Clearance of 25-hydroxyvitamin D3 During Vitamin D3 Supplementation
Phase: PHASE1
Conditions: Chronic Kidney Disease
Sponsor: University of Washington

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Clinical Research Protocol 
 
Clearance of 25-hydroxyvitamin D3 during vitamin D3 supplementation 
 
 
Protocol Number: 
CLEAR-PLUS-01 
Version: 
1.0 
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 (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 
11 
1.1 Overview of Clinical Studies ..................................................................................11 
2 STUDY RATIONALE 
11 
2.1 Risk / Benefit Assessment ......................................................................................14 
3 STUDY OBJECTIVES 
14 
3.1 Primary Objective ...................................................................................................14 
3.2 Secondary Objectives ..............................................................................................15 
4 STUDY DESIGN 
15 
5 CRITERIA FOR EVALUATION 
15 
5.1 Primary Efficacy Endpoint .....................................................................................15 
5.2 Secondary Efficacy Endpoints ................................................................................15 
5.3 Safety Evaluations ..................................................................................................16 
6 SUBJECT SELECTION 
17 
6.1 Study Population .....................................................................................................17 
6.2 Inclusion Criteria ....................................................................................................17 
6.3 Exclusion Criteria ...................................................................................................17 
7 CONCURRENT MEDICATIONS 
18 
8 STUDY TREATMENTS 
19 
8.1 Method of Assigning Subjects to Treatment Groups ..............................................19 
8.2 Blinding...................................................................................................................19 
8.3 Formulation of Test and Control Products..............................................................19 
8.4 Supply of Study Drug at the Site ............................................................................19 
8.5 Supply of Study Drug at the Site ............................................................................20 
8.6 Study Drug Accountability .....................................................................................20 
8.7 Measures of Treatment Compliance .......................................................................21 
9 STUDY PROCEDURES AND GUIDELINES 
21 
9.1 Clinical Assessments ..............................................................................................21 
9.2 Clinical Laboratory Measurements .........................................................................22 
9.3 Pharmacokinetic Measurements .............................................................................22 
9.4 Research Laboratory Measurements .......................................................................22 
10 EVALUATIONS BY VISIT 
23 
10.1 Screening Visit (Study Visit 1) ...............................................................................23 
10.2 Baseline Visit (Study Visit 2, Day 0) ......................................................................23 
10.3 Study Visit 3 (Day 1) ..............................................................................................24 
10.4 Study Visit 4 (Day 4) ..............................................................................................24 
10.5 Study Visit 5 (Day 7) ..............................................................................................24 

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10.6 Study Visits 6-10 (Day 14, 21, 28, 42, and 56) ......................................................24 
11 ADVERSE EXPERIENCE REPORTING AND DOCUMENTATION 
24 
11.1 Adverse Events .......................................................................................................24 
11.2 Serious Adverse Experiences (SAE) ......................................................................26 
12 DISCONTINUATION AND REPLACEMENT OF SUBJECTS 
27 
12.1 Early Discontinuation of Study Drug .....................................................................27 
12.3 Withdrawal of Subjects from the Study ..................................................................28 
12.4 Replacement of Subjects .........................................................................................28 
13 PROTOCOL VIOLATIONS 
28 
14 STATISTICAL METHODS AND CONSIDERATIONS 
29 
14.1 Data Sets Analyzed .................................................................................................29 
14.2 Demographic and Baseline Characteristics ............................................................29 
14.3 Analysis of Primary Endpoint .................................................................................29 
14.4 Analysis of Secondary Endpoints ...........................................................................29 
14.5 Interim Analysis ......................................................................................................29 
14.6 Sample Size and Randomization ............................................................................29 
15 DATA COLLECTION, RETENTION AND MONITORING 
30 
15.1 Data Collection Instruments ...................................................................................30 
15.2 Data Management Procedures ................................................................................30 
15.3 Data Quality Control and Reporting .......................................................................30 
15.4 Archival of Data ......................................................................................................30 
15.5 Availability and Retention of Investigational Records ...........................................30 
15.6 Monitoring ..............................................................................................................31 
15.7 Subject Confidentiality ...........................................................................................31 
16 ADMINISTRATIVE, ETHICAL, REGULATORY CONSIDERATIONS 
31 
16.1 Protocol Amendments .............................................................................................31 
16.2 Institutional Review Boards and Independent Ethics Committees .........................31 
16.3 Informed Consent Form ..........................................................................................32 
16.4 Publications .............................................................................................................32 
16.5 References ...............................................................................................................33 
 

