Conditions: Sickle Cell Anemia (HbSS, or HbSβ-thalassemia0)
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 46
Healthy volunteers: no

Brief summary:
In this feasibility trial, the investigators will compare participants treated with montelukast and hydroxyurea to those treated with placebo and hydroxyurea for a total of 8 weeks.

Detailed description:
The primary hypothesis for this trial is that montelukast adds efficacy to hydroxyurea therapy for improving vaso-occlusion when compared to hydroxyurea alone. The following specific aims will be tested in adolescents and adults with sickle cell disease (SCD):

Aim 1. To determine whether montelukast versus placebo added to hydroxyurea will improve markers of vaso-occlusion-associated tissue injury in adolescents and adults with sickle cell disease.

Aim 2. To evaluate physiologic effects of montelukast versus placebo added to hydroxyurea in adolescents and adults with sickle cell disease.

Subaim 2A. To determine if montelukast versus placebo added to hydroxyurea will improve lung function in adolescents and adults with sickle cell disease.

Subaim 2B. To determine if montelukast versus placebo added to hydroxyurea will improve forearm microvascular blood flow in adolescents and adults with sickle cell disease, respectively.

Funding Source - FDA OOPD

Arms:
arm 1: Oral montelukast therapy taken daily for eight weeks with current hydroxyurea regiment arm 2: Oral placebo taken daily for eight weeks with current hydroxyurea regiment

Interventions: intervention 1: Montelukast added to Hydroxyurea intervention 2: Placebo added to Hydroxyurea
Intervention descriptions: intervention 1: None intervention 2: None

Protocol document:
Prot_SAP_000.pdf: 
Montelukast Trial in Sickle Cell Anemia 
NCT01960413
October 10, 2016


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Montelukast Trial in Sickle Cell Anemia  
Protocol Version 10.0 
10/27/2016Principal Investigator: Michael DeBaun, MD, MPH and Joshua Field, MD, 
MS 
Sub-Investigators: Cheryl Hillery, MD, Pippa Simpson, PhD, Adetola Kassim, MD and 
Nancy Wandersee, PhD, Emmanuel Volanakis, MD 
Biostatistician: Pippa Simpson, PhD 
Study Coordinators: Zora Jovanovic, BSN, RN , Debora Nischik, RN Valencia Bryant, 
CRT  
Project Manager: Dionna O. Roberts, MS, MPH 
 
Conducted by: 
 Vanderbilt-Meharry Center for Excellence in Sickle Cell Disease, Nashville, Tennessee 
Froedtert Hospital Adult Sickle Cell Clinic, Milwaukee, Wisconsin 
 
IND# 76,997 
NCT #01960413 
 
 
 
 
 
 
 
 
 
 


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Background and Significance  
SCD is an autosomal recessive disorder that affects one of every 400 African-
American newborns in the United States1. The basis for SCD is a point mutation in 
the sixth codon of the β-globin gene. Under hypoxic conditions, the resulting 
hemoglobin S polymerizes inducing rigid, dense and deformed erythrocytes. In a 
multi-cellular process, sickle erythrocytes interact with non-sickle erythrocytes, 
leukocytes, platelets and endothelial cells causing recurrent microvascular 
occlusion, tissue ischemia and ultimately end-organ damage. Chronic vasculopathy 
is complicated by acute vaso-occlusive episodes, which are the pathogenic basis for 
the two most common morbidities in SCD, pain2 and acute chest syndrome (ACS)3 
episodes. Hydroxyurea is the only FDA-approved drug for preventing vaso-occlusive 
episodes and has become standard medical care for most adolescents and adults 
with SCD. 
 
The largest study examining the relationship between pain, ACS and asthma was 
performed in a cohort of 291 children identified from the Cooperative Study of Sickle 
Cell Disease (CSSCD)4. Of these children, 49 (16.8%) had a diagnosis of asthma 
noted in the record. Children with asthma had twice as many episodes of pain (1.39 
episodes per patient-year versus 0.47 episodes per patient-year, p<0.001) and twice 
as many episodes of ACS (0.39 v. 0.20 events per patient-year, p<0.001) when 
compared to children without asthma.   
 
In a separate study of 1,963 children and adults with SCD enrolled in the CSSCD, 
those with asthma and SCD died at younger ages when compared to individuals 
with SCD without asthma.  The median survival for individuals with asthma was 52.5 
years in comparison to 64.3 years in individuals without asthma. After controlling for 
previously identified risk factors for death5, individuals with asthma had a more than 
2-fold increase in risk of death (Hazard Ratio: 2.36 by Cox regression, 95% CI 1.21 
to 4.62, p=0.01).  Taken together, these data indicate that asthma is a significant 
cause of acute vaso-occlusive episodes and premature mortality.  Since asthma 
worsens the course of SCD, examining mechanisms that are important to asthma 
pathogenesis may provide therapeutic targets for individuals with SCD. 
 
Inhibition of the leukotriene pathway is an effective strategy for prevention of asthma 
symptoms, another pro-inflammatory disease.  Leukotrienes are a family of lipid 
mediators that cause bronchoconstriction and are involved in inflammatory 
responses. Two types of leukotrienes exist: 1) CysLTs – leukotrienes C4 (LTC4), D4 
(LTD4), and E4 (LTE4),-that are peptide-conjugated lipids and prominent products of 
activated eosinophils, basophils, mast cells and macrophages and 2) leukotriene B4   
(LTB4), a leukocyte chemoattractant and activator. CysLTs bind to receptors on 
vascular endothelial cells and bronchial smooth muscle.  In the vasculature, CysLTs 
mediate vasoconstriction 6, upregulation of cell adhesion molecules7 and increased 
vascular permeability8.  In the airway, CysLTs promote bronchoconstriction, airway 
remodeling, increased mucous production and smooth muscle proliferation 8-10.  


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Levels of CysLTs correlate with asthma severity11, asthma exacerbations12, allergen 
challenge 13,14 and exercise-induced bronchoconstriction15. 
 
LTB4 is a 5-lipoxygenase metabolite of arachidonic acid in neutrophils that acts as a 
chemoattractant and enhances neutrophil adhesion to endothelium. In a cross-
sectional study, Setty et al. assessed plasma levels of this metabolite in controls (n = 
9) and in individuals with SCD both in basal "steady state" (n = 37) and during 
episodes of pain (n = 10) and ACS (n = 5)16. LTB4 levels were markedly increased in 
patients with SCD in basal steady state (P <.003) compared with those in controls. 
Levels were further increased during pain (P <.04) and ACS (P <.0001) episodes. 
Taken together, these data suggest that the study of leukotriene pathogenesis and 
perhaps leukotriene receptor inhibition may dampen the anti-inflammatory state and 
lessen the impact of the co-morbid conditions associated with SCD.  
 
Inhibition of leukotriene activity is an anti-inflammatory target for asthma. 
Therapeutic benefit of the leukotriene receptor antagonist montelukast is well 
documented. Montelukast is associated with an increased exercise-induced 
bronchoprotection17, reduced asthma exacerbations18 and has no evidence of 
tolerance with prolonged use19. Further, montelukast has been well tolerated with 
relatively few reported toxicities20.  In contrast, the 5-lipooxygenase inhibitor, 
zileuton, has been associated with hepatotoxicity, limiting the utility of this drug21. 
 
