Assessment of a Novel Fixed-dose Combination (FDC) Drug VR-AD-1005 for the Treatment of Acute Watery Diarrhea in Cholera: a Phase II, Randomized, Placebo-controlled, Double-blinded Efficacy and Safety Trial
Phase: PHASE2
Conditions: Cholera
Sponsor: Hunazine Biotech S.L.

22 
4.0 Research Design and Methods 
Describe the research design and methods and procedures to be used in achieving the specific aims of the research project. If applicable, mention the 
type of personal protective equipment (PPE), use of aerosol confinement, and the need for the use BSL2 or BSL3 laboratory for different part of the 
intended research in the methods.. Define the study population with inclusion and exclusion criteria, the sampling design, list the important outcome 
and exposure variables, describe the data collection methods/tools, and include any follow-up plans if applicable. Justify the scientific validity of the 
methodological approach (biomedical, social, gender, or environmental).  
Also, discuss the limitations and difficulties of the proposed procedures and sufficiently justify the use of them. 
This study is a phase II, randomized, placebo controlled, double blind efficacy and safety trial. The 
study is designed for the assessment of a novel fixed dose combination (FDC) drug VR-AD-1005 for 
the treatment of acute watery diarrhea in adult cholera patients. Adult (18-65 years) cholera patients 
admitted to icddr,b Dhaka Hospital will be randomized to intervention and placebo groups with 75 
patients per group.  
Group A (intervention product arm): Patients will receive VR-AD-1005 treatment for three days, 
or less if recovered earlier, in addition to the general cholera protocol treatment. 
Group B (placebo arm): Patients will receive placebo for three days, or less if recovered earlier, 
in addition to the general cholera protocol treatment. 
Key aspects of the trial design and Trial Schema are given below in table 2 and figure 2. 
Table 2: Key aspects of the trial design 
Intervention Model: 
Parallel arm intervention model with 2 
Arms: Group A (intervention product arm) 
and Group B (placebo arm) 
Population Type: 
[Adult patient] 
Control: 
Intervention: 
[placebo] 
[VR-AD-1005] 
Population 
Diagnosis or 
Condition: 
[Vibrio cholerae 
infection] 
Active Comparator: [not applicable] 
Population Age: 
Minimum: [18y.o.] 
– Maximum:
[65y.o.]
Trial 
Intervention 
Assignment 
Method:  
Double-blind 
randomization 
Site Distribution: 
Single site 
NCT06193408- 20-November-2023

23 
 
Figure 2-Trial Schema 
 
 
 
4.1. Rationale for Trial Design 
The double-blind randomized controlled trial (RCT) is accepted by the authorities as objective 
scientific methodology that, when ideally performed, produces knowledge untainted by bias. The 
most important design techniques for avoiding bias in clinical trials are blinding and randomization, 
and these should be normal features of most controlled clinical trials intended to be included in a 
marketing application. Most such trials follow a double-blind approach, in which treatments are pre-
packed in accordance with a suitable randomization schedule and supplied to the trial center(s) 
labelled only with the subject number and the treatment period so that no one involved in the conduct 
of the trial is aware of the specific treatment allocate to any particular subject, not even as a code 
letter. The baseline characteristics will be summarized using descriptive statistics to facilitate 
evaluation of possible differences between treatment groups, which might bias the comparison of 
treatments. Study medication compliance, protocol violations and reasons for dropouts will be 
examined both as indicators of treatment differences and to evaluate any biases in the proposed 
treatment comparisons. 
 
 
4.2. Trial Population 
 
4.2.1. Selection of Trial Population 
Adults, both sexes, aged 18-65 years of age. 
 
4.2.2. Rationale for Trial Population 
Cholera predominantly affects children and young adults20. The paediatric population is outside of 
the scope of the current protocol, however, during the outbreak all age groups are at risk of infection. 
With this consideration, capping the age at 65 years will facilitate the recruitment, while allowing to 
reduce the burden and complications of co-managing chronic diseases associated with elderly 
populations. 
 

24 
 
4.2.3. Inclusion Criteria 
• 
Signed informed consent. 
• 
Adults, both genders aged 18-65 years. 
• 
Acute watery diarrhea (defined as passage of three or more liquid stools within the 24 hours 
before admission) with severe dehydration on arrival. 
• 
Detection of V. cholerae by rapid diagnostic assay (e.g. dark field microscopy). 
 
4.2.4. Exclusion Criteria 
• 
Known or suspected hypersensitivity to trial product(s) or related products. 
• 
Subjects with passage of bloody stools or muco-purulent stools. 
• 
Subjects with chronic diarrhea (>4 weeks of Diarrhea). 
• 
Clinically significant concomitant systemic disease (i.e. cardiovascular diseases including 
heart failure, acute kidney injury, sepsis or life-threatening malignant cancer). 
• 
Mental incapacity, unwillingness, or language barriers, precluding adequate understanding 
or cooperation. 
• 
History of receiving antimicrobial or antidiarrheal drugs within 6 hours prior to admission. 
• 
Positive urine pregnancy test for all female patients 
• 
Failure to obtain informed consent. 
• 
Failure to definitively diagnose cholera via culture or RT-PCR 
 
4.2.5. Screen Failures 
Patients with acute diarrhea, who go through the screening but do not fit the inclusion criteria or 
present with criteria excluding them from the clinical trial as per sections 4.2.3 and 4.2.4 above 
would be labelled as screen failures and will not receive the study treatment and will receive standard 
treatment in the hospital. Screen failures are not expected to contribute to the analysis of efficacy 
and safety. Screen failures who have medical conditions will be referred to the appropriate level of 
care. 
 
 
4.3. Sequence of events 
• 
All prospective patients visiting the facilities aged 18-65 years experiencing acute watery diarrhea 
(duration 24 hours or less) and severe dehydration will be assessed. Assessment will be done 
primarily by the study nurses (at least three nurses will rotate for the study duration). Study 
doctors will also assist with the assessment. 
• 
Consent will be sought from suspected patients to enroll cases. Confirmatory microbiology for 
cholera will be done. Eligibility of the participants will be identified using the screening tool. This 
study will not include vulnerable participants (refer to inclusion and exclusion criteria in sections 
4.2.3 and 4.2.4). 
• 
Trained nurses/study doctors will take the patients (who fulfil all inclusion criteria and do not 
meet  any of the exclusion criteria) or guardian for critically ill patients through the consenting 
process. This will involve explaining in plain language what the study is about and the risk/ 
benefits of participating. Participants will be informed that participation in the trial is voluntary 
and that they can withdraw at any time during the 3 days of inpatient care. They will be required 
to sign applicable consent forms, one form will be kept by investigators and another will be given 
to participants. 
• 
Upon consenting, randomization will be done by the independent researcher and treatment 
started by the study nurses in consultation and supervision by study doctors, in addition to 
standard management for V. Cholerae for patients. 
• 
All patients will be admitted in the dedicated wards of the study site for all the 3 days of 
treatment. Early discharges will not be permitted for enrolled study participants due to safety 
and endpoint measurement reasons. To facilitate data analysis, and in particular safety analysis 
(development of possible adverse events, while undergoing treatment with VR-AD-1005), all 

25 
 
participants with different level of severity of the disease must follow the same treatment 
protocol. Having variations in the treatment strategy, such as early discharge, may complicate 
the conduct of the trial. An example of such complication is the development of unrelated AEs 
which stem from changes in the lifestyle of participants after discharge, for example, change in 
diet or a community-acquired disease, which will be prevented and avoided by having all 
participants as in-patients. 
• 
Patients will receive their first dose administered by the study nurse immediately after the 
consenting process and randomization is completed. The treatment is in a capsule form and 
participants will be instructed by the study nurse to swallow the treatment whole without 
chewing with a glass of water or oral rehydration solution. The volume of liquid used for ingesting 
the treatment will be recorded in patient water intake charts for monitoring and analysis 
purposes. Subsequent doses will be timed based on the first dose (i.e. second dose is 3 hours 
after first dose). The study nurses will record the timing for each patient’s medication and will 
keep strictly to the timing between shifts by handing over study patient folder records after each 
shift. Study nurses will additionally monitor for vomiting or accidental loss of treatment dose (for 
example, dropped dose) and to re-administer, if vomiting occurs within 30 minutes of treatment 
administration or if the pill is clearly visible in the vomit. 
• 
Stool samples will be taken to the laboratory for culture and identification of V. cholerae and 
blood samples taken for study laboratory assessments. Laboratory assessments will be 
conducted at the icddr,b Immunobiology, Nutrition and Toxicology Laboratory and/or diagnostics 
laboratories. Any other necessary study laboratory test may be performed at any of these 
laboratories depending on capacity and efficiency of study procedures.  
• 
During the three days of admission, participants will be monitored daily by study doctors and 
nurses. Treatment will be given as per scheduled doses each day, monitoring and measurement 
of water intake, weighing of vomiting and stool output using calibrated scales (scales will be 
calibrated on daily basis) and all other study procedures. For recruited patients whose culture 
comes back negative for cholera, treatment will be stopped (culture results expected by day 
three) and they will not be included in the final analysis. 
• 
In the event of SAE’s during treatment or after discharge, participants will be referred to the 
appropriate clinic for comprehensive management. 
• 
Participants will only be discharged when the study doctors deem so after the three days of 
treatment. Participants who are not discharged after 3 days will not receive further treatment 
but will be monitored by study staff. 
• 
After discharge, participants will be followed up with weekly phone calls to check on their health 
and wellbeing and also ascertain, if they are experiencing any side effects of the treatment. For 
participants without phone, the phone number of their guardian or neighbor will be taken for 
the follow up. Where participant cannot be reached via phone, their personal address will be 
followed to conduct the follow up. After three attempts of phone calls and one home visit, 
participants that cannot be reached will be declared lost to follow up. 
 
 
4.4. Trial Intervention 
 
4.4.1. Description of Trial Intervention 
Investigational product is a capsule formulation designed for oral administration. 
 
4.4.2. 
Preparation, Handling, Storage and Accountability 
Acquisition and accountability: Investigational products will be supplied to the site by approved 
carrier services. CRO will perform oversight over the drug supplies to the site, drug storage, usage 
and return of unused product to the sponsor for destruction. 
Formulation appearance, packaging: VR-AD-1005 oral solid dose is an investigational product 
containing two active substances, Sainzsubadine (sloe berry powder) and Sucralfate. 

26 
 
Primary packaging material: VR-AD-1005 oral dose will be packaged in 10 count adult 
configurations. The primary packaging for the finished product is a PVC/ACLAR®, laminated and 
alfoil/PVC push-through blister. 
Product storage and stability: Store below 30 °C protect from moisture and direct sunlight. Store 
the product in the original containers. 
 
4.4.3. Route of Trial Intervention  
Intervention is taken orally. Cholera is a gastrointestinal disease; the oral route is by far the most 
common route of drug administration in gastrointestinal diseases. 
 