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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 D3 during vitamin D3 
supplementation 
SPONSOR 
Ian H. de Boer, MD, MS 
FUNDING 
ORGANIZATION 
National Institute of Diabetes and Digestive and Kidney 
Diseases (R01DK099199) 
NUMBER OF SITES 
Single-site study 
RATIONALE 
Vitamin D clearance is an important and often overlooked 
aspect of vitamin D biology that has relevant implications for 
the clinical assessment and treatment of vitamin D deficiency. 
The circulating concentration of 25-hydroxyvitamin D 
[25(OH)D] is the most widely accepted measure of vitamin D 
status, and vitamin D supplements are often prescribed and 
titrated based on 25(OH)D concentration. However, circulating 
concentrations of 25(OH)D are necessarily determined by both 
25(OH)D production and 25(OH)D clearance. Therefore, 
25(OH)D clearance likely plays an important role in the 
assessment of vitamin D status and the response to vitamin D 
supplementation. 
The goal of this study is to determine how 25(OH)D3 clearance 
is affected by vitamin D3 supplementation using a gold 
standard pharmacokinetic approach. We expect that this study 
will enhance interpretation of available diagnostic tests, inform 
the results of ongoing large clinical trials of vitamin D 
supplements, and help develop new strategies to target vitamin 
D to improve health. 
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 in 
subjects that supplement their diet with vitamin D3. These data 
will be compared with previously acquired “un-supplemented” 
measurements obtained from the same patients. This study will 
only enroll subjects who previously participated in the CLEAR 
or CLEAR-CF protocol and from whom un-supplemented 
measurements of 25(OH)D3 clearance were obtained.  The 
CLEAR and CLEAR-CF protocols are both included under  
IND 115016. In this study, a repeat measurement of 25(OH)D3 
clearance will be obtained from eligible subjects who are 
supplementing their diet with  a stable dose of Vitamin D3, 
defined as cholecalciferol 2000 IU daily. The patterns of 
25(OH)D3 clearance in these patients, with and without 
vitamin D3 supplementation, will be assessed.  

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Allocation: Non-Randomized 
Intervention Model: Single Group Assignment 
Masking: Open Label  
PRIMARY OBJECTIVE 
The goal of this study is to determine if 25(OH)D3 clearance is 
affected by vitamin D3 supplementation. 
SECONDARY 
OBJECTIVES 
• To explore whether disease status (chronic kidney disease 
or cystic fibrosis) modify the 25(OH)D3 clearance response 
to vitamin D3 supplementation.  
• To evaluate biomarkers of 25(OH)D3 clearance during 
vitamin D3 supplementation.  
NUMBER OF SUBJECTS 
Up to 20 
SUBJECT SELECTION 
CRITERIA 
Inclusion Criteria:  
• Successful completion of related protocol CLEAR or 
CLEAR-CF, defined as: 
o Previous administration of D6-25-
hydroxyvitamin D3 Solution under the CLEAR 
or CLEAR-CF protocol, 
o Completion of at least 7 CLEAR or CLEAR-CF 
study visits, and  
o Absence of adverse events related to D6-25-
hydroxyvitamin D3 Solution administration 
during participation in CLEAR or CLEAR-CF 
 
Exclusion Criteria: 
• Primary hyperparathyroidism 
• Gastric bypass 
• Tuberculosis or sarcoidosis 
• Current pregnancy 
• Child-Pugh Class B or C cirrhosis (i.e. cirrhosis with 
ascites, hepatic encephalopathy, bilirubin >=2 mg/dL, 
serum albumin <=3.5 g/dL, or PT >= 4 seconds) 
• History of kidney transplantation (unless failed transplant 
now treated with hemodialysis) 
• Use of 1,25(OH)2D3 or an analogue, calcimimetics, or 
medications known to induce CYP24A1 within 4 weeks 
(wash-out allowed) 
• Serum calcium > 10.1 mg/dL 
• Hemoglobin < 9 g/dL 
TEST PRODUCT, DOSE, 
AND ROUTE OF 
ADMINISTRATION 
We will administer D6-25-hydroxyvitamin D3 Solution for 
Injection, 20 µg/mL intravenously. The deuterated 25(OH)D3 
is formulated using cosolvents to enhance solubility.  Each mL 
contains 20 µg D6-25(OH) D3, 60% propylene glycol, 10% 
ethanol (ETOH) and water (30%) for injection, to volume. To 

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ensure reliable detection of circulating deuterated 25(OH)D3, 
without administering a dose that might alter the underlying 
vitamin D metabolism, we aim to 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 6 months 
Screening and vitamin D3 supplement run-in:  3 months 
Re-screening and scheduling:  approximately 1 month 
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 18 
months for subject recruitment and an additional 12 weeks for 
final subject follow-up. 
CONCOMMITANT 
MEDICATIONS 
Required: 
• Vitamin D3 (cholecalciferol) 2000 IU daily by mouth from 
start of run-in through completion of follow-up, provided 
by study team 
Allowed: 
• Additional vitamin D supplements (cholecalciferol or 
ergocalciferol, alone or contained in multivitamins) not to 
exceed a mean daily dose of 400 IU 
Prohibited (from 4 weeks prior to D6-25-hydroxyvitamin D3 
until completion of the study): 
• 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 