Inhibiting the leukotriene pathway may attenuate vaso-occlusion by two potential 
mechanisms. First, blocking the actions of leukotrienes may decrease vascular 
inflammation by decreasing leukocyte migration and the expression of cell adhesion 
molecules on vascular endothelium. Second, inhibiting CysLTs may improve airway 
mechanics leading to better oxygen delivery to the microvasculature. Importantly, 
reducing vascular inflammation and improving airway mechanics are not mutually 
exclusive mechanisms and, if we show a benefit to montelukast therapy, both may 
contribute to clinical improvement. 
 
2. Study Objectives 
 2.1 
Primary Objective: To determine whether montelukast versus placebo 
added to hydroxyurea will improve markers of vaso-occlusion/tissue injury in 
adolescents and adults with SCD.  
 2.2 
Secondary Objectives:  
1) To determine if montelukast versus placebo added to hydroxyurea will 
improve lung function in adolescents and adults with SCD.  
2) To determine if montelukast versus placebo added to hydroxyurea will 
improve blood flow in adolescents and adults with SCD. 
3. Study Population 


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3.1 
Enrollment Inclusion Criteria:  
1) Diagnosis of HbSS, or HbSβ-thalassemia0, confirmed by hemoglobin 
analysis  
2) Males and females age 16 years to 70 years old 
3) Greater than 2 episodes of pain in the last 12 months 
4) On a stable dose of hydroxyurea for at least 2 months and a stable 
hemoglobin 
 
 
3.2 
 Enrollment Exclusion Criteria:  
 
1) Judged not likely to be study compliant by his/her hematologist  
2) History of adverse reaction to montelukast or any of the components of 
montelukast 
3) Have used medications known to interact with montelukast such as 
rifampin, phenobarbital, and gemfibrozil within 4 weeks of enrollment 
4) Currently being treated with a leukotriene antagonist (montelukast or 
zileuton) or have used montelukast/zileuton within the last 60 days   
5) Chronic blood transfusion therapy defined as regularly scheduled 
transfusions. 
6) Hemoglobin A greater than15% on hemoglobin analysis 
7) Individuals with a current physician diagnosis of asthma (within last 12 
months) or requires continuous supplemental oxygen, or predicted or 
current use of asthma medications (inhaled corticosteroids, but 
participants taking bronchodilators will be allowed to participate). 
8) Current participation in another therapeutic trial for SCD 
9) Known current pregnancy 
10) Known history of HIV 
11) Serum creatinine greater than 3 times the site’s upper limit of normal 
 
4. Trial Enrollment 
4.1 
Selection of Subjects 
Subjects will be recruited in Nashville and Milwaukee. The newly created 
Vanderbilt - Meharry Center for Excellence for SCD, currently follows about 
50 pediatric patients between 16 to 21 years of age and 150 adult patients 
between 21 to 70 years of age. The Center Director, M. DeBaun, also has a 
collaborative relationship with Tennessee Oncology, and they actively follow 
180 adults with SCD in the Middle Tennessee region and have agreed to refer 
patients for participation in this feasibility trial. The Froedtert Hospital Adult 
Sickle Cell Clinic in Milwaukee currently serves approximately 75 children (≥ 


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16 years) and 200 adults with HbSS/HbSβ-thalassemia0 between 21 to 70 
years of age.  
Clinical manifestations of sickle cell disease change with the age of the 
patient and as a result the subject’s age is taken into consideration for study 
participation. In regards to painful vaso-occlusive crises, the incidence rate 
increases with age and contributing factors to vaso-occlusive crises may be 
different at age extremes. We chose to exclude very young and older 
patients with SCD from our study to reduce the potential confounding effects 
of age.   
Currently there are few other trials available to this patient population and 
many of these individuals are currently on hydroxyurea. In our experience 
>50% of adult patients with HbSS/HbS β-thalassemia0 will have ≥ 3 pain 
episodes per year (including those that did not result in healthcare 
utilization). We recognize that this is a higher rate of pain than has been 
previously reported in the literature, however, the clinic populations in the 
academic medical centers at Nashville and Milwaukee provide care for 
individuals with more severe disease compared to community-based 
centers. For this phase II study, we conservatively estimate the dropout rate 
at 20%. However, we will use an intent-to-treat analysis and so have 50 for 
the primary outcome, but 63 for all analyses. Additionally, we will 
conservatively estimate that four patients will need to be approached for 
each one that participates (4:1 ratio). Therefore, we will plan to screen 252 
eligible patients and consent 63 individuals in order to accrue 50 study 
subjects. Based on data from Washington University in St. Louis, 35% of 
adults with HbSS/Sβ-thalassemia0 were prescribed hydroxyurea. Assuming 
similar rates of hydroxyurea administration in Milwaukee and Nashville and 
that all adults prescribed hydroxyurea will have >2 pain episodes per year, 
approximately 271 adolescents and adults with SCD will be available for 
screening. Decreasing the entry criteria to ≥ 2 pain episodes per year will 
further increase the number of potentially eligible patients. Randomization 
will occur after the 2 week lead-in period. Randomization will be done using 
the software BREEZE and will occur in blocks of 10 to allow for any potential 
seasonal differences. Individuals who complete 70% of diary days in the 
lead-in period will be eligible for randomization. Patients will be recruited in 
clinic with direct contact by the study physicians and/or study personnel. 
Patients may also be contacted with a phone call prior to scheduled visits to 
inform them of the study. 


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Definitions 
Baseline: The subject is well, reporting no more than baseline pain and at least 
30 days from a hospitalization or emergency department visit for any reason. 
Pain: An episode of acute pain with no cause other than a vaso-occlusive 
event lasting at least 24 hours that requires the administration of oral or 
parenteral opioids in a medical facility or by the patient25. 
 
4.2 
Study Design 
For this double blind placebo controlled phase II trial a total of 63 subjects will 
be recruited from the Sickle Cell Clinics at two national sites; Vanderbilt 
University in Nashville, Tennessee and Froedtert Hospital in Milwaukee.  A 
20% subject dropout rate is expected therefore it is anticipated approximately 
50 subjects will complete the study.   
 
We are investigating montelukast as an adjuvant therapy with hydroxyurea. 
Individuals with asthma will be excluded from this study because the benefit 
of montelukast for the treatment of asthma is well established22-24. Although 
the best therapy for individuals with SCD and asthma is not known, the 
present study is designed to evaluate the effect of montelukast on chronic 
vasculopathy and not on asthma-related morbidity. 
 
Study subjects will be randomized to receive either: 
 
• 
Oral montelukast therapy taken daily for eight weeks with current 
hydroxyurea regiment 
                      OR 
• 
Oral placebo taken daily for eight weeks with current hydroxyurea 
regiment 
 
 
After informed consent is obtained, subjects will begin the study activities. 
Subjects, family members, health personnel, study investigators and team 
members will be blinded to treatment allocation. The study 
pharmacy/pharmacy staff will be unblinded to treatment allocation. The DSMB 
with be unblended at the time of DSMB reporting by the biostatistician. 
Unblinding would only occur in cases of adverse events where unblinding is 
essential for subject safety and will be at the discretion of the principal 
investigator or DSMB. Subjects whose treatment is unblinded would be taken 
off study drug and would be discontinued from the study.  
 
