin definition, albeit without the PEEP
specification, which implies mechanical ventilation. See last paragraph of study rationale for
reasoning (Section 2.1).
**To ensure flexible adaptation of products approved by the FDA for the treatment of severe
COVID-19, standard of care is defined as the NIH Current COVID-19 Treatment Guidelines.[53]
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5.2 Exclusion Criteria
Exclusion from study enrollment includes meeting one or more of the following criteria:
1. Vulnerable populations such as pregnant patients, children, individuals with severe
physical or mental disabilities who cannot provide meaningful consent.
2. Active malignancy requiring treatment within the last five years.
3. Major physical trauma in the last 5 days, including motor vehicle accidents, assaults,
mechanical falls with sequalae of significant bleeding or craniofacial bruising, and
surgeries.
4. Active tuberculosis or cystic fibrosis.
5. Severe chronic respiratory disease including chronic obstructive pulmonary disease or
pulmonary fibrosis requiring home oxygen > 5L/min.
6. Use of extracorporeal membrane oxygenation (ECMO) during the current hospitalization.
7. Pre-existing pulmonary hypertension.
8. Severe pre-existing hepatic impairment (presence of cirrhosis, liver function tests (LFTs)
≥ 6x baseline, INR ≥ 2.0).
9. Pre-existing Chronic Kidney Disease (CKD) stage IIIb or End Stage Renal Disease (ESRD)
prior to onset of COVID-19 (stage I, II, and IIIa are acceptable)
10. Irreversible coagulopathy (e.g., frequently occluded vascular access despite
anticoagulation, precipitous platelet drops concurrent with end-organ damage suggesting
consumptive process) or irreversible bleeding disorder (e.g., frequent bleeding from
vascular access, endotracheal tubes, and foley).
11. Pneumonia clearly attributable to a non-COVID-19 related process, including aspiration
pneumonia or pneumonia that is exclusively bacterial, or originating from a diagnosed
alternative virus (e.g., influenza).
12. Patients who are not full code.
13. Endotracheal intubation duration ≤ 24 hours.
14. Moribund—expected survival < 24 hours.
15. Severe metabolic disturbances on presentation (e.g., ketoacidosis, pH < 7.3)
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5.3 Screen Failures
Screen failures are defined as participants who consent to participate in the clinical trial but are
not subsequently randomized to the study intervention or entered in the study.
5.4 Strategies for Recruitment and Retention
1. Anticipated number of research sites in the US = 2-15 inpatient hospital settings.
2. Potential participants will be identified in the emergency room, floor, stepdown, and
intensive care units and approached by one of the study investigators or resident physicians
who have had protocol training on the inclusion and exclusion criteria.
3. Recruitment strategy is primarily based on direct discussion with the patient and/or
healthcare proxy.
4. The recruiting physician’s ability to establish transparency and direct communication with
the participant or healthcare proxy is one of the major factors in improving recruitment and
retaining participants, particularly historically under-represented populations.
5. Retention can also be improved via telephone and email reminder of follow-up calls at
Days 15 and 29 and obtaining multiple contact numbers for the participant prior to hospital
discharge.
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6. STUDY INTERVENTION
6.1 Study Intervention(s) Administration
6.1.1 Study Intervention Description
A central randomization service will randomize each participant to one of the following:
Treatment Arm 1: PLACEBO: 100 mL of 0.9% sodium chloride IV on Days 1 and 4.
Treatment Arm 2: IP (ExoFlo) 10 ml in 90 mL of 0.9% sodium chloride IV on Days 1 and 4.
Treatment Arm 3: IP (ExoFlo) 15 ml in 85 mL of 0.9% sodium chloride IV on Days 1 and 4.
6.1.2 Dosing and Administration
The study intervention will be administered intravenously over 60 minutes on Days 1 and 4.
6.1.3 Monitoring of study intervention
During the intervention, the patient will be on continuous 3-lead cardiac monitoring and
continuous pulse oximetry measuring heart rate (HR) and SpO2 with mean arterial pressure (MAP)
obtained via either intermittent noninvasive blood pressure (NIBP) or continuously via an arterial
line. Baseline vitals, including HR, MAP, respiratory rate (RR), and SpO2 will be obtained and
recorded 5 minutes prior to the infusion of IP/Placebo and every 5 minutes for the first 15 minutes
of the infusion and every 15 minutes thereafter for the remainder of the 60-minute total infusion
of IP and every hour thereafter for a total of 4 hours following the infusion, and every 3 hours
thereafter for the first 24 hours.
The temperature will be obtained and recorded 5 minutes prior to
the infusion of the IP/Placebo and repeated every 15 minutes over the course of the infusion.
During the infusion, measurements +/- 2 minutes of specified times for q5min vitals and +/- 5
minutes of specified times for q15min vitals are considered permissible; following the infusion,
vital sign measurements are permissible +/-15 minutes of the specified times.
6.1.4 Detailed Treatment Plan in the Event of an Infusion Reaction
If the patient demonstrates signs and symptoms including but not limited to hypotension,
tachycardia, fever or temperature increase ≥1 degree Celsius (1°C), chills, nausea, shortness of
breath, or urticaria, stop the infusion.
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A.
Respiratory distress is a sign of significant clinical instability, consistent with severe
infusion reactions such as anaphylaxis or septic infusion reaction from bacterial contamination.
Direct a staff member to call for the hospital code team. Be ready to start Advanced Cardiac
Life Support (ACLS).
B.
If appropriate, ACLS should be implemented as per standard procedures.
C.
If the patient is responsive, remain vigilant and focus on airway, breathing, and circulation.
Cycle the NIBP every 1-5 minutes if the patient does not have an arterial line.
1. RESPIRATORY: If there is respiratory distress in a non-intubated patient, ask another
staff member to call for the anesthesiologist on-call. Make sure that the patient is at least
on a NRB. If the patient is hypoventilating with stridor, assist ventilation with AmbuBag
connected to oxygen and ask respiratory technician to set-up noninvasive positive pressure
ventilation (NIPPV). If the patient shows rapid clinical deterioration and demonstrates
complete respiratory failure, ask the anesthesiologist to help with endotracheal intubation.
2. If anaphylaxis is suspected, administer IM Epinephrine into the lateral thigh 1:10000 IM.
May add IV Hydrocortisone 4 mg/kg and Albuterol nebulizer. When patients have severe
bronchospasms, rescue maneuvers may be inadequate and clinical scenario reassessment
is always needed.
3. CIRCULATION: If the patient is hypotensive, bolus IV normal saline 250-500 mL and
can consider IM Adrenaline to be administered into the lateral thigh.
D.
The nurse or other responsible provider should notify the Principal Investigator (PI)
immediately. Tryptase level will be sent to evaluate for possible anaphylactic reaction per
institutional standard of care.
E.
Patient should be on continuous EKG and pulse oximetry. If there is no arterial line, the
NIBP should be cycled every 5 minutes.
F.
Patients may be given acetaminophen 1000 mg for temperature and diphenhydramine 25 mg
oral or IV for urticaria.
If the patient does not demonstrate signs and symptoms of a severe transfusion reaction and only
has minor symptoms such as temperature elevation less than 1°C and urticaria, the study physician
should remain vigilant as infusion reactions can be delayed. The above treatment plan for an
infusion reaction is a guide that can be followed to the greatest extent possible; however, it is
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understood that hemodynamic fluctuations and other clinical events may necessitate deviation
from this standard regimen. Doses and times as detailed in the current protocol are suggested
guidelines to be followed by the physician and team caring for the subject. The actual doses and
times administered are at the discretion of the physician, based on the clinical status of the subject
and will not be considered protocol deviations if not given exactly as described in the protocol.
6.2 Preparation/Handling/Storage/Accountability
6.2.1 Acquisition and accountability
After written informed consent is completed by the participant or the participant’s healthcare proxy,
the co-investigator will contact the central randomization service for randomized allocation to the
study intervention on Day 1. The same randomization code will be provided for the study
intervention and used for ordering of the study intervention—the same identifier will be used for
Days 1 and 4. Once the study intervention order is received, the hospital pharmacist will call the
Interactive Response Technology (IRT), reporting the randomization code and receiving in return
the unblinded allocation. The unblinded hospital pharmacist will prepare the study intervention,
labeling with the randomization code and without the study identity. The blinded study intervention
will be then be delivered via the hospital transport system to the participant’s nurse, who will
perform a series of safety checks including directly confirmation with the hospital site principal
investigator prior to the intravenous administration.
6.2.2 Formulation, Appearance, Packaging, and Labeling
The IP, the formulation of extracellular vesicles isolated from human donor bone marrow using
proprietary technology from Direct Biologics LLC and processed under cGMP standards, is
currently available in glass vials containing 1 mL, 2 mL or 5 mL frozen liquid product. The IP is
aseptically processed. The sterile biologic product is sealed inside the glass vial. Both the glass
vial and product box are labeled with the manufacturer’s lot number, product volume, and
expiration date (month/day/year). The IP product box includes instructions for use, tissue
traceability postcard (pre-addressed & postage paid) and 6 chart stickers. The product box is sealed
with Direct Biologics, LLC, logo sticker and labeled with the manufacturer’s lot number, the
manufacturer’s catalog number, product volume, and expiration date (month/day/year); in addition
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to the contact information for Direct Biologics, LLC, the external packaging exhibits the widely
used symbols for single use only, limited to use by a physician, temperature limits (≤ -40°C) to
which the product can be safely stored prior to immediate use. At room temperature, the IP thaws
within 5 minutes; once properly thawed, The IP is a clear liquid that is visually identical to normal
saline, which is used as placebo in this study.
6.2.3 Product Storage and Stability
The IP is stored ≤ -40°C in ultra-low temperature freezers for up to 5 years at designated Direct
Biologics, LLC, storage facilities. It can be stored between -20°C and -40°C for up to 6 months or
at room temperature for 6 hours. The product must be stored while it is still frozen and once thawed,
the IP is NOT recommended for sterilization or refreezing for future use. The research site directors
and hospital pharmacists are instructed to refuse the product and inform Direct Biologics, LLC, if
the dry ice has sublimated. The product boxes will be kept sealed and stored within the pharmacy
medication freezer between - 20°C and - 40°C no more than 6 months prior to administration within
2-3 weeks of shipping. The IP manual may be referred to for further details.
6.2.4 Study Treatment Preparation
The pharmacist will check the expiration date, discrepancies in the label information, intact seal
on the product box, and intact seal on the glass vial. The pharmacist will use standard practices for
handling and disposal of human tissue. While the outer packaging (product box and HDPE [plastic]
vial card) is not sterile, the biologic product inside the glass vial is considered sterile. The hospital
pharmacist will let the IP thaw completely before drawing the IP out of the vial with a sterile needle
into a syringe. For treatment arm 2 study intervention, 10 mL of 0.9% sodium chloride will be
withdrawn sterilely from a 100 mL 0.9% sodium chloride and discarded; IP from two 5 mL glass
vials will be withdrawn (10 mL total) and sterilely injected into and mixed with 90 mL of 0.9%
sodium chloride. For treatment arm 3 study intervention, 15 mL of 0.9% sodium chloride will be
withdrawn sterilely from a 100 mL 0.9% sodium chloride and wasted; IP from three 5 mL glass
vials will be withdrawn (15 mL total) and sterilely injected into and mixed with 85 mL of 0.9%
sodium chloride. For placebo, 100 ml of 0.9% sodium chloride will be used.
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After receiving the study treatment, the participant’s nurse will perform the following checks:
(1)
Appropriate venous access,
(2)
The patient’s name, DOB, MRN
(3)
The study intervention randomization code on the sticker label matching the
randomization code on the IP order,
(4)
Expiration time for use has not passed,
(5)
Direct verbal confirmation with the attending physician in case there are any significant
that would change study eligibility.
