Clinical trial NCT03104855
Conditions: Cystic Fibrosis
Keywords: cystic fibrosis vitamin d catabolism
Sponsor: University of Washington
Start date: 2017-04-03; Completion date: 2023-09-06
Enrollment: 10 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The goal of this study is to define 25(OH)D3 catabolism in CF patients using gold standard pharmacokinetics studies. Specifically, the investigators will evaluate the metabolic clearance of 25(OH)D3 among participants with CF and matched control subjects. The goal of this work is to provide the first comprehensive characterization of vitamin D metabolism in CF patients and promote novel hypotheses for subsequent studies.

Detailed description:

Arms: arm 1: None
Interventions: intervention 1: d6-25-hydroxyvitamin D3
Intervention descriptions: intervention 1: intravenous administration of stable isotope-labeled D6-25(OH)D3

Protocol documents:
Prot_SAP_000.pdf: 
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` 
Clinical Research Protocol 
Clearance of 25-hydroxyvitamin D in cystic fibrosis 
 
Protocol Number: 
CLEAR-CF-01 
Version: 
0.2 
Investigational Product: 
Deuterated 25-dihydroxyvitamin D3  
IND Number: 
115016 
Development Phase: 
Phase 1 
Sponsor:  
 
Ian H. de Boer, MD, MS  
Funding Organization: 
National Institute of Diabetes and Digestive and Kidney 
Diseases (P30DK089507)  
National Institute of Diabetes and Digestive and Kidney 
Diseases (R01DK099199) 
Principal Investigator: 
Name: Ian H. de Boer, MD, MS 
Telephone: (206) 616-5403   
Fax: (206) 685-9399 
E-mail: deboer@u.washington.edu  
 


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TABLE OF CONTENTS 
 
1 
BACKGROUND 
9 
1.1 
Overview of Clinical Studies ............................................................................................. 9 
2 
STUDY RATIONALE 
9 
2.1 
Risk / Benefit Assessment ................................................................................................. 11 
3 
STUDY OBJECTIVES 
12 
3.1 
Primary Objective .............................................................................................................. 12 
3.2 
Secondary Objectives ........................................................................................................ 12 
4 
STUDY DESIGN 
12 
5 
CRITERIA FOR EVALUATION 
12 
5.1 
Primary Efficacy Endpoint ................................................................................................ 12 
5.2 
Secondary Efficacy Endpoints ........................................................................................... 13 
5.3 
Safety Evaluations ............................................................................................................. 13 
6 
SUBJECT SELECTION 
14 
6.1 
Study Population ................................................................................................................ 14 
6.2 
Inclusion Criteria ............................................................................................................... 14 
6.3 
Exclusion Criteria .............................................................................................................. 14 
7 
CONCURRENT MEDICATIONS 
15 
8 
STUDY TREATMENTS 
15 
8.1 
Method of Assigning Subjects to Treatment Groups ......................................................... 15 
8.2 
Blinding ............................................................................................................................. 15 
8.3 
Formulation of Test and Control Products ........................................................................ 16 
8.4 
Supply of Study Drug at the Site ....................................................................................... 16 
8.5 
Supply of Study Drug at the Site ....................................................................................... 17 
8.6 
Study Drug Accountability ................................................................................................ 17 
8.7 
Measures of Treatment Compliance .................................................................................. 17 
9 
STUDY PROCEDURES AND GUIDELINES 
17 
9.1 
Clinical Assessments ......................................................................................................... 17 
9.2 
Clinical Laboratory Measurements .................................................................................... 18 
9.3 
Pharmacokinetic Measurements ........................................................................................ 19 
9.4 
Research Laboratory Measurements .................................................................................. 19 
10 EVALUATIONS BY VISIT 
19 
10.1 Screening Visit (Study Visit 1) .......................................................................................... 19 
10.2 Baseline Visit (Study Visit 2, Day 0) ................................................................................ 19 
10.3 Study Visit 3 (Day 1) ......................................................................................................... 20 
10.4 Study Visit 4 (Day 4) ......................................................................................................... 20 
10.5 Study Visit 5 (Day 7) ......................................................................................................... 20 
10.6 Study Visits 6-10 (Day 14, 21, 28, 42, and 56) ................................................................. 20 
11 ADVERSE EXPERIENCE REPORTING AND DOCUMENTATION 
21 
11.1 Adverse Events .................................................................................................................. 21 


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11.2 Serious Adverse Experiences (SAE) ................................................................................. 22 
12 DISCONTINUATION AND REPLACEMENT OF SUBJECTS 
24 
12.1 Early Discontinuation of Study Drug ................................................................................ 24 
12.3 Withdrawal of Subjects from the Study ............................................................................. 24 
12.4 Replacement of Subjects.................................................................................................... 24 
13 PROTOCOL VIOLATIONS 
25 
14 STATISTICAL METHODS AND CONSIDERATIONS 
25 
14.1 Data Sets Analyzed ............................................................................................................ 25 
14.2 Demographic and Baseline Characteristics ....................................................................... 25 
14.3 Analysis of Primary Endpoint ........................................................................................... 25 
14.4 Analysis of Secondary Endpoints ...................................................................................... 25 
14.5 Interim Analysis ................................................................................................................ 26 
14.6 Sample Size and Randomization ....................................................................................... 26 
15 DATA COLLECTION, RETENTION AND MONITORING 
26 
15.1 Data Collection Instruments .............................................................................................. 26 
15.2 Data Management Procedures ........................................................................................... 26 
15.3 Data Quality Control and Reporting .................................................................................. 26 
15.4 Archival of Data ................................................................................................................ 26 
15.5 Availability and Retention of Investigational Records ...................................................... 27 
15.6 Monitoring ......................................................................................................................... 27 
15.7 Subject Confidentiality ...................................................................................................... 27 
16 ADMINISTRATIVE, ETHICAL, REGULATORY CONSIDERATIONS 
27 
16.1 Protocol Amendments ....................................................................................................... 27 
16.2 Institutional Review Boards and Independent Ethics Committees .................................... 28 
16.3 Informed Consent Form ..................................................................................................... 28 
16.4 Publications ....................................................................................................................... 29 
16.5 References ......................................................................................................................... 29 
 


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LIST OF ABBREVIATIONS 
AE 
adverse event 
ALT 
alanine aminotransferase 
AST 
aspartate aminotransferase 
BUN 
blood urea nitrogen 
CFR 
Code of Federal Regulations 
CRF 
case report form 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HIPAA 
Health Insurance Portability and Accountability Act of 1996 
ICF 
informed consent form 
ICH 
International Conference on Harmonisation 
IEC 
Independent Ethics Committee 
IRB 
Institutional Review Board 
IV 
intravenous 
PI 
Principal Investigator 
PK 
pharmacokinetic 
SAE 
serious adverse experience 
SGOT 
serum glutamic oxaloacetic transaminase 
SGPT 
serum glutamate pyruvate transaminase 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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PROTOCOL SYNOPSIS 
TITLE 
Clearance of 25-hydroxyvitamin D in cystic fibrosis 
SPONSOR 
Ian H. de Boer, MD, MS 
FUNDING 
ORGANIZATION 
National Institute of Diabetes and Digestive and Kidney 
Diseases (P30DK089507, R01DK099199) 
NUMBER OF SITES 
Single-site study 
RATIONALE 
Cystic fibrosis (CF) is among the most common autosomal 
recessive disorders and leads to serious, life threatening 
respiratory, infectious, and bone complications. Vitamin D 
deficiency is highly prevalent among CF patients, is unusually 
resistant to standard vitamin D supplementation regimens, and 
may contribute to osteoporosis and immune dysfunction. The 
prevalence of vitamin D deficiency, determined by circulating 
concentrations of 25-hydroxyvitmain D (25(OH)D), remains as 
high as 90% in CF patients despite modern practices of vitamin 
D supplementation and pancreatic enzyme replacement.  
A number of plausible hypotheses may explain the marked 
vitamin D deficiency intrinsic to CF. Pancreatic insufficiency, 
the most common gastrointestinal CF complication, leads to 
malabsorption of fat soluble vitamins A, D, E, and K. 
Hepatobiliary disease may impair the uptake of vitamin D2 and 
D3 into the liver for conversion into 25(OH)D, or may interfere 
with enterohepatic recirculation to enhance loss of vitamin D 
metabolites. Small intestinal bacterial overgrowth may further 
obstruct the gastrointestinal absorption of vitamin D. Finally, 
urinary losses of vitamin D binding protein and albumin, the 
major carriers of vitamin D metabolites, may contribute to 
lower circulating concentrations of vitamin D metabolites.  
Proposed mechanisms to explain vitamin D deficiency in CF 
lack empiric supportive data. In this study, we will determine 
specific vitamin D metabolic disturbances that are intrinsic to 
CF patients and to explore their biologic sequelae. Identifying 
the causes of vitamin D deficiency in CF could suggest 
alternative treatments, such as intramuscular vitamin D 
preparations, activated vitamin D agents, or higher doses of 
supplements, to improve clinical outcomes that have been 
linked with vitamin D deficiency, such as bone disease and 
infection. 
STUDY DESIGN 
This is a single-dose, open-label pharmacokinetic study that 
uses intravenous administration of a stable deuterium-labeled 
25(OH)D3 to evaluate the metabolic clearance of 25(OH)D3.  


