Conditions: Cystic Fibrosis Dysbiosis
Keywords: Cystic Fibrosis Gastrointestinal Dysbiosis Vitamin D Prebiotic Inulin Gut microbiome Intestinal inflammation Intestinal absorption
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 40
Healthy volunteers: no

Brief summary:
The study will assess if administration of high-dose vitamin D and a commonly used prebiotic (inulin) is effective to reduce gastrointestinal dysbiosis and to improve critical intestinal functions in Cystic Fibrosis with the additive or synergistic effects of the combination of vitamin D + inulin.

Detailed description:
Cystic fibrosis (CF) is the most common life-shortening genetic condition among Caucasians in the United States. Individuals with CF have an altered gastrointestinal (GI) microbiota, which may be a result of chronic systemic inflammation and infection, frequent use of antibiotics, and/or medically prescribed and habitual high-fat/high-calorie diets.

The study will assess if administration of high-dose vitamin D and a commonly used prebiotic (inulin) is effective to reduce gastrointestinal dysbiosis and to improve critical intestinal functions in Cystic Fibrosis.

Arms:
arm 1: Vitamin D3 50,000 IU/week and 12 g/day chicory-derived prebiotic inulin for 12 weeks arm 2: Vitamin D3 50,000 IU/week and 12 g/day corn-derived maltodextrin/day as the prebiotic placebo for 12 weeks arm 3: Matching to Vitamin D3 placebo capsules, and 12 g/day chicory-derived prebiotic inulin for 12 weeks arm 4: Matching to Vitamin D3 placebo capsules, and 12 g/day corn-derived maltodextrin/day as the prebiotic placebo for 12 weeks

Interventions: intervention 1: Vitamin D3 intervention 2: Placebo vitamin D3 intervention 3: Inulin intervention 4: Placebo Inulin
Intervention descriptions: intervention 1: High-dose vitamin D3 50,000 IU /week for 12 weeks intervention 2: Matching to Vitamin D3 placebo capsules for 12 weeks intervention 3: Chicory-derived prebiotic inulin 12 g/day for 12 weeks intervention 4: Corn-derived maltodextrin 12g/day as the prebiotic placebo for 12 weeks

Protocol document:
Prot_SAP_000.pdf: 
Title: Vitamin D and Prebiotics for Intestinal Health in Cystic Fibrosis
NCT #: NCT04118010
Date: Oct/31/2022


IRB Research Protocol
1. Title: Vitamin D and Prebiotics for Intestinal Health in Cystic Fibrosis
Short title: Vitamin D and Prebiotics in Cystic Fibrosis
Principal Investigator: Vin Tangpricha, MD, PhD
Funded as a “Pilot and Feasibility Study” by the Cystic Fibrosis Foundation
Funding start date: 9/1/2019. Notice of Award Received July 2019.
Version 10/31/22
Co-Investigators:
Thomas R. Ziegler, MD
William Hunt, MD
Jose Binongo, PhD
Jessica A. Alvarez, PhD, RD
2. Abstract
Patients with CF have dysfunctional intestinal microbiota. We hypothesize that administration of 
high-dose vitamin D and a potent prebiotic will be effective and simple strategies to reduce CF-
related intestinal dysbiosis and improve critical intestinal functions.  We further hypothesize that 
the combination of vitamin D + inulin will be additive or synergistic in this regard. We therefore 
propose a pilot and feasibility, 2 x 2 factorial design, placebo-controlled, randomized controlled 
trial to test the feasibility of our approach in a larger study. We will test our hypothesis with the 
following aims: 1. Determine the impact of vitamin D and the prebiotic inulin on gut microbiota 
richness and diversity in stable outpatient adults with CF. We will assess the change in major gut 
microbiome characteristics from baseline and targeted microbiome-dependent metabolites 
(secondary endpoints) in 40 randomized CF adults block-randomized to either: 1) high-dose 
vitamin D3 (50,000 IU weekly plus prebiotic placebo; 2) prebiotic (inulin) plus placebo vitamin 
D3); 3) combined vitamin D3 and inulin; and 4) double vitamin D and prebiotic placebo. Aim 2. 
Our primary endpoint will be changes in gut and sputum microbiome.
3. Introduction and Background:
Cystic fibrosis (CF) is the most common life-shortening genetic condition among 
Caucasians in the United States.  Individuals with CF have an altered gastrointestinal (GI) 
microbiota, which may be a result of chronic systemic inflammation and infection, frequent use 
of antibiotics, and/or medically prescribed and habitual high-fat/high-calorie diets1.  Limited 
studies to date document that the GI microbiota of CF individuals is disordered—resulting in a 