4.4.4. Dosing and Administration 
Cholera typically presents with voluminous liquid stool due to hypersecretion of fluids by the intestine 
21. VR-AD-1005 is not absorbed and acts at the level of intestinal mucosa. Thus, first dose of the 
drug is dissolved in much larger than normal volume of fluid presented in the intestine of a patient 
recently admitted for cholera. In addition, due to the diarrhea, first dose of the drug is evacuated 
with stool faster than subsequent doses. In order to maintain working concentration and counteract 
dilution, first dose of the drug is tripled, representing the “loading dose” drug administration tactics. 
The remaining doses are single doses for the three days which is in line with average duration of the 
cholera episode and thus gives sufficient time for the drug to exert it’s effect. The treatment will be 
administered to participants under the supervision of trained study personnel, i.e. study nurse. 
Nurses will be instructed to monitor for vomiting or accidental loss of treatment dose (for example, 
dropped dose). The participants will be instructed to swallow the treatment whole without chewing 
with a glass of water or oral rehydration solution. The volume of the liquid will be registered in the 
water intake charts. 
 
Administration begins when the patient is able to swallow the tablet. If the patient vomits less than 
30 minutes after administration of dose, the dose will have to be re-administered after about 
15 minutes. If the patient vomits after 40 minutes, the clinician will check the vomitus to see if the 
medication is in there, if the medication is not visible then the dose is not re-administered. If the 
medication is visible, then the dose is re-administered after 15 minutes. Both arms will receive extra 
dosses of the medication for these purposes. In the event of lost dose, the pharmacy will have a 
backup of the medication doses for emergency purposes. 
 
VR-AD-1005 oral dose will be administered orally 4 times per day (approximately every 4-5 hours 
with overnight break to allow for participants rest and recovery) with first administration of 3 capsules 
as loading dose. Dosing is indicated in the table below (Table 3), with reasonable timing of 
administration at 7:00, 12:00, 17:00 and 21:00 hours ± 1 hour. 
 
Table 3: VR-AD-1005 doses and dosing schedule 
Time 
Day 1 
Day 2 
Day 3 
Time 0 hr 
3 doses 
1 dose 
1 dose 
Time +5:00 hr 
1 dose 
1 dose 
1 dose 
Time +10:00 hr 
1 dose 
1 dose 
1 dose 
Time +15:00 hr 
1 dose 
1 dose 
1 dose 
 
4.4.5. 
Access to Trial Intervention After End of Trial 
After the end of trial, IP will no longer be accessible to the participants. Cholera is an acute 
infectious disease, and after resolution of cholera episode treatment is no longer warranted. 
 

27 
 
4.4.6 Participant Assignment, Randomization and Blinding 
4.4.6.1. Participant Assignment 
During recruitment, in addition to rehydration/antibiotics, each eligible participant will receive a 
numbered individual package of treatment, with package number being unique to the particular 
participant. After the end of the study for the purpose of data analysis the randomization tables will 
be supplied to the biostatisticians and data from all participants who have completed the trials will 
be assigned to the Group A (VR-AD-1005 intervention) or Group B (placebo) for analysis. 
 
4.4.6.2. Participant Randomization 
This trial is a double-blind, placebo-controlled trial. IP and placebo randomization will be performed 
by the IP manufacturer using uneven-block randomization strategy with the following considerations: 
• 
A computer-generated randomization list that provides an associated unique study identification 
number is produced by a researcher unrelated to the study/trial 
• 
The randomization list is saved as a document in a format that assigns appropriate identifiers to 
the study subjects. This document is safely and securely kept by the Sponsor along with a log 
of the code used to generate the list. 
• 
The randomization list is sent to the assigned person to preserve any and all blinding and 
concealment. The blinded investigators/research team should not see the randomization list 
during the running of the trial (see 6.6.3 for blinding). 
• 
Randomization is double-blind, and the clinical trial site is not aware of the type of intervention 
(IP or placebo) that a particular patient will receive. 
A procedure for emergency breaking the code is implemented by the sponsor and the icddr,b Data 
Safety Monitoring Board (DSMB) in case there is a need for emergency access for the benefit of the 
participant.  
 
4.4.6.3. Blinding and Unblinding 
The blinding/randomization of the medication is determined when the individual patient study 
supplies are packaged by the IP manufacturer. Standard randomization methods are used, i.e. 
uneven block randomization. The study center is required to dispense the patient medication in 
chronological order to each of the patients entering the study. A sealed envelope containing the 
randomization code breaks will be given to the Investigator for safe keeping enabling easy access in 
the event of an emergency, requiring the code to be broken. 
 
Management of Emergency Code Break: 
The emergency code breaks will be placed by Hunazine Biotech in a sealed envelope and kept at the 
study center. The code must only be broken in the case of medical events which the 
investigator/physician in charge of the patient feels cannot be adequately treated without knowing 
the identity of the study medication. 
Sponsor should be contacted before breaking the code. If it is not possible, and the situation is an 
emergency, the investigator may break the code and contact the Sponsor as soon as possible. The 
following information needs to be recorded in the event of an Emergency Code Break: 
• 
Date of Code Break 
• 
Identification of person(s) requesting the Code Break 
• 
Reason for breaking the code 
• 
Investigator Signature. 
The person who broke the code must sign and date the envelope which, if opened at study site, will 
be returned to Safety Board (SB) by the SB site monitor. The patient will be withdrawn from the 
study. 
 

28 
 
4.4.6.4. Trial Intervention Compliance 
During the study, the field monitor will visit the site regularly to check the completeness of patient 
records, the accuracy of entries on the Case Report Form (CRF), the adherence to the protocol and 
to Good Clinical Practice (GCP), the progress of enrollment, and to ensure that study investigational 
product is being stored, dispensed, and accounted for according to specifications. Key study 
personnel must be available to assist the field monitor during these visits. 
The investigator must maintain source documents for each patient in the study, consisting of case 
and visit notes (hospital or clinic medical records) containing demographic and medical information, 
laboratory data and the results of any other tests or assessments. All information on CRFs must be 
traceable to these source documents in the patient's file. The investigator must also keep the original 
informed consent form signed by the patient (a signed copy is given to the patient).  
The investigator must give the monitor access to all relevant source documents to confirm their 
consistency with the CRF entries. The investigator must give the monitor access to all relevant 
follow-up source documents for safety monitoring purposes and safety database updates. 
 
 
4.5. Trial Assessments and Procedures 
4.5.1. Screening/Baseline Assessments and Procedures 
• 
Medical history: All patients admitted with secretory diarrhea with severe dehydration will be 
offered to participate in the study. 
• 
Informed consent. Severely ill patients unable to sign the consent form will be resuscitated 
according to the proper SOPs, until they are able to make an informed decision regarding the 
enrollment. 
• 
Randomization and assignment to treatment Group. IP assignment. 
• 
Concomitant medication review. 
• 
Physical examination: height, body weight, temperature, thirst, HEENT (note and record signs 
of dehydration, such as sunken eyes), skin (note and record signs of dehydration, such loss 
of turgor), level of consciousness using AVPU (active, response on voice or pain, unconscious) 
criteria, vomiting. 
• 
Hydration status should be assessed in each participant including assessment of IV fluids, 
oral ORS intake and oral water intake. 
• 
Vital signs: body temperature, heart rate, respiratory rate, blood pressure (including 
orthostatic blood pressure measurement when possible), urine output. 
• 
Blood collection (5 ml) for Complete Blood Count (CBC), Basic Metabolic Panel (BMP) including 
serum electrolytes and creatinine.  
• 
Stool collection for V. cholerae RDT (dark field microscopy) and culture. 
• 
Initiate Re-hydration and antibiotic treatment in accordance with the SOPs for cholera 
patients until recovery.  
• 
Initiate IP administration as soon as participants can tolerate oral intake. 
 
4.5.2. Efficacy Assessments and Procedures 
• 
Medications review. 
• 
Physical examination: Body weight, thirst, HEENT (note and record signs of dehydration, such 
as sunken eyes), skin (note and record signs of dehydration, such loss of turgor), level of 
consciousness, vomiting. 
• 
Hydration status should be assessed in each participant including assessment of IV fluids, 
oral ORS intake and oral water intake. The volume of rehydration solutions received by the 
participant is recorded continuously for both enteral and parenteral rehydration for the entire 
duration of the treatment. Access to unaccounted water (i.e. tap water) should be restricted, 
and measured volume of ORS or water should be provided for free access instead, with the 
entire volume of consumed liquid measured and recorded in the charts. 

29 
 
• 
Vital signs: body temperature, heart rate, respiratory rate, blood pressure (including 
orthostatic blood pressure measurement when possible), and urine output. 
• 
Blood collection (5 ml) for Complete Blood Count (CBC), Basic Metabolic Panel (BMP) including 
serum electrolytes and creatinine at treatment dose #5. 
• 
Stool collections for efficacy analysis starts immediately after participant receives the first 
dose of IP and continues for the entire duration of the treatment. Stool samples are collected 
continuously, and weighed at 12-hour intervals. Number of stool passages and stool weight 
is recorded in CRFs. 
• 
At the end of the treatment the last physical exam is performed as per the schedule of events 
and stool sample will be sent for V. cholerae culture to confirm elimination of the pathogen. 
During the pre-treatment and treatment periods the data will be recorded into provided CRFs 
by the PI or his designee. 
 
4.5.3 Safety Assessments and Procedures 
• 
Physical examination: height, body weight, thirst, HEENT (note and record signs of 
dehydration, such as sunken eyes), skin (note and record signs of dehydration, such loss of 
turgor), level of consciousness, vomiting. 
• 
Vital signs: body temperature, heart rate, respiratory rate, blood pressure (including 
orthostatic blood pressure measurement when possible), and urine output. 
• 
Clinical Laboratory assessments: Complete Blood Count (CBC), Basic Metabolic Panel (BMP) 
Procedures and assessments may be performed as part of the subject’s standard medical care; 
however, data for these assessments should remain in the subject’s medical record and should not 
be provided to HB, unless specifically requested by the sponsor. Laboratory assessments will be done 
at the on-site laboratories.  
 
Schedule of activities during screening and three days of treatment period is given in appendix 1 
 
 
4.6. Start of Trial and End of Trial 
Trial starts once the participant has signed the informed consent form. The end of trial is defined as 
database lock after completion of the study by the last patient. A participant is considered to have 
completed the study if he or she has completed all phases of the study including the last visit or the 
last scheduled procedure shown in Appendix I. 
 
