Daily Vitamin D for Sickle-cell Respiratory Complications
Phase: PHASE2
Conditions: Sickle Cell Disease, Anemia, Sickle Cell, Anemia, Hemolytic, Congenital, Respiratory Tract Diseases, Respiration Disorders, Acute Chest Syndrome, Lung Diseases, Asthma, Respiratory Tract Infections, Nutrition Disorders, Deficiency Diseases Vitamin, Vitamin D Deficiency
Sponsor: Columbia University

 
 
 
 
 
 
 
 
 
 
 
 
 
Official Title: Daily Vitamin D for Sickle-cell Respiratory Complications (ViDAS-2) 
ClinicalTrials.gov ID: NCT04170348 
Version 4: 06/29/2022 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 

IND Study Protocol, Revision-4 
 
3. Introductory Statement: This Phase 2 clinical trial, ViDAS-2, will determine if daily oral vitamin D can reduce 
the risk of respiratory complications in children with sickle-cell disease. Sickle-cell disease, an orphan disease 
affecting an estimated 100,000 individuals in the United States,1, 2 is an inherited red blood cell disorder with 
acute vaso-occlusive complications and chronic multi-organ damage resulting from hemolysis-induced 
endothelial dysfunction and vasculopathy.3, 4 Respiratory complications are the leading cause of morbidity and of 
death.5, 6 Our earlier ViDAS-1 clinical trial provided evidence that monthly bolus oral vitamin D3 supplementation 
in children with sickle cell disease reduced the annual rate of respiratory complications by more than 50% but 
only after a year of treatment and with no significant difference between vitamin D3 given as standard- (12,000 
IU/mo) or high- (100,000 IU/mo) dose therapy (Figure 1). The observed reductions are both statistically 
significant (P<0.0001) and clinically important. Respiratory infections or asthma attacks that would have no 
lasting effects in individuals with no sickle hemoglobinopathy can trigger severe or even fatal manifestations in 
those with sickle cell disease. 
 
Figure 1: Annual rates of respiratory events (infections, exacerbations of asthma, and the acute chest syndrome) 
in children with sickle cell disease treated with no supplementation during a baseline year and monthly oral doses 
of vitamin D3 given under observation during Years 1 and 2. 
Vitamin D, in addition to its role in calcium and bone homeostasis, is a multifunctional regulator of both innate 
and adaptive immune responses.7-11 The scientific premise of the proposed clinical trial is that circulating 
concentrations of vitamin D3, the parent compound, are the principal determinant of the anti-infective and 
immunomodulatory effects of supplementation.12-14  With monthly bolus oral vitamin D3, the parent compound is 
cleared from the circulation within days.vxc12, 13, 15, 16  In our earlier clinical trial monthly bolus oral vitamin D3, 
neither the standard- nor high-dose treatments would produce sustained increases in circulating vitamin D3, 
potentially explaining the uniformity of response to the two treatments. Apparently, a year of treatment with bolus 
dosing was needed to permit sufficient amounts of vitamin D3 to accumulate within cells of the immune system 
and produce the observed reductions in the rates of respiratory events. Our hypothesis is that daily oral vitamin 
D3 will rapidly reduce the rate of respiratory complications during Year 1. We propose a 2-year controlled, double-
blind, randomized Phase 2 clinical trial comparing the efficacy in reducing the rate of respiratory events in sickle-
cell disease of daily oral vitamin D3 (3,333 IU/d) with monthly bolus oral vitamin D3, (100,000 IU/mo) as a control. 
Baseline concentrations of 25-hydroxyvitamin D in our population (mean ~14 ng/mL) guide the choice of the 
high-dose daily treatment for the proposed trial and are too low to permit inclusion of a placebo control. 
This Phase 2 clinical trial, designed to be a minimal risk study, has three specific aims: 
(1) to determine whether daily oral vitamin D3 (3,333 IU/d) given to children and adolescents with sickle-
cell disease, compared to monthly bolus oral vitamin D3, (100,000 IU/mo) will more rapidly reduce the 
rate of respiratory events, defined as respiratory infections, exacerbations of asthma, and episodes 
of the acute chest syndrome; 
(2) to evaluate the effects of daily oral vitamin D3 on pulmonary function; and 
(3) to examine the effects of monthly oral vitamin D3 on immune function, using measures of T-cell 
effector and regulatory function and of systemic inflammation. 
 
 
The results of our clinical trial could offer a simple and low-cost intervention to help avert serious respiratory 
complications in sickle-cell disease that could lead to important reductions in morbidity and death in 
children with sickling disorders and to major changes in the management of their vitamin D status.  