Then the study treatment will be administered intravenously over 60 minutes on an IV pump.
6.3 Measures to Minimize Bias: Randomization and Blinding
This research study involves blinding of both participant and research investigators as well as
randomized and concealed allocation of each participant into one of the three treatment arms by
Interactive Response Technology (IRT). Stratified block randomization will be used to minimize
bias and equally distribute the confounding variable between the treatment groups following
stratification by research site. Randomization code will be provided by IRT. The hospital
pharmacists will not be blinded to the intervention as they must prepare the correct allocated study
intervention; therefore, the hospital pharmacists must be provided random treatment assignment
code directly from the IRT and are not allowed to reveal the study intervention identity until the
study is completed and the database is locked.
Planned Unblinding will occur after the data is fully collected, the source is verified, and the
database is locked. Planned early unblinding due to the IP’s superior efficacy may occur following
day = 7 for the final patient randomized. Organizational model for planned early and final
unblinding are included in this protocol.
Unplanned Unblinding: Emergency unblinding may be required to protect the participant’s safety
if knowing the participant’s treatment assignment would affect immediate medical management.
For emergency unblinding, the investigator may call:
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Phone: 1-888-ASK-BIO2 Option 2, Then Option 1 (Email: support@bioclinica.com)
The investigator must then inform both the sponsor and the DSMB of the emergency unblinding.
Early unblinding of a participant’s treatment assignment may occur for non-urgent reasons.
Contact the medical monitor for any non-urgent unblinding. The non-urgent cases will be
reviewed by the DSMB on a case-by-case basis and the decision to unblind will be rendered on a
need-to-know basis with the fewest number of people informed as possible. In the event of non-
urgent early unblinding of participant for medical/safety reasons, a case report form (CRF)
capturing the self-reported and subjective data must be entered into the study database and the
event of unblinding is reported to both the sponsor and the DSMB.
All SAEs will be reported to the safety CRO and the data will be reviewed by the DSMB
periodically. Intentional and unintentional breaking of the blind, such as accidental reveal of the
study intervention by the hospital pharmacist both directly or in the patient chart, will be reported
to and recorded by the DSMB, who will determine the necessity of also notifying the PI and/or the
study sponsor. Inadvertent blinding by laboratory measures is reduced by the lack of standard
direct or indirect detection assay for the therapeutic intervention.
6.4 Staggering Protocol
BATCH 1: Nine patients will be enrolled across sites*. There will be a delay of 24 hours prior to
the next enrollment batch following the redosing of the randomly assigned study treatment to
allow for assessment of any redosing-related adverse reaction.
BATCH 2: Approximately fifty-one additional patients will be enrolled across sites—the IRT is
alerted specifically following Day 7 for the 60th patient randomized overall into the study. There
will be a delay of 24 hours.
BATCH 3: The remainder of the patients will be enrolled across sites*.
*Patients are not necessarily enrolled on the same day within the same batch.
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6.5 Study Intervention Compliance
The site principal investigator is responsible for ensuring all study staff are appropriately trained:
• Residents and medical attendings on obtaining informed consent if delegated to do so.
• Unblinded pharmacist on the proper preparation of the study infusions.
• Nursing staff on safety checks and correct monitoring during and after the study infusions.
The staff will be trained on the protocol, IP manual, and the IRT system. The site PI will be
responsible for either in-person or electronic daily rounds with the clinical team—evaluating for
(1) any immediate infusion related adverse reaction (< 4 hours), (2) SAE, (3) any other meaningful
events or observations, and (4) any questions or delinquencies with not adhering to the schedule
of activities.
6.6 Concomitant Medications
Concomitant medications are permitted if they are considered standard of care according to the
updated NIH guidelines for COVID-19, which may be accessed via the following link:
https://files.covid19treatmentguidelines.nih.gov/guidelines/covid19treatmentguidelines.pdf
With regard to concomitant medications taken during the study, we will conduct analyses to
compare usage between treatment groups and to assess the potential effects of their use on the
primary endpoint. Concomitant use of investigational agents for COVID-19 which have not been
established as standard of care will need to be discontinued for 24 hours prior to receiving the first
dose of the study administration.
6.7 Rescue Medications for Infusion Reaction
Although no targeted “rescue medication” or antidote exists for the therapeutic intervention, the
study site may administer generic rescue medications when indicated that will be obtained locally
and will consist of standard management for infusion reactions, including but not limited to:
(1)
Fever or suspected hypersensitivity reaction: Acetaminophen 1000 mg PO or IV for fever or
temperature increase ≥ 1°C.
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(2)
Suspected hypersensitivity reaction: diphenhydramine 25 to 50 mg PO or IV for acute reaction.
The date and time of rescue medication administration as well as the name and dosage regimen of
the rescue medication must be recorded in the case report form.
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7. STUDY INTERVENTION & PARTICIPANT DISCONTINUATION
7.1 Discontinuation of Study Intervention:
Discontinuation or pausing of the study intervention may occur for many reasons, including
physiologic changes during the study intervention suggesting potential infusion reaction,
unfavorable physiologic changes within 24 hours following the study intervention, stopping rules,
voluntary patient withdrawal. Unique and unanticipated scenarios will be evaluated by the
investigators and the DSMB on a case-by-case basis.
7.1.1 Clinical Criteria* for Slowing or Suspension of Study Intervention:
The following criteria is included for guidance rather than as strict clinical algorithms. The guiding
principle is that the clinical team should be assessing for signs and symptoms consistent with an
immunologic reaction following initiation of a study intervention:
A)
Criteria* for slowing product administration (by reducing flow rate by at least 50%) include
itchiness, elevation in temperature > 1°C, significant changes in heart rate and MAP consistent
with significant vasodilation possibly leading to distributive shock.
B)
Criteria* for suspending product administration include criteria for slowing product
administration plus additional symptoms of acute infusion; rigors, nausea, abdominal pain,
chills, worsening dyspnea or hypoxia, hypotension, tachycardia, facial swelling, generalized
swelling, profuse bleeding from vascular access sites, significant changes in heart rate and
MAP consistent with distributive shock.
7.1.2 Clinical Criteria for Not Receiving the Second Study Intervention:
Patient should not receive the second study intervention if the oxygenation improves to a level of
recovery, as defined by SpO2 ≥ 93% on room air (or P/F ratio ≥ 300 mmHg) or pre-COVID-19
baseline O2 support for ≥ 4 hours. Patient should not receive the next study intervention if patient
has developed SUSAR to the investigational product within 4 hours of intravenous administration.
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7.1.3 Stopping Rules:
Sequential stopping rules for safety will be adopted by the DSMB. Because SAEs are expected in
the critically ill population of study, the safety data will be examined intermittently for potential
disproportionate incidence of SAEs in one treatment arm versus another. Based on the initial first
clinical study of ExoFlo in COVID-19 subjects, mortality rate was reported to be 16% (4 of 24
patients) and there were no reports of serious adverse events attributable to the investigational
product. The DSMB will be conducting interval review of cumulative unblinded data to determine
if excessive risks/adverse events occur in the study and if stopping rules should be applied in
accordance with the DSMB charter. In addition, the DSMB will continuously receive SAE reports
for information.
In each data review cycle, the DSMB biostatistician will compare the patient-level incidence of
Grade 3 adverse events of special interests and of Grade 4 adverse events of special interests
within 72 hours of the study administration between the treatment group (i.e., the two active
arms pooled) and the control group. The study will be stopped if either of the following criteria is
met: (A) The difference in incidence of Grade 3 related adverse events of special interests within
72 hours of the study administration in the treatment group as compared to the control group is
statistically significantly greater than 0.30. (B) The difference in incidence of related Grade 4
adverse events of special interests within 72 hours of the study administration as compared to the
control group is statistically significantly greater than 0.20. Tests for statistical significance will
be performed using one-sided Wald tests conducted at the 0.10 level of significance. If the
stopping rules are met, enrollment and treatment will be paused by the DSMB. The DSMB will
convene and review at the intervals based on the data available with one planned safety and
interim analysis following Day 7 after the 60th patient (50% of N) randomized overall into the
trial.
Adverse Events of Special Interests:
Grade 5 events
Any deaths (Grade 5 events) unless it is unequivocally not due to treatment.
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Grade 4 events
Generally, life-threatening; urgent intervention required for the following events:
• Allergic reaction- urgent intervention required
• Anaphylaxis/Anaphylactic reaction
• Alanine aminotransferase/ or alkaline phosphatase/ or aspartate aminotransferase increase
≥ 20.0 x ULN if baseline was normal or ≥ 20 times baseline if baseline was abnormal)
• Creatinine increased ≥ 6.0 x ULN
• UOP decreased: anuria ≤ 240 ml in 24 hours
• ARDS—intubation or urgent intervention required
• Hypoxia—airway compromise, urgent intubation or trach required
• Hypotension- life threatening; urgent intervention required
• Thromboembolic event—hemodynamic or neurologic instability requiring urgent
intervention
• Vasculitis—evidence of peripheral or visceral ischemia; urgent intervention indicated
Grade 3 events
Allergic reaction—bronchospasm; intravenous medications indicated Anaphylaxis—
symptomatic bronchospasms with or without urticaria
Alanine aminotransferase or alkaline phosphatase or aspartate aminotransferase increase – > 5.0 –
20.0 x ULN if baseline was normal or > 5 up to 20 times baseline if baseline was abnormal
Creatinine increased: > 3.0 x baseline or > 3.0-6.0 x ULN
UOP decreased—oliguria < 80 ml in 8 hour
Hypotension—medical intervention indicated but not immediately life threatening
Thromboembolic event—medical intervention indicated but not immediately life threatening
Vasculitis—severe symptoms; medical intervention, i.e. steroids, indicated but not immediately
life threatening
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7.1.4 Discontinuation of the Study Intervention:
Discontinuation of the study intervention does not mean discontinuation from the study. In this
modified intention-to-treat analysis, all participants who were randomized and received any
portion of the study intervention will be followed according to the schedule of activities and will
be followed for primary, secondary, and exploratory end points until day 61. A participant who
was randomized but then opted out either due to personal choice or did not receive any study
intervention will still be followed for primary and secondary endpoints. A dedicated CRF will be
used for all instances in which a study intervention is discontinued; the CRF will record the date
and the specific underlying reasons for discontinuation/withdrawal.
7.2 Participant Discontinuation/Withdrawal from the Study
Participants are free to withdraw from the study at any time upon request. An investigator may
discontinue or withdraw a participant from the study for the following reasons:
• Pregnancy
• If the participant meets an exclusion criterion (either newly developed or not previously
recognized) that precludes further study participation.
• If any clinical AE, laboratory abnormality, or other medical condition or situation
occurs such that continued participation in the study would not be in the best interest
of the participant.
• Participant unable to receive the study intervention for 7 days following enrollment due
to clinical instability.
The reason for participant discontinuation or withdrawal from the study will be recorded on the
CRF. Subjects who sign the informed consent form and are randomized but do not receive the
study intervention may be refilled. Subjects who sign the informed consent form and are
randomized and receive any portion of the study intervention, and subsequently withdraw, or are
withdrawn or discontinued from the study, will not be refilled.
7.3 Lost to Follow-Up
A participant will not be lost to follow-up if still hospitalized at Days 29 and 61. However, once
the participant is discharged, he or she will be considered lost to follow-up if he or she fails to
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respond to at least three telephone attempts on day 29 and 61 and at least three telephone attempts
on day 29 and 61. The following actions must be taken if a participant fails to return the follow-
up call:
• Before a participant is deemed lost to follow-up, the investigator or designee will make
every effort to regain contact with the participant (where possible, at least 3 telephone calls
and, if necessary, a certified letter to the participant’s last known mailing address or local
equivalent methods). These contact attempts should be documented in the participant’s
medical record or study file.