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Allocation: Non-Randomized 
Intervention Model: Single Group Assignment 
Masking: Open Label  
PRIMARY OBJECTIVE 
The goal of this study is to define 25(OH)D3 catabolism in CF 
patients using gold standard pharmacokinetics studies. 
Specifically, we will evaluate the metabolic clearance of 
25(OH)D3 among participants with CF and matched control 
subjects. The goal of this work is to provide the first 
comprehensive characterization of vitamin D metabolism in CF 
patients and promote novel hypotheses for subsequent studies. 
SECONDARY 
OBJECTIVES 
• To explore the biochemical pathways through which 
1,25(OH)D3 is metabolized, and the effects of CKD and 
race on these pathways, by evaluating the appearance and 
disappearance of labeled metabolites of 25(OH)D3.  
• To determine clinical and biochemical factors that are 
correlated with the metabolic clearance of 25(OH)D3. 
• To evaluate biomarkers of 25(OH)D3 clearance.  
• To compare monocyte expression of vitamin D-related 
genes in CF patients versus healthy controls 
NUMBER OF SUBJECTS 
Up to 20 
SUBJECT SELECTION 
CRITERIA 
Inclusion Criteria:  
• Age ≥ 18 years 
• Serum total 25(OH)D 10-50 ng/mL 
 
Exclusion Criteria: 
• Primary hyperparathyroidism 
• Gastric bypass 
• Tuberculosis or sarcoidosis 
• Current pregnancy 
• Use of vitamin D3 or vitamin D2 supplements exceeding a 
mean daily dose of 400 IU within 3 months (wash-out 
allowed) 
• Use of 1,25(OH)2D3 or an analogue, calcimimetics, or 
medications known to induce CYP24A1 within 3 months 
(wash-out allowed) 
• Serum calcium > 10.1 mg/dL 
• Hemoglobin < 10 g/dL 
TEST PRODUCT, DOSE, 
AND ROUTE OF 
ADMINISTRATION 
We will administer deuterated 25(OH)D3-d6 
[26,26,26,27,27,27-d6] intravenously. The labeled 25(OH)D3 
will be prepared in a co-solvent formulation of ethanol (10%) 
and propylene glycol (40%). To ensure reliable detection of 
circulating deuterated 25(OH)D3, without administering a dose 
that might alter the underlying vitamin D metabolism, we aim to 


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administer a dose that results in a peak deuterated 25(OH)D3 
concentration of approximately 5 ng/mL. The administered dose 
will be calculated as the targeted peak serum deuterated 
25(OH)D3 (5 ng/mL) multiplied by blood volume. 
CONTROL PRODUCT, 
DOSE AND ROUTE OF 
ADMINISTRATION 
There is no control product.   
DURATION OF SUBJECT 
PARTICIPATION AND 
DURATION OF STUDY 
Subjects will be on study for approximately 12 weeks 
Screening:  approximately 4 weeks 
Treatment:  1 day (subjects to the University of Washington 
Clinical Research Center) 
Follow-up:  8 weeks 
The total duration of the study is expected to be up to 3 years 
for subject recruitment and an additional 12 weeks for final 
subject follow-up. 
CONCOMMITANT 
MEDICATIONS 
Allowed: 
• Vitamin D supplements (cholecalciferol, ergocalciferol) not 
to exceed a mean daily dose of 400 IU 
Prohibited (within last 3 months, wash-out allowed): 
• 1,25(OH)2D3 or an analogue (e.g. paricalcitol, hectorol) 
• Calcimimetics (e.g. cinaclacet) 
• Medications known to potently induce or inhibit CYP24A1 
or CYP3A4  
EFFICACY 
EVALUATIONS 
None 
PRIMARY ENDPOINT 
• 
Metabolic clearance of labeled 25(OH)D3 (administered 
dose/AUC) 
SECONDARY ENDPOINTS 
• 
AUC of labeled 25(OH)D3  
• 
Terminal half-life of labeled 25(OH)D3 
• 
Volume of distribution in the central compartment of 
labeled 25(OH)D3 
• 
Monocyte transcription of CYP27B1, CYP24A1, VDR, and 
cathelicidin 
OTHER EVALUATIONS 
Metabolic formation clearance (metabolite/parent AUC ratio) 
for metabolites of labeled 25(OH)D3 
SAFETY EVALUATIONS 
Change in the serum concentrations of calcium, creatinine, 
AST, and ALT and from baseline to 7 days after 25(OH)D3 
administration 
Incidence of adverse events 


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PLANNED INTERIM 
ANALYSES  
This is not a clinical trial with an efficacy outcome, and it is 
neither randomized nor blinded. For interim analyses are not 
planned.  
STATISTICS 
Primary Analysis Plan 
We will examine the distribution of 25(OH)D3 clearance by CF 
status using box plots and summary statistics. We will test 
associations of CF status with 25(OH)D3 clearance (continuous 
outcome variable) using linear regression. Models will be 
adjusted for age, gender, body size, baseline 25(OH)D 
concentration, and other covariates strongly related to 
25(OH)D3 clearance.  
Rationale for Number of 
Subjects 
For this descriptive study, our sample size of 20 was based on 
feasibility and funding considerations. 
 


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1 
BACKGROUND  
Cystic fibrosis (CF) is among the most common autosomal recessive disorders and leads to 
serious, life threatening respiratory, infectious, and bone complications. Vitamin D deficiency is 
highly prevalent among CF patients, is unusually resistant to standard vitamin D 
supplementation regimens, and may contribute to osteoporosis and immune dysfunction. The 
prevalence of vitamin D deficiency, determined by circulating concentrations of 25-
hydroxyvitmain D (25(OH)D), remains as high as 90% in CF patients despite modern practices 
of vitamin D supplementation and pancreatic enzyme replacement. A number of plausible 
hypotheses may explain the marked vitamin D deficiency intrinsic to CF. Pancreatic 
insufficiency, the most common gastrointestinal CF complication, leads to malabsorption of fat 
soluble vitamins A, D, E, and K. Hepatobiliary disease may impair the uptake of vitamin D2 and 
D3 into the liver for conversion into 25(OH)D, or may interfere with enterohepatic recirculation 
to enhance loss of vitamin D metabolites. Small intestinal bacterial overgrowth may further 
obstruct the gastrointestinal absorption of vitamin D. Finally, urinary losses of vitamin D binding 
protein and albumin, the major carriers of vitamin D metabolites, may contribute to lower 
circulating concentrations of vitamin D metabolites. Proposed mechanisms to explain vitamin D 
deficiency in CF lack empiric supportive data. In this study, we will determine specific vitamin 
D metabolic disturbances that are intrinsic to CF patients and to explore their biologic sequelae. 
Identifying the causes of vitamin D deficiency in CF could suggest alternative treatments, such 
as intramuscular vitamin D preparations, activated vitamin D agents, or higher doses of 
supplements, to improve clinical outcomes that have been linked with vitamin D deficiency, such 
as bone disease and infection.  
1.1 
Overview of Clinical Studies 
Intravenous deuterated 25(OH)D3 has been used previously in humans in one published study, 
and intravenous tritiated 25(OH)D3 has been used in humans in several previous studies.1-5 In 
addition deuterium is used extensively in the study of metabolism and is considered safe.6 
2 
STUDY RATIONALE 
Vitamin D deficiency is overwhelmingly prevalent in cystic fibrosis. Vitamin D deficiency is the 
most common and persistent nutritional inadequacy in CF.7 Vitamin D is normally generated in 
the skin as vitamin D3 (cholecalciferol) or consumed in the diet as either vitamin D3 or vitamin 
D2 (ergocalciferol).8 Vitamin D3 and vitamin D2 are next converted in the liver to 25-
hydroxyvitamin D3 (25(OH)D3) and 25-hydroxyvitamin D2 (25(OH)D2), respectively, which 
together are referred to as total 25-hydroxyvitamin D (25(OH)D). Circulating 25(OH)D 
concentrations are the accepted clinical markers of vitamin D status and are markedly reduced in 
CF patients. The prevalence of vitamin D deficiency in CF is as high as 90%, depending on the 
patient population and 25(OH)D threshold used to define deficiency (20-30 ng/mL).9-12 Given 
the role of vitamin D in calcium homeostasis and bone health, the measurement and treatment of 
vitamin D deficiency is an integral component of clinical CF care. However, CF patients 
inadequately respond to vitamin D supplementation, such that 25(OH)D concentrations remain 
insufficient in many CF patients despite treatment.12-14 
The cause of vitamin D deficiency in cystic fibrosis is unclear.  Malabsorption of fat soluble 
vitamins, including vitamin D, is likely a major contributor to vitamin D deficiency in CF.15,16 


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Pancreatic insufficiency is the most plausible mechanism of fat malabsorption; the chloride 
channel cystic fibrosis transmembrane conductance regulator (CFTR), which is present in 
enterocytes, does not appear to directly contribute to malabsorption. CF patients have variable 
and often poor plasma 25(OH)D responses to dietary vitamin D supplementation, which could be 
due in part to reduced bioavailability.12-16 However, in studies administering parenteral vitamin 
D2 and ultraviolet light (one study each), plasma 25(OH)D response was also markedly 
impaired.7,17 In addition, higher than usual antibiotic doses are often required in CF patients, 
suggesting induction of metabolizing enzymes that may overlap with those which metabolize 
vitamin D.18-21 Together, these findings suggest additional defects in vitamin D metabolism in 
CF patients. For full hormonal activity, 25(OH)D must be converted to the active vitamin D 
hormone, 1,25-dihydroxyvitamin D (1,25(OH)2D). In addition, 25(OH)D can undergo a series of 
catabolic steps leading to its elimination (Figure 1).22 The most common route of 25(OH)D3 
catabolism begins with conversion to 24,25-dihydroxyvitamin D3 by the cytochrome P450 
enzyme CYP24A1. Drs. Lin and Thummel (Co-investigators) have discovered that 25(OH)D3 
also undergoes hydroxylation at the 4-position and sulfation at the 3-O-position. The resulting 
metabolites, 4β,25(OH)2D3 and 
25(OH)D3-3-sulfate, respectively, are 
generated in enterocytes and 
hepatocytes by CYP3A4 and 
SULT2A1.23,24 Vitamin D metabolic 
enzymes are regulated by parathyroid 
hormone (PTH) and fibroblast growth 
factor-23 (FGF-23) as well as 
1,25(OH)2D itself. Our group has 
contributed to an expanding 
knowledge of dysregulated vitamin D 
metabolism in chronic kidney disease, 
in which both vitamin D activation and 
catabolism are impaired.22,25 However, 
little is known about the vitamin D 
metabolic axis in CF in terms of 
vitamin D activation, vitamin D 
catabolism, or the hormonal response to vitamin D deficiency. Clearance of 25(OH)D may be 
affected in CF by altered enterohepatic function of CYP3A4 and SULT2A1. In addition, 
enterohepatic vitamin D circulation, in which sulfated 25-hydroxyvitamin D is secreted in bile 
and delivered to target enterocytes, may be disrupted, leading to loss of conjugated vitamin D in 
the stool and altered enteric metabolism. Small bowel bacterial overgrowth due to impaired 
gastrointestinal mobility may further reduce absorption of vitamin D. Finally, vitamin D binding 
protein (DBP) and albumin, which carry circulating vitamin D metabolites, may be lost in the 
urine.26 Defining which of these potential metabolic defects is clinically relevant may lead to 
improved methods of vitamin D deficiency diagnosis and treatment. 
Impaired vitamin D metabolism may have broad adverse health effects in cystic fibrosis. 
1,25(OH)2D3 binds to the intracellular vitamin D receptor to modulate transcription of genes 
involved in bone health, inflammation, and blood pressure. Vitamin D is a central determinant of 
calcium and phosphate absorption from gastrointestinal tract to provide mineral content for 
growing bones.27 Moreover, vitamin D deficiency may directly impair muscle strength, thereby 