less diverse, pro-inflammatory GI microbiota profile (dysbiosis) compared to a healthy, non-
inflammatory GI microbiota composition in individuals without CF.  Further, the severity of the 
CFTR mutation impacts the degree of the GI dysbiosis, which begins early in childhood and 
persists into adulthood2. In addition, GI dysbiosis has been associated with significant CF 
morbidity, including impaired growth in children3, nutrient malabsorption4, GI tract 
inflammation4, decreased pulmonary function5, and increased need for IV antibiotics5. Thus, 
efforts to correct or improve GI microbiota dysbiosis may result in improved CF clinical 
outcomes. 
Studies designed to correct GI dysbiosis with probiotics to reduce pulmonary 
exacerbation and GI tract inflammation have been inconclusive to date, in part due to limited 
sample sizes and study quality issues6. Prebiotics, which are non-digestible fermentable 
oligosaccharides, such as fructo-oligosaccharides (e.g. inulin) and resistant starches (e.g. pectin), 
occur naturally in various plants and are routinely used as dietary supplements and effectively 
modulate and diversify the GI microbiota in humans7.  Also, relevant to this proposal, vitamin D 
deficiency has been associated with GI dysbiosis in rodent models of CF and in clinical studies 
with individuals with CF8. We recently conducted a pilot, double-blind, randomized controlled 
trial (RCT) examining the impact of high-dose vitamin D3 supplementation on GI microbiota in 
adults with CF and concomitant vitamin D deficiency.  
Vitamin D3 supplementation decreased indexes of GI dysbiosis, particularly in subjects 
with prevalent vitamin D deficiency.  Subjects receiving vitamin D3 (vs. placebo) demonstrated 
decreased abundance of potentially pathogenic bacteria while less pathogenic bacteria were 
enriched9. In a separate study using high-resolution plasma metabolomics, high-dose vitamin D 
in adults with CF during an acute pulmonary exacerbation altered metabolic pathways potentially 
related to gut microbial metabolism10.  Favorable changes to the GI microbiota have also been 
observed with vitamin D supplementation in adults without CF11.  Therefore, we hypothesize that 
administration of high-dose vitamin D and a commonly used prebiotic (inulin) will be effective 
strategies to reduce GI dysbiosis and to improve critical intestinal functions in CF with the 
additive or synergistic effects of the combination of vitamin D + inulin. 
To address our hypotheses, we propose a pilot and feasibility, placebo-controlled, 2 x 2 
factorial, double blind, randomized controlled trial (RCT) of vitamin D ± prebiotic in adults with 
CF and insufficient vitamin D status.
Aim 1. Determine the impact of high-dose oral vitamin D and the prebiotic inulin on gut 
microbiota richness and diversity, and associated metabolomic changes, in stable 
outpatient adults with CF. In a 12-week RCT, we will assess the feasibility of a 2x2 factorial 
design RCT (primary-endpoint) to change major gut 
microbiome characteristics from baseline (Shannon 
diversity index) and targeted microbiome-dependent 
metabolites in 40 CF adults block-randomized to either: 1) 
high-dose vitamin D3 50,000 IU weekly (plus prebiotic 
placebo); 2) inulin (12 g/day) prebiotic (plus vitamin D 
placebo); 3) combined vitamin D3 and inulin; and 4) 
vitamin D placebo and prebiotic placebo. At baseline and 