 
4. General investigational plan 
Acute respiratory illness is a principal cause of morbidity and mortality worldwide17 and can be devastating for 
children with sickle cell disease, an inherited red blood cell disorder affecting an estimated 100,000 Americans,3 
predominantly of African ancestry. Sickle cell disease is characterized by a shortened life expectancy, hemolytic 
anemia, acute episodes of vaso-occlusive pain, and recurrent, chronic damage to vital organs.3, 4 Pulmonary 
manifestations are common and often severe. Respiratory infections or asthma attacks that would have no 
lasting effects in individuals with no sickle hemoglobinopathy can trigger severe or even fatal manifestations in 
those with sickle cell disease.18  For instance, children with influenza and sickle cell disease are hospitalized at 
a rate more than 50-fold greater than children without sickle cell disease.19 Asthma is common, affecting 15 to 
28% of children with sickling disorders.5 Unique to sickle cell disease is a respiratory complication characterized 
by fever, respiratory symptoms and a new pulmonary infiltrate, known as “acute chest syndrome.”6 Commonly 
precipitated by respiratory infections and asthma, the acute chest syndrome is the leading cause of death in 
sickle cell disease.5, 6 The pathogenesis of sickle cell lung disease is unclear, but involves microvascular 
occlusion, hemolysis-induced endothelial dysfunction and vasculopathy that produce a chronic inflammatory 
state, often exacerbated by an infectious trigger.3, 20, 21 Compromised immunity from functional asplenia may 
contribute to the risks of respiratory infections and pulmonary disease.22 Improved vaccination and penicillin 
prophylaxis have greatly reduced the risk of bacterial pathogens but are ineffective against viruses and atypical 
organisms that now predominate as risks for acute chest syndrome.23  
Vitamin D, in addition to its role in calcium and bone homeostasis, is a multifunctional regulator of innate and 
adaptive immune responses and of inflammation.7-11 Vitamin D acts, in part, through its metabolite, 1,25-
dihydroxyvitamin D (1,25(OH)2D),24, 25 which binds to the vitamin D receptor to function as a transcription factor 
inducing vitamin D responsive genes that are present in most if not all cells of the immune system.26 1,25(OH)2D 
mediates the innate immune host response against respiratory tract pathogens by stimulating expression of 
cathelicidin (hCAP18/LL37), an antimicrobial peptide with activity against viral, bacterial and fungal pathogens.27, 
28 1,25(OH)2D regulates the adaptive immune system by modulating T-lymphocyte proliferation and function, 
and by down-regulating the inflammatory response and cytokine expression.29 In addition to these functions of 
1,25(OH)2D, the parent compound vitamin D itself is a potent and general mediator of endothelial stability and 
barrier function.30 A recent Cochrane review of randomized clinical trials identified high quality evidence that 
vitamin D supplementation reduced the risk of asthma exacerbations, although too few pediatric trials were 
included to permit definitive conclusions for children.31, 32 While observational studies consistently have found 
significant associations between low vitamin D levels and increased susceptibility to respiratory infections, 
randomized clinical trials examining the effects of vitamin D supplementation on the risk of respiratory infections 
have had conflicting findings due, in part, to differences in the baseline status of the participants, the duration of 
the studies and the amount and frequency of dosing.33-36 
Children with sickle cell disease are at high risk for vitamin D deficiency due to limited sun exposure, dark skin 
color and poor nutrition. Our own and other observational studies have found that most children with sickle cell 
disease have low vitamin D status.37, 38 Contrary to an earlier hypothesis,39 the low levels of vitamin D in black 
Americans are not explained by differences in the concentration of vitamin D-binding protein.40, 41 Considering 
that individuals with low vitamin D levels seem to derive the most benefit from supplementation36 and that children 
with sickle cell disease have a heightened susceptibility to respiratory infections and asthma together with a 
greatly increased vulnerability to complications, we conducted the ViDAS-1 study, a 2-year active-controlled, 
double-blind, randomized clinical trial comparing high-dose oral vitamin D3, 100,000 IU, given once a month 
under observation (daily equivalent 3,333 IU/day), with standard-dose oral vitamin D3, 12,000 IU, given once a 
month under observation (daily equivalent 400 IU/day),42 to determine if oral vitamin D3 (cholecalciferol) can 
reduce the risk of respiratory complications in children and adolescents, 3 to 20 years old, with sickle cell disease 
(ViDAS-1 study). Because of the established risks of inadequate mineralization of the skeleton associated with 
vitamin D deficiency,9, 42-44 a placebo group could not be included in the study design.  
The ViDAS-1 trial results have been presented in our annual reports and are summarized here to clarify the 
rationale for the ViDAS-2 trial. Seventy sickle cell subjects, ages 3-20 years, with baseline records of respiratory 
events over one year before randomization, underwent screening. Sixty-two subjects with 25-hydroxyvitamin D 
levels 5-60 ng/mL were randomly assigned in a 1:1 ratio to oral vitamin D3 100,000 IU (n=31) or 12,000 IU (n=31), 
under observed administration once monthly for two years. The primary outcome was the annual rate of 
respiratory events (respiratory infection, asthma exacerbation, acute chest syndrome) ascertained by use of a 

validated questionnaire administered biweekly. Analysis included 62 children, mean (SD) age 9.9 (3.9) years, 
32 (52%) females, predominantly HbSS (87%), with mean baseline 25-hydroxyvitamin D of 14.3 ng/mL. The 
mean (SEM) 25-hydroxyvitamin D concentrations during Treatment Years 1 and 2 are shown in Figure 2. A total 
of 291 adverse events were reported, including 55 serious adverse events in 20 subjects, most of which were 
expected for sickle cell disease; none were attributed to the study drug. There were 17 abnormal safety 
laboratory values; all abnormal laboratory values were determined to be clinically insignificant, without requiring 
withholding or discontinuation of study treatment. 
The annual rates of respiratory events at baseline, intervention year-1, and year-2 with the high-dose were 
4.34±0.35 (SEM), 4.28±0.36, and 1.49±0.37, and, with the standard-dose, 3.91±0.35, 3.34±0.37, and 1.54±0.37, 
respectively (see Figure 1 above). In pediatric patients with sickle cell disease, two-year monthly oral vitamin D3 
was associated with a >50% reduction in the rate of respiratory illness during the second year, with similar 
decreases from high- and standard-dose treatment. 
In summary, in children and adolescents with sickle cell disease who received monthly bolus doses of oral 
vitamin D3 under observation for two years, the annual rates of respiratory events (respiratory infections, asthma 
exacerbations and acute chest syndrome) decreased by more than 50% during the second year of treatment, 
with similar reductions in the groups treated with 100,000 IU/month and with 12,000 IU/month. The treatment 
groups did not differ significantly with respect to pulmonary function, hand-grip strength, fever or episodes of 
vaso-occlusive pain. Both doses were safe with no significant differences in adverse events.  
 
 
Figure 2: Mean serum 25-hydroxyvitamin D concentrations by treatment group: High-dose: 100,000 IU (2.5 mg) 
vitamin D3 given once a month under observation (daily equivalent 3,333 IU/day); Standard-dose, 12,000 IU 
(0.3 mg) vitamin D3 given once a month under observation (daily equivalent 400 IU/day). 
This ViDAS-1 study is the first randomized clinical trial to examine the potential benefit of vitamin D for preventing 
respiratory complications in sickle cell disease. We chose monthly dosing to allow for directly observed 
administration and avoid non-adherence with daily dosing. We provided prolonged treatment to allow for 
seasonal variation. Our primary outcome measure was collected biweekly using a validated questionnaire45 to 
adequately capture all respiratory events, and verified by review of medical records.  Because we could not 
include a placebo group, given the known risks of skeletal harm with vitamin D deficiency,9, 42-44 the decrease in 
the annual rates of respiratory events during Year 2 cannot be ascribed unequivocally to vitamin D 
supplementation. Nonetheless, no significant differences in the mean weekly rates of influenza-like illnesses in 
New York City during the baseline and study years (2012-2015) were found by the New York State Department 
of Health Influenza-Like Illness Surveillance Program (ILINet).46 Moreover, because recruitment for the trial 
extended over 18 months, overlap between study participants receiving supplementation during Year 1 and 2 
was considerable. For example, Year 2 treatment of patients enrolled in months 1 to 6 of the trial coincided with 
Year 1 treatment of patients enrolled in months 12 to 18.  
In the ViDAS-1 study, the effects of standard- and high-dose vitamin D on respiratory events were statistically 
indistinguishable. Conceivably, these results could be interpreted as evidence that current recommendations for 
vitamin D supplementation are sufficient to maintain respiratory health in patients with sickle cell disease. In the 