• Should the participant continue to be unreachable, he or she will be considered to have
withdrawn from the study with a primary reason of lost to follow-up.
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8. STUDY ASSESSMENTS AND PROCEDURES
8.1 Study Procedures By Visit
SCREENING VISIT (Day 0 | Can be Day 1)
Basic Evaluation: Patient’s age, gender, race/ethnicity, height and weight (which will be used to
calculate BMI, should be obtained following hospital admission but does not have to be repeated
to be Days 0 or 1), medical history, concomitant medications, days symptomatic of COVID-19
prior to admission, date of admission (which will be used to calculate number of days from
admission to the first study treatment), date of intubation if the patient is currently intubated with
an endotracheal tube (which will be used to calculate number of days of intubation—NOTE,
patient cannot be intubated more than > 24 hours for enrollment), physical examination, serum
pregnancy test for women of childbearing potential. EKG should be obtained following hospital
admission but does not have to be repeated to be either Day 0 or 1.
Respiratory Evaluation: Qualitative SARS-CoV-2 RT PCR, prone positioning (if used and if
so, the frequency—both ventilated and nonventilated patients may be intermittently “proning”
per hospital site protocol), PaO2/FiO2 (see appendix 11.1 for imputation if no ABG or arterial
line—please note that the use of ABG or insertion of arterial line is dependent on clinical need
only and should not be performed in the sole interest of the clinical trial), radiographic imaging
of the chest (CXR or CT chest), and oxygen support (i.e. RA vs NC vs FM vs NRB vs BIPAP or
CPAP vs HFNC O2 vs MV; FiO2 setting should be recorded for BIPAP, CPAP, HFNC, and
MV; L/min of O2 should be recorded for NC and FM; PEEP should be recorded for MV,
BIPAP, or CPAP.
Labs: BMP, CBC with differential, LFTs, PT/INR, PTT; CRP, D-dimer, Ferritin will be
collected. Interleukin-6 and tumor necrosis factor alpha do not have to be collected prior to
enrollment.
Microbiology: Urinalysis, urine culture, blood culture (2 sets), and sputum culture should be
collected following current hospital admission—they do not have to be repeated to be on Day 0
or 1.
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Rule out tests: Mycoplasma IgM, QuantiFERON Gold, Legionella Antigen, Strep. Pneumoniae
Antigen, Influenza A/B PCR should be obtained following current hospital admission—they do
not have to be repeated to be on Day 0 or 1.
Adverse event review: Review of adverse events accumulated over the course of
hospitalization thus far should be performed prior to study enrollment.
Study Evaluation and Consent: Participant is considered for the study if patient meets
eligibility criteria. Informed consent is obtained if patient or proxy agrees following description
of the study and the informed consent process. The informed consent form is collected, and the
patient is considered enrolled.
VISIT DAY 1
Study Re-Evaluation and Randomization & IV Study infusion: Participant’s eligibility criteria
will be re-evaluated to make sure there are no significant interim changes that would disqualify
him or her from the study. Women of childbearing potential should have a urine dipstick obtained
4 hours prior to the first dose of study treatment if it has been more than 24 hours since the serum
pregnancy test was collected. If patient remains as an eligible participant for the trial, the study
investigator calls the Interactive Response Technology. The hospital pharmacist(s) will be notified
of the study infusion order and can call the Interactive Response Technology with the
randomization code in order to receive the study infusion identity and prepare the infusion
appropriately. The study infusion is labeled with the randomization code but not with the infusion
identity; the nurse administers the study infusion over the course of 60 minutes. Monitoring is
already specified in section 6.1.3.
Basic Evaluation, Respiratory Evaluation, Standard Labs, Acute Phase Reactants, Metrics,
Microbiology (if indicated) are to be performed according to the Schedule of Activities (Section
1.3).
Flow Cytometry Labs: T-lymphocyte panel (CD3+, CD4+, CD8+); NK cell count (defined as
CD3- and CD56+ subset of a light scatter characterization of lymphocytes. These labs should be
drawn within 4-6 hours prior to the first study infusion.
Adverse Event Review: Interim adverse events should be reviewed and recorded. SAE(s) if any
are continuously monitored and reported within 24 hours of knowledge.
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VISIT DAY 4
Study Re-Evaluation & IV Study Infusion: Participant’s eligibility criteria will be re-
evaluated primarily for placement on ECMO and change in code status—these are the only two
interim changes that would disqualify him or her from the second study administration aside
from: Patient should not receive the second study infusion if there was a SUSAR within 4 hours
following the first dose.
Patient also should not receive the second study infusion if he or she
has SpO2 ≥ 93% on RA or returned to baseline SpO2 and O2 requirement if patient has chronic
lung disease. Otherwise, the patient may receive the second study infusion, which is the same
identity and dosing as the first study infusion, also administered over 60 minutes.
Basic Evaluation, Respiratory Evaluation, Standard Labs, Acute Phase Reactants, Metrics,
Microbiology (if indicated) are to be performed according to Schedule of Activities.
Flow cytometry labs: T-lymphocyte panel (CD3+, CD4+, CD8+); NK cell count (defined as
CD3- and CD56+ subset of a light scatter characterization of lymphocytes. These labs should be
drawn within 4-6 hours prior to the study infusion.
Adverse event review: Interim adverse events should be reviewed and recorded. SAE(s) if any
are continuously monitored and reported within 24 hours of knowledge.
VISIT DAY 7, 10, 15, 29, 61
Basic Evaluation, Respiratory Evaluation, Standard Labs, Acute Phase Reactants, Metrics,
Microbiology (if indicated) are to be performed according to Schedule of Activities. Regarding
metrics, SOFA score (Days 1, 15, and 29) will only be tabulated on inpatients and the nonvisual
portion of EQ-5D-5L will be collected over the phone on Days 29 and 61 on outpatients.
Flow cytometry labs: T-lymphocyte panel (CD3+, CD4+, CD8+); NK cell count (defined as
CD3- and CD56+ subset of a light scatter characterization of lymphocytes). These labs should be
drawn within 4-6 hours prior to the study infusion and performed according to Schedule of
activities Section 1.3.
Adverse event review: Any adverse events /serious adverse event should be reviewed and
recorded per Schedule of activities and requirements for reporting SAEs.
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8.2 Efficacy Assessments
Evaluation of the patient’s charts will include baseline demographics including age, gender,
race/ethnicity, BMI. There will be detailed review of the medical comorbidities, admission
documentation, noting days of illness prior to admission, in addition to past medical history and
presenting symptoms, concomitant medications and therapies, daily progress note, and the most
recent progress note in the chart, noting both the subjective interval history and critical events in
addition to the highlights of the vitals and labs. The day and time of the study intervention
administration will be verified via the MAR; from this day and time, it will be calculated how
many days following the first treatment the patient was hospitalized prior to treatment and/or how
many the days the patient was intubated prior to the treatment. The date and time of other events
such as patient expiration, patient’s intubation or extubation, patient’s recovery as defined by SpO2
≥ 93% on room air for longer than 4 hours, and hospital discharge will be tabulated. Improvement
in P/F ratio from pre-infusion baseline (Day 0) to Day 7 will be analyzed as a primary endpoint in
addition to time to recovery, incidence of SAE, and all-cause will be analyzed as secondary
endpoints. The exploratory endpoints will be trended according to the Schedule of Activities in
Section 1.3.
8.3 Safety and Data Safety Monitoring Board
Stopping rules are established in this study to determine if the serious or severe adverse events
have met a pre-defined statistical parameter. If stopping rules are met, the enrollment will halt for
the DSMB to review and adjudicate the cause of the AE and to determine if study should be
modified for continuation.
8.4 Adverse Events and Serious Adverse Events
8.4.1 Definition of Adverse Events
Any medical condition that is present at the time that the participant is screened will be
considered as baseline medical history and not reported as an AE. However, if the study
participant’s condition deteriorates at any time during the study after signing the informed
consent, it will be recorded as an AE.
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An AE is any untoward medical occurrence in a subject or clinical investigation subject
administered a pharmaceutical product and which does not necessarily have to have a causal
relationship with the intervention. An AE can therefore be:
• Any unfavorable and unintended sign, symptom, or disease temporally associated with
the use of a medicinal (investigational) product, whether or not considered related to the
product
• Any new disease or exacerbation of a pre-existing disease (a worsening in the nature,
frequency, or severity of a medical condition)
• Recurrence of medical conditions that are not present at baseline
• Any changes in laboratory value or other clinical tests (e.g., AST, ALT, CPK) that are
associated with symptoms, or that lead to a change in study treatment or additional
concomitant treatment, or that result in discontinuation from the study drug
8.4.2 Definition of Serious Adverse Events
An AE or suspected adverse reaction is considered "serious" if, in the view of either the
investigator or sponsor, it results in any of the following outcomes: death, a life-threatening AE,
inpatient hospitalization or prolongation of existing hospitalization, a persistent or significant
incapacity or substantial disruption of the ability to conduct normal life functions, or a congenital
anomaly/birth defect. Important medical events that may not result in death, may be life-
threatening, or require hospitalization, may be considered serious when, based upon appropriate
medical judgment, they jeopardize the participant, possibly requiring medical or surgical
intervention to prevent one of the outcomes listed in this definition. Examples of such medical
events include allergic bronchospasm requiring intensive treatment in an emergency room or at
home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the
development of drug dependency or drug abuse.
A serious adverse event (SAE) is any untoward medical occurrence that meets one of the
following criteria:
• Fatal (results in the outcome death)
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• Life-threatening*
• Requires hospitalization or prolongation of existing hospitalization. (Hospitalizations for
elective medical or surgical procedures or treatments planned before the signing of
informed consent in the study are not SAEs by this criterion).
• Results in persistent or significant disability/incapacity or substantial disruption of the
subject’s ability to conduct normal life functions
• Is a congenital anomaly/birth defect in a child or fetus of a subject who has been exposed
to the molecule or study treatment regimen before conception or during pregnancy.
• Is medically significant meaning the AE did not meet any of the above criteria but could
have jeopardized the subject and might have required medical or surgical intervention to
prevent one of the outcomes listed above or involves suspected transmission via a
medicinal product of an infectious agent.
*Note: The term “life-threatening” in the definition of “serious” refers to an event in which the
subject was at risk of death at the time of the event; it does not refer to an event which
hypothetically might have caused death if it were more severe.
8.4.3 Follow-Up Reporting
As further information regarding the SAE becomes available, follow-up information should be
provided and emailed to the Sponsor (or designee), including any records during hospitalization
and tests performed that were necessary to evaluate the SAE.
All SAEs that have not resolved by the end of study, or that have not resolved upon
discontinuation of the subject’s participation in the study, must be followed until any of the
following occurs:
• The event resolves
• The event stabilizes
• The event returns to baseline, if baseline value/status is available
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• The event can be attributed to agents other than the study drug or to factors unrelated to
study conduct
It becomes unlikely that any additional information can be obtained (subject or health care
practitioner refusal to provide additional information, lost to follow-up after demonstration of
due diligence with follow-up efforts).
8.4.4 Definition of Adverse Events of Special Interest (AESI)
As per Council for International Organizations of Medical Sciences (CIOMS) VI, an adverse
event of special interest (serious or non-serious) is one of scientific and medical concern specific
to the sponsor’s product or program, for which ongoing monitoring and rapid communication by
the investigator to the sponsor may be appropriate. Such events may require further investigation
in order to characterize and understand them. Depending on the nature of the event, rapid
communication by the trial sponsor to other parties may also be needed. In the case of ExoFlo,
there are presently no anticipated AESIs. However, as with AEs, possible AESIs will be
reviewed and assessed individually and in aggregate on a continuous basis to identify and track
any emerging safety signals. Suspicion for AESIs could be triggered by non-serious events that
may be prodromes of serious medical conditions and will be evaluated and reported on a case-
by-case basis. See Stopping rule Section 7.1.3.