12 weeks, we will determine changes in GI microbiota composition, diversity, and richness using 
16S rRNA gene sequencing and microbiome-dependent metabolites pathways in stool and 
plasma using high-resolution metabolomics analysis.
3.1 Scholarly rationale and prior literature 
Intestinal Microbiota in CF
Patients with CF have aberrant intestinal microbiota, as evidenced by decreased bacterial 
diversity and overabundance of pathogenic bacteria starting in infancy12,13. Many factors in CF 
may lead to aberrant intestinal microbiota including frequent use of antibiotics, small bowel 
bacterial overgrowth, disturbed bowel motility, alterations in mucin clearance, pancreatic 
insufficiency, and intestinal inflammation14,15,16.  It is also possible that the habitual high-fat CF 
diet further promotes dysbiosis17. In a study of adolescents with CF, the severity of the CFTR 
mutation (presence of F508del) was associated with more abundant pathogenic bacteria such as 
E. coli and E. biforme, less abundant beneficial bacteria, and less microbial diversity18, 
suggesting an inherent dysbiosis caused by the CFTR defect. Rodent models of CF have 
mirrored the GI dysbiosis found in patients with CF in that rodents with mutations in the CFTR 
have less bacterial diversity and richness compared to wild type (WT) animals19,20. It is, 
therefore, clear that GI dysbiosis exists in patients with CF; however, the clinical significance of 
this state is unknown.  
Antibiotics alter intestinal microbiota
Patients with CF often receive combination antibiotic therapy to ensure coverage against 
the highly pathogenic bacteria Pseudomonas aeruginosa21.  Broad spectrum antibiotic therapy 
alters the intestinal microbiota by disrupting local host-microbe communities, leading to 
decreased bacterial richness and diversity22. In a study of adults with CF during a period of 
stability, the number of IV antibiotic courses was inversely related to the intestinal microbiota 
diversity23. A longitudinal study of a single patient with CF found that courses of antibiotic 
therapy decreased intestinal diversity and richness24. Therefore, wide spectrum antibiotic therapy 
remains a major factor in the intestinal dysbiosis found in patients with CF.
Probiotics in cystic fibrosis
Early, generally small, studies suggested that probiotics may improve GI health and 
nutrition in patients with CF. A 6-month RCT of a Lactobacillus reuteri-based probiotic given to 
adults with CF found increased GI diversity and decreased abundance of the potentially 
pathogenic Proteobacteria phylum compared to placebo25. Bruzzese et al found Lactobacillus 
GG given to children with CF experienced reduced abundance of potentially pathogenic bacteria 
and improved intestinal inflammation as assessed by fecal calprotectin26. Jafari et al also found 
that probiotics given to children improved quality of life and rate of pulmonary exacerbations 
compared to placebo27. In contrast, a large study of Lactobacillus GG in children up to age 16 
found no impact of probiotics on pulmonary exacerbations or other clinical outcomes such as 
BMI and FEV128.  There are currently too few well designed RCTs investigating the role of 
probiotics in patients with CF to recommend this therapy for all patients with CF29,30. 
Furthermore, probiotics have been reported to cause bacteremia in immunocompromised patients 


which is of concern in patients with CF31,32.  Thus, the efficacy and safety of probiotics to 
improve GI dysbiosis and subsequent clinical CF outcomes is not well-established.
Prebiotics, gut microbiota and risk respiratory infections
Prebiotics are non-digestible dietary fibers that include inulin-type fructans and 
oligosaccharides that promote the formation of beneficial bacteria in the gut33. Prebiotics do not 
contain bacteria and thus are not associated with bacteremia.  A number of RCTs have 
confirmed that prebiotics can change gut microbiota in adults and children without CF34,35,36. 
Additionally, some RCTs have demonstrated that changes in gut microbiota were associated with 
improved lung health.  Ranucci et al reported in infants without CF enrolled in a 24 month RCT 
that an oligosaccharide-enriched formula protected against respiratory infections and increased 
the abundance of Bifidobacteria, a genera associated with protection against respiratory 
infections37.   Another RCT examining the prebiotics galacto-oligosaccharides and polydextrose 
found in children without CF found that the prebiotic formula was associated with fewer 
episodes of respiratory infections compared to children ingesting a standard formula38. Although 
prebiotics have demonstrated efficacy in promoting beneficial gut microbiota and improving 
respiratory infections in children without CF, no studies have been published using prebiotics in 
children and adults with CF. Prebiotics potentially have a better safety profile than probiotics 
since they are not associated with risk of bacteremia.
Preliminary Results: Vitamin D status and intestinal microbiota in CF
Others have shown that vitamin D deficient mice or vitamin D receptor KO mice, have increased 
intestinal dysbiosis and GI inflammation39. We explored the role for vitamin D in altering the 
intestinal microbiota in a pilot randomized placebo-controlled trial of adults with CF. We 
evaluated 41 adults with CF who were clinically stable and were not on recent IV antibiotics.  
Prior to any intervention, we found that vitamin D insufficient subjects displayed a higher 
abundance of potentially more pathogenic bacteria in the stool from the class of 
Gammaproteobacteria (class representing gram- bacteria such as Pseudomonas) (Figure 2).
We then randomized 23 subjects were 
vitamin D insufficient to vitamin D3 50,000 
IU weekly for 12 wks or placebo.  We 
found that treatment with vitamin D3 
demonstrated significant changes in the 
clustering of microbial populations 
compared to placebo (Figure 3).  Subjects 
who received vitamin D had significant 
Figure 2 Relative abundance of 
Gammaproteobacteria in the stool of 
adults with CF based on vitamin D status: 
vitamin D (25(OH)D) insufficient (<30 
ng/mL) or vitamin D sufficient (≥30 ng/mL).  
Subjects with vitamin D insufficiency had more 
abundance of this class of potentially 
pathogenic bacteria compared to those with 
vitamin D sufficiency.
Figure 3 Principal 
coordinate (PC) analysis 
based on unweighted 
UniFrac distance matrices 
generated with stool 16S 
rRNA gene sequencing. 
Adults with CF and vitamin D 
insufficiency (25(OH)D < 30 
ng/mL) were randomized to 
vitamin D (blue) or placebo 
(red).  Stool was collected at 
baseline (panel A) and 12 
weeks of intervention (panel 
B). Principal coordinate 
analysis demonstrates 
differential clustering of 
bacterial communities before 
and after treatment.  