5. Intervention 


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5.1 
Study Drug Formulation 
Montelukast 10 mg and matching placebo capsules will be supplied by the 
Vanderbilt University Medical Center Investigational Drug Service Pharmacy.  
Please see appendix 3.  
 
5.1.1  Packaging 
Montelukast and placebo will be supplied by Froedtert Hospital Investigational 
Drug Service for the Froedtert Hospital Adult Sickle Cell Clinic.  The 
Vanderbilt-Meharry Center for Excellence in Sickle Cell Disease will be 
supplied by Vanderbilt University Medical Center Investigational Drug Service 
Pharmacy.  Each bottle will contain 30 tablets of montelukast 10 mg capsules 
or placebo. 
 
5.1.2  Storage 
Store montelukast and placebo at room temperature, 25˚C (77˚F).  
Excursions permitted to 15-30˚C (59-86˚F).  Subjects will be instructed to 
keep the medication in the original container, tightly closed, and out of the 
reach of children. 
 
5.1.3  Administration 
Montelukast 10 mg or placebo will be taken once daily at approximately the 
same time every day, with or without food.  Subjects will continue to take 
montelukast or placebo daily for 8 weeks.  Each subject will receive 2 bottles 
of montelukast or placebo.  One bottle dispensed on day 0 and one bottle 
dispensed at week 4. Hospitalizations for pain or ACS are expected outcomes 
in SCD trial. Montelukast/Placebo will not be stopped for hospitalizations 
unless a causal relationship is suspected by the PI. If the subject is 
hospitalized or records an acute pain episode within two weeks of completion 
of study drug the final lab measurements will be delayed for an additional four 
weeks and subject will continue to take the study drug for the additional two 
weeks. 
 
5.1.4  Compliance Monitoring 
The study coordinator will inventory the subject’s usage of montelukast or 
placebo during study visits at weeks 4, 7 and 8.  The study coordinator will 
attempt to contact the subject by phone daily Monday through Friday to 
remind and encourage subjects to take study drug and complete the diary. 
 
5.1.5  Drug Accountability and Ordering 
Drug accountability logs will be maintained for all study drugs.  At a minimum, 
these logs must contain quantity of drug received and dispensed, date 
received and dispensed, subject number, expiration date, dose, quantity 
returned, balance forward, and initials of individual dispensing the study drug.  
Used medication vials can be discarded according to institutional standards.  


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Fax completed order form to Froedtert Hospital Investigational Drug Service, 
 for the Froedtert Hospital Adult Sickle Cell Clinic. Contact the 
Vanderbilt University Medical Center Investigational Drug Service Pharmacy 
at 
 for the Vanderbilt-Meharry Center for Excellence in Sickle 
Cell Disease.  Allow 5 business days for shipment.   
 
        
 
5.1.6 Prohibited Concomitant Medications 
Concomitant use of rifampin, phenobarbital, or gemfibrozil should be avoided 
during the study. 
 
The use of a leukotriene inhibitor or any other investigational agent is 
prohibited during the treatment period of this study. 
 
At each clinic visit after screening, the investigator will ask the patient whether 
any medications (other than study drug), including over-the-counter 
medications, were taken since the previous visit. 
 
 
5.2 
Montelukast Diary 
The research coordinator will pre-fill the subject’s opioid medications and 
medication amounts into the diary.  
Study subjects will be asked to complete the following daily: 
• List the date and day of the week 
• Rate their maximum daily pain score on a scale of 0-9 (0 is 
no pain and 9 is greatest pain) 
• Complete the questions on how pain has affected their 
general activity  
• Record daily opioid use  
 
5.3 
Pulmonary Function Test 
Spirometry, diffusion capacity of carbon monoxide (DLCO), and lung volumes 
will be tested with according to American Thoracic Society criteria26,27.  We 
will also obtain pre- and post-bronchodilator measurements (Albuterol, 4 puffs 
from a metered dose inhaler administered by an aerochamber).  A calibrated 
spirometer will be used. While seated, the subject will be encouraged to 
perform between 5 and 8 maneuvers to obtain 3 acceptable tracings. Up to 
five maneuvers will be repeated after inhalation of an Albuterol inhaler. The 
adequacy of the blows will be judged using the following Epidemiologic 


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Standardization Criteria: a minimum duration of 6 seconds, back extrapolated 
volume < 5%, FVC within 5% or 200cc (whichever is less), and assessment 
by the technician as to the adequacy of the effort.  For lung volumes, patients 
will be tested with body plethysmography. To ensure American Thoracic 
Society criteria are met across the two participating sites for spirometry, test 
results will be over-read by a single pulmonologist.  
 
 
5.4 
Blood Measurements 
Blood samples for inflammatory markers will be collected at the following 
study time points: 
 
• Pre-randomization 
• Randomization 
• Week 4  
• Week 8   
 
Whole blood samples will be collected in a 5mL light blue top citrate tube. 
Samples will be centrifuged at 1500 g for 15 minutes. Plasma will be 
aliquoted into four eppendorf tubes (about 600L each) and immediately 
frozen at –80oC as per a routine coagulation reference sample. Samples will 
be batched and stored frozen at -80oC until shipped. Samples will be shipped 
on dry ice overnight by Fed-X to the laboratory of Dr. Nancy Wandersee, 
Sickle Cell Disease Laboratory, 
 Milwaukee, 
Wisconsin, 53226 for testing.  
 IL-1, IL-6, TNFα and IFN- will be measured with the human-specific Bio-
Plex cytokine assay kit. To determine plasma levels of the human-specific 
soluble VCAM-1, soluble E-selectin, and soluble P-selectin, ELISA kits 
DVC00, BBE2B, and BBE6, will be used respectively,   
 
5.5 
Urine Analysis 
 A urine specimen will be collected at the following study time point: 
 
• Pre-randomization 
• Randomization 
• Week 8 
 
Subject will provide a clean catch urine sample by standard methods of urine 
collection that are non-invasive and do not require catheterization. A 
maximum of 4 ml of urine will be saved and processed within 2 hours of 
collection.  Urine will be centrifuged to remove particulates. Urine specimen 


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will be aliquoted and frozen at -80oC. Samples will be batched and stored 
frozen at -80oC until shipped. Samples will be shipped on dry ice overnight by 
Fed-X to the laboratory of Dr. Nancy Wandersee, Sickle Cell Disease 
Laboratory, 
 Milwaukee, Wisconsin, 53226for 
testing.  
Urine leukotriene E4 levels will be quantified by liquid chromatography-mass 
spectrometry. Samples will be analyzed by using LC-ESI-MS (Agilent 1100 
LC/MSD, SL model).  For quantitative measurements, the m/z = 440 will be 
used for LTE4 and the m/z = 445 is used for [2H5]-LTE4 (the internal standard, 
at retention time of 13.38 min), respectively. The detection limit is 5 pg of 
LTE4. The standard curves will be constructed over the range of 5 pg to 2500 
pg of LTE4 per injection. The concentrations of LTE4 in the samples will be 
calculated by comparing the ratios of peak areas LTE4:[2H5]-LTE4 to the 
standard curves. 
 