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indirectly reducing bone density. Monocytes metabolize vitamin D and require 1,25(OH)2D3 for 
robust immune function. For example, within monocytes, autocrine metabolism of 25(OH)D3 to 
1,25(OH)2D3 by CYP27B1 is required for the expression of cathelicidin and resulting activity 
against mycobacterium tuberculosis.28,29 Addition of 1,25(OH)2D3 to cultured monocytes reduces 
expression of pro-inflammatory cytokines IL-1, IL-6, IL-8, and TNF-α, and promotes generation 
of regulatory Th2 lymphocytes. Disruption of the vitamin D receptor directly stimulates the 
renin-angiotensin-aldosterone system, promoting hypertension and arteriosclerosis. In large 
population studies, vitamin D deficiency, defined by lower circulating 25(OH)D concentrations, 
is associated with cardiovascular disease events, fractures, and cancer. The clinical consequences 
of vitamin D deficiency in CF patients are not yet clearly defined; however, bone disease and 
infections represent major clinical complications of CF that are plausibly influenced by vitamin 
D deficiency. A more detailed understanding of the relationships between vitamin D metabolism 
and bone disease and infections in CF may provide new opportunities to reduce these important 
clinical complications. 
Multiple vitamin D interventions are widely available, but their proper use in CF awaits 
identification of specific vitamin D metabolic disturbances. Supplementation with vitamin D3 or 
D2 may be improved by administration of higher oral dosage programs or intramuscular 
injections. Ultraviolet light exposure has been used in small studies of CF patients and may 
augment concurrent vitamin D supplementation. If identified, cytochrome P-450 enzymes with 
altered activities in CF could be targeted for intervention. Finally, vitamin D receptor agonists 
(1,25(OH)2D and its analogues) are widely used in patients with kidney disease and could play a 
role in the treatment of vitamin D deficiency in CF. 
2.1 
Risk / Benefit Assessment 
This study will not provide any direct benefit to the study subjects. Individual subjects may have 
a modestly reduced chance of adverse health events because their care will be monitored more 
closely than it might otherwise. Future patients may benefit from these studies by virtue of 
knowledge gained about the pathophysiology and ascertainment of impaired vitamin D 
metabolism. 
The risks of administering deuterated 25(OH)D3 in tracer quantities are low. 25(OH)D3 is a 
naturally occurring substance and generally circulates in concentrations of 10-50 ng/mL. The 
deuterated 25(OH)D3 we propose to administer differs from the naturally occurring form only by 
the substitution of 6 hydrogen atoms with deuterium. This isotope is stable and has metabolic 
and biologic characteristics identical to the naturally occurring form. We will administer 
quantities intended to make small changes in total circulating 25(OH)D3 concentration, i.e., an 
increase of 5 ng/mL.  
There is some risk of hypercalcemia with the administration 25(OH)D3, but this risk is 
minimized by excluding participants with baseline 25(OH)D > 50 ng/mL and by administering 
quantities that raise 25(OH)D by only approximately 5 ng/mL. We will monitor for 
hypercalcemia during our research. If instances of hypercalcemia are observed, we will change 
our study protocol accordingly to prevent hypercalcemia in future participants. This study also 
includes placement of peripheral intravenous catheters and blood draws. Risks include 
discomfort and minor bleeding associated with catheter placements and anemia due to blood 
sampling. 


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3 
STUDY OBJECTIVES 
3.1 
Primary Objective 
The goal of this study is to define 25(OH)D3 catabolism in CF patients using gold standard 
pharmacokinetics studies. Specifically, we will evaluate the metabolic clearance of 25(OH)D3 
among participants with CF and matched control subjects. The goal of this work is to provide the 
first comprehensive characterization of vitamin D metabolism in CF patients and promote novel 
hypotheses for subsequent studies. 
3.2 
Secondary Objectives 
• To explore the biochemical pathways through which 25(OH)D3 is metabolized, and the 
effects of CF on these pathways, by evaluating the appearance and disappearance of labeled 
metabolites of 25(OH)D3.  
• To determine clinical and biochemical factors that are correlated with the metabolic 
clearance of 25(OH)D3. 
• To evaluate potential biomarkers of 25(OH)D3 
• To compare monocyte expression of vitamin D-related genes in CF patients versus healthy 
controls 
4 
STUDY DESIGN 
This is a cross-sectional, observational study that uses the intravenous administration of stable 
isotope-labeled 25(OH)D3 to evaluate the metabolic clearance of 25(OH)D3. It is a single-dose, 
open-label PK study of 25(OH)D3. 
We will recruit a total of up to 20 study subjects:  10 with and 10 without CF. Subjects who 
provide written informed consent and qualify at screening will receive a single dose of 
intravenous labeled 25(OH)D3. Subsequent blood draws and urine collections will be used to 
determine the metabolic clearance of 25(OH)D3 and related parameters.  
5 
CRITERIA FOR EVALUATION 
5.1 
Primary Efficacy Endpoint 
The primary outcome of interest is the metabolic clearance of labeled 25(OH)D3.  
We will measure serum concentrations of 24,25(OH)2D3, 25(OH)D3, 25(OH)D2, 1,25(OH)2D3 
and 1,25(OH)2D2 at the University of Washington by mass spectrometry For each participant and 
time point, a single serum aliquot is used to measure this panel of vitamin D metabolites using by 
immunoaffinity extraction and HPLC-mass spectrometry (Xevo TQ, Waters Corp., Milford, 
MA) with deuterated internal standards.25,30-32 
For each subject, non-compartmental analysis of plasma concentration versus time data will be 
performed using Phoenix software (Pharsight, Cary, NC). Clearance will be calculated as 
administered 25(OH)D3 dose divided by 25(OH)D3 AUC. We focus on clearance as our primary 
outcome because it reflects the metabolism of 25(OH)D3 accounting for circulating 25(OH)D3 
concentration (units of volume/time, akin to creatinine clearance). Clearance is independent of 
volume of distribution, in contrast to t1/2, which will be evaluated as a secondary outcome. 
Clearance will be evaluated with and without adjustment for body size.   


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5.2 
Secondary Efficacy Endpoints 
• AUC of labeled 25(OH)D3  
• Terminal half-life of labeled 25(OH)D3 
• Volume of distribution in the central compartment of labeled 25(OH)D3 
• Metabolic formation clearance (metabolite/parent AUC ratio) for metabolites of labeled 
25(OH)D3 
• Monocyte transcription of CYP27B1, CYP24A1, VDR, and cathelicidin 
5.3 
Safety Evaluations 
An abnormal clinical laboratory value will be documented as an adverse event if one of the 
following applies: 
• The abnormality is not contradicted by a repeat test to confirm the abnormality. 
• The abnormality suggests a disease and/or organ toxicity. 
• The abnormality is of a degree that requires active management (e.g., requires a medication 
change, more frequent follow-up, or further diagnostic evaluation). 
Change in clinical laboratory findings (if there are specific labs, then why they are appropriate to 
measure, e.g., BUN or Creatinine for an aminoglycoside) 
An adverse event (AE) is any symptom, sign, illness or experience that develops or worsens in 
severity during the course of the study.  Intercurrent illnesses or injuries should be regarded as 
adverse events.  Abnormal results of diagnostic procedures are considered to be adverse events if 
the abnormality: 
• results in study withdrawal 
• is associated with a serious adverse event 
• is associated with clinical signs or symptoms 
• leads to additional treatment or to further diagnostic tests 
• is considered by the investigator to be of clinical significance 
A serious adverse event is any AE that is:  
• fatal 
• life-threatening 
• requires or prolongs hospital stay 
• results in persistent or significant disability or incapacity 
• a congenital anomaly or birth defect 
• an important medical event 
Important medical events are those that do not fit the other definitions of serious adverse events, 
but the event may jeopardize the patient and may require treatment to prevent one of the listed 
serious adverse events. 
Adverse events will be reported if they occur between the time of informed consent and 30 days 
after the last study visit. All unresolved adverse events will be followed by the PI until 
resolution, the adverse event is otherwise explained, or the participant is lost to follow-up. At the 
last study visit, the investigator will instruct each participant to report any subsequent event that 


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the participant, or participant’s personal physician, reasonably believes may be related to the 
study. The investigator will notify the study sponsor of any death or adverse event occurring 
after the participant has discontinued participation if the event can be reasonably related to the 
study. 
6 
SUBJECT SELECTION 
6.1 
Study Population 
We will target enrollment of 10 patients with CF and 10 healthy controls matched on age, 
gender, and race. To recruit the 10 CF patients, we will first turn to observational studies of CF 
for which participants have already granted consent for contact for additional research studies. If 
sufficient participants are not identified through this route, we will recruit directly from the 
University of Washington CF clinic. The 10 matched healthy controls will be drawn from the 
Healthy Kidney Study. Clinical data and contact information for these control subjects are 
contained in the Kidney Research Institute biorepository. 
6.2 
Inclusion Criteria 
We will study only adults (Table 6.2). We will limit to a range of total serum 25(OH)D. 
 