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• 
Volume of distribution in the central compartment of 
labeled 25(OH)D3 
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 
PLANNED INTERIM 
ANALYSES  
This is not a clinical trial with an efficacy outcome, and it is 
neither randomized nor blinded. Formal interim analyses are 
not planned.  
STATISTICS 
Primary Analysis Plan 
We will compare the distribution of D6-25(OH)D3 clearance 
measured during this study to the distribution of D6-25(OH)D3 
clearance measured in the same participants in the un-
supplemented state during CLEAR or CLEAR-CF. We will use 
the paired t-test to test differences in these distributions. We 
will use linear regression with multiplicative interaction terms 
to explore whether CKD or CF modify the D6-25(OH)D3 
clearance response to vitamin D3 supplementation. 
Rationale for Number of 
Subjects 
With 20 participants, we can detect differences in D6-
25(OH)D3 clearance that are small (17.5-55 mL/min depending 
on assumptions) compared to the mean value observed in 
CLEAR (351 mL/min) and compared to the change in 
clearance we expect based on preliminary data (>50%). 
 

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1 
BACKGROUND  
Vitamin D supplementation improves bone mineral density, helps prevent fracture in high-
risk populations, and may promote other aspects of health.1 The broad potential health benefits of 
vitamin D are currently being tested in large clinical trials, such as the Vitamin D and Omega-3 
Trial (VITAL). Circulating concentrations of 25-hydroxyvitamin D [25(OH)D] are currently the 
most widely accepted tool used to gauge the need for and response to vitamin D supplements. 
However, large inter-individual differences in circulating 25(OH)D concentration and the 
25(OH)D response to vitamin D supplementation are well documented. Furthermore, 25(OH)D 
is itself relatively inactive, and circulating 25(OH)D concentrations reflect only substrate 
available for conversion to the 1,25-dihydroxyvitamin D [1,25(OH)2D], the active vitamin D 
hormone.  
Vitamin D clearance is an important and often overlooked aspect of vitamin D biology. 
Known clinical determinants of circulating 25(OH)D concentration, including dietary intake, sun 
exposure, adiposity, and race, focus almost entirely on vitamin D intake/production. These 
factors explain a minority of variability in circulating 25(OH)D concentration and the response 
to vitamin D supplementation. By definition, steady-state circulating concentrations of 25(OH)D 
are determined by both intake/production and clearance. Genetic heterogeneity in CYP24A1, the 
cytochrome P450 enzyme primarily responsible for 25(OH)D clearance, is also a known 
determinant of circulating 25(OH)D concentration. Our preliminary data suggest that vitamin D 
supplementation induces CYP24A1-mediated 25(OH)D clearance, and that kidney function may 
be an important determinant of the 25(OH)D clearance response to vitamin D supplementation. 
The proposed studies will provide insight into the biology of 25(OH)D clearance that is 
likely to enhance interpretation of available diagnostic tests, inform the results of ongoing large 
clinical trials of vitamin D supplements, and help develop new strategies to target vitamin D to 
improve health. 
1.1 
Overview of Clinical Studies 
Deuterated and tritiated 25(OH)D3  have been used previously to study metabolism of vitamin 
D3 in humans.2-6 In addition deuterium is has been used extensively to study metabolism and is 
considered safe.7 
2 
STUDY RATIONALE 
Vitamin D may promote health through pleiotropic actions. Traditionally understood as a 
calcium regulatory hormone, 1,25-dihydroxyvitamin D (1,25(OH)2D, the potent hormonal form 
of vitamin D) binds to its target receptor to regulate the transcription of hundreds of genes.1-8 
Vitamin D receptors are present in all nucleated cells in the body, and activation of vitamin D 
receptors modulates cellular processes related to proliferation, differentiation, fibrosis, cancer, 
cardiovascular diseases, and other chronic diseases.6-10 For each of these processes, large effects 
have been demonstrated in cell culture and animal-experimental models, and clinical 
epidemiology studies have observed associations of circulating vitamin D metabolite 
concentrations with clinical health outcomes. As an example, for cardiovascular diseases, 
1,25(OH)2D and analogues promote nitric oxide-mediated endothelial vasodilation,11-16 down-
regulate vascular smooth muscle cell proliferation and migration into the intima,17 inhibit 
osteogenic differentiation of vascular smooth muscle cells,18 inhibit macrophage cholesterol 
uptake and foam cell formation,19-21 promote a shift from atherogenic Th1 to anti-atherogenic 