standard-dose group, the estimated mean dietary vitamin D of 228.6 IU/day, together with the monthly 
supplement providing about 400 IU/day, would meet the current Estimated Average Requirement for vitamin D 
of 600 IU/day.9 Nonetheless, 25-OHD concentrations were at insufficient or deficient levels for skeletal health in 
75% of the standard-dose group while in the sufficient range in 98% of the high-dose group.  
Alternatively, both (i) the timing of the observed decreases in the annual rates of respiratory events and (ii) the 
uniformity in the magnitudes of the reductions with the standard- and high-dose treatments potentially could be 
explained by the monthly bolus dosing schedule for vitamin D used in the trial. Since the design of our study, 
new evidence has accumulated that circulating concentrations of vitamin D3, the parent compound, rather than 
those of 25-OHD, may be vital determinants of the anti-infective, immunomodulatory and other extra-skeletal 
effects of supplementation.12, 13, 30, 47 To be active, supplemental vitamin D3 must undergo two hydroxylations, 
first to 25-OHD and then to the functional metabolite, 1,25-dihydroxyvitamin D, that binds to the vitamin D 
receptor to function as a transcription factor inducing vitamin D responsive genes. For the principal endocrine 
functions of vitamin D, bone mineralization together with calcium and phosphate homeostasis, the first 
hydroxylation takes place using a 25-hydroxylase in the liver,24, 25, 48 producing 25-OHD that enters the circulation. 
The second hydroxylation occurs in the kidney, using the 1-alpha- hydroxylase CYP27B1 to yield 1,25-
dihydroxyvitamin D.24, 26 By contrast, for extra-skeletal autocrine and paracrine activities, vitamin D3, the parent 
compound, enters cells more easily than 25-OHD, and both hydroxylations may take place intracellularly.12-14 
Both CYP27B1 and 25-hydroxylases are widely expressed and the vitamin D receptor is present in most, if not 
all, cells of the immune system.26 In addition to these functions of 1,25(OH)2D, the parent compound vitamin D 
itself is a potent and general mediator of endothelial stability and barrier function.30 
With a single oral bolus dose of vitamin D3,12,000 IU, the half-life of 25-OHD is on the order of weeks while that 
of vitamin D3 is about one day. With a single oral bolus dose of vitamin D3,100,000 IU, 25-OHD remains elevated 
for more than 3 months while vitamin D3 returns to near baseline in less than a week.12, 13, 15, 16 The repeated oral 
bolus doses of vitamin D3 resulted in the plateaus in 25-OHD shown in Figure 2. While we did not measure 
vitamin D3 concentrations in our study, neither the standard- nor high-dose treatments would produce sustained 
increases in circulating vitamin D3, potentially explaining the uniformity of response to the two treatments. Vitamin 
D3 in blood seems to be in diffusional equilibrium with vitamin D3 in adipose tissue.12 With bolus doses, vitamin 
D3 in excess of use will accumulate in fat and gradually raise circulating vitamin D3 concentrations. Apparently, 
a year of treatment with bolus dosing was needed to permit sufficient amounts of vitamin D3 to circulate, 
accumulate within cells of the immune system, and produce the observed reductions in the rates of respiratory 
events. In shorter trials of bolus oral doses of vitamin D3, insufficient durations of treatment could explain the 
lack of effect on respiratory infections,36, 49 perhaps in conjunction with other possible explanations for the lack 
of a protective effect of bolus doses of vitamin D.50-54  
Children with sickle cell disease typically have low vitamin D status,37, 38 heightened susceptibility to respiratory 
infections and asthma, and a greatly increased vulnerability to potentially fatal complications of respiratory 
illness. Our study results provide evidence for a substantial protective effect of vitamin D against sickle cell 
respiratory complications that was manifest only after a year of bolus administration. While our results cannot 
prove that vitamin D was responsible for the substantial and significant reduction in respiratory illness, this 
hypothesis is consistent with the known facts.  
The ViDAS-2 trial is designed to determine if daily oral administration of vitamin D will produce sustained 
increases in the concentration of circulating vitamin D3 and provide children and adolescents with sickle cell 
disease with protection against respiratory infections, asthma exacerbations and acute chest syndrome. Daily 
supplementation with 2000–6000 IU/d vitamin D3 results, within three weeks, in stable circulating concentrations 
of vitamin D3 within the range of 10–40 ng/mL in a linear fashion.13, 55-57 Our primary study hypothesis is that daily 
oral vitamin D3 (cholecalciferol), 3,333 IU/d (0.083 mg/d), will reduce the annual rate of respiratory events 
(infections, exacerbations of asthma, and the acute chest syndrome) by 50% or more during Treatment Year 1 
compared with monthly bolus oral vitamin D3, 100,000 IU/mo (25 mg/d) as a control. 
The control treatment with monthly bolus administration should provide an estimate of the annual rates of 
respiratory events that would be expected with placebo while protecting against the skeletal risks associated 
with insufficient or deficient vitamin D levels (25(OH)D<20 ng/mL).9, 42-44 
5. Investigator’s brochure: Not required for a Sponsor-Investigator Investigational New Drug (IND) application.  
 
6. Protocols:  

 
6a. 
Study protocol: This ViDAS-2 study is a 2-year active-controlled, double-blind, parallel-group, 
randomized Phase 2 clinical trial comparing the efficacy in reducing the rate of respiratory events in children and 
adolescents with sickle cell disease of daily oral vitamin D3, 3,333 IU/d (0.083 mg/d) with monthly bolus oral 
vitamin D3, 100,000 IU/mo (2.5 mg/mo), as a control. A profile of the Phase 2 clinical trial design is shown 
schematically in the Figure.  
The study population for the ViDAS-2 randomized trial will be recruited from among approximately 200 patients, 
ages 3-20 years old, followed in the Columbia University Medical Center Pediatric Sickle Cell Program. Subjects 
will be recruited and screened based on medical history, physical examination, and laboratory evaluation (serum 
25-hydroxyvitamin D, parathyroid hormone, chemistry profile including calcium, phosphate, magnesium, BUN, 
creatinine, and liver function tests, urinalysis, and urinary calcium/creatinine ratio). Patients who complete the 
screening visit will then return and their potential inclusion in the trial will be determined by the serum 25-
hydroxyvitamin D concentration: 
 (i)   
Patients found to be severely vitamin D deficient, with a screening serum 25-hydroxyvitamin D 
concentration below 5 ng/mL, will not be randomized but will be eligible to continue participation in the study by 
being assigned to treatment with daily oral vitamin D3, 3,333 IU/d (0.083 mg/d), in an open-label manner and by 
completing all other study procedures. Management of these patients will consist of oral administration of vitamin 
D3, 100,000 IU, initially, followed by oral vitamin D3, 100,000 IU, every other week for 2 months, and then daily 
oral vitamin D3, 3,333 IU/d (0.083 mg/d), thereafter for the duration of the study, with follow-up by the participant’s 
attending physician. This schedule of vitamin D administration for treatment of vitamin D deficiency is consistent 
with the recommendations for Stoss therapy of vitamin D deficiency by the Drug and Therapeutics Committee of 
the Lawson Wilkins Pediatric Endocrine Society.42  
 (ii)   
Patients with excessive levels of vitamin D, as determined by a screening serum 25-hydroxyvitamin D 
concentration above 100 ng/mL, will be excluded from participation in the trial and referred to their attending 
physician for follow-up and management. We do not anticipate that any of our patients will have excessive levels 
of vitamin D but include this provision for exclusion as a precautionary measure. 
 (iii)    Patients with levels of vitamin D already within the range found in sun-rich environments, as determined 
by a screening serum 25-hydroxyvitamin D concentration of 61-100 ng/mL, will not be randomized because no 
supplemental vitamin D would be indicated. We do not anticipate that any of our patients will have levels of 
vitamin D in this range but if any are identified, they would be eligible to continue participation in the study and 
complete all other study procedures. They would not receive supplemental vitamin D within the study. 
 (iv)   Patients with a screening serum 25-hydroxyvitamin D concentration of 5 - 60 ng/mL, will be randomized 
within a month in a gender- and age-stratified (3 to 9 and 10 to 20 years of age) manner to treatment with either 
(1) daily oral vitamin D3, 3,333 IU/d (0.083 mg/d) or (2) 100,000 IU (2.5 mg) oral vitamin D3 once monthly with 
daily placebo. 
Prior to initiation of treatment, subjects will have baseline assessments that will include: (i) pulmonary function 
test (limited to age above 5y/o); and (ii) blood tests for biomarkers of inflammation (complete blood count, C-
reactive protein, and Th1/Th2 cytokines). Participants will be seen monthly in person or remotely in a telehealth 
visit for confirmation of administration of study drug,accounting of unused study drug, interval history including 
respiratory events and adverse event monitoring. Laboratory safety assessments will include serum albumin-
corrected calcium and spot urinary calcium-creatinine ratios to be obtained immediately before study drug 
administration, and performed at intervals as shown in the Table (Schedule of Study Evaluations) to monitor 
hypercalcemia and hypercalcuria. Abnormal laboratory values will be reviewed by the Clinical Trial Safety Officer 
to determine clinical significance and need for intervention, as described in detail below.  