5.6 
 Six-Minute Walk 
The six-minute walk test will be performed in accordance with a modified 
version of the American Thoracic Society Six-Minute Walk Guidelines as 
listed in Appendix One.  Subjects will be asked to walk at a brisk pace for 6 
minutes along a marked 30 meter path. Subjects will be asked to assess their 
fatigue level and shortness of breath using the Borg scale in Appendix Two 
prior to the 6 minute-walk and immediately after completion of the 6 minute- 
walk. 
 
5.7 
Unblinding 
Investigators will remain blind to treatment assignment until the entire study is 
complete.  Only research pharmacist (and designated pharmacy staff) at each 
study site, the DSMB and the designated study statistician at Medical College 
of Wisconsin should be aware of the treatment that is assigned to each 
subject. However, concerns regarding serious toxicity or safety may arise, 
prompting unblinding to become necessary.  
 
 
If an investigator at a participating institution deems it necessary to unblind, it 
must be submitted in writing to the Principal Investigator, Michael DeBaun, 
MD, MPH with the reason. Requests for unblinding should also be submitted 
to the Study Coordinator.  If the PI agrees, the request will be forwarded to 
the study statistician by e-mail. The treatment assignment is then relayed to 
the Study Coordinator who communicates with the site. It will be the 
responsibility of each institution to withdraw the participant from the study and 
complete a Date Off Treatment/Off Study Form with the date the patient is 
unblinded as the date off-treatment. 
 


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If a participant needs to be unblinded for urgent clinical care, the investigator 
or the site monitor physician at the participating institution may withdraw the 
participant from the study and the PI can find out the participant’s treatment 
assignment from the respective pharmacy. The investigator must notify the 
Principal Investigator, Michael DeBaun, MD, MPH, in writing within one week 
of the unblinding and withdrawal of the subject from the study. 
 
5.8  
Discontinuation Criteria 
Subjects may be removed from the study, or treatment stopped by the 
Investigator for any of the following reasons: 
1. Occurrence of CTCAE Criteria of grade 3 or higher that are deemed 
attributable to the drug per the opinion of the PI. 
2. . Subject makes the decision to withdraw.  
3. . PI decides to withdraw the subject due to noncompliance.  
4. Withdrawal by the PI or subject because of treatment side effects or 
complications.  
5. The subject’s study treatment is unblinded.  
 
6. Study Procedures  
 
 
6.1 
Pre-randomization (two weeks prior to randomization, plus or minus 
3 days) 
• Collection of subject’s demographics 
• Subject is instructed on how to complete Montelukast diary and is 
given written directions for diary. Instruct subject to bring diary to all 
clinic visits 
• Education for Montelukast diary reviewed 
• Montelukast diary pain section completed daily by subject with first 
entry completed in clinic with study staff 
• Urine pregnancy test will be performed on women of child bearing 
age 
• CBC with differential that include: 
✓ White blood cell count 
✓ Hemoglobin 
✓ Hematocrit 
✓ Mean corpuscular volume (MCV) 
✓ Platelet count 


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✓ Eosinophil count  
• Blood Chemistry  
✓ Creatinine 
• Laboratory  blood measures that include: 
✓ Reticulocyte count 
✓ Lactate dehydrogenase  (LDH) 
• Lab Sample for the following inflammatory mediators sent to Dr. 
Wandersee’s Lab: 
✓ sVCAM-1 
✓ sP-selectin 
✓ IL-1, IL-6 
✓ TNFα  
✓ IFN-  
• Urine specimen collected for send out analysis of LTE4 
• Study staff determines eligibility status by completing Eligibility 
Study form 
• If eligible subject is randomized by: 
✓ Study staff sends eligibility form to pharmacy with assigned 
unique study ID number  
✓ Pharmacy will be supplied with study drug kits for both 
treatment arms and will assign study drug kit to subject 
✓ Pharmacist will be unblinded to study treatment assignment 
Pharmacist will confirm randomization by returning signed 
Eligibility form to study staff to confirm randomization 
 
 
6.1.1 Randomization Criteria 
After the consent is signed study subjects will record a daily pain score, and 
daily opioid use, in the Montelukast diary during the pre-randomization period. 
If the subject completes 70% of the Montelukast diary entries during the pre-
randomization period and also meets study eligibility criteria then the subject 
will be considered eligible for randomization.  
 
6.1.2 Hospitalization during Pre-randomization 
If the subject is hospitalized for a pain crisis prior to randomization then study 
participation is discontinued. Two weeks after the pain crisis resolves subjects 
may be re-screened and restart the study if eligibility criteria are met. If 
greater than 30 days has passed since the date the consent was signed the 
individual will be re-consented. 
 
6.2 
Randomization and Procedures (Day 0)  
 
• Montelukast diary reviewed by study staff for compliance in order to 
determine if eligible to be randomized 


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After confirmation of randomization the following procedures will be 
performed: 
• Urine pregnancy test will be performed on all women of child 
bearing age 
• Physical examination 
• Collection of medical history  
• Montelukast questionnaire completed 
• Concomitant medications reviewed by study staff 
• All laboratory blood measurements including inflammatory 
mediators for  Pre-randomization are repeated 
• Urine specimen collected for send out analysis of LTE4 
• Pulmonary function test 
• Six minute walk  
• 4 week supply of montelukast or placebo dispensed to subject 
• Subject takes and records first dose of montelukast or placebo, 
records daily pain score, opioid usage, in Montelukast Diary in clinic 
to demonstrate understanding of diary usage 
• The first dose of montelukast or placebo is to be taken one hour 
(but no longer than 90 minutes) prior to the pulmonary function test 
• Subject instructed to bring diary and study drug to each visit 
 
6.3 
Week 4 (post day of randomization plus or minus 3 days) 
• Study staff monitors Montelukast diary and inventories study drug 
for compliance  
• Assessment  for toxicities by physician/ nurse practitioner  
• Physical examination 
• Collection of medical history  
• All laboratory blood measurements including inflammatory 
mediators for pre-randomization are repeated (see pre-
randomization) 
• Urine pregnancy test will be performed on all women of child 
bearing age 
• Concomitant medications reviewed by study staff 
• 4 week supply of montelukast/placebo dispensed to subject 
• Subject continues recording daily pain, opioid usage, and  records 
receipt of study medication in Montelukast Diary 
 
 
6.4 
Week 7 (post day of randomization plus or minus 3 days) 
• Study Staff monitors Montelukast diary and inventories study drug 
for compliance 


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• Subject continues recording daily pain, opioid usage, and  records 
receipt of study medication in Montelukast Diary  
• Concomitant medications reviewed by study staff 
• Collection of medical history 
 
 
6.5 
End of Active Study Visit Week 8 (post day of randomization plus or 
minus 3 days) 
•  
• Study coordinator collects Montelukast  diary and study drug 
container from subject 
•  Assessment for study toxicities  
• Physical examination  
• Collection of medical history 
• All laboratory blood measurements including inflammatory 
mediators for pre-randomization are repeated (see pre-
randomization) 
• Urine pregnancy test will be performed on all women of child 
bearing age 
• Pulmonary function test 
• Six minute walk  
• Urine specimen collected for send out analysis of LTE4 
 
6.6  Post-study Follow up Period (weeks 9-12 plus or minus 3 days) 
• Subjects will be contacted by phone weekly (plus or minus 3 days) 
for 4 weeks following the conclusion of montelukast or placebo 
therapy to assess for any potential toxicities related to study drug  
 
6.7  
Hospitalizations during active drug treatment 
 
In the event that a subject is hospitalized for a sickle cell pain crisis prior to 
the end of the 8-week treatment period, the study endpoint will be extended 
for 2 additional weeks, post-inpatient discharge, during which time the study 
subject will continue to take montelukast/placebo. In this case, all 
aforementioned tests to be performed at weeks 7 and 8 will be rescheduled to 
weeks 9 and 10, respectively.  
 