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Th2 lymphocytes,22  reduce the development and progression of ventricular hypertrophy,23-27 
mitigate cardiac fibrosis,27-30 and suppress the renin-angiotensin system in animal-experimental 
models.31-33 Cardioprotective biological actions of vitamin D are further supported by 
associations of low serum 25-hydroxyvitamin D [25(OH)D] and 1,25(OH)2D concentrations 
with clinical hypertension, myocardial infarction, stroke, left ventricular hypertrophy, cancer, 
and mortality.34-46 The totality of evidence for vitamin D has motivated large clinical trials, 
currently underway, to test whether vitamin D treatment will reduce the risks of cancer, 
cardiovascular disease, and other clinical health outcomes in the general population. 
Inter-individual differences in serum 25(OH)D concentration and the serum 25(OH)D 
response to vitamin D supplements are large. The circulating concentration of 25(OH)D is 
widely accepted as a biomarker of total intake of vitamins D3 and D2 (cholecalciferol and 
ergocalciferol) from cutaneous synthesis and dietary consumption.1-3 Serum or plasma 25(OH)D 
concentration increases robustly to vitamin D supplementation or exposure to ultraviolet light. 
Circulating 25(OH)D concentration is also inversely correlated with a number of health 
outcomes, including bone mineral density, risk of hip fracture, incident cancer, and 
cardiovascular events. Therefore, circulating 25(OH)D concentration is used to define vitamin D 
sufficiency and deficiency. There is a known large variation across individuals and populations 
in circulating 25(OH)D concentration. Moreover, published trials demonstrate substantial 
heterogeneity in the biologic response to vitamin D treatment, defined by changes in parathyroid 
hormone (PTH), intestinal calcium absorption, bone turnover, and bone mineral density.47 While 
clinical characteristics such as race (presumably due to differences in cutaneous vitamin D 
production due to skin pigmentation), sun exposure, dietary intake of vitamin D, and adiposity 
(presumably due to vitamin D volume of distribution/fat sequestration) are known to be 
associated with circulating 25(OH)D concentration, they explain a minority of this variation. 
Additional reasons for differences in 25(OH)D and the 25(OH)D response to supplementation 
are poorly understood. 
Vitamin D clearance plays an important role in vitamin D biology. Concentrations of vitamin 
D metabolites in blood and tissues must necessarily represent a balance of production and 
clearance. In health, CYP24A1 is the major enzyme responsible for catabolism of both 
25(OH)D3 and 1,25(OH)2D3 (Figure 1).4,48-54 CYP24A1 is found in most tissues in the body and 
is rapidly induced by 1,25(OH)2D3 to prevent 1,25(OH)2D3 intoxication.55-57 Polymorphisms in 
CYP24A1 were found to be associated with circulating 25(OH)D concentration in a genome-
wide association study (GWAS).58 Similarly, in another GWAS, a polymorphism in CYP24A1 
showed the strongest association with the circulating concentration of PTH, a hormone known to 
regulate and be regulated by 
1,25(OH)2D.59 In unpublished data, we 
found a CYP24A1 polymorphism to also 
be most strongly associated with the 
serum concentration of fibroblast growth 
factor-23, another hormone known to 
regulate and be regulated by 1,25(OH)2D. 
Inactivating mutations in CYP24A1 have 
been found to cause “idiopathic” infantile 
hypercalcemia, characterized by excess 
1,25(OH)2D-mediated intestinal calcium 
absorption and hypercalcemia, 

