Clinical trial NCT04914377
Conditions: Covid19
Keywords: TQ Formula Covid 19
Sponsor: Novatek Pharmaceuticals
Start date: 2021-06-08; Completion date: 2022-01-01
Enrollment: 55 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This is a randomized (1:1), double-blind, placebo-controlled phase 2 study to assess safety and efficacy of Total TQ Formula (3 g daily dose of Nigella Sativa Oil) capsules versus placebo in treating patients who have tested positive for novel Coronavirus 2019 (Covid-19) in the outpatient setting.

Patients will be treated at a dose of 500 mg, 3 capsules, two times a day for 14 days from date of randomization. Quantitative viral load as measured by RT-PCR will be evaluated at baseline and on days 7 and 14. Covid-19 symptoms will be measured throughout the study using Modified FLU-PRO Plus.

Detailed description:
Study Population: Up to 60 participants will be randomized 1:1 to receive either TQ Formula Capsules + Standard of Care (SOC) or placebo +SOC

Phase: Phase II

Approximately 2-4 centers in the United States

TQ Formula (Nigella Sativa) 500 mg, 3 capsules, BID, taken orally

Study Duration: 6 months

Participant Duration: Up to 45 days

Arms: arm 1: Capsules containing TQ Formula arm 2: Capsules containing corn oil
Interventions: intervention 1: TQ Formula/Tab
Intervention descriptions: intervention 1: TQ Formula will be administered as an oral dose daily. The dose planned in this study is 3 g per day (3 500mg capsules to be taken twice daily).

Protocol documents:
Prot_000.pdf: 
 
 
  
A Randomized, Double-Blind, Placebo-Controlled Study 
to Evaluate the Safety and Efficacy of TQ Formula in  
Treating Participants who have Tested Positive for Novel 
Coronavirus 2019 (BOSS-Covid-19)  
Protocol Number: BOSS-001  
  
Principal Investigators:   
Nahla Salem, MD                                             
Hassan Bencheqroun, MD  
  
IND Sponsor: Novatek Pharmaceuticals IND 
Number: 152687  
  
CRO: Tranquil Clinical Research  
  
  
Version Number:  v 4.0  
25 March 2021  
  
Approved by:    
 A. O. Kaseb 
 Professor 
A. O. Kase  b 
A. O. Kaseb (Mar 25, 2021 17:18 CDT) 
 Mar 25, 2021 
Name  
Title  
Signature  
Date  
  
  
  
Table of Contents  
STATEMENT OF COMPLIANCE ....................................................................................................................... 5  


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1 
PROTOCOL SUMMARY ......................................................................................................................... 6  
1.1 
Synopsis ......................................................................................................................................... 6  
1.2 
Schema .......................................................................................................................................... 9  
1.3 
Schedule of Activities (SoA) ....................................................................................................... 10  
2 
INTRODUCTION ................................................................................................................................... 11  
2.1 
Background .................................................................................................................................. 11  
2.2 
Rationale and Risk/Benefit Assessment .................................................................................... 14  
2.2.1 
Known Potential Benefits ......................................................................................... 17  
2.2.2 
Known Potential Risks .............................................................................................. 23  
2.2.3 
Assessment of Potential Risks and Benefits .......................................................... 25  
3 
OBJECTIVES AND ENDPOINTS ......................................................................................................... 25  
4 
STUDY DESIGN .................................................................................................................................... 27  
4.1 
Overall Design ............................................................................................................................. 27  
4.2 
Scientific Rationale for Study Design ......................................................................................... 30  
4.3 
Justification for Dose and Formulation....................................................................................... 31  
4.4 
End of Study Definition................................................................................................................ 32  
5 
STUDY POPULATION .......................................................................................................................... 33  
5.1 
Inclusion Criteria .......................................................................................................................... 33  
5.2 
Exclusion Criteria ......................................................................................................................... 33  
5.3 
Lifestyle Considerations .............................................................................................................. 34  
5.4 
Screen Failures............................................................................................................................ 34  
5.5 
Strategies for Recruitment and Retention ................................................................................. 34  
6 
STUDY INTERVENTION ...................................................................................................................... 35  
6.1 
Study Intervention(s) Administration .......................................................................................... 35  
6.1.1 
Study Intervention Description ................................................................................. 35  
6.1.2 
Dosing and Administration ....................................................................................... 35  
6.2 
Preparation/Handling/Storage/Accountability ............................................................................ 35  
6.2.1 
Acquisition and accountability .................................................................................. 35  
6.2.2 
Formulation, Appearance, Packaging, and Labeling ............................................. 35  
6.2.3 
Product Storage and Stability .................................................................................. 35  
6.2.4 
Preparation ................................................................................................................ 36  
6.3 
Measures to Minimize Bias: Randomization and Blinding........................................................ 36 
6.4 Study Intervention Compliance................................................................................................... 
36  
 6.5  
Concomitant and Prohibited Therapy ........................................................................................ 37  
6.3.1 
Rescue Medicine ...................................................................................................... 37  
7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT  
DISCONTINUATION/WITHDRAWAL .............................................................................................................. 37  
7.1 
Discontinuation of Study Intervention ........................................................................................ 37  
7.2 
Participant Treatment Discontinuation/Withdrawal from the Study ......................................... 38  
7.3 
Lost to Follow-Up ......................................................................................................................... 38  
8 
STUDY ASSESSMENTS AND PROCEDURES ................................................................................. 39  
8.1 
Efficacy Assessments ................................................................................................................. 39  
8.2 
Safety and Other Assessments .................................................................................................. 40  


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8.3 
Adverse Events and Serious Adverse Events ........................................................................... 40  
8.3.1 
Definition of Adverse Events (AE) ........................................................................... 40  
8.3.2 
Definition of Serious Adverse Events (SAE) ........................................................... 40  
8.3.3 
Classification of an Adverse Event .......................................................................... 41  
8.3.4 
Time Period and Frequency for Event Assessment and Follow-Up ..................... 42  
8.3.5 
Adverse Event Reporting ......................................................................................... 43  
8.3.6 
Serious Adverse Event Reporting ........................................................................... 43  
8.3.7 
Reporting Events to Participants ............................................................................. 44  
8.3.8 
Events of Special Interest ........................................................................................ 44  
8.3.9 
Reporting of Pregnancy............................................................................................ 44  
8.4 
Unanticipated Problems .............................................................................................................. 44  
8.4.1 
Definition of Unanticipated Problems (UP) ............................................................. 44  
8.4.2 
Unanticipated Problem Reporting............................................................................ 45  
8.4.3 
Reporting Unanticipated Problems to Participants ................................................. 45  
8.5 
Drug Induced Liver Injury Plan ................................................................................................... 45  
9 
STATISTICAL CONSIDERATIONS ..................................................................................................... 47  
9.1 
Statistical Hypotheses ................................................................................................................. 47  
9.2 
Sample Size Determination ........................................................................................................ 48  
9.3 
Populations for Analyses ............................................................................................................ 48  
9.4 
Statistical Analyses ..................................................................................................................... 49  
9.4.1 
General Approach ..................................................................................................... 49  
9.4.2 
Analysis of the Primary Efficacy Endpoint(s) .......................................................... 49  
9.4.3 
Analysis of the Secondary Endpoint(s) ................................................................... 50  
9.4.4 
Safety Analyses ........................................................................................................ 51  
9.4.5 
Baseline Descriptive Statistics ................................................................................. 51 
9.4.6 Planned Interim Analyses 
........................................................................................ 51  
9.4.7 
Sub-Group Analyses ................................................................................................ 52  
9.4.8 
Tabulation of Individual participant Data ................................................................. 52  
9.4.9 
Exploratory Analyses ................................................................................................ 52  
10 
SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS ............................. 52  
10.1 
Regulatory, Ethical, and Study Oversight Considerations........................................................ 52  
10.1.1 
Informed Consent Process....................................................................................... 52  
10.1.2 
Study Discontinuation and Closure ......................................................................... 53  
10.1.3 
Confidentiality and Privacy ....................................................................................... 54  
10.1.4 
Future Use of Stored Specimens and Data ............................................................ 54  
10.1.5 
Key Roles and Study Governance .......................................................................... 54  
10.1.6 
Safety Oversight ....................................................................................................... 55 
10.1.7 Clinical Monitoring 
.................................................................................................... 55  
10.1.8 Quality Assurance and Quality Control ................................................................... 55 10.1.9 Data 
Handling and Record Keeping ........................................................................ 56  
10.1.10 
Protocol Deviations ................................................................................................... 57  
10.1.11 
Publication and Data Sharing Policy ....................................................................... 58  


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10.1.12 
Conflict of Interest Policy .......................................................................................... 58  
10.2 
Additional Considerations ........................................................................................................... 58  
10.3 
Abbreviations ............................................................................................................................... 59  
10.4 
Protocol Amendment History ...................................................................................................... 61 
11 REFERENCES 
...................................................................................................................................... 63  
12 
APPENDIX 1 – Common Terminology Criteria for Adverse Events (CTCAE) v5.0 .......................... 72  
13 
Appendix 2 – Modified FLU-PRO Plus©Questionnaire ...................................................................... 73  
14 
Appendix 3 – BASELINE COVID-19 SEVERITY CATEGORIZATION * ........................................... 75  
15 
Appendix 4 – Prohibited Medications* ................................................................................................. 76  
16 
Appendix 5 - Common substrates for CYP2C9 by therapeutic class ............................................... 79  
  
  
  
 


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STATEMENT OF COMPLIANCE  
  
The trial will be conducted in accordance with International Conference on Harmonisation Good Clinical  
Practice (ICH GCP), and applicable United States (US) Code of Federal Regulations (CFR). The Principal 
Investigator will assure that no deviation from, or changes to the protocol, will take place without prior 
agreement from the Investigational New Drug (IND) sponsor, and documented approval from the 
Institutional Review Board (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 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 IRB before the changes are implemented to the study.  All changes to the consent form 
will be IRB approved; in the event that a new consent form is developed and approved by IRB, a 
determination will be made regarding whether a new consent needs to be obtained from existing 
participants who already provided consent, using a previously approved consent form.  
  
This document contains information that is privileged or confidential. As such, it may not be disclosed 
unless specific prior permission is granted in writing by the sponsor or such disclosure is required by 
federal or other laws or regulations. Persons to whom any of this information is to be disclosed must 
first be informed that the shared information is confidential. These restrictions on disclosure will apply 
equally to all future information supplied, which is indicated as privileged or confidential.  
  
The investigator agrees to comply with the ICH-GCP, World Medical Association Declaration of Helsinki 
(and relevant updates) and applicable local regulations.  The investigator agrees that all staff members 
involved in the conduct of this study are informed about their obligations in meeting the above 
commitments.  
  
  
Principal Investigator:  
  
Printed Name: _________________________________  
  
Signature: _____________________________________  
  
Date: _________________________________________  
  
Institution Name: _______________________________  
  
  
  
  


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Objectives:  
  
Primary Objective:  
  
  
  
1  
 
PROTOCOL SUMMARY  
1.1  
SYNOPSIS   
Title:  
A Randomized, Double-Blind, Placebo-Controlled Study to Evaluate the  
Safety and Efficacy of TQ Formula in Treating Participants who have Tested  
 
Positive for Novel Coronavirus 2019 ( BOSS-Covid-19)  
Study Description:  
This is a randomized (1:1), double-blind, placebo-controlled phase 2 study  
to assess safety and efficacy of Total 3 g daily dose of Black Seed Oil (‘TQ 
Formula ’ this point forward) capsules versus placebo in treating patients 
who have tested positive for novel Coronavirus 2019 (Covid-19) in the 
outpatient setting.   
  
Patients will be treated at a dose of 500 mg, 3 capsules, two times a day 
for 14 days from date of randomization.   
  
Quantitative viral load as measured by RT-PCR will be evaluated at baseline 
and on days 7 and 14. Covid-19 symptoms will be measured throughout 
the study using Modified FLU-PRO Plus.    
  
A detailed schematic describing all visits and a schedule of assessments is 
included in the Schema and Schedule of Activities, Sections 1.2 and 1.3, 
respectively.  
To evaluate if treatment with 3 g TQ Formula (500 mg per capsule, 
3 capsules BID) given orally on outpatient basis can significantly 
reduce median time to sustained clinical response compared to 
placebo in participants with COVID-19 infection treated in the 
outpatient setting.  Sustained clinical response is defined as a 
reduction of scores to </= 2 on all symptoms of the Modified 
FLUPRO Plus.  
  
Secondary Objectives:    
1. To compare the viral load profile over time (from baseline through 
to Day 14) between treatment with 3 g TQ Formula (500 mg per 
capsule, 3 capsules BID) given orally on outpatient basis and 
placebo in participants with COVID-19 infection treated in the 
outpatient setting  
2. To compare the percentage of RT-PCR negative/undetectable (i.e., 
viral clearance) on Day 7 and Day 14 in participants taking 3 g TQ 
Formula (500 mg per capsule, 3 capsules BID) versus participants 
taking placebo   
3. To compare the duration and severity of symptoms (measured by  


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Modified FLU-PRO Plus) over time from Day 1 through Day 14 in  
 
total FLU-PRO  Plus symptom severity score overall and in 
subdomain 
scores 
(namely, 
Nose, 
Throat, 
Eyes, 
Chest/Respiratory, 
Gastrointestinal, 
 
Body/Systemic, 
Taste/Smell),  between treatment with 3 g TQ Formula (500 mg 
per capsule, 3 capsules BID) given orally on outpatient basis and 
placebo in participants with COVID-19 infection  
4. To investigate if there exists an association between viral load and 
symptom severity by study arm and if such associations change 
overtime  
5. To evaluate the safety and tolerability of TQ Formula (500 mg oral 
capsule, 3 capsules BID) when given to participants with COVID-19 
infection  
  
Exploratory Objectives:    
1. 
To evaluate the basic pharmacokinetics of TQ Formula active 
ingredient (thymoquinone) at the same time points (Days 1, 7, and 
14) in participants with COVID-19 infection.  
2. 
To explore the effect of TQ Formula on inflammatory cytokines, 
coagulation factors and effector immune cells at same time points 
(Days 1, 7, and 14) in participants with COVID-19 infection.  
  
  


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Endpoints:  
Primary Endpoint:  
Measurement of the difference in median time to sustained clinical 
response in participants taking 3 g TQ Formula (500 mg per capsule, 3 
capsules BID) versus participants taking placebo.  Sustained clinical 
response is defined as a reduction of scores to </= 2 on all symptoms of 
the Modified FLU-PRO Plus.  
  
Secondary Endpoints:   
1. Measurement of change in quantitative viral load from baseline, 
Day 7, and Day 14 using RT-PCR in participants taking 3 g TQ 
Formula (500 mg per capsule, 3 capsules BID) versus participants 
taking placebo with COVID-19 infection.  
2. Percentage of negative/undetectable RT-PCR (i.e., viral clearance) 
on Day-7 and Day-14 in participants taking 3 g TQ Formula (500 mg 
per capsule, 3 capsules BID) versus participants taking placebo  
3. Measurement of severity of, and change in, Covid-19 symptoms per 
total score as well as sub-scores (Nose, Throat, Eyes,  
Chest/Respiratory, Gastrointestinal, Body/Systemic, Taste/Smell) 
measured through Modified FLU-PRO Plus from Day 1 through Day  
 
14 in participants with COVID-19 infection treated either with 3 g 
TQ Formula (500 mg per capsule, 3 capsules BID) or placebo.  
4. Correlation Coefficient of quantitative viral load and symptom 
severity at baseline, at Day 7, and Day 14 in participants taking 3 g  
TQ Formula (500 mg per capsule, 3 capsules BID) versus 
participants taking placebo   
5. Number of overall adverse events, related adverse reactions, and 
hospitalizations reported in participants taking 3 g TQ Formula (500 
mg per capsule, 3 capsules BID) versus participants taking placebo.  
All AEs/SAEs will be captured throughout the study as per schedule 
of assessments.  
  
Exploratory Endpoints:  
1. Measurement of thymoquinone and metabolites’ concentration in 
the plasma on day 1, Day 7 and 14 using HPLC in patients treated 
with TQ Formula.  
2. Measurement of the inflammatory cytokine production, 
coagulation factors and the various effector immune cell subsets in 
the Peripheral Blood Mononuclear Cells (PBMC) of all patients on 
Day 1, Day 7 and Day 14 using FACS.  


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Study Population:  
Up to 60 participants will be randomized 1:1 to receive either TQ Formula  
Capsules + Standard of Care (SOC) or placebo +SOC   
There should be a good representation of senior population (65 years of age 
and above), as well as ethnic and racial variability  
Phase:  
Phase II  
Study Sites/Facilities 
Enrolling Participants:  
Approximately 2-4 centers in the United States    
Description of Study 
Intervention:  
TQ Formula  (Nigella Sativa) 500 mg, 3 capsules, BID, taken orally  
Study Duration:  
6 months  
Participant Duration:  
Up to 45 days  
  
  
  


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Protocol # BOSS-001  
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1.2  
SCHEMA  
  
  
  
Screening  
Visit  
  
 
  
  
 
  
Visit Days  
4, 7, 10, 14   
Total N:  Obtain informed consent. Screen potential participants by inclusion and exclusion 
criteria; obtain history, complete physical exam, vitals, blood draws, …etc., and  
document.  See Section 1.3, Schedule of Activities  
Follow-up assessments of study endpoints and safety See 
Section 1.3, Schedule of Activities  
  
Visit 1  
Day 1  
Baseline  
  
  
  
  
  
  
   
  
  
  
  
Perform baseline assessments.  
  
 See  Section 1.3, Schedule of Activities  
  
Placebo   
Up to 30 participants  
Black Seed Oil  
Up to 30 participants  
Administer initial study intervention.   
Randomize  
  
  
  
  
  
Final Study  
Visit/ Day 21  
  
  
  
  
  
End of Study FU   
Phone Call  
  
Final Assessments  
See  Section 1.3, Schedule of  
Activities  
Safety Follow Up  
Phone Call  


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1.3  
SCHEDULE OF ACTIVITIES (SOA)  
  
Procedures  
Screen  
Visit  
Baseline d 
Visit  
Study  
Visit 2 f   
Study  
Visit 3g  
Study  
Visit 4 f  
Study  
Visit 5g  
Final  
Study  
Visit f  
Safety Follow Up 
Phone Call  
   
Day -3 to 
-1  
Visit 1,   
Day 1  
Day 4  
+/-1 day  
Day 7 +/- 1 
day  
Day 
10 
+/-1 day  
Day 14 +/1 
day  
Day 21 +/1 
day  
Day 45 +/- 1 day  
Informed consent  
X  
   
   
   
   
   
   
  
Demographics  
X  
   
   
   
   
   
   
  
Medical history  
X  
   
   
   
   
   
   
  
Inclusion/Exclusion Criteria 
Evaluation  
X  
X  
   
   
   
   
   
  
Rapid Antigen Test for Covid-19  
Xc  
  
  
  
  
  
  
  
RT-PCR sample for quantitative 
viral load analysis  
  
Xc  
  
X  
  
X  
  
  
Serum Collection (Blood sample 
for immune monitoring)  
  
X  
  
X  
  
X  
  
  
  
Physical exam   
X  
   
   
X  
   
X  
 
  
Vital signs  
X  
X  
  
X  
  
X  
  
  
Pulse Oximetry  
X  
  
  
  
  
  
  
  
Walking Oximetry  
X  
  
  
  
  
  
  
  
Height  
X  
   
   
   
   
   
   
  
Weight  
X  
   
   
   
   
   
   
  
  
Hematology   
X  
   
Xe  
X  
Xe  
X  
  
  
Serum chemistry a  
X  
   
Xe  
X  
Xe  
X  
 
  
Pregnancy test b  
X  
   
   
   
   
   
   
  
Randomization  
   
X  
   
   
   
   
   
  
Administer study intervention  
   
X….................................................................................X  
   
  
  
Concomitant medication review  X  
X  
X  
X  
X  
X  
X  
  
Adverse event 
review/evaluation  
X  
X  
X  
X  
X  
X  
X  
X  
Patient 
Reported 
Outcomes 
(Modified FLU-PRO Plus)*  
X+  
X………………………………………………………………………………………………….X  
  
Complete Case Report Forms 
(CRFs)  
X  
X  
X  
X  
X  
X  
X  
  
a: Albumin, alkaline phosphatase, total bilirubin, direct bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, glucose, LDH, phosphorus, potassium, total 
protein, AST, ALT, sodium, CBC/diff, LDH, , CRP, prothrombin time, activated partial prothrombin time, , D-dimer b: Serum pregnancy test (women of 
childbearing potential).  
c:  Covid-19 Confirmatory test in addition to quantitative viral load   


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d: Screening and Baseline can be combined   
e: If clinically indicated at the discretion of the investigator  
f: Visits at Day 4 (visit 2), Day 10 (visit 4), and Day 21 (final visit) will be telehealth and can be changed to in-person at the discretion of the investigator g: In the 
event of a complete lockdown due to Covid, visits at Day 7(Visit 3) and Day 14 (Visit 5) could be replaced by home health visit for sample collection and telehealth 
visit for clinical evaluation.  
*Administered everyday  
+ Modified FLU-PRO Plus at Screen will be completed on site to determine if patient meets inclusion criterion  
  
  
  
2  
  
INTRODUCTION  
  
2.1  
BACKGROUND   
  
In December of 2019, a series of acute atypical respiratory disease cases occurred in Wuhan, China which 
quickly spread to other areas. It was discovered that a distinctive coronavirus was responsible which was 
named the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, 2019-nCoV) as a result of its 
high similarity (~80%) to SARS-CoV, which caused acute respiratory distress syndrome (ARDS) and high 
mortality in the years 2002–2003 [91]. This virus has resulted in a global pandemic with an increasing 
death toll. It is easily transmittable from human to human. The outbreak of SARS-CoV-2 was considered 
to have originally started via a zoonotic transmission associated with the seafood market in Wuhan, 
China. It was later determined that human to human transmission played a major role in the subsequent 
outbreak [92]. The disease caused by this virus was called Coronavirus disease 19 (COVID-19) and a 
pandemic was declared by the World Health Organization (WHO). COVID-19 has infected a large number 
of people worldwide, being reported in roughly 200 countries [22,93]. The lack of a targeted therapy 
continues to be a problem.   
  
Until today, six distinct strains of Human coronaviruses (HCoVs) had been described, in addition to the 
newly emerged COVID-19 [46,47,48,]. Coronaviruses are enveloped, positive-sense, single-stranded RNA 
viruses of ~30 kb. They infect a broad array of host species [1]. They are essentially categorized into four 
genera; α, β, γ, and δ based on their genomic structure. α and β coronaviruses infect only mammals [2]. 
Human coronaviruses such as 229E and NL63 are responsible for the common cold and croup; they 
belong to the group of alpha coronaviruses. In contrast, SARS-CoV, OC43, Middle East respiratory 
syndrome coronavirus (MERS-CoV) and SARS-CoV-2 are classified as β coronaviruses. SARS and MERS 
HCoV are the most aggressive strains of coronaviruses, leaving about 800 deaths each. SARS HCoV has a 
10% mortality rate, while MERS HCoV has a 36% mortality rate, according to the WHO [47].  
  