increases in the taxa of Lactococcus (generally accepted as beneficial bacteria) and significant 
decreases in Veillonella and Erysipelotrichaceae (potentially more pathogenic bacteria) (Figure 
4)
In a separate pilot study of CF patients hospitalized for acute pulmonary exacerbation, high-
resolution plasma metabolomics indicated that butanoate and tryptophan metabolism, among 
several other pathways, were altered by high-dose vitamin D compared to placebo40. Butanoate 
(butyrate) is a major metabolite arising from the bacterial fermentation of dietary fiber, and 
tryptophan metabolism is heavily influenced by the gut microbiota41,42. Together, these data 
provide strong preliminary data for a potential effect of vitamin D treatment on gut microbiota, 
as well as for the utility of high-resolution metabolomics to assess metabolic responses to an 
intervention. 
4.1) Specific Aims and Objectives
We hypothesize that administration of high-dose 
vitamin D and a commonly used prebiotic 
(inulin) will be effective strategies to reduce GI dysbiosis and to improve critical intestinal 
functions in CF with the additive or synergistic effects of the combination of vitamin D + 
inulin. 
To address our hypotheses, we propose a pilot and feasibility, placebo-controlled, 2 x 2 
factorial, double blind, randomized controlled trial (RCT) of vitamin D ± prebiotic in 
adults with CF and insufficient vitamin D status. 
4.2) Specific aims:
Aim 1. Determine the impact of high-dose oral vitamin D and the prebiotic inulin on gut 
microbiota richness and diversity, and associated metabolomic changes, in stable 
outpatient adults with CF. In a 12-week RCT, we will assess the feasibility of a 2x2 factorial 
design RCT (primary-endpoint) to change major gut microbiome characteristics from baseline 
(Shannon diversity index) and targeted microbiome-dependent metabolites in 40 CF adults 
block-randomized to either: 1) high-dose vitamin D3 50,000 IU weekly (plus prebiotic placebo); 
2) inulin (12 g/day) prebiotic (plus vitamin D placebo); 3) combined vitamin D3 and inulin; and
4) vitamin D placebo and prebiotic placebo.
At baseline and 12 weeks, we will determine changes in GI microbiota composition, diversity, 
and richness using 16S rRNA gene sequencing and microbiome-dependent metabolites pathways 
in stool and plasma using high-resolution metabolomics analysis.
Figure 4 Linear discriminant analysis Effect Size 
(LefSe) in adults with CF and vitamin D deficiency 
randomized to vitamin D (green) or placebo (red).  
The LefSe analysis demonstrates genera and species 
substantially and differentially abundant in the stool 
samples from subjects randomized with vitamin D 
compared to those randomized to placebo.


Primary outcome measure: To compare phylotype richness of the respiratory and gastrointestinal 
tract microbiota before and after supplementation with high dose vitamin D ± inulin. These will 
be measured using ecological diversity measures including the Shannon index and the Species 
Richness index.
5. Study design and methods
5. 1 Overview of study design (Figure)
Study synopsis: 60 adults will sign consent for 
screening with 40 randomized with CF and 
vitamin D intake less than 2000 IU daily will be 
randomized in a 12-week RCT to: 1) high-dose 
vitamin D3 (50,000 IU weekly plus 12 g corn-
derived maltodextrin/day as the prebiotic placebo);       
2) Chicory-derived inulin (12 g/day) prebiotic plus
placebo vitamin D3); 3) combined vitamin D3 and 
inulin; and 4) placebo vitamin D3 and placebo prebiotic. Stool and serum will be collected at baseline and 
at 12 weeks in the Clinical Research Center (CRC) of the NIH-funded Georgia Clinical and Translational 
Science Alliance (Georgia CTSA). We will assess the feasibility of this approach and collect preliminary 
data on changes in gut microbiota diversity (Shannon Index as primary), composition, and richness 
from baseline over time using 16S rRNA gene sequencing to inform future studies. Additional 
measures will include high-resolution stool and plasma metabolomics.
a.
Enrollment:
1. Patient Selection: We will screen for study patients from adults with CF seen for routine
care at the Emory CF Center. Inclusion Criteria: 1) male and female patients (age > 18
years) with confirmed CF by genetic mutation and/or sweat chloride testing, 2) stable
pulmonary function (no greater than 5% difference in FEV1% predicted from previous
measurement within the past year), 3) not currently on oral or systemic antibiotics for
pulmonary exacerbation 4) use of CFTR modulator therapy is allowed Exclusion
Criteria: 1) active GI disease, abdominal pain and/or diarrhea, 2) chronic kidney disease
worse than stage 3 (eGFR < ml/min per 1.73 m2), 3) any vitamin D supplement use
>2,000 IU or vitamin D analogue, (patients who are taking more than 2,000 IU of
vitamin D must agree to stop the vitamin D for 6 weeks and take less than 2,000 IU
of vitamin D during the study) 4) use of immunosuppressants or history of organ
transplantation, or 5) current use of probiotics or prebiotics.
2. Subject identification and initial screening: We propose to consent 60 subjects at
least to randomize 40 subjects in the following arms. We propose to study 40 subjects
(10 in each arm) in this 2x2 factorial design study (statistical considerations below).