Table 6.2. Participant Eligibility – Inclusion 
criteria 
Age ≥ 18 years 
Serum 25(OH)D concentration 10 – 50ng/ml 
 
6.3 
Exclusion Criteria 
Exclusion criteria will be inability to give informed consent, pregnancy, active hepatitis or 
cirrhosis, hemoglobin <10mg/dL, serum calcium >10mg/dL, use of ergocalciferol or 
cholecalciferol (> 400 IU/day) within 3 months, use of active vitamin D receptor agonists (e.g. 
calcitriol) or cinacalcet within 4 weeks, or use of a cytochrome P-450 (CYP) inhibitor or inducer 
within 4 weeks (table 2 and table 3). If people do not meet eligibility criteria based on 
medication use, they will be allowed to participate in the study after an appropriate washout 
period if their primary physician agrees. For ergocalciferol and cholecalciferol, the washout 
period will be 3 months. For active vitamin D compounds, cinacalcet, and CYP inhibitors and 
inducers the washout period will be 4 weeks. 
 
Table 6.3. Patient Eligibility – Exclusion 
criteria 
Ergocalciferol in last 3 months, cholecalciferol 
>400IU/day in last 3 months, active vitamin D 
receptor agonist (e.g. calcitriol) or cinacalcet in 
last 4 weeks,  
Active hepatitis or cirrhosis 
Hemoglobin < 10mg/dl 
Serum calcium concentration > 10.1 mg/dl 


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Pregnancy 
Inability to give informed consent 
Medications known to strongly induce or 
suppress the CYP enzymes which metabolize 
vitamin D (see example list below) 
 
Table 6.4. CYP inhibitors and inducers 
CYP Inhibitors 
CYP Inducers 
Amprenavir 
Imatinib 
Avasimibe 
Aprepitant 
Indinavir 
Bosentan 
Atazanivir 
Itraconazole 
Carbamazepine 
Casopitant 
Ketoconazole 
Efavirenz 
Cimetidine 
Lopinavir 
Etravirine 
Ciprofloxacin 
Nefazodone 
Modafinil 
Clarithromycin 
Nelfinavir 
Nafcillin 
Conivaptan 
Posaconazole 
Phenobarbital 
Darunavir 
Ritonavir 
Phenytoin 
Diltiazem 
Saquinavir 
Rifabutin 
Dronedarone 
Shisandra 
Rifampin 
Elvitegravir 
Telithromycin 
St. John’s 
Wort 
Erythromycin 
Tipranavir 
Fluconazole 
Verapamil 
Grapefruit 
Juice 
Voriconazole 
 
7 
CONCURRENT MEDICATIONS 
All subjects should be maintained on the same medications throughout the entire study period, as 
medically feasible, with no introduction of new chronic therapies. Specific allowed and 
disallowed medications are detailed in Section 6 (Eligibility Criteria). 
8 
STUDY TREATMENTS 
8.1 
Method of Assigning Subjects to Treatment Groups 
All consenting and qualified participants will be assigned to the single treatment arm. 
8.2 
Blinding 
Neither participants nor study investigators will be blinded to the study intervention. 


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8.3 
Formulation of Test and Control Products 
We will administer labeled 25(OH)D3 intravenously. Deuterated 25-dihydroxyvitamin D3 (26, 
26, 26, 27, 27, 27 – d6) will be manufactured under Good Manufacturing Practice by the Sigma-
Aldrich ISOTEC Stable Isotope Division (Miamisburg, OH). The deuterated 25(OH)D3 
produced by Sigma-Aldrich will be provided in solid form and formulated for IV administration 
by the University of Iowa Pharmaceuticals. 
8.3.1 Formulation of Test Product 
The labeled 25(OH)D3 prepared by Sigma-Aldrich will be formulated in a co-solvent of ethanol 
(10%) and propylene glycol (40%) by University of Iowa Pharmaceuticals. The formulated drug 
product will be aliquoted into single-use vials and frozen for storage. 
8.3.2 Packaging and Labeling 
Individual doses of formulated 25(OH)D3 will be prepared for intravenous administration by the 
University of Washington Investigational Drug Services. The prepared dose of 25(OH)D3 will be 
calculated according to body size and labeled with the participant’s full name, date of birth, and 
medical record number along with the drug contents. 
8.4 
Supply of Study Drug at the Site 
Single-use vials of formulated 25(OH)D3 will be stored at the University of Washington 
Investigational Drug Services. Additional vials of formulated 25(OH)D3 will be kept at 
University of Iowa Pharmaceuticals for stability testing. 
8.4.1 Dosage/Dosage Regimen 
Each participant will receive a single intravenous dose of deuterated 25(OH)D3. We will use 
body size-based dosing to target a peak achieved deuterated 25(OH)D3 concentration of 5 
ng/mL. The target of 5 ng/mL was selected because it will allow precise tracking of circulating 
deuterated 25(OH)D3 concentration throughout follow-up (based on known limits of detection 
for our assay) without substantially perturbing underlying vitamin D status. Theoretically, the 
dose required to achieve a 5 ng/mL increment in deuterated 25(OH)D3 concentration can be 
calculated as the targeted peak  concentration multiplied by the volume of distribution, which for 
25(OH)D3 is expected to be equal to blood volume. We will use the formula of Nadler et al 
(1962) to estimate blood volume for each participant. We will begin the study by administering 
to each participant a dose of 5 ng/mL x estimated blood volume. A typical dose will be 25-30 
mcg. We will monitor achieved 25(OH)D3 concentrations real time. If our initial approach fails 
to achieve peak 25(OH)D3 concentrations near 5 ng/mL, we will revise our approach for future 
participants, as indicated. For example, if mean peak achieved 25(OH)D3 concentrations are near 
2.5 ng/mL with our initial approach, we will increase our dose by a factor of two. We will not 
exceed a dose of 100 mcg for any participant. 
8.4.2 Dispensing 
All study drug will be dispensed by the University of Washington Investigational Drug Services. 


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8.4.3 Administration Instructions  
The study drug will be administered intravenously on a single occasion over a period of 5 
minutes. 
8.5 
Supply of Study Drug at the Site 
Single-use vials of formulated 25(OH)D3 will be stored at the University of Washington 
Investigational Drug Services.  The vials will be labeled as investigational product by the 
University of Iowa Pharmaceuticals, the manufacturer of formulated product. 
8.5.1 Storage 
Study drug will be stored at a temperature <-60°C at the University of Washington 
Investigational Drug Services. 
8.6 
Study Drug Accountability 
An accurate and current accounting of the dispensing and return of study drug for each subject 
will be maintained on an ongoing basis by the University of Washington Investigational Drug 
Services.  The number of study drug dispensed and returned by the subject will be recorded on 
the Investigational Drug Accountability Record. 
8.7 
Measures of Treatment Compliance 
Each study drug administration will be directly observed by study staff. 
9 
STUDY PROCEDURES AND GUIDELINES 
A Schedule of Events representing the required testing procedures to be performed for the 
duration of the study is provided in Section 10 (Evaluations by Visit) below and diagrammed in 
Appendix 1. 
Prior to conducting any study-related activities, written informed consent and the Health 
Insurance Portability and Accountability Act (HIPAA) authorization must be signed and dated 
by the subject or subject’s legal representative.  If appropriate, assent must also be obtained prior 
to conducting any study-related activities. 
9.1 
Clinical Assessments 
9.1.1 Concomitant Medications 
All concomitant medication and concurrent therapies will be documented at Screening, Baseline 
(Study Day 0), and the final Study Visit (Study Visit 10).  Dose, route, unit frequency of 
administration, and indication for administration and dates of medication will be captured. 
9.1.2 Demographics  
Demographic information (date of birth, gender, race) will be recorded at Screening. 


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9.1.3 Medical History  
Relevant medical history, including history of current disease, other pertinent history, and 
information regarding underlying diseases will be recorded at Baseline (Study Day 0). 
9.1.4 Physical Examination 
Height and weight will be measured as the Screening visit and at Baseline (Study Day 0). When 
prompted by reports or suspicion of potential adverse effects during follow-up, physical exam 
findings will be documented by qualified staff (MD, NP, RN, or PA) and will be followed by a 
physician or other qualified staff immediately or at the next scheduled visit, as indicated. 
9.1.5 Vital Signs 
Body temperature, blood pressure, pulse and respirations will be measured after resting for 
5 minutes at Baseline (Study Day 0), prior to administration of study drug. 
9.1.6 Other Clinical Procedures 
Physical activity will be ascertained by questionnaire and accelerometry between Study days 1 
and 7. Vitamin D intake will be estimated by food frequency questionnaire at Baseline. Serum 
and plasma will be collected at Baseline to measure kidney and liver function, calcium and 
phosphorus, parathyroid hormone, fibroblast growth factor-23, and vitamin D binding protein. 
Whole blood will also be collected at baseline to isolate monocytes for RNA expression 
quantification. Two 24-hour urine collections will be obtained, immediately preceding and 
following deuterated 25(OH)D3 administration, to quantify urinary excretion of albumin and the 
metabolites of deuterated 25(OH)D3, respectively. Adiposity will be measured by Dual energy 
X-ray absorptiometry at the baseline visit.  
9.1.7 Adverse Events 
Information regarding occurrence of adverse events will be captured throughout the study. 
Duration (start and stop dates and times), severity/grade, outcome, treatment and relation to 
study drug will be recorded on the case report form (CRF). 
9.2 
Clinical Laboratory Measurements 
9.2.1 Hematology 
A complete blood count will be obtained at the Screening Visit. 
9.2.2 Blood Chemistry Profile 
Serum sodium, potassium, chloride, bicarbonate, random glucose, BUN, creatinine, aspartate 
aminotransferase (AST/SGOT), alanine aminotransferase (ALT/SGPT), albumin, calcium, and 
phosphorous will be obtained at the Screening Visit.   
Repeat serum sodium, potassium, chloride, bicarbonate, random glucose, BUN, creatinine, 
aspartate aminotransferase (AST/SGOT), alanine aminotransferase (ALT/SGPT), albumin, 
calcium, and phosphorous will be obtained at Baseline  and Study Day 7. 