All coronaviruses contain specific genes in ORF1 (Open Reading Frames) downstream regions that 
encode proteins for viral replication, nucleocapsid and spikes formation [48]. The glycoprotein spikes on 
the outer surface of coronaviruses are responsible for the attachment and entry of the virus to host cells. 
MERS-coronavirus employs dipeptidyl peptidase 4 (DPP4), while HCoV-NL63 and SARS-coronavirus 
require angiotensin-converting enzyme 2 (ACE2) as a key receptor [49]. A number of host-cell receptors 
are reported to be recognized by the viral spike protein of SARS-Cov-2, among which is the cell-surface 
Heat Shock Protein A5 (HSPA5), also termed GRP78 or BiP [65]. In a fluorescent study, it was confirmed 
that the SARS-CoV-2 also uses the same ACE2 (angiotensin-converting enzyme 2) cell receptor and 
mechanism for the entry to host cell which is also used by the SARS-CoV [49,50,51,].  
The life cycle of the virus within the host consists of the following 5 steps: attachment, penetration, 
biosynthesis, maturation and release. Once viruses bind to host receptors (attachment), they enter host 


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cells through endocytosis or membrane fusion (penetration). Once viral contents are released inside the 
host cells, viral RNA enters the nucleus for replication. Viral mRNA is used to make viral proteins 
(biosynthesis). Then, new viral particles are made (maturation) and released. Coronaviruses consist of 
four structural proteins; Spike (S), membrane (M), envelop (E) and nucleocapsid (N) [3]. Spike is 
composed of a transmembrane trimetric glycoprotein protruding from the viral surface, which 
determines the diversity of coronaviruses and host tropism. Spike comprises of two functional subunits; 
S1 subunit is responsible for binding to the host cell receptor and S2 subunit is for the fusion of the viral 
and cellular membranes. Angiotensin converting enzyme 2 (ACE2) has previously been identified as a 
functional receptor for SARS-CoV [4]. Structural and functional analysis shows that the spike for 
SARSCoV-2 also binds to ACE2 [[5], [6], [7]]. ACE2 expression is high in the lung, heart, ileum, kidney and 
bladder [8]. In the lung, ACE2 is highly expressed on its epithelial cells. Following the binding of SARS- 
CoV-2 to the host protein, the spike protein undergoes protease cleavage. After the cleavage at the S1/S2 
cleavage site, S1 and S2 subunits remain non-covalently bound and the distal S1 subunit contributes to 
the stabilization of the membrane anchored S2 subunit at the prefusion state [6]. Subsequent cleavage at 
the S2 site presumably activates the spike for membrane fusion via irreversible, conformational changes.  
The symptoms of patients infected with SARS-CoV-2 range from mild symptoms to severe respiratory 
failure with multiple organ failure. On Computerized tomography (CT) scan, characteristic pulmonary 
ground glass opacification can be seen even in asymptomatic patients [12]. Because ACE2 is highly 
expressed on the apical side of lung epithelial cells in the alveolar space [13,14], this virus can likely 
enter and destroy them. This matches with the fact that the early lung injury is often seen in the distal 
airway. Epithelial cells, alveolar macrophages and dendritic cells (DCs) are three main components for 
innate immunity in the airway [15]. DCs reside underneath the epithelium. Macrophages are located at 
the apical side of the epithelium. DCs and macrophages serve as innate immune cells to fight against 
viruses till adaptive immunity is involved.  
T cell responses against coronaviruses are initiated by antigen presentation via DCs and macrophages. 
DCs and macrophages can phagocytize apoptotic cells infected by virus [16]. For example, virus-infected 
apoptotic epithelial cells can be phagocytized by DCs and macrophages, which leads to antigen 
presentation to T cells. SARS-CoV can also bind to dendritic-cell specific intercellular adhesion molecule- 
3-grabbing nonintegrin (DC-SIGN) and DC-SIGN-related protein (DC-SIGNR, L-SIGN) in addition to ACE2 
[[17], [18], [19]]. DC-SIGN is highly expressed on dendritic cells and macrophages. These antigen 
presenting cells move to the draining lymph nodes to present viral antigens to T cells. CD4+ and CD8+ T 
cells play a critical role. CD4+ T cells activate B cells to promote the production of virus-specific antibody, 
while CD8+ T cells can kill viral infected cells.  
Patients with severe diseases show lymphopenia, particularly the reduction in peripheral blood T cells 
[20,21]. Patients with severe diseases have been reported to have increased plasma concentrations of 
proinflammatory cytokines, including interleukin (IL)-6, IL-10, granulocyte-colony stimulating factor 
(GCSF), monocyte chemoattractant protein 1 (MCP1), macrophage inflammatory protein (MIP)1α, and 
tumor necrosis factor (TNF)-α [20,21]. The more severe conditions patients are in, the higher their IL-6 
levels are. CD4+ and CD8+ T cells are activated in those patients as suggested by higher expression of  
CD69, CD38 and CD44. Higher percentage of checkpoint receptor Tm3+PD-1+ subsets in CD4+ and CD8+ 
T cells show that T cells are also exhausted. NK group 2 member A (NKG2A), another marker for 
exhaustion is elevated on CD8+ T cells [22]. Exhaustion of T cells could have led to the progression of the 
disease. Another interesting finding is that aberrant pathogenic CD4+ T cells with co-expressing 
interferon (IFN)-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF) are seen in COVID19 
patients with severe disease [20]. GM-CSF can help to differentiate innate immune cells and augment T 


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cell function, but it can initiate tissue damage at excess [23,24]. GM-CSF+IFN-γ+ CD4+ T cells were 
previously seen upon strong T cell receptor (TCR) responses in experimental autoimmune 
encephalomyelitis (EAE) models, where CD8+ T cells expressing GM-CSF were found at higher percentage 
and secreted IL-6.   
The study of SARS-CoV shows that virus infected lung epithelial cells produced IL-8 in addition to IL-6 
[15]. IL-8 is a well-known chemoattractant for neutrophils and T cells. Infiltration of a large number of 
inflammatory cells are observed in the lungs from severe COVID-19 patients [25,26], and these cells 
presumably consist of a constellation of innate immune cells and adaptive immune cells. Among innate 
immune cells, the majority would be expected to be neutrophils. Neutrophils can act as double-edged 
sword as neutrophils can induce lung injury [27,28,29]. The majority of the observed infiltrating adaptive 
immune cells are likely T cells. CD8+ T cells are primary cytotoxic T cells. Severe patients also show 
pathological cytotoxic T cells derived from CD4+ T cells [30]. These cytotoxic T cells can kill virus but also 
contribute to lung injury [31]. Circulating monocytes respond to GM-CSF released by these pathological T 
cells. CD14+CD16+ inflammatory monocyte subsets are also found at significantly higher percentage in 
COVID-19 patients. These inflammatory CD14+CD16+ monocytes have high expression of IL-6, which 
likely accelerate the progression of systemic inflammatory response.  
An interesting note is that ACE2 is significantly expressed on innate lymphoid cells (ILC)2 and ILC3. NK 
cells are a member of ILC1, which constitute a large portion of ILCs in the lung (~95%). ILC2 and ILC3 
work for mucous homeostasis.   
In addition to respiratory symptoms, thrombosis and pulmonary embolism have been observed in severe 
diseases. This is in line with the finding that elevated d-dimer and fibrinogen levels are observed in 
severe diseases. The function of the endothelium includes promotion of vasodilation, fibrinolysis, and 
anti-aggregation. Because endothelium plays a significant role in thrombotic regulation [32], 
hypercoagulable profiles seen in severe diseases likely indicate significant endothelial injury. Endothelial 
cells also express ACE2 [33]. Of note, the endothelial cells represent the one third of lung cells [35]. 
Microvascular permeability as a result of the endothelial injury can facilitate viral invasion.  
Interestingly, studies have shown correlation between viral load and disease severity. Liu et al [109] 
surmised that patients with severe COVID-19 tend to have a high viral load and a long virus-shedding 
period and as such, the viral load of SARS-CoV-2 might be a useful marker for assessing disease severity 
and prognosis. Tan et al. [110] in a 2020 study discovered that non-survivors of COVID-19 infection had 
obviously higher levels of viral load, indicated by ORF1ab Ct values, as compared to survivors.  
There is currently no effective antiviral treatment for COVID-19. Existing drug alternatives as a result of 
clinical management of earlier discovered coronaviruses have been utilized in the treatment of COVID19 
patients.  
The growing interest in phytomedicine brings along the issue of their safety, and the requirements to 
meet health standards. It has been shown that the seeds and oil of NS plant are characterized by a very 
low degree of toxicity [39].  
A detailed review of risk/benefits and findings from non-clinical studies and clinical studies can be found 
in sections 2.3.1 and 2.3.2.   
  
2.2  
RATIONALE AND RISK/BENEFIT ASSESSMENT    


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Black seed oil offers promising potential as a therapeutic option in management of SARS-CoV-2 as it has 
been shown in studies to possess beneficial properties like anti-viral effects (see sections 2.2.1 and 
2.2.2).  
  
A number of synthetic compounds initially thought to have shown promise in COVID-19 therapy, 
including hydroxychloroquine and chloroquine phosphate [54,55] (which act through several 
mechanisms, including alkalization of the host cell phagolysosomes), and newer antiviral medications 
such as lopinavir [56] have subsequently been shown to have little or no effect on hospitalized COVID19, 
as indicated by overall mortality, initiation of ventilation and duration of hospital stay [113].   
Remdesivir [57,58] which also showed promise is the first FDA approved COVID antiviral therapy [114]. 
Currently (January 2021), two vaccines are authorized and recommended to prevent COVID-19 [116]:  
  
• 
Pfizer-BioNTech COVID-19 vaccine  
• 
Moderna’s COVID-19 vaccine  
  
Traditional herbal medicines and purified natural products may guide the development of novel antiviral 
drugs. In other words, more efficient drugs can often be designed based on the structure of natural 
compounds that exhibit the desired activity. Classic examples of this drug discovery pathway include 
emetine, an isoquinoline alkaloid isolated from Cephaelis ipecacuanha and used as an amoebicidal drug; 
quinine, derived from the bark of Cinchona trees; and numerous other drugs modified from natural 
compounds, such as  aspirin, morphine and paclitaxel, an antineoplastic drug used for the treatment of 
cancer [59]. Indeed, half of all drugs approved between 1981 and 2014 were derived from or mimicked a 
natural compound [60].  
  
Traditional herbal medicines and purified natural products may guide the development of novel antiviral 
drugs. In other words, more efficient drugs can often be designed based on the structure of natural 
compounds that exhibit the desired activity. Classic examples of this drug discovery pathway include 
emetine, an isoquinoline alkaloid isolated from Cephaelis ipecacuanha and used as an amoebicidal drug; 
quinine, derived from the bark of Cinchona trees; and numerous other drugs modified from natural 
compounds, such as aspirin, morphine and paclitaxel, an antineoplastic drug used for the treatment of 
cancer [59]. Indeed, half of all drugs approved between 1981 and 2014 were derived from or mimicked a 
natural compound [60].  
  
Among various medicinal plants, Nigella sativa (N. sativa) (Family Ranunculaceae) is emerging as an herb 
which many research studies have revealed its wide spectrum of pharmacological potential. N. sativa is 
commonly known as black seed. N. sativa is native to Southern Europe, North Africa and Southwest Asia 
and it is cultivated in many countries in the world like Middle Eastern Mediterranean region, South 
Europe, India, Pakistan, Syria, Turkey, Saudi Arabia [36]. Nigella sativa is a widely used medicinal plant 
throughout the world. It is very popular in various traditional systems of medicine like Unani and Tibb, 
Ayurveda and Siddha. Seeds and oil have a long history of folklore usage in various systems of medicines 
and food. The seeds of N. sativa have been widely used in the treatment of different diseases and 
ailments [42].   
  
N. sativa has been extensively studied for its biological activities and therapeutic potential and shown to 
possess wide spectrum of activities which include (among a host of others) antibacterial antifungal, 


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antiviral, antiparasitic, immunomodulatory, analgesic, antimicrobial, antiviral, anthelmintics, 
antiinflammatory, gastroprotective, hepatoprotective, renal protective [42]. Most of the therapeutic 
properties of this plant are due to the presence of thymoquinone which is major bioactive component of 
the essential oil [42]. Thymoquinone, the most prominent constituent of N. sativa seeds essential oil has 
been intensively investigated.  
  
  
Thymoquinone may block the SARS-CoV-2 entry via ACE2 in pneumocytes. Abdel-Fattah et al. found that 
N. sativa oil and thymoquinone produce antinociceptive effects through indirect activation of supraspinal 
μ1- and κ-opioid receptor subtypes [103]. In addition, Takai et al. suggested that brain endogenous 
angiotensin II was involved in central nociceptive mechanisms by its antagonistic interaction with the 
endogenous opioid system [1104. Furthermore, Lantz et al. showed that opioid active peptides such as 
hemorphins have inhibitory effect on ACE [105]. The above line of evidence suggests that opioid 
receptors and ACE share similar inhibitory molecules and as such Rahman in a write up suggested that it 
is possible that thymoquinone might also block ACE2 [106].  
  
Recently, and in collaboration with CODEX Bio Labs, Black Seed Oil and Thymoquinone were tested for 
their effect on viral entry and viral protein translation using Codex’s Murine Leukemia Virus (MLV) 
particles pseudotyped (PP) with a SARS-CoV-2 Spike (unpublished data). In summary, various 
combinations/concentrations of black seed oil and thymoquinone were tested against SARS-CoV-2 MLV 
pseudovirus particles (pp). Luciferase activities were measured with Firefly Luciferase Assay Kit 
(CB80552-010, Codex BioSolutions Inc). It was observed that both Blackseed Oil and Thymoquinone 
seemed to block viral infection (see graphs below).  


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Fig. A Effect of Blackseed oil and Thymoquinone to block SARS-CoV-2-PP infecting the cells. X-axis: 
compound concentration. Y-axis, relative luminescence unit (RLU), reflecting the luciferase activity and 
the viral infectivity  
However, it was found that at high concentrations, Blackseed Oil and Thymoquinone caused cell death 
which indicates that both may have cell toxicity. To confirm it, cell growth assay was performed in the 
presence of Blackseed Oil or Thymoquinone with Codex’s EnerCount cell growth assay kit which 
measures the ATP levels inside the cells  
  


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Fig. B Cell toxicity of Blackseed oil and Thymoquinone on Expi-293F-ACE2 cells. X-axis: compound 
concentration. Y-axis, relative luminescence unit (RLU), reflecting the luciferase activity and the cell 
number  
Conclusion: By comparing Fig. A and Fig. B, it was concluded that at certain concentrations, Blackseed oil  
(0.06%-0.6%) and Thymoquinone (1-10 µ M) can block the SARS-CoV-2 MLV-PP infection without any cell 
toxicity.  
  
 
  
Clinical Studies  
Abdel-Moneim  et al. in a study conducted on HCV patients were able to demonstrate that extracts of NS 
and Zingiber officinale, alone and together (500 mg of NS and/or Z. officinale twice daily for one month), 
improved liver function and decreased viral load in HCV patients [88]. Decreased viral load and improved 
liver function were similarly reported in another study by Barakat et al., where HCV patients received 
capsules of NS oil (450 mg) three times a day over a 3-month period [86].   
2.2.1   KNOWN POTENTIAL BENEFITS   


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Onifade et al. reported reduction of viral load to an undetectable level in 3 months, elevation of the CD4 
count, alleviation of the symptoms, and a sustained sero-reversion in a sero-positive human 
immunodeficiency virus (HIV) infected man treated with NS (10 mL twice/day for six months) [87]. 
Similarly, another study conducted on a sero-positive HIV infected woman receiving NS and honey 
therapy (10 mL thrice/day for 1 year) demonstrated sustained sero-reversion which the author ascribed 
to the probable virucidal activity of NS [88].   
  
A study by Salem et al. stated that, in a four-week course, the efficacy of NS powder (2 g/day) 
administered together with omeprazole to eradicate an H. pylori infection in non-ulcer dyspeptic 
patients was relatively the same as that of triple therapy [82].   
  
Akhtar et al. demonstrated the efficacy of single oral administration of NS powdered seeds and ethanolic 
extract (40 mg/kg body weight) in reducing the percentage of fecal eggs per gram in children who were 
infected with cestodes [83].  
  
Fard et al. conducted a randomized trial in 100 women with Candida albicans vaginitis. The therapeutic 
effects of black seed capsules (500 mg twice daily) used together with clotrimazole vaginal cream were 
compared with those of placebo capsules (500 mg twice daily) used in combination with the same 
vaginal cream. After a 7-day course, symptoms of the infection such as vaginal itching, discharge, 
irritation were reduced more in the cohort receiving black seed capsules used with clotrimazole vaginal 
cream [84].  
  
Blackseed study in Covid-19 patients:  
In an investigator-initiated, open-label-placebo and randomized controlled trial conducted from April 30 
to July 29, 2020 in four medical care facilities in Pakistan, 313 COVID-19 positive patients were stratified 
into two groups: mild to moderate (cough, fever, sore throat, nasal congestion, malaise and/or shortness 
of breath) and severe (fever and/or cough along with pneumonia, severe dyspnea, respiratory distress, 
tachypnea (>30 breaths/min) or hypoxia (SpO2 <90% on room air) (210 and 103 patients respectively) 
using the Clinical Management Guidelines for COVID-19 by the Ministry of National Health Services, 
Pakistan [114]. The patients within each of the two groups were randomly assigned to the treatment and 
control groups (Honey plus Nigella Sativa [HNS] and Placebo). The HNS group received honey (1 gm) plus 
Nigella sativa seeds (80mg) per kg body weight orally in 2-3 divided doses daily for upto 13 days while 
the control group received placebo (empty capsules). The primary outcomes were viral clearance 
(negative RT-PCR for the SARS-CoV-2 RNA), alleviation of clinical symptoms and the lowering of CGS on 
day 6. Secondary outcomes included reduction in fever degree (day 4), CRP levels (day 6), severity of 
symptoms (day 8), CGS score (day 10) and mortality on day 30. The study results showed that HNS 
helped with symptoms alleviation and viral clearance and reduced mortality in patients with moderate 
and severe disease. It was discovered that alleviation of COVID-19 symptoms for patients in the HNS 
groups occurred earlier than control groups: 4 versus 7 days for the moderate patients and 6 versus 13 
days for the severe disease patients. Viral clearance (being negative for the SARS-CoV-2 RTPCR test) 
occurred 4 days sooner in the HNS group for both moderate and severe cases. A significant reduction in 
degree of fever was observed in the severe cases on day 4 (OR: 0.21; 95% CI: 0.09-0.46; P=0.0001). CRP 
levels decreased significantly (P <0.0001) on day 6 in both the HNS groups compared with their 
respective control groups. As per median degree of symptom severity on day 8, 98.13% patients were 
asymptomatic in HNS treated moderate cases in comparison to 56.31% in the control group (OR: 0.009; 
95% CI: 0.001-0.08; P<0.0001). In severe cases, more patients were asymptomatic in the HNS group 


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while more had moderate symptoms (median) in the control arm (OR: 0.1; 95% CI: 0.040.24). By day 10, 
96.26% of the moderate cases patients fully resumed normal activities with HNS compared to 68.93% in 
control group (OR: 0.07; 95% CI: 0.02-0.21). For the severe group, the median CGS at day 10 revealed 
that HNS cases resumed normal activities while control patients were still hospitalized requiring oxygen 
therapy (OR:0.05; 95% CI: 0.02-0.15). Thirty-day morality was 18.87% in control group and 4% with HNS 
therapy (OR: 0.18 95% CI: 0.02-0.92)[115].  
  
 


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A. Mean oxygen saturation spO2 over time in severe cases; Kaplan-Meier probability curves for time 
taken (in days) for alleviation of symptoms in moderate (B) and severe cases (C); Kaplan-Meier 
probability curves for time taken (in days) for viral clearance in moderate (D) and severe cases (E).ns = 
non-significant, *= P<0.05, **=P<0.001,***=P<0.0001.  
  
To mask the after taste/unpleasant taste of the black seed oil in the TQ Formula, enteric coating will be 
applied on the drug (and the placebo). Previous clinical studies using fish oil have shown enteric coating 


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to be safe with the added benefit of improving compliance and enhancing gastric absorption by transient 
protection against gastric acidity [112].  
  
Non-Clinical Studies  
  
A molecular docking and molecular stimulation study by Elfiky et al., where natural product compounds 
were tested against the HSPA5 substrate binding domain, showed that the active components of 
cinnamon and the seeds of Nigella sativa may tightly bind to cell-surface HSPA5 (one of the host cell 
receptors recognized by the viral spike protein) and could be successful in contradicting SARS-CoV-2 spike 
recognition and attachment [63].  
  
In a study by Ulasli et al., extracts prepared from the flowers and buds of Anthemis hyalina DC.  
(Asteraceae), the seeds of Nigella sativa L. (Ranunculaceae), and the peels of Citrus sinensis L. (Rutaceae) 
were tested against MHV-A59 type of betacoronavirus. The maximum non-toxic dose was found to be 
with 1/50 dilution of the extracts. Exposure of the extracts in virus-infected HeLa- 
CEACAM1a (HeLa-epithelial carcinoembryonic antigen-related cell adhesion molecule 1a) cells instigated 
a decrease in virus load. Following Ns treatment, the number of viruses was very low at 6 h post 
infections and it was 1/10th of the control amount by 8 h post infections. However, the reason for the 
decreased viral load is not known.[89] Salem et al. investigated the antiviral effect of black seed oil (BSO) 
from Nigella sativa using murine cytomegalovirus (MCMV) as a model. The viral load and innate 
immunity during early stage of the infection were analyzed. Intraperitoneal administration of BSO to 
BALB/c mice inhibited the virus titers in spleen and liver on day 3 of infection. This effect coincided with 
an increase in serum level of IFN-gamma. On day 10 of infection, the virus titer was undetectable in 
spleen and liver of BSO-treated mice, while it was detectable in control mice. The oil treatment increased 
IFN- gamma production and augmented numbers of CD4+ helper T cells, suppressor function and 
numbers of macrophages [90].  
  
  
Umar et al. looked at the protective and antiviral activities of Nigella sativa against avian influenza (H9N2) 
in 130 non-vaccinated turkeys using 5 experimental groups of 30 turkeys each. Group A was kept as non-
infected and a non-treated negative control while group B was kept as infected and non-treated positive 
control. Turkeys in groups A and B received normal commercial feed while turkeys of groups C, D and E 
were fed on diets containing 1.5%, 4% and 6% NS seeds, respectively, from day one through the entire 
experiment period. All groups were challenged with H9N2 AIV at 4th week of age except group A.  
Turkeys in group B showed more pronounced virus secretion than the turkeys in other groups receiving 
different levels of NS. Moreover, significantly higher antibody titer against H9N2 AIV in turkeys fed 6% NS 
seeds showed the immunomodulatory nature of NS. Similarly, increased cytokine gene expression 
suggested antiviral behavior of NS especially in a dose-dependent manner, leading to suppressed 
pathogenesis of H9N2 viruses. However, reduced virus shedding and enhanced immune responses were 
more pronounced in those turkeys received NS at the rate of 4% and 6% showing that supplement of NS 
would significantly enhance immune responsiveness and suppress pathogenicity of influenza viruses in 
turkeys [107].  
  
In another study by Zaher et al., effect of Nigella sativa was tested using antiviral assays. Results showed 
that N Sativa has antiviral activity against Infectious Laryngotracheitis Virus (ILTV) at a concentration of 
35µM [108].  
  


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In a study by Shahzad et al. [80], rats sensitized to ovalbumin and challenged intranasally with ovalbumin 
to induce an allergic inflammatory response were compared to ovalbumin-sensitized, intranasally 
ovalbumin-exposed rats pretreated with intraperitoneally administered black seed oil and to control rats. 
The levels of IgE, IgG1 and ova-specific T-cell proliferation in spleen were measured by ELISA. The pro-
inflammatory cytokine IL-4, IL-5, IL-6 and TGF-beta1 mRNA expression levels were measured by reverse 
transcription polymerase chain reaction. The intraperitoneal administration of black seed oil inhibited 
the Th2 type immune response in rats by preventing inflammatory cell infiltration and pathological 
lesions in the lungs. It significantly decreased the nitric oxide production in BALF, total serum IgE, IgG1 
and OVA-specific IgG1 along with IL-4, IL-5, IL-6 and TGF-beta1 mRNA expression [80].  
  
In a similar study, Parlar et al. [61] looked at allergic airway inflammation induced by ovalbumin (OVA) 
challenge in sensitized-rats and effect of thymoquinone using 3 groups: 1. Unsensitized control group 
(Control); 2. Sensitized and challenged group (OVA): Rats were sensitized and challenged with OVA; 3. TQ 
administrated group (TQ): Sensitized rats were treated with TQ, three times as 30 min, one and two days 
before the OVA provocation (Three doses of TQ- 1, 10, and 30 mg/kg). Inflammatory cells, interleukin 
(IL)-6 and TNF-α in bronchoalveolar lavage (BAL) fluid, and lipid peroxidation (LPO) in lung tissue were 
measured. Total inflammatory cells were significantly elevated in the OVA group compared with control 
group, mainly eosinophils and neutrophils. TQ treatment resulted in significantly reduced numbers of 
total inflammatory cells in the BALF from rat with OVA-induced allergic asthma, especially eosinophils 
and neutrophils. IL-6 level in BAL fluid was not significantly changed both in OVA and TQ groups as 
compared to control group. However, TNF-α level in BAL fluid was significantly elevated in the OVA group 
as compared to the control group (p=0.011), while TNF-α level in BAL fluid was lower in the TQ group 
than those in the OVA group.  
  
In an in vitro study by Vaillancourt et al., thymoquinone significantly abolished LPS-induced 
proinflammatory cytokines such as interleukin1beta (IL-1β), tumor necrosis factor-alpha (TNF-α), 
metalloproteinase-13 (MMP-13), COX-2, and prostaglandin E2 in an in vitro model of rheumatoid 
arthritis [62].  
  