Patients with CF who are scheduled for routine outpatient appointments will be pre-
screened for eligibility by reviewing the electronic medical record.  
3. Informed consent and subject enrollment: Informed consent will be obtained from the
subject or the legally authorized representative. Amid the coronavirus crisis, informed
consent will be obtained via secure videochat via Emory Zoom. The patient’s identity
will be confirmed with two forms of identification with full name and date of birth.  The
research coordinator or Drs. Alvarez, Ziegler, or Tangpricha will obtain informed consent
from the eligible subjects. The informed consent will be signed securely via DocuSign
and returned via encrypted email. If the subject can not return via DocuSign, the subject
will take a photo of the signed informed consent and return via encrypted email and mail
in the wet signature. Once the DocuSign form is received or the photo/wet signature is
received, the subject will be mailed the kits for stool and sputum collection as well as the
vitamin D/prebiotic/inulin/placebo.
4. Baseline studies: Stool samples will be collected using a stool kit provided to the subject.
Subjects will be provided in person or via return mail with a stool kit and a prepaid
envelope to have the sample mailed directly to the study lab within the next 48 hours.
These will be stored at -80 degree C. Subjects will provide a sputum sample in person or
via return mail.  Sputum samples will be collected via a sputum kit on day of visit and
stored at -80 degree C. Relevant medical information will be extracted from the medical
record and from questions to the participant.
5. Intervention:
Vitamin D dose: We have conducted a number of studies demonstrating very effective 
use of vitamin D3 at a dose of 50,000 IU weekly in the adult CF population.  Our study 
in 2009 demonstrated that vitamin D3 was superior to vitamin D2 or UV lamp therapy in 
CF43. Subjects receiving weekly doses of oral vitamin D3 50,000 IU for 12 weeks raised 
mean serum 25(OH)D concentrations from 21 ± 10 to 47 ± 20 ng/mL (p<0.001) with 
100% of subjects achieving a serum 25(OH)D greater than 30 ng/mL. We conducted a 
more recent study examining our dosing regimen of vitamin D3 50,000 IU vs placebo 
weekly for 12 weeks on gut microbiota in patients with CF and found that serum 
25(OH)D increased significantly in the vitamin D3 group from a mean of 24.99 ± 5.05 to 
45.29 ± 21.01 (p<0.05) compared to no change in placebo.  Therefore, for this study we 
will use our established vitamin D dosing regimen of vitamin D3 50,000 IU once weekly 
for 12 weeks. As in past studies, we will obtain the vitamin D3 and matching placebo 
capsules from Bio-Tech Pharmacal, Inc.
Inulin: Inulin is non-digestible fiber found in many food products. Inulin is 
alsoavailable as an over the counter supplement. Inulin will be provided from the brand 
name “Now Foods”. This is a commercially available inulin supplement that is available 
in powder form. We have been in contact with the investigational pharmacy who will 
make sachets of 12 grams of Jetsu inulin for the active inulin group and 12 grams of 