8.4.5 Classification of Severity of Adverse Events and Relationship to Study Treatment
All clinical Aes encountered during the study will be reported on the AE page of the CRF.
Intensity of Aes will be graded based on the CTCAE, Version 5.0 and reported in detail as
indicated on the CRF. For any Aes not found in the CTCAE, the following descriptions of
intensity grading can be used:
Mild – Events require minimal or no treatment and do not interfere with the participant’s
daily activities.
Moderate – Events result in a low level of inconvenience or concern with the therapeutic
measures. Moderate events may cause some interference with functioning.
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Severe – Events interrupt a participant’s usual daily activity and may require systemic drug
therapy or other treatment. Severe events are usually potentially life-threatening or
incapacitating.
All Aes must have their relationship to study intervention assessed by the clinician who examines
and evaluates the participant based on temporal relationship and his/her clinical judgment. The
degree of certainty about causality will be graded using the categories below.
Definitely Related – There is clear evidence to suggest a causal relationship, and other
possible contributing factors can be ruled out. The clinical event, including an abnormal
laboratory test result, occurs in a plausible time relationship to study intervention
administration and cannot be explained by concurrent disease or other drugs or chemicals.
The response to withdrawal of the study intervention should be clinically plausible. The event
must be pharmacologically or phenomenologically definitive.
Probably Related – There is evidence to suggest a causal relationship, and the influence of
other factors is unlikely. The clinical event, including an abnormal laboratory test result,
occurs within a reasonable time after administration of the study intervention, is unlikely to
be attributed to concurrent disease or other drugs or chemicals, and follows a clinically
reasonable response on withdrawal.
Possibly Related – There is some evidence to suggest a causal relationship (e.g., the event
occurred within a reasonable time after administration of the trial medication). However,
other factors may have contributed to the event (e.g., the participant’s clinical condition,
other concomitant events). Although an AE may rate only as “possibly related” soon after
discovery, it can be flagged as requiring more information and later be upgraded to “probably
related” or “definitely related”, as appropriate.
Unlikely to be related – A clinical event, including an abnormal laboratory test result, whose
temporal relationship to study intervention administration makes a causal relationship
improbable (e.g., the event did not occur within a reasonable time after administration of the
study intervention) and in which other drugs or chemicals or underlying disease provides
plausible explanations (e.g., the participant’s clinical condition, other concomitant
treatments).
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Not Related – The AE is completely independent of study intervention administration,
and/or evidence exists that the event is definitely related to another etiology. There must be
an alternative, definitive etiology documented by the clinician.
8.4.6 Expected Adverse Reactions
Expected adverse reactions are Aes that are known to occur for the study intervention being
studied. Expectedness is assessed based on the awareness of Aes previously observed, not on the
basis of what might be anticipated from the properties of the study intervention. Thus far based on
the preliminary data, there is no expected adverse reaction from the IP. All treatment emergent
adverse event would be considered unexpected for regulatory reporting purpose.
8.4.7 Time Period and Frequency for Event Assessment and Follow-up
The occurrence of an AE or SAE may come to the attention of study personnel during study visits
and interviews of a study participant presenting for medical care, or upon review by a study
monitor. All Aes including local and systemic reactions not meeting the criteria for SAEs will be
captured on the appropriate CRF. Information to be collected includes event description, time of
onset, clinician’s assessment of severity, relationship to study product (assessed only by those with
the training and authority to make a diagnosis), and time of resolution/stabilization of the event.
All Aes occurring while on study must be documented appropriately regardless of relationship. All
Aes will be followed to adequate resolution. Any medical condition that is present at the time that
the participant is screened will be considered as baseline and not reported as an AE. However, if the
study participant’s condition deteriorates at any time during the study, it will be recorded as an AE.
Changes in the severity of an AE will be documented to allow an assessment of the duration of the
event at each level of severity to be performed. Aes characterized as intermittent require
documentation of onset and duration of each episode. The investigators, the study coordinator, will
record all adverse event events with start and stop dates occurring any time after informed consent
is obtained to 30 days (for SAEs) after the last dose of IP. Events will be followed for outcome
information until resolution or stabilization. Any death occurring within 30 days of the last dose
of IP, regardless of attribution to the IP/intervention, requires reporting to sponsor/designee within
24 hours. Deaths due to COVID-19 should be recorded as an outcome on CRT.
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8.4.8 Adverse Event Reporting
According to 21 CFR 312.64(b), the investigator must record nonserious Aes and report them to
the sponsor. This will be performed per schedule of activity (See Section 1.3).
8.4.9 Serious Adverse Event Reporting
The study investigator will report to the sponsor, any SAE, whether or not considered study
intervention related within 24 hours.
Any SAE must be reported by the site to:
• The Sponsor (or designee) within 24 hours of becoming aware of the event
• The investigational site’s IRB/Ecs by the investigator in accordance with their regulations
If an investigational site becomes aware of a new SAE or has follow-up information to a
previously reported SAE, the site must notify the Sponsor (or designee) within 24 hours of
becoming aware. Notification can be accomplished by completing the SAE report form
submitting it to the Sponsor via email:
Email: PHV_ DB-EF-PHASEII-001_SO@IQVIA.com
Reporting instructions and the SAE Report Form are provided in the Study Manual.
8.5 Unanticipated Problems
Unanticipated problems (Ups) are problems that involve risks to participants or others to include,
in general, any incident, experience, or outcome that meets all following criteria:
• Unexpected in terms of nature, severity, or frequency given (1) the research procedures
that are described in the protocol-related documents, such as the IRB-approved research
protocol and ICF; and (2) the characteristics of the participant population being studied;
• Related or possibly related to participation in the research (“possibly related” means there
is a reasonable possibility that the incident, experience, or outcome may have been caused
by the procedures involved in the research); and
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• Suggests that the research places participants or others at a greater risk of harm (including
physical, psychological, economic, or social harm) than was previously known or
recognized.
The investigator will report Ups to the reviewing IRB. The UP report will include the following
information: protocol identifying information: protocol title and number, PI’s name, and the IRB
project number; detailed description of the event, incident, experience, or outcome; an explanation
of the basis for determining that the event, incident, experience, or outcome represents an UP; a
description of any changes to the protocol or other corrective actions that have been taken or are
proposed in response to the UP.
To satisfy the requirement for prompt reporting, Ups will be reported using the following timeline:
(1)
Ups that are SAEs will be reported to the IRB within 24 hours of the investigator becoming
aware of the event.
(2)
Any other Ups will be reported to the IRB within 7 days of the investigator becoming aware
of the problem.
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9. STATISTICAL CONSIDERATIONS
9.1 Statistical Hypotheses
Primary Endpoint:
(1)
The primary efficacy endpoint, Improvement in partial pressure of arterial oxygen to fraction
of inspired oxygen (PaO2/FiO2) ratio from pre-infusion baseline (Day 0) to Day 7, will be
analyzed using the Analysis of Variance (ANOVA) adjusting for an intubation status. No
improvement (+0 mmHg) will be assigned to subjects who die or had negative change in P/F
ratio from baseline. The PaO2/FiO2 ratio of patients discharged prior to Day 7 will be imputed
as 380. A difference between 2 mean changes from the baseline to Day 7 will be tested at one-
sided significance level of 0.075 (overall Type I error rate ≤ 0.1) and estimated with 85%
confidence interval.
Ho: µ0 ≥ µ1
Ha: µ0 < µ1
µ0: Mean improvement from baseline to Day 7 in the control arm 1
µ1: Mean improvement from baseline to Day 7 in the selected experimental arm 2 or mean
improvement from baseline to Day 7 in the selected IP arm, or arm 3 if both arms
remain open after planned interim safety review with 60 patients. No statistical
comparison will be performed for the unselected IP arm.
Secondary Endpoints:
(1)
Time to recovery as defined by from the first study treatment until return of oxygenation
saturation (SpO2) ≥ 93% on room air (or PaO2/FiO2 ≥ 300 mmHg) will be estimated using
the Kaplan-Meier (KM) method and 95% confidence interval by arm. Median time to
recovery and a percentage of recovered patients at Days 14 and 28 will be displayed by
arm. A recovery odds ratio between the selected IP and control arm will also be estimated
using a cox regression model.
(2)
Incidence of Serious Adverse Events (SAEs) will be estimated by arm and displayed by
severity and relationship to the study drug. Incidence of SAEs that led to not receiving full
doses of the study treatment (IP+saline or saline alone) will also be summarized by am.
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(3)
All-cause mortality (or overall survival) will be estimated using the KM method as survival
rates at Day 14, 28 and 60 by arm. The hazard ratio (HR) between the selected IP and
control arms will also be estimated using a cox regression model.
Exploratory Endpoints:
All exploratory endpoints will be analyzed and compared among arms descriptively and estimated
at the following time points as listed in Schedule of Activities in Section 1.3.
9.2 Sample Size Determination
For the selected experimental IP arm, approximately 30% higher improvement of P/F ratio from
pre-infusion baseline to Day 7 and ≥ a higher recovery rate by 10 percentage point for selected
experimental arm is assumed compared to those achieved by the control arm at Day 7. The
underlying assumption is that the difference between the two mean changes (experimental and
placebo arms) from the baseline to Day 7 is approximately 72 mmHg (see Table 1) with a
standard deviation of 15012. Total of 80 (40 x 2) subjects for the final 2-arm comparison will
generate approximately 80% power with 1-sided alpha of 0.075 using ANOVA adjusting for
intubation status.
Table 1. Assumptions used to estimate mean changes from baseline to Day 7
ExlFlo Arm
Approximate
% Patients
Estimated Day 7 Status
Mean
Changed
from Baseline
to Day 7
Overall
Mean
ExoFlo
Moderate
50%
Recovered
55%
315
Non-recovery
45%
90
Severe
50%
Recovered
35%
406
Non-recovery
65%
116
216
Control
Moderate
50%
Recovered
45%
235
Non-recovery
55%
64
Severe
50%
Recovered
25%
320
Non-recovery
75%
88
144
Difference:
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9.3 Analysis Population
This modified intention-to-treat (mITT) Analysis set will be used for primary and secondary
efficacy endpoints and defined as patients who are randomized and received at least a partial
dose of the randomly allocated study treatment. Patients will be analyzed according to the
randomized treatment arm. Safety Analysis set is defined as patients who received any dose of
the study treatment (IP/saline or saline alone) and analyzed by the treatment arm/dose they
actually received. Per-protocol Analysis set is defined as patients who are randomized and
received full doses of both randomly allocated study treatment infusions (i.e., days 1 and 4). The
Per-Protocol Analysis set will be used as a secondary analysis set for the primary and the first
secondary efficacy endpoints.
9.4 Statistical Analyses
All estimated results will be presented with 95% confidence intervals except the primary
endpoint of the comparison of P/F ratios after the final unblinding occurs.
9.4.1 Analysis of the Safety Endpoint
All safety analyses are based on the Safety Analysis set. Patients will be grouped according to the
treatment which they received and summarized for treatment emergent AEs/SAE by severity and
relationship to the study drug. All deaths and any AEs that led to not receiving full doses of the
study treatment will be listed. All AE incidence rates will be estimated with 95% CI when needed.