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particularly with vitamin D supplementation.49 These observations demonstrate that vitamin D 
clearance is critical for maintaining proper vitamin D and calcium homeostasis, perhaps most 
crucially in the context of vitamin D supplementation. 
Serum 24,25(OH)2D3 (or its ratio with 25(OH)D3) maybe a useful novel biomarker of 
functional vitamin D status. Because CYP24A1 is found in most tissues in the body and is 
rapidly induced by 1,25(OH)2D3,55-57 CYP24A1 expression is commonly used as a marker of 
tissue 1,25(OH)2D3 activity in basic science.64-80 However, the ability of vitamin D catabolism to 
reflect 1,25(OH)2D3 function has not been similarly harnessed for clinical use. Evaluation of 
circulating 24,25(OH)2D3 concentration may offer this opportunity. 24,25(OH)2D3 is the most 
abundant product of 25(OH)D3 catabolism by CYP24A1.49 When viewed in the context of 
prevailing 25(OH)D3, 24,25(OH)2D3 reflects CYP24A1 activity. 24,25(OH)2D3 circulates at 
high concentrations (generally 1-10 ng/mL)81,82 and has a circulating half-life of approximately 7 
days.83 Circulating 24,25(OH)2D3 appears to come from both renal and non-renal sources.54,67,84-
86 In the kidney proximal tubule, CYP24A1 transcription is induced by fibroblast growth factor-
23 and suppressed by PTH.56,87 In our work, we have found chronic kidney disease (CKD, 
specifically low estimated glomerular filtration rate, or eGFR) to be strongly correlated with 
serum 24,25(OH)2D3 concentration.  
Our preliminary data suggest that vitamin D supplementation increases vitamin D 
catabolism. We recently published a study assessing the effects of vitamin D2 (ergocalciferol) 
supplementation on circulating vitamin D metabolites and vitamin D regulatory hormones.88 We 
enrolled 25 participants with CKD 
(eGFR <60 mL/min/1.73m2) and 44 
participants with eGFR ≥60 
mL/min/1.73m2 and treated each with 
vitamin D2 50,000 IU twice weekly for 
5 weeks. We measured vitamin D 
metabolites at baseline and the end of 
treatment using the precise mass 
spectrometry methods described in 
Section 4e of Approach. Among other 
findings, we reported that vitamin D2 
supplementation significantly increase 
serum 24,25(OH)2D3 to 25(OH)D3 
ratio (Table 2.1), suggesting induction 
of 25(OH)D3 clearance. We 
hypothesized that induced clearance 
contributed to maintenance of normal circulating 1,25(OH)2D concentrations. Notably, the 
magnitude of increase in 24,25(OH)2D3 to 25(OH)D3 ratio was blunted among participants with 
CKD, suggesting that the decreased 25(OH)D3 clearance suggested by low 24,25(OH)2D3 
concentrations in our cross-sectional studies extends to the setting of vitamin D2 
supplementation. The use of vitamin D2 as the supplement allowed us to differentiate exogenous 
from endogenous vitamin D metabolites, but an important limitation (to be addressed with the 
proposed studies) was that we could not differentiate true induction of clearance from a shift in 
available CYP24A1 substrate from 25(OH)D3 to 25(OH)D2.  
Table 2.1. Effects of vitamin D2 supplementation 
on serum 24,25(OH)2D3:25(OH)D3 ratio 
 
Participants 
with CKD 
(N=25) 
Participants with 
normal eGFR 
(N=44) 
Baseline 
72 (9) 
116 (8) 
End of 
study 
110 (9) 
195 (8) 
Change 
+38 (21 to 56) 
+80 (65 to 94) 
p-value 
<0.001 
<0.001 
Cell contents are mean (SD) or mean change with 
supplementation (95% CI) (in pg/ng), or p-value 

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In summary, despite widespread interest in vitamin D an intervention to improve diverse 
aspects of health, little attention has been paid to the role of clearance in vitamin D biology. 
Clearance is a fundamental component of vitamin D homeostasis and is likely to be an important 
determinant of both steady state 25(OH)D concentrations and the biologic response to vitamin D 
supplementation. Our preliminary data suggest that vitamin D clearance induces CYP24A1-
mediated 25(OH)D clearance, and that clinical characteristics (e.g. kidney function) modify this 
response. Our preliminary data further support the concept that circulating 24,25(OH)2D3 
concentration is a useful biomarker of CYP24A1-mediated 25(OH)D clearance. However, most 
studies performed to date, including our own, have used biomarkers to evaluate the effects of 
vitamin D supplements on vitamin D clearance. In this study, we will use gold standard 
pharmacokinetic studies to assess the 25(OH)D3 clearance response to vitamin D3 
supplementation. 
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. Moreover, this 
protocol will only enroll participants who successfully completed a related protocol (CLEAR or 
CLEAR-CF) without adverse effect. This study also includes placement of peripheral 
intravenous catheters and blood draws. Risks include discomfort at the site of administration, 
minor bleeding associated with catheter placement, and anemia due to blood sampling. 
3 
STUDY OBJECTIVES 
3.1 
Primary Objective 
The goal of this study is to determine whether 25(OH)D3 clearance is affected by vitamin D3 
supplementation. The data obtained in this study will be compared to previously obtained “un-
supplemented” measurements taken in the same patients. Only subjects who previously 
participated in the CLEAR or CLEAR-CF protocol and from whom un-supplemented, 
measurements of 25(OH)D3 clearance were obtained will be enrolled in this study.   

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3.2 
Secondary Objectives 
• To explore whether disease status (chronic kidney disease or cystic fibrosis) modify the 
25(OH)D3 clearance response to vitamin D3 supplementation.  
• To evaluate biomarkers of 25(OH)D3 clearance during vitamin D3 supplementation. 
4 
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 in subjects 
supplementing their diet with a stable dose of vitamin D3. This study will enroll up to 20 subjects 
who previously participated in the CLEAR or CLEAR-CF protocol and from whom un-
supplemented measurements of 25(OH)D3 clearance were obtained.   
Subjects who qualify and provide written informed consent will be prescribed a stable, standard 
dose of vitamin D3 supplement (cholecalciferol, 2000 IU daily by mouth), to be taken throughout 
the duration of the study (approximately 6 months).  
After 3 months of vitamin D3 run-in, participants will be re-screened for eligibility (prior to 
receiving the intravenous labeled 25(OH)D3 study drug). Participants who remain eligible at re-
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. The metabolic clearance of 25(OH)D3 during stable vitamin D3 supplementation 
(obtained from this protocol) will be compared to the metabolic clearance of 25(OH)D3 in the 
un-supplemented state (obtained for the same participants during CLEAR or CLEAR-CF) to 
assess the effects of vitamin D3 supplementation. 
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.53-56 
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.   
5.2 
Secondary Efficacy Endpoints 
• AUC of labeled D6-25(OH)D3  
• Terminal half-life of labeled D6-25(OH)D3 