maltodextrin which is a food additive and commonly used as a control to inulin.  
Subjects will be asked to dissolve a sachet daily into a beverage of their choice (coffee, 
tea, water) and to ingest this daily.
 All study drugs will be dispensed by the Investigational Drug Pharmacy.   
6. Follow-up studies: Patients will be followed throughout the duration of the study for 3
months. Subjects will provide their stool and sputum sample either in person or return by
mail. For those subjects seen in person, participants will be seen for study follow-up
visits at 3 months (+/- 4 weeks) as scheduled for their usual medical care (routine out-
patient follow-up visits). Interim phone calls will be made to participants on a bi-weekly
basis after week 1 to remind about taking pills. This will be done by the co-investigator.
At the end of 3 months, we will obtain repeat stool and sputum samples from the entire
cohort.
Procedures to be performed:
a.
A history and physical exam will be performed as routine standard of care for patients
presenting to the Cystic Fibrosis Center. Relevant medical information will be extracted
from the medical record and from questions to the participant
b.
Stool collection: Participants will be asked to provide a stool sample in a collection
container for analysis of stool microbiota. This will be done upon enrollment (baseline)
and at 3 month follow-up.
c.
Sputum collection: Participants will be asked to expectorate sputum into a collection
container for analysis of airway microbiota. This will be done upon enrollment (baseline)
and at 3 month follow-up.
d.
Dietary history will be taken at baseline and at the end of the study to determine if there
are any changes in dietary pattern pre and post intervention.
7. Risk/benefits:
Potential risk: The risk of participating in this study is minimal.  There is the question of
withholding treatment in subjects who are vitamin D insufficient and randomized to receive
placebo however, there is currently no evidence that short duration (3 month) period of not
receiving supplemental vitamin D affects morbidity and mortality in CF. Moreover, at the
end of the 3 months, patients in both groups will be treated with 1000 IU daily of vitamin D
to ensure that the group that didn’t get vitamin D will be treated. Inulin is not systematically
absorbed so there should be no systemic side effects. However, inulin can cause


gastrointestinal side effects such as bloating, diarrhea and excess gas. We will monitor for 
this potential side effect during our biweekly calls for adherence to the study drug.
Additional risks will be associated with confidentiality issues surrounding administration of 
the patient questionnaire and collection/recording of data, but steps will be taken to minimize 
these risks as discussed below. 
Potential benefits:  There may be no specific benefit to patients participating in the study.  If 
our hypotheses are confirmed, and vitamin D plus/minus inulin supplementation improves 
the beneficial microbiota in CF. Future studies will investigate the cause of dysbiosis and 
other potential clinical outcomes. Therefore, the anticipated benefits of the proposed research 
study outweigh the potential risks to participating subjects. 
8. Data collection
The following chart demonstrates when and from where data items will be collected.  All of the 
sputum, and stool samples that are collected at enrollment and at the 3 month follow-up visit will 
be stored for future analysis of microbiota.  Samples will be stored for long term storage for 
future studies only if subjects provide written informed consent with a second informed consent 
to grant long term storage of samples.
 Data Items
Source and timeline of collection
Demographic Information
Self-report; Electronic Medical Record
Medical history (e.g., CF genotype , Dietary 
supplement and medication intake and 
antibiotic treatment and pulmonary 
exacerbations in the past 3 months)
Completed at enrollment.  
Self-report; Electronic Medical Record 
Sputum microbiota analysis 
Sputum Sample collected at baseline and 
month 3 during standard follow-up visit to 
the CF center for routine care.
Stool microbiota analysis
Stool Sample collected at baseline and month 
3 during standard follow-up visit to the CF 
center for routine care.
Spirometry (including FEV1 and FVC) – as 
standard of care by inpatient and outpatient 
hospital team.
Obtained from electronic medical Record as 
pulmonary function tests are measured at 
baseline and 3 months as part of routine care.
8.1 Specimens to be collected: Specimens collected at baseline and follow-up visits will include 
stool and sputum. 


8.2 Randomization: Subjects will be randomized to two interventions (vitamin D3 vs placebo) 
and (prebiotic vs placebo). Treatment assignments will be performed by blinded allocation 
according a schedule maintained by biostatisticians of the Georgia CTSA. The subjects’ 
physicians will be informed of their participation in the trial, and the electronic medical record at 
Emory will be flagged indicating that the patient is participating in this trial to prevent the use of 
vitamin D or pre/probiotics. Our primary analysis will be intention-to-treat. If any subjects 
receive therapies that impact the microbiome such as antibiotics or immunosuppressant drugs, 
they will be analyzed in the groups to which they were randomized. In a secondary analysis we 
will make note of the subjects who these therapies and determine if there were any between 
group differences in the initiation of these therapies.  The Investigational Drug Pharmacy will 
provide the randomization scheme. 
8.3 Archived specimens: Study subjects may sign an additional consent form for long term 
storage of samples.  In addition to immune function, vitamin D has been linked to several non-
skeletal disease states and conditions in the general population that may be applicable to CF. One 
example is diabetes, a common co-morbidity in patients with CF.  It is, therefore, possible that 
remaining specimens will be banked for future study related to vitamin D in CF but beyond the 
specific scope of the current study.  Any stored samples will be de-identified with a specific code 
whose identity can only be accessed by authorized study personnel appointed by the PI.  
Specimens will be stored in a -80° freezer at the Vitamin D and Bone Research Laboratory at the 
Emory University School of Medicine Division of Endocrinology and Metabolism.
9. Participant selection
9.1 Requested sample size and expected refusal or withdrawal rate:  We propose to recruit 
60 subjects to sign consent in order to have 40 randomized at the study site.  This is a pilot study 
which will provide critically needed data to adequately power a larger study. The CF Foundation 
funded this as a pilot and feasibility study.
9.2 Inclusion/exclusion criteria:
We will screen for study patients from adults with CF seen for routine care at the Emory CF 
Center. 
Inclusion Criteria: 1) male and female patients (age > 18 years) with confirmed CF by genetic 
mutation and/or sweat chloride testing, 2) stable pulmonary function (no greater than 5% 
difference in FEV1% predicted from previous measurement within the past year), 3) not 
currently on oral or systemic antibiotics for pulmonary exacerbation, and 4) use of CFTR 
modulator therapy is allowed 
Exclusion Criteria: 1) active GI disease, abdominal pain and/or diarrhea, 2) chronic kidney 
disease worse than stage 3 (eGFR < ml/min per 1.73 m2), 3) any vitamin D supplement use 
>2,000 IU or vitamin D analogue, 4) use of immunosuppressants or history of organ 
transplantation, or 5) current use of probiotics or prebiotics.