 
4.7. 
Adverse Events and Serious Adverse Events 
 
4.7.1. Definitions of AE, SAE and ADR 
Terminology according to ICH S2A: 
 
Adverse event: An adverse event (AE) can therefore be any unfavourable and unintended sign 
(including an abnormal laboratory finding, for example), symptom, or disease temporally associated 
with the use of a medicinal product, whether or not considered related to the medicinal product. 
Adverse Drug Reaction (ADR):  In the pre-approval clinical experience with a new medicinal 
product or its new usages, particularly as the therapeutic dose(s) may not be established: 
Unexpected Adverse Drug Reaction: An adverse reaction, the nature or severity of which is not 
consistent with the applicable product information (e.g., Investigator's Brochure for an unapproved 
investigational medicinal product). 
Serious Adverse Event or Adverse Drug Reaction: During clinical investigations, adverse events 
may occur which, if suspected to be medicinal product-related (adverse drug reactions), might be 
significant enough to lead to important changes in the way the medicinal product is developed (e.g., 

30 
 
change in dose, population, needed monitoring, consent forms). This is particularly true for reactions 
which, in their most severe forms, threaten life or function. Such reactions should be reported 
promptly to regulators. 
Serious adverse event (experience) or reaction is any untoward medical occurrence that at any dose:  
* results in death,  
* is life-threatening,  
NOTE: The term "life-threatening" in the definition of "serious" refers to an event in which 
the patient was at risk of death at the time of the event; it does not refer to an event which 
hypothetically might have caused death if it were more severe.  
* Requires inpatient hospitalisation or prolongation of existing hospitalisation,  
* results in persistent or significant disability/incapacity, or  
* is a congenital anomaly/birth defect. 
 
4.7.2. Time Period and Frequency for Collecting AE, SAE and ADRs Information 
 
• Single Cases of Serious, Unexpected ADRs  
All adverse drug reactions (ADRs) that are both serious and unexpected are subject to expedited 
reporting. This applies to reports from spontaneous sources and from any type of clinical or 
epidemiological investigation, independent of design or purpose. It also applies to cases not 
reported directly to a sponsor or manufacturer (for example, those found in regulatory authority-
generated ADR registries or in publications). The source of a report (investigation, spontaneous, 
other) should always be specified.  
 
Expedited reporting of reactions which are serious but expected will ordinarily be inappropriate. 
Expedited reporting is also inappropriate for serious events from clinical investigations that are 
considered not related to study product, whether the event is expected or not. Similarly, non-
serious adverse reactions, whether expected or not, will ordinarily not be subject to expedited 
reporting.  
 
Information obtained by a sponsor or manufacturer on serious, unexpected reports from any 
source should be submitted on an expedited basis to appropriate regulatory authorities if the 
minimum criteria for expedited reporting can be met.  
 
Causality assessment is required for clinical investigation cases. All cases judged by either the 
reporting health care professional or the sponsor as having a reasonable suspected causal 
relationship to the medicinal product qualify as ADRs. For purposes of reporting, adverse event 
reports associated with marketed drugs (spontaneous reports) usually imply causality. Many 
terms and scales are in use to describe the degree of causality (attributability) between a 
medicinal product and an event, such as certainly, definitely, probably, possibly or likely related 
or not related. Phrases such as "plausible relationship," "suspected causality," or "causal 
relationship cannot be ruled out" are also invoked to describe cause and effect. However, there 
is currently no standard international nomenclature. The expression "reasonable causal 
relationship" is meant to convey in general that there are facts (evidence) or arguments to 
suggest a causal relationship. 
 
• Other Observations  
There are situations in addition to single case reports of "serious" adverse events or reactions 
that may necessitate rapid communication to regulatory authorities; appropriate medical and 
scientific judgement should be applied for each situation. In general, information that might 
materially influence the benefit-risk assessment of a medicinal product or that would be sufficient 
to consider changes in medicinal product administration or in the overall conduct of a clinical 
investigation represents such situations. Examples include:  
 

31 
 
a. For an "expected," serious ADR, an increase in the rate of occurrence which is judged to be 
clinically important.  
 
b. A significant hazard to the patient population, such as lack of efficacy with a medicinal product 
used in treating life-threatening disease.  
 
c. A major safety finding from a newly completed animal study (such as carcinogenicity). 
 
4.7.3. 
Identifying AE, SAE and ADRs 
Principal investigator will be responsible for determining whether an adverse event (AE) is expected 
or unexpected. 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. Time period 
and frequency for event assessment and follow-up: 
The occurrence of an adverse event (AE) or serious adverse event (SAE) or adverse drug reaction 
(ADR) may come to the attention of study personnel during study weeks and interviews of a study 
participant presenting for medical care, or upon review by a study monitor. 
Any medical condition that is present at the time that the participant is screened will be considered 
as baseline and not reported as an AE. However, if the study participant’s condition deteriorates at 
any time during the study, it will be recorded as an AE. Changes in the severity of an AE will be 
documented to allow an assessment of the duration of the event at each level of severity to be 
performed. AEs characterized as intermittent require documentation of onset and duration of each 
episode. 
 
4.7.4. 
Recording AE, SAE and ADRs 
All AEs, including local and systemic reactions not meeting the criteria for AE/SAEs/ADRs, will be 
captured on the appropriate Adverse Event Form. Information to be collected includes event 
description, time of onset, clinician’s assessment of severity, relationship to study product (assessed 
only by those with the training and authority to make a diagnosis), and time of resolution/stabilization 
of the event. 
All AEs occurring while on study must be documented appropriately regardless of relationship. All 
AEs will be followed to adequate resolution. 
Any medical condition that is present at the time that the participant is screened will be considered 
as baseline and not reported as an AE. However, if the study participant’s condition deteriorates at 
any time during the study, it will be recorded as an AE. 
Changes in the severity of an AE will be documented to allow an assessment of the duration of the 
event at each level of severity to be performed. AEs characterized as intermittent require 
documentation of onset and duration of each episode. 
 
4.7.5. 
Reporting of Fatal or Life Threatening or Serious Unexpected ADRS 
 
• Fatal or Life-Threatening Unexpected ADRs  
All SAEs should be reported to local DSMB within 24 hours of known information by the 
PI/investigators. 
Certain ADRs may be sufficiently alarming so as to require very rapid notification to regulators 
in countries where the medicinal product or indication, formulation, or population for the 
medicinal product are still not approved for marketing, because such reports may lead to 
consideration of suspension of, or other limitations to, a clinical investigations program. Fatal or 
life-threatening, unexpected ADRs occurring in clinical investigations qualify for very rapid 
reporting. Regulatory agencies should be notified (e.g., by telephone, facsimile transmission, or 
in writing) as soon as possible but no later than 7 calendar days after first knowledge by the 
sponsor that a case qualifies, followed by as complete a report as possible within 8 additional 

32 
 
calendar days. This report must include an assessment of the importance and implication of the 
findings, including relevant previous experience with the same or similar medicinal products.  
 
• All Other Serious, Unexpected ADRs  
Serious, unexpected reactions (ADRs) that are not fatal or life-threatening must be filed as 
soon as possible but no later than 15 calendar days after first knowledge by the sponsor that 
the case meets the minimum criteria for expedited reporting. 
 
• Minimum criteria for reporting  
Information for final description and evaluation of a case report may not be available within the 
required time frames for reporting outlined above. Nevertheless, for regulatory purposes, initial 
reports should be submitted within the prescribed time as long as the following minimum criteria 
are met: an identifiable patient; a suspect medicinal product; an identifiable reporting source; 
and an event or outcome that can be identified as serious and unexpected, and for which, in 
clinical investigation cases, there is a reasonable suspected causal relationship. Follow-up 
information should be actively sought and submitted as it becomes available. 
 
• How to Report  
The CIOMS-I form has been a widely accepted standard for expedited adverse event reporting. 
However, no matter what the form or format used, it is important that certain basic 
information/data elements, when available, be included with any expedited report, whether in a 
tabular or narrative presentation. The listing in Attachment 1 addresses those data elements 
regarded as desirable; if all are not available at the time of expedited reporting, efforts should 
be made to obtain them.  
All reports must be sent to those regulators or other official parties requiring them (as 
appropriate for the local situation) in countries where the drug is under development. 
 
 
4.8. 
Safety Oversight - Data Safety Monitoring Board (DSMB)  
A Data and Safety Monitoring Board (DSMB) will be formed by the Ethical Review Committee to 
evaluate and assess the safety of study participants. The DSMB will be composed of 3-5 members, 
three of which are nominated by the ERC chairperson. The PI also has the option of nominating 1 or 
2 members with clinical expertise. The DSMB will convene two times, at the beginning before the 
trial is started, and at the end of the trial. Medical monitor will review the safety and have the option 
of convening a DSMB meeting if there is a safety issue that requires additional interpretation. The PI 
can also request a DSMB meeting depending upon the study complexity or in light of safety concerns. 
The DSMB will review all unanticipated problems involving risk to the participants or others, serious 
adverse events, and all participant deaths associated with the protocol. The DSMB will advise the PI 
of its findings and provide recommendations. The PI will inform the Sponsor in detail about the 
discussions of those meetings. 
 
 
4.9. 
Case management 
From screening to discharge, patients’ safety will be ensured through following procedures, which 
are also indicated in Table 4: schedule of activities. Screening procedures will be started only 
after obtaining written informed consent. During screening, medical and medication history will be 
collected and patients’ condition will be monitored through physical examination, obtaining vital 
signs, assessment of stool and urine output vomiting, dehydration status, water intake, ORS intake 
and IV fluid infused, and testing for complete blood count (CBC) and serum electrolytes and 
creatinine. For female of child bearing age pregnancy will also be evaluated as a part of the exclusion 
criteria. All the inclusion and exclusion criteria will be strictly maintained for enrollment of the patients 
in the study, and no vulnerable participants will be enrolled. After randomization and administration 

33 
 
of IP/placebo, physical examination will be performed and vital signs will be obtained for initial three 
hours followed by at least 8 hourly for the whole treatment period and also at the end of treatment. 
Assessment of hydration status, including dehydration, vomiting, urine output, water intake, ORS 
intake or IV fluid infusion will be performed throughout the treatment period. Concomitant 
medication review will also be performed at least once in a day. Blood testing for the re-evaluation 
of the patient will be done according to the clinical condition of the patient and once at the end of 
treatment. Beside giving study specific treatment, all patients will be provided standard care and 
treatments. Standard of care (SoC) for adult cholera patients having severe dehydration includes 
rehydration with IV cholera saline (100ml/kg over 3 hours) and Doxycycline 300 mg after initial 
hydration; SoC also includes replacement of ongoing purging with ORS (if no persistent vomiting or 
no high purging) or IV fluid (if there is persistent vomiting or high purging). Adverse events, including 
SAE will be closely monitored, and appropriate treatment will be provided; if needed, patients will be 
referred to specialized clinics or hospitals. Recovery from diarrhoea will be assessed continuously 
through stool output and frequency, and stool culture will be performed to confirm the absence of 
V. cholerae. Feeding with a normal diet will be continued /initiated based on clinical feasibility; 
feeding with a normal diet will be resumed when vomiting has stopped and patients are able to 
tolerate oral intake. Patients will be fed meals prepared and dispensed under hygienic conditions. 
Street food will be prohibited due to the high risk of bacterial contamination, which may hinder case 
management and also invalidate the results of this study. Patients will receive 2 to 3 cooked meals 
per day. Even after discharge, health and wellbeing of the participants will be followed up with weekly 
phone calls or home visits; it will be ascertained if they are experiencing any side effects of the 
treatment or any other AE or SAE.  
 