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Intact parathyroid hormone (PTH) will be measured at Visits 2 and 5 (Day 0 and 7 gauged from 
administration of deuterated 25(OH)D3). 
9.2.3 Pregnancy Test 
A urine pregnancy test will be obtained from female subjects who are of childbearing age at the 
Baseline Visit prior to administration of the study drug. 
9.3 
Pharmacokinetic Measurements 
Blood for determination of serum concentrations of deuterated 25(OH)D3 and its metabolites will 
be collected at Study Visits 2-10.  
At Baseline, blood will be drawn prior to administration of study drug as well as 5 minutes and 4 
hours after administration. 
9.4 
Research Laboratory Measurements  
Novel products of 25(OH)D3 catabolism, including 4β,25(OH)2D3 and 25(OH)D3-3-sulfate will 
be measured using LC-MS/MS, as previously described.33  25(OH)D3-3-sulfate will be extracted 
from plasma. 
10 EVALUATIONS BY VISIT 
10.1 Screening Visit (Study Visit 1)  
The following activities will occur during the screening visit:   
• 
Collect signed consent form 
• 
Perform focused medical history restricted to study eligibility criteria 
• 
Complete medication inventory through EMR and patient report. 
• 
Record height and weight of patient 
• 
Collect non-fasting blood sample for measurement of complete blood count and total 
25(OH)D. 
• 
Collect non-fasting blood sample for serum sodium, potassium, chloride, bicarbonate, 
random glucose, BUN, creatinine, AST/SGOT, ALT/SGPT, albumin, calcium, and 
phosphorous. 
• 
Collect urine for measurement of albumin and creatinine (except for hemodialysis patients). 
• 
Collect urine for pregnancy test for women of childbearing potential. 
• 
Participants who meet eligibility criteria at the screening visit will be invited to return for the 
remaining study visits.  
• 
Provide selected subjects with supplies required for first 24-hour urine collection 
10.2 Baseline Visit (Study Visit 2, Day 0)  
Visit 2 will take place at the University of Washington Clinical Research Center (CRC) or a 
suitable alternate facility and will last approximately 5 hours. Visit 2 will take place no more 
than 90 days after Visit 1. If more than 90 days elapse between Visits 1 and 2, screening 
procedures may be repeated to verify continued eligibility. The following activities will occur 
during the baseline visit: 
• Perform a complete medical history 


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• Review current medications.  
• Complete physical activity and food frequency questionnaires.  
• Collect blood prior to administration of study drug for measurement of kidney and liver 
function, basic chemistries, vitamin D metabolites, parathyroid hormone (PTH), and other 
measurements related to vitamin D metabolism and for isolation of monocytes for RNA 
expression analyses.  
• Collect urine sample from first 24-hour collection period.  Sample will be used for 
measurement of albumin, creatinine, vitamin D binding protein, and vitamin D metabolites:   
• Perform dual energy x-ray absorptiometry (DEXA) to measure adiposity.   
• Administer deuterated 25(OH)D3 intravenously as a 5 mL IV bolus.  
• Collect blood 5 minutes and 4 hours post-infusion for measurement of vitamin D metabolites.  
• Distribute supplies for second 24-hour urine collection. 
10.3 Study Visit 3 (Day 1)   
This visit is expected to last less than one hour.  The following activities will occur during Visit 
3: 
• Perform an abbreviated medical history 
• Collect urine sample from second 24-hour collection period.   
• Collect blood for measurement of serum vitamin D metabolite concentrations.  
• Distribute Accelerometer for measurement of physical activity  
10.4 Study Visit 4 (Day 4) 
This visit is expected to last less than one hour.  The following activities will occur during the 
Visit 4: 
• Perform an abbreviated medical history 
• Collect blood for measurement of serum vitamin D metabolite concentrations.  
10.5 Study Visit 5 (Day 7) 
This visit is expected to last less than one hour.  Clinical activities will occur in the following 
order: 
• Perform an abbreviated medical history 
• Collect blood for measurement of serum vitamin D metabolite concentrations.  
• Collect blood for serum sodium, potassium, chloride, bicarbonate, random glucose, BUN, 
creatinine, aspartate aminotransferase (AST/SGOT), alanine aminotransferase (ALT/SGPT), 
albumin, calcium, and phosphorous at Study Visit 5 (Day 7) to monitor study safety.  
• Collect Accelerometer 
10.6 Study Visits 6-10 (Day 14, 21, 28, 42, and 56) 
Each of these visits is expected to last less than one hour.  Clinical activities will occur in the 
following order: 
• Perform an abbreviated medical history 
• Collect blood for measurement of serum vitamin D metabolite concentrations.  


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11 ADVERSE EXPERIENCE REPORTING AND DOCUMENTATION 
11.1 Adverse Events 
An adverse event (AE) is any symptom, sign, illness or experience that develops or worsens in 
severity during the course of the study.  Intercurrent illnesses or injuries should be regarded as 
adverse events.  Abnormal results of diagnostic procedures are considered to be adverse events if 
the abnormality: 
• results in study withdrawal 
• is associated with a serious adverse event 
• is associated with clinical signs or symptoms 
• leads to additional treatment or to further diagnostic tests 
• is considered by the investigator to be of clinical significance  
11.1.1 AE Severity 
The National Cancer Institute’s Common Terminology Criteria for Adverse Events (CTCAE) 
Version 3.0 should be used to assess and grade AE severity, including laboratory abnormalities 
judged to be clinically significant. The modified criteria can be found in the study manual.  If the 
experience is not covered in the modified criteria, the guidelines shown in Table 11.1 below 
should be used to grade severity.  It should be pointed out that the term “severe” is a measure of 
intensity and that a severe AE is not necessarily serious. 
Table 11.1  AE Severity Grading 
Severity 
(Toxicity 
Grade) 
Description 
Mild (1) 
Transient or mild discomfort; no limitation in activity; no medical 
intervention or therapy required. The subject may be aware of the sign or 
symptom but tolerates it reasonably well. 
Moderate (2) 
Mild to moderate limitation in activity, no or minimal medical 
intervention/therapy required. 
Severe (3) 
Marked limitation in activity, medical intervention/therapy required, 
hospitalizations possible. 
Life-threatening 
(4) 
The subject is at risk of death due to the adverse experience as it occurred. 
This does not refer to an experience that hypothetically might have caused 
death if it were more severe. 
 
11.1.2 AE Relationship to Study Drug 
The relationship of an AE to the study drug should be assessed using the following the guidelines 
in Table 11.2. 


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Table 11.2  AE Relationship to Study Drug 
Relationship 
to Drug 
Comment 
Definitely 
Previously known toxicity of agent; or an event that follows a reasonable 
temporal sequence from administration of the drug; that follows a known or 
expected response pattern to the suspected drug; that is confirmed by stopping or 
reducing the dosage of the drug; and that is not explained by any other 
reasonable hypothesis. 
Probably 
An event that follows a reasonable temporal sequence from administration of the 
drug; that follows a known or expected response pattern to the suspected drug; 
that is confirmed by stopping or reducing the dosage of the drug; and that is 
unlikely to be explained by the known characteristics of the subject’s clinical 
state or by other interventions. 
Possibly 
An event that follows a reasonable temporal sequence from administration of the 
drug; that follows a known or expected response pattern to that suspected drug; 
but that could readily have been produced by a number of other factors. 
Unrelated 
An event that can be determined with certainty to have no relationship to the 
study drug. 
11.2 Serious Adverse Experiences (SAE) 
An SAE is defined as any AE occurring at any dose that results in any of the following 
outcomes: 
• death 
• a life-threatening adverse experience 
• inpatient hospitalization or prolongation of existing hospitalization 
• a persistent or significant disability/incapacity 
• a congenital anomaly/birth defect  
Other important medical events may also be considered an SAE when, based on appropriate 
medical judgment, they jeopardize the subject or require intervention to prevent one of the 
outcomes listed.  
A serious adverse event is any AE that is:  
• fatal 
• life-threatening 
• requires or prolongs hospital stay 
• results in persistent or significant disability or incapacity 
• a congenital anomaly or birth defect 
• an important medical event 
Important medical events are those that do not fit the other definitions of serious adverse events, 
but the event may jeopardize the patient and may require treatment to prevent one of the listed 
serious adverse events. 


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Adverse events will be collected if they occur between the time of informed consent and up to 30 
days after the last study visit. All unresolved adverse events will be followed by the PI until 
resolution, the adverse event is otherwise explained, or the participant is lost to follow-up. At the 
last study visit, the investigator will instruct each participant to report any subsequent event that 
the participant, or participant’s personal physician, reasonably believes may be related to the 
study. The investigator will notify the study sponsor of any death or adverse event occurring 
after the participant has discontinued participation if the event can be reasonably related to the 
study. 
11.3 Abnormal lab values  
An abnormal clinical laboratory value will be documented as an adverse event if one of the 
following applies: 
1. The abnormality is not contradicted by a repeat test to confirm the abnormality. 
2. The abnormality suggests a disease and/or organ toxicity. 
3. The abnormality is of a degree that requires active management (e.g., requires a 
medication change, more frequent follow-up, or further diagnostic evaluation). 
 