9.3 Subject recruitment plan: Recruitment will be performed through referral from the 
outpatient CF Clinic.  
9.4 Screening for eligibility: All patients with CF will be screened for eligibility at presentation 
to the CF clinic for routine visit by reviewing the subject’s vitamin D intake. Screening will be 
completed daily by the research coordinator. Patients who have met the above inclusion criteria 
and have met none of the exclusion criteria on our initial assessment will be offered the 
opportunity to participate in our study.  The study personnel will explain the study protocol in 
detail in the consent form as well as verbally.  Any additional information regarding questions or 
concerns from potential or confirmed study participants will be provided by the study 
coordinator.
9.5 Withdrawal from study: Participants may withdraw from the study at any time.  Automatic 
withdrawal occurs when a participant moves away from the center or refuses providing further 
blood, stool or sputum sample.
10. Statistical analysis
Statistical Considerations for Pilot Study: 
We received this funding from the CF Foundation to test the feasibility of this 2x2 study design.
We will evaluate the feasibility of recruitment, retention, assessment and implementation of the 
proposed interventions. We wish to identify any potential flaws in the study design and potential 
challenges in the subsequent large-scale study. Our measures of feasibility will focus on metrics 
of recruitment, retention, acceptability & completion as adopted from the NIH/NCCIH . Our 
target metric of success is compared to our previous single intervention study with vitamin D 
(Table).
Feasibility 
Question
Feasibility Metric
Prior Rate
Target Metric of 
Success
Recruitment
Number of subjects 
screened a month
16.2/month
>15/month
Recruitment
Number of Enrolled/Screen 
Fails
1:4.05
1:4 Enrolled/Screened
Retention
Number of dropouts and 
reasons for dropout
13% (3 out of 23 
enrolled)
<15%
Adherence
Number of subjects who 
adhere to assignment
Not recorded
>80%
Acceptability
Subject ratings for 
acceptability during and 
after participation
Not recorded
>3 on 5 point Likert 
scale
Completion
Number of subjects who 
complete all study 
procedures
73%
>70%
Completion
Duration from initiation to 
completion of pilot study
52 subjects in 15 
months
40 randomized subjects 
in 15 months


The pilot sample size of 40 randomized patients is based on the pragmatics of recruitment, 
patient flow, and budgetary constraints. We have preliminary data on vitamin D; however, we do 
not have data on prebiotics which necessitates this pilot study to adequately power a larger study. 
For the vitamin D arm, our preliminary data suggest a standard deviation of 1.2 of the diversity 
index. In our proposed pilot study consisting of 20 subjects in each vitamin D arm, we have 29% 
power to detect a between-group difference of 0.5 and 56% power to detect a difference of 0.75 
in the diversity index using a significance level of 0.05. For the prebiotic arm, we do not have 
preliminary data. However, if vitamin D and pre-biotic have a synergistic effect, there may be 
greater power to detect a between-group difference of 0.5 (or 0.75).  
The CF Foundation acknowledges that we are underpowered as a pilot study. However, our goal 
is not to evaluate efficacy of the proposed interventions but to examine the feasibility of 
conducting this pilot in a 2x2 factorial study of a larger scale. Moreover, the pilot data from this 
study will give us a first impression of the variability and effect sizes of the Shannon diversity 
index in the patient population of interest in each of the 4 treatment arms and thus assist in the 
sample size and power calculation of the subsequent larger study. In addition, these pilot data 
will inform power calculations for our secondary outcomes. Finally, the data that we collect from 
this pilot will help identify modifications needed in the planning and design of the subsequent 
study. We will also determine between-group differences, but given that low power is 
characteristic of pilot studies, statistical significance is not the focus of the analysis. We will also 
summarize patient characteristics at baseline and during follow-up using mean ± SD (or median 
and interquartile range, as appropriate) for continuous variables and frequency (percentage) for 
categorical variables. Statistics on the diversity index will also be reported at baseline and at 12 
wks for each of the 4 treatment combinations. A qualitative review will also be provided, and 
this will include, among others, an evaluation of the representativeness of the subjects, a 
discussion on how to refine the data collection tools, how to track patients better, and how to 
improve implementation of the proposed interventions.
11. Adverse event reporting:
All adverse events will be documented and reviewed by the PI. These will be enumerated and 
reported according to IRB policies. Any serious adverse events will be reported within 24 hours 
to the IRB. They will be reviewed by all investigators and a disposition determined before 
proceeding with further enrollment. 
12. Data and safety monitoring plan (DSMP)
The procedures associated with this research trial are associated with minimal risk. Therefore, no 
formal data safety monitoring board will be appointed.  Our data safety and monitoring plan will 
be as follows:  The physical risks associated with this study are associated with venipuncture, 
any adverse events associated with venipuncture will be monitored by the site study coordinator 
and reported to the study PI, Dr. Tangpricha. Adverse events will then be reported to the Emory 
IRB within the timeframe stipulated by IRB regulations.