4.10. Concomitant Therapy  
4.10.1. Prohibited Concomitant Therapy 
Medications that have shown a positive effect on any aspect of diarrhea will not be permitted 
during the trial, including: 
• 
Cholestyramine 
• 
EGF/urogastrone 
• 
Loperamide 
• 
Racecadotril 
• 
Somatostatin 
• 
Alosetron 
• 
Mytesi (Crofelemer) 
• 
Atropine/dyphenoxilate 
 
4.10.2. Permitted Concomitant Therapy 
Concomitant therapy should be administered to all patients in accordance with the icddr,b 
guidelines on the treatment of cholera patients with acute diarrhea. Important steps are 
listed below (Table 5). 
 
Table 5-Assessment of dehydration status (10) 
Assess  
Condition* 
Normal  
Irritable/Less active*  Lethargic / Comatose*  
 
Eyes 
Normal  
Sunken  
Sunken  
 
Tongue 
Normal  
Dry  
Dry  
 
Thirst* 
Normal  
Thirsty (drinks 
eagerly)  
Unable to drink*  
 
Skin pulse* 
Normal  
Goes back slowly*  
Goes back very 
slowly*  
 
Radial 
pulse* 
Normal  
Reduced*  
Uncountable or 
absent*  

34 
 
Diagnosis  
 
No sign of 
dehydration  
If at least 2 signs 
including one (*) 
sign is present, 
diagnose.  
Some Dehydration  
If some dehydration 
plus one of the (*) 
signs are present, 
diagnose Severe 
Dehydration  
Management 
 
A 
B 
C 
 
A. No sign of dehydration – ORS  
• 
50 ml ORS per kg body weight over 4-6 hrs plus ongoing losses  
• 
Send patient to home with 4 packets of ORS  
• 
Continue feeding, including breast milk for infants and young children on  
 
B. Some dehydration – ORS  
• 
80 ml ORS per kg body weight over 4 – 6 hours plus ongoing losses  
• 
Observe patients for 6 - 12 hours.  
• 
Continue feeding, including breast milk for infants and young children on breastfeeding  
• 
Reassess patients and dehydration status frequently - hourly.  
In case of frequent vomiting (>3 times in 1 hour) or some dehydration persists ≥6 hours even 
after oral rehydration therapy: Treat with I/V fluid 
 
C. Severe dehydration – I/V solution containing sodium, potassium, chloride and bicarbonate 
(e.g. Ringer’s Lactate/ cholera saline)  
 Start I/V fluid immediately: 
• 
100 ml / kg Adult and Children > 1 year  
30 ml / kg in first 1/2 hour  
70 ml / kg in next 2.5 hours  
• 
Encourage the patient to take ORS solution as soon as he/she is able to drink without 
vomiting. 
• 
Antibiotic, if needed, after full rehydration. 
• 
Zinc-20 mg/day for 10 days in children 6 months-5 yr old. 
 
 
 
4.10.3. Antibiotic Therapy  
Stool samples for V. cholerae dark field microscopy and culture should be collected before the 
onset of antibiotic therapy for presumed cases of cholera (Profuse watery stools, typically looking 
like ‘rice water’, resulting dehydration; many individuals affected in the same locality).  
• 
Adults : Azithromycin, 1 gram single dose orally 
• 
Repeat dose if vomiting within 1/2 hour of taking the medicine  
Do not use azithromycin when a patient is 60-65 years or older or at any age with an established 
heart disease to avoid adverse effects, specially conduction defects. 
 In such a case, Doxycycline 300 mg single dose is recommended.  
Antibiotics should be administered no sooner than 2 hours after taking VR-AD-1005. 
 
 
4.11. Discontinuation of Trial Intervention and Participant Withdrawal from 
Trial 
 
Participants may withdraw from a research study for any reason, even without mentioning any 
reason. The participants will be able to receive standard treatment of the hospital without any 
penalty. For evaluation and reporting purposes, researchers may ask participants for their reasons 
for early withdrawal.  

35 
 
 
An investigator may discontinue trial intervention or withdraw a participant from the study for the 
following reasons: 
• 
Significant study intervention non-compliance. 
• 
If any adverse event (AE), Serious adverse events (SAE), laboratory abnormality, or other 
medical condition or situation occurs, 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. 
• 
Participant unable to receive study intervention for 3 days. 
• 
Reporting over-dose 
• 
protect a participant from excessive risk or risk with a demonstrated lack of benefits (e.g., a 
participant experiences serious side-effect without the anticipated therapeutic effects), or 
• 
maintain the integrity of the data (e.g., a participant is not following study procedures or may 
be deliberately providing false information). 
 
Whenever an investigator terminates a subject's participation in research, the investigator must 
explain to the participant the reasons for the termination and, as appropriate, other treatment 
options available to the participant. 
The data to be collected at the time of study intervention discontinuation will include the following: 
• 
Physical examination 
• 
Vital signs 
• 
Complete blood count (CBC) 
• 
Blood Basic metabolic panel, including Serum electrolytes and creatinine 
• 
Assessment of vomiting 
• 
Assessment of stool output 
• 
Assessment of dehydration 
• 
Assessment of IV and oral ORS and oral water intake 
• 
Assessment of urine output 
• 
Administration of questionnaires or other instruments helping participants to register 
symptoms, such as stool charts. 
• 
Assessment of adverse events. 
Follow-up calls will be made with patients who completed the protocol with questionnaires focusing 
on the side effects. Calls well be made once a week during the 28-day follow-up period. 
 
If any serious adverse events occur before treatment phase, study treatment may be postponed at 
the researcher's decision. 
 
The reason for participant discontinuation or withdrawal from the study will be recorded on the CRF. 
Subjects who sign the informed consent form and are randomized but do not receive the study 
intervention may be replaced. Subjects who sign the informed consent form, and are randomized 
and receive the study intervention, and subsequently withdraw, or are withdrawn or discontinued 
from the study, will not be replaced. 
 
 
4.12. Lost to Follow-up 
A participant will be considered lost to follow-up if he or she fails to return for three out of four 
scheduled phone calls or one missed in-person visit (for withdrawals) 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: The site will attempt to contact the participant and reschedule the missed 
visit within 7 days 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. 

36 
 
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/or electronic messages, 
one home visit). 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 been lost to 
follow-up and will be excluded from the long-term safety data analysis. 
Since follow-up occurs after the completion of the treatment course, loss to follow-up is not critical 
for the primary and secondary endpoints and treatment data obtained during the course of the trial 
will remain valid. 
 
 
4.13. Protocol Deviations 
A deviation is any noncompliance with the clinical trial protocol, GCP, or manual of procedures 
requirements. The noncompliance may be either on the part of the participant, the investigator, or 
the study site staff. Deviations from the protocol should be avoided. If deviations occur, The 
implications of the deviation must be reviewed and discussed. Investigator must document and 
explain protocol deviations by stating the reason, date, and the corrective action(s) taken. Deviations, 
for which no corrections are relevant, can be acknowledged and confirmed via edit checks in the 
CRF. The documentation for the protocol deviations must be kept in the Investigator's trial file and 
Hunazine Biotech trial master file. Major deviations from the protocol, as defined as deviations that 
impact participant safety or endpoints, will be promptly reported to the DSMB and Sponsor. Minor 
deviations from the protocol will be recorded on a source document and corresponding eCRF, and 
included in the annual report. Examples of minor deviations are events outside the control of the 
investigators (e.g., participant missed visit window or study parameters that are not part of the 
primary endpoint was not performed at a visit).  
 
 
4.14. Study monitoring plan (monitoring visits, level of source data verification, 
etc.) 
Trial monitoring activities will be provided by the CRO monitor(s), which will be conducted prior to 
beginning, at initiation, during, and at closeout of the trial by the monitor(s). At the beginning of the 
study, the monitor(s) will (1) verify the physical facilities of the study site. (2) ensure that the study 
team including the investigators and study staff are qualified and experienced. (3) provide training 
to study staff. During the course of the study, the monitor will visit the clinical site at regular intervals 
to verify compliance to the protocol; completeness, accuracy, and consistency of the data; and 
adherence to local regulations and requirements, including GCP on the conduct of clinical research. 
At a minimum the monitor(s) will do the following: (1) ensure that study staffs are performing their 
duties as per the delegation of authority log. (2) ensure that the study is being performed according 
to the protocol and applicable regulatory requirements. (3) review adherence to the protocol-specific 
SOPs. (4) verify source documents and eCRF are completed accurately and perform verification of 
eCRF against the available source documents. The monitor should have access to participant medical 
records and other study-related records needed to verify the entries on the eCRFs. (5) check that all 
AEs / SAEs and deviations have been appropriately reported. (6) ensure that each participant or 
parents/guardians have signed the approved ICF. (7) Ensure adequacy of study drugs and clinical 
trial supplies at the site. (8) review different study logs, e.g. ‘delegation of authority’, ‘IP 
accountability log’, ‘temperature log’ etc. (9) review visit findings with PI / investigator(s) and provide 
action items for the site personnel. (10) provide refresher training if necessary. The PI and the 
monitor must agree to cooperate to ensure that any problems detected in the course of these 
monitoring visits, including eCRF completion and query resolution, are resolved. Close-out monitoring 
visit includes (1) Final review of the source documents and eCRF, and resolution of missing and 
questionable data (2) Final ‘IP accountability’; returning remaining IPss to the sponsor or destruction 
of remaining IPs at study site. (3) Final ‘specimen accountability’. (5) Review and record site close-
out report submitted to sponsor, IRB and regulatory authorities. 

37 
 
 
 
4.15. Regulatory Agency inspection 
The PI must be aware that representatives from regulatory authorities (e.g. DGDA) or the IRB may 
wish to inspect the eCRFs and associated study records. The PI and study coordinator must make 
the relevant records available for inspection and will be available to respond to reasonable requests 
and audit queries made by authorized representatives of regulatory agencies. 
 
4.16. General Considerations: Risk Management and Quality Assurance 
4.16.1. Quality Tolerance Limits 
A QTL is a trial-level parameter, not a patient-level parameter. Examples include 
inclusion/exclusion protocol violations, incomplete/missing endpoint data, and AEs/SAEs of 
special interest. 
 
4.16.2. Data Quality Assurance 
QA is a systematic and independent assessment of all the clinical trial activities and records. It 
checks for following aspects: Generation, recording, analysis, and reporting of the clinical data 
are in accordance with the protocol, Standard Operating Procedures (SOPs) and Good Clinical 
Practices (GCPs). 
 
4.16.3. Source Data 
The executive order on GCP defines source data as any information in original records and 
certified copies of original records of clinical findings, observations or other activities in a 
clinical trial necessary for the reconstruction and evaluation of the trial. 
 