 
Figure: Profile of the Phase 2 ViDAS-2 clinical trial design. 

Blood specimens for 25(OH)D and vitamin D3 will be collected every third month for the first six months, then at 
months 6, 12, 18 and 24, stored at -112°F (-80°C) freezer, and analyzed in a single batch at the end of the study 
by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (See below for Vitamin D assay).  
Adverse events will be determined according to the Common Terminology Criteria of Adverse Events version 
4.0. Pulmonary function tests will be repeated at 12-months and 24-months after initiation of treatment, while the 
biomarkers of inflammation, the health-related quality of life questionnaire and the Block Calcium and Vitamin D 
screener and sun exposure questionnaires will be collected every 6 months. Table 3 below outlines the schedule 
of study evaluations. 
 
Primary Outcome Measure: Respiratory Events 
A Respiratory Events Questionnaire, modified from established instruments,58, 59 will be used to track respiratory 
events. The questionnaire, to be administered in person during monthly outpatient study visits and biweekly 
telephone interviews, includes questions about the following six symptoms: difficulty breathing, wheezing, fever, 
cough, runny nose, and sore throat. If any of the above symptoms is present, additional questions about severity 
and duration of symptom, medication use, visit to a medical provider, hospitalization, and missed school day/s 
due to illness will be asked. History of outpatient sick visits and hospital admissions will be verified by review of 
medical records. All completed questionnaires will be reviewed and adjudicated in a blinded fashion by a 
designated Investigator to determine whether a respiratory event had occurred. 
(Study Questionnaire is attached in Appendix) 
 
Definitions of Respiratory Events: 
A “respiratory event” will be considered if at least one of the six symptoms occurred, except for fever alone 
without accompanying respiratory symptoms, or a runny nose accompanied by itchy/watery eyes suggestive of 
allergy. A new event will be counted if at least seven days had elapsed since the last counted event. Patients 
with prolonged respiratory symptom/s lasting more than two weeks will be evaluated by the study pulmonologist, 
whose final diagnosis will determine whether the symptom/s comprised a respiratory event.  
 “Respiratory infection” will be defined as: (i) runny nose, cough, or sore throat for at least 2 days or any of these 
symptoms for at least 1 day accompanied by fever, not otherwise attributed to allergy; (ii) physician diagnosed 
pharyngitis, otitis media, or influenza; or (iii) physician-diagnosed croup, bronchiolitis, or pneumonia. The study 
questionnaire was validated in our study population and found to have 80% over-all accuracy for identifying 
respiratory tract pathogens60. 
“Asthma exacerbation” will be defined by a physician-diagnosed acute asthma attack that required treatment 
with corticosteroids, emergency room treatment, or hospital admission.  
“Acute chest syndrome” will be defined according to the standard definition, as an acute illness characterized by 
fever and/or respiratory symptoms, accompanied by a new pulmonary infiltrate on a chest x-ray.61 
In instances when there is an overlap of these diagnoses within a single duration of symptoms, such as when 
respiratory infection preceded an asthma exacerbation or acute chest syndrome, or when infection or acute 
asthma occurred with an acute chest syndrome, the separate diagnoses will be counted as a single event.   
 
Vitamin D assay:  
Serum 25-hydroxyvitamin D2 (25-OH-D2) and 25-hydroxyvitamin D3 (25-OH-D3) will be measured and 
summed as total 25-hydroxyvitamin D. Assays will be performed in the Biomarkers Core Laboratory within the 
Irving Institute for Clinical and Translational Research using Ultra Performance Liquid Chromatography-Tandem 
Mass Spectrometry (LC-MSMS).  
  
Sample preparation:  
25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 will be extracted from serum samples using liquid-liquid 
extraction. Briefly, 150uL of human serum will be spiked with 10uL of internal standard and vortexed, followed 
by the addition of 150uL of 0.2 M zinc sulfate. After mixing, 300uL of methanol is added followed by 750uL of 
hexane with vortexing in between. The mixture is again vortexed for 30 seconds followed by centrifugation at 
13,000 rpm for 5 minutes. The hexane layer is transferred to LC vials, dried down under nitrogen and dissolved 
in 100uL of 70% methanol.  
 