 
7.  Subject Remuneration 
 
For their time and efforts in study activities, research subjects will receive $50 for each 
of the following patient visits: 
• Pre-randomization  
• Day of randomization visit 


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• Study week 4 visit 
• Study week 7 visit 
• End of active study week 8 visit 
 
A total of $250.00 will be paid to subjects completing all study visits.  
 
7.1 
Potential benefits to subjects 
There may be no direct benefit to subjects for taking part in the study; 
however, it may benefit others with SCD.  Novel therapies for the prevention 
of pain episodes in adults with SCD are desperately needed. Hydroxyurea 
therapy was a major advance for the treatment of SCD-related morbidity; 
however, many adults continue to suffer repeated episodes of pain despite 
hydroxyurea therapy.  Abundant pre-clinical and clinical data suggests that 
CysLT contribute to the pathogenesis of painful episodes and preliminary 
data suggesting that montelukast may be an effective preventative therapy. 
This phase II trial may lead to a larger, multi-center, phase III trial if 
montelukast has some efficacy for improving markers of vaso-occlusion. 
 
 


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Table 1. Study Schedule of Procedures  
Procedure 
Pre-Rand./ 
Week -2 
Randomization 
(Day 0) 
Daily 
Week 4 
Week 7† 
Week 8† 
Montelukast Diary 
X 
X 
X 
X 
X 
X 
Urine pregnancy test will 
be performed on women of 
child bearing age 
X 
X 
 
X 
 
X 
Six Minute Walk 
 
X 
 
 
 
X 
Physical Examination 
 
X 
 
X 
 
X 
Documentation-medical 
history, medications, 
transfusions,  & clinical 
data  
 
X 
 
X 
X 
X 
Montelukast Questionnaire 
 
X 
 
 
 
 
Blood draw- routine labs,*  
blood chemistry & 
inflammatory mediators** 
X 
X 
 
X 
 
X 
Urine analysis of LTE4 
X 
X 
 
 
 
X 
Pulmonary Function Test 
 
X 
 
 
 
X 
Coordinator  calls subject 
& records  (Mon-Fri) 
administration of  study 
drug  
 
X†† 
X 
X 
X 
X 
Subject records opioid use 
in montelukast diary 
 
X 
X 
 
 
 
Coordinator inventories 
drug usage and monitors 
diary compliance 
 
X 
 
X 
X 
X 
Montelukast/Placebo 
dispensed to subject 
 
X 
 
X 
 
 
*CBC, Reticulocyte count, eosinophil count, LDH 
** sVCAM-1, sP-selectin, IL-1, IL-6, TNFα and IFN- 
† delayed by 2 weeks, post-discharge, if hospitalized for 
sickle cell pain crisis 
†† First montelukast/placebo dose to be taken and recorded in clinic


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Follow-up procedures Subjects will be contacted by phone weekly (plus or minus 3 days) for 4 weeks 
following the conclusion of montelukast or placebo therapy to assess for potential toxicities  
 
 
8.  Human Subjects Protection 
 
8.1 
Informed Consent: Subjects are required to sign an informed consent prior 
to screening, and before undergoing any study procedures or assessments, 
in accordance with ICH E6; 4.8, “Informed Consent of Trial Subjects.”  When 
substantial modifications are made to the informed consent, the IRB may 
require that all subjects currently enrolled in the study will be re-consented; 
ICH E6; 4.8 guidelines would still apply.   
 
Subjects will be provided with a copy of the signed informed consent form 
used in the study, procedures, and assessments.  Subjects will also be 
provided with the contact telephone numbers of the investigator and qualified 
personnel who can assist with their questions and concerns.   
 
8.2  
Protected Health Information 
 
Protected health information that will be collected as part of this study 
includes name, date of birth and dates for hospitalization.  This information 
will be stored for 10 years beyond the completion of the study. After blood is 
used for study testing it will be discarded.  
 
8.3 
Confidentiality 
Loss of confidentiality is always a potential risk when collecting protected 
health information. Research records will remain confidential. Subject’s 
records will be available to the study staff and to each site’s IRB and for audit 
purposes. Only authorized personnel will have access to the protected health 
information and research records.  In order that confidentiality can be 
maintained, the PI/study staff will keep records in locked cabinets/room and 
results of tests will be coded to prevent association with volunteers’ names. 
All electronic data will be entered into a secured, website data management 
system that requires training and is password protested. Identifying 
information will not be included on laboratory samples, faxes, or 
correspondence. Laboratory samples will be shipped with only unique study 
numbers that will be linked to a key kept by the site PI.   
All study team members are trained in HIPAA privacy regulations and other 
applicable site privacy policies. No information will be released, nor will 
participation in the research be acknowledged, to any party except where 
compulsory according to law or intuitional policy. The results of the research 


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study may be published, but volunteers’ names or identities will not be 
revealed.  
 
9. Adverse Event Monitoring 
 
9.1 
Definitions 
 
Adverse Event (AE) – Any untoward or unfavorable medical occurrence in a 
human subject, including any abnormal sign (for example, abnormal physical 
exam or laboratory finding), symptom, or disease, temporally associated with 
the subject’s participation in research, whether or not considered related to 
the subject’s participation in research. Pain episodes documented in the diary 
will not be reported as adverse events. 
 
Serious Adverse Event (SAE) – Any adverse event temporally associated 
with the subject’s participation in research that meets any of the following 
criteria: 
• Results in death; 
• Is life-threatening (places subject at immediate risk of death from 
the event as it occurs); 
• Requires inpatient hospitalization; 
• Results in a persistent significant/incapacity; 
• Results in a congenital anomaly/birth defect; or 
• Any other adverse event that, based upon appropriate  medical 
judgment, may jeopardize the subject’s health and may require 
medical or surgical intervention to prevent on the other outcomes 
listed in this definition 
Note that seriousness and severity are separate concepts. The term “severe” 
refers to the intensity of a specific event; a severe event may be of minor 
medical significance (e.g., a severe leg cramp). The term “serious” is based 
on an outcome or action criteria that are usually associated with events that 
pose a threat to the patient’s life or functioning. An event that is mild in 
severity is serious if it leads to one of the outcomes defined above. 
 