In a review done by Gholamnezhad et al. in 2018, anti-inflammatory effect of Thymoquinone was 
demonstrated in allergic lung inflammation- It was reported that TQ decreased IL-4, IL-5, and IL-13 but 
increased IFN-γ in BALF and lung homogenates [40] .   
  
An in-vitro study in 2018, Cobourne-Duval et al. conducted a comparative quantitative proteomic 
analysis of LPS/IFNγ-activated BV-2 microglial cells (immortalized murine microglial cell line) with and 
without TQ treatment. The LPS/IFNγ-activated BV-2 cells showed significantly higher protein expression 
of several inflammatory cytokines compared to the controls: IL-2 = 127%, IL-4 = 151%, IL-6 = 670%, IL-10  
= 133%, and IL-17a = 127%. The protein expression of the same inflammatory cytokines in the TQ 
treated, LPS/IFNγ-activated cells were reduced significantly (P < .0001) compared to the protein 
expression levels activated cells without TQ treatment: IL-2 = 38%, IL-4 = 19%, IL-6 = 83%, IL-10 = 23%, 
and IL-17a = 29% [43] Findings from the study showed TQ reduced the expression of several 
inflammatory cytokines in the LPS/ IFNγ activated BV-2 microglial cells, exhibiting an inhibitory effect on 
the expression of IL-2, IL-4, IL-6, IL-10, and IL-17a [43].  
  
  
 
  
2.2.2   KNOWN POTENTIAL RISKS   


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The acute and sub-acute toxicity of N. sativa has been examined in various in-vitro and in-vivo studies. 
The seed extract and its constituents appear to have a low level of toxicity.   
  
Ong et al. in 2016 examined the acute and subacute toxicity profiles of orally administered 
thymoquinone and thymoquinone-loaded nanostructured lipid carrier in BALB/c mice [37]. In this study, 
animal survival, body weight, organ weight-to-body weight ratio, hematological profile, biochemistry 
profile, and histopathological changes were analyzed.  For acute toxicity study, twenty-seven female 
BALB/c mice were randomly assigned into nine groups (n=3), which were the control, two vehicle groups, 
and six treatment groups of TQNLC and TQ at three fixed doses (5, 50, and 300 mg/kg of body weight). 
The control received tap water. The two vehicle groups received olive oil and blank NLC, respectively, of 
the same volume as the treated groups. For treatment groups, TQ was diluted in olive oil, while TQNLC 
was diluted in deionized water. All the treatments were injected via oral administration on the first day 
only. For the subacute toxicity study, ninety BALB/c mice were randomly divided into nine groups (five 
males and five females): the control group, two vehicle groups, and six treatment groups of TQNLC and 
TQ with three escalating doses each (1, 10, and 100 mg/kg of body weight). The control received tap 
water, while the two vehicle groups received olive oil and blank NLC, respectively, at the same volume as 
the treated groups. For treatment groups, TQ was diluted in olive oil while TQNLC was diluted in 
deionized water. TQ and TQNLC were orally administered by gavage for 28 days and the mice were 
observed for any changes in the general physical conditions, such as appearance, fur condition, behavior, 
and mortality. For the acute toxicity study, 300mg/kg caused mortality to all the mice within 24 hours for 
TQ and only 1 mouse for TQNLC. No significant gross organ changes or weight changes were observed.  
For the subacute study, oral administration of TQNLC and TQ for 28 days did not cause any behavioral 
abnormalities of the mice at any time point. No mortality was observed during the experimental period. 
No significant gross organ changes or weight changes were observed. There were no significant changes 
in the hematological profiles of either male or female mice. No alterations were observed in the kidneys 
for all the treated groups in both sexes. The liver of both male and female mice treated with 100 mg/kg 
TQ and 100 mg/kg TQNLC showed areas of pyknotic nucleus and cell degeneration. Although the liver 
biochemical data were within the normal range, this suggests that the high dose of TQ and TQNLC may 
cause some toxic effects to the liver but not to the extent of altering the functions of the organ [37].  
  
A study by Abukhader in 2012 showed that the MTD for the oral ingestion of TQ in both male and female 
rats was 250 mg/kg. With respect to rats which received oral ingestion of TQ, data collected from 
experimental observations and analysis suggested that single oral ingestion of TQ in doses lower than 
500 mg/kg body weight could be nonlethal. Signs of transient toxicity were observed with 300 and 500 
mg/kg such as weight loss, slight abdominal distention [44].  
  
Zaoui et al. in 2002 investigated the toxicity of Nigella sativa L seeds in mice and rats through 
determination of LD50. The results on the acute toxicity study suggested that Nigella sativa oil has a large 
window of safety with a LD50 > 2000 mg/kg [38,39].  
Datau et al. in a study performed on 39 centrally obese men demonstrated that intake of NS seeds (3 
g/day for 3 months) had no detectable side effects [65]. Administration of 2g/day of NS seed for 6 weeks 
had no adverse effect on serum ALT or serum Cr in adult subjects [66]. Dehkordi et al. reported that the 
intake of NS extract (200 or 400 mg/day) for 2 months caused no observable complications in patients 
with mild hypertension [67]. NS at doses of 1, 2 and 3 g/day for 3 months had no adverse effect on either 
the renal or the hepatic functions of diabetic patients [68]. Patients with allergic rhinitis treated with NS 
seeds (250mg/day) for 2 weeks had no adverse effects [70], though some patients with allergic rhinitis 


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when treated using nasal drops of NS oil showed nasal dryness [73]. NS oil intake (equivalent to oil 
obtained from 0.7 g of seeds) for 40 days showed reasonable kidney and liver safety in patients with type 
2 diabetes mellitus (DM) [74]. Whereas no severe side effects were reported in administration of NS oil 
(5 mL/day) to functional dyspeptic patients, some mild adverse impacts were observed, including 
nausea, bloating, and burning sensation [73]. Three controlled studies investigated the adjuvant use of 
NS in type-2 DM [68,77,78]. Nigella sativa administered at the daily dosages of 2 g/day and 3 g/day 
appeared to be well tolerated and was not associated with any adverse renal or hepatic functions 
throughout the study period.    
In a patient suffering from DM, along with coronary artery disease and hypertension receiving NS at 
2,000-2,500 mg/day, acute renal failure was reported to have occurred 6 days after the start of 
treatment [74]. However, it was surmised that this adverse effect could not have been related to NS 
since other clinical trials with many more human subjects had demonstrated the safety of NS in higher 
doses over longer periods of intake and that the adverse effect was probably attributable to 
contamination of the tablets with other products [75].   
In yet another study by Tubesha et al., the acute toxicity of a nanoemulsion preparation of 
Thymoquinone was studied in rats. A test dose limit of 20ml of thymoquinone-rich fraction 
nanoemulsion (TQRFNE) (containing 44.5 mg TQ)/kg. TQRFNE and distilled water (DW) as a control were 
administered orally to both sexes of rats on Day 0 and observed for 14 days. All the animals appeared 
normal and healthy throughout the study. There was no observed mortality or any signs of toxicity 
during the experimental period [64].  
  
Ali M. Al-Amri et al. conducted a phase I safety and clinical activity study of thymoquinone in patients 
with advanced refractory malignant disease. Patients who were at least 18 years of age with an Eastern 
Cooperative Oncology Group performance status (ECOG) of </= 2 received thymoquinone orally at a 
starting dose level of 3, 7, or 10mg/kg/day. Dose escalation proceeded according to a modified Fibonacci 
design. All 21 patients received at least one week of treatment, with a median of 3.71 weeks (range 1 
week to 20 weeks). No side effects were reported though the maximum tolerated dose (MDT) was not 
identified [45].  
  
  
2.2.3 ASSESSMENT OF POTENTIAL RISKS AND BENEFITS   
  
To our knowledge, this will be the first IND study of TQ Formula (Black Seed Oil) in patients with positive 
Covid-19 diagnosis.  However, Black Seed Oil/Nigella Sativa has been studied extensively over many 
years, and has been found to be relatively safe, with very few side effects.    
  
Furthermore, Black seed (Black Cumin or Nigella Sativa) has been categorized by FDA under “Spices and 
other natural seasonings and flavorings that are generally recognized as safe for their intended use, 
within the meaning of section 409 of the Act” Title 21, Chapter I, Subchapter B, Sec. 182.10 Spices and 
other natural seasonings and flavorings.  
Website: https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?fr=182.10  
  
The aim of this study is to provide a potential treatment for outpatients with a positive Covid-19 
diagnosis, where the current standard of care (SOC) is limited to pain/symptom management such as 
acetaminophen.  Any potential decrease in symptom longevity will benefit this population; and the 
potential benefits outweigh the minor risks in this case.  In addition, this small, randomized phase II 


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study will have a minimal number of patients exposed to the investigational product, and for a minimum 
duration.  
  
3  
OBJECTIVES AND ENDPOINTS  
  
  
OBJECTIVES/Purpose  
ENDPOINTS/Outcome Measures  
JUSTIFICATION FOR 
ENDPOINTS  
Primary  
  
  
To evaluate if treatment with 3 g  
TQ Formula (500 mg per capsule, 3 
capsules BID) given orally on 
outpatient basis can significantly 
reduce median time to sustained 
clinical response compared to 
placebo in participants with 
COVID-19 infection treated in the 
outpatient setting.  
  
Measurement of the difference in 
median time to sustained clinical 
response in participants taking 3 g 
TQ Formula (500 mg per capsule, 3 
capsules BID) versus participants 
taking placebo.  Sustained clinical 
response is defined as a reduction 
of scores to </= 2 on all symptoms 
of the Modified FLU-PRO Plus.  
A reduction in time to 
sustained clinical response is 
a direct measure of 
treatment effectiveness.  
Secondary  
  
  
1. To compare the viral load profile 
over time (from baseline through 
to Day 14) between treatment 
with 3 g TQ Formula  (500 mg per 
capsule, 3 capsules BID) given 
orally on outpatient basis and 
placebo in participants with 
COVID-19 infection.  
Measurement of change in 
quantitative viral load from 
baseline, Day-7, and Day-14 using 
RT-PCR in participants taking 3 g  
TQ Formula  (500 mg per capsule, 
3 capsules BID) versus participants 
taking placebo with COVID-19 
infection.  
Faster decline in viral load is 
hypothesized to lead to 
faster recovery from the 
illness and less infectivity  
[109, 110]  
  
  
OBJECTIVES/Purpose  
ENDPOINTS/Outcome Measures  
JUSTIFICATION FOR 
ENDPOINTS  


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2. To compare the percentage of 
RTPCR negative/undetectable (i.e., 
viral clearance) on Day 7 and Day 
14 in participants taking 3 g TQ 
Formula  (500 mg per capsule, 3 
capsules BID) versus participants 
taking placebo  
Percentage of  
negative/undetectable RT-PCR (i.e., 
viral clearance) on Day-7 and  
Day-14 in participants taking 3 g  
TQ Formula  (500 mg per capsule, 3 
capsules BID) versus participants 
taking placebo    
Treatment with 3 g TQ 
Formula  (500 mg per 
capsule, 3 capsules BID) 
given orally on outpatient 
basis is hypothesized to 
result in higher percentages 
of viral clearance by RT-PCR 
Faster decline in viral load is 
hypothesized to lead to 
faster recovery from the 
illness and less infectivity  
[109, 110]  
  
3. To compare the duration and 
severity of symptoms (measured 
by Modified FLU-PRO Plus) 
overtime from Day 1 through Day 
14 in total Modified FLU-PRO Plus 
symptom severity score overall 
and in sub-domain scores (namely, 
Nose, Throat, Eyes,  
Chest/Respiratory,  
Gastrointestinal, Body/Systemic, 
Taste/Smell) , between treatment 
with 3 g TQ Formula  (500 mg per 
capsule, 3 capsules BID) given 
orally on outpatient basis and 
placebo in participants with  
COVID-19 infection  
Measurement of severity of and 
change in Covid-19 symptoms per 
total score as well as sub-scores  
(Nose, Throat, Eyes,  
Chest/Respiratory,  
Gastrointestinal, Body/Systemic,  
Taste/Smell) measured through 
Modified FLU-PRO Plus from Day-1 
through Day-14 in participants with 
COVID-19 infection  treated either 
with 3 g TQ Formula  (500 mg per 
capsule, 3 capsules BID) or placebo  
FLU-PRO is a validated 
measure that has been used 
on multiple virus studies 
[111].  The Modified FLUPRO 
Plus version has additional 
questions (Taste/Smell) that 
are  
Covid-19 specific.  The 
Modified FLU-PRO Plus has 
been shortened to reduce 
number of symptoms.  
  
4. To investigate if there exists an 
association between viral load and 
symptom severity by study arm 
and if such associations change 
overtime  
Correlation Coefficient of 
quantitative viral load and 
symptom severity at baseline, at 
Day-7, and Day-14 in participants 
taking 3 g TQ Formula  (500 mg per 
capsule, 3 capsules BID) versus 
participants taking placebo   
Decreases in viral load are 
hypothesized to be 
correlated with better 
clinical outcomes [109, 110].  
5. To evaluate the safety and 
tolerability of TQ Formula  (500 mg 
oral capsule, 3 capsules BID) when 
given to participants with COVID-
19 infection.  
Number of overall adverse events, 
related adverse reactions, and 
hospitalizations reported in 
participants taking 3 g TQ Formula   
(500 mg per capsule, 3 capsules 
BID) versus participants taking 
placebo.  All AEs/SAEs will be 
captured throughout the study as 
per schedule of assessments.  
Comparing the number of 
AEs and SAEs and any 
relationship to IP is 
important for determining 
the safety profile of TQ 
Formula.  
  


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OBJECTIVES/Purpose  
ENDPOINTS/Outcome Measures  
JUSTIFICATION FOR 
ENDPOINTS  
Tertiary/Exploratory   
  
  
1. To evaluate the basic 
pharmacokinetics of TQ Formula  
main active ingredient 
(thymoquinone) at same time 
points (Days 1, 7, and 14) in 
participants with COVID-19 
infection.  
  
Measurement of thymoquinone 
and metabolites’ concentration in 
the plasma on day 1, Day 7 and 14 
using HPLC in patients treated with 
TQ Formula .  
  
Measurement of the inflammatory 
cytokine production, coagulation 
factors and the various effector 
immune 
cell 
subsets 
in 
the 
Peripheral 
Blood 
Mononuclear 
Cells (PBMC) of these patients on 
Day 1, Day 7 and Day 14 using FACS.  
Thymoquinone is the main 
active ingredient of TQ 
Formula 
 
and 
the 
pharmacokinetics 
of 
thymoquinone 
and 
its’ 
metabolites in the plasma of 
treated patients are used to 
correlate with  
effectiveness of treatment   
  
The inflammatory cytokines 
and immunological markers 
are of importance because 
of their correlation with 
disease severity in COVID19.  
2. To explore the effect of TQ Formula  
on 
inflammatory 
cytokines, 
coagulation factors and effector 
immune cells at same time points 
(Days 1, 7, and 14) in participants 
with COVID-19 infection.  
  
  
4  
STUDY DESIGN   
  
4.1  
OVERALL DESIGN  
  
This clinical research trial aims to demonstrate the safety of TQ Formula , as well as preliminary efficacy 
in treatment of Covid-19.  The hypothesis is that treatment with TQ Formula will significantly reduce 
median time to sustained clinical response, defined as a reduction of scores to </= 2 on all symptoms of 
the Modified FLU-PRO Plus, compared with placebo.  In addition, it is hypothesized that treatment with 
TQ formula will significantly reduce viral load and symptom burden from baseline to Day-7 compared to 
treatment with placebo.  
  
This is a randomized, double-blind, placebo-controlled phase II study to assess the safety and efficacy of 
TQ Formula versus placebo in the treatment of Covid-19 in an outpatient setting.  Participants will be 
randomized 1:1 to receive either 3 g per day of TQ Formula  or placebo (identical in appearance).  The 
participants will receive up to 14 days of dosing.  
  
Screening Visit  
  
The following evaluations should be completed at the Screening visit:  
  
Informed Consent  


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The purpose and procedures of the study will be fully explained to potential participants. Those wishing 
to enroll in the study will sign a written informed consent prior to initiating any study related evaluations 
or procedures.   
  
Demographics  
Participant demographics, including age, race and gender will be captured and documented in the source 
and the electronic case report form (eCRF).  
  
Medical History  
Participant medical history, including any surgical procedures, will be captured and documented in the 
source and the electronic case report form (eCRF).  
  
Inclusion/Exclusion Criteria Evaluation  
Participants will be assessed to ensure that they meet all inclusion criteria and do not meet any of the 
exclusion criteria as listed in sections 5.1 and 5.2   
  
Covid-19 Rapid Test (Antigen)  
Participants will be given a CLIA waived rapid antigen Covid-19 test (per site standard procedures) in 
order to obtain same days results to use for randomization of participants.  The test may be performed 
prior to informed consent as part of standard of care at the clinic.  
  
Concomitant Medication Review  
All concomitant medications, including vitamins and supplements, currently being taken by participants 
will be documented in source and in the eCRF.  
  
Physical Exam   
A complete physical exam of the following systems will be performed by the investigator or appropriately 
qualified designee and documented in the source and in the eCRF at Screen, Day 7 and Day 14.  A 
complete physical examination, performed at screening and other specified visits, should include an 
evaluation of the head, eyes, ears, nose, and throat, and the cardiovascular, dermatologic, respiratory, 
gastrointestinal, and neurologic systems.  Any abnormality identified at baseline should be recorded in 
the patient’s medical records. Limited, symptom-directed physical examinations should be performed at 
specified postbaseline visits and as clinically indicated.  Changes from baseline abnormalities should be 
recorded in patient notes.  New or worsened clinically significant abnormalities should be recorded as 
adverse events in the patient’s medical records.  
  
Vital Signs  
Vital signs, including blood pressure (BP), temperature and heart rate will be collected and documented 
in source and in the eCRF.  
  
Pulse and Walking Oximetry  
SpO2 and 6-minute walk test (walking oximetry) will be performed at screening.  
  
Height and Weight  
Height and weight of patient will be taken at screening.  
  


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Laboratory Evaluations (Hematology and Serum Chemistry)  
Blood samples (approximately 10 ml in total) will be taken for the following laboratory evaluations: 
Albumin, alkaline phosphatase, total bilirubin, direct bilirubin, bicarbonate, BUN, calcium, chloride, 
creatinine, glucose, LDH, phosphorus, potassium, total protein, AST, ALT, sodium, CBC/diff, LDH, , CRP, 
prothrombin time, activated partial prothrombin time, D-dimer  
  
Pregnancy Test  
Females of child-bearing potential will have a serum pregnancy test completed.  
  
  
Baseline Visit  
  
The following evaluations should be completed at the Baseline visit:  
  
Inclusion/Exclusion Criteria Evaluation  
Participants will be re-assessed to ensure that they meet all inclusion criteria and do not meet any of the 
exclusion criteria as listed in sections 5.1 and 5.2   
  
Vital Signs  
Vital signs, including blood pressure (BP), temperature and heart rate will be collected and documented 
in source and in the eCRF.  
  
Sample for quantitative viral load testing (RT-PCR) and confirmation of Covid-19 positive result Participants 
will have a sample taken for RT-PCR quantitative viral testing.  
  
Serum Collection (Immune monitoring)  
Blood samples (approximately 15 ml) will be collected for immune monitoring   
  
Randomization  
Participants will be randomized to receive either TQ Formula  + Standard of Care (SOC) or placebo +SOC  
  
Administer Study Intervention  
Participants will be provided with study treatment kit, with enough capsules for 14 days of treatment, 3 
capsules BID.  
  
Concomitant Medications and Adverse Events Review  
Participants will be assessed for any new adverse events and any changes in concomitant medications  
  
Patient Reported Outcomes   
Participants will receive the Modified FLU-PRO Plus questionnaire to complete on a daily basis for the 
duration of the study, from randomization to final study visit (Day 21).  
  
  
Subsequent Study Visits (Including Day 21 Final Visit)  
  
Concomitant Medications and Adverse Events Review  


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Participants will be assessed for any new adverse events and any changes in concomitant medications at 
each study visit  
  
Physical Exam   
A complete physical exam will be performed by the investigator or appropriately qualified designee and 
documented in the source and in the eCRF at Day 7 and Day 14. A complete physical examination, 
performed at screening and other specified visits, should include an evaluation of the head, eyes, ears, 
nose, and throat, and the cardiovascular, dermatologic, respiratory, gastrointestinal, and neurologic 
systems.  Any abnormality identified at baseline should be recorded in the patient’s medical records. 
Limited, symptom-directed physical examinations should be performed at specified postbaseline visits 
and as clinically indicated.  Changes from baseline abnormalities should be recorded in patient notes.  
New or worsened clinically significant abnormalities should be recorded as adverse events in the 
patient’s medical records.  
  
Vital Signs  
Vital signs, including blood pressure (BP), temperature and heart rate will be collected and documented 
in source and in the eCRF at each study visit.  
  
Serum Collection (Blood sample for immune monitoring)  
Blood samples (approximately 15 ml) will be collected for immune monitoring at Day 7 and Day 14, in 
addition to the baseline visit.  
  
Sample for quantitative viral load testing (RT-PCR)  
Participants will have a sample taken for RT-PCR quantitative viral testing at Day 7 and again at Day 14, in 
addition to the baseline visit.  
  
Laboratory Evaluations (Hematology and Serum Chemistry)  
Blood samples (approximately 10ml in total) will be taken for safety laboratory evaluations (as listed in 
screening above) at Day 7 and 14 and as clinically indicated at any other study visits  
  
Patient Reported Outcomes   
Participants will receive the Modified FLU-PRO Plus questionnaire to complete on a daily basis for the 
duration of the study, from randomization to final study visit (Day 21).  
  
Safety Follow Up Phone Call (Day 45)  
  
Adverse Events Review  
Participants will be called to assess status for any ongoing adverse events and/or serious adverse events   
  
  
  
4.2  
SCIENTIFIC RATIONALE FOR STUDY DESIGN  
  


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Blackseed oil has been shown to possess anti-inflammatory properties, and anti-corona virus properties, 
based on our extensive literature and our pre-clinical studies that showed its activity in blocking viral 
entry. See sections 2.2 and 2.3 for details.  
This study design (study drug versus placebo) was chosen as there are currently no known outpatient 
treatments for Covid-19.  Participants can maintain their regular standard of care, irrespective of 
treatment allocation, and are allowed to take any additional vitamins or supplements.    
  
The randomization of participants will allow researchers to determine any significant differences 
between treatment groups.  Participants will be blinded to their treatment, in order to remove any 
reporting bias on the patient reported outcome questionnaire.  
  
4.3  
JUSTIFICATION FOR DOSE AND FORMULATION  
  
TQ Formula will be administered as an oral dose daily. The dose planned in this study is 3 g per day (3 
500mg capsules to be taken twice daily). The dosing decision is based on the mechanism of action, the 
pharmacology in non-human studies (the drug has extensive pre-clinical pharmacokinetic and 
pharmacodynamic testing) and human data for completed studies across a number of therapeutic 
indications (see references in toxicity section above). The proposed dose of 3 g per day has been used in 
a number of human trials reporting side effects ranging from absence of any side effects to mild side 
effects; otherwise, the drug has been well tolerated. Mahdavi et al. conducted a double-blind 
placebocontrolled randomized clinical trial to determine the effects of NS oil combined with a calorie-
restricted diet on systemic inflammatory biomarkers in obese women. 90 volunteer obese women were 
enrolled. The intervention group received 3 g/day NS oil soft gel capsules (one capsule, three times a 
day, 30 minutes before each main meal), and the placebo group received similar amounts of sunflower 
oil as a placebo for eight weeks. Subjects reported no side effects during the intervention except mild 
gastrointestinal problems [81]. In a study by Bamosa et al., patients with type 2 diabetes mellitus were 
randomly divided into 3 groups to receive Nigella Sativa seed extracts at 1 gm, 2 gms and 3 gms per day 
respectively for 12 weeks. In the study, generally, the three doses of Nigella sativa were well tolerated 
with only three patients who experienced a mild epigastric discomfort that settled down after taking the 
capsules post meals [68]. In a study by Datau et al. performed on 39 centrally obese men assigned to 
receive NS seeds (3 g/day for 3 months) or placebo, subjects reported subjective complaints related to 
central obesity (body ache, weakness, sleeplessness, belching, decreased libido). All subjective 
complains reported by subjects in the treatment group disappeared in the first week of the study, but 
they all still persisted in the control group through the end of the study. [65].  
  
Enteric coated formulation   
To mask the after taste/unpleasant taste of black seed oil in TQ Formula, enteric coating will be applied 
on the drug (and the placebo). Previous clinical studies using fish oil have shown enteric coating to be 
safe with the added benefit of improving compliance and enhancing gastric absorption by transient 
protection against gastric acidity [112].  
  