Source data may be both electronic and on paper. The following list includes examples of 
where source data may be stored: 
 
• 
medical records 
• 
laboratory reports 
• 
diaries 
• 
dispensing logs 
• 
Any research records 
• 
Case Report Forms (CRF) 
• 
ECG printouts 
• 
X-ray images 
• 
radiological reports, etc. 
• 
communication: verbal                                               

38 
 
5.0 Sample Size Calculation and Outcome (Primary and Secondary) Variable(s) 
Clearly mention your assumptions. List the power and precision desired. Describe the optimal conditions to attain the sample size. Justify the 
sample size that is deemed sufficient to achieve the specific aims. 
 
Planned screening is 500 participants. ITT (intention to treat) group is 150 patients. Enrollment will 
stop once 150 participants are recruited, or once 100% of per-protocol dataset (PP) is achieved. 
Sample size was calculated based on the mean volume of stool output in cholera patients. Mean 
stool output was assumed at 280±156 g/kg 22–24. Assuming 33% reduction (alternative hypothesis) 
vs no reduction (null hypothesis), sample size was calculated using GraphPad Prizm software for 
two-tailed t-test with alpha 5% and power 0.8. Using recruitment of 50 participants per arm (100 
total), power level of 0.82 can be achieved. Although, this is a sufficient level of power for the 
projected study, data points lost due to participant drop-out, cross-over to other study arms, 
withdrawal and missing data will affect the power of the study. We calculated that recruitment of up 
to 75 patients per study arm would allow power of 0.94.  
 
Endpoints associated with primary and secondary objectives, and justification for endpoints are given 
below (Table 1). 
 
Table 1-Primary and Secondary Objectives and Endpoints 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Primary 
• To assess the efficacy of 
VR-AD-1005 for the 
treatment of acute 
diarrhea in cholera 
patients in combination 
with standard treatment 
(rehydration and 
antibiotics, please see 
attached SoP for more 
details) versus standard 
treatment with placebo. 
Efficacy is measured as 
reduction in stool output 
between the start of 
treatment until final 
diarrheal stool before 
clinical recovery 
(defined as 
disappearance of watery 
stool and two 
consecutive passages of 
soft stool) or end of 
study treatment 
(treatment duration 72 
hours), which is earlier. 
• Reduction in stool output volume (ml/hour/kg) 
over a treatment period measured as stool 
weight and recalculated to stool volume using 
1:1 w/v ratio, and plotted as stool output 
quantity per each 12-hour period for the 
duration stool collection. 6 points are expected 
for a 72-hour stool collection. Measurement of 
stool output will be performed by collecting the 
stool from a participant into the pre-weighed 
vessel. The vessel with stool will be weighed on 
the calibrated scale, the weight of the vessel 
would be subtracted, and the resulting stool 
weight recorded into the stool diary form. 
During weighing of stool, Infection Prevention 
and control (IPC) standards will 
be upheld by 
implementing protection of personnel via using 
appropriate personal protective equipment. 
Hand washing before and after stool weighing 
will be strictly adhered to thereby preventing 
infection of study personnel. After weighing, 
stool will be disposed of as biohazardous waste 
at study sites and the weighing vessel 
appropriately disinfected before next 
measurement or discarded, if disposable. 
• Primary endpoint allows 
to monitor and 
characterize the efficacy 
of VR-AD-1005 for 
reduction of stool 
output. Over-secretion 
of hypotonic fluid occurs 
predominantly in the 
small intestine, and fluid 
is lost with stool. The 
volume of fluid loss in 
cholera patients can be 
life-threatening due to 
rapid dehydration. Thus, 
the primary efficacy 
endpoint is designed to 
demonstrate potential of 
VR-AD-1005 treatment 
to reduce fluid loss with 
stool. 
Secondary 
• Assessments of 
improvement of 
hydration status, fluid 
input and duration of 
• 
Time until the stool volume output reduction 
to 200 ml/hour or less. Duration of stool 
output in excess of 200 ml/hour per patient 
will be recorded between the start and end of 
• Secondary end-points 
are designed to 
supplement the primary 
end-point by providing a 

39 
 
diarrhea following VR- 
AD-1005 administration. 
 
• Evaluation of safety and 
tolerability of VR-AD-
1005 in addition to 
standard of care (SoC) 
versus SoC with placebo 
in adults with 
symptomatic cholera via 
documentation of 
adverse events, physical 
examination, and vital 
signs. 
the treatment for both treatment groups. 
• 
Time until clinical recovery (defined as 
disappearance of watery stool and two 
consecutive passages of soft stool). Duration 
of diarrhea until the passage of first non-
watery stool (soft/loose) or no stool for 12 
hours or the end of treatment (72 hours) will 
be derived from date and time records in 
daily stool charts. 
• 
Number of subjects with clinical recovery as 
above within treatment group. The number 
of subjects with clinical recovery will be 
recorded as total for the entire treatment 
duration, and as number of recoveries per 
each 24-hour period of treatment. 
• 
Number of unscheduled IV rehydration 
episodes per treatment. The number of 
unscheduled IV rehydration episodes per 
treatment will be recorded for the entire 
duration of the treatment. Unscheduled IV 
rehydration is defined as “receipt of IV fluid 
during the maintenance phase of hydration 
due to development of severe dehydration or 
some dehydration with high purging (>15 
ml/kg/hour) or persistent vomiting (>3 
episodes/hour), or failure to take ORS for any 
reason” 
• 
Duration of IV rehydration for the entire 
treatment period per treatment group and 
per each 24- hour period of treatment. 
• 
Safety will be measured by the proportion of 
patients that will present with AE/SAE as 
defined in Appendix II following 
administration of VR-AD- 1005. 
measure of hydration 
status of the 
participants, as well as 
providing a measure of 
the duration of the 
illness. Dehydration is 
the primary cause of 
death in cholera; 
duration of illness is the 
primary measure of the 
burden on the 
healthcare system and 
quality of 
life/productivity loss for 
cholera patients. 
Assessment of reduction 
in the need for 
unscheduled 
intravenous rehydration 
and time until switch to 
oral rehydration therapy 
will allow to assess the 
benefit of VR-AD-1005 
for in-patient 
management of cholera 
cases. Thus, secondary 
end-points allow to 
characterize the 
benefits related to 
potential improvements 
in the clinical course of 
the disease, case 
management and 
quality of life for cholera 
patients.  
 
 
 
 
 
 

40 
 
6.0 Data Analysis 
Describe plans for data analysis, including stratification by sex, gender and diversity. Indicate whether data will be analysed by the investigators 
themselves or by other professionals. Specify what statistical software packages will be used and if the study is blinded, when the code will be 
opened. For clinical trials, indicate if interim data analysis will be required to determine further course of the study. 
 
Data analysis must comply with ICH E9 Guideline and ICH E9(R1) Guideline. All relevant data 
collected in the trial should be considered in the statistical considerations section. 
The analysis will be conducted on all participant data at the time the trial ends.  
 
Statistics for primary end-point data and parametric secondary end-point data will be presented via 
means (alternatively, LSMEANS) with standard deviations; primarily two-tailed Student’s t-test will 
be used for analysis with significance level of p≤0.05. For non-parametric analysis, such as lifetime 
data, Kaplan-Meier analysis will be used with significance level of p≤0.05. Some data might be 
expressed and analyzed as % values, such as the occurrence of unscheduled IV rehydration. 
 
Analysis of secondary endpoints will be performed through the same method of the primary endpoint, 
which is found via a one- sided two sample unequal variance Student’s t-test. The scale for each 
secondary endpoint will be nominal and will be determined at a single endpoint, which is at the end 
of intervention. The results will be presented by LSMEANS (Least-squares Means) with standard 
errors. 
 
Primary Endpoint: 
Reduction in stool output volume (ml/hour) over a treatment period measured and plotted as total 
stool output during 12-hour stool collection at every 12 hours for 72 hours.  
 
Secondary Endpoints: 
• 
Time until the stool volume output of 200mL/hour or less. Duration of stool output in excess 
of 200mL/hour per patient will be recorded between the start and end of the treatment for 
both treatment groups. 
• 
Time until recovery. Duration of diarrhea until the passage of first non-watery stool or no 
stool for 12 hours or the end of treatment (72 hours) will be derived from date and time 
records in daily stool charts. 
Number of unscheduled IV rehydration episodes per treatment. The number of unscheduled 
IV rehydration episodes per treatment will be recorded for the entire duration of the 
treatment. Unscheduled IV rehydration is defined as “receipt of IV fluid during the 
maintenance phase of hydration due to development of severe dehydration or some 
dehydration with high purging (>15 ml/kg/hour) or persistent vomiting (>3 episodes/hour), 
or failure to take ORS for any reason” 
• 
Duration of IV rehydration for the entire treatment period per treatment group and per each 
24-hour period of treatment. 
• 
Number of recovered subjects within treatment group. The number of recovered subjects will 
be recorded as total for the entire treatment duration, and as number of recoveries per each 
24-hour period of treatment. 
• 
Safety will be measured by the proportion of patients that will present with AE following 
administration of VR-AD-1005. 
 
 
6.1. Analysis set 
Data for VR-AD-1005 intervention vs. placebo groups will be analyzed in the following comparison 
data sets: 
A. Stool output comparison  
B. Comparison of the number of unscheduled hydration episodes (both IV and oral, as defined 
above). 

41 
 
C. Comparison of the total volume of the rehydration solutions used during the study. 
D. Duration of diarrheal illness will be compared between the groups. 
E. Duration of stool output in excess of 200ml/hour will be compared. 
F. Comparison for the number of AE/SAE. 
G. Comparisons for the base-line descriptive statistics. 
 
 
6.2. 
Analysis Supporting Primary Objectives 
6.2.1. 
Statistical Model, Hypothesis, and Method of Analysis 
 
Primary Endpoint: 
Reduction in stool output volume over a treatment period. 
Primary Endpoint 1., Hypothesis 1. Treatment with VR-AD-1005 reduces stool output in patients 
with diarrhea due to V. cholerae infection. Null hypothesis is stated as no statistically significant 
difference in stool output volume as compared to placebo; alternative Hypothesis 1 is stated as 
statistically significant reduction in stool output volume in patients treated with VR-AD- 1005. 
Two analytical approaches are proposed. Approach 1 will be based on the total quantity of stool 
purged by participant during the intervention administration period. Stool output will be expressed 
as g/kg body weight based on the stool charts, and the means will be compared between intervention 
and placebo groups using Student’s unpaired t-test. In approach 2, stool purging is measured and 
plotted as total stool output in ml/kg/12 hours based on the individual stool collection charts. Weight 
of stool will be converted to volume using 1:1 weight/volume ratio. Diarrhea of cholera resolves 
rapidly, and majority of patients are expected to recover within 30-50 hours after randomization.12-
hour analysis increments allow a better insight into the onset and timing of the effect of study 
intervention as compared to cumulative data over the duration of the treatment. Means will be 
compared for each 12-hour period after first intervention administration using unpaired Student’s t-
test. Normal distribution is assumed for both cases. Analysis will be conducted in the ITT, MITT and 
PP groups. Missing data and outliers will be handled using imputation technique. Non-adherence and 
lost to follow-up patients will be categorized into the appropriate group and handled accordingly. No 
subtractions will be made from the ITT group after randomization assignment. 
 