Grade 4 and 5 events will always be considered Serious Adverse Events. 
Many Grade 3 and some Grade 1 and 2 events may meet the definition of a 
Serious Adverse Event. 
 
Unexpected Adverse Event- Any adverse event occurring in one or more 
subjects in a research protocol, the nature, the severity, or frequency of which 
is not consistent with either: 
• The known of foreseeable risk of adverse events associated 
with the procedures involved in the research that are described 
in; 


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o  the protocol related documents or the current IRB-
approved informed consent document and;  
o other relevant sources of information, such as a product 
labeling or package insert or; 
• The expected natural progression of any underlying disease, 
disorder, or condition of the subject(s) experiencing the adverse 
event and the subject’s predisposing risk factor profile for the 
adverse event. 
 
Unanticipated problem involving risks to subjects or others (UP) - Any incident, 
experience or outcome that meets all the following criteria: 
• Unexpected (in terms of nature, severity, or frequency) given: 
o The research procedures that are described in the protocol 
related-documents, such the IRB-approved research 
protocol and the informed consent form document and, 
o The characteristics of the subject population being 
studied;  
• Related or possible related to the subject participation in 
research; and 
• Suggests that the research places subjects or others at a 
greater risk of harm (including physical, psychological, 
economic, or social harm) related to the research than was 
previously known or recognized. 
 
Grading of Adverse Events – Events will be graded by using the Common 
Terminology Criteria for Adverse Events (CTCAE) Criteria version 4.0. 
 
Attribution – the determination and documentation of whether an adverse 
event is related to a medical procedure. 
 
Attribution Categories: 
1. Not Related –Event clearly related to other factors (e.g., 
clinical state, other therapies; concomitant drugs) 
2. Possible Related – Sequence of event is compatible with 
study drug, device, or procedure, but could have been 
produced by other factors 
3. Probably Related - Sequence of event is compatible with 
study drug, or procedure and cannot be explained by other 
factors without much doubt 
4. Definitely Related - Sequence of event is compatible with 
study drug, or procedure and beyond doubt cannot be 
explained by other factors 
 
 


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9.2 
Study Staff 
Information on all adverse events, whether reported by the subject, directly 
observed, or detected by physical examination, laboratory test or other 
means, will be collected, recorded, followed, and reported as described in the 
following sections.  
The PI will monitor and review adverse events monthly or more often as 
needed.   
 
9.3 
Timeline for Reporting of Adverse events, Serious Adverse Events, 
Unexpected Adverse Events and Unanticipated Problems 
  
  
All serious adverse events, all unexpected adverse events, and all 
unanticipated problems will be reported to the DSMB within 24 hours of 
learning of the event’s occurrence/or within 24 hours of the business day by 
phone, email, or SAE form regardless of attribution to the study drug.  
 
 
9.4 
Reporting Events to Local Institutional Review Boards 
All serious adverse events will be reported to the institutional review board 
(IRB). Each site will comply with their institution’s standard operating 
procedures for reporting adverse events. Subjects will be instructed to report 
any adverse event(s) during the study and post-study .The investigator will 
notify the IRB and any other applicable regulatory agency of any 
unanticipated death or adverse event occurring after a participant has 
discontinued or terminated study participation that may reasonably be related 
to the study.  
 
9.5 
Data Safety Monitoring Board (DSMB) 
The principal investigator will appoint three outside physicians and one local 
biostatistician from the MCW site with research experience as members of 
the DSMB group. They will monitor accruing data in order to confirm that the 
patients in the trial are being cared for safely. The DSMB will be responsible 
for:  
1) Reviewing and analyzing the progress of the study;  
2) Approving amendments to the trial protocol, if warranted;  
3) Monitoring the safety of the study treatments and diagnostic 
procedures;  
4) Ensuring data quality;  


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5) Reviewing interim analyses and recommending early stopping or 
continuation of the trial; and  
6) Reviewing recruitment and event rates.  
7) Provide a summary of their findings in a written report 
 
The DSMB will meet every 6 months by phone, or in person, or more often if 
warranted to review serious adverse events. 
 
Local Data Safety Monitoring Plan -The local principal investigator and/or 
members of the research team will monitor the study for inclusion/exclusion 
criteria, accrual/withdrawal rates, and breech of confidentially annually prior to 
submission to the IRB. Any adverse events will be reviewed immediately with 
the Principal Investigator and will be reported to the local respective IRB per 
site IRB regulations. All study events will be summarized once a year for the 
IRB, during annual continuing review progress report. 
 
 
9.6 
Potential Risks to Volunteer 
 
Likely:   
Blood Sampling: Venipuncture can cause pain and discomfort at the needle 
puncture site. 
Spirometry:  Breathing out hard and fast can cause coughing, mild chest 
discomfort or lightheadedness.   
Albuterol: CNS stimulation can cause restlessness, irritability, and 
nervousness
 
Less Likely: 
Blood Sampling: Venipuncture can cause, bruising, fainting or local infection 
at the needle site. 
Montelukast: Upper respiratory infection, fever, headache, pharyngitis, 
cough, abdominal pain, diarrhea, otitis media, influenza, rhinorrhea, sinusitis 
Albuterol:  Hypertension, tremor, Insomnia, headache, muscle cramps, 
irritation of the oropharynx (hoarseness). 
 


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Rare:  
Blood Sampling: In rare circumstances venipuncture can cause blood clots, 
peripheral nerve injury or an arterial puncture.   
In addition to the above possible risks, there is always the risk of developing 
previously unknown side effects. 
Montelukast: Neuropsychiatric events including: 
• 
Aggression 
• 
Hostility 
• 
Anxiousness 
• 
Disorientation 
• 
Insomnia 
• 
Depression 
• 
Restlessness 
• 
Irritability 
• 
Dream abnormalities 
• 
Somnambulism 
• 
Hallucinations 
• 
Suicidal thinking  
• 
Behavior changes 
• 
Tremor 
• 
Death 
Albuterol:  
• 
Paradoxical bronchospasm 
• 
Hypersensitivity reaction (including urticaria, angioedema, rash) 
• 
Hypokalemia 
• 
Arrhythmias/ Angina  
• 
fast, pounding, or irregular heartbeat 
• 
chest pain 
 
 


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10. Data 
10.1 Data Management 
The data gathered will be entered into REDCap data system with each ID 
having a unique identifier for each participant. REDCap is a secure, web-
based application for building and managing online databases. All data will be 
de-identified and samples will only have a unique identifier that will link the 
unique identifier with the patient’s name.  
The REDCap clinical database will be maintained by the Department of 
Quantitative Health Sciences, at the Children's Research Institute under the 
direction of Pippa Simpson, PhD.    
10.2 Data Collection 
Research information, including consent forms and Montelukast Daily Diary 
will be maintained secure as described in the Confidentiality section 8.3. 
 Each participant will be identified with a unique identifier number in REDCap. 
This unique identifier will be linked to the same unique identifier for a 
biological specimen sample stored in Dr. Nancy Wandersee’s SCD Lab. 
 