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• Volume of distribution in the central compartment of labeled D6-25(OH)D3 
• Metabolic formation clearance (metabolite/parent AUC ratio) for metabolites of labeled D6-
25(OH)D3 
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 
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 
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6 
SUBJECT SELECTION 
6.1 
Study Population 
Only participants who successfully completed the CLEAR or CLEAR-CF protocol will be 
eligible for this study. CLEAR and CLEAR-CF are single-center studies that recruit adults with 
and without chronic kidney disease (for CLEAR) or with or without cystic fibrosis (for CLEAR-
CF) from the University of Washington system and related institutions. CLEAR or CLEAR-CF 
participants will be approached for participation in this study at the time of their last CLEAR or 
CLEAR-CF visit, or within 18 months of completing the CLEAR or CLEAR-CF protocol. 
6.2 
Inclusion Criteria 
The main inclusion criterion is the successful completion of related protocol CLEAR or CLEAR-
CF, defined as: 
• Receipt of D6-25-hydroxyvitamin D3 Solution under the CLEAR or CLEAR-CF 
protocol, 
• Completion of at least 7 CLEAR or CLEAR-CF study visits, and  
• Absence of adverse events related to D6-25-hydroxyvitamin D3 Solution administration 
during participation in CLEAR or CLEAR-CF 
CLEAR and CLEAR0CF enrolled only adults, with additional eligibility criteria related to race, 
disease status (stage of chronic kidney disease or presence of cystic fibrosis), and screening 
serum 25(OH)D concentration (10-50 ng/mL). 
6.3 
Exclusion Criteria 
Exclusion criteria will be: 
• Inability to give informed consent 
• Primary hyperparathyroidism 
• Gastric bypass 
• Tuberculosis or sarciodosis 
• pregnancy 
• Child-Pugh Class B or C cirrhosis (i.e. cirrhosis with ascites, hepatic encephalopathy, 
bilirubin >=2 mg/dL, serum albumin <=3.5 g/dL, or PT >= 4 seconds) 
• History of kidney transplantation (unless failed transplant now treated with hemodialysis) 
• hemoglobin <9 mg/dL 
• serum calcium >10.1 mg/dL 
• use of active vitamin D receptor agonists (e.g. 1,25(OH)2D3 or an analogue, calcitriol) or 
cinacalcet within 4 weeks 
• use of a cytochrome P-450 (CYP) inhibitor or inducer within 4 weeks (table 6.4) 
• Unwilling to take vitamin D3 (cholecalciferol) 2000 IU daily by mouth, as provided by 
the study 
 
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 
active vitamin D compounds, cinacalcet, and CYP inhibitors and inducers the washout period 
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Table 6.3. Patient Eligibility – Exclusion criteria 
Active vitamin D receptor agonist (e.g. calcitriol) or cinacalcet 
in last 4 weeks 
Child-Pugh Class B or C cirrhosis 
Hemoglobin < 9 mg/dl 
Serum calcium concentration > 10.1 mg/dl 
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 
 