6.2.2. 
Handling of Missing Data 
Missing data other than protocol-planned baseline values will be classified as missing completely at 
random (MCAR) or missing at random (MAR). MCAR can be confirmed by partitioning the data into 
two parts: one set containing the missing values, and the other containing the non-missing values.  
After partitioning the data, the t-test of mean difference is carried out in order to check whether 
there exists any difference in the sample between the two data sets. If the number of missing values 
is small, i.e. if the number of the cases is less than 5% of the sample, then the researcher can drop 
them. Otherwise, when analyzing using ITT population, model based multiple imputation (MI) will 
be used for both primary and secondary outcomes. The number of imputations will be the largest 
value of: 
• 
10, or 
• 
The proportion of missing values for the actual variable * 100. 
200 burn in iterations will be used for all analyses. Trace plots and distribution plots will be created 
to check the accuracy of imputations. 
 
 
6.3. 
Analysis Supporting Secondary Objectives 
Analysis of secondary end-points is not dependent on the analysis of the primary end-point. 
 
Secondary Endpoint 1, Hypothesis 2. Treatment with VR-AD-1005 improves hydration control 
in patients with diarrhea due to V. cholerae infection. Null hypothesis is stated as no statistically 

42 
 
significant difference in the number of unscheduled hydration episodes as compared to placebo; 
alternative hypothesis 2 is stated as statistically significant improvement in hydration control, 
expressed as number of unscheduled rehydration episodes over the treatment period; in patients 
treated with VR-AD-1005. Unscheduled IV rehydration is defined as “ 
“receipt of IV fluid during the maintenance phase of hydration due to development of severe 
dehydration or some dehydration with high purging (>15 ml/kg/hour) or persistent vomiting (>3 
episodes/hour), or failure to take ORS for any reason”  
 
Secondary Endpoint 2, Hypothesis 3. Treatment with VR-AD-1005 shortens duration of diarrhea 
in patients with diarrhea due to V. cholerae infection. Null hypothesis is stated as no statistically 
significant difference in the duration of diarrhea as compared to placebo; alternative hypothesis 2 is 
stated as statistically significant reduction in the duration of diarrhea, expressed as number of days 
till normalization of stool, in patients treated with VR-AD-1005. 
 
Secondary Endpoint 3, Hypothesis 4. Treatment with VR-AD-1005 shortens duration of stool 
output in excess of 200ml/hour in patients with diarrhea due to V. cholerae infection. Null hypothesis 
is stated as no statistically significant difference in the duration of stool output >200ml/hour as 
compared to placebo; alternative hypothesis 2 is stated as statistically significant reduction in the 
duration of diarrheal output >200ml/hour, expressed in hours, in patients treated with VR-AD-1005. 
 
Analysis: 
Secondary Endpoint 1, assessment of hydration control. There are two measurable parameters to 
be assessed in the patient hydration. Parameter 1 is the number of unscheduled hydration episodes. 
The numbers will be derived from the individual rehydration records, and the number of patients 
receiving IV rehydration will be expressed in % and compared for control and study intervention 
group. Alternatively, total number of IV rehydration episodes cand be expressed as means for control 
and study intervention group and analyzed using Student’s t-test. Parameter 2 is the volume of the 
oral rehydration solution received during the course of interventional treatment. Total volume of 
rehydration solution (oral and IV where applicable) will be derived from the individual rehydration 
records, expressed in ml/kg of patient weight, and analyzed as means using unpaired Student’s t-
test. Analysis subgroups will include 1) total hydration received over the course of study after first 
administration of interventional medication, and 2) hydration received during first 12 hours and 3) 
hydration received during first 24 hours. 
 
Secondary Endpoint 2. Assessment of duration of diarrhea. Total duration of diarrheal illness is 
an important parameter in the assessment of antidiarrheal treatment efficacy. Study intervention is 
expected to reduce the overall duration of the diarrhea. Duration of diarrhea is assessed from first 
administration of study intervention until the termination of diarrhea, which is defined as first formed 
stool, or no stool for 12 consecutive hours. Duration of diarrhea will be derived from individual stool 
charts. Kaplan-Meier survival curves will be built to compare duration of diarrhea between control 
and intervention groups. 
 
 
Secondary Endpoint 3. Duration of stool output in excess of 200ml/hour. As stated above, duration 
of diarrheal disease in cholera is limited, averaging 30-35 hours24. This could mask the beneficial 
effect of antidiarrheal treatment (as measured solely by stool quantification) if high initial stool output 
terminates abruptly. In order to establish the time course of the study intervention’s effect, in 
addition to analyzing the total duration of diarrhea in patients we need to introduce a parameter that 
combines the measured quantity of stool output and the time factor, addressing the question weather 
interventional medication can reduce the severity of diarrhea in the early stage of administration of 
study intervention. For analysis, individual stool charts will be used to calculate stool output per 
patient per 12-hour period and express it as stool volume in ml/hour. Duration of time when stool 

43 
 
output is in excess of 200ml/hour will be calculated for each participant, expressed as means for 
control and study intervention group and analyzed using Student’s t-test. 
 
Normal distribution is assumed for Endpoints 1 and 3. Non-parametric statistics is used in the analysis 
of Endpoint 2. Analysis will be conducted in the ITT, MITT and PP groups. Missing data and outliers 
will be handled using imputation technique. Non-adherence and lost to follow-up patients will be 
categorized into the appropriate group and handled accordingly. No subtractions will be made from 
the ITT group after randomization assignment. 
 
6.4. 
Safety Analyses 
Safety analysis is not the primary or secondary objective of this study, thus safety will be analyzed 
as summary statistics during treatment. AEs will be coded per Medical Dictionary for Regulatory 
Activities (MedDRA), and calculated. Each AE will be counted once only for a given participant, and 
presented using standard available criteria for severity, frequency, and relationship of AEs to study 
intervention. Start date, stop date, severity, relationship, expectedness, outcome, and duration will 
be reported for each SAE. Adverse events leading to premature discontinuation from the study 
intervention and serious treatment-emergent AEs will be listed. Proportion of the patients presented 
with AE or SAE will be calculated for IP and Placebo arms as a fraction of the total number of 
participants per arm and expressed as percent. Percentage of AE and SAE occurrence will be 
compared between IP and placebo groups to provide information regarding the safety of the 
investigational product. 
 
6.5. 
Other Analyses 
In addition to the overall analysis of the intervention vs. placebo, it is important to elucidate the 
efficacy of the IP as a function of the severity of the diarrheal disease. Thus, participants will be 
segregated into three subgroups based on the volume of stool output. All subgroups will be analyzed 
using both ITT and PP populations. moderate subgroup (stool volume 3-8 liters per 24 hours) and 
severe subgroup (stool volume >8 liters per 24 hours). Subgroups are intended for analytical 
purposes and will not affect randomization. Within the subgroups the analysis will be performed as 
above. 
 
6.6. 
Interim Analyses 
No interim analysis is currently planned. 
 

44 
 
7.0. Data Safety Monitoring Plan (DSMP) 
All clinical investigations (research protocols testing biomedical and/or behavioural intervention(s)) should include the Data and Safety Monitoring 
Plan (DSMP). The purpose of DSMP is to provide a framework for appropriate oversight and monitoring of the conduct of clinical trials to ensure 
the safety of participants and the validity and integrity of the data. It involves involvement of all investigators in periodic assessments of data 
quality and timeliness, participant recruitment, accrual and retention, participant risk versus benefit, performance of trial sites, and other factors 
that can affect study outcome. 
 
7.1. Data Protection 
All study data and records will be treated confidentially and used solely for research purposes; We 
will ensure that, only the researcher will have access to the collected data/information obtained from 
the participant’s records. We will also make sure that the data that are gathered are saved and well 
encrypted under password to prevent unauthorized access. If the subject and parents or the subject's 
legal representative withdraws the previously given informed consent or refuses to consent to 
continue in the trial, or if the subject dies and no consent is available from a subject's legal 
representative, or if the subject is lost to follow up then the subject's data will be handled as follows:  
• 
Data collected will be retained by Hunazine Biotech and entered into the database. 
• 
Safety events will be reported to the department responsible for global product safety, 
Hunazine Biotech/regulatory authorities according to local/national requirements. 
• 
If data is used, it will always be in accordance with local law and IRB/IEC procedures. 
 
7.2. Reports and Publications 
Information obtained during the conduct of this trial is considered confidential and can be used by 
Hunazine Biotech for regulatory purposes and for the general development of the trial product. All 
information supplied and obtained by Hunazine Biotech in connection with this trial shall remain the 
sole property of Hunazine Biotech and is to be considered confidential information. No confidential 
information shall be disclosed to others without prior written consent from Hunazine Biotech. Such 
information shall not be used except in the performance of this trial. The information obtained during 
this trial may be made available to other physicians who are conducting other clinical trials with the 
trial product, if deemed necessary by Hunazine Biotech. One principal Investigator will be appointed 
to review and sign the CTR (Signatory Investigator) on behalf of all participating Investigators. The 
signatory Investigator will be appointed based upon the criteria defined by the International 
Committee of Medical Journal Editors (ICMJE) for research publications. The protocol will be 
registered in a publicly accessible database such as clinicaltrials.gov, CTIS/EudraCT, WHO Pan African 
Clinical Trial Registry (PACTR), or other applicable registers. In addition, after study completion (i.e., 
LPLV) and finalization of the study report the results of this trial will be submitted for publication and 
posted in a publicly accessible database of clinical trial results. 
 
Hunazine Biotech commits to communicating, and otherwise making available for public disclosure, 
results of trials regardless of outcome. Public disclosure includes publication of a paper in a scientific 
journal, abstract submission with a poster or oral presentation at a scientific meeting, or by other 
means. 
 
Hunazine Biotech reserves the right to not release data until specified milestones, e.g. when the 
clinical trial report is available. This includes the right to not release interim results of clinical trials, 
because the release of such information can invalidate the results of the entire trial. 
 
At the end of the trial, one or more manuscripts for publication will be prepared collaboratively 
between Investigator(s) and Hunazine Biotech. Hunazine Biotech reserves the right to postpone 
publication and/or communication for less than 60 days to protect intellectual property. 
 
The results of this trial will be subject to public disclosure at external web sites according to 
international regulations, which is reflected in Hunazine Biotech Code of Conduct for Clinical Trial 
Disclosure. 
 

45 
 
In all cases, the trial results shall be reported in an objective, accurate, balanced, and complete 
manner, with a discussion of the strengths and limitations of the trial. Screen In the event of any 
disagreement about the content of any publication, both the Investigators' and Hunazine Biotech's 
opinions shall be fairly and sufficiently represented in the publication. 
 
Hunazine Biotech maintains the right to be informed of any Investigator plans for publication and to 
review any scientific paper, presentation, communication or other information concerning the 
investigation described in this protocol. Any such communication must be submitted in writing to the 
Hunazine Biotech Trial Manager prior to submission for comments. Comments will be given within 
four weeks from receipt of the planned communication. 
 