Table. SCHEDULE OF STUDY EVALUATIONS AND PARTICIPANT TIMELINE 

 
Screening 
Randomize¶ 
Month 1 
Month 2 
Month 3 
Month 4 
Month 5 
Month 6 
Month 7 
Month 8 
Month 9 
Month 10 
Month 11 
Month 12 
Month 13 
Month 14 
Month 15 
Month 16 
Month 17 
Month 18 
Month 19 
Month 20 
Month 21 
Month 22 
Month 23 
Month 24 
Medical history 
X   
 
 
 
 
 
 
 
 
 
 
 
X   
 
 
 
 
 
 
  
 
 
 
X 
Physical exam  
X  
 
 
 
 
 
 
 
 
 
 
 
X  
 
 
 
 
 
 
 
 
 
 
X 
Anthropometry 
X   
 
 
 
 
 
 
 
 
 
 
 
X   
 
 
 
 
 
 
 
 
 
 
X 
Respiratory 
events  
X X X X X X X X X 
X X X X X X X X X X X X X X X X X 
Oral vitamin D3  
 
X X X X X X X X 
X X X X X 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 X X X X X X X X X X X X 
HRQL, Diet/Sun 
exposure 
 
X  
 
 
 
 
X  
 
 
 
 
X   
 
 
 
 
X  
  
 
 
 
X 
LABORATORY EVALUATIONS 
Serum 25-OHD 
X X  
 
X  
 
X   
 
  
 
 
X   
  
 
   
  
X   
  
 
  
  
X 
Serum D3 
 
X  
 
X  
 
X   
 
  
 
 
X   
  
 
   
  
X   
  
 
  
  
X 
Serum Calcium, 
albumin 
X X  
 
X  
 
X  
 
 
 
 
X   
 
 
 
 
X  
  
 
 
 
X 
CMP, Mg, PO4 
X  
 
 
 
 
 
X  
 
 
 
 
X  
 
 
 
 
X  
 
 
 
 
X 
Urinalysis 
X X  
 
 
 
 
X  
 
 
 
 
X  
 
 
 
 
X  
 
 
 
 
X 
Urinary Ca, Cr 
 
X  
 
X  
 
X  
 
X  
 
X  
 
X  
 
X  
 
X  
 
X 
VDBP 
 
X  
 
 
 
 
 
 
 
 
 
 
X   
 
 
 
 
 
 
  
 
 
 
X 
Serum PTH 
  
X   
  
  
  
 
X  
  
 
  
 
X   
 
  
 
 
X  
  
  
 
 
X 
β-hCG 
X  
 
 
 
 
 
X  
 
 
 
 
X  
 
 
 
 
X  
 
 
 
 
X 
AIRWAY EVALUATIONS (aim 2) 
PFT 
 
X  
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
  
 
 
 
X 
IMMUNE SYSTEM CHANGES (aim 3) 
T effector function 
 IL-4, IL-5, IL-
13, IFNγ, IL-2 
 
X  
 
 
 
 
X  
 
 
 
 
X   
 
 
 
 
X  
 
 
 
 
X 
T regulatory function 
IL-10, TGF-β 
 
X  
 
 
 
 
X  
 
 
 
 
x    
 
 
 
 
X  
 
 
 
 
X 
Systemic inflammation 
CBC/plts 
 
X  
 
 
 
 
X  
 
 
 
 
X  
 
 
 
 
X  
 
 
 
 
X 
Serum CRP 
 
X  
 
 
 
 
X  
 
 
 
 
X   
 
 
 
 
X  
 
 
 
 
X 
IL-1α, IL-1β, 
TNFα 
 
X  
 
 
 
 
X  
 
 
 
 
X  
 
 
 
 
X  
 
 
 
 
X 
Abbreviations and explanations: Respiratory events: questionnaire for respiratory events during preceding two 
weeks; Oral vitamin D3: 100,000 IU (2.5 mg) oral vitamin D3 once monthly with daily placebo or daily oral vitamin 
D3 (3,333 IU/d); Diet/Sun exposure: Block Calcium and Vitamin D screener questionnaire and sun exposure 
questionnaire; HRQL: child and parent reported; PedsQLTM Generic Core Scale, and PedsQLTM Sickle Cell 
Disease Module; PFT: Pulmonary function tests; Serum 25(OH)D: Serum 25-hydroxyvitamin D concentration; 
Serum D3: serum concentrations of vitamin D3, the parent compound; Ca: calcium; CMP: comprehensive 
metabolic panel; PO4: phosphate; Mg: magnesium; VDBP: vitamin D-binding protein concentration; Urinary Ca, 
Cr: Urinary calcium and creatinine; Serum PTH: Serum intact parathyroid hormone; β-hCG: β- Human chorionic 
gonadotropin; T-effector function: T-helper 1,2 cytokines interleukin (IL)-10, 4,5,13, 2 and interferon (IFN)γ. T-
regulatory function by cytokines IL-10, transforming growth factor (TGF)β; Systemic inflammation assessed by 
CBC: complete blood count with leukocyte and platelet (plt) counts; Serum CRP: Serum high-sensitivity C-
reactive protein; and assessed by IL-1α, β, tumor necrosis factor(TNF)α). For all assessments, a protocol window 
of ± 7 days is specified. 

LC-MSMS:  
LC-MSMS analysis will be performed on a UPLC-MS/MS platform comprising a triple quadruple Agilent 6410 
mass spectrometer (Agilent, Santa Clara, CA) integrated to Agilent UPLC 1290 series controlled by MassHunter 
quantitative analysis software (version B 04.00). Chromatographic separation will be performed by injecting 10uL 
of the extract onto an Agilent Poroshell 120 EC-C18 column (3.0 x 50mm, 2.7 µm) maintained at 50°C. The flow 
rate is maintained at 500µl/min. The initial flow conditions are 30% solvent A (water containing 0.1% formic acid) 
and 70% solvent B (methanol containing 0.1% formic acid). Solvent B is raised to 90% linearly over 4min and to 
98% in 4.1min, held for 1.4min and back to initial conditions by 5.6min with a total run time of 6.5min. The mass 
spectrometer is operated under multiple reaction monitoring (MRM) mode with positive electrospray ionization. 
MRM transitions will be m/z 413->395 for 25(OH)D2, 401->383 for 25(OH)D3 and 407->389 for d6-25(OH)D3. 
Retention times 2.89 min for 25(OH)D3 and 3.04 min for 25(OH)D2. Lower limit of quantitation for the assay for 
both 25(OH)D3 and 25(OH)D2 is 1.25 ng/ml. Intra-day precision is 2.4% for 25(OH)D2 and 3.5% for 25(OH)D3. 
Inter-day precision is 8.1% for 25(OH)D2 and 5.5% for 25(OH)D3. Calibrators are standardized against the NIST 
standards. 
 
Serum vitamin D3, the parent compound, will be measured by LC-MSMS by Heartland Assays, Ames, Iowa.  
 
STUDY DRUG 
The 3,333 IU (0.083 mg) oral vitamin D3, the 100,000 IU (2.5 mg) oral vitamin D3, and placebo dosage forms will 
be custom manufactured as single batches from Tishcon Laboratories Inc, (Westbury, NY) a Good-
Manufacturing-Practice (GMP) facility, in identical capsule formulations but for the concentration of vitamin D3. 
The content of active ingredient will be analyzed in random samples from the batch in Tishcon Laboratories by 
high-performance liquid chromatography using United States Pharmacopeia methods.  
 
For study participants randomized to daily treatment, the 3,333 IU (0.083 mg) oral vitamin D3 will be taken daily, 
with the dose at monthly in-person visits administered by the Clinical Research Coordinator or directly observed 
during a telehealth visit. For study participants randomized to control treatment, placebo will be taken daily and 
the 100,000 IU (2.5 mg) oral vitamin D3 dose will be administered by the Clinical Research Coordinator at monthly 
in-person visits or directly observed during a telehealth visit. 
 