Dosing Modifications  
Based upon data from previous studies using blackseed oil capsules, the doses proposed in this protocol 
are within the maximum tolerated doses. However, dose reductions will be allowed per protocol as 
below (Table 4.3.1).   


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If a patient experiences a Grade 3 or greater drug-related AE after a dose of blackseed capsules upon 
evaluation per protocol, then the next dose will not be administered. The patient may receive additional 
doses of blackseed capsules, at the discretion of the PI, if the Grade 3 or greater event has resolved or 
has been reduced sufficiently as assessed by the treating PI prior to administration of the additional 
doses of blackseed capsules and dose reduction will be allowed as per Table 4.3.1 below.  
  
Table 4.3.1: Dose modifications  
Agent  
Starting Dose (0 Dose  
Level)  
-1 Dose Level  
-2 Dose Level  
Blackseed Oil  
Capsules/Placebo  
500 mg, 3 capsules, orally 
twice a day (total  
3g daily)  
500 mg, 2 capsules, orally 
twice a day (total  
2g daily)  
500 mg, 1 capsule, orally 
twice a day (total  
1g daily)  
  
For Grade 3 or greater treatment related toxicity that cannot be controlled with symptomatic treatment 
within 48 hours, such as nausea and/or vomiting that can be controlled by antiemetics or diarrhea that 
can be controlled by anti-diarrheal medicines, the dose level will be decreased to dose level -1. Similarly, 
patients who develop Grade 3 treatment related toxicity that cannot be controlled with symptomatic 
treatment within 48 hours at dose level -1 will have further decrease in dosage to dose level -2. Finally, 
patients who develop Grade 3 treatment related toxicity that cannot be controlled with symptomatic 
treatment within 48 hours at dose level -2 will discontinue therapy. If the patient experiences significant 
toxicity requiring a dose reduction, the patient will remain at the lower dose level for subsequent cycles, 
i.e., dose re escalation is not allowed.  
Patients should terminate study treatment if further reduction is indicated beyond the –2 level. 
Treatment may be delayed up to 10 days to allow for recovery from treatment-related toxicities. If the 
patient has not recovered after a 10 day delay, all study treatment will be discontinued.  Participant will 
remain on study for safety follow up per schedule of assessments.  
  
  
Resuming therapy may not be allowed until treatment-related toxicities have resolved to baseline, unless 
the toxicities are considered by the Investigator unlikely to develop into serious or lifethreatening events. 
In this case, treatment may be continued at the same dose without reduction or interruption.  
  
  
  
4.4  
END OF STUDY DEFINITION  
  
A participant is considered to have successfully completed the study if he or she has completed all 
phases of the study including the last visit shown in the Schedule of Activities (SoA), Section 1.3.  An end 
of study form will be completed in EDC.  Participants who terminate early will also have an end of study 
form completed.  
  
  
5  
STUDY POPULATION  


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5.1  
INCLUSION CRITERIA  
  
In order to be eligible to participate in this study, an individual must meet all of the following criteria:  
  
1. Provision of signed and dated informed consent form  
2. Stated willingness to comply with all study procedures and availability for the duration of the 
study   
3. Male or female, aged 18 and over, presenting with recent mild to moderate clinical symptoms of 
Covid-19 infection (per FDA guidance – see appendix 3)  
4. Positive COVID-19 infection confirmed with a rapid antigen test at screening and confirmed with 
a RT-PCR test at baseline  
5. A score of ≥3 on a minimum of 2 symptoms on the Modified FLU-PRO Plus  
6. Ability to take oral medication and be willing to adhere to the dosing regimen (Twice a day – BID 
for 14 days)  
7. For females of reproductive potential: negative pregnancy test at screening and use of highly 
effective contraception method during study participation and for an additional 4 weeks after 
the end of study drug administration   
8. For males of reproductive potential: use of condoms or other methods to ensure effective 
contraception with partner during study participation  
9. Agreement to adhere to Lifestyle Considerations (see section 5.3) throughout study duration  
  
  
5.2  
EXCLUSION CRITERIA  
  
An individual who meets any of the following criteria will be excluded from participation in this study:  
  
1. Current or recent (within 4 weeks) treatment with any corticosteroids; however, high-dose 
inhaled steroids, which are used to treat acute or chronic bronchial inflammation, will be 
permitted    
2. Current or recent (within 4 weeks) treatment with any antivirals   
3. Room air oxygen saturation (SaO2) < 94% at screen  
4. Walking oximetry < 90% or participant unable to complete 6-minute walking oximetry test at 
screen  
5. Severe Covid-19 symptoms (severe per FDA classification – see appendix 3)  
6. Requires immediate admission to hospital for any reason  
7. Pregnancy or lactation  
8. Known allergic reactions to components of black seed oil, or thymoquinone  
9. Treatment with another investigational drug or other investigational intervention within 2 weeks 
of study start and throughout study duration.  
10. Significant hepatic disease (ALT/AST> 4 times the ULN); any laboratory parameter >/= 4 times the 
ULN  
11. History of severe chronic kidney disease or history of liver disease  


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12. Patients with inflammatory bowel disease (such as Crohn’s) that could affect the intestinal 
absorption of TQ Formula  enteric coated capsules.  
13. Known HIV or Hepatitis C infection  
14. Influenza diagnosis (confirmed by testing) during screening or within prior 14 days  
15. Any uncontrolled condition(s) or diagnosis, both physical or psychological, or physical exam 
finding that precludes participation, as per investigator  
  
5.3  
LIFESTYLE CONSIDERATIONS  
  
  
It is recommended that doses be taken with food, approximately 12 hours apart.  Participants may 
continue with their normal standard of care, including any supplements or vitamins.    
  
5.4  
SCREEN FAILURES  
  
Screen failures are defined as participants who consent to participate in the clinical trial but are not 
subsequently randomly assigned to the study intervention or entered in the study. A minimal set of 
screen failure information is required to ensure transparent reporting of screen failure participants, to 
meet the Consolidated Standards of Reporting Trials (CONSORT) publishing requirements and to respond 
to queries from regulatory authorities. Minimal information includes demography, screen failure details, 
eligibility criteria, and any serious adverse event (SAE).  
  
Individuals who do not meet the criteria for participation in this trial (screen failure) because of an initial  
negative Covid-19 test may be rescreened. Rescreened participants should be assigned a new participant 
number, with a source note indicating that the participant is being re-screened.  
  
  
5.5  
STRATEGIES FOR RECRUITMENT AND RETENTION  
  
Participants will be identified through Covid-19 screening tests at participating study sites and potential 
referral centers.  Individuals who test positive for Covid-19 will be contacted and asked if they wish to 
participate in a research study.  Upon signing of the informed consent, each patient will undergo 
screening procedures to determine eligibility for the study.  Each study site may develop a site-specific 
recruitment plan if required.  
  
Up to 60 participants will be equally randomized to receive either TQ Formula  3g (3x 500 mg capsules, 
BID) or matching placebo capsules (See section 9.2 for sample size determination details).   There will be 
between 2-4 US sites participating in this study.  The sites will be outpatient clinics that are currently 
testing/treating Covid-19 patients.  
  
It is anticipated that each site will recruit approximately 4-5 participants per week.  Based on this 
anticipated accrual rate, 2 sites should be able to complete recruitment for this study within 
approximately 4-5 weeks.  
  


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Participants will be compensated a reasonable amount for their study time and out of pocket costs such 
as parking, transportation or food on visit days will be reimbursed.  
  
6  
STUDY INTERVENTION  
  
  
6.1  
STUDY INTERVENTION(S) ADMINISTRATION  
  
6.1.1 STUDY INTERVENTION DESCRIPTION  
  
The study intervention is TQ Formula  capsules, 500 mg per capsule.  The control product is placebo 
capsules, identical in appearance to TQ Formula  capsules but with no active ingredients.  
  
Black seed oil is currently available as a food supplement, with 500 mg capsules being sold over the 
counter.  For the purposes of this trial, both TQ Formula  and placebo capsules will be manufactured as 
over-encapsulated capsules.  A specific batch and lot will be produced, with appropriate stability testing.  
 
  
All study participants will receive the same dose of either TQ Formula  (total dosing of 3 g per day) or 
placebo for the duration of the study.  Study participants should take 3 capsules orally (500 mg each), 
approximately 12 hours apart, two times per day (BID).   Each dose should be taken preferably with food.  
Missed doses will not be made up; however, the window for dosing is +/- 6 hours.  If longer than 6 hours 
since missed dose, participants should continue with next scheduled dose.  There are no dose limiting 
effects anticipated with this investigational drug.  
  
6.2  
PREPARATION/HANDLING/STORAGE/ACCOUNTABILITY  
  
6.2.1 ACQUISITION AND ACCOUNTABILITY  
  
A central IP depot will provide the pre-labelled, pre-packaged investigational product to the study sites.  
Sites will receive an initial shipment and possibly a second shipment (if required).  All used, unused 
and/or expired product will be returned to the IP depot or destroyed onsite if study site meets regulatory 
requirements for destruction and can provide appropriate documentation for sponsor.  
  
6.2.2 FORMULATION, APPEARANCE, PACKAGING, AND LABELING  
  
The investigational drug consists of 500 mg of black seed oil per capsule.  Placebo consists of similar oil 
without active ingredient.  Investigational drug will be undistinguishable from placebo; the appearance 
of the capsules and all packaging will be identical.  Investigational product will be packaged as 500 
mg/placebo capsules; each participant will receive a total of 84 capsules in order to take 6 capsules per 
day for the 14-day study treatment period.  Investigational product will be labelled per CFR regulations.  
  
6.1.2   DOSING AND ADMINISTRATION  


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6.2.3 PRODUCT STORAGE AND STABILITY  
  
Both study and placebo product capsules should be stored at ambient temperature (15°C to 25°C) and 
protected from sunlight exposure.  Investigational product should be stored in a locked, limited-access 
room.   Only designated study personnel should dispense investigational product.  
  
  
 
  
There is no preparation of study or placebo product required by the study site.  All pre-packaged and 
pre-labelled kits with appropriate number of capsules for 14 days of dosing for each patient are provided 
to the sites.  Sites will be provided with a randomization number for each subject that will correspond to 
a kit number on site.  
  
6.3  
MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING  
  
Study participants will be randomized in a one to one fashion (1:1) to receive either 3 g/day of TQ  
Formula  (500 mg capsules x 3, BID) or placebo capsules (identical in appearance to active product) for 14 
days.  Participants will receive the full study supply (14-day medication) at randomization.  Study 
capsules should be stored at ambient temperature (15°C to 25°C), away from direct sunlight.  
  
The study is double blinded; all subjects, investigators, and study personnel involved in the conduct of 
the study, including data management and primary biostatistician, will be blinded to treatment 
assignment.  There will be an independent unblinded statistician .  The unblinded study statistician will 
not otherwise participate in study procedures or data analysis.  
  
A randomization list will be prepared by the unblinded statistician and provided to the investigational 
product (IP) depot for labelling.  Sponsor and CRO study staff will remain blinded to study treatment.   
Randomization numbers will be assigned through  the Electronic Data Capture (EDC) system.  
  
Participants will complete a daily questionnaire on Covid-19 symptoms, which will be used to determine 
the primary endpoint for the study.  For this reason, it is important that participants remain blinded to 
treatment allocation.  Participants will, however, have access to their viral test results after Day 14, if a 
test is completed as part of standard of care.   
  
Unblinding  
In the unlikely event that unblinding is required, the principal investigator can contact the unblinded 
statistician to unblind treatment.  Treatment unblinding is discouraged if knowledge of the treatment 
assignment will not materially change the planned management of a medical emergency.  
Unblinding should be discussed in advance with the medical monitor if possible. If the investigator is not 
able to discuss treatment unblinding in advance, then he/she must notify the medical monitor as soon as 
possible about the unblinding incident without revealing the participant’s treatment assignment.   
  
  
6.4  
STUDY INTERVENTION COMPLIANCE  
6.2.4   PREPARATION  


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Investigators will assess compliance to study treatment dosing during each visit with participants.  In 
addition, participants will be asked to return all investigational product with them to each on-site visit for 
compliance verification.  Investigator or designee will perform IP accountability and document on 
appropriate study logs and source documentation on site.  Patient compliance must remain between 
80% and 120% throughout the course of the study.  Patients who miss more than 6 consecutive doses 
will be labelled as non-compliant and will not be included in the per protocol population.  These patients 
will be included in the safety analyses.  
  
6.5  
CONCOMITANT AND PROHIBITED THERAPY  
  
  
For this protocol, a prescription medication is defined as a medication that can be prescribed only by a 
properly authorized/licensed clinician. Medications to be reported in the Electronic Case Report Form 
(eCRF) are concomitant prescription medications, over-the-counter medications and supplements.  
  
Concomitant medication usage will be collected at screening and throughout the course of the study at 
each study visit.  Any medication currently being taken at the time of screening will be recorded at the 
screening visit.  Any changes in concomitant medication usage will be documented throughout the 
course of the study during study visits.  
  
All standard of care medication to treat Covid-19 is allowed per investigator discretion.  
  
Prohibited Medications  
All standard of care medication is allowed during the course of the clinical trial.  The use of any drugs 
approved to treat severe Covid-19 requiring hospitalization, such as: corticosteroids, antivirals,  
Hydroxychloroquine is prohibited for entry into the study.  The use of these medications is considered 
inpatient regimen for severe Covid-19, and these patients are excluded from this trial.  If any participant 
requires these medications due to worsening of Covid-19 symptoms, study treatment will be stopped 
and participant will continue to be followed for safety (See Appendix 4).  
  
Investigators should carefully assess patients who are currently being treated with any drugs that are 
metabolized by CYP2C9 due to potential for interaction with Thymoquinone (See Appendix 5).  
  
6.5.1 RESCUE MEDICINE  
This section is not applicable.  
  
7  
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT DISCONTINUATION/WITHDRAWAL  
  
  
  
7.1  
DISCONTINUATION OF STUDY INTERVENTION  
  


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Discontinuation of investigational product does not mean discontinuation from the study, and remaining 
study procedures should be completed as indicated by the study protocol.  If a clinically significant 
finding is identified (including, but not limited to changes from baseline) after enrollment, the 
investigator or qualified designee will determine if any change in participant management is needed.  
Any new clinically relevant finding will be reported as an adverse event (AE).  
  
The data to be collected at the time of study intervention discontinuation will include the following:  
• 
Reason for discontinuation  
• 
Adverse events  
• 
Changes to concomitant medications  
  
If a participant discontinues investigational product, all study visits will continue as per the Schedule of 
Assessments (SOA).  
  
Participants who are randomized but who subsequently have a negative RT-PCR Covid-19 test, will 
discontinue investigational product, but will continue to be followed up for safety and efficacy.    
  
7.2  
PARTICIPANT TREATMENT DISCONTINUATION/WITHDRAWAL FROM THE STUDY  
  
  
Participants are free to withdraw from participation in the study at any time upon request.  
An investigator may discontinue or withdraw a participant from the study treatment for the following 
reasons:  
  
• 
Pregnancy  
• 
Significant study intervention non-compliance   
• 
If any clinical adverse event (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  
• 
Disease progression which requires discontinuation of the study intervention  
• 
If the participant meets an exclusion criterion (either newly developed or not previously 
recognized) that precludes further study participation  
The reason for participant treatment discontinuation or withdrawal from the study will be recorded in 
the source documents and on the Electronic Case Report Form (eCRF).   
For any participants who withdraw from the study, all attempts should be made to ensure that a safety 
follow up visit is completed (Day 21 procedures).    
  
7.3  
LOST TO FOLLOW-UP  
  
A participant will be considered lost to follow-up if he or she fails to attend 3 consecutive scheduled visits 
(either telehealth or in person) and is unable to be contacted by the study site staff.   
  
The following actions must be taken if a participant fails to return to the clinic for a required study visit:  


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• 
The site will attempt to contact the participant and reschedule the missed visit within 1 day and 
counsel the participant on the importance of maintaining the assigned visit schedule and 
ascertain if the participant wishes to and/or should continue in the study.  
• 
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, 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.  
  
  
8  
STUDY ASSESSMENTS AND PROCEDURES  
  
Administrative Procedures  
Informed consent  
Inclusion/exclusion criteria  
Demographics and medical history  
Prior and concomitant medication review  
Randomization and Study intervention  
Modified FLU-PRO Plus Questionnaire  
Complete case report forms  
Clinical Procedures/Assessments  
Review adverse events and concomitant medications  
Physical examination  
Height, weight, and vital signs (T, P, RR, BP)  
Pulse and Walking Oximetry  
LOCAL Laboratory Assessments  
Covid-19 Rapid Antigen Test   
Pregnancy test (only for females of child-bearing potential)  
Hematology (CBC with differential)  
Chemistry panel  
CENTRAL Laboratory Assessments  
RT-PCR sample for quantitative viral load analysis  
Whole blood for biomarker/immune studies (serum and plasma)  
  
  
  
  
8.1  
EFFICACY ASSESSMENTS   


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Covid-19 Quantitative Viral Load (RT-PCR)  
Participants will have an RT-PCR sample taken at baseline, day 7 and day 14 to measure quantitative viral 
load and viral load clearance. These samples will be sent to a central laboratory for analysis.  
  
Patient Reported Outcomes   
Participants will receive the Modified FLU-PRO Plus questionnaire (Appendix 2) to complete on a daily 
basis for the duration of the study, from randomization to final study visit.  The responses will be used to 
measure severity and change in Covid-19 symptoms from baseline.  
  
Serum Collection (Blood sample for immune monitoring)  
Participants will have blood collected for immune monitoring.  This testing will explore pharmacokinetics 
and inflammatory cytokines, coagulation factors and effector immune cells in samples.  These samples 
will be sent to a central laboratory for analysis.  
  
8.2  
SAFETY AND OTHER ASSESSMENTS  
  
Physical Exam  
A complete physical exam of the following systems will be performed by the investigator or  
appropriately qualified designee and documented in the source and in the eCRF at Screening, Day 7 and 
Day 14.  A complete physical examination, performed at screening and other specified visits, should 
include an evaluation of the head, eyes, ears, nose, and throat, and the cardiovascular, dermatologic, 
respiratory, gastrointestinal, and neurologic systems.    
  
Vital Signs  
Vital signs, including blood pressure (BP), temperature, respiratory rate and heart rate will be collected 
throughout the course of the study.  
  
Laboratory Evaluations (Hematology and Serum Chemistry)  
The following laboratory evaluations will be tested locally at screening, Day 7 and Day 14: Albumin, total 
protein, alkaline phosphatase, total bilirubin, direct bilirubin, ALT, AST, sodium, potassium, chloride, 
bicarbonate, BUN, creatinine, phosphorus, calcium, glucose, CBC/diff, LDH, CRP, prothrombin time, 
activated partial prothrombin time, D-dimer.  In the event of elevated liver enzymes, please see section 
8.5 for the drug induced liver injury plan.  
  
Adverse Event Review and Evaluation  
Participants will be assessed for any new adverse events throughout the course of the study.  Participants 
will be encouraged to contact the study site immediately if they observe any worsening symptoms or 
health concerns.  
    
8.3  
ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS  
  
  


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8.3.1 DEFINITION OF ADVERSE EVENTS (AE)  
  
Adverse event is defined as any untoward medical occurrence associated with the use of an intervention 
in humans, whether or not considered intervention-related (21 CFR 312.32 (a)).  
  
For this study, adverse events will be captured from the time of informed consent until 7 days after last 
dose of investigational product (30 days after last dose for SAEs)  
  
8.3.2 DEFINITION OF SERIOUS ADVERSE EVENTS (SAE)   
  
An adverse event (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 adverse event  
 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, be life-threatening, or require hospitalization may 
be considered serious when, based upon appropriate medical judgment, they may jeopardize the 
participant and may require 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.  
  
SAEs will be captured from the time of informed consent until 30 days after the last dose of 
investigational product.  For information on SAE reporting and associated procedures and forms, refer to 
the study Safety Plan.  
  
8.3.3  
  
CLASSIFICATION OF AN ADVERSE EVENT  
8.3.3.1 SEVERITY OF EVENT  
  
All AEs will be assessed for severity by the investigator or designated sub-investigator.  The severity of all 
AEs will be graded according to the National Cancer Institute Common Terminology Criteria for Adverse 
Events (NCI CTCAE v. 5.0)  
https://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/CTCAE_v5_Quick_Reference 
_5x7.pdf  
  
For adverse events (AEs) not included in the CTCAE, the following guidelines will be used to describe 
severity.   
  
• 
Mild – Events require minimal or no treatment and do not interfere with the participant’s daily 
activities.   


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• 
Moderate – Events result in a low level of inconvenience or concern with the therapeutic 
measures. Moderate events may cause some interference with functioning.  
• 
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.  Of note, the term “severe” does not necessarily equate to “serious”.  
  
  
8.3.3.2 RELATIONSHIP TO STUDY INTERVENTION  
  
All adverse events (AEs) must have their relationship to study intervention assessed by the Investigator 
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. In a clinical 
trial, the study product must always be suspect.   
  
• 
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 (dechallenge) should be clinically plausible. The event must be 
pharmacologically or phenomenologically definitive, with use of a satisfactory rechallenge 
procedure if necessary.  
• 
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 (dechallenge). Rechallenge information is not required to fulfill this definition.  
• 
Possibly Related – A clinical event, including an abnormal laboratory test result, whose temporal 
relationship to study intervention administration makes a causal relationship possible, but 
improbable, and in which other drugs or chemicals or underlying disease may provide plausible 
explanations.  There is some evidence to suggest a causal relationship (e.g., the event occurred 
within a reasonable time after administration of the trial medication) or a temporal relationship 
cannot be ruled out. However, other factors may have contributed to the event (e.g., the 
participant’s clinical condition, other concomitant events).     
Note: 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.  
• 
Not Related – The AE is completely independent of study intervention administration (e.g., the 
event did not occur within a reasonable time after administration of the study intervention), 
and/or evidence exists that the event is definitely related to another etiology. There must be an 
alternative, definitive etiology documented by the clinician.  
  
  
 
  
The principal investigator will be responsible for determining whether an adverse event (AE) is expected 
or unexpected; however, sponsor will make the final determination for reporting purposes to regulatory 
8.3.3.3   EXPECTEDNESS   


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authorities.  An AE will be considered unexpected if the nature, severity, or frequency of the event is not 
consistent with the risk information previously described for the study intervention.  
  
8.3.4 TIME PERIOD AND FREQUENCY FOR EVENT ASSESSMENT AND FOLLOW-UP  
  
The occurrence of an adverse event (AE) or serious adverse event (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 electronic case report form (eCRF). 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 medical history 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 a new 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, severity, and duration of each episode.  
  
The principal investigator or designee will record all reportable events with start dates occurring any time 
after informed consent is obtained until 7 (for non-serious AEs) or 30 days (for SAEs) after the last dose 
of study drug.  At each study visit, the investigator will inquire about the occurrence of AE/SAEs since the 
last visit.  Events will be followed for outcome information until resolution or stabilization.  A final follow-
up phone call will be performed at Day 45 in order to assess status of any ongoing AEs or SAEs.  Any AEs 
that are considered unresolved at Day 45 will be followed clinically by the investigator until resolution or 
stabilization.  
  
  
 
  
Adverse Event  
An AE is any unfavorable and unintended sign, symptom, or disease temporally associated with the use 
of an investigational medicinal product (IMP) or other protocol-imposed intervention, regardless of 
attribution.  This includes the following:  
1. 
AEs not previously observed in the subject that emerge during the protocol-specified AE 
reporting period, including signs or symptoms that were not present prior to the AE reporting 
period  
2. 
Pre-existing medical conditions judged by the investigator to have worsened in severity 
or frequency or changed in character during the protocol-specified AE reporting period  
8.3.5   ADVERSE EVENT REPORTING   


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3. 
Abnormal laboratory values or test results constitute adverse events only if they induce 
clinical signs or symptoms and are considered clinically significant  
  
8.3.6 SERIOUS ADVERSE EVENT REPORTING   
  
Generally, any AE considered serious by the PI or Sub-investigator or which meets the definition of an 
SAE included in Section 8.3.2, Definition of Serious Adverse Events must be submitted on an SAE form 
to the Tranquil Clinical Research pharmacovigilance group (PVG).     
  
The principal investigator or designee must immediately report to the sponsor any serious adverse event, 
whether or not considered drug related, including those listed in the protocol or investigator brochure 
and must include an assessment of whether there is a reasonable possibility that the drug caused the 
event [21 CFR 312.64(b)].   
  