11.4 Hospitalization, Prolonged Hospitalization or Surgery  
Any adverse event that results in hospitalization or prolonged hospitalization will be documented 
and reported as a serious adverse event unless specifically instructed otherwise in this protocol.  
Any condition responsible for surgery will be documented as an adverse event if the condition 
meets the criteria for and adverse event. Neither the condition, hospitalization, prolonged 
hospitalization, nor surgery are reported as an adverse event in the following circumstances: 
1. Hospitalization or prolonged hospitalization for diagnostic or elective surgical procedures 
for a preexisting condition.  Surgery should not be reported as an outcome of an adverse 
event if the purpose of the surgery was elective or diagnostic and the outcome was 
uneventful. 
2. Hospitalization or prolonged hospitalization required to allow efficacy measurement for 
the study. 
3. Hospitalization or prolonged hospitalization for therapy of the target disease of the study, 
unless it is a worsening or increase in frequency of hospital admissions as judged by the 
clinical investigator. 
11.2.1 Serious Adverse Experience Reporting 
The PI will document all SAEs that occur (whether or not related to study drug) on an SAE 
Report Form.  The collection period for all SAEs will begin after informed consent is obtained 
until all procedures for the final study visit have been completed. 
All SAE Report Forms will be reviewed by the PI.  SAE reports that are both related and 
unexpected will be sent to FDA within one business day of learning of the event.  SAE reports 
that are both related and unexpected will also be forwarded to the UW Institutional Review 
Board, in accordance with UW Standard Operating Procedures.  Because this is an observational 


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study, no data safety and monitoring board will be created for this trial. Adverse events and 
recruitment will be monitored by the PI and reported to the UW IRB, as described above.  
12 DISCONTINUATION AND REPLACEMENT OF SUBJECTS 
12.1 Early Discontinuation of Study Drug  
A subject may be discontinued from study treatment at any time if the subject, the investigator, 
or the Sponsor feels that it is not in the subject’s best interest to continue.  The following is a list 
of possible reasons for study treatment discontinuation:  
• Subject withdrawal of consent  
• Subject is not compliant with study procedures 
• Adverse event that in the opinion of the investigator would be in the best interest of the 
subject to discontinue study treatment 
• Protocol violation requiring discontinuation of study treatment 
• Lost to follow-up 
• Sponsor request for early termination of study 
• Positive pregnancy test (females) 
If a subject is withdrawn from treatment due to an adverse event, the subject will be followed 
and treated by the Investigator until the abnormal parameter or symptom has resolved or 
stabilized.   
All subjects who discontinue study treatment should come in for an early discontinuation visit as 
soon as possible and then should be encouraged to complete all remaining scheduled visits and 
procedures. 
All subjects are free to withdraw from participation at any time, for any reason, specified or 
unspecified, and without prejudice. 
Reasonable attempts will be made by the investigator to provide a reason for subject 
withdrawals.  The reason for the subject’s withdrawal from the study will be specified in the 
subject’s source documents Refer to Section 10 for early termination procedures. 
12.3 
Withdrawal of Subjects from the Study 
A subject may be withdrawn from the study at any time if the subject, the investigator, or the 
Sponsor feels that it is not in the subject’s best interest to continue.   
All subjects are free to withdraw from participation at any time, for any reason, specified or 
unspecified, and without prejudice. 
Reasonable attempts will be made by the investigator to provide a reason for subject 
withdrawals.  The reason for the subject’s withdrawal from the study will be specified in the 
subject’s source documents.  Subjects who withdraw after Visit 2 but prior to Visit 10 will be 
encouraged to come in for a final visit (and the procedures to be followed would include those 
for their next scheduled visit).   
12.4 
Replacement of Subjects 
Subjects who withdraw from the study treatment will be replaced.   


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Subjects who withdraw from the study will be replaced.   
13 PROTOCOL VIOLATIONS 
A protocol violation occurs when the subject or Sponsor-Investigator fails to adhere to 
significant protocol requirements affecting the inclusion, exclusion, subject safety and primary 
endpoint criteria.  Protocol violations for this study include, but are not limited to, the following: 
• Failure to meet inclusion/exclusion criteria 
• Use of a prohibited concomitant medication 
• Failure to comply with Good Clinical Practice (GCP) guidelines will also result in a protocol 
violation.  The Sponsor-Investigator will determine if a protocol violation will result in 
withdrawal of a subject. 
When a protocol violation occurs, it will be discussed with the Sponsor-Investigator and a 
Protocol Violation Form detailing the violation will be generated. This form will be signed by a 
Sponsor-Investigator.   A copy of the form will be maintained in the regulatory binder and in the 
Sponsor-Investigator’s files. 
14 STATISTICAL METHODS AND CONSIDERATIONS 
14.1 Data Sets Analyzed 
All participants who receive the study drug (deuterated vitamin D) will be included in the safety 
analysis.    
14.2 Demographic and Baseline Characteristics 
The following demographic variables at screening will be summarized by CKD status and race: 
age, gender, medical comorbidities, medications used, height, weight, body mass index, 
laboratory values (including calcium, phosphorus, albumin, vitamin D binding protein, 
parathyroid hormone, fibroblast growth factor-23), and adiposity measured by DXA.   
14.3 Analysis of Primary Endpoint 
We will examine the distribution of 25(OH)D3 clearance by CF status using box plots and 
summary statistics. We will explore the distribution of 25(OH)D3 clearance by key covariates 
(age, gender, and baseline concentrations of 25(OH)D, PTH, FGF-23, and vitamin D binding 
protein) using locally weighted scatterplot smoothing (LOWESS), cubic splines, and box plots. 
We will pay particular attention to the associations of PTH and FGF-23 with 25(OH)D3 
clearance, including multivariable modeling to determine independent associations. We will test 
associations of CF status with 25(OH)D3 clearance (continuous outcome variable) using linear 
regression. Models will be adjusted for age, gender, body size, baseline 25(OH)D concentration, 
and other covariates strongly related to 25(OH)D3 clearance.  
14.4 Analysis of Secondary Endpoints 
Analyses of secondary endpoints will parallel those of the primary endpoint. 


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14.5 Interim Analysis 
This is not a clinical trial with an efficacy outcome, and it is neither randomized nor blinded. No 
formal interim analyses are planned. 
14.6 Sample Size and Randomization 
For this descriptive study, our sample size of 20 was based on feasibility and funding 
considerations. 
15 DATA COLLECTION, RETENTION AND MONITORING 
15.1 Data Collection Instruments 
The PI will prepare and maintain adequate and accurate source documents designed to record all 
observations and other pertinent data for each subject treated with the study drug. Study 
personnel at each site will enter data from source documents corresponding to a subject’s visit 
into the protocol-specific electronic Case Report Form (eCRF) when the information 
corresponding to that visit is available.  Subjects will not be identified by name in the study 
database or on any study documents to be collected by the Sponsor (or designee), but will be 
identified by a subject number. The Investigator is responsible for all information collected on 
subjects enrolled in this study.  All data collected during the course of this study must be 
reviewed and verified for completeness and accuracy by the Investigator.   
15.2 Data Management Procedures 
The data will be entered into a validated database.  Database lock will occur once quality 
assurance procedures have been completed. 
All procedures for the handling and analysis of data will be conducted using good computing 
practices meeting FDA guidelines for the handling and analysis of data for clinical trials. 
15.3 Data Quality Control and Reporting 
After data have been entered into the study database, a system of computerized data validation 
checks will be implemented and applied to the database on a regular basis. Queries are entered, 
tracked, and resolved through the EDC system directly.  The study database will be updated in 
accordance with the resolved queries.  All changes to the study database will be documented. 
15.4 Archival of Data 
The database is safeguarded against unauthorized access by established security procedures; 
appropriate backup copies of the database and related software files will be 
maintained.  Databases are backed up by the database administrator in conjunction with any 
updates or changes to the database.   
At critical junctures of the protocol (e.g., production of interim reports and final reports), data for 
analysis is locked and cleaned per established procedures. 


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15.5 Availability and Retention of Investigational Records 
The PI will make study data accessible to the UW monitor, other authorized representatives of 
the University of Washington IRB, and Regulatory Agency (e.g., FDA) inspectors upon request.  
A file for each subject will be maintained that includes the signed Informed Consent and HIPAA 
Authorization and copies of all source documentation related to that subject.  The Sponsor-
Investigator will ensure the reliability and availability of source documents from which the 
information on the CRF was derived. 
All study documents (patient files, signed informed consent forms, copies of CRFs, Study File 
Notebook, etc.) will be maintained for at least two years after the study is completed.   
15.6 Monitoring 
Study Monitoring Plan.  This study will be monitored according to the safety guidelines outlined 
above.  The PI will allocate adequate time for such monitoring activities.  The PI will also ensure 
that any compliance or quality assurance reviewer is given access to all the above noted study-
related documents and study related facilities (e.g. diagnostic laboratory), and has adequate space 
to conduct a monitoring visit, if requested.  
Monitoring visits will be conducted by representatives of the University of Washington 
according to the U.S. CFR Title 21 Parts 50, 56, and 312 and ICH Guidelines for GCP (E6).  
15.7 Subject Confidentiality 
In order to maintain subject confidentiality, only a subject number will identify all study subjects 
on CRFs and other documentation submitted to the Sponsor.  
16 ADMINISTRATIVE, ETHICAL, REGULATORY CONSIDERATIONS 
The study will be conducted according to the Declaration of Helsinki, Protection of Human 
Volunteers (21 CFR 50), Institutional Review Boards (21 CFR 56), and Obligations of Clinical 
Investigators (21 CFR 312). 
To maintain confidentiality, all laboratory specimens, evaluation forms, reports and other records 
will be identified by a coded number and initials only.  All study records will be kept in a locked 
file cabinet and code sheets linking a patient’s name to a patient identification number will be 
stored separately in another locked file cabinet.  Clinical information will not be released without 
written permission of the subject, except as necessary for monitoring by the FDA.  The 
Investigator must also comply with all applicable privacy regulations (e.g., Health Insurance 
Portability and Accountability Act of 1996, EU Data Protection Directive 95/46/EC). 
16.1 Protocol Amendments 
Any amendment to the protocol will be written by the Investigator-Sponsor.  Protocol 
amendments cannot be implemented without prior written IRB approval except as necessary to 
eliminate immediate safety hazards to patients.  A protocol amendment intended to eliminate an 
apparent immediate hazard to patients may be implemented immediately, provided the IRBs are 
notified within five working days. 