* Greater than 8 ounces per day 
7 
CONCURRENT MEDICATIONS 
All subjects will be asked to take vitamin D3 (cholecalciferol) 2000 IU daily by mouth, provided 
by the study. Vitamin D3 is a common form of vitamin D supplement that is readily available by 
prescription or over the counter. Vitamin D3 is administered orally, and 2000 IU daily is a 
common dose that is well below the tolerable upper intake level advised by the Institute of 
Medicine (4000 IU daily). This form and dose of vitamin D supplement is being actively studied 
in large clinical trials with clinical outcomes, such as the Vitamin D and Omega-3 Trial 
(VITAL), which will increase the utility of the data generated from this study. Hypercalcemia is 
a rare but known potentially complication of vitamin D3 supplementation (more commonly at 
higher doses), so serum calcium will be checked after 3 months of vitamin D3 supplementation 
(at the re-screening visit), prior to administration of the deuterated 25(OH)D3 study drug. 
Vitamin D3 will be obtained from Carlson Laboratories; supporting information is supplied in 
the protocol appendix. Participants will be allowed to take up to 400 IU daily additional vitamin 
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the 2000 IU daily provided by the study. Non-study vitamin D supplements exceeding 400 IU 
daily will be disallowed. 
Other specific allowed and disallowed medications are detailed in Section 6 (Eligibility Criteria). 
All subjects should be maintained on the same medications throughout the entire study period, as 
medically feasible, with no introduction of new chronic therapies. 
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. 
8.3 
Formulation of Test and Control Products 
We will administer labeled D6-25(OH)D3 Solution for Injection, 20 µg/mL intravenously. D6-
25(OH) D3 Solution for Injection, 20 µg/mL  has been manufactured according to current Good 
Manufacturing Practices by SRI International (Palo Alto, CA). 
8.3.1 Formulation of Test Product 
The labeled D6-25(OH)D3 Solution for Injection, 20 µg/mL prepared by SRI International is 
formulated in cosolvents to enhance solubility.  Each mL contains 20 µg D6-25(OH) D3, 60% 
propylene glycol (60%), 10% ethanol (ETOH) and water (30%) for injection, to volume.   
8.3.2 Packaging and Labeling 
The formulated drug product has been aliquoted into single-use vials by SRI International and 
frozen at <60°C for storage. Vial labels include the drug product name, concentration, batch 
number, manufacture date, and storage conditions, similar to the draft label shown below. 
 
8.4 
Supply of Study Drug at the Site 
Single-use vials will be stored at <60°C at the University of Washington Investigational Drug 
Services or Kidney Research Institute. Individual doses of D6-25(OH) D3 Solution for Injection 
will be prepared for intravenous administration by the University of Washington Investigational 
Drug Services by thawing and drawing the appropriate volume into a syringe. The prepared dose 
of will be calculated according to estimated blood volume based on body weight. The prepared 

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dosing syringe will be labeled with the participant’s full name, date of birth, and medical record 
number along with the drug contents. Once thawed, the drug product will be infused within eight 
hours of preparation. 
8.4.1 Dosage/Dosage Regimen 
Each participant will receive a single intravenous dose of D6-25(OH) D3 Solution for Injection, 
20 µg/mL. We will use estimated blood volume based on body weight to achieve a target 
concentration of 5 ng/mL D6-25(OH) D3. The target of 5 ng/mL was selected because it will 
allow precise tracking of circulating deuterated D6-25(OH) D3concentration 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 would be 32 µg (1.6 mL) for an 85 kg individual. We have verified in 
CLEAR and CLEAR-CF that we achieve target D6-25(OH) D3 concentrations with this 
approach. 
8.4.2 Dispensing 
All study drug will be dispensed by the University of Washington Investigational Drug Services. 
8.4.3 Administration Instructions  
The study drug will be administered intravenously on a single occasion over a period of 5 
minutes. Subsequent to thawing, the drug product vial may be stored refrigerated (2-8 °C) for up 
24 hours prior to preparation for administration in a syringe.  Once prepared for administration 
and held at room temperature, the solution should be administered within 8 hours. 
8.5 
Supply of Study Drug at the Site 
All single-use vials of formulated D6-25(OH)D3 will be shipped overnight on dry ice to the 
University of Washington Investigational Drug Services, with temperature monitoring. The 
University of Washington is the sole clinical site for this study.   
8.5.1 Storage 
Study drug will be securely stored at a temperature <-60°C at the University of Washington 
Investigational Drug Services or Kidney Research Institute. 
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 
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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, Re-
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 data will be abstracted from data provided during CLEAR or CLEAR-CF.  
9.1.3 Medical History  
Relevant medical history, including history of current disease, other pertinent history, and 
information regarding underlying diseases, will be abstracted from data provided during CLEAR 
or CLEAR-CF.  
9.1.4 Physical Examination 
Height and weight will be measured at the Re-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, vitamin D intake, and body composition measured by dual energy X-ray 
absorptiometry will be abstracted from data provided during CLEAR or CLEAR-CF. Serum and 
plasma will be collected at Baseline to measure calcium and phosphorus, intact parathyroid 
hormone, fibroblast growth factor-23, and vitamin D binding protein. Two 24-hour urine 
collections will be obtained, immediately preceding and following administration of D6-25(OH) 