Upon identification, all SAEs will be reported by the site within 24 hours using the Initial SAE  
Report Form. The Initial SAE Report Form should be submitted Tranquil Clinical Research (CRO) within 24 
hours either by e-mail (preferable) or by phone using the following contact information:  
  
Email : Safety@tranquilconsulting.com Phone 
: +1 713-898-2137  
  
  
All serious adverse events (SAEs) will be followed until satisfactory resolution or until the site investigator 
deems the event to be chronic or the participant is stable. Other supporting documentation of the event 
may be requested by the study sponsor and should be provided as soon as possible.  
  
The study sponsor will be responsible for notifying the Food and Drug Administration (FDA) of any 
unexpected fatal or life-threatening suspected adverse reaction as soon as possible, but in no case later 
than 7 calendar days after the sponsor's initial receipt of the information.    
  
In addition, the sponsor must notify FDA and all participating investigators in an Investigational New Drug 
(IND) safety report of potential serious risks, from clinical trials or any other source, as soon as possible, 
but in no case later than 15 calendar days after the sponsor becomes aware and determines that the 
information qualifies for reporting.  
  
8.3.7 REPORTING EVENTS TO PARTICIPANTS   
  
Participants will be made aware of any significant changes in safety information that results in a change 
to the informed consent form.  All study participants will be re-consented with the new form as soon as 
possible.  
  
 
8.3.8   EVENTS OF SPECIAL INTEREST   
This section is not applicable.  
  
8.3.9   REPORTING OF PREGNANCY   


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In the event that a participant becomes pregnant while on study, study treatment will be discontinued, 
and a pregnancy form will be completed.  
  
  
8.4  
UNANTICIPATED PROBLEMS  
  
8.4.1 DEFINITION OF UNANTICIPATED PROBLEMS (UP)  
  
The Office for Human Research Protections (OHRP) considers unanticipated problems involving risks to 
participants or others to include, in general, any incident, experience, or outcome that meets all of the 
following criteria:  
  
• 
Unexpected in terms of nature, severity, or frequency given (a) the research procedures that are 
described in the protocol-related documents, such as the Institutional Review Board 
(IRB)approved research protocol and informed consent document; and (b) 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  
• 
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.  
  
An incident, experience, or outcome that meets the definition of an UP noted above may warrant 
consideration of changes to the protocol or consent in order to protect the safety, welfare, or rights of 
participants or others.  
  
8.4.2  UNANTICIPATED PROBLEM REPORTING   
  
The investigator will report unanticipated problems (UPs) to the reviewing Institutional Review Board 
(IRB) and to the Sponsor/CRO. The UP report will include the following information:  
  
• 
Protocol identifying information: protocol title and number, PI’s name, and the IRB project 
number;  
• 
A 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:    
  
• 
UPs that are serious adverse events (SAEs) will be reported according to the timelines noted in 
section 8.3.6.  
• 
Any other UP will be reported to the IRB (per IRB policy) and to the CRO/study sponsor within a 
reasonable time period of the investigator becoming aware of the problem.   


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• 
All UPs should be reported to appropriate institutional officials (as required by an institution’s 
written reporting procedures), the supporting agency head (or designee), and the Office for 
Human Research Protections (OHRP) as applicable, within the appropriate timeline (per 
institution policy) of the IRB’s receipt of the report of the problem from the investigator.  
  
  
8.4.3 REPORTING UNANTICIPATED PROBLEMS TO PARTICIPANTS   
  
If an unanticipated problem results in a change to the informed consent form, all study participants will 
be provided with the updated information and re-consented with the new form.  
  
8.5  
DRUG INDUCED LIVER INJURY PLAN  
  
If patient reports symptoms compatible with DILI (early and nonspecific symptoms include anorexia, 
nausea, fatigue, right upper abdominal discomfort, vomiting), liver enzyme measurements should be 
made immediately, regardless of when the next visit or monitoring interval is scheduled.  
  
As part of the study screening and monitoring criteria, liver enzymes are assessed at screening, Day 7 
(Study Visit 3) and Day 14 (Study Visit 5). Finding of the following will require further follow up:  
Finding of 3-fold or greater elevations above 
the ULN of ALT or AST in subjects  
 
with normal values before drug  
Repeat testing within 48 to 72 hours of  
 
exposure OR  
all four of the usual serum measures  
(ALT, AST, ALP, and TBL) to confirm the  
2-fold increases of ALT or AST above  abnormalities and to determine if they baseline values for 
subjects with AT  
are increasing or decreasing values between 3 and 4 times ULN before drug 
exposure 
  
  
If symptoms persist or repeat testing shows AT >3xULN for subjects with normal baseline measures or 
2fold increases above baseline values for subjects with elevated values before drug exposure, it is 
appropriate to initiate close observation to determine whether the abnormalities are improving or 
worsening as follows:  
 
  
Repeating liver enzyme and serum  
bilirubin tests two or three times  
weekly.  
Frequency of retesting can decrease to  
once a week or less if abnormalities  
stabilize or the trial drug has been  
discontinued and the subject is  
asymptomatic. 


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• 
Obtaining a more detailed history of symptoms and prior or concurrent diseases.  
• 
Obtaining a history of concomitant drug use (including non-prescription medications and herbal 
and dietary supplement preparations), alcohol use, recreational drug use, and special diets.  
• 
Ruling out acute viral hepatitis types A, B, C, D, and E; autoimmune or alcoholic hepatitis; NASH 
(Non-alcoholic steatohepatitis); hypoxic/ischemic hepatopathy; and biliary tract disease.  
• 
Obtaining a history of exposure to environmental chemical agents.  
• 
Obtaining additional tests to evaluate liver function, as appropriate (e.g., INR, direct bilirubin).  
• 
Considering gastroenterology or hepatology consultations  
  
Discontinuation of treatment will be considered if:  
• 
ALT or AST >8xULN  
• 
ALT or AST >5xULN for more than 2 weeks  
• 
ALT or AST >3xULN and (TBL >2xULN or INR >1.5)  
• 
ALT or AST >3xULN with the appearance of fatigue, nausea, vomiting, right upper quadrant pain 
or tenderness, fever, rash, and/or eosinophilia (>5%)   
  
  
9  
STATISTICAL CONSIDERATIONS   
  
A separate Statistical Analysis Plan will be written to describe the statistical analysis procedures 
addressing each primary, secondary and exploratory aims in detail.  
  
9.1  
STATISTICAL HYPOTHESES  
  
The primary statistical hypotheses are:  
1. Treatment with 3 g TQ Formula  (500 mg per capsule, 3 capsules BID) given orally on 
outpatient basis (study drug) will significantly reduce median time to sustained clinical 
response compared to placebo in participants with COVID-19 infection treated in the 
outpatient setting.  Sustained clinical response is defined as a reduction of scores to </= 2 on 
all symptoms of the Modified FLU-PRO Plus.    
  
The secondary statistical hypotheses are as follows:  
1. Study drug will significantly reduce quantitative viral load by Day-14 compared to placebo in 
participants with COVID-19 infection treated in the outpatient setting.  
  
2. Study drug will significantly increase the percentage of negative/undetectable RT-PCR viral 
load at Day 7 and Day 14 in participants taking 3 g TQ Formula  (500 mg per capsule, 3 
capsules BID) versus participants taking placebo in participants with COVID-19 infection  
  
3. Study drug will significantly reduce duration and severity of symptoms overall and within 
each symptom domain overtime from Day 1 to Day 14 compared to placebo in participants 
with COVID-19 infection  
  
4. There is a significantly positive correlation between quantitative viral load and symptom 
severity at baseline, at Day 7, and Day 14 in patients with COVID-19 infection  
  


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5. Study drug will lead to acceptable AE and SAE profiles compared to placebo   
  
• 
Primary Efficacy Endpoint(s):   
  
1. Measurement of the difference in median time to sustained clinical response in participants 
taking 3 g TQ Formula  (500 mg per capsule, 3 capsules BID) versus participants taking 
placebo.  
  
  
• 
Secondary Efficacy Endpoint(s):  
  
1. Measurement of change in quantitative viral load from baseline, Day 7, and Day 14 using 
RTPCR in participants taking 3 g TQ Formula  (500 mg per capsule, 3 capsules BID) versus 
participants taking placebo with COVID-19 infection.  
  
2. Percentage of negative/undetectable RT-PCR (i.e., viral clearance) on Day-7 and Day-14 in 
participants taking 3 g TQ Formula  (500 mg per capsule, 3 capsules BID) versus participants 
taking placebo  
3. Measurement of severity of and change in Covid-19 symptoms per total score as well as sub-
scores (Nose, Throat, Eyes, Chest/Respiratory, Gastrointestinal, Body/Systemic, Taste/Smell) 
measured through Modified FLU-PRO Plus from Day 1 through Day 14 in participants with 
COVID-19 infection  treated either with 3 g TQ Formula  (500 mg per capsule, 3 capsules BID) 
or placebo  
  
4. Correlation Coefficient of quantitative viral load and symptom severity at baseline, at Day 7, 
and Day 14 in participants taking 3 g TQ Formula  (500 mg per capsule, 3 capsules BID) 
versus participants taking placebo  
  
5. Number of overall adverse events, related adverse reactions, and hospitalizations reported 
in participants taking 3 g TQ Formula  (500 mg per capsule, 3 capsules BID) versus 
participants taking placebo.  All AEs/SAEs will be captured throughout the study as per 
schedule of assessments.  
  
9.2  
SAMPLE SIZE DETERMINATION  
  
For statistical analysis purposes, sustained clinical response is defined as a reduction to a score of </= 2 
on all symptom scores on the Modified FLU-PRO Plus questionnaire.  
  
Based on the median time to sustained clinical response reported by CDC [122], the expected median 
time to response in the placebo arm will be approximately 16 days from the onset of symptoms in 
outpatients. With a sample size of 20 in each arm, a difference in median time to sustained clinical 
response of 10 days between the intervention arm and the placebo arm will be able to be detected with 
81.5% statistical power with 5% Type-1 error rate. Median time to sustained clinical response will be 
estimated based on 21-day follow-up. With an attrition expectation of around 20%, the planned accrual 
is 26 patients in each arm and potentially up to 30 patients.   


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Patients will be randomly assigned to one of the two study arms, using a block-randomization with a 
block size of 4, stratified by the enrolling sites. No other stratifications will be used. Randomization block 
size will be kept fixed at 4 as this is a small study and making the block size random as well is not needed. 
An unblinded statistician will generate a randomization list.   
  
Definition of Evaluability for Primary Objectives: The double blind, placebo controlled two-arm clinical 
trial is designed to enroll up to 60 patients equally randomized between the two arms of the study. An 
evaluable patient is defined as a patient who has received at least one dose of the intervention drug or 
placebo.    
  
9.3  
POPULATIONS FOR ANALYSES  
  
• 
Full Analysis Dataset: The Full Analysis Set (FAS) is defined as all randomized participants who 
receive at least one dose of study intervention.   
• 
Final Dataset for Primary Objectives (FDPO): FDPO is defined as all evaluable participants 
according to the evaluability definition above.  
• 
Safety Analysis Dataset: The Safety Analysis Set is defined as participants who take at least one 
dose of study intervention.  
• 
Per-Protocol Analysis Dataset: The Per-Protocol Analysis Set (PP) is defined as all participants in 
the FAS who complete all visits up to and including Day 14, are compliant with study medication 
and do not have any major protocol violation. Major protocol violations will be identified prior to 
breaking the blind.  
  
9.4  
STATISTICAL ANALYSES  
  
 
  
Categorical variables will be presented as frequencies and percentages with column and row percentages 
in 2x2 or RxC tables as appropriate. Association between two nominal variables will be investigated using 
Chi-Square tests and/or Fisher’s Exact test as needed. Cochran Armitage trend-test may be included 
among the possible tests to investigate the possible trend between one ordinal factor and one binary 
factor. Association between two ordinal variables may be investigated through Non-zero Correlation test.    
Continuous variables will be presented as mean and standard deviation as well as five-number summary 
(minimum, first quartile, median, third quartile, and maximum). Range and inter-quartile range may also 
be explicitly provided as needed. Distribution of a continuous variable among the levels of a binary or 
polynomial factor will be investigated graphically through boxplots and through Wilcoxon-MannWhitney 
test and/or Kruskal Wallis tests. Association between two continuous variables will be described through 
scatter plots and Pearson’s and Spearman’s correlation coefficients as appropriate. When needed, 
normality assumption of a given continuous variable will be tested using Shapiro-Wilk Normality test.  
The details of the statistical models addressing each and every primary, secondary, and exploratory 
objectives along with primary predictors and control variables will be provided in a corresponding 
Statistical Analysis Plan (SAP). In this protocol document, a general analysis and statistical testing 
approach will be provided, and the details will be left for the study SAP.  
9.4.1   GENERAL APPROACH  


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In all comparisons and tests, a two-sided p-value will be used and a Type-1 error rate of 0.05 will be used 
to indicate significance. No multiplicity correction will be employed for statistical tests addressing the 
two primary objectives. Similarly, no multiplicity correction will be employed for statistical tests 
addressing the secondary and exploratory objectives as the results from these objectives must be 
considered within the hypotheses generating context.  
  
9.4.2 ANALYSIS OF THE PRIMARY EFFICACY ENDPOINT(S)  
  
  
The primary endpoint of this clinical trial is the estimate of the median time to sustained clinical 
response for the treatment and placebo arms. Kaplan-Meier method will be used to estimate the time to 
sustained clinical response distribution and log-rank test will be utilized to compare the time to sustained 
clinical response distributions between the two study arms. Events will be defined as cases that attain 
clinical response while on the follow-up window and time to event will be calculated as the days from 
symptom onset to the resolution of symptoms to zero based on the Modified FLU-PRO Plus 
questionnaire. Patients who do not attain to clinical response while on the follow-up window will be 
censored on Day-21 or on the day they are off study before Day-21 due to other reasons.    
Although all analyses will be conducted based on the intent-to-treat context, sensitivity analyses based 
on the adherence to the protocol regimen will also be carried out for the primary as well as secondary 
objectives.  
When appropriate, Last-Value-Carried-Forward (LVCF) imputation approach will be utilized for the 
missing values. As this is a small study that has a short span of enrollment and treatment windows, we 
do not anticipate excessive missing values; however, missingness structure will still be analyzed as to 
whether or not the missingness is Missing Completely at Random (MCAR), or Missing at Random (MAR), 
or Not-Missing at Random (NMAR). In the even of MCAR and MAR, sensitivity analyses will be carried 
out by using Multiple Imputation (MI) techniques.   
  
9.4.3 ANALYSIS OF THE SECONDARY ENDPOINT(S)  
  
None of the following endpoints depend on the findings of the primary endpoint.  
The first secondary endpoint of this clinical trial is the differential change of viral load distribution 
overtime between the two-study arm. As the viral load will be measured at Baseline, on Day-7, and on 
Day-14 (and potentially for some patients in Day-4 and Day-10 visits), the outcome variable of interest 
will be a longitudinal interval-scale measure. To assess the differential behaviour of viral load overtime 
between the two study arms, Random Coefficients Models will be utilized where viral load will be the 
dependent variable and study arm and time will be main predictors. Viral load measures may be 
subjected to some data transformation (e.g., log transformation) to improve the model fit and 
assumptions as necessitated by model diagnostics.  In addressing this objective, all available data will be 
utilized; that is, in addition to the evaluable patients, data from non-evaluable patients such as data only 
at baseline visit or data  at baseline and  Day-10 for example will be used to improve the local fit of the 
underlying model. To assess differential behavior in total symptom severity score, time*study-arm 
interaction will be investigated as well.  
  
The percentages of RT-PCR negative/undetectable viral loads between the two study arms will be 
compared using Fisher’s Exact test at Day-7 and Day-14 visits.   
  


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The third secondary endpoint of this clinical trial is the differential change of total and subdomain 
symptom severity score measured by Modified FLU-PRO Plus overtime between the two-study arm. As 
the total and subdomain symptom severity score will be measured at Baseline (Day-1) and on each day 
throughDay-14, the outcome variables of interest will be a longitudinal interval-scale measures. To assess 
the differential behaviour of total and subdomain symptom scores overtime between the two study 
arms, Random Coefficients Models will be utilized where total and each of the subdomain symptom 
severity score will be the dependent variables and study arm and time will be main predictors. Total and 
subdomain symptom severity scores may be subjected to some data transformation (e.g., log 
transformation) to improve the model fit and assumptions as necessitated by model diagnostics.  In 
addressing this objective, all available data will be utilized; that is, in addition to the evaluable patients, 
data from non-evaluable patients such as data only at baseline visit or data just at baseline and  Day-10 
for example will be used to improve the local fit of the underlying model. To assess differential behavior 
in total symptom severity score, time*study-arm interaction will be investigated as well. When 
scientifically indicated, aggregated symptom severity scores may be defined through combining a set of 
subdomains such as Nose, Eyes. And Throat.  
  
Association between quantitative viral load and symptom severity will be investigated through regression 
models, where total symptom severity score will be the dependent variable predicted by the quantitative 
viral load measures in the model at each visit. Study-Arm*viral load interaction will be added to the 
model to investigate if any association between viral load and symptom severity is mitigated by the study 
treatment.   
  
Last but not the least secondary endpoint is the comparison of Adverse Events (AE) and Serious Adverse 
Events (AE) between the two study arms. AEs and SAEs are nominal variables described by grade and 
attribution to the protocol therapy. For the presentation purposes, we will define ‘Toxicity’ as an AE with 
at least possible attribution of the therapy. Summary tables of frequencies and percentages of AEs will 
be generated as number of episodes within number of patients (N-episodes/N-subjects) by grade and 
attribution. When frequencies allow, percentages of patients experiencing an AE, or a set of AEs will be 
compared between the two arms using Fisher’s Exact test. In doing so, some aggregation can be done at 
the grade level such as ‘Grade 3+’ AEs or categories along these lines. Such comparisons will be done for 
toxicities of interest as well.  
  
Analysis details for the exploratory aims will be provided in the study SAP.  
  
  
 
Although this is not a dose-escalation trial, the following safety monitoring scheme will be employed in 
the initial enrollment phase of the trial:  
The safety monitoring phase of the study will consist of up to 12 patients, whose adverse event profiles 
will be monitored continuously. If two or less number of patients in this initial cohort of 12 patients 
experience Grade 3+ adverse events attributable to the study regimen and not resolved through 
symptomatic treatments within 48 hours, the safety monitoring phase will be considered complete and 
the enrollment will continue. If three or more patients in this initial cohort of 12 patients experience  
Grade 3+ adverse events attributable to the study regimen and not resolved through symptomatic 
treatments within 48 hours, then the enrollment will be halted to discuss the safety measure of the 
protocol and possible amendments will be developed to address the safety concerns. As the adverse 
event profiles of the patients in this safety monitoring phase are followed closely, the safety monitoring 
9.4.4   SAFETY ANALYSES  


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phase will be halted once the number of patients with Grade 3+ adverse events attributable to the study 
regimen and not resolved through symptomatic treatments within 48 hours go beyond two cases; 
therefore, the patient enrollment may be halted for safety evaluation before enrolling 12 patients. The 
above safety monitoring rules will be employed regardless of patient evaluability for the primary 
endpoints; however, to be included in the safety monitoring, it will be required that the patient received 
at least one dose of the study regimen and attribution of a given adverse event to the study regiment is 
clearly established as ‘at least possibly attributable to the study regimen’.  
  
9.4.5 BASELINE DESCRIPTIVE STATISTICS  
  
9.4.6 PLANNED INTERIM ANALYSES   
  
This section is not applicable for the analysis of the primary and secondary objectives; however, there 
will be safety interim analyses after the initial cohort of 12 patients have been enrolled and have 
received at least one dose of the assigned drug.  
  
 
  
In analyses for primary and secondary endpoints, age, gender, race and other demographics and clinical 
factors and markers may be used as control variables as appropriate; however, the statistical design of 
this study does not include any planned subgroup analyses. All subgroup analyses if found appropriate 
will be considered exploratory to generate hypotheses for future prospective studies.  
  
9.4.8 TABULATION OF INDIVIDUAL PARTICIPANT DATA  
  
Patient specific data will be presented by study arm, including patient demographics and baseline clinical 
factors and measures.   
  
 
  
The study includes exploratory objectives for the PK profile of the study drug and measurement of the 
immune response through cytokine production, coagulation factors and various effector immune cell 
subsets in the PBMC of these patients on Day 1, Day 7 and 14 using FACS. The details of the analytic 
approaches for these exploratory objectives will be shared in the study SAP.  Analyses to address these 
two exploratory aims will be detailed in the study SAP.  
  
10 SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS  
  
10.1 REGULATORY, ETHICAL, AND STUDY OVERSIGHT CONSIDERATIONS  
  
  
9.4.7   SUB-GROUP ANALYSES  
9.4.9   EXPLORATORY ANALYSES  


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In obtaining and documenting informed consent, the investigator must comply with applicable regulatory 
requirements (e.g., 45 CFR Part 46, 21 CFR Part 50, 21 CFR Part 56) and should adhere to ICH GCP.   
  
Prior to the beginning of the trial, the investigator should have the IRB’s written approval for the protocol 
and the written informed consent form(s) and any other written information to be provided to the 
participants.   
  
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.    
  
10.1.1.2 CONSENT PROCEDURES AND 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 Institutional Review 
Board (IRB)-approved and the participant will be asked to read and review the document. The 
investigator 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 
informed consent document 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.  
  
  
10.1.2 STUDY DISCONTINUATION AND CLOSURE  
  
This study may be temporarily 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, investigator, the Investigational 
New Drug (IND) sponsor and regulatory authorities.    
  
If the study is prematurely terminated or suspended, the Principal Investigator (PI) will promptly inform 
study participants, the Institutional Review Board (IRB), and sponsor and will provide the reason(s) for 
10.1.1   INFORMED CONSENT PROCESS  
  
  
10.1.1.1   CONSENT/ASSENT AND OTHER INFORMATIONAL DOCUMENTS PROVIDED TO  
PARTICIPANTS  


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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 i.e.- If three or 
more patients in the initial cohort of 12 patients experience  Grade 3+ adverse events 
attributable to the study regimen and not resolved through symptomatic treatments within 48 
hours, then the enrollment will be halted to discuss the safety measure of the protocol and 
possible amendments will be developed to address the safety concerns.  
• 
Demonstration of efficacy that would warrant stopping     
• 
Insufficient compliance to protocol requirements  
• 
Data that are not sufficiently complete and/or evaluable  
• 
Determination that the primary endpoint has been met  
• 
Determination of futility  
  
Study may resume once concerns about safety, protocol compliance, and data quality are addressed, and 
satisfy the sponsor, IRB and/or Food and Drug Administration (FDA).  
  
  
 
  
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, or the data 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 Institutional 
Review Board (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, medical records (office, clinic, or hospital) 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 and scientific reporting, will 
be transmitted to and stored in the study EDC system. 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/Sponsor research staff will be secured and password protected. At the end of 
the study, all study databases will be de-identified and archived by the Sponsor.  
10.1.3   CONFIDENTIALITY AND PRIVACY   


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10.1.4 FUTURE USE OF STORED SPECIMENS AND DATA   
  
Samples collected for this study will be analyzed and stored at an immune monitoring laboratory.  After 
the study is completed, the de-identified, archived data will be transmitted to and stored by the study 
Sponsor for use by other researchers including those outside of the study. Permission to transmit 
samples to the immune monitoring laboratory will be included in the informed consent.   
  
  
When the study is completed, access to study data and/or samples will be provided through the study 
Sponsor.  
  
  
10.1.5 KEY ROLES AND STUDY GOVERNANCE  
  
Principal Investigator (s)  
Medical Monitor  
Nahla Salem, MD                                             
Hassan Bencheqroun, MD  
Emmanuel Iyoha, MD  
Tranquil Clinical Research  
E-mail: emmanueli@tranquilconsulting.com  
  
  
  
 
  
Safety oversight will be under the direction of the medical monitor and the principal investigators at the 
sites.  A Data and Safety Monitoring Board (DSMB) will not be used for this study, due to the short 
recruitment/treatment periods and the pre-established safety profile for Black Seed Oil.  
  
 
  
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 International Conference 
on Harmonisation Good Clinical Practice (ICH GCP), and with applicable regulatory requirement(s).   
  
• 
Monitoring for this study will be performed by Tranquil Clinical Research.   
• 
On-site monitoring visits will occur at pre-defined intervals.  In the event that on-site monitoring 
is prohibited, remote monitoring will be performed at the same intervals.  Comprehensive 100% 
source data verification will be performed by the clinical research associates (CRAs) or designees.    
10.1.6   SAFETY OVERSIGHT  
10.1.7   CLINICAL MONITORING  


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• 
Details of clinical site monitoring are documented in a Clinical Monitoring Plan (CMP). The CMP 
describes in detail who will conduct the monitoring, at what frequency monitoring will be done, 
at what level of detail monitoring will be performed, and the distribution of monitoring reports.  
  