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16.2 Institutional Review Boards and Independent Ethics Committees 
The protocol and consent form will be reviewed and approved by the University of Washington 
Institutional Review Board prior to study initiation.  Serious adverse experiences regardless of 
causality will be reported to the IRB in accordance with the standard operating procedures and 
policies of the IRB, and the Investigator will keep the IRB informed as to the progress of the 
study.  The Investigator will obtain assurance of IRB compliance with regulations. 
Any documents that the IRB may need to fulfill its responsibilities (such as protocol, protocol 
amendments, Investigator’s Brochure, consent forms, information concerning patient 
recruitment, payment or compensation procedures, or other pertinent information) will be 
submitted to the IRB.  The IRB”s written unconditional approval of the study protocol and the 
informed consent form will be in the possession of the Investigator before the study is initiated.  
The IRBs unconditional approval statement will be transmitted by the Investigator to the Sponsor 
or designee prior to the shipment of study supplies to the site.  This approval must refer to the 
study by exact protocol title and number and should identify the documents reviewed and the 
date of review. 
Protocol and/or informed consent modifications or changes may not be initiated without prior 
written IRB approval except when necessary to eliminate immediate hazards to the patients or 
when the change(s) involves only logistical or administrative aspects of the study.  Such 
modifications will be submitted to the IRB and written verification that the modification was 
submitted and subsequently approved should be obtained.   
The IRB must be informed of revisions to other documents originally submitted for review; 
serious and/or unexpected adverse experiences occurring during the study in accordance with the 
standard operating procedures and policies of the IRB; new information that may affect 
adversely the safety of the patients of the conduct of the study; an annual update and/or request 
for re-approval; and when the study has been completed. 
16.3 Informed Consent Form  
Informed consent will be obtained in accordance with the Declaration of Helsinki, ICH GCP, US 
Code of Federal Regulations for Protection of Human Subjects (21 CFR 50.25[a,b], CFR 50.27, 
and CFR Part 56, Subpart A), the Health Insurance Portability and Accountability Act (HIPAA, 
if applicable), and local regulations. 
The Investigator will prepare the informed consent form, assent and HIPAA authorization and 
provide the documents to the Sponsor or designee for approval prior to submission to the 
IRB/IEC.  The consent form generated by the Sponsor-Investigator must be approved by the 
IRB/IEC.  The written consent document will embody the elements of informed consent as 
described in the International Conference on Harmonisation and will also comply with local 
regulations.  
A properly executed, written, informed consent will be obtained from each subject prior to 
entering the subject into the trial.  Information should be given in both oral and written form and 
subjects  must be given ample opportunity to inquire about details of the study.  If a subject is 
unable to sign the informed consent form (ICF) and the HIPAA authorization, a legal 
representative may sign for the subject.   A copy of the signed consent form (and assent) will be 
given to the subject and the original will be maintained with the subject’s records. 


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16.4 Publications  
The publication or presentation of any study results shall comply with all applicable privacy 
laws, including, but not limited to, the Health Insurance Portability and Accountability Act of 
1996.  
16.5 References 
1. 
Vicchio D, Yergey A, O'Brien K, Allen L, Ray R, Holick M. Quantification and kinetics 
of 25-hydroxyvitamin D3 by isotope dilution liquid chromatography/thermospray mass 
spectrometry. Biol Mass Spectrom. 1993;22(1):53-8. doi: 10.1002/bms.1200220107. PubMed 
PMID: 8431502. 
2. 
Compston JE, Merrett AL, Ledger JE, Creamer B. Faecal tritium excretion after 
intravenous administration of 3H-25-hydroxyvitamin D3 in control subjects and in patients with 
malabsorption. Gut. 1982;23(4):310-5. PubMed PMID: 7076008; PubMed Central PMCID: 
PMCPMC1419735. 
3. 
Davies M, Heys SE, Selby PL, Berry JL, Mawer EB. Increased catabolism of 25-
hydroxyvitamin D in patients with partial gastrectomy and elevated 1,25-dihydroxyvitamin D 
levels. Implications for metabolic bone disease. J Clin Endocrinol Metab. 1997;82(1):209-12. 
PubMed PMID: 8989260. 
4. 
Batchelor AJ, Compston JE. Reduced plasma half-life of radio-labelled 25-
hydroxyvitamin D3 in subjects receiving a high-fibre diet. Br J Nutr. 1983;49(2):213-6. doi: 
S0007114583000318 [pii]. PubMed PMID: 6299329. 
5. 
Gray RW, Weber HP, Dominguez JH, Lemann J. The metabolism of vitamin D3 and 25-
hydroxyvitamin D3 in normal and anephric humans. J Clin Endocrinol Metab. 1974;39(6):1045-
56. PubMed PMID: 4372244. 
6. 
Speakman JR. The history and theory of the doubly labeled water technique. Am J Clin 
Nutr. 1998;68(4):932S-8S. PubMed PMID: 9771875. 
7. 
Mailhot G. Vitamin D bioavailability in cystic fibrosis: a cause for concern? Nutrition 
reviews. 2012;70(5):280-93. doi: 10.1111/j.1753-4887.2012.00471.x. PubMed PMID: 
22537214. 
8. 
Rosen CJ. Clinical practice. Vitamin D insufficiency. The New England journal of 
medicine. 2011;364(3):248-54. Epub 2011/01/21. doi: 10.1056/NEJMcp1009570. PubMed 
PMID: 21247315. 
9. 
Rovner AJ, Stallings VA, Schall JI, Leonard MB, Zemel BS. Vitamin D insufficiency in 
children, adolescents, and young adults with cystic fibrosis despite routine oral supplementation. 
Am J Clin Nutr. 2007;86(6):1694-9. PubMed PMID: 18065588. 
10. 
Wolfenden LL, Judd SE, Shah R, Sanyal R, Ziegler TR, Tangpricha V. Vitamin D and 
bone health in adults with cystic fibrosis. Clin Endocrinol (Oxf). 2008;69(3):374-81. doi: 
10.1111/j.1365-2265.2008.03216.x. PubMed PMID: 18284636; PubMed Central PMCID: 
PMC2851223. 
11. 
Stephenson A, Brotherwood M, Robert R, Atenafu E, Corey M, Tullis E. Cholecalciferol 
significantly increases 25-hydroxyvitamin D concentrations in adults with cystic fibrosis. Am J 
Clin Nutr. 2007;85(5):1307-11. PubMed PMID: 17490967. 


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12. 
Boyle MP, Noschese ML, Watts SL, Davis ME, Stenner SE, Lechtzin N. Failure of high-
dose ergocalciferol to correct vitamin D deficiency in adults with cystic fibrosis. Am J Respir 
Crit Care Med. 2005;172(2):212-7. doi: 10.1164/rccm.200403-387OC. PubMed PMID: 
15860755; PubMed Central PMCID: PMC2718468. 
13. 
West NE, Lechtzin N, Merlo CA, Turowski JB, Davis ME, Ramsay MZ, Watts SL, 
Stenner SP, Boyle MP. Appropriate goal level for 25-hydroxyvitamin D in cystic fibrosis. Chest. 
2011;140(2):469-74. Epub 2011/03/12. doi: 10.1378/chest.10-2114. PubMed PMID: 21393390. 
14. 
Green DM, Leonard AR, Paranjape SM, Rosenstein BJ, Zeitlin PL, Mogayzel PJ, Jr. 
Transient effectiveness of vitamin D2 therapy in pediatric cystic fibrosis patients. J Cyst Fibros. 
2010;9(2):143-9. Epub 2010/02/02. doi: 10.1016/j.jcf.2010.01.002. PubMed PMID: 20117062. 
15. 
Lo CW, Paris PW, Clemens TL, Nolan J, Holick MF. Vitamin D absorption in healthy 
subjects and in patients with intestinal malabsorption syndromes. Am J Clin Nutr. 
1985;42(4):644-9. PubMed PMID: 4050723. 
16. 
Lark RK, Lester GE, Ontjes DA, Blackwood AD, Hollis BW, Hensler MM, Aris RM. 
Diminished and erratic absorption of ergocalciferol in adult cystic fibrosis patients. Am J Clin 
Nutr. 2001;73(3):602-6. PubMed PMID: 11237938. 
17. 
Khazai NB, Judd SE, Jeng L, Wolfenden LL, Stecenko A, Ziegler TR, Tangpricha V. 
Treatment and prevention of vitamin D insufficiency in cystic fibrosis patients: comparative 
efficacy of ergocalciferol, cholecalciferol, and UV light. J Clin Endocrinol Metab. 
2009;94(6):2037-43. doi: 10.1210/jc.2008-2012. PubMed PMID: 19336509; PubMed Central 
PMCID: PMC2690417. 
18. 
Hutabarat RM, Smith AL, Unadkat JD. Disposition of drugs in cystic fibrosis. VII. 
Acetylation of sulfamethoxazole in blood cells: in vitro-in vivo correlation and characterization 
of its kinetics of acetylation in lymphocytes. Clin Pharmacol Ther. 1994;55(4):427-33. Epub 
1994/04/01. PubMed PMID: 8162669. 
19. 
Hutabarat RM, Unadkat JD, Kushmerick P, Aitken ML, Slattery JT, Smith AL. 
Disposition of drugs in cystic fibrosis. III. Acetaminophen. Clin Pharmacol Ther. 
1991;50(6):695-701. Epub 1991/12/01. PubMed PMID: 1752114. 
20. 
Hutabarat RM, Unadkat JD, Sahajwalla C, McNamara S, Ramsey B, Smith AL. 
Disposition of drugs in cystic fibrosis. I. Sulfamethoxazole and trimethoprim. Clin Pharmacol 
Ther. 1991;49(4):402-9. Epub 1991/04/01. PubMed PMID: 2015729. 
21. 
Wang JP, Unadkat JD, al-Habet SM, O'Sullivan TA, Williams-Warren J, Smith AL, 
Ramsey B. Disposition of drugs in cystic fibrosis. IV. Mechanisms for enhanced renal clearance 
of ticarcillin. Clin Pharmacol Ther. 1993;54(3):293-302. Epub 1993/09/01. PubMed PMID: 
8375124. 
22. 
Bosworth C, de Boer IH. Impaired vitamin D metabolism in CKD. Seminars in 
nephrology. 2013;33(2):158-68. Epub 2013/03/08. doi: 10.1016/j.semnephrol.2012.12.016. 
PubMed PMID: 23465502; PubMed Central PMCID: PMC3592201. 
23. 
Wang Z, Lin YS, Dickmann LJ, Poulton EJ, Eaton DL, Lampe JW, Shen DD, Davis CL, 
Shuhart MC, Thummel KE. Enhancement of hepatic 4-hydroxylation of 25-hydroxyvitamin D3 
through CYP3A4 induction in vitro and in vivo: implications for drug-induced osteomalacia. 
Journal of bone and mineral research : the official journal of the American Society for Bone and 
Mineral Research. 2013;28(5):1101-16. Epub 2012/12/06. doi: 10.1002/jbmr.1839. PubMed 
PMID: 23212742; PubMed Central PMCID: PMC3609874. 