7.3. Authorship 
Authorship of publications should be in accordance with guidelines from The International Committee 
of Medical Journal Editors' Uniform Requirements (sometimes referred to as the Vancouver Criteria). 
The Investigator(s) offered authorship will be asked to comment and approve the publication. No 
permission to publish will be granted to any clinical research organization involved in the trial 
described in this protocol. 
 
 
 

46 
 
8.0. Ethical Assurance for Protection of Human rights 
Describe the justifications for conducting this research in human participants. If the study needs observations on sick individuals, provide sufficient 
reasons for using them. Indicate how participants’ rights will be protected, and if there would be benefit or risk to each participants of the study. 
Discuss the ethical issues related to biomedical and social research for employing special procedures, such as invasive procedures in sick children, 
use of isotopes or any other hazardous materials, or social questionnaires relating to individual privacy. Discuss procedures safeguarding participants 
from injuries resulting from study procedures and/or interventions, whether physical, financial or social in nature. [Please see Guidelines] 
 
8.1. Regulatory and Ethical Considerations 
The trial will be conducted in compliance with ICH GCP, applicable regulatory requirements, and in 
accordance with the Declaration of Helsinki. The sponsor and investigator will comply with all 
applicable regulations. In addition, the investigator will follow national and institutional requirements 
including, but not limited to Investigational Medicinal Product (IMP), clinical research, Informed 
Consent Forms (ICF) and Institutional Review Board (IRB) regulations. The approval of the trial 
protocol will be obtained from Ethical Review Committee (ERC) of icddr,b and Directorate General of 
Drug Administration (DGDA), which is functioning under the Ministry of Health & Family Welfare, 
Government of the People's Republic of Bangladesh. The investigator will be aware that 
representatives from DGDA or ERC may wish to inspect if the trial is in compliance to the protocol 
and adhered to GCP and local regulatory requirement. They may also look at eCRFs and associated 
study records to check the accuracy and consistency of the data. 
 
All subjects included in the trial will be treated according to the icddr,b SoC for the treatment of 
cholera (attached) in addition to either VR-AD-1005 (75 subjects) or placebo (75 subjects). Subjects 
assigned to the trial will be transferred to a treatment regimen anticipated to be better than or equal 
to the treatment they receive at the time they enter the trial. However, they will have to spend some 
extra time, as additional visits to the clinic are required and some of these tests performed during 
the trial are outside normal practice. 
 
There is no information available today indicating any risks in connection with the use of VR- AD-
1005 FDC as compared to other products from the same pharmaceutical category. Data available for 
individual API indicate low risk which is far outweighed by the benefits. 
 
All subjects participating in the trial will be monitored closely by the site with frequent visits and 
telephone contacts. When the subjects' participation in the trial ends, the subjects will consult their 
Investigator to decide on the best available marketed treatment. 
 
8.2. Committees 
Prior to commencement of the trial the protocol, any protocol amendments, subject 
information/informed consent form, any other written information to be provided to the subject and 
parents/subject's legal representative, subject recruitment procedures (incl. advertisement), if any, 
IB, available safety information, information about payments and compensation available to subjects 
if not mentioned in the subject information, the Investigator's current curriculum vitae (CV) and/or 
other documentation evidencing qualifications, and other documents as required by the local 
Institutional Review Board/ Independent Ethics Committee (IRB/IEC) should be submitted. The 
submission letter should clearly identify the trial identification number, version, European Union Drug 
Regulating Authorities Clinical Trials (EudraCT) number, title and/or the date of the documents that 
have 
been submitted to the IRB/IEC. Written approval favorable opinion must be obtained from 
IRB/IEC prior to commencement of the trial. During the trial, the Investigator or sponsor, as 
applicable, must promptly report the following to the IRB/IEC, in accordance with local requirements: 
updates to IB, unexpected SAEs where a causal relationship cannot be ruled out, substantial protocol 
amendments to the protocol, non- substantial protocol amendments according to local requirements, 
deviations to the protocol implemented to eliminate immediate hazards to the subjects, new 
information that may affect adversely the safety of the subjects or the conduct of the trial (including 
new risk/benefit analysis in case it will have an impact on the planned follow-up of the subjects), 

47 
 
annually written summaries of the trial status, and other documents as required by the local IRB/IEC 
and Ethics Review Committee. 
Substantial protocol amendments must not be implemented before approval/favorable opinion, 
unless necessary to eliminate immediate hazards to the subjects. 
 
8.3. Informed Consent Process 
All potential study participants will be informed on the purpose of the study, procedures, risks and 
benefits of participating in the study, participant’s expectations, and the compensation plan for 
their participation. It will be ensured that all information collected will be stored under lock and key 
or on the researchers’ computer under password to prevent unauthorized access and used only for 
the purpose for which the data was collected. We will guarantee data privacy and confidentiality of 
all data obtained by ensuring that personal identifiers collected from participant folders would not 
be used during the analysis in the study. The participants will be encouraged to ask questions 
about the study, have the questions answered and then be given time to decide if they would 
participate in the study.  It will also be emphasized that participation is voluntary, and that the 
participant has the right to withdraw themselves from the study at any time without prejudice. An 
impartial witness will be present during the informed consent discussions with an illiterate 
participant. A person who can only write his/her name and cannot read or write in Bangla will be 
considered as illiterate for the study purposes. The Investigator must obtain voluntary, personally 
signed and dated informed consent from participants before any study-related procedure.  The 
original, signed ICF must be kept in the site study file; one copy of the ICF will be given to the 
participant / parents. 
 
 

48 
 
Use of Animals 
Describe if and the type and species of animals to be used in the study. Justify with reasons the use of particular animal species in the research and 
the compliance of the animal ethical guidelines for conducting the proposed procedures. 
 
No animals will be used in this study. 
 
Collaborative Arrangements 
Describe if this study involves any scientific, administrative, fiscal, or programmatic arrangements with other national or international organizations 
or individuals. Indicate the nature and extent of collaboration and include a letter of agreement between the applicant or his/her organization and the 
collaborating organization. 
 
Hunazine Biotech will serve as the sponsor of the study and will provide the Clinical Trial Material 
for the phase II study. Hunazine Biotech will be responsible for cGMP manufacture of the 
investigational product. 
 
 
Facilities Available 
Describe the availability of physical facilities at site of conduction of the study. If applicable, describe the use of Biosafety Level 2 and/or 3 laboratory 
facilities. For clinical and laboratory-based studies, indicate the provision of hospital and other types of adequate patient care and laboratory support 
services. Identify the laboratory facilities and major equipment that will be required for the study. For field studies, describe the field area including 
its size, population, and means of communications plus field management plans specifying gender considerations for community and for research 
team members. 
 
• The trial will be conducted at icddr,b Dhaka Hospital. It has adequate facilities for the management 
of acutely ill patients, particularly those are suffering from diarrheal illnesses. The patients are 
used to manage in short stay unit of the hospital. Those who require hospital stay for>48 hours 
with comorbidities require admission in longer stay unit. Additionally, the hospital has a dedicated 
intensive care unit for the management of seriously ill patients, like those with septic shock. 
 
• The Clinical Haematology and Microscopy Lab, the Clinical Biochemistry Lab, and the Clinical 
Microbiology and Immunology Lab under the Clinical and Diagnostic Services, Office of Executive 
Director support clinical research activities beside performing routine diagnosis. We have the 
facility for sample processing and storage. We have refrigerator (4°C) and deep freezer ((-20°C 
and- 80°C) and backup generator for 24 hours supply. 
 

49 
 
Literature Cited 
Identify all cited references to published literature in the text by number in parentheses. List all cited references sequentially as they appear in the 
text. For unpublished references, provide complete information in the text and do not include them in the list of Literature Cited. There is no page 
limit for this section, however, exercise judgment in assessing the “standard” length. 
 
1. Nutrition & Clinical Services Division, icddr,b. Treatment for Cholera and other Diarrheas. 
Published online June 24, 2018. 
2. Firth A, Prathapan P. Azithromycin: The First Broad-spectrum Therapeutic. Eur J Med Chem. 
2020;207:112739. doi:10.1016/j.ejmech.2020.112739 
3. Thiagarajah J, Verkman A. CFTR pharmacology and its role in intestinal fluid secretion. Curr 
Opin Pharmacol. 2003;3(6):594-599. doi:10.1016/j.coph.2003.06.012 
4. Tradtrantip L, Namkung W, Verkman AS. Crofelemer, an Antisecretory Antidiarrheal 
Proanthocyanidin Oligomer Extracted from Croton lechleri , Targets Two Distinct Intestinal 
Chloride Channels. Mol Pharmacol. 2010;77(1):69-78. doi:10.1124/mol.109.061051 
5. Hollander D, Tytgat GNJ, eds. Sucralfate: From Basic Science to the Bedside. Springer US; 
1995. doi:10.1007/b102476 
6. Blumenthal M, Busse WR, Bundesinstitut für Arzneimittel und Medizinprodukte (Germany), eds. 
The Complete German Commission E Monographs. Therapeutic Guide to Herbal Medicines. 
American Botanical Council ; Integrative Medicine Communications; 1998. 
7. ANSM. BLACKTHORN  FOR HOMOEOPATHIC PREPARATIONS. In: French Pharmacopoeia. ; 
2007. 
8. Hollman PCH. Absorption, Bioavailability, and Metabolism of Flavonoids. Pharm Biol. 
2004;42(sup1):74-83. doi:10.3109/13880200490893492 
9. Zhong S, Sandhu A, Edirisinghe I, Burton‐Freeman B. Characterization of Wild Blueberry 
Polyphenols Bioavailability and Kinetic Profile in Plasma over 24‐h Period in Human Subjects. 
Mol Nutr Food Res. 2017;61(12):1700405. doi:10.1002/mnfr.201700405 
10. Czank C, Cassidy A, Zhang Q, et al. Human metabolism and elimination of the anthocyanin, 
cyanidin-3-glucoside: a 13C-tracer study. Am J Clin Nutr. 2013;97(5):995-1003. 
doi:10.3945/ajcn.112.049247 
11. Nomi, Iwasaki-Kurashige, Matsumoto. Therapeutic Effects of Anthocyanins for Vision and Eye 
Health. Molecules. 2019;24(18):3311. doi:10.3390/molecules24183311 
12. Balta I, Sevastre B, Mireşan V, et al. Protective effect of blackthorn fruits (Prunus spinosa) 
against tartrazine toxicity development in albino Wistar rats. BMC Chem. 2019;13(1):104. 
doi:10.1186/s13065-019-0610-y 
13. Sung JL, Yu JY, Wang TH, Wang CY, Chen DS. A placebo-controlled, double-blind study of 
sucralfate in the short-term treatment of duodenal ulcer. Scand J Gastroenterol Suppl. 
1983;83:21-24. 
14. Elsborg L, Boysen K, Bruusgaard A, Reinicke V. Sucralfate versus placebo treatment in 
duodenal and prepyloric ulcer: a clinical endoscopic, double-blind controlled investigation. 
Hepatogastroenterology. 1984;31(6):269-271. 