9.4.2 Analysis of the Primary and Secondary Efficacy as well as Exploratory Endpoints
The primary analysis for primary and secondary efficacy endpoints is based on mITT set. All
efficacy analyses for an unselected IP arm vs control arm and secondary/exploratory analyses
will be descriptive and no formal statistical comparison will be performed. P-values may be
displayed for a descriptive purpose only except for the primary endpoint for the final 2-arm
comparison. Patients will be grouped according to the treatment to which they were randomized.
Appropriate analysis sets for exploratory endpoints will be defined in the statistical analysis plan
(SAP).
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9.4.3 AE Analysis
Clinical and laboratory AEs will be coded using the Medical Dictionary for Regulatory Activities
(MedDRA) as well as Common Terminology Criteria for Adverse Events (CTCAE) v5.0. System
organ class, high-level group term, high-level term, preferred term, and lower-level term will be
attached to the clinical database. Within each System Organ Class, AE are listed and accompanied
by descriptions of severity (Grade 1-5). All AEs will be summarized, including any infusion-
related adverse reactions within 4 hours.
9.4.4 Baseline and Disposition Summaries
Demographic and baseline characteristic measurements will be summarized using descriptive
methods. Demographic summaries will include sex, race/ethnicity, and age.
Disposition summary will include the portion of patients who did not receive full doses by their
primary reason and the portion of patients who did not reach Day 60 by their primary reason of
exiting the study early.
9.4.5 Interim Analysis
The IRT will notify the unblinded DSMB such that an interim analysis can be held following day
7 of 60th patient randomized overall (50% of total N of 120), which should also correspond to
approximately 20 patients per each treatment arm.
An efficacy analysis will also be performed by the unblinded DSMB. With the primary efficacy
variable being the improvement in P/F ratio from pre-infusion baseline (Day 0) to Day 7, an
interim analysis will be performed with a 1-sided p-value threshold of 0.025. All randomized
patients receiving at least 1 dose of study intervention will be analyzed. If the selected treatment
arm containing IP demonstrates superiority over placebo, meeting the allocated Type I error rate
as specified, then the DSMB may advise the sponsor to unblind and stop enrollment or unblind
and continue enrollment.
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9.4.6 Planned Early Unblinding Analysis
Planned final unblinding analysis for the primary endpoint will be performed following day = 7
for last patient randomized. All randomized patients receiving at least 1 dose of study
intervention will be analyzed.
9.4.7 Sub-Group Analyses
Sub-group analysis of primary and secondary efficacy will be performed on baseline ventilation
status to see if treatment with the IP has more of an effect on outcome when administered to
intubated ARDS patients versus non-intubated ARDS patients.
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10. REGULATORY ETHICAL AND STUDY OVERSIGHT CONSIDERATIONS
10.1 Informed Consent Process
Consent forms describing in detail the study intervention, study procedures, and risks are given to
the participant and written documentation of informed consent is required prior to starting
intervention/administering study intervention.
10.2 Consent Procedures & Documentation
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in the
study and continues throughout the individual’s study participation. Consent forms will be IRB-
approved, and the participant will be asked to read and review the document. The investigator or
trained proxy of the investigator (resident physician, attending physician) will explain the research
study to the participant and answer any questions that may arise. A verbal explanation will be
provided in terms suited to the participant’s comprehension of the purposes, procedures, and
potential risks of the study and of their rights as research participants. Participants will have the
opportunity to carefully review the written consent form and ask questions prior to signing. The
participants should have the opportunity to discuss the study with their family or surrogates or
think about it prior to agreeing to participate. The participant will sign the informed consent
document prior to any procedures being done specifically for the study. Participants must be
informed that participation is voluntary and that they may withdraw from the study at any time,
without prejudice. A copy of the ICF will be given to the participants for their records. The
informed consent process will be conducted and documented in the source document (including
the date), and the form signed, before the participant undergoes any study-specific procedures. The
rights and welfare of the participants will be protected by emphasizing to them that the quality of
their medical care will not be adversely affected if they decline to participate in this study.
10.3 Study Discontinuation and Closure
Study drug dosing in an individual subject will be placed on hold and may be discontinued,
following a review of all available clinical data by the medical monitor and discussion with the
investigator, if any of the following occurs: EITHER Any SAE or ≥ Grade 3 AE suspected to be
an adverse reaction to the IP within 4 hours of administration OR patient has already recovered
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with SpO2 ≥ 93% on room air. This study may be suspended or prematurely terminated if there is
sufficient reasonable cause. Written notification, documenting the reason for study suspension or
termination, will be provided by the suspending or terminating party to study participants,
investigators, IRB, FDA and CBER. If the study is prematurely terminated or suspended, the PI
will promptly inform study participants, the IRB, and sponsor and will provide the reasons for the
termination or suspension. Study participants will be contacted, as applicable, and be informed of
changes to study visit schedule. Circumstances that may warrant termination or suspension include
but are not limited to: determination of unexpected, significant, or unacceptable risk to participants,
demonstration of efficacy that would warrant stopping, insufficient compliance to protocol
requirements, data that is not sufficiently complete and/or analyzable, determination that the
primary endpoint has been met, and/or determination of futility. Study may resume once concerns
about safety, protocol compliance, and data quality are addressed, and satisfy the sponsor, IRB
and/or FDA.
10.4 Confidentiality and Privacy
Participant confidentiality and privacy is strictly held in trust by the participating investigators,
their staff, and the sponsor(s) and their interventions. This confidentiality is extended to cover
testing of biological samples and genetic tests in addition to the clinical information relating to
participants. Therefore, the study protocol, documentation, data, and all other information
generated will be held in strict confidence. No information concerning the study will be released
to any unauthorized third party without prior written approval of the sponsor. All research activities
will be conducted in as private a setting as possible. The study monitor, other authorized
representatives of the sponsor, representatives of the IRB, regulatory agencies or pharmaceutical
company supplying study product may inspect all documents and records required to be
maintained by the investigator, including but not limited to, hospital medical records, and
pharmacy records for the participants in this study. The clinical study site will permit access to
such records. The study participant’s contact information will be securely stored at each clinical
site for internal use during the study. At the end of the study, all records will continue to be kept
in a secure location for as long a period as dictated by the reviewing IRB, Institutional policies, or
sponsor requirements. Study participant research data, which is for purposes of statistical analysis
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and scientific reporting, will be transmitted to and stored at the CRO. This will not include the
participant’s contact or identifying information. Rather, individual participants and their research
data will be identified by a unique study identification number. The study data entry and study
management systems used by clinical sites and by CRO will be secured and password protected.
10.5 Data Storage
Data collected for this study will be analyzed and stored at the independent CRO. After the study
is completed, the de-identified, archived data will be transmitted to and stored at the Direct
Biologics, LLC, for use by other researchers including those outside of the study. During the
conduct of the study, an individual participant can choose to withdraw consent to have biological
specimens stored for future research.
10.6 DSMB Safety Oversight
The DSMB is an independent safety oversight committee that will operate on “need-to-know”
principles and rules. For example, all individuals involved in the conduct of the trial aside from
the unblinded pharmacist and the independent CRO will remain unaware of information related to
the planned early unblinding analysis, DSMB deliberations, recommendations, and date/time of
the meetings. Members of the DSMB will be selected strictly by credentials and expertise.
Individuals with a personal or professional interest in the outcome of the trial will be excluded
from consideration. The DSMB closed sessions will be attended by members of the DSMB and
unblinded supporting statisticians, who will be unblinded to the study intervention for proper safety
oversight given the high incidence of adverse events reported in patients with COVID-19
associated ARDS. Only in closed sessions will the presented data be deliberately and privately
reviewed. The DSMB will be required to keep detailed meeting minutes, kept in a confidential file
inaccessible to sponsors.
10.7 Clinical Monitoring
Clinical site monitoring is conducted to ensure that the rights and well-being of trial participants
are protected, that the reported trial data are accurate, complete, and verifiable, and that the
conduct of the trial is in compliance with the currently approved protocol/amendment(s), with
the International Conference on Harmonisation (ICH) Good Clinical Practice (GCP) guidelines,
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and with applicable regulatory requirements. The first clinical monitoring visit will occur within
one week of the first dosed subject to verify eligibility and compliance; then routine monitoring
visits will occur on a 2-week interval until the database is locked.
10.8 Quality Assurance and Quality Control
Each clinical site will perform internal quality management of study conduct, data and biological
specimen collection, documentation, and completion. An individualized quality management plan
will be developed to describe a site’s quality management. Quality control (QC) procedures will
be implemented beginning with the data entry system and data QC checks that will be run on the
database will be generated. Any missing data or data anomalies will be communicated to the sites
for clarification/resolution. Following written Standard Operating Procedures (SOPs), the
monitors will verify that the clinical trial is conducted and data are generated and biological
specimens are collected, documented (recorded), and reported in compliance with the protocol,
ICH GCP, and applicable regulatory requirements including current GMP and Good Tissue
Practice (GTP).
10.9 Protocol Deviation
A protocol deviation is any noncompliance with the clinical trial protocol, ICH GCP, or Manual
of Procedures (MOP) requirements. The noncompliance may be either on the part of the
participant, the investigator, or the study site staff. As a result of deviations, corrective actions are
to be developed by the site and implemented promptly. These practices are consistent with ICH
GCP:
• 4.5 Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3
• 5.1 Quality Assurance and Quality Control, section 5.1.1
• 5.20 Noncompliance, sections 5.20.1, and 5.20.2.
It is the responsibility of the site investigator to use continuous vigilance to identify and report
deviations within 5 working days of identification of the protocol deviation, or within 5 working
days of the scheduled protocol-required activity. All deviations must be addressed in study source
documents, reported to CBER and CRO. Protocol deviations must be sent to the reviewing IRB
per their policies. The site investigator is responsible for knowing and adhering to the reviewing
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IRB requirements. Further details about the handling of protocol deviations will be included in the
MOP.
10.10 Public Access
This study will be conducted in accordance with the following publication and data sharing policies
and regulations: NIH Public Access Policy, which ensures that the public has access to the
published results of NIH funded research. It requires scientists to submit final peer-reviewed
journal manuscripts that arise from NIH funds to the digital archive PubMed Central upon
acceptance for publication. This study will comply with the NIH Data Sharing Policy and Policy
on the Dissemination of NIH-Funded Clinical Trial Information and the Clinical Trials
Registration and Results Information Submission rule. As such, this trial will be registered at
ClinicalTrials.gov, and results information from this trial will be submitted to ClinicalTrials.gov.
In addition, every attempt will be made to publish results in peer-reviewed journals.
10.11 Conflict of Interest Policy
The independence of this study from any actual or perceived influence, such as by the
pharmaceutical industry, is critical. Therefore, any actual conflict of interest of persons who have
a role in the design, conduct, analysis, publication, or any aspect of this trial will be disclosed and
managed. Furthermore, persons who have a perceived conflict of interest will be required to have
such conflicts managed in a way that is appropriate to their participation in the design and conduct
of this trial.
10.12 Protocol Amendment History
Amendment
Personnel
Name
Changes
Amendment
1, 05 August
2020
Sascha
Sengupta,
MD
1)
Sections were reformatted in accordance with GCP standards. Study contact
information removed.
2)
ExoFlo was removed from the title. The terminology Extracellular Vesicles
was used in the title and study description.
3)
Protocol authorization page now has signature spaces for both the Chief
Medical Officer and the Chief Scientific Officer.
4)
Investigator’s Agreement was transferred from the end to the section after
Protocol authorization.
5)
List of Index Tables added after Table of Contents.
6)
Both sections 1.2 and 1.3 emphasize that the second infusion is not
administered if patient recovers by day = 4.
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7)
Section 1.3 was compiled with all evaluations, labs, and procedures required
itemized as rows.