The rationale for the choice of oral vitamin D3 dosage forms is based on the results of our earlier ViDAS-1 trial. 
In our study population of children and adolescents with sickle cell disease, the majority of the participants (77%) 
had insufficient or deficient vitamin D levels (25(OH)D<20 ng/mL),42, 43 with an overall mean baseline 25(OH)D 
of 14.3 (median 14.6, range 5-28 ng/mL). Because of the established risks of inadequate mineralization of the 
skeleton associated with vitamin D insufficiency and deficiency,9, 42-44 a placebo group could not be included in 
the study design. At the end of our earlier ViDAS-1 trial, 25(OH)D concentrations were at insufficient or deficient 
levels for skeletal health in 75% of those who had received 2 years of standard-dose treatment (400 IU/day 
vitamin D supplement together with an estimated mean dietary vitamin D of 228.6 IU/day, providing the 
current Institute of Medicine Recommended Dietary Allowance (RDA) for vitamin D of 600 IU/day, considered to 
meet the requirement of 97.5% of the population9, 62). By contrast, in study participants who received 100,000 IU 
(2.5 mg) oral vitamin D3 monthly (daily equivalent 3,333 IU/day), 25(OH)D concentrations were in the sufficient 
range in 98%. Nonetheless, in the study participants receiving bolus 100,000 IU (2.5 mg) oral vitamin D3 monthly 
during ViDAS-1 Treatment Year 1, the annual rates of respiratory events (respiratory infections, asthma 
exacerbations and acute chest syndrome) were not significantly different from the year-long ViDAS-1 baseline 
period with no supplemental vitamin D (baseline rate = 4.34±0.35 (SEM), Treatment Year 1 rate = 4.28±0.36). 
Accordingly, in the current trial, control treatment with bolus administration of 100,000 IU (2.5 mg) oral vitamin D3 
monthly should provide an estimate of the annual rates of respiratory events that would be expected with placebo 
while protecting against the skeletal risks associated with insufficient or deficient vitamin D levels (25(OH)D<20 
ng/mL).9, 42-44 
 
The scientific premise of the proposed clinical trial is that circulating concentrations of vitamin D3, the parent 
compound, are the principal determinant of the anti-infective and immunomodulatory effects of supplementation. 
With monthly bolus oral vitamin D3, the parent compound is cleared from the circulation within days.55 In contrast, 
daily supplementation with 2000–6000 IU/d vitamin D3 results, within three weeks, in stable circulating 
concentrations of vitamin D3 within the range of 10–40 ng/mL in a linear fashion.13, 55-57 The study hypothesis is 
that participants randomized to daily treatment with the 3,333 IU (0.083 mg) oral vitamin D3 dose will develop 

stable circulating concentrations of vitamin D3 that will provide rapid protection against respiratory infection, 
exacerbations of asthma and the risk of acute chest syndrome. As shown in our earlier ViDAS-1 trial, during 
Treatment Year 1, monthly bolus oral vitamin D3 with 100,000 IU (2.5 mg) oral vitamin D3 (daily equivalent 3,333 
IU/day) failed to decrease the annual rate of respiratory events compared to the baseline period with no vitamin 
D supplementation. The continuation of the trial into Treatment Year 2 will determine (i) if any reduction in the 
rate of respiratory illness with daily vitamin D3 seen during Treatment Year 1 is sustained and (ii) if monthly bolus 
oral vitamin D3 again reduces the rate of respiratory events by 50% or more during Treatment Year 2. To our 
knowledge, this study will provide the first direct comparison of the effects on respiratory illness of daily and 
monthly doses of vitamin D that deliver approximately equivalent amounts of vitamin D per month. 
 
Drug Accountability: Once a randomization request has been completed and at least 2 days before the 
scheduled Baseline study visit), the Study Coordinator will coordinate dispensing of the study medication by the 
Research Pharmacy. The Research Pharmacy is responsible for determining which assignment (A or B) is active 
study-dose treatment (vitamin D3 3,333 IU/capsule to take daily except during the monthly study visit when 3,333 
IU/capsule x 1 capsule and placebo x 1 capsule will be administered by study coordinator on-site or self-
administered under direct observation by study coordinator during telehealth visit), and which assignment is 
control-dose treatment (placebo capsule to take daily except during monthly study visit when vitamin D3 50,000 
IU/capsule x 2 capsules will be administered by study coordinator on-site or self-administered under direct 
observation vy study coordinator during telehealth visit). Study medication labels will include the prescription 
number, subject ID, and randomization ID as recorded in the Research Pharmacy Medication Dispensing Log. 
The initial dose will be administered during the Baseline study visit after all the baseline procedures have been 
completed. Two capsules containing either study-dose (3,333 IU/capsule x 1 capsule and placebo x 1 capsule) 
or control-dose (vitamin D3 50,000 IU/capsule x 2 capsules) will be given orally. Capsules can either be 
swallowed or for those subjects who are unable to swallow capsules, contents of the capsules can be sprinkled 
in apple sauce or pudding and consumed in its entirety. Subjects will be instructed to take one capsule daily until 
the time of the next study visit. Subjects will be asked to count and dispose any unused capsules observed on 
video by study coordinator at the following monthly visit. The number of unused capsules will be recorded by the 
study coordinator in the Research Pharmacy Drug Return Log. These procedures will be repeated at each 
monthly study visit. Month 24 will serve as the Study Exit visit; study drug will not be administered or dispensed 
at this visit. 
 
STUDY SUBJECTS 
Inclusion criteria: 
 
1) Diagnosis of sickle cell disease (Hb SS, Hb SC, Hb S-Beta-thalassemia) 
 
 
2) Age 3-20 y/o 
 
 
Exclusion criteria:  
 
1) Patient unwilling or unable to provide written informed consent (and assent, if applicable) 
 
2) Patient unable or unwilling to comply with requirements of the clinical trial  
 
3) Participation in another clinical trial 
 
4) Current diagnosis of rickets  
5) History of hypercalcemia or diagnosis of any medical condition associated with hypercalcemia, 
including primary hyperparathyroidism, malignancy, sarcoidosis, tuberculosis, granulomatous 
disease, familial hypocalciuric hypercalcemia 
 
6) Current use of corticosteroids, excluding inhaled steroids  
 
7) Current use of anticonvulsants (phenytoin, phenobarbital, carbamazepine)  
 
 
8) Therapy with thiazide diuretics or lithium carbonate  
 
9) Known liver or renal disease 
10) Patients taking medications for pulmonary complications of sickle cell disease not on a stable dose of 
medications, as defined by a change in medications or doses within the three months prior to study 
entry 
11) Patients on chronic red blood cell transfusion therapy 
12) Pregnancy 
 
Subject Justification:  

This study will enroll about 80 children and adolescents, ages 3-20 years, with sickle cell disease. Participation 
of children in the proposed research is essential to answer the research question, because pulmonary 
complications in sickle cell disease develop early in life. The study aims to determine whether vitamin D 
supplementation can reduce the risk of respiratory complications during childhood and prevent deterioration in 
lung function. The proposed research will predominantly include subjects of African origin, including African 
Americans and those from the Caribbean Islands (Dominican Republic, Puerto Rico, West Indies, Cuba, and 
Haiti), because sickle cell disease primarily affects people of African descent.3 
 
Subject Randomization:  
Within one month after screening procedures have been completed, subjects will be randomized. Using 
computer-generated randomization, participants with 25-hydroxyvitamin D concentrations of 5-60 ng/mL will be 
randomly assigned using randomly permuted blocks of size 2, 4 or 6 assignments with separate schema 
prepared for stratification by age (3-9 and 10-20), gender and hydroxyurea therapy. Randomization will be 
performed by the Research Pharmacy; all other research staff and participants will be blinded to allocation. 
 