11. Statistical Considerations 
11.1    Description of Endpoints: 
11.1.1 Primary Endpoint: Our primary endpoint is a 30% reduction in sVCAM.  
The study will conclude 8 weeks after initiation of montelukast or placebo 
treatment, at which time markers of vaso-occlusion/tissue injury will be 
compared to baseline in individuals with SCD.  
11.1.2 Secondary Endpoints:  
1) Other inflammatory markers (sP-selectin, sE-selectin, IL-1, IL-6, 
TNFα, IFN- and LTE4) 
2) Daily pain score 
3) Proportion of pain free days to days with pain 
4) Opioid use, as reported in the diary 
5) Pulmonary function as measured by FEV1.  
6) Six-minute walk 


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11.2 Statistical Analysis 
 
Summary statistics such as mean, median, standard deviation, range and 
correlation and plots will be used to examine distributions and 
interrelationships. Where necessary, for parametric assumption, we will 
employ appropriate transformations with justifications. For example, the 
log of some of the biomarkers data may be used since cytokine growth 
tends to be multiplicative. We will model relationships using regression 
trees and regression models (linear and non linear). We will use random 
effects model to investigate changes over time, where patient is random. 
Initially we will use an auto correlation structure for the variance 
covariance matrix but will explore other structures. In models covariates 
such as group, age, gender, WBC, hemoglobin, % fetal hemoglobin and 
interactions of group with other covariates will be used.  Statistical 
software will be employed for data analysis: SAS version 9.13, Salford 
System CART for trees, and SPSS Version 18.0. We plan to accrue 25 
participants in each arm. From preliminary data, the mean sVCAM level is 
353 ng/ml with a standard deviation of 104 ng/ml. If we have similar 
variability in the proposed study, we will be able to detect a change of ≥ 62 
ng/ml (or 18%) with 80% power. Our primary outcome measure is based 
on a 30% reduction, which would be ~106 ng/ml reduction. The study was 
designed with 25 in each group in order to explore all three aims and 
potential confounders. The 95% confidence interval for detecting a 30% 
difference is between 204 ng/ml and 290 ng/ml (or an 18-42% reduction in 
sVCAM). Importantly, the lower limit of the 95% confidence interval (18%) 
is still, in our opinion, a clinically relevant reduction in sVCAM. Thus, if we 
detect a 30% or larger difference in sVCAM in this study, we will be 
assured that, based on the 95% confidence interval, these data are 
clinically important28.    
 
11.3 Follow-up and Record Retention 
 
The current proposal involves on-going data collection for the duration of the 
study at each site. The records will be maintained per site policy. 
 
 
 
 
 
 
 


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References 
1. 
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Castro O, Brambilla DJ, Thorington B, et al. The acute chest syndrome in sickle 
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Boyd JH, Macklin EA, Strunk RC, DeBaun MR. Asthma is associated with acute 
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Rabinovitch N, Zhang L, Gelfand EW. Urine leukotriene E4 levels are associated 
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Lynch KR, O'Neill GP, Liu Q, et al. Characterization of the human cysteinyl 
leukotriene CysLT1 receptor. Nature. 1999;399:789-793. 
11. 
Centers for Disease Control. Asthma prevalence and control characteristics by 
race/ethnicity -- United States, 2002. MMWR. 2004;53:145-148. 
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Chavis C, van Vyve T, Chanez P, et al. Leukotriene E4 plasma levels in adult 
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Manning PJ, Rokach J, Malo JL, et al. Urinary leukotriene E4 levels during early 
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Kumlin M, Dahlen B, Bjorck T, Zetterstrom O, Granstrom E, Dahlen SE. Urinary 
excretion of leukotriene E4 and 11-dehydro-thromboxane B2 in response to 
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15. 
Kikawa Y, Miyanomae T, Inoue Y, et al. Urinary leukotriene E4 after exercise 
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Setty BN, Stuart MJ. Eicosanoids in sickle cell disease: potential relevance of 
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17. 
Kemp JP, Dockhorn RJ, Shapiro GG, et al. Montelukast once daily inhibits 
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Knorr B, Matz J, Bernstein JA, et al. Montelukast for chronic asthma in 6- to 14-
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Villaran C, O'Neill SJ, Helbling A, et al. Montelukast versus salmeterol in patients 
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Bisgaard H. Leukotriene modifiers in pediatric asthma management. Pediatrics. 
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Mansuy D. Molecular structure and hepatotoxicity: compared data about two 
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Reiss TF, Chervinsky P, Dockhorn RJ, Shingo S, Seidenberg B, Edwards TB. 
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Noonan MJ, Chervinsky P, Brandon M, et al. Montelukast, a potent leukotriene 
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Montelukast Asthma Study Group. Eur Respir J. 1998;11:1232-1239. 
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Laviolette M, Malmstrom K, Lu S, et al. Montelukast added to inhaled 
beclomethasone in treatment of asthma. Montelukast/Beclomethasone Additivity 
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25. 
Ataga KI, Reid M, Ballas SK, Yasin Z, Bigelow C, James LS, Smith WR, 
Galacteros F, Kutlar A, Hull JH, Stocker JW; ICA-17043-10 Study Investigators. 
Improvements in haemolysis and indicators of erythrocyte survival do not 
correlate with acute vaso-occlusive crises in patients with sickle cell disease: a 
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channel blocker senicapoc (ICA-17043). Br J Haematol. 2011 Apr;153(1):92-104. 
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Standardization of Spirometry, 1994 Update. American Thoracic 
Society. Am J Respir Crit Care Med. 1995;152:1107-1136. 
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American Thoracic Society. Single-breath carbon monoxide diffusing capacity 
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28. 
Goodman SN, Berlin JA. The use of predicted confidence intervals when 
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Intern Med. 1994;121:200-206. 
29. 
Amercian Thoracic Society. Official statement of the ATS Guidleines for the Six-
Minute Walk Test AmJ Respir Crit Care Med 2002;166:111-117. 
 
 
 
 
 
Appendix One 


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Six Minute Walk 
Location 
The 6MWT should be performed indoors, along a long, flat, straight, enclosed corridor 
with a hard surface that is seldom traveled. The walking course must be 30 meters in 
length. A 100-ft hallway is required. The length of the corridor should be marked every 3 
meters. The turnaround points should be marked with a cone. A starting line, which 
marks the beginning and end of each 60-m lap, should be marked on the floor using 
brightly colored tape.  
Required Equipment  
1. Countdown timer or stopwatch 
2. Two small cones to mark the turnaround points  
3. A chair that can be easily moved along the walking course  
4. Worksheets on a clipboard  
5. Meter tape measure and colored tape 
Subject Preparation  
1. Comfortable clothing should be worn.  
2. Appropriate shoes for walking should be worn.  
3. Subjects should use their usual walking aids during the test (cane, walker, etc.).  
4. Subjects should not have exercised vigorously within 2 hours of beginning the 
test.  
Measurements  
1. A "warm-up" period before the test should not be performed.  
2. The subject should sit at rest in a chair, located near the starting position, for at 
least 10 minutes before the test starts. During this time, check for 
contraindications, measure pulse and blood pressure, and make sure that 
clothing and shoes are appropriate. Complete the first portion of the worksheet.  
3. Have the subject stand and rate their baseline dyspnea and overall fatigue 
using the Borg scale.  
4. Set the timer to 6 minutes. Assemble all necessary equipment (timer, 
clipboard, Borg Scale, worksheet) and move to the starting point.  
5. Instruct the subject as follows:  
"The object of this test is to walk as far as possible for 6 minutes. You will walk back and 
forth in this hallway. Six minutes is a long time to walk, so you will be exerting yourself. 
You will probably get out of breath or become exhausted. You are permitted to slow 