8)
Section 1.3 footnote specified that patients are to be monitored for 72 hours
following the first and the second infusion. If the patient meets the hospital’s
criteria for discharge and recovers from a respiratory standpoint, there is no
need to prolong the inpatient monitoring following day = 7 if the patient
received 2 infusions or following day = 4 if the patient received only 1
infusion.
9)
Changed terminology of day =0 to day =1 throughout the protocol. Day = 3
changed to day =4 and so forth throughout the protocol.
10)
Concomitant medication was removed from the exclusion criteria. Standard of
care was updated so that patients with severe COVID-19 will be allowed to
remain concurrently on Remdesivir and Dexamethasone. Remdesivir and
Dexamethasone were specifically mentioned as part of Standard of Care.
11)
Statistical section 9.2 was updated with new assumptions about the
distribution curve (given that we’re trying to prove the additive effect of
ExoFlo on Remdesivir) and percentages of patients recovered by day. The
sample size required was 66 for power of 0.8 and 84 for power of 0.9; thus,
expanded sample size to N=75 and updated this throughout the protocol.
12)
Staggering protocol was updated to simplify it into 3 stages.
13)
Statistical basis for stopping rules was changed to Bayesian technique; safety
review cycle was specified in a table and changed to reflect a practical but still
frequent meeting schedule.
Amendment
II, 31
August 2020
Sascha
Sengupta,
MD
Vikram
Sengupta,
MD
1)
Endpoints were revised and restructured in the Section 1.1 protocol synopsis,
Section 3 Objectives & Endpoints, and Section 9.1 Statistical Hypothesis.
Primary endpoint was changed to % recovered by day =5. Safety endpoints
were simplified as Incidence of Serious Adverse Events and moved into
Secondary Endpoints. VFDs, PaO2/FiO2 which was clarified as improvement
in P/F ratio, as well as all-cause mortality was also renamed a secondary
endpoint. Median time to recovery was removed. Secondary efficacy
endpoints were renamed Exploratory endpoints.
2)
NK degranulation assay remains in Section 1.3 Schedule of Activities. Limited
this test to day = 1, 4, 7, and 15 given the logistics of the external lab involved.
Specified that EKG, microbiology, and rule out labs do not have to be repeated
on day=0 if obtained following hospital admission. Specified that flow
cytometry labs do not have to be obtained on day = 0. Specified that Il-6 and
TNF-alpha do not have to be obtained on day =0.
3)
Time windows for vital sign measurements were specified in Section 1.3 as well
as Section 6.1.3.
4)
EMR removed from the Study Intervention (all of section 6) per CRO request as
individual hospital sites may have different protocol.
5)
Statistical sample size calculation in Section 9.2 was updated using current
primary endpoint. Target N remains 75.
6)
Steering committee and specifics of interaction with DSMB are removed from
the protocol per CRO request as there are differences with their current
Standard Operating Procedures
7)
Modified criteria for slowing and criteria for suspending study intervention in
section 7.1.1. Original criteria of using 10% of baseline for slowing infusion
was very restrictive per CRO.
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8)
Study Procedure by Visit section 8.1 was added per request by CRO for
clarification.
9)
Definition Adverse Events of Special Interests (AESI) was added as section
8.4.3.
10)
Reporting events to participants in section 8.4.9 was updated to reflect that the
CRO typically only informs patients when they have an unexpected or severe
AE.
11)
Data Safety Review Cycles are updated in the Section 7.1.4 Stopping Rules.
There will be 3 safety review cycles for which the DSMB will convene, also
specified in section 10.6: The first is following day =5 for the 9th enrolled
participant; the second is following day =5 for the 37th enrolled patient. The
third is following day = 15 for the 75th enrolled participant (stopping rules does
not apply here as 100% of target N is enrolled). Additional times for meeting(s)
if any will be based on the DSMB charter.
12)
Interim analysis following day =5 enrollment of the 37th patient was added as
section 9.4.6. Planned early unblinding was updated in section 9.4.7 to specify
that unblinding may occur if there’s superiority with either a primary or
secondary endpoint.
Amendment
III, 07
October
2020
Sascha
Sengupta,
MD
1)
Range of research sites increased to 2-5 in section 1.1.
2)
N corrected to 75 in section 1.2
3)
Quantitative SARS-CoV-2 RT PCR unavailable at sites; changed to qualitative
SARS-CoV-2 RT PCR for screening, day=15, 29, & 61. Updated in Sections
1.1, 1.3, 3, 8.1, and 8.2.
4)
NK degranulation assay removed throughout the protocol due to lack of site
feasibility. Updated in Sections 1.1, 1.3, 3, 8.1, 8.2 and 9.1.
5)
Section 7.1.3 on stopping rules was revised to a non-Bayesian version of the
previous stopping rules due to request of the biostatisticians.
6)
Due to site request to clarify section 7.1.2 as all patients will be selected for the
study due to overall clinical deterioration including respiratory deterioration, the
clinical criteria for not receive the repeat study administration has been specified
as the scenario in which a serious and suspected adverse reaction occurred in
response to the Investigational Product within 4 hours of administration.
Amendment
IV
2 November
2020
Sascha
Sengupta,
MD
1)
Range of research sites increased to 2-15 in section 1.1 and 4.1.
2)
Days of exploratory endpoints updated in section 1.1 to match 1.3.
3)
Primary endpoint was updated from proportion of patients recovered by day =5
back to median time to recovery in days given uncertainty regarding anticipated
recovery distributions. This was updated in Sections 1.1, 1.3, 4, 5, 9.1. Sample
size calculation was performed for the primary endpoint without change in N and
updated in Section 9.2.
4)
Ventilator-free days removed as a secondary endpoint given unpredictable
patient characteristics at active RCT site. This was updated in Sections 1.1, 1.3,
4, 9.1.
5)
Schedule of Activities 1.3 updated to reflect EDC design, which is PaO2/FiO2
will remain a daily record in the EDC. While the hospital site will routinely
requiring O2 support recording in the EMR, for ease of use of the EDC, O2
support will only be recorded for days of interest in the EDC.
6)
Overall study design in Section 4.1 was updated to remove intubation status from
the stratification blocks. Due to need for increased enrollment and
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unpredictability of the COVID-19 ARDS patient demographic, patients will be
randomized to treatment arm and stratified by research site only.
7)
Inclusion criteria in Section 5.1 was revised to remove redundant language. For
example, it is unnecessary to state again that patients must be rapidly clinically
deteriorating when screening for EXIT COVID-19. Inclusion criteria already
includes the modified definition of ARDS as well as hypoxia requiring
noninvasive or invasive oxygen support despite Standard of Care, which can
include Dexamethasone and Remdesivir.
8)
All concomitant medications should be logged into the EDC due to CRO
preference. This was updated in Section 6.6.
9)
Section 10.6 was update such that board members of the DSMB were changed
from blinded to unblinded in order to provide proper safety oversight.
10)
In Section 7.1.3, stopping rules were updated with change in day from 5 to 15 for
the trial milestones used for data safety review cycle.
11)
In Section 7.1.3, AEs of special interests are revised to count towards the
stopping rules only if occurring within 72 hours of IP administration. To employ
this disproportionate incidence of grade 3, 4, and 5 SAE between treatment arms
without specifying the acute time frame will be to exercise unnecessary bias
against most novel products in general—suppose if a new biologic is protective
and extends survival (thus possibly hospitalization) for some patients, the
biologic treatment arms will accumulate disproportionate incidence of grade 3, 4,
and 5 SAE compared to placebo simply due to the survival of the treated patients
and the nature of the severe COVID-19 disease state itself, which has been
shown to be prothrombotic and hyperinflammatory in nature.
12)
AEs of Special Interests were revised in Section 7.1.3. Given that multiorgan
failure and cytokine storm are well known and frequently reported sequalae of
refractory COVID-19, these two particular AE of special interests were removed
to reduce false signals in the COVID-19 patient population. DSMB will still be
able to make assessments on data provided and take action based on clinical risk.
13)
Language in Section 8.3 was corrected to reflect that it is the hospital site
research staff that collects data at the site, not the CRO.
Amendment
V
31
December
2020
Sascha
Sengupta,
MD
1)
Primary endpoint was changed from median time to recovery to improvement in
P/F ratio from pre-infusion baseline (Day 0) to Day 7 in Sections 1.1, 3, and 9.1.
Time to recovery was revised to a secondary endpoint.
2)
EQ-5D-5L will be collected only on Days 29 and 61. This was updated in
Sections 1.1, 1.3, 3, and 9.1.
3)
Schedule of Activities in Section 1.3 was updated to reflect the addition to days
of COVID-19 symptoms prior to the current admission in addition to flexibility
with SARS-CoV-2 qualitative PCR given the shortage of testing at certain
hospital sites. In the event of test shortage, documentation of positive SARS-
CoV-2 qualitative or quantitative within 14 days prior to admission will suffice
for Day 0 or 1. The frequency of temperature monitoring was reduced to 5
minutes prior to the infusion and every 15 minutes during the infusion to reduce
staff exposure.
4)
Schedule of Activities in Section 1.3 was updated to reflect the increase in
sample size from N=75 to 120. Please note that while the initial IRT plan will
be to randomize to the three treatment arms 1:1:1—following review of safety
after the 60th patient has been randomized overall and assuming no significant
safety issues (i.e. SUSAR), the unblinded DSMB may request that the IRT be
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reprogrammed to randomize to Treatment Arm 1 and selected experimental Arm
only, such that the final sample size can be reduced from 120 to 100 with, for
example: Arm 1 (N=40), Arm 2 (N=20), and Arm 3 (N=40). This strategy is
justified in the calculation of sample size Section 9.2 and permits N to remain
appropriately low in number for a Phase II Clinical Trial.
5)
Schedule of Activities in Section 1.3 was also updated to clarify a common site
question. Screening and dosing can be on the same day. Therefore, screening
can occur on day =0 or day =1.
6)
Stratification in Overall Study Design in Sections 4.1 and 6.3 was changed back
to stratifying by both research site and intubation status.
7)
Exclusion Criteria in section 5.3 revised to reduce the duration of intubation
from 72 hours to 24 hours.
8)
To allow for flexible adoption of medications approved by the FDA for Severe
COVID-19, Standard of Care was updated with link to NIH treatment guidelines
for COVID-19 in Sections 5.1 and 6.6. Concomitant Medications Section 6.6
was specifically updated with the NIH treatment guideline for COVID-19 link.
9)
Method of notification was updated to the appropriate telephone and/or email
for both sections 6.3 and 8.4.9.
10)
Criteria for slowing or suspending the study administration in Section 7.1.1 was
broadened more as clinical guidance rather than strict clinical algorithms.
11)
The Staggering Protocol in Section 6.4 was updated such that a 24-hour pause is
implemented following day 7 following the 60th patient randomized overall.
This corresponds to the updated Interim Analysis (Section 9.4.5). The Stopping
Rules (Section 7.1.3) was also updated to reflect this important trial milestone.
12)
Methods for Statistical Analysis in Section 9.1, 9.2, and 9.3 were revised given
the change in primary endpoint and further refined overall.
13)
Interim Analysis (Section 9.4.5) was revised to reflect the efficacy analysis will
be performed at Day 7 following the 60th patient randomized overall into the
trial.
14)
Planned Early Unblinding (Section 9.4.6) was revised to reflect that efficacy
analysis will be performed at Day 7 following the final patient randomized
overall into the trial.
Amendment
VI 8
February
2021
Sascha
Sengupta,
MD
1)
IRT plan for randomizing only to treatment arm 1 and treatment arm 3 was
removed from Sections 1.1, 1.3, and 9.2 such that target N is up to 120 for the
overall study.
2)
Acceptable IP for clinical trials were updated with a second lot # P-441-2004-
C5, which was included in previous CMC administrative amendment
submission to the FDA, demonstrating comparable data and laboratory analysis.