Subject Compensation:  
Study participants will receive $40 per monthly study visit, with the exception of the following 3 study visits: 1) 
Baseline/Randomization visit, 2) Month 12 visit, and 3) Month 24 Study exit visit, for which participants will 
receive $100. The modest compensation will be provided to compensate for participant's time, transportation 
expense, and completion of study-required procedures. Compensation will be in the form of cash or gift card and 
paid in installment during each monthly visit. Participants will not be required to provide receipts for 
reimbursement. A total of $540 per calendar year is compensated to each subject who completes all study-
related visits. 
 
STATISTICAL ANALYSIS PLAN 
Our primary study hypothesis is that daily oral vitamin D3 (cholecalciferol), 3,333 IU/d (0.083 mg/d), will reduce 
the annual rate of respiratory events (infections, exacerbations of asthma, and the acute chest syndrome) by 
50% or more during Treatment Year 1 compared with monthly bolus oral vitamin D3, 100,000 IU/mo (25 mg/d) 
as a control. As detailed above, control treatment with bolus administration of 100,000 IU (2.5 mg/d) oral vitamin 
D3 monthly should provide an estimate of the annual rates of respiratory events that would be expected with 
placebo treatment.  
 
Our primary study analysis will examine the annual rate of respiratory events (the sum of respiratory infections, 
asthma exacerbations and episodes of acute chest syndrome) in children with sickle cell disease randomized in 
a double-blind manner to the daily and monthly doses of oral vitamin D3. The primary statistical analysis will be 
by “intention to treat” and will use a semiparametric regression method to estimate the between group difference 
in intensity (events per subject per unit of time at risk) with the assumption that covariates have a multiplicative 
effect on the mean and rate functions. This approach takes account of the skewed distribution of the respiratory 
events. The results of this analysis will be summarized as a regression parameter estimate, with the standard 
error of the estimate, a chi-square test with associated p-value, and a hazard ratio with confidence limits 
expressing the rate difference of the daily relative to the monthly group (as described in Lin, DY, Wei, LJ, Yang, 
I, and Ying, Z. “Semiparametric regression for the mean and rate functions of recurrent events,” J R Stat Soc 
Series B Stat Methodol, 2000; 62:711–730).63 This approach can adjust for group differences in time-
independent covariates and estimate rate ratios at specific values of covariates and at specific times of follow 
up. The annualized rate of events and the average inter-event interval (in two-week intervals, the observation 
protocol) will be entered as time-independent covariates. After testing the initial hypothesis of overall group 
difference, we will sequentially test the between-treatment group differences in the study design enrollment 
criteria strata: age, gender and therapy (hydroxyurea). Secondary analyses using the same statistical framework 
will separately test the study hypotheses at the one-year time point. The functional form of covariates and 
proportional hazards assumptions will be checked for all models and cumulative hazard plots and cumulative 
residual plots will be used to display group differences in the evolution of events and model fit, respectively. 
 
Subsidiary analyses will be made to examine a number of secondary study hypotheses with respect to the effects 
of vitamin D3 on changes in pulmonary function and airway inflammation, and on immune system, but these 
comparisons have not entered into the sample size calculation. Data will be summarized as means ± standard 

deviation or as counts and proportions. Study Group differences in continuous measures at baseline will be 
compared using independent t tests and differences in dichotomous categorical measures at baseline will be 
compared using Fisher’s exact test.  
 
Sample size 
For sample size estimation, we base our estimate of the annual rate of respiratory events on the rate observed 
in the earlier ViDAS-1 trial in our study population, a mean annual baseline respiratory event rate of 4.1. For our 
estimate of the treatment effect, we use the observed reduction in annual mean respiratory event rate in the 
earlier ViDAS-1 trial for monthly bolus oral vitamin D3, 100,000 IU/mo (2.5 mg/mo), from Treatment Year 1 to 
Treatment Year 2 from 4.28±0.36 (SEM) to 1.49±0.37, or a decrease of about 65 percent. Using a conservative 
estimate of a treatment effect of 50 percent reduction in the rate of respiratory events as the minimal clinically 
significant difference to be detected, 26 subjects in each group will be sufficient to detect an effect size of 
this magnitude with a two-sided significance level of alpha = 0.025 and 90 percent power. Allowing for 
as much as a 15 percent loss of participants during the two-year study (due to clinical events, such as 
initiating chronic red blood cell transfusion therapy, failure of compliance or exclusion or withdrawal for 
any reason), then our sample size estimate is 26/0.85 = 31 participants in each treatment group, or a total 
of 62 participants. Although the secondary analyses planned have not entered into the calculation of the sample 
size required for the proposed study, the conservative specification for the treatment effect; 90 percent power; 
and a generous allowance for loss of participants, should help provide adequate power for subsidiary 
comparisons.  
 
Annualized Event Rate and Inter-Event-Interval (IEI) 
Individual participant rates of events during the treatment phase (Treatment Years 1 and 2) will be annualized 
by dividing the number of days of observation by 365.25. Individual participant inter-event-interval (IEI) will be 
the average of the number of days between the first day of observation and the day of the first event and the 
number of days separating the resolution of one event to the onset of the subsequent event. The treatment 
phase IEI will be calculated as the average of the number of days separating the resolution of one event to the 
onset of the subsequent event, the number of days from randomization to the first event, and the days between 
the last event and final day of study participation. If a participant is in the midst of an event episode on the day 
of randomization, the calculation of the average IEI will start with the date the event resolved.  
 