50 
 
15. Danielsson G, Jungbeck C, Peterson K, Norgren L. A Randomised Controlled Trial of Micronised 
Purified Flavonoid Fraction vs Placebo in Patients with Chronic Venous Disease. Eur J Vasc 
Endovasc Surg. 2002;23(1):73-76. doi:10.1053/ejvs.2001.1531 
16. Olszewski W. Clinical Efficacy of Micronized Purified Flavonoid Fraction (MPFF) in Edema. 
Angiology. 2000;51(1):25-29. doi:10.1177/000331970005100106 
17. Yang L, Ling W, Du Z, et al. Effects of Anthocyanins on Cardiometabolic Health: A Systematic 
Review and Meta-Analysis of Randomized Controlled Trials. Adv Nutr. 2017;8(5):684-693. 
doi:10.3945/an.116.014852 
18. Xu Z, Xie J, Zhang H, et al. Anthocyanin supplementation at different doses improves 
cholesterol efflux capacity in subjects with dyslipidemia-a randomized controlled trial. Eur J Clin 
Nutr. 2021;75(2):345-354. doi:10.1038/s41430-020-0609-4 
19. Kakkos SK, Nicolaides AN. Efficacy of micronized purified flavonoid fraction (Daflon®) on 
improving individual symptoms, signs and quality of life in patients with chronic venous 
disease: a systematic review and meta-analysis of randomized double-blind placebo-controlled 
trials. Int Angiol. 2018;37(2). doi:10.23736/S0392-9590.18.03975-5 
20. Troeger C, Blacker BF, Khalil IA, et al. Estimates of the global, regional, and national morbidity, 
mortality, and aetiologies of diarrhoea in 195 countries: a systematic analysis for the Global 
Burden of Disease Study 2016. Lancet Infect Dis. 2018;18(11):1211-1228. doi:10.1016/S1473-
3099(18)30362-1 
21. Das R, Nasrin S, Palit P, et al. Vibrio cholerae in rural and urban Bangladesh, findings from 
hospital-based surveillance, 2000–2021. Sci Rep. 2023;13(1):6411. doi:10.1038/s41598-023-
33576-3 
22. Alam NH, Ahmed T, Khatun M, Molla AM. Effects of food with two oral rehydration therapies: a 
randomised controlled clinical trial. Gut. 1992;33(4):560-562. doi:10.1136/gut.33.4.560 
23. Alam N, Majumder R, Fuchs G. Efficacy and safety of oral rehydration solution with reduced 
osmolarity in adults with cholera: a randomised double-blind clinical trial. The Lancet. 
1999;354(9175):296-299. doi:10.1016/S0140-6736(98)09332-5 
24. Alam NH. Efficacy and tolerability of racecadotril in the treatment of cholera in adults: a double 
blind, randomised, controlled clinical trial. Gut. 2003;52(10):1419-1423. 
doi:10.1136/gut.52.10.1419 
 
 
 
 
 
 

51 
 
List of Abbreviations 
 
AE 
Adverse Event 
ANCOVA 
Analysis of Covariance 
AS 
Active Substance 
AWD 
Acute Watery Diarrhea 
BMP 
Basic Metabolic Panel 
Caco-2 
Heterogeneous 
human 
epithelial 
colorectal adenocarcinoma 
cell line 
cAMP 
Cylic adenosin monophosphate 
Ca2+ 
Calcium ion 
CBC 
Complete Blood Count 
CFTR 
Cystic Fibrosis Transmembrane Conductance Regulator 
cGMP 
Cyclic Guanosine Monophosphate 
CFR 
Code of Federal Regulations 
CICC 
Calcium-induced chloride channel 
Cl- 
Chloride ion 
CLIA 
Clinical Laboratory Improvement Amendments 
CMP 
Clinical Monitoring Plan 
COC 
Certificate of Confidentiality 
CONSORT 
Consolidated Standards of Reporting Trials 
CRF 
Case Report Form 
CRO 
Contract Research Organization 
CTC 
Cholera Treatment Centres 
CTx 
cholera toxin 
CV 
Cardiovascular 
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 
EM 
Electron Microscopy 
FDA 
Food and Drug Administration 
FDAAA 
Food and Drug Administration Amendments Act of 2007 
FDC 
Fixed-Dose Combination 
FFR 
Federal Financial Report 
GCP 
Good Clinical Practice 
GLP 
Good Laboratory Practices 
GMP 
Good Manufacturing Practices 
GRS 
Generally Recognized as Safe 
GI 
Gastrointestinal 
GWAS 
Genome-Wide Association Studies 
HIPAA 
Health Insurance Portability and Accountability Act 
IB 
Investigator’s Brochure 
ICD 
International Classification of Disease 
ICH 
International Conference on Harmonisation 
ICMJE 
International Committee of Medical Journal Editors 
IDE 
Investigational Device Exemption 

52 
 
IND 
Investigational New Drug Application 
IP 
Investigational Product 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
ISO 
International Organization for Standardization 
ITT 
Intention-To-Treat 
Kg 
Kilogram 
LSMEANS 
Least-squares Means 
MedDRA 
Medical Dictionary for Regulatory Activities 
ml 
Milliliter 
MOP 
Manual of Procedures 
MQAE 
N-[ethoxycarbonylmethyl]-6-methoxy-quinolinium bromide 
MSDS 
Material Safety Data Sheet 
NDC 
National Drug Code 
Na+ 
Sodium ion 
NCT 
National Clinical Trial 
NHE3 
Sodium-hydrogen exchanger 3 
NIH 
National Institutes of Health 
NIH IC 
NIH Institute or Center 
OHRP 
Office for Human Research Protections 
ORS 
Oral Rehydration Salt 
ORT 
Oral Rehydration Therapy 
PBS 
Phosphate buffered saline 
PI 
Principal Investigator 
QA 
Quality Assurance 
QC 
Quality Control 
RCT 
Randomized Controlled Trial 
RDT 
Rapid Detection Test 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SMC 
Safety Monitoring Committee 
SOA 
Schedule of Activities 
SOC 
System Organ Class 
SOP 
Standard Operating Procedure 
UP 
Unanticipated Problem 
US 
United States 
WT 
Wild type 
WHO 
World Health Organization 
 

53 
 
Appendix I 
 
Schedule of activities 
Procedures 
Screenin
g Hour -
4 
Day1 
Hour 
0:00 
Day 1 
Time +5:00 
hr 
Day 1 
Time+1
0:00 hr 
Day 1 
Ho Time 
+15:00 hr 
Day 2 
Hour 0:00 
Day 2 
Time +5:00 
hr 
Day 2 
Time 
+10:00 hr 
Day 2 
Time 
+15:00 hr 
Day 3 Hour 
0:00 
Day 3  
 Time +5:00 
hr 
Day 3  
Time 
+10:00 hr 
Day 3   
Time 
+15:00 hr 
Ho
9 00
End of 
treatment 
Informed 
consent 
X 
Demographics 
X 
Medical history 
X 
Pregnancy 
screening 
X 
Randomization 
and 
dispensation 
of numbered 
IP 
X 
 
Concomitant 
medication 
review 
X 
X 
 
 
 
X 
 
 
 
X 
Physical exam 
(including 
Height and 
weight) 
X 
X 
 
X 
 
X 
X 
Vital signs 
BP, pulse, 
temperature 
X 
X 
X 
X 
X 
Blood collection 
for CBC  
X 
X 
X 
X 
X 
Blood collection 
for BMP 
X 
X 
X 
Assessment of 
stool output 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Assessment of 
vomiting 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Assessment of 
rehydration 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Assessment of 
dehydration 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Assessment of 
IV fluid infused 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Assessment of 
water intake 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Assessment of 
ORS intake 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Assessment of 
urine output 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 

54 
 
Collection of 
stool samples 
for V. Cholerae 
confirmation 
X 
X 
Stool 
collection for 
electrolytes 
X 
X 
X 
Administer 
study 
intervention 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Initiate 
rehydration 
and/or 
antibiotics 
treatment 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Adverse Events 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Complete Case 
Report Forms 
(CRFs) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 

55 
 
Appendix II : ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS – DEFINITIONS, 
SEVERITY, AND CASUALTY 
 
Definitions 
Adverse event: An untoward medical occurrence after exposure to a medicine, which is not 
necessarily caused by that medicine. 
 
Serious Adverse event: An adverse reaction that results in death, is life-threatening, 
requires hospitalisation or prolongation of existing hospitalisation, results in persistent or 
significant disability or incapacity, or is a birth defect 
 
Assessment of severity 
The terms “severe” and “serious” are not synonymous. Severity refers to the intensity of an 
adverse event (rated as mild, moderate, or severe, or according to a pre-defined grading 
criterion; the event itself may be of relatively minor medical significance (such as severe 
headache without any further findings). 
 
Severity and seriousness need to be independently assessed for each adverse event recorded 
on the CRF. 
 
Serious adverse events are required to be reported by the Investigator to the Sponsor 
immediately (i.e., no more than 24 hours after learning of the event). 
 
The assessing adverse event severity table below will be used for assessing severity for 
adverse events. 
 
Table -Adverse Event Severity Grading Scale 
 
Grade 
Severity 
1 
Mild; asymptomatic or mild symptoms; clinical or diagnostic observations 
only; or intervention not indicated 
2 
Moderate; minimal, local, or non-invasive intervention indicated; or limiting 
age-appropriate instrumental activities of daily livinga 
3 
Severe or medically significant, but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; or 
limiting self-care activities of daily livingb, 
4 
Life-threatening consequences or urgent intervention indicatedd 
5 
Death related to adverse eventd 
Note:  
a Instrumental activities of daily living refer to preparing meals, shopping for groceries or 
clothes, using the telephone, managing money, etc. 
b Examples of self-care activities of daily living include bathing, dressing and undressing, 
feeding oneself, using the toilet, and taking medications, as performed by patients who 
are not bedridden. 
c If an event is assessed as a "significant medical event," it must be reported as a serious 
adverse event  
d Grade 4 and 5 events must be reported as serious adverse events  
 
Assessment of causality 
Investigators should use their knowledge of the participant, the circumstances 
surrounding the event, and an evaluation of any potential alternative causes to determine 
whether or not an adverse event is considered to be related to the study treatment, 

56 
 
indicating "yes" or "no" accordingly. The following guidance should be taken into 
consideration: 
 
• Temporal relationship of event onset to the initiation of study treatment. 
• Course of the event, considering especially the effects of dose reduction,   discontinuation 
of study treatment, or reintroduction of study treatment 
• Known association of the event with the study treatment or with similar treatments. 
• Known association of the event with the disease under study. 
• Presence of risk factors in the participant or use of concomitant medications known to 
increase the occurrence of the event. 
• The presence of non-treatment-related factors that are known to be associated with the 
occurrence of the event. 
• For participants receiving combination therapy, causality will be assessed individually for 
each protocol-mandated therapy.