Lot numbers will continue to be logged per IP manual. This was updated in
RCT Protocol sections 1.1, 2.24, and 4.3.1.
3)
Language in Section 8.1 clarified and specified such that the hospital sites did
not misinterpret the assessment as conflicting with Section 7.1.2. To be clear,
on Day 4, patients’ eligibility criteria are reassessed such that only a change of
code status and placement of ECMO (aside from criteria clarified in 7.1.2)
would then disqualify patients from receiving the patient from receiving the
repeat study administration.
4)
Stratification in Overall Study Design in Sections 4.1 and 6.3 was changed back
to stratifying by research site only.
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11. APPENDICES
11.1 Imputing PaO2 from SpO2 and Estimating FiO2
The relationship between PaO2 and SpO2 is sigmoidal. While many studies employed linear or
log-linear regression modeling, the following equation, which is technically the Ellis inversion of
the Severinghaus equation, provides a non-linear method for imputing PaO2 from SpO2 and was
proven to be superior in accuracy in analysis of data from three ARDS Network Studies with
total N = 1,184 as well as in prospective, observational study with N = 703.[89, 90]
2 1/2 1/3
11,700 11,700
𝑃𝑂
2
= {
+ [50
3
+ (
)
]
}
(1⁄𝑆 − 1) 1⁄𝑆 − 1
2 1/2 1/3
11,700 11,700
+
{
− [50
3
+ (
)
]
}
(1⁄𝑆 − 1) 1⁄𝑆 − 1
Non-linear*
PO2 = PaO2; S = SaO2 or SpO2; F=FiO2
The following table is the lookup table for PaO2 for given SpO2 and is derived from the
supplemental data of the 2017 prospective, observational study, where * is generally considered
unreliable on the basis of the sigmoidal shape of the hemoglobin-oxygen dissociation curve and
§ is based on SpO2 99.5%.[89]
Measured SpO2 (%)
Imputed PaO2 (mmHg)
100*
167*§
99*
132*
98*
104*
97*
91*
96
82
95
76
94
71
93
67
92
64
91
61
90
59
89
57
88
55
87
53
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Measured SpO2 (%)
Imputed PaO2 (mmHg)
86
51
85
50
84
49
83
47
82
46
81
45
80
44
79
43
78
42
77
42
76
41
75
40
74
39
73
39
72
38
71
37
70
37
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The following table for imputing PaO2 from SpO2 and FiO2 is from the retrospective analysis of
ARDSnet data.[90] Overall, these tables allow for (1) calculation of estimated PaO2/FiO2 when an
ABG or arterial line (A-line) is not available, (2) practical use of PaO2/FiO2 as a standardized
measure of oxygenation when there is institutional and physician variability in mode of oxygen
support, aggressiveness of early intubation, and speed of ventilator weaning. Although an
imputed PaO2/FiO2 may not be as precise as ABG derived PaO2/FiO2, it is still more precise than
other metrics, such as ordinal scales of oxygen support.
The following table for estimating FiO2 from mode of oxygen support is from International
Symposium on Intensive Care and Emergency Medicine
(https://www.intensive.org/epic2/Documents/ Estimation%20of%20PO2%20and%20FiO2.pdf).
Method
O2 (L/min)
Estimated FiO2 (%)
Nasal Cannula
1
24
2
28
3
32
4
36
5
40
6
44
Face Mask
5
40
6-7
50
7-8
60
Nonrebreather
10
95
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11.2 Sample EQ-5D-5L and Scoring
The 5-level EQ-5D version (EQ-5D-5L) consists of a descriptive system, including the
5 dimensions of mobility, self-care, usual activities, pain/discomfort, and anxiety/depression, as
well as EQ visual analogue scale (EQ-VAS). Because the EQ-5D will be administered over the
phone, only the descriptive system will be administered and not the EQ-VAS. Each dimension
has 5 levels: no problems, slight problems, moderate problems, severe problems and extreme
problems. The patient will be asked to indicate his/her health state verbally. This decision results
in a 1-digit number that expresses the level selected for that dimension. The digits for the
5 dimensions can be combined into a 5-digit number that describes the patient’s health state.
Valuation study for EQ-5D-5L has already been performed in the US in 2019.
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e-Table 1. Inclusion and Exclusion Criteria for EXIT COVID-19
Inclusion Criteria – must have all of the following:
• Male or female aged 18-85.
• COVID-19 positive as defined by positive Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) SARS-
CoV-2.
• Hypoxia requiring noninvasive oxygen support such as Nasal Cannula (NC), Nonrebreather (NRB), Bilevel
Positive Airway Pressure (BIPAP), Continuous Positive Airway Pressure (CPAP), high flow nasal cannula
oxygen (HFNC O2) or mechanical ventilation (MV) despite initiating standard of care.
Inclusion Criteria for Subgroup Analysis – must also meet the following specification:
• Moderate to severe ARDS as defined by modified Berlin definition (non-ventilated patients did not have to
have PEEP specifications), which includes timing within 1 week of known clinical insult or new or
worsening respiratory symptoms; bilateral opacities not fully explained by effusions, or lung collapse;
respiratory failure not fully explained by cardiac failure or fluid overload; PaO2/FiO2 ≤ 200 mm Hg.
Exclusion Criteria - one or more of the following
• Vulnerable populations such as pregnant patients, children, individuals with severe physical or mental
disabilities who cannot provide meaningful consent.
• Active malignancy requiring treatment within the last five years.
• Major physical trauma in the last 5 days, including motor vehicle accidents, assaults, mechanical falls with
sequalae of significant bleeding or craniofacial bruising, and surgeries.
• Active tuberculosis or cystic fibrosis.
• Severe chronic respiratory disease including chronic obstructive pulmonary disease or pulmonary fibrosis
requiring home oxygen > 5L/min.
• Use of extracorporeal membrane oxygenation (ECMO) during the current hospitalization.
• Pre-existing pulmonary hypertension.
• Severe pre-existing hepatic impairment (presence of cirrhosis, liver function tests (LFTs) ≥ 6x baseline, INR
≥ 2.0).
• Pre-existing Chronic Kidney Disease (CKD) stage IIIb or End Stage Renal Disease (ESRD) prior to onset of
COVID-19 (stage I, II, and IIIa are acceptable)
• Irreversible coagulopathy (e.g., frequently occluded vascular access despite anticoagulation, precipitous
platelet drops concurrent with end-organ damage suggesting consumptive process) or irreversible
bleeding disorder (e.g., frequent bleeding from vascular access, endotracheal tubes, and foley).
• Pneumonia clearly attributable to a non-COVID-19 related process, including aspiration pneumonia or
pneumonia that is exclusively bacterial, or originating from a diagnosed alternative virus (e.g., influenza).
• Patients who are not full code.
• Endotracheal intubation duration ≤ 24 hours.
• Moribund—expected survival < 24 hours.
• Severe metabolic disturbances on presentation (e.g., ketoacidosis, pH < 7.3)
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e-Table 2. Demographic and Clinical Characteristics of the Patients at Baseline (ITT Analysis Set)
Statistics
ExoFlo 15 mL
(N=34)
ExoFlo 10 mL
(N=34)
Placebo
(N=34)
Age
n
34
34
34
Mean (SD)
56.8 (14.97)
62.1 (13.47)
58.5 (11.76)
Min, Max
24, 81
29, 79
32, 78
Age ≥ 65
n (%)
8 (23.5)
14 (41.2)
10 (29.4)
Age < 65
n (%)
26 (76.5)
20 (58.8)
24 (70.6)
Gender
Male
n (%)
22 (64.7)
21 (61.8)
24 (70.6)
Female
n (%)
12 (35.3)
13 (38.2)
10 (29.4)
Race
American Indian or Alaska Native
n (%)
0
0
0
Asian
n (%)
4 (11.8)
1 (2.9)
1 (2.9)
Black or African American
n (%)
5 (14.7)
1 (2.9)
4 (11.8)
Native Hawaiian or Other Pacific
Islander
n (%)
0
0
0
White
n (%)
21 (61.8)
31 (91.2)
26 (76.5)
Unknown or Other
n (%)
4 (11.8)
1 (2.9)
3 (8.8)
BMI (kg/m2) [1]
n
33
34
34
Mean (SD)
34.98 (9.459)
35.63 (10.939)
34.24 (8.526)
Min, Max
19, 60.6
18.9, 75.5
21.5, 63.5
Respiratory Rate (breaths/min) [1]
n
34
34
34
Mean (SD)
23.8 (5.38)
24.4 (5.41)
25.2 (7.90)
Min, Max
17, 37
14, 39
16, 45
Intubated Prior to Enrolling the
Study
n (%)
2 (5.9)
1 (2.9)
4 (11.8)
Time from the First COVID-19
Diagnosis to First ExoFlo Dose Date
(days)
n
34
34
34
Mean (SD)
10.0 (6.55)
9.1 (4.36)
9.5 (4.12)
Min, Max
2, 38
1, 23
2, 18
Total SOFA Score [1]
n
34
34
33
Mean (SD)
3.2 (1.78)
2.9 (1.20)
3.2 (1.88)
Min, Max
2, 9
0, 6
2, 9
PaO2/FiO2 Ratio (mmHg) [1]
n
17
19
18
Mean
(SD)
115.202
(61.5299)
113.824
(48.5386)
102.952
(43.0002)
Min, Max
50, 211.1
60, 208.8
61, 205
PaO2/FiO2 Ratio < 100 mmHg
n (%)
10 (29.4)
9 (26.5)
10 (29.4)
PaO2/FiO2 Ratio ≥ 100 mmHg
n (%)
7 (20.6)
10 (29.4)
8 (23.5)
Prior Therapy [2]
n
30
27
30
Remdesivir
n (%)
17 (50.0)
21 (61.8)
23 (67.6)
Plasma
n (%)
7 (20.6)
9 (26.5)
9 (26.5)
Dexamethasone
n (%)
26 (76.5)
25 (73.5)
27 (79.4)
[1] Baseline is the last measure prior to the first dose of ExoFlo (Day 0 or Day 1, 5 min before dosing)
[2] Started prior to the first dose of ExoFlo regardless of its end date.