Treatment Outcome Analysis 
All primary and secondary outcome analyses will be performed under the intent-to-treat principle. Crude 
estimates of group differences in the annualized event rate and average inter-event-interval during Treatment 
Year 1 and Treatment Year 2 will be analyzed by linear mixed models for repeated measures with fixed effects 
for group, time and group-by-time interaction, and an AR(1) covariance structure. Analyses of secondary 
outcomes for pulmonary function testing will also use this approach. Further analysis of treatment group 
differences will be assessed using a variation of the Cox Proportional Hazards model with a counting process 
input of start and stop times for each event and the robust sandwich covariance estimator of Lin.63 This approach 
models hazard for recurrence under the assumption of proportional mean processes and generates hazard ratios 
with 95% confidence intervals and Wald Chi-Square statistics for testing the null hypothesis. We will evaluate 
the influence of the event rate, average IEI as covariates and calculate between treatment group hazard ratios. 
We will also evaluate the influence of randomization stratifying variables as covariates of the estimate of the 
between treatment group difference: sex (female vs male), age (child vs adolescent) and hydroxyurea use (use 
vs non-use). For participants in each treatment group who withdraw from the study prior to completion of the 
protocol, we will repeat the primary intent-to-treat analysis with the subset of participants who completed the 
two-year study in a “per protocol” analysis. Finally, treatment group differences in adverse events of least grade 
2 severity will be assessed with chi-square or Fisher’s Exact test. We will only present comparisons of events 
where three or more instances are reported over the two-year treatment period.  
 
 
DATA AND SAFETY MONITORING 
Adherence to protocol: Conduct of the clinical trial will be in accordance with this new protocol for the ViDAS-2 
study in our Investigational New Drug application. This protocol includes the appointment of a Clinical Trial Safety 
Officer to review laboratory results as they become available, and the formation of a Data and Safety Monitoring 

Board (DSMB) consisting of an expert in pediatric sickle cell disease, a biostatistician, and a Pediatric 
Endocrinologist with expertise in vitamin D metabolism. The DSMB will meet bi-annually. 
 
Safety monitoring and review plan: For the first month after initiation of study treatment, the Clinical Research 
Coordinator  will telephone each study participant (or their parent or guardian) weekly to ask about any adverse 
experiences or any change from baseline status. To maintain blinding of participant treatment assignment during 
safety monitoring, a Clinical Trial Safety Officer, a physician expert in Pediatric Endocrinology and vitamin D 
metabolism, will be appointed to review laboratory results as they are performed during the course of the clinical 
trial to detect any abnormality requiring intervention. Abnormalities that require dose adjustment or other 
intervention will be reported immediately to the Sponsor-Investigator and summarized for annual reports to the 
U.S. Food and Drug Administration (FDA), the DSMB, and the Columbia University Institutional Review Board 
(CU IRB). 
 
Reporting of adverse events:  All identified Adverse Events will be reported to the Sponsor-Investigator. The 
Sponsor-Investigator will determine whether the event is serious or an Unexpected Suspected Adverse Reaction 
and requires expedited reporting. Serious Adverse Events (SAEs) that meet criteria for FDA and/or CU IRB 
reporting, i.e., unexpected, suspected to be related to study drug (FDA requirements), or unanticipated problems 
(CU IRB requirement) will be reported within 24 hours to the Sponsor-Investigator for review. Other SAEs that 
do not meet the above criteria will be reviewed by Clinical Trial Safety Officer with reports to the Sponsor-
Investigator quarterly. The Sponsor-Investigator must also report SAEs that meet criteria for FDA and/or CU IRB 
reporting to the DSMB promptly within one week, while other SAEs that do not meet the above criteria will be 
reported to the DSMB Chair quarterly. The Sponsor-Investigator is required to report to the FDA serious, 
unexpected, suspected adverse event within 15 days, and within 7 days if fatal. A serious, unexpected suspected 
adverse reaction is a serious adverse event not previously observed with the drug and that has a reasonable 
possibility of having been caused by it. The CU IRB requires reporting of all Unanticipated Problems promptly 
and not later than one week, as well as during continuing annual review. An Unanticipated Problem is defined 
as any incident, outcome or experience involving risks to subjects or others in any human subject research that 
is unexpected, related or possibly related to research participation, and suggests that the research places 
subjects or others at a greater risk of harm (physical, psychological, economic or social harm) than was 
previously known or recognized. Adverse Events that do not meet the criteria of a SAE will be reported the DSMB 
during biannual review.  
 
Clinical Trial Data and Safety Monitoring Board review: The Trial Statistician and Trial Epidemiologist will prepare 
a report for the DSMB at the end of Treatment Year 1 and Treatment Year 2. Data will be presented in a blinded 
fashion and study subjects will be identified only in the case of a safety concern. Biannual (and, if needed, more 
frequent) safety reviews will be conducted. Consideration for early termination of the trial will include: (i) failure 
of enrolled participants to meet entry criteria, (ii) differences in the baseline characteristics of the two study 
groups (despite gender- and age-stratified randomization), (iii) excessive or unbalanced loss of study 
participants, and (iv) other considerations to be determined by the DSMB. 
 
Data management and quality: The Sponsor-Investigator or a designated Study Investigator will review all 
completed data collection forms biannually for protocol compliance and for completeness and accuracy of data. 
A statement of the results of this review will be included in the annual report to the FDA Orphan Products 
Program, in the annual IND report to the FDA, the CU IRB, and in the biannual reports to the Clinical Trial Data 
and Safety Monitoring Board and. 
 
Reviews of clinical trial progress:  Progress in the conduct of the clinical trial will be reviewed at least annually 
and more frequently as required by the FDA and the CU IRB, and biannually by the Clinical Trial Data and Safety 
Monitoring Board, 
 
Records to be kept: Case Report Forms (CRF) will be provided for each subject.  Subjects will not be identified 
by name on any study documents. After patient and subject identifiers have been replaced by study identifiers, 
the results of all clinical and laboratory assessments will be recorded on Case Report Forms for each subject. In 
a similar manner, demographic data and data from the medical history, physical examination will be recorded on 
Case Report Forms. All data on the Case Report Forms will be legibly recorded in black ink or typed. Corrections 
will be made by striking through the incorrect entry with a single line and then entering the correct information 

adjacent to it. The correction will be initialed and dated by the Sponsor-Investigator or a designated, qualified 
staff member. Any requested information that is not obtained as specified in the protocol will have an explanation 
noted on the Case Report Forms as to why the required information was not obtained. Data for the Case Report 
Forms will be entered into a computerized database and entered into a Columbia University Certified 
Environment encrypted and protected with a strong password, in accordance with Columbia University 
encryption policies 
 
Confidentiality of study data:  The Sponsor-Investigator will be responsible for the accumulation and 
maintenance of appropriate data files and the preservation of the confidentiality and security of these files. The 
need for strict confidentiality of all study records will be emphasized to the staff of the study. Subjects will not be 
identified by name on any study documents and will be identified solely by a Study Identification Number. All 
reports and summaries prepared by the Investigator will be presented in such a way that no individual participant 
can be identified. The data collected in this study will remain under the control of the Investigator and will be kept 
confidential with the sole exception that Hospital or Government authorities may have access to records 
containing the identity of study volunteers. If publications result, volunteer names will not be used. All forms, 
computer tapes and diskettes will be kept in locked cabinets.  All computerized files will be encrypted and 
protected with a strong password, in accordance with Columbia University encryption policies. 
 
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