  
  
10.1.8 QUALITY ASSURANCE AND QUALITY CONTROL  
  
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 site(s) for clarification/resolution. When appropriate, Last-Value-Carried-Forward 
(LVCF) imputation approach will be utilized for the missing values. As this is a small study that has a short 
span of enrollment and treatment windows, we do not anticipate excessive missing values; however, 
missingness structure will still be analyzed as to whether or not the missingness is Missing Completely at 
Random (MCAR), or Missing at Random (MAR), or Not-Missing at Random (NMAR). In the even of MCAR 
and MAR, sensitivity analyses will be carried out by using Multiple Imputation (MI) techniques.   
  
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, International Conference on Harmonisation Good Clinical 
Practice (ICH GCP), and applicable regulatory requirements (e.g., Good Laboratory Practices (GLP), Good 
Manufacturing Practices (GMP)).   
  
The investigational site will provide direct access to all trial related sites, source data/documents, and 
reports for the purpose of monitoring and auditing by the sponsor and/or designee, and inspection by 
local and regulatory authorities.  
  
It is recommended that each site have SOPs for quality management.  Some of the SOPs may describe 
the following:    
• 
How data and biological specimens (when applicable) will be evaluated for compliance with the 
protocol, ethical standards, regulatory compliance, and accuracy in relation to source 
documents.  •  The documents to be reviewed (e.g., CRFs, clinic notes, product accountability 
records, specimen tracking logs, questionnaires, audio or video recordings), who is responsible, 
and the frequency for reviews.    
• 
Who will be responsible for addressing QA issues (e.g., correcting procedures that are not in 
compliance with protocol) and QC issues (e.g., correcting errors in data entry).   
• 
Staff training methods and how such training will be tracked.  
• 
If applicable, calibration exercises conducted prior to and during the study to train examiners 
and maintain acceptable intra- and inter-examiner agreement.  
  
  
  
 
  
10.1.9   DATA HANDLING AND RECORD KEEPING   
  
10.1.9.1   DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES   


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Each participating site will maintain appropriate medical and research records for this trial, in compliance 
with ICH GCP and regulatory and institutional requirements for the protection of confidentiality of 
participants.  Each site will permit authorized representatives of the sponsor, CRO, and regulatory 
agencies to examine (and when permitted by applicable law, to copy) clinical records for the purposes of 
quality assurance reviews, audits, and evaluation of the study safety, progress, and data validity. Describe 
in this section who will have access to records.    
  
Source data are all information, original records of clinical findings, observations, or other activities in a 
clinical trial necessary for the reconstruction and evaluation of the trial.  Electronic source data are data 
initially recorded in electronic form. Examples of source data include, but are not limited to, hospital 
records, clinical and office charts, laboratory notes, memoranda, participants’ memory aids or evaluation 
checklists, pharmacy dispensing records, audio recordings of counseling sessions, recorded data from 
automated instruments, copies or transcriptions certified after verification as being accurate and 
complete, microfiches, photographic negatives, microfilm or magnetic media, x-rays, and participant files 
and records kept at the pharmacy, at the laboratories, and medico-technical departments involved in the 
clinical trial.   
  
Data collection is the responsibility of the clinical trial staff at the site under the supervision of the site 
investigator. The investigator is responsible for ensuring the accuracy, completeness, legibility, and 
timeliness of the data reported.  
  
All source documents should be completed in a neat, legible manner to ensure accurate interpretation 
of data.  Source data should meet ALCOAC standards: attributable, legible, contemporaneous, original, 
accurate and complete.  
  
Data recorded in the electronic case report form (eCRF) derived from source documents should be 
consistent with the data recorded on the source documents.   
  
Clinical data (including but not limited to patient demographics, vital signs, medical history, adverse 
events (AEs), concomitant medications, and expected adverse reactions data) and clinical laboratory data 
as per the schedule of assessments will be entered into a 21 CFR Part 11-compliant electronic data 
capture system. The data system includes password protection and internal quality checks, such as 
automatic range checks, to identify data that appear inconsistent, incomplete, or inaccurate. Clinical data 
will be entered directly from the source documents.  
  
  
 
  
Study documents should be retained for a minimum of 2 years after the last approval of a marketing 
application in an International Conference on Harmonisation (ICH) region and until there are no pending 
or contemplated marketing applications in an ICH region or until at least 2 years have elapsed since the 
formal discontinuation of clinical development of the study intervention. These documents should be 
retained for a longer period, however, if required by local regulations. No records will be destroyed 
without the written consent of the sponsor, if applicable. It is the responsibility of the sponsor to inform 
the investigator when these documents no longer need to be retained.  
  
10.1.9.2   STUDY RECORDS RETENTION   


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A protocol deviation is any noncompliance with the clinical trial protocol or International Conference on 
Harmonisation Good Clinical Practice (ICH GCP) 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 
in a timely manner and when possible in advance of the scheduled protocol-required activity.   
All deviations must be addressed in study source documents and reported to site CRA and/or sponsor.   
Protocol deviations must be sent to the reviewing Institutional Review Board (IRB) per their policies. The 
site investigator is responsible for knowing and adhering to the reviewing IRB requirements. Further details 
about the handling of protocol deviations will be included in the monitoring and/or project plans.   
  
There will be no waivers for protocol deviations granted for this study.  
  
  
10.1.11  
PUBLICATION AND DATA SHARING POLICY  
  
This study will be conducted in accordance with the following publication and data sharing policies and 
regulations:  
  
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.  
Data from this study may be requested from other researchers 5 years after the completion of the 
primary endpoint by contacting the sponsor.   
  
The sponsor will be responsible for developing publication procedures and resolving authorship issues.  
Please refer to your specific contract, grant, and/or Clinical Trials Agreements.    
  
  
  
 
  
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.   
  
10.1.10   
PROTOCOL DEVIATIONS   
10.1.12   
CONFLICT OF INTEREST POLICY  


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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.  The 
study leadership has established policies and procedures for all study group members to disclose all 
conflicts of interest and will establish a mechanism for the management of all reported dualities of 
interest.  
  
10.2 ADDITIONAL CONSIDERATIONS  
  
This section is not applicable.  
  
  


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10.3 ABBREVIATIONS  
  
AE  
Adverse Event  
ANCOVA  
Analysis of Covariance  
CDC  
Centers for Disease Control  
CFR  
Code of Federal Regulations  
CLIA  
Clinical Laboratory Improvement Amendments  
CMP  
Clinical Monitoring Plan  
COC  
Certificate of Confidentiality  
CONSORT  Consolidated Standards of Reporting Trials  
CRF  
Case Report Form  
DCC  
Data Coordinating Center  
DHHS  
Department of Health and Human Services  
DSMB  
Data Safety Monitoring Board  
DRE  
Disease-Related Event  
EC  
Ethics Committee  
eCRF  
Electronic Case Report Forms  
FAS  
Full Analysis Set  
FDA  
Food and Drug Administration  
FDAAA  
Food and Drug Administration Amendments Act of 2007  
FDPO  
Final Dataset for Primary Objectives  
FFR  
Federal Financial Report  
GCP  
Good Clinical Practice  
GLP  
Good Laboratory Practices  
GMP  
Good Manufacturing Practices  
GWAS  
Genome-Wide Association Studies  
HIPAA  
Health Insurance Portability and Accountability Act   
IB  
Investigator’s Brochure  
ICH  
International Conference on Harmonisation   
ICMJE  
International Committee of Medical Journal Editors  
IDE  
Investigational Device Exemption  
IND  
Investigational New Drug Application  
IRB  
Institutional Review Board  
ISM  
Independent Safety Monitor  
ISO  
International Organization for Standardization  
ITT  
Intention-To-Treat  
LSMEANS  Least-squares Means  
LCVF  
Last-Value-Carried-Forward   
MAR  
Missing at Random   


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MCAR  
Missing Completely at Random  
MedDRA  
Medical Dictionary for Regulatory Activities  
MI  
Multiple Imputation  
MOP  
Manual of Procedures  
MSDS  
Material Safety Data Sheet  
NCT  
National Clinical Trial  
NIH   
National Institutes of Health  
NIH IC  
NIH Institute or Center  
NMAR  
Not-Missing at Random  
OHRP  
Office for Human Research Protections  
NMAR  
Not-Missing at Random  
PBMC  
Peripheral Blood Mononuclear Cells  
PI  
Principal Investigator  
QA  
Quality Assurance  
QC  
Quality Control  
RT-PCR  
Reverse transcription polymerase chain reaction  
SAE  
Serious Adverse Event  
SAP  
Statistical Analysis Plan  
SMC  
Safety Monitoring Committee  
SOA  
Schedule of Activities  
SOC  
Standard of Care  
SOP  
Standard Operating Procedure  
UP  
Unanticipated Problem  
US  
United States  
  
  
  
 


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10.4 PROTOCOL AMENDMENT HISTORY  
  
Version  
Date  
Description of Change   
Brief Rationale  
1.0  
13-Nov-2020  
Initial Final Version  
  
2.0  
  
Primary endpoint changed per FDA 
recommendations  
Influenza test and RT-PCR test at 
screen removed to reduce patient 
burden  
Replacement of patients removed 
per FDA recommendation  
Modified FLU-PRO Plus - modified 
to reduce the number of symptoms 
and ensure that the scale is 
consistent across all symptoms and 
to reduce patient burden  
Drug Induced Liver Injury plan 
added   
  
Changes per FDA Pre-IND 
response recommendations  
  
  
  
  
  
  
  
  
  
  
  
  


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66. 
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Mohtashami R, Huseini HF, Heydari M, Amini M, Sadeqhi Z, Ghaznavi H, Mehrzadi S 
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Arslan E, Sayin S, Demirbas S, Cakar M, Somak NG, Yesilkaya S, Saglam K A case study 
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Al-Jenoobi FI, Al-Thukair AA, Abbas FA, Ansari MJ, Alkharfy KM, Al-Mohizea AM, Al-
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in human liver microsomes and healthy human subjects. Drug Metab Lett. 2010 Jan; 
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10: e0113486.  
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Hosseini MS, Mirkarimi SA, Amini M, Mohtashami R, Kianbakht S, et al. (2013) Effects of 
Nigella sativa L. seed oil in type II diabetic patients: a randomized, double-blind, 
placebo-controlled clinical trial. J Med Plants 12: 93-99.  
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Gelot P, Bara-Passot C, Gimenez-Arnau E, Beneton N, Maillard H, Celerier P [Bullous 
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Shahzad M, Yang X, Raza Asim MB, et al. Black seed oil ameliorates allergic airway 
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Mahdavi R, Namazi N, Alizadeh M, Farajnia S. Nigella sativa oil with a calorie-restricted 
diet can improve biomarkers of systemic inflammation in obese women: A randomized 
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EM Salem, Yar T, Bamosa AO, Al-Quorain A, Yasawy MI, Alsulaiman RM, et al. 
Comparative study of Nigella Sativa and triple therapy in eradication of Helicobacter 
Pylori in patients with nonulcer dyspepsia. Saudi J Gastroenterol. 2010;16(3):207-14.  
83. 
Akhtar MS, Riffat S. Field trial of Saussurea lappa roots against nematodes and Nigella 
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Fard FA, Zahrani ST, Bagheban AA, Mojab F. Therapeutic effects of Nigella Sativa Linn 
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Abdel-Moneim A, Morsy BM, Mahmoud AM, Abo-Seif MA, Zanaty MI. Beneficial 
therapeutic effects of Nigella sativa and/or Zingiber officinale in HCV patients in Egypt. 
Excli J. 2013; 12:943- 
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86. 
Barakat EMF, El Wakeel LM, Hagag RS. Effects of Nigella sativa on outcome of hepatitis C 
in Egypt. World J Gastroenterol. 2013;19(16):2529-36.  
87. 
Onifade AA, Jewell AP, Adedeji WA. Nigella sativa concoction induced sustained 
seroreversion in HIV patient. Afr J Tradit Complement Altern Med. 2013;10(5):332-5.  
88. 
Onifade AA, Jewell AP, Okesina AB. Seronegative conversion of an HIV positive subject 
treated with Nigella sativa and honey. Afr J Infect Dis. 2015;9(2):47-50.  
89. 
Ulasli M, Gurses SA, Bayraktar R, Yumrutas O, Oztuzcu S, Igci M, Igci YZ, Cakmak EA, 
Arslan A The effects of Nigella sativa (Ns), Anthemis hyalina (Ah) and Citrus sinensis (Cs) 
extracts on the replication of coronavirus and the expression of TRP genes family. Mol 
Biol Rep. 2014 Mar; 41(3):1703-11.  
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Salem ML, Hossain MS. Protective effect of black seed oil from Nigella sativa against 
murine cytomegalovirus infection. International Journal of Immunopharmacology. 2000 
Sep;22(9):729740. DOI: 10.1016/s0192-0561(00)00036-9.  
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2020; 215:108427. doi: 10.1016/j.clim.2020.108427  
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Li Q., Guan X., Wu P., Wang X., Zhou L., Tong Y., Ren R., Leung K.S.M., Lau E.H.Y., Wong 
J.Y., Xing X., Xiang N., Wu Y., Li C., Chen Q., Li D., Liu T., Zhao J., Liu M., Tu W., Chen C., Jin 
L., Yang R.,  
Wang Q., Zhou S., Wang R., Liu H., Luo Y., Liu Y., Shao G., Li H., Tao Z., Yang Y., Deng Z., Liu B., Ma 
Z., Zhang Y., Shi G., Lam T.T.Y., Wu J.T., Gao G.F., Cowling B.J., Yang B., Leung G.M., Feng Z. Early 
transmission dynamics in Wuhan, China, of Novel Coronavirus-infected pneumonia. N Engl J 
Med. 2020; 382:1199–1207.  
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Zhang J., Litvinova M., Wang W., Wang Y., Deng X., Chen X., Li M., Zheng W., Yi L., Chen 
X., Wu Q., Liang Y., Wang X., Yang J., Sun K., Longini I.M., Jr., Halloran M.E., Wu P., 
Cowling B.J., Merler S., Viboud C., Vespignani A., Ajelli M., Yu H. Evolving epidemiology 
and transmission dynamics of coronavirus disease 2019 outside Hubei province, China: 
a descriptive and modelling study. Lancet Infect Dis. 2020 doi: 10.1016/S1473-
3099(20)30230-9.  
94. 
William B. Grant et al. Evidence that Vitamin D Supplementation Could Reduce Risk of 
Influenza and COVID-19 Infections and Deaths. Nutrients April 2020.  
95. 
Raharusun, P.; Priambada, S.; Budiarti, C.; Agung, E.; Budi, C. Patterns of COVID-19 
Mortality and Vitamin D: An Indonesian Study. SSRN 2020, doi:10.2139/ssrn.3585561.  
96. 
Zhou F, Yu T, Du R, et al. Clinical course and risk factors for mortality of adult inpatients 
with  
COVID-19 in Wuhan, China: a retrospective cohort study. Lancet. 2020;395(10229):1054-1062  


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Metlay JP, Waterer GW, Long AC, et al. Diagnosis and treatment of adults with 
communityacquired pneumonia. An official clinical practice guideline of the American 
Thoracic Society and Infectious Diseases Society of America. Am J Respir Crit Care Med. 
2019;200(7): e45-e67.  
98. 
Jean M. Connors, Jerrold H. Levy; COVID-19 and its implications for thrombosis and 
anticoagulation. Blood 2020; 135 (23): 2033–2040  
99. 
Institute of Medicine. Food and Nutrition Board. Dietary Reference Intakes: Vitamin C, 
Vitamin E, Selenium, and Carotenoids external link disclaimer. Washington, DC: National 
Academy Press, 2000.  
100. 
Corrigan JJ Jr. The effect of vitamin E on warfarin-induced vitamin K deficiency. Ann N Y 
Acad Sci. 1982; 393:361-368. doi:10.1111/j.1749-6632. 1982.tb31275.x  
101. 
Brown BG, Zhao X-Q, Chait A, Fisher LD, Cheung MC, Morse JS, et al. Simvastatin and 
niacin, antioxidant vitamins, or the combination for the prevention of coronary disease. 
N Engl J Med 2001; 345:1583-92.  
102. 
Forouzanfar F, Bazzaz BS, Hosseinzadeh H Black cumin (Nigella sativa) and its constituent 
(thymoquinone): a review on antimicrobial effects. Iran J Basic Med Sci. 2014 Dec; 
17(12):929-38.  
103. 
Abdel-Fattah A.M., Matsumoto K., Watanabe H. Antinociceptive effects of Nigella sativa 
oil and its major component, thymoquinone, in mice. Eur. J. Pharmacol. 2000; 400:89–
97. doi:  
10.1016/s0014-2999(00)00340-x  
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Takai S., Song K., Tanaka T., Okunishi H., Miyazaki M. Antinociceptive effects of 
angiotensin-converting enzyme inhibitors and an angiotensin II receptor antagonist in 
mice. Life Sci. 1996;59:PL331–336. doi: 10.1016/0024-3205(96)00527-9.  
105. 
Lantz I., Glamsta E.L., Talback L., Nyberg F. Hemorphins derived from hemoglobin have 
an inhibitory action on angiotensin converting enzyme activity. FEBS Lett. 1991; 287:39–
41. doi:  
10.1016/0014-5793(91)80011-q.  
106. 
Rahman MT. Potential Benefits of Combination of Nigella sativa and Zn Supplements to 
Treat COVID-19 [published online ahead of print, 2020 Jun 24]. J Herb Med. 
2020;100382. doi:  
10.1016/j.hermed.2020.100382  
107. 
Umar, S., Munir, M. T., Subhan, S., Azam, T., & Nisa, Q. (2016). Protective and antiviral 
activities of Nigella sativa against avian influenza (H9N2) in turkeys. Journal of the Saudi 
Society of Agricultural Sciences.  
108. 
Zaher, K. S., Ahmed, W. M., & Zerizer, S. N. (2008). Observations on the biological effects 
of black cumin seed (Nigella sativa) and green tea (Camellia sinensis). Global Vet, 2(4), 
198–204.  
109. 
Liu Y, Yan LM, Wan L, et al. Viral dynamics in mild and severe cases of COVID-19. Lancet  


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Infect Dis. 2020;20(6):656-657. doi:10.1016/S1473-3099(20)30232-2  
110. 
Tan L, Kang X, Ji X, et al. Validation of Predictors of Disease Severity and Outcomes in  
COVID-19 Patients: A Descriptive and Retrospective Study [published online ahead of print, 2020  
May 19]. Med (N Y). 2020; 10.1016/j.medj.2020.05.002. doi: 10.1016/j.medj.2020.05.002  
111. 
Powers JH 3rd, Bacci ED, Leidy NK, et al. Performance of the inFLUenza Patient-Reported 
Outcome (FLU-PRO) diary in patients with influenza-like illness (ILI). PLoS One. 
2018;13(3): e0194180. Published 2018 Mar 22. doi: 10.1371/journal.pone.0194180  
112. 
Belluzzi A, Brignola C, Campieri M, Pera A, Boschi S, Miglioli M. Effect of an 
entericcoated fish-oil preparation on relapses in Crohn's disease. N Engl J Med. 1996 Jun 
13;334(24):1557-60. doi: 10.1056/NEJM199606133342401. PMID: 8628335.  
113. 
Repurposed antiviral drugs for COVID-19 –interim WHO SOLIDARITY trial results WHO  
Solidarity trial consortium, Hongchao Pan, Richard Peto, Quarraisha Abdool Karim, Marissa  
Alejandria, Ana Maria Henao-Restrepo, César Hernández García, Marie-Paule Kieny, Reza  
Malekzadeh, Srinivas Murthy, Marie-Pierre Preziosi, Srinath Reddy, Mirta Roses Periago, Vasee  
Sathiyamoorthy, John-Arne Røttingen, Soumya Swaminathan, as the members of the Writing 
Committee, assume responsibility for the content and integrity of this article medRxiv  
2020.10.15.20209817; doi: https://doi.org/10.1101/2020.10.15.20209817  
114. 
Lamb, Y.N. Remdesivir: First Approval. Drugs 80, 1355–1363 (2020). 
https://doi.org/10.1007/s40265-020-01378-w  
115. 
Sohaib Ashraf, Shoaib Ashraf, Muhammad Ahmad Imran, Moneeb Ashraf, Larab  
Kalsoom, Uzma Nasim Siddiqui, Muhammad Ghufran, Nighat Majeed, Iqra Farooq, Zaighum  
Habib, Abubakar Hilal, Zain-ul-Abdin, Ayesha Khaqan, Muhammad Kiwan Akram, Sidra Ashraf,  
Rutaba Akmal, Sundas Rafique, Khawar Nawaz, Shahroze Arshad, Sohail Ahmad, Kanwal Hayat,  
Ali Arshad, Muhammad Faisal Nadeem, Muhammad Hassan, Abeer-bin-Awais, Muhammad 
Azam, Muhammad Suhail, Sibgha Zulfiqar, Imran Anwar, Saulat Sarfraz, Ayesha Hamayoun,  
Amber Malik, Hui Zheng, Talha Mahmood, Mahmood Ayyaz, Ali Ahmad, Muhammad Ashraf, 
Qazi Abdul Saboor, Mateen Izhar Efficacy of honey and Nigella sativa against COVID-19: 
HNSCOVID-PK Trial medRxiv 2020.10.30.20217364; doi: 
https://doi.org/10.1101/2020.10.30.20217364, 
https://www.medrxiv.org/content/10.1101/2020.10.30.20217364v2  
116. 
Tenforde MW, Kim SS, Lindsell CJ, Rose EB, Shapiro NI, Files DC, Gibbs KW, Erickson HL, 
Steingrub JS, Smithline HA, Gong MN. Symptom duration and risk factors for delayed 
return to usual health among outpatients with COVID-19 in a multistate health care 
systems network— United States, March–June 2020. Morbidity and Mortality Weekly 
Report. 2020 Jul 31;69(30):993.  
  