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down, to stop, and to rest as necessary. You may lean against the wall while resting, but 
resume walking as soon as you are able.  
You will be walking back and forth around the cones. You should pivot briskly around 
the cones and continue back the other way without hesitation. Now I'm going to show 
you. Please watch the way I turn without hesitation."  
Demonstrate by walking one lap yourself. Walk and pivot around a cone briskly.  
Remember that the object is to walk AS FAR AS POSSIBLE for 6 minutes, but don't run 
or jog.  
Start now or whenever you are ready.  
1. Position the subject at the starting line. You should also stand near the starting 
line during the test. Do not walk with the subject. As soon as the subject starts 
to walk, start the timer.  
2. Do not talk to anyone during the walk. Use an even tone of voice when using 
the standard phrases of encouragement. Watch the subject. Do not get 
distracted and lose count of the laps. Each time the participant returns to the 
starting line, count the lap on the worksheet. Let the participant see you do it.  
After the first minute, tell the subject the following (in even tones): "You are doing well. 
You have 5 minutes to go."  
When the timer shows 4 minutes remaining, tell the subject the following: "Keep up the 
good work. You have 4 minutes to go."  
When the timer shows 3 minutes remaining, tell the subject the following: "You are 
doing well. You are halfway done."  
When the timer shows 2 minutes remaining, tell the subject the following: "Keep up the 
good work. You have only 2 minutes left."  
When the timer shows only 1 minute remaining, tell the subject: "You are doing well. 
You have only 1 minute to go."  
Do not use other words of encouragement (or body language to speed up).  
If the subject stops walking during the test and needs a rest, say this: "You can lean 
against the wall if you would like; then continue walking whenever you feel able." Do not 
stop the timer. If the subject stops before the 6 minutes are up and refuses to continue 


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(or you decide that they should not continue), wheel the chair over for the subject to sit 
on, discontinue the walk, and note on the worksheet the distance, the time stopped, and 
the reason for stopping prematurely.  
When the timer is 15 seconds from completion, say this: "In a moment I'm going to tell 
you to stop. When I do, just stop right where you are and I will come to you."  
When the timer rings (or buzzes), say: "Stop!" Walk over to the subject. Consider taking 
the chair if they look exhausted. Mark the spot where they stopped by placing a bean 
bag or a piece of tape on the floor.  
1. Post-test: Record the post walk Borg dyspnea and fatigue levels and ask 
this: "What, if anything, kept you from walking farther?"  
2. If using a pulse oximeter, measure SpO2 and pulse rate from the oximeter 
and then remove the sensor.  
3. Record the number of laps from the counter (or tick marks on the 
worksheet).  
4. Record the additional distance covered (the number of meters in the final 
partial lap) using the markers on the wall as distance guides. Calculate the 
total distance walked, rounding to the nearest meter, and record it on the 
worksheet.  
5. Congratulate the subject on the good effort and offer a drink of water.  


Medical Sciences Institute – Blood Center of Wisconsin 
Montelukast Clinical Trial Protocol, Version 10.0 –  October 27, 2016PRO 
Page 30 of 32 
 
 
Appendix Two 
Borg Scale 
At the beginning of the of the 6-minute exercise show the scale to the subject and ask 
the subject this: “please grade your level of shortness of breath using this scale” Then 
ask this: Please grade your level of fatigue using this scale. Document the grade 
chosen. 
At the end of the walk, remind the subject of the breathing number they chose before 
the walk and ask the subject to grade their breathing level again. Then ask the subject 
to grade their level of fatigue, after reminding them of their grade before the walk.   
Document the grade chosen. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
One to Ten  
BORG SCALE 
0 
Nothing at all 
1 
Very slight 
2 
Slight (light) 
3 
Moderate 
4 
Somewhat severe 
5 
Severe (heavy) 
6 
 
7 
VERY Severe 
8 
 
9 
 
10 
Very, very severe (maximal) 


Medical Sciences Institute – Blood Center of Wisconsin 
Montelukast Clinical Trial Protocol, Version 10.0 –  October 27, 2016PRO 
Page 31 of 32 
 
 
Appendix Three  
 
MONTELUKAST 10 MG CAPSULES 
SIZE “1”  IRB# 130794 
 
Ingredients:  
 
Montelukast 10 mg tablets   
 
# 90 
 
 
 
 
Microcrystalline cellulose  
 
to cover 
 
 
Yield  
 
 
90 capsules 
 
Starting Materials:  
Capsule machine   
 
Capsule cleaning towel 
 
 
 
 
Size “1” dark green caps  
 
Compounding: 
 
1. Don appropriate attire for compounding. 
 
 
 
 
2. Fill capsule machine with empty capsules, remove tops. 
3. Cut one tablet into two halves (if necessary) and place in 
each empty capsule. 
                                           4.  Obtain verification from a pharmacist. 
5.  Cover with microcrystalline, replace tops and clean 
capsules. 
                                           6.  Package and label appropriately. 
                                           7.  Log in Investigational Drug Accountability Record. 
 
References:  
 
USP795 
 
 
  
Expiration Date: 
 
6 months 
 
Storage: 
 
 
Room Temperature 
 
Auxiliary Labels:  
Investigational Use Only 
 
  
Sample Labels: 
                                           Vanderbilt Investigational Drug Service 
 
            
 
Montelukast 10 mg caps           # 90 
 
 
           
 
 Lot# IDS 0000 Exp: xx/xx/xx     xx/xx 
 
           
 
 IRB # 130794      Dr. DeBaun 
 
 


Medical Sciences Institute – Blood Center of Wisconsin 
Montelukast Clinical Trial Protocol, Version 10.0 –  October 27, 2016PRO 
Page 32 of 32 
 
 
PLACEBO FOR MONTELUKAST 10 MG CAPSULES 
SIZE “1” IRB# 130794 
 
Ingredients: 
Microcrystalline cellulose 
to fill 
 
 
Yield 
100 capsules 
 
Starting Materials: 
Capsule machine  
Capsule cleaning towel 
 
Size “1” dark green caps  
 
Compounding: 
1. Don appropriate attire for compounding. 
 
2. Fill capsule machine with empty capsules, remove tops. 
 
3. Fill each capsule with microcrystalline. 
                                          4.  Replace tops and clean capsules. 
                                          6.  Package and label appropriately. 
                                          7.  Log in Investigational Drug Accountability Record. 
 
References: 
USP795 
 
  
Expiration Date: 
6 months 
 
Storage: 
 
Room Temperature 
 
Auxiliary Labels:  
Investigational Use Only 
 
  
Sample Labels: 
                                          Vanderbilt Investigational Drug Service 
 
          Placebo for Montelukast 10 mg caps   # 90 
 
 
          Lot# IDS 0000 Exp: xx/xx/xx     xx/xx 
 
          IRB # 130794      Dr. DeBaun