Journal Pre-proof

e-Table 3. Summary of Efficacy by ARDS Status (Moderate to Severe ARDS in ITT Analysis Set)
Moderate*
Severe*
Study Endpoints
Statistics
IP 15 mL
(N=6)
IP 10 mL
(N=9)
Placebo
(N=7)
IP 15 mL
(N=10)
IP 10 mL
(N=9)
Placebo
(N=10)
Subjects Discharged
n (%)
3 (50.0)
3 (33.3)
3 (42.9)
4 (40.0)
4 (44.4)
3 (30.0)
Median Time to Discharge (KM) [1]
n
6
9
7
10
9
10
Median
NR
NR
NR
NR
NR
NR
(1st, 3rd
Quartiles)
(8.0, NR)
(27.0, NR)
(24.0, NR)
(24.0, NR)
(22.0, NR)
(21.0, NR)
Mean Time to Discharge (Restricted
to Discharged Subjects)
n
3
3
3
4
4
3
Mean (SD)
7.3 days (1.15)
16.3 days
(10.50)
21.3 days (6.43)
24.0 days
(25.46)
18.0 days
(10.55)
11.7 days (8.14)
Min, Max
6, 8
6, 27
14, 26
6, 60
8, 31
6, 21
Subjects Who Died Within 30 Days
n (%)
1 (16.7)
2 (22.2)
4 (57.1)
4 (40.0)
3 (33.3)
3 (30.0)
Subjects Who Died Within 60 Days
n (%)
1 (16.7)
4 (44.4)
4 (57.1)
5 (50.0)
5 (55.6)
7 (70.0)
Median Time to Death (KM)
Median
NR
41.0 days
19.0 days
NR
42.0 days
40.5 days
Mortality Rate at 15 Days (KM)
%
16.7
28.6
42.9
30.0
25.0
10.0
Mortality Rate at 30 Days (KM)
%
16.7
28.6
57.1
40.0
37.5
30.0
Mortality Rate at 60 Days (KM)
%
16.7
76.2
57.1
50.0
62.5
70.0
Mean Time to Death (Restricted to
Subjects Who Died)
n
1
4
4
5
5
7
Mean (SD)
6.0 days (NE)
26.8 days
(17.06)
10.5 days (5.80)
19.2 days
(10.33)
25.0 days
(17.10)
30.1 days
(11.25)
Min, Max
6, 6
11, 42
6, 19
11, 36
10, 51
14, 41
P/F Ratio Increase from Baseline to
Day 7 (mmHg) [2]
n
6
9
7
10
8
10
Mean (SD)
66.333
(124.8113)
41.874
(65.7479)
5.457 (9.3842)
54.573
(64.7043)
49.443
(40.8447)
84.214
(91.7971)
Journal Pre-proof

Moderate*
Severe*
Study Endpoints
Statistics
IP 15 mL
(N=6)
IP 10 mL
(N=9)
Placebo
(N=7)
IP 15 mL
(N=10)
IP 10 mL
(N=9)
Placebo
(N=10)
Min, Max
0, 311
0, 176
0, 21
0, 171.9
0, 121.2
0, 303.16
#Ventilation-Free Days (within 60
Days)
n
6
9
7
10
9
10
Mean (SD)
41.2 (24.42)
13.2 (19.72)
25.0 (31.02)
32.6 (28.19)
20.7 (21.45)
25.1 (27.44)
Min, Max
0, 61
0, 61
0, 61
3, 61
2, 61
1, 61
Median Time to Recovery (KM) [3]
n
6
9
7
10
9
10
Median
NR
NR
NR
NR
NR
NR
(1st, 3rd
Quartiles)
(7.0, NR)
(NR, NR)
(15.0, NR)
(NR, NR)
(18.0, NR)
(25.0, NR)
Mean Time to Recovery (Restricted
to Recovered Subjects)
n
2
2
2
1
3
4
Mean (SD)
6.0 days (1.41)
13.5 days
(13.44)
13.5 days (2.12)
24.0 days (NE)
15.0 days (3.61)
18.8 days (9.95)
Min, Max
5, 7
4, 23
12, 15
24, 24
11, 18
5, 27
*According to modified Berlin definition, moderate ARDS is defined as 100 mmHg < P/F ratio <= 200 mmHg, while severe ARDS is defined as P/F ratio <= 100
mmHg.
KM = Kaplan Meier method, NE = Not Evaluable, NR = Not Reached
[1] Subjects who died or discontinued from the study due to a reason other than discharge before reaching 60 days (Day 61) are censored at Day 61.
[2] P/F ratio: All treated subjects with baseline and at least one P/F ratio measured at Day 4 or 7. For missing Day 7 data, 380 mmHg was assigned for
discharged patients, and no change (0) was assigned to patients with negative change from the baseline or died before Day 7.
#Ventilation-free days: days when patients are not on mechanical ventilation within 60 days of follow-up.
[3] Subjects who died or discontinued from the study due to a reason other than recover before reaching 60 days (Day 61) are censored at Day 61.
Journal Pre-proof

e-Table 4. Summary of Efficacy by Age Group (ITT Analysis Set)
Age ≥ 65
Age < 65
Study Endpoints
Statistics
IP 15 mL
(N=8)
IP 10 mL
(N=14)
Placebo
(N=10)
IP 15 mL
(N=26)
IP 10 mL
(N=20)
Placebo
(N=24)
Subjects Discharged
n (%)
2 (25.0)
8 (57.1)
6 (60.0)
18 (69.2)
10 (50.0)
11 (45.8)
Median Time to Discharge (KM) [1]
n
8
14
10
26
20
24
Median
NR
12.0 days
23.5 days
13.0 days
NR
NR
(1st, 3rd
Quartiles)
(NR, NR)
(6.0, NR)
(6.0, NR)
(6.0, NR)
(9.0, NR)
(6.5, NR)
Subjects Who Died Within 30 Days
n (%)
5 (62.5)
4 (28.6)
3 (30.0)
4 (15.4)
6 (30.0)
9 (37.5)
Subjects Who Died Within 60 Days
n (%)
5 (62.5)
6 (42.9)
4 (40.0)
5 (19.2)
8 (40.0)
12 (50.0)
IP 15 mL vs Placebo
P-value [2]
0.3428
0.0218
Median Time to Death (KM)
Median
13.0 days
NR
NR
NR
NR
41.0 days
Mortality Rate at 15 Days (KM)
%
71.4
22.6
20.0
7.7
21.8
25.9
Mortality Rate at 30 Days (KM)
%
71.4
30.4
30.0
15.4
33.8
39.0
Mortality Rate at 60 Days (KM)
%
71.4
45.8
40.0
19.4
47.0
52.1
P/F Ratio Increase from Baseline to
Day 7 (mmHg) [3]
n
3
9
6
14
9
12
Mean (SD)
32.690 (47.3584)
28.219
(41.8297)
21.947
(39.7214)
60.404 (93.2238)
57.604
(61.8153)
62.388
(90.4587)
Min, Max
0, 87
0, 133
0, 102
0, 311
0, 176
0, 303.16
#Ventilation-Free Days (within 60
Days)
n
8
14
10
26
20
24
Mean (SD)
20.8 (25.27)
34.7 (27.77)
42.8 (25.50)
47.6 (22.85)
30.1 (25.64)
30.3 (28.75)
Min, Max
0, 61
0, 61
0, 61
3, 61
0, 61
0, 61
Journal Pre-proof

Age ≥ 65
Age < 65
Study Endpoints
Statistics
IP 15 mL
(N=8)
IP 10 mL
(N=14)
Placebo
(N=10)
IP 15 mL
(N=26)
IP 10 mL
(N=20)
Placebo
(N=24)
IP 15 mL vs Placebo
P-value [4]
0.0792
0.0455
KM = Kaplan Meier method, NR = Not Reached
[1] Subjects who died or discontinued from the study due to a reason other than discharge before reaching 60 days (Day 61) are censored at Day 61.
[2] Chi-square test for 60-day mortality rates. P-value is displayed for a descriptive purpose.
[3] P/F ratio: All treated subjects with baseline and at least one P/F ratio measured at Day 4 or 7. For missing Day 7 data, 380 mmHg was assigned for
discharged patients, and no change (0) was assigned to patients with negative change from the baseline or died before Day 7.
#Ventilation-free days: days when patients are not on mechanical ventilation within 60 days of follow-up.
[4] Wilcoxon rank-sum test. P-value is displayed for a descriptive purpose.
Journal Pre-proof

e-Table 5. Summary of Efficacy by ARDS Status (Moderate to Severe ARDS aged 18 to 65 in ITT Analysis Set)
Moderate*
Severe*
Study Endpoints
Statistics
IP 15 mL
(N=4)
IP 10 mL
(N=4)
Placebo
(N=4)
IP 15 mL
(N=9)
IP 10 mL
(N=6)
Placebo
(N=7)
Subjects Discharged
n (%)
2 (50.0)
2 (50.0)
1 (25.0)
4 (44.4)
3 (50.0)
2 (28.6)
Median Time to Discharge (KM) [1]
n
4
4
4
9
6
7
Median
NR
NR
NR
NR
NR
NR
(1st, 3rd
Quartiles)
(7.0, NR)
(21.5, NR)
(NR, NR)
(24.0, NR)
(22.0, NR)
(8.0, NR)
Mean Time to Discharge (Restricted
to Discharged Subjects)
n
2
2
1
4
3
2
Mean (SD)
7.0 days (1.41)
21.5 days (7.78)
24.0 days (NE)
24.0 days
(25.46)
20.3 days
(11.59)
7.0 days (1.41)
Min, Max
6, 8
16, 27
24, 24
6, 60
8, 31
6, 8
Subjects Who Died Within 30 Days
n (%)
0
1 (25.0)
3 (75.0)
3 (33.3)
0
2 (28.6)
Subjects Who Died Within 60 Days
n (%)
0
1 (25.0)
3 (75.0)
4 (44.4)
2 (33.3)
5 (71.4)
Median Time to Death (KM)
Median
NR
NR
14.0 days
NR
NR
40.0 days
Mortality Rate at 15 Days (KM)
%
0.0
33.3
50.0
22.2
0.0
14.3
Mortality Rate at 30 Days (KM)
%
0.0
NR
75.0
33.3
0.0
28.6
Mortality Rate at 60 Days (KM)
%
0.0
NR
75.0
44.4
40.0
71.4
Mean Time to Death (Restricted to
Subjects Who Died)
n
0
1
3
4
2
5
Mean (SD)
13.0 days (NE)
12.0 days (6.08)
20.3 days
(11.62)
42.0 days
(12.73)
28.8 days
(12.40)
Min, Max
13, 13
8, 19
11, 36
33, 51
14, 41
P/F Ratio Increase from Baseline to
Day 7 (mmHg) [2]
n
4
4
4
9
5
7
Mean (SD)
77.750
(155.5000)
44.000
(88.0000)
5.250 (10.5000)
59.407
(66.6868)
68.488
(38.7443)
103.951
(100.5286)
Journal Pre-proof

Moderate*
Severe*
Study Endpoints
Statistics
IP 15 mL
(N=4)
IP 10 mL
(N=4)
Placebo
(N=4)
IP 15 mL
(N=9)
IP 10 mL
(N=6)
Placebo
(N=7)
Min, Max
0, 311
0, 176
0, 21
0, 171.9
33.6, 121.2
0, 303.16
#Ventilation-Free Days (within 60
Days)
n
4
4
4
9
6
7
Mean (SD)
47.0 (16.67)
25.3 (27.74)
13.3 (25.84)
34.6 (29.17)
26.5 (24.66)
19.6 (28.44)
Min, Max
28, 61
0, 61
0, 52
3, 61
2, 61
1, 61
Median Time to Recovery (KM) [3]
n
4
4
4
9
6
7
Median
NR
NR
NR
NR
NR
NR
(1st, 3rd
Quartiles)
(NR, NR)
(13.5, NR)
(NR, NR)
(NR, NR)
(16.0, NR)
(18.0, NR)
Mean Time to Recovery (Restricted
to Recovered Subjects)
n
1
2
1
1
3
3
Mean (SD)
5.0 days (NE)
13.5 days
(13.44)
15.0 days (NE)
24.0 days (NE)
15.0 days (3.61)
16.0 days
(10.15)
Min, Max
5, 5
4, 23
15, 15
24, 24
11, 18
5, 25
*According to modified Berlin definition, moderate ARDS is defined as 100 mmHg < P/F ratio <= 200 mmHg, while severe ARDS is defined as P/F ratio <= 100
mmHg.
KM = Kaplan Meier method, NE = Not Evaluable, NR = Not Reached
[1] Subjects who died or discontinued from the study due to a reason other than discharge before reaching 60 days (Day 61) are censored at Day 61.
[2] P/F ratio: All treated subjects with baseline and at least one P/F ratio measured at Day 4 or 7. For missing Day 7 data, 380 mmHg was assigned for
discharged patients, and no change (0) was assigned to patients with negative change from the baseline or died before Day 7.
#Ventilation-free days: days when patients are not on mechanical ventilation within 60 days of follow-up.
[3] Subjects who died or discontinued from the study due to a reason other than recover before reaching 60 days (Day 61) are censored at Day 61.
Journal Pre-proof