  
 


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12 APPENDIX 1 – COMMON TERMINOLOGY CRITERIA FOR ADVERSE EVENTS (CTCAE)  
V5.0  
  
https://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/CTCAE_v5_Quick_Reference 
_5x7.pdf  
  
  
  
 


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13 APPENDIX 2 – MODIFIED FLU-PRO PLUS©QUESTIONNAIRE  
  
  


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14 APPENDIX 3 – BASELINE COVID-19 SEVERITY CATEGORIZATION *  
  
SARS-CoV-2 infection without symptoms  
• Positive testing by standard reverse transcription polymerase chain reaction (RT-PCR) assay or 
equivalent test  
• No symptoms  
   
Mild COVID-19  
• Positive testing by standard RT-PCR assay or equivalent test  
• Symptoms of mild illness with COVID-19 that could include fever, cough, sore throat, malaise, 
headache, muscle pain, gastrointestinal symptoms, without shortness of breath or dyspnea    
• No clinical signs indicative of Moderate, Severe, or Critical Severity  
  
Moderate COVID-19  
• Positive testing by standard RT-PCR assay or equivalent testing  
• Symptoms of moderate illness with COVID-19, which could include any symptom of mild illness or 
shortness of breath with exertion   
• Clinical signs suggestive of moderate illness with COVID-19, such as respiratory rate ≥ 20 breaths per 
minute, saturation of oxygen (SpO2) > 93% on room air at sea level, heart rate ≥ 90 beats per minute  
• No clinical signs indicative of Severe or Critical Illness Severity  
   
Severe COVID-19  
• Positive testing by standard RT-PCR assay or an equivalent test  
• Symptoms suggestive of severe systemic illness with COVID-19, which could include any symptom of 
moderate illness or shortness of breath at rest, or respiratory distress  
• Clinical signs indicative of severe systemic illness with COVID-19, such as respiratory  rate ≥ 30 per 
minute, heart rate ≥ 125 per minute, SpO2 ≤ 93% on room air at sea level or  PaO2/FiO2 < 300  
• No criteria for Critical Severity  
  
Critical COVID-19   
• 
Positive testing by standard RT-PCR assay or equivalent test    
• 
Evidence of critical illness, defined by at least one of the following:    
– Respiratory failure defined based on resource utilization requiring at least one of the 
following:   
Endotracheal intubation and mechanical ventilation, oxygen delivered by high- flow nasal cannula 
(heated, humidified, oxygen delivered via reinforced nasal  cannula at flow rates > 20 L/min with 
fraction of delivered oxygen ≥ 0.5),  noninvasive positive pressure ventilation, ECMO, or clinical 
diagnosis of respiratory failure (i.e., clinical need for one of the preceding therapies, but  preceding 
therapies not able to be administered in setting of resource limitation)   


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– Shock (defined by systolic blood pressure < 90 mm Hg, or diastolic blood pressure <  60 
mm Hg or requiring vasopressors)    – Multi-organ dysfunction/failure    
  
NOTE: A clinical diagnosis of respiratory failure (in the setting of resource limitation) in which  the 
management deviates from standard of care should be recorded as part of formal data collection. 15 
APPENDIX 4 – PROHIBITED MEDICATIONS*  
Corticosteroids  
• 
hydrocortisone (Cortef)  
• 
cortisone  
• 
ethamethasoneb (Celestone)  
• 
prednisone (Prednisone Intensol)  
• 
prednisolone (Orapred, Prelone)  
• 
triamcinolone (Aristospan Intra-Articular, Aristospan Intralesional, Kenalog)  
• 
Methylprednisolone (Medrol, Depo-Medrol, Solu-Medrol)  
• 
dexamethasone (Dexamethasone Intensol, DexPak 10 Day, DexPak 13 Day, DexPak 6 Day  
• 
betamethasone  
Antivirals  
• 
Abacavir  
• 
Acyclovir (Aciclovir)  
• 
Adefovir  
• 
Amantadine  
• 
Ampligen  
• 
Amprenavir (Agenerase)  
• 
Umifenovir (Arbidol)  
• 
Atazanavir  
• 
Atripla   
• 
Baloxavir marboxil (Xofluza)  
• 
Biktarvy  
• 
Bulevirtide  
• 
Cidofovir  
• 
Cobicistat (Tybost)  
• 
Combivir  
• 
Daclatasvir (Daklinza)  


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• 
Darunavir  
• 
Delavirdine  
• 
Descovy  
• 
Didanosine  
• 
Docosanol  
• 
Dolutegravir  
• 
Doravirine (Pifeltro)  
• 
Edoxudine  
• 
Efavirenz  
• 
Elvitegravir  
• 
Emtricitabine  
• 
Enfuvirtide  
• 
Entecavir  
• 
Etravirine (Intelence)  
• 
Famciclovir  
• 
Fomivirsen  
• 
Fosamprenavir  
• 
Foscarnet  
• 
Ganciclovir (Cytovene)  
• 
Ibacitabine  
• 
Ibalizumab (Trogarzo)  
• 
Idoxuridine  
• 
Imiquimod  
• 
Imunovir  
• 
Indinavir  
• 
Lamivudine  
• 
Letermovir (Prevymis)  
• 
Lopinavir  
• 
Loviride  
• 
Maraviroc  
• 
Methisazone  
• 
Moroxydine  
• 
Nelfinavir  
• 
Nevirapine  
• 
Nexavir (Kutapressin)  


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• 
Nitazoxanide  
• 
Norvir  
• 
Oseltamivir (Tamiflu)  
• 
Penciclovir  
• 
Peramivir  
• 
Penciclovir  
• 
Peramivir (Rapivab)  
• 
Pleconaril  
• 
Podophyllotoxin  
• 
Raltegravir  
• 
Remdesivir (Veklury)  
• 
Ribavirin  
• 
Rilpivirine (Edurant)  
• 
Rilpivirine  
• 
Rimantadine  
• 
Ritonavir  
• 
Saquinavir  
• 
Simeprevir (Olysio)  
• 
Sofosbuvir  
• 
Stavudine  
• 
Taribavirin (Viramidine)   
• 
Telaprevir  
• 
Telbivudine (Tyzeka)  
• 
Tenofovir alafenamide  
• 
Tenofovir disoproxil  
• 
Tipranavir  
• 
Trifluridine  
• 
Trizivir HIV  
• 
Tromantadine  
• 
Truvada  
• 
Umifenovir  
• 
Valaciclovir (Valtrex)  
• 
Valganciclovir (Valcyte)  
• 
Vicriviroc  
• 
Vidarabine  


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• 
Zalcitabine  
• 
Zanamivir (Relenza)  
• 
Zidovudine  
Hydroxychloroquine  
Baricitinib (Olumiant)  
Convalescent Plasma  
This listing is not intended to be exhaustive.  
  
  
16 APPENDIX 5 - COMMON SUBSTRATES FOR CYP2C9 BY THERAPEUTIC CLASS   
  
Class  
Antiinflammatories  Oral  
Hypoglycemics  
Oral  
Anticoagulants  
Diuretics and 
uricosurics  
Angiotensin II 
blockers  
Substrates  
Flurbiprofen  
Diclofenac  
Naproxen  
Piroxicam  
Suprofen  
Ibuprofen  
Mefenamic acid  
Celecoxib  
Tolbutamide  
Glyburide  
Glipizide  
Glimepiride  
(S)-Warfarin  
(S)-
Acenocoumarol  
(Phenprocoumon)
 
  
Torsemide  
Ticrynafen 
  
Sulfinpyrazone 
sulfide  
  
Losartan  
Irbesartan  
Candesartan  
Class  
(con’t.)  
Antiasthmatics  
Anticonvulsants  Anticancer agents  
Endogenous 
compounds  
Miscellaneous  


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Substrates  
(con’t)  
Zafirlukast  
(Zileuton)  
Phenytoin  
(Phenobarbital)  
(Trimethadione)  
Cyclophosphamide  
(Tamoxifen)  
Arachidonic acid  
5-
Hydroxytryptamine 
Linoleic acid  
Mestranol  
Fluvastatin  
_9-
Tetrahydrocannabinol  
(Benzopyrene)  
(Pyrene)  
(Fluoxetine)  
(Sildenafil)  
(Rosiglitazone)  
  
This listing is not intended to be exhaustive.  
Parentheses indicates that (where known) other P450s or metabolic pathways play a major role in clearance.  
  


 
 


 
 
Black Seed Oil_Protocol_V4.0_25Mar2021_Fina 
l 
 
Final Audit Report 
2021-03-25 
Created: 
2021-03-25 
By: 
Deborah Bates (deborahb@tranquilconsulting.com) 
Status: 
Signed 
Transaction ID: 
CBJCHBCAABAAPJg0g1eCGJb7k8iFw71Mie-WfsOaEVlx 
"Black Seed Oil_Protocol_V4.0_25Mar2021_Final" History 
 Document created by Deborah Bates (deborahb@tranquilconsulting.com) 
2021-03-25 - 3:11:20 PM GMT- IP address: 184.147.32.224 
 Document emailed to A. O. Kaseb (akaseb99@gmail.com) for signature 
2021-03-25 - 3:12:31 PM GMT 
 Email viewed by A. O. Kaseb (akaseb99@gmail.com) 
2021-03-25 - 10:18:02 PM GMT- IP address: 74.125.214.15 
 Document e-signed by A. O. Kaseb (akaseb99@gmail.com) 
Signature Date: 2021-03-25 - 10:18:34 PM GMT - Time Source: server- IP address: 143.111.84.100 
 Agreement completed. 


 
 
2021-03-25 - 10:18:34 PM GMT 
 


 
 
 

SAP_001.pdf: 
BOSS-Covid-19 Study (BOSS-001)  
  
SAP BOSS-001 Study Version 2.0 09/30/2021             Confidential and Proprietary                                                          Page 1 of 22  
Statistical Analysis Plan  
  
  
TRIAL FULL TITLE  
A Randomized, Double-Blind, Placebo-Controlled Study to  
Evaluate the Safety and Efficacy of TQ Formula in Treating  
Participants who have Tested Positive for Novel Coronavirus  
2019 (BOSS-Covid-19)  
SAP VERSION  
Rev. 2.0  
SAP VERSION DATE  
09/30/2021  
TRIAL STATISTICIAN  
Mehmet Kocak, Ph.D.  
PRINCIPAL  
INVESTIGATORS  
Yasir Ahmed, M.D.; Hassan Bencheqroun, M.D.; Enrique Villa,  
M.D.                                 
SAP AUTHOR  
Mehmet Kocak, Ph.D.  
  
  
Author Signature:  
Name: Dr. Mehmet Kocak 
Date: 9/30/2021 
 
Sponsor Signature:  
Name: Dr. Ahmed Kaseb 
Date: 9/30/2021 
 
          
  
Contents  
1  Abbreviations ..................................................................................................................... 3 
2  Introduction ........................................................................................................................ 5 
2.1  Preface ........................................................................................................................ 5 
2.2  Purpose ....................................................................................................................... 5 
3  Study Design ...................................................................................................................... 6 


BOSS-Covid-19 Study (BOSS-001)  
  
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3.1  Overview ...................................................................................................................... 6 
3.2  Sample size ................................................................................................................. 6 
3.3  Inclusion/Exclusion Criteria .......................................................................................... 7 
3.3.1  Inclusion Criteria ........................................................................................... 7 
3.3.2  Exclusion Criteria .......................................................................................... 8 
3.4  Randomization, Blinding, and Patient Selection ........................................................... 8 
3.5  Study Calendar and Data Collection ............................................................................ 9 
3.6  Primary Objective....................................................................................................... 11 
3.7  Secondary Objective .................................................................................................. 12 
4  Study Endpoints ............................................................................................................... 13 
4.1  Efficacy Endpoints ..................................................................................................... 13 
4.1.1  Primary Efficacy Endpoint: ........................................................................... 13 
4.1.2  Secondary Efficacy Endpoints ...................................................................... 13 
4.1.3  Other Efficacy Endpoints ............................................................................. 13 
4.2  Safety Endpoints ........................................................................................................ 13 
5  Statistical Methods ........................................................................................................... 14 
5.1  General Statistical Methods ....................................................................................... 14 
5.2  Analysis Sets ............................................................................................................. 14 
5.3  Statistical Analyses .................................................................................................... 14 
5.3.1  Demographic and Baseline Characteristics .................................................... 14 
5.3.2  Primary Analyses ........................................................................................ 15 
5.3.3  Secondary Analyses .................................................................................... 16 
5.3.4  Other Analyses ........................................................................................... 18 
5.3.5  Covariate and Subgroup Analyses ................................................................ 19 
5.3.6  Safety Analyses .......................................................................................... 19 
6  Definitions for Clinical Evaluations .................................................................................... 19 
6.1  Follow-Up Time Points ............................................................................................... 19 
6.2  Points of Enrollment ................................................................................................... 19 
6.3  Definitions for Clinical Evaluation ............................................................................... 19 
7  General Considerations .................................................................................................... 20 


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7.1  Missing Data and Outliers: ......................................................................................... 20 
7.2  Protocol Violations and Deviations ............................................................................. 20 
7.3  Disposition of Subjects and Withdrawals .................................................................... 20 
7.4  Sensitivity Analysis .................................................................................................... 21 
7.5  Changes to Planned Analysis after Blind Review of Data and Unblinding Consideration (if 
Applicable) ................................................................................................................................ 21 
7.6  Interim Analysis and Data Monitoring ......................................................................... 21 
7.7  Timing with Respect to Database Locking ................................................................. 21 
7.8  Analysis Software ...................................................................................................... 22 
7.9  List of Tables, Figures and Listings ............................................................................ 22 
8  References ....................................................................................................................... 22 
8.1 Applicable Regulatory Guidelines: .............................................................................. 22 
• ICH E9: Statistical Principles for Clinical Trials (September 1998) .................................. 22 
8.2  Other applicable Documents ...................................................................................... 22 
 
                 
  
1  
Abbreviations  
AE  
Adverse Event  
 
IND  
Investigational New Drug 
Application  
ANCOVA  
Analysis of Covariance  
CDC  
Centers for Disease Control  
IRB  
Institutional Review Board  
CFR  
Code of Federal Regulations  
ISM  
Independent Safety Monitor  
CLIA  
Clinical Laboratory Improvement  
Amendments  
ISO  
International Organization for 
Standardization  
CMP  
Clinical Monitoring Plan  
ITT  
Intention-To-Treat  
COC  
Certificate of Confidentiality  
LSMEANS  Least-squares Means  
CONSORT  Consolidated Standards of 
Reporting Trials  
LCVF  
Last-Value-Carried-Forward   
MAR  
Missing at Random   
CRF  
Case Report Form  
MCAR  
Missing Completely at Random  
DCC  
Data Coordinating Center  
MedDRA  
Medical Dictionary for Regulatory 
Activities  
DHHS  
Department of Health and Human 
Services  
MI  
Multiple Imputation  
DSMB  
Data Safety Monitoring Board  
MOP  
Manual of Procedures  
DRE  
Disease-Related Event  
MSDS  
Material Safety Data Sheet  
EC  
Ethics Committee  
NCT  
National Clinical Trial  
eCRF  
Electronic Case Report Forms  
NIH   
National Institutes of Health  
FAS  
Full Analysis Set  
NIH IC  
NIH Institute or Center  


BOSS-Covid-19 Study (BOSS-001)  
  
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FDA  
Food and Drug Administration  
NMAR  
Not-Missing at Random  
FDAAA  
Food and Drug Administration 
Amendments Act of 2007  
OHRP  
Office for Human Research 
Protections  
FDPO  
Final Dataset for Primary 
Objectives  
NMAR  
Not-Missing at Random  
PBMC  
Peripheral Blood Mononuclear 
Cells  
FFR  
Federal Financial Report  
GCP  
Good Clinical Practice  
PI  
Principal Investigator  
GLP  
Good Laboratory Practices  
QA  
Quality Assurance  
GMP  
Good Manufacturing Practices  
QC  
Quality Control  
GWAS  
Genome-Wide Association 
Studies  
RT-PCR  
Reverse transcription polymerase 
chain reaction  
HIPAA  
Health Insurance Portability and 
Accountability Act   
SAE  
Serious Adverse Event  
SAP  
Statistical Analysis Plan  
IB  
Investigator’s Brochure  
SMC  
Safety Monitoring Committee  
ICH  
International Conference on 
Harmonisation   
SOA  
Schedule of Activities  
SOC  
Standard of Care  
ICMJE  
International Committee of 
Medical Journal Editors  
SOP  
Standard Operating Procedure  
UP  
Unanticipated Problem  
IDE  
Investigational Device Exemption  
US  
United States  
 
  
  
  


BOSS-Covid-19 Study (BOSS-001)  
  
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2  
Introduction  
2.1   Preface  
In December of 2019, a series of acute atypical respiratory disease cases occurred in Wuhan, 
China which quickly spread to other areas. It was discovered that a distinctive coronavirus was 
responsible which was named the severe acute respiratory syndrome coronavirus-2 (SARS-CoV2, 
2019-nCoV) as a result of its high similarity (~80%) to SARS-CoV, which caused acute respiratory 
distress syndrome (ARDS) and high mortality in the years 2002–2003. This virus has resulted in a 
global pandemic with an increasing death toll. It is easily transmittable from human to human. The 
outbreak of SARS-CoV-2 was considered to have originally started via a zoonotic transmission 
associated with the seafood market in Wuhan, China. It was later determined that human to human 
transmission played a major role in the subsequent outbreak. The disease caused by this virus 
was called Coronavirus disease 19 (COVID-19) and a pandemic was declared by the World Health 
Organization (WHO). COVID-19 has infected a large number of people worldwide, being reported 
in roughly 200 countries. The lack of a targeted therapy continues to be a problem.  
There is currently no effective antiviral treatment for COVID-19. Existing drug alternatives as a 
result of clinical management of earlier discovered coronaviruses have been utilized in the 
treatment of COVID-19 patients.  
The growing interest in phytomedicine brings along the issue of their safety, and the requirements 
to meet health standards. It has been shown that the seeds and oil of NS plant are characterized 
by a very low degree of toxicity. Black seed oil offers promising potential as a therapeutic option in 
management of SARS-CoV-2 as it has been shown in studies to possess beneficial properties like 
anti-viral effects.  
The aim of this study is to provide a potential treatment for outpatients with a positive Covid-19 
diagnosis, where the current standard of care (SOC) is limited to pain/symptom management such 
as acetaminophen.  Any potential decrease in symptom longevity will benefit this population; and 
the potential benefits outweigh the minor risks in this case.  In addition, this small, randomized 
phase II study will have a minimal number of patients exposed to the investigational product, and 
for a minimum duration.  
  
2.2  Purpose  
This Statistical Analysis Plan (SAP) describes the planned analysis and reporting of BOSS-001 
study, which is a randomized, double-blind, place-controlled study of Black Seed Oil in treating 
Covid-19 patient on an outpatient basis.  


BOSS-Covid-19 Study (BOSS-001)  
  
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Also, exploratory analyses not necessarily identified in this SAP and/or specified in the 
corresponding protocol may be performed, should the investigating team and/or EC deem it 
necessary.  Any post-hoc, or unplanned analyses not identified in this SAP will be clearly identified 
in the respective Clinical Study Report (CSR).  
The following documents were reviewed and used as guiding references in preparation of this 
SAP:  
• 
Clinical Research Protocol: BOSS-001 Version 6.0  dated 17 May 2021  
• 
ICH E9: Statistical Principles for Clinical Trials (September 1998)  
• 
E9 Statistical Principles for Clinical Trials Guidance document by FDA   
Although we have quoted the relevant sections of the protocol in this SAP for convenience, the 
reader of this SAP is encouraged to also read the entire clinical protocol for details on the 
intervention and conduct of this study, and the operational aspects of clinical assessments and 
their timings subjects in this study.  
  
3  
Study Design  
3.1  Overview  
This is a randomized (1:1), double-blind, placebo-controlled phase 2 study to assess safety and 
efficacy of Total 3 g daily dose of Black Seed Oil (‘TQ Formula ’ this point forward) capsules versus 
placebo in treating patients who have tested positive for novel Coronavirus 2019 (Covid-19) in the 
outpatient setting.   
Patients will be treated at a dose of 500 mg, 3 capsules, two times a day for 14 days from date of 
randomization.   
Quantitative viral load as measured by RT-PCR will be evaluated at baseline and on days 7 and 
14.  
Covid-19 symptoms will be measured throughout the study using Modified FLU-PRO Plus.   
3.2  Sample size  
The total sample for this study is a minimum of 52 and up to 60 patients equally split between the 
placebo arm and the treatment arm.  
The sample size is driven by the primary objective to compare the median time to sustained clinical 
response between the two arms.   
For statistical analysis purposes, sustained clinical response is defined as a reduction to a score of 
</= 2 on all symptom scores on the Modified FLU-PRO Plus questionnaire and remaining </= 2 for 


BOSS-Covid-19 Study (BOSS-001)  
  
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at least three days. Time to sustained clinical response will be calculated from the date of testing to 
the first day all symptoms are ≤ 2.  
Power calculation will be based only on the first primary objective of the trial.  Based on the median 
time to sustained clinical response reported by CDC [122], the expected median time to response in 
the placebo arm will be approximately 16 days from the onset of symptoms in outpatients. With a 
sample size of 20 in each arm, a difference in median time to sustained clinical response of 10 days 
between the intervention arm and the placebo arm will be able to be detected with 81.5% statistical 
power with 5% Type-1 error rate. Median time to sustained clinical response will be estimated based 
on 21-day follow-up. With an attrition expectation of around 20%, the planned accrual is 26 patients 
in each arm and potentially up to 30 patients.  
   
3.3  Inclusion/Exclusion Criteria    
3.3.1 Inclusion Criteria  
In order to be eligible to participate in this study, an individual must meet all of the following criteria: 
1. Provision of signed and dated informed consent form  
2. Stated willingness to comply with all study procedures and availability for the duration of the 
study   
3. Male or female, aged 18 and over, presenting with recent mild to moderate clinical 
symptoms of Covid-19 infection (per FDA guidance – see appendix 3)  
4. Positive COVID-19 infection confirmed with a rapid antigen test at screening (or RT-PCR 
within the last 3 days) and confirmed with a RT-PCR test at baseline  
5. A score of ≥3 on a minimum of 2 symptoms on the Modified FLU-PRO Plus  
6. Ability to take oral medication and be willing to adhere to the dosing regimen (Twice a day – 
BID for 14 days)  
7. For females of reproductive potential: negative pregnancy test at screening and use of 
highly effective contraception method during study participation and for an additional 4 
weeks after the end of study drug administration   
8. For males of reproductive potential: use of condoms or other methods to ensure effective 
contraception with partner during study participation  
9. Agreement to adhere to Lifestyle Considerations (see section 5.3) throughout study 
duration  
  


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3.3.2 Exclusion Criteria  
An individual who meets any of the following criteria will be excluded from participation in this 
study:  
  
 
Current or recent (within 4 weeks) treatment with any corticosteroids; however, high-dose 
inhaled steroids, which are used to treat acute or chronic bronchial inflammation, will be 
permitted    
 
Current or recent (within 4 weeks) treatment with any antivirals   
 
Room air oxygen saturation (SaO2) < 94% at screen  
 
Walking oximetry < 90% or participant unable to complete 6-minute walking oximetry test at 
screen  
 
Severe Covid-19 symptoms (severe per FDA classification – see appendix 3)  
 
Requires immediate admission to hospital for any reason  
 
Pregnancy or lactation  
 
Known allergic reactions to components of black seed oil, or thymoquinone  
 
Treatment with another investigational drug or other investigational intervention within 2 
weeks of study start and throughout study duration.  
 
Significant hepatic disease (ALT/AST> 4 times the ULN); any laboratory parameter >/= 4 
times the ULN  
 
History of moderate to severe CKD, (i.e. an estimated glomerular filtration rate less than 45 
mL/min) at the time of enrollment or history of liver disease  
 
Patients with inflammatory bowel disease (such as Crohn’s) that could affect the intestinal 
absorption of TQ Formula  enteric coated capsules.  
 
Known HIV or Hepatitis C infection  
 
Influenza diagnosis (confirmed by testing) during screening or within prior 14 days  
 
Any uncontrolled condition(s) or diagnosis, both physical or psychological, or physical exam 
finding that precludes participation, as per investigator  
 
Current treatment with CYP2C9 substrates  
  
3.4  Randomization, Blinding, and Patient Selection  
Study participants will be randomized in a one to one fashion (1:1) using a block randomization 
with a block size of 4 independently within each center to receive either 3 g/day of TQ Formula  
(500 mg capsules x 3, BID) or placebo capsules (identical in appearance to active product) for 14 


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days.  Participants will receive the full study supply (14-day medication) at randomization.  Study 
capsules should be stored at ambient temperature (15°C to 25°C), away from direct sunlight. The 
study is double blinded; all subjects, investigators, and study personnel involved in the conduct 
of the study, including data management and primary biostatistician, will be blinded to treatment 
assignment. There will be an independent unblinded statistician. The unblinded study statistician 
will not otherwise participate in study procedures or data analysis.  
A randomization list will be prepared by the unblinded statistician and provided to the  
investigational product (IP) depot for labelling.  Sponsor and CRO study staff will remain blinded to 
study treatment. Randomization numbers will be assigned through the Electronic Data Capture 
(EDC) system.  
Participants will complete a daily questionnaire on Covid-19 symptoms, which will be used to 
determine the primary endpoint for the study. For this reason, it is important that participants 
remain blinded to treatment allocation.   
  
Unblinding  
In the unlikely event that unblinding is required, the principal investigator can contact the unblinded 
statistician to unblind treatment.  Treatment unblinding is discouraged if knowledge of the 
treatment assignment will not materially change the planned management of a medical emergency. 
Unblinding should be discussed in advance with the medical monitor if possible. If the investigator 
is not able to discuss treatment unblinding in advance, then he/she must notify the medical monitor 
as soon as possible about the unblinding incident without revealing the participant’s treatment 
assignment.  
Final unblinding for the data analysis will not take place until and unless all data queries from data 
quality assessments are adequately addressed by each participating site.  
  