Oversight of the progress and safety of the trial will be provided by the PI. Adverse events are 
not anticipated, but any occurring will be documented and reported according to Emory IRB 
policies and procedures. Cumulative adverse events and study progress summary will be 
communicated to the IRB at the time of continuing review.
Anticipated Adverse Events:
Untreated vitamin D deficiency: Subjects enrolled in the placebo arm of this study may have 
25(OH)D less than 30 ng/mL which is considered vitamin D insufficient by the present CFF 
guidelines. Vitamin D deficiency does not generally result in clinical symptoms. The study will 
be completed over the course of 3 months and thus long term consequences of low vitamin D 
would be unlikely. There is currently no evidence that short duration (3 month) period of not 
receiving supplemental vitamin D affects morbidity and mortality in CF. Moreover, at the end of 
the 3 months, both groups will be treated with 1000 IU Vitamin D daily to ensure that the group 
that did not get vitamin D will be treated.
Vitamin D Toxicity/Hypercalcemia: One of the potential risks of taking high amounts of vitamin 
D is vitamin D toxicity. This would manifest as hypercalcemia.  The risk of this will be minimal 
since the dose of vitamin D that has been proposed in the intervention arm is 50,000 IU once a 
week for 3 months which has been recommended by the CF foundation as standard of care for 
treatment of vitamin D deficiency. 
Gastrointestinal Discomfort from Inulin: This is a dietary fiber and there could be some side 
effects as a result of the inulin administration including diarrhea, bloating, excess gas. We will 
record these reports as part of the study.
Patient Monitoring: will be performed by the P.I. and Co-Investigators
Plans for protecting subject confidentiality:
Recruitment and Informed Consent: Potential subjects will be approached during routine 
clinic visit in person or via telemedicine at the Emory cystic fibrosis center after discussion and 
approval of the subject’s care team.  Participation in our study is voluntary and their decision 
does not affect their healthcare in their CF center.  The site-PI or designee will discuss the study 
with each potential subject and ask questions to determine whether the subject (and legal 
guardian) comprehends the risks and benefits of the research.  If the subject (and legal guardian) 
decides to participate, they will provide written informed consent.
Confidentiality: All paper records and case report forms will be kept in locked file cabinets 
prior to data entry into the electronic database.  All electronic data will be coded, de-identified 
and stored on password protected computers and will be accessible only to the investigative 
team.  All data maintained in the computerized database will be accessible only with a login and 
protected password by members of the investigative team. After the study is completed, all data 
will be kept according to NIH and FDA regulations in a locked file. 
13. If applicable: pharmaceutical, biologic, and device information


We will obtain the vitamin D and matching placebo capsules from BioTech Pharmacal Inc and 
inulin from Now Foods.  At the initiation and at the end of 3 months, we will test the capsule for 
vitamin D content by an independent laboratory to ensure that the dosage is correct and remains 
stable throughout the duration of the study.
Our clinical trial using vitamin D and inulin is exempt from IND regulations [21 CFR 312.2 (b)].  
These is based on the fact that we are using standard supplementation protocols that have been 
recommended by the CF foundation for treatment of vitamin D Deficiency. There are no 
guidelines for inulin dosing. Moreover, our trial is not intended to support a new indication for 
use, will not support a change in advertising, does not change the route of administration, will be 
conducted in compliance with an IRB and informed consent and will not be represented as safe 
or effective for the purposes under investigation in this proposal.  
11. References and appendices
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