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24. 
Wang Z, Schuetz EG, Xu Y, Thummel KE. Interplay between vitamin D and the drug 
metabolizing enzyme CYP3A4. The Journal of steroid biochemistry and molecular biology. 
2013;136:54-8. Epub 2012/09/19. doi: 10.1016/j.jsbmb.2012.09.012. PubMed PMID: 22985909; 
PubMed Central PMCID: PMC3549031. 
25. 
Bosworth CR, Levin G, Robinson-Cohen C, Hoofnagle AN, Ruzinski J, Young B, 
Schwartz SM, Himmelfarb J, Kestenbaum B, de Boer IH. The serum 24,25-dihydroxyvitamin D 
concentration, a marker of vitamin D catabolism, is reduced in chronic kidney disease. Kidney 
international. 2012;82(6):693-700. Epub 2012/06/01. doi: 10.1038/ki.2012.193. PubMed PMID: 
22648296; PubMed Central PMCID: PMC3434313. 
26. 
Thrailkill KM, Jo CH, Cockrell GE, Moreau CS, Fowlkes JL. Enhanced excretion of 
vitamin D binding protein in type 1 diabetes: a role in vitamin D deficiency? The Journal of 
clinical endocrinology and metabolism. 2011;96(1):142-9. Epub 2010/10/15. doi: 
10.1210/jc.2010-0980. PubMed PMID: 20943786; PubMed Central PMCID: PMC3038488. 
27. 
Ott SM, Aitken ML. Osteoporosis in patients with cystic fibrosis. Clin Chest Med. 
1998;19(3):555-67. Epub 1998/10/06. PubMed PMID: 9759556. 
28. 
Fabri M, Stenger S, Shin DM, Yuk JM, Liu PT, Realegeno S, Lee HM, Krutzik SR, 
Schenk M, Sieling PA, Teles R, Montoya D, Iyer SS, Bruns H, Lewinsohn DM, Hollis BW, 
Hewison M, Adams JS, Steinmeyer A, Zugel U, Cheng G, Jo EK, Bloom BR, Modlin RL. 
Vitamin D is required for IFN-gamma-mediated antimicrobial activity of human macrophages. 
Sci Transl Med. 2011;3(104):104ra2. Epub 2011/10/15. doi: 10.1126/scitranslmed.3003045. 
PubMed PMID: 21998409; PubMed Central PMCID: PMC3269210. 
29. 
Liu PT, Stenger S, Li H, Wenzel L, Tan BH, Krutzik SR, Ochoa MT, Schauber J, Wu K, 
Meinken C, Kamen DL, Wagner M, Bals R, Steinmeyer A, Zugel U, Gallo RL, Eisenberg D, 
Hewison M, Hollis BW, Adams JS, Bloom BR, Modlin RL. Toll-like receptor triggering of a 
vitamin D-mediated human antimicrobial response. Science (New York, NY. 
2006;311(5768):1770-3. PubMed PMID: 16497887. 
30. 
Laha TJ, Strathmann FG, Wang Z, de Boer IH, Thummel KE, Hoofnagle AN. 
Characterizing antibody cross-reactivity for immunoaffinity purification of analytes prior to 
multiplexed liquid chromatography-tandem mass spectrometry. Clin Chem. 2012;58(12):1711-6. 
doi: 10.1373/clinchem.2012.185827. PubMed PMID: 22968104; PubMed Central PMCID: 
PMC3731945. 
31. 
Strathmann FG, Laha TJ, Hoofnagle AN. Quantification of 1alpha,25-dihydroxy vitamin 
D by immunoextraction and liquid chromatography-tandem mass spectrometry. Clinical 
Chemistry. 2011;57(9):1279-85. Epub 2011/07/20. doi: 10.1373/clinchem.2010.161174. 
PubMed PMID: 21768219; PubMed Central PMCID: PMC3261234. 
32. 
de Boer IH, Sachs MC, Chonchol M, Himmelfarb J, Hoofnagle AN, Ix JH, Kremsdorf 
RA, Lin YS, Mehrotra R, Robinson-Cohen C, Siscovick DS, Steffes MW, Thummel KE, Tracy 
RP, Wang Z, Kestenbaum B. Estimated GFR and circulating 24,25-dihydroxyvitamin D3 
concentration: a participant-level analysis of 5 cohort studies and clinical trials. Am J Kidney 
Dis. 2014;64(2):187-97. doi: 10.1053/j.ajkd.2014.02.015. PubMed PMID: 24703961; PubMed 
Central PMCID: PMC4111986. 
33. 
Wang Z, Senn T, Kalhorn T, Zheng XE, Zheng S, Davis CL, Hebert MF, Lin YS, 
Thummel KE. Simultaneous measurement of plasma vitamin D(3) metabolites, including 
4beta,25-dihydroxyvitamin D(3), using liquid chromatography-tandem mass spectrometry. Anal 


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Biochem. 2011;418(1):126-33. Epub 2011/07/26. doi: 10.1016/j.ab.2011.06.043. PubMed 
PMID: 21784054; PubMed Central PMCID: PMC3164754. 


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APPENDIX 1. SCHEDULE OF STUDY PROCEDURES 
Procedure 
VISIT 1 
SCREENI
NG 
VISIT 2 
BASELINE 
(DAY 0) 
VISIT 3 
DAY 1 
VISIT 4 
DAY 4 
VISIT 5 
DAY 7 
VISIT 6 
DAY 14 
VISIT 7 
DAY 21 
VISIT 8 
DAY 28 
VISIT 9 
DAY 42 
VISIT 10 
DAY 56 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
Focused Medical 
History 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
Medical History  
 
X 
 
 
 
 
 
 
 
 
Physical Exam 
and Vital Signs 
X 
X 
 
 
 
 
 
 
 
 
Medications 
Inventory 
X 
X 
 
 
 
 
 
 
 
X 
Blood Samples* 
X 
X 
X  
X 
X 
X 
X 
X 
X 
X 
Urine collections 
X 
 
 
 
 
 
 
 
 
 
24-hour urine 
collections 
 
X 
X 
 
 
 
 
 
 
 
DNA collection 
 
X 
 
 
 
 
 
 
 
 
Physical activity 
and food 
frequency 
questionnaires 
 
X 
 
 
 
 
 
 
 
 
Accelerometer 
 
 
X  
X 
X 
 
 
 
 
 
DXA 
 
X 
 
 
 
 
 
 
 
 
Study Drug 
Administration 
 
X 
 
 
 
 
 
 
 
 
*Blood Draws Detailed in Appendix 2. 
 


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APPENDIX 2. DRAFT BLOOD DRAW TYPES AND VOLUMES 
Blood sample 
VISIT 1 
SCREEN
ING 
VISIT 2 
BASELI
NE (DAY 
0) 
VISIT 3 
DAY 1 
VISIT 4 
DAY 4 
VISIT 5 
DAY 7 
VISIT 6 
DAY 14 
VISIT 7 
DAY 21 
VISIT 8 
DAY 28 
VISIT 9 
DAY 42 
VISIT 
10 
DAY 56 
TOTAL 
VOLUM
E (ML) 
Clinical lab: CBC  
(3 mL EDTA-plasma 
[purple top]) 
X 
 
 
 
 
 
 
 
 
 
3 
Clinical lab: chemistries  
(3 mL serum [lime green 
top, PST]) 
X 
X 
 
 
X 
 
 
 
 
 
9 
Clinical lab: total 
25(OH)D 
(3 mL serum [lime green 
top, PST]) 
X 
 
 
 
 
 
 
 
 
 
3 
Clinical lab: PTH* 
 
X 
 
 
X 
 
 
 
 
 
0* 
Vitamin D metabolites 
(10 mL serum [red top]) 
 
X 
X 
X 
X 
X 
X  
X 
X 
X 
90 
Stored blood 
(10 mL serum [red top], 
20 mL EDTA-plasma 
[purple top], 4 mL citrate-
plasma [blue top]) 
 
X 
 
 
 
 
 
 
 
 
34 
DNA** 
 
X 
 
 
 
 
 
 
 
 
0** 
RNA 
 
3 
 
 
 
 
 
 
 
 
 
Total volume (mL) 
9 
50 
10 
10 
13 
10 
10 
10 
10 
10 
142 
Notes: * PTH measured with chemistries, requires no additional volume; **DNA extracted from EDTA plasma vacutainers, requires no additional 
volume