3.5  Study Calendar and Data Collection  
The schedule of the study visits and data to be collected are shown below.    
Table 1. Schedule of events   
 Procedures  
Day 
-3 
to -1  
Visit 1,   
Day 1  
Day 4 
+/-1 day 
Day 7  
+/- 1 
day  
Day 10 
+/-1 day  
Day 14 
+/-1 day  
Day  
21 +/1 
day  
Day 45  
+/- 1 day 
Informed consent  
X  
   
   
   
   
   
   
  
Demographics  
X  
   
   
   
   
   
   
  
Medical history  
X  
   
   
   
   
   
   
  


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Inclusion/Exclusion Criteria Evaluation  
X  
X  
   
   
   
   
   
  
Rapid Antigen Test for Covid-19  
Xc  
  
  
  
  
  
  
  
RT-PCR sample for quantitative viral 
load analysis  
  
Xc  
  
X  
  
X  
  
  
Serum Collection (Blood sample for 
immune monitoring)  
  
X  
  
X  
  
X  
  
  
  
Physical exam   
X  
   
   
X  
   
X  
 
  
Vital signs  
X  
X  
  
X  
  
X  
  
  
Pulse Oximetry  
X  
  
  
  
  
  
  
  
Walking Oximetry  
X  
  
  
  
  
  
  
  


BOSS-Covid-19 Study (BOSS-001)  
  
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 Procedures  
Day 
-3 
to -1  
Visit 1,   
Day 1  
Day 4 
+/-1 day 
Day 7  
+/- 1 
day  
Day 10 
+/-1 day  
Day 14 
+/-1 day  
Day  
21 +/1 
day  
Day 45  
+/- 1 day 
Height  
X  
   
   
   
   
   
   
  
Weight  
X  
   
   
   
   
   
   
  
  
Hematology   
X  
   
Xe  
X  
Xe  
X  
  
  
Serum chemistry a  
X  
   
Xe  
X  
Xe  
X  
 
  
Pregnancy test b  
X  
   
   
   
   
   
   
  
Randomization  
   
X  
   
   
   
   
   
  
Administer study intervention  
   
X…......................................................................X  
   
  
  
Concomitant medication review  
X  
X  
X  
X  
X  
X  
X  
  
Adverse event review/evaluation  
X  
X  
X  
X  
X  
X  
X  
X  
Patient Reported Outcomes (Modified 
FLU-PRO Plus)*  
X+  
X…………………………………………………………....X  
  
Complete Case Report Forms (CRFs)  
X  
X  
X  
X  
X  
X  
X  
  
a: Albumin, alkaline phosphatase, total bilirubin, direct bilirubin, bicarbonate, BUN, calcium, 
chloride, creatinine, glucose, LDH, phosphorus, potassium, total protein, AST, ALT, sodium, 
CBC/diff, LDH, , CRP, prothrombin time, activated partial prothrombin time, , D-dimer b: 
Serum pregnancy test (women of childbearing potential).  
c:  Covid-19 Confirmatory test in addition to quantitative viral load  d: 
Screening and Baseline can be combined   
e: If clinically indicated at the discretion of the investigator  
f: Visits at Day 4 (visit 2), Day 10 (visit 4), and Day 21 (final visit) will be telehealth and can be 
changed to in-person at the discretion of the investigator  
g: In the event of a complete lockdown due to Covid, visits at Day 7(Visit 3) and Day 14 (Visit 5) 
could be replaced by home health visit for sample collection and telehealth visit for clinical 
evaluation.  
*Administered everyday  
+ Modified FLU-PRO Plus at Screen will be completed on site to determine if patient meets 
inclusion criterion  
  
3.6  Primary Objective  
Primary Objectives:  


BOSS-Covid-19 Study (BOSS-001)  
  
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1. To evaluate if treatment with 3 g TQ Formula (500 mg per capsule, 3 capsules BID) given 
orally on outpatient basis can significantly reduce median time to sustained clinical 
response compared to placebo in participants with COVID-19 infection treated in the 
outpatient setting.  Sustained clinical response is defined as a reduction of scores to </= 2  
on all symptoms of the Modified FLU-PRO Plus and should be remaining </= 2 for at least 
three days.  
2. To evaluate the safety and tolerability of TQ Formula (500 mg oral capsule, 3 capsules BID) 
when given to participants with COVID-19 infection  
  
3.7  Secondary Objective  
1. To compare the viral load profile over time (from baseline through to Day 14) between 
treatment with 3 g TQ Formula (500 mg per capsule, 3 capsules BID) given orally on 
outpatient basis and placebo in participants with COVID-19 infection treated in the 
outpatient setting  
2. To compare the percentage of RT-PCR negative/undetectable (i.e., viral clearance) on Day 
7 and Day 14 in participants taking 3 g TQ Formula (500 mg per capsule, 3 capsules BID) 
versus participants taking placebo   
3. To compare the duration and severity of symptoms (measured by Modified FLU-PRO Plus) 
over time from Day 1 through Day 14 in total FLU-PRO  Plus symptom severity score 
overall and in sub-domain scores (namely, Nose, Throat, Eyes, Chest/Respiratory,  
Gastrointestinal,  Body/Systemic, Taste/Smell),  between treatment with 3 g TQ Formula 
(500 mg per capsule, 3 capsules BID) given orally on outpatient basis and placebo in 
participants with COVID-19 infection  
4. To investigate if there exists an association between viral load and symptom severity by 
study arm and if such associations change overtime.  
  
Exploratory objectives:  
1. 
To evaluate the basic pharmacokinetics of TQ Formula active ingredient (thymoquinone) 
at the same time points (Days 1, 7, and 14) in participants with COVID-19 infection.  
2. 
To explore the effect of TQ Formula on inflammatory cytokines, coagulation factors and 
effector immune cells at same time points (Days 1, 7, and 14) in participants with 
COVID19 infection.  


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4  
Study Endpoints  
4.1  Efficacy Endpoints  
4.1.1 Primary Efficacy Endpoint:   
Measurement of the difference in median time to sustained clinical response in participants taking 
3 g TQ Formula (500 mg per capsule, 3 capsules BID) versus participants taking placebo.   
Sustained clinical response is defined as a reduction of scores to </= 2 on all symptoms of the 
Modified FLU-PRO Plus and sustained </= 2 for at least three days.  
4.1.2 Secondary Efficacy Endpoints  
1. Measurement of change in quantitative viral load from baseline, Day 7, and Day 14 using 
RT-PCR in participants taking 3 g TQ Formula (500 mg per capsule, 3 capsules BID) 
versus participants taking placebo with COVID-19 infection.  
2. Percentage of negative/undetectable RT-PCR (i.e., viral clearance) on Day-7 and Day-14 in 
participants taking 3 g TQ Formula (500 mg per capsule, 3 capsules BID) versus 
participants taking placebo  
3. Measurement of severity of, and change in, Covid-19 symptoms per total score as well as 
sub-scores (Nose, Throat, Eyes, Chest/Respiratory, Gastrointestinal, Body/Systemic, 
Taste/Smell) measured through Modified FLU-PRO Plus from Day 1 through Day 14 in 
participants with COVID-19 infection treated either with 3 g TQ Formula (500 mg per 
capsule, 3 capsules BID) or placebo.  
4. Correlation Coefficient of quantitative viral load and symptom severity at baseline, at Day 7, 
and Day 14 in participants taking 3 g TQ Formula (500 mg per capsule, 3 capsules BID) 
versus participants taking placebo  
4.1.3 Other Efficacy Endpoints   
The endpoints for the exploratory aims are the following:  
1. Measurement of thymoquinone and metabolites’ concentration in the plasma on day 1, Day 
7 and 14 using HPLC in patients treated with TQ Formula.  
2. Measurement of the inflammatory cytokine production, coagulation factors and the various 
effector immune cell subsets in the Peripheral Blood Mononuclear Cells (PBMC) of all 
patients on Day 1, Day 7 and Day 14 using FACS.  
If other analyses are needed beyond the endpoints mentioned above, this plan will be revised.  
4.2  Safety Endpoints  
Number of overall adverse events, related adverse reactions, and hospitalizations reported in 
participants taking 3 g TQ Formula (500 mg per capsule, 3 capsules BID) versus participants 


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taking placebo.  All AEs/SAEs will be captured throughout the study as per schedule of 
assessments.  
5  
Statistical Methods   
5.1  General Statistical Methods  
Summary tables (descriptive statistics and/or frequency tables) will be provided for all baseline  
and on study variables, efficacy variables, and safety variables, as appropriate. Continuous 
variables will be summarized with descriptive statistics (n, mean, standard deviation, range, and 
median). Frequency counts and percentage of subjects within each category will be provided for 
categorical data when appropriate in frequency and contingency tables.  
5.2  Analysis Sets  
The following are the main analysis datasets defined in the study protocol:  
• 
Full Analysis Dataset: The Full Analysis Set (FAS) is defined as all randomized participants 
who receive at least one dose of study intervention.   
• 
Final Dataset for Primary Objectives (FDPO): FDPO is defined as all randomized evaluable 
participants regardless of whether or not they received the assigned treatment regimen 
according to the evaluability definition above.  
• 
Intent-to-treat (ITT) dataset: ITT dataset is defined as all randomized patients regardless of 
their adherence to or completion of the study regimen. This dataset includes all randomized 
participants even if they do not receive any dose or they withdraw from the study without 
completing the regimen or experience protocol violations.  
• 
Safety Analysis Dataset: The Safety Analysis Set is defined as participants who take at 
least one dose of study intervention.  
• 
Per-Protocol Analysis Dataset: The Per-Protocol Analysis Set (PP) is defined as all 
participants in the FAS who complete all visits up to and including Day 14, are compliant 
with study medication and do not have any major protocol violation. Major protocol 
violations will be identified prior to breaking the blind.  
  
5.3  Statistical Analyses  
5.3.1 Demographic and Baseline Characteristics  
Demographic and baseline characteristics data will be summarized with descriptive statistics. 
Continuous variables will be summarized with n, mean, standard deviation, minimum, maximum 
and median. Frequency counts and percentage of subjects within each category will be provided 
for categorical data.  


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5.3.2 Primary Analyses  
Primary estimand-1 is defined to be the difference of median time to sustained clinical response 
between participants taking 3 g TQ Formula  (500 mg per capsule, 3 capsules BID) and 
participants taking placebo regardless of whether or not they actually receive their assigned 
therapy (ITT) and assuming that their hospitalization and treatment discontinuation rates will be 
similar. Possible intercurrent events for this estimand can be, though expected to be quite rare, 
hospitalization (i.e., transitioning from outpatient status to inpatient status) and treatment 
discontinuation, both of which may lead to potential missing data or change in the primary 
measures. The study participants will be reminded to submit their Modified FLU-PRO Plus 
questionnaires even after these intercurrent events to avoid missing data as much as practically 
possible. Hospitalization-free, while-on-study and per-protocol treatment versions of the above 
estimand will also be considered within each population definitions above.  
Median time to sustained clinical response for the treatment and placebo arms will be estimated 
using the Kaplan-Meier method and Log-rank test will be utilized to compare the time to sustained 
clinical response distributions between the two study arms using LIFETEST procedure in SAS 
Version 9.4 ® (SAS Institute, Cary, North Carolina, USA). Events will be defined as cases that 
attain sustained clinical response while on study and time to event will be calculated as the days 
from the date of symptoms onset to the date that all symptoms are below Level-2 (‘A little bit’) on 
the Modified FLU-PRO Plus questionnaire. Patients who do not attain to clinical response while on 
study will be censored on Day-21 or on the day they are off study before Day-21 due to other 
reasons.    
Although all analyses will be conducted based on the intent-to-treat principle, sensitivity analyses 
based on the adherence to the protocol regimen will also be carried out for the primary as well as 
secondary objectives. Sensitivity analyses will also include hospitalization-free, while-on-study, and 
per-protocol treatment cohorts as described above.  
Primary Estimand-2 for the safety objective is the difference of frequencies of Adverse Events and 
Serious Adverse Events between participants taking 3 g TQ Formula (500 mg per capsule, 3 
capsules BID) and participants taking placebo regardless of whether or not they actually receive 
their assigned therapy (ITT) and assuming that their hospitalization and treatment discontinuation 
rates will be similar. The above mentioned intercurrent events are also applicable for this safety 
estimand. Therefore, hospitalization-free, while-on-study and per-protocol treatment versions of the 
above estimand will also be considered within each population definitions above.  
.  


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5.3.3 Secondary Analyses  
Analysis plan for Secondary Aim-1:  
Secondary estimand-1 is defined to be the slope difference of viral load change overtime between 
participants taking 3 g TQ Formula  (500 mg per capsule, 3 capsules BID) and participants taking 
placebo regardless of whether or not they actually receive their assigned therapy (ITT) and 
assuming that their hospitalization and treatment discontinuation rates will be similar.    
The first secondary endpoint of this clinical trial is the differential change of viral load distribution 
overtime between the two-study arm. As the viral load will be measured at Baseline, on Day-7, and 
on Day-14 (and potentially for some patients in Day-4 and Day-10 visits), the outcome variable of 
interest will be a longitudinal interval-scale measure. To assess the differential behaviour of viral 
load overtime between the two study arms, Random Coefficients Models will be utilized where viral 
load will be the dependent variable and study arm and time will be main predictors. Viral load 
measures may be subjected to some data transformation (e.g., log transformation) to improve the 
model fit and assumptions as necessitated by model diagnostics. In addressing this objective, all 
available data will be utilized; that is, in addition to the evaluable patients, data from non-evaluable 
patients such as data only at baseline visit or data at baseline and  Day-10 for example will be 
used to improve the local fit of the underlying model. To assess differential behavior in total 
symptom severity score, time*study-arm interaction will be investigated as well. The above 
mentioned intercurrent events, namely, hospitalization (i.e., transitioning from outpatient status to 
inpatient status) and treatment discontinuation, are applicable for Secondary estimand-1 as well. 
Therefore, hospitalization-free, while-on-study and per-protocol treatment versions of the above 
estimand will also be considered within each population definitions above, namely, Full Analysis 
Dataset (FAS), Final Dataset for Primary Objectives (FDPO), and Per-Protocol Analysis Dataset 
(PPAS). The above analysis will be carried out using the MIXED procedure of SAS software.  
  
Analysis plan for Secondary Aim-2:  
Secondary estimand-2 is defined to be the proportion difference of patients with RT-PCR 
negative/undetectable viral loads between participants taking 3 g TQ Formula  (500 mg per 
capsule, 3 capsules BID) and participants taking placebo regardless of whether or not they actually 
receive their assigned therapy (ITT) and assuming that their hospitalization and treatment 
discontinuation rates will be similar. To address this estimand, the percentages of RT-PCR 
negative/undetectable viral loads between the two study arms will be estimated and compared 
using Fisher’s Exact test at Day-7 and Day-14 visits.  The above mentioned intercurrent events for 


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the primary endpoint are applicable for Secondary estimand-1 as well. Therefore, 
hospitalizationfree, while-on-study and per-protocol treatment versions of the above estimand will 
also be considered within each population definitions above. Beyond the single test of 
independence at each time point, Logistic Regression models will also be constructed to 
investigate the interaction of the study arms with other factors if interest such as gender, age, etc.   
  
Analysis plan for Secondary Aim-3:  
Secondary estimand-3 is defined to be the difference of total and subdomain symptom scores 
measured through Modified FLU-PRO Plus questionnaire between participants taking 3 g TQ 
Formula  (500 mg per capsule, 3 capsules BID) and participants taking placebo regardless of 
whether or not they actually receive their assigned therapy (ITT) and assuming that their 
hospitalization and treatment discontinuation rates will be similar. To address this estimand, the 
differential change of total and subdomain symptom severity score measured by Modified 
FLUPRO Plus overtime between the two-study arm will be estimated and compared between 
the two study arms. As the total and subdomain symptom severity score will be measured at 
Baseline (Day-1) and on each day through Day-14, the outcome variables of interest will be a 
longitudinal interval-scale measures. To assess the differential behaviour of total and 
subdomain symptom scores overtime between the two study arms, Random Coefficients 
Models will be utilized where total and each of the subdomain symptom severity score will be 
the dependent variables and study arm and time will be main predictors. Total and subdomain 
symptom severity scores may be subjected to some data transformation (e.g., log 
transformation) to improve the model fit and assumptions as necessitated by model 
diagnostics.  In addressing this objective, all available data will be utilized; that is, in addition 
to the evaluable patients, data from non-evaluable patients such as data only at baseline visit 
or data just at baseline and  Day-10 for example will be used to improve the local fit of the 
underlying model. To assess differential behavior in total symptom severity score, time*study-
arm interaction will be investigated as well. When scientifically indicated, aggregated symptom 
severity scores may be defined through combining a set of subdomains such as Nose, Eyes. 
And Throat.  The above mentioned intercurrent events are applicable for Secondary estimand-
1 as well. Therefore, hospitalization-free, while-on-study and per-protocol treatment versions 
of the above estimand will also be considered within each population definitions above. For the 
above analyses, the MIXED procedure of SAS software will be utilized.  
  


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Analysis plan for Secondary Aim-4:  
Secondary estimand-4 is defined to be the assessment of whether or not a significant correlation 
between quantitative viral load and symptom severity exists and if so, whether or not such a linear 
association is dependent on the study arm. To address this estimand, the association between 
quantitative viral load and symptom severity will be investigated through linear regression models 
using the GLM procedure of SAS software, where total symptom severity score will be the 
dependent variable predicted by the quantitative viral load measures in the model at each visit. 
Study-Arm*viral load interaction will be added to the model to investigate if any association 
between viral load and symptom severity is mitigated by the study treatment. The above 
mentioned intercurrent events are applicable for Secondary estimand-1 as well. Therefore, 
hospitalization-free, while-on-study and per-protocol treatment versions of the above estimand will 
also be considered within each population definitions above.   
   
5.3.4 Other Analyses  
Analysis plan for Exploratory Aim-1:  
Descriptive and graphical statistics will be used to present the distribution of metabolites’ 
concentration markers in the plasma on day 1, Day 7 and 14 using HPLC within each study arm.  
These markers will also be compared for differential distribution between the arms using Wilcoxon- 
Mann-Whitney test at each visit. To compare the change over time in these markers, Random 
Coefficients Models will be utilized where each of these markers will be the dependent variable 
and study arm and time will be main predictors.   
  
Analysis plan for Exploratory Aim-2:  
Descriptive and graphical statistics will be used to present the distribution of the inflammatory 
cytokine production, coagulation factors and the various effector immune cell subsets in the  
Peripheral Blood Mononuclear Cells (PBMC) of all patients on Day 1, Day 7 and Day 14 using 
FACS within each study arm. These markers will also be compared for differential distribution 
between the arms using Wilcoxon-Mann-Whitney test. Like above, to compare the change over 
time in these markers, Random Coefficients Models will be utilized where each of these markers 
will be the dependent variable and study arm and time will be main predictors.  
  
In addition to the primary analysis described in Section 5.3.2 above, Response Free Survival 
(RFS) distributions will be compared using Cox-Proportional Hazards Models between the two 


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study arms controlling for other factors of interest such as gender, baseline viral load, age etc. The 
interaction of the study arm with these covariates will be also investigated. Considering the small 
size of the study, number of covariates to be added to such multivariable models will be limited.  
We expect that only 2-3 covariates will be added to these models in addition to the study arm.   
  
5.3.5 Covariate and Subgroup Analyses  
Covariate and subgroup analyses will be presented when applicable and supported by the 
available data, considering that the sample size of each study arm is already limited by up to 30 
subjects per arm.  
5.3.6 Safety Analyses  
Safety will be evaluated by summary of adverse events. Adverse event incidence rates will be 
summarized for each study arm, namely, Active Drug and Placebo Arms, in terms of severity and 
its relationship to study drug. A tabulation of Serious Adverse Events (SAEs) will be provided within 
each study arm with the number of patients experiencing a given AE or ADE, and the number of 
episodes while on study. Similar reports will be generated for AE monitoring purposes for the entire 
study cohort without unblinding of the study participants while the study is still ongoing.  
  
6  
Definitions for Clinical Evaluations  
6.1  Follow-Up Time Points  
The treatment time for each participant is 14 days and the follow-up time is for 7 days or until 
ongoing adverse events are resolved up to 45 days as part of safety follow-up.   
6.2  Points of Enrollment   
Subjects will be considered enrolled in the study when they have been informed of all aspects of the 
study, had ample time to review the Informed Consent document, signed the Informed Consent 
document, satisfied the Inclusion/Exclusion Criteria, and randomized.  
6.3  Definitions for Clinical Evaluation  
Clinical evaluation will be done using Flu-Pro Plus questionnaire modified for Covid-19 patients 
treated in an outpatient setting. The participants will fill out the questionnaire at the time of 
Screening, Enrollment, and from Day-1 to Day-21 post randomization each day using online data 
capture forms.   


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7  
General Considerations  
If a subject is not able to complete the study after being enrolled, the Investigator, or designee will 
document the last contact with the subject.  All attempts to contact the subject will be documented 
in the source documentation.  Missed or out-of-range visits will be considered protocol deviations.  
.  
7.1  Missing Data and Outliers:   
In this study, the primary objective is driven by data collected during the baseline visit and up to 
Day-21; considering the short treatment and follow-up time, missing data is expected to be 
minimal. We do not anticipate excessive missing values; however, missingness structure will still 
be analyzed as to whether or not the missingness is Missing Completely at Random (MCAR), or 
Missing at Random (MAR), or Not-Missing at Random (NMAR). In the event of MCAR and MAR, 
sensitivity analyses will be carried out by using Multiple Imputation (MI) techniques when 
appropriate. Specifically for the Modified FLU-PRO Plus data, Last-Value-Carried-Forward (LVCF) 
imputation approach will be utilized as this assessment will be for Day-1 through Day-21 and LVCF 
approach would be appropriate to impute intermittent missing values in these time series type 
data.  
The data quality checks will be an ongoing effort for the study and potential outliers will be detected 
and resolved as much as possible while the study is ongoing. In the analysis phase, the outliers 
will be evaluated based on whether or not they are influential values in given statistical model a 
given outlier may be perfectly in line with the model built and does not necessarily mean 
problematic for modeling assumptions. If they are found to be influential, then possible data 
transformation approaches such as log-transformation will be considered.  
7.2  Protocol Violations and Deviations   
Missed visits and out of range visits will be considered protocol deviations while such participants 
will still be deemed evaluable for the primary objective. Investigators will not deviate from the 
clinical protocol except to deliver emergency care or to eliminate an immediate hazard to the 
subject. All study deviations from the clinical protocol will be captured in the study CTMS database. 
All deviations with the potential to affect subject safety, rights, or well-being will also be reported as 
required by the IRB/EC and to sponsor.  
7.3  Disposition of Subjects and Withdrawals  
All subjects who provide informed consent and are enrolled will be accounted for in this study. 
Subjects maintain the right to discontinue their participation in the study at any point.  If a subject 


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exercises this right, the Investigator will make every effort to ascertain and document the reason 
for withdrawal.  
7.4   Sensitivity Analysis  
 Sensitivity analysis is not applicable for this study.  
7.5  
Changes to Planned Analysis after Blind Review of Data and 
Unblinding Consideration (if Applicable)  
Not applicable.  
7.6  Interim Analysis and Data Monitoring  
There is no planned interim analysis for the primary objective of the study; however, there will be 
safety interim analyses after the initial cohort of 12 patients have been enrolled and have received 
at least one dose of the assigned drug.  This safety interim analysis will be conducted without 
unblinding the patients. As part of this safety interim analysis, if two or less number of patients in 
this initial cohort of 12 patients experience Grade 3+ adverse events attributable to the study 
regimen and not resolved through symptomatic treatments within 48 hours, there will be no need 
for unblinding the safety cohort; however, if there are 3 or more cases with Grade 3+ adverse 
events attributable to the study regimen and not resolved through symptomatic treatments within 
48 hours, the enrollment will be halted, this initial cohort of patients will be unblinded to evaluate 
the distribution of the AEs between the placebo and the study drug, and the possible corrective 
amendments will be considered.    
7.7  Timing with Respect to Database Locking  
The data quality checks will be an ongoing effort while the study is still on. When all patients are 
enrolled and complete their modified Flu-Pro Plus assessment to Day 21, a complete 
dataextraction will be requested for an efficacy data quality control. Once the efficacy data is 
deemed to be complete and accurate, the efficacy data will be locked and another complete data 
extraction will be requested to generate a preliminary efficacy report. As part of this preliminary 
efficacy analysis, the study statistician will be unblinded but all other member of the study team will 
remain blinded and the preliminary safety report will not contain any individual level data. Upon the 
completion of the 45-day safety follow-up of the last enrolled patient,, there will be a final data 
quality assessment finalizing the safety parameters, generating final set of queries for the study 
sites to correct/modify/update. Once all data quality check queries are adequately addressed, the 
study database will be ordered to be locked and the entire study team will be unblinded to patient 
randomization.  
  


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7.8  Analysis Software  
All analysis will be performed using SAS® Software version 9.4 or later. When appropriate, 
Rpackage will be used as well.  
7.9  List of Tables, Figures and Listings  
Demographic characteristics table, AE and SAE tables, tables of Covid-19 test results both from 
the rapid test and RT-PCR, the distribution statistics table for the viral load assessments, any other 
secondary objective measure tables. Modified FLU-PRO Plus assessments will be shown 
graphically as time series plot with a reference line at Level-2 (‘Somewhat’) where the y-axis will 
show the scale from ‘Not at all (Level-0)’ to ‘Very much (Level-4)’.  
8  
References  
8.1  Applicable Regulatory Guidelines:   
 
•  ICH E9: Statistical Principles for Clinical Trials (September 1998)  
  
8.2  Other applicable Documents  
Not Applicable