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D3, to quantify urinary excretion of albumin and the metabolites of deuterated D6-25(OH) D3, 
respectively. Dialysis patients will be exempt from all urine collections. 
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 Re-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 Re-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. 
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. For anuric (dialysis) participants, blood 
pregnancy tests will be obtained to rule out pregnancy during the week preceding the 
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.57  25(OH)D3-3-sulfate will be extracted 
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10 EVALUATIONS BY VISIT 
10.1 Screening Visit (Study Visit 0)  
The following activities will occur during the screening visit:   
• 
Collect signed consent form 
• 
Confirm successful completion of CLEAR or CLEAR-CF protocol 
• 
Perform focused medical history restricted to study eligibility criteria 
• 
Complete medication inventory through EMR and patient report 
• 
Participants who meet eligibility criteria at the screening visit will be prescribed vitamin D3, 
2000 IU daily by mouth 
10.2 Re-screening Visit (Study Visit 1)  
The Re-screening Visit will take place approximately 90 days after the Screening Visit 
(allowable range 60-180 days). The following activities will occur during the re-screening visit:   
• 
Confirm adherence (≥80%) to study vitamin D3 (by self-report and pill count) 
• 
Repeat focused medical history restricted to study eligibility criteria 
• 
Complete medication inventory through EMR and patient report. 
• 
Physical Exam (height and weight), and Vital Signs (blood pressure, heart rate) 
• 
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. 
• 
Participants who continue to meet eligibility criteria, at the screening visit and who are 
adherent (≥80%) to study vitamin D3, will be invited to return for the remaining study visits.  
• 
Provide selected subjects with supplies required for first 24-hour urine collection 
10.3 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 at least 90 
days after Visit 0 and 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 
• Review current medications.  
• Physical Exam (height and weight), and Vital Signs (blood pressure, heart rate) 
• Complete physical activity and food frequency questionnaires.  
• Pregnancy test for women of childbearing potential 
• Collect blood prior to administration of study drug for measurement of basic chemistries, 
vitamin D metabolites, parathyroid hormone (PTH), and other measurements related to 
kidney disease and vitamin D metabolism.  
• 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:   
• Administer D6-25(OH) D3 intravenously.  

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• Collect blood 5 minutes and 4 hours post-infusion for measurement of vitamin D metabolites.  
• Distribute supplies for second 24-hour urine collection. 
10.4 Study Visit 3 (Day 1)   
This visit is expected to last less than one hour.  The following activities will occur during Visit 
3: 
• Collect urine sample from second 24-hour collection period.   
• Collect blood for measurement of serum vitamin D metabolite concentrations.  
10.5 Study Visit 4 (Day 4) 
This visit is expected to last less than one hour. A window of one day before or after the 
scheduled date will be allowed when needed. Blood will be collected for measurement of serum 
vitamin D metabolite concentrations.  
10.6 Study Visit 5 (Day 7) 
This visit is expected to last less than one hour. A window of one day before or after the 
scheduled date will be allowed when needed. The following activities will occur during Visit 7: 
• 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.  
10.7 Study Visits 6-10 (Day 14, 21, 28, 42, and 56) 
Each of these visits is expected to last less than one hour. A window of up to 3 days before or 
after the scheduled date will be allowed when needed. Blood will be collected at each visit for 
measurement of serum vitamin D metabolite concentrations. Medication inventory will be 
repeated at Visit 10 (Day 56). 
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  

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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. 
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. 

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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. 
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. 

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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 
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 

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• 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.   
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 

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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 (D6-25(OH) D3 Solution for Injection) will be 
included in the safety analysis.    
14.2 Demographic and Baseline Characteristics 
The following variables collected at re-screening will be summarized: 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 compare the distribution of D6-25(OH)D3 clearance measured during this study to the 
distribution of D6-25(OH)D3 clearance measured in the same participants in the unsupplemented 
state during CLEAR or CLEAR-CF. We will use the paired t-test to test differences in these 
distributions. We will use linear regression with multiplicative interaction terms to explore 
whether CKD or CF modify the D6-25(OH)D3 clearance response to vitamin D3 
supplementation.. 
14.4 Analysis of Secondary Endpoints 
Analyses of secondary endpoints will parallel those of the primary endpoint. 
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 
The sample size of 20 participants gives us excellent power to detect even modest differences in 
25(OH)D3 clearance with vitamin D3 supplementation. To determine detectable effect sizes 
using a fixed sample size of 20 participants, a pre-post design using the paired t-test, 90% power, 
and a type 1 error rate of 5%, we assumed that the standard deviation of baseline deuterated 
25(OH)D3 clearance observed among our first four actual participants (319, 322, 332, and 431 
mL/day) was the same as that of supplemented clearance. We calculated the minimal detectable 
differences in deuterated 25(OH)D3 clearance (i.e. the smallest differences we can detect with 
90% power; power is >90% for larger differences),over a range of correlation for baseline and 
supplemented clearance (a factor that is unknown). Table 14.1 shows that we can detect 
differences in clearance that are small compared to the mean value for our first four participants 
(351 mL/day) and compared to the change in clearance we expect (>50% increase, as per 
preliminary study reported in Table 2.1). 

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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. 
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 
Table 14.1. Study power 
Correlation of 
baseline and 
supplemented 
clearances 
Minimal 
detectable 
difference with 
90% power 
(mL/day) 
r = 0 
55 
r = 0.25 
48 
r = 0.5 
39 
r = 0.75 
27.5 
r = 0.9 
17.5 

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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.  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 will not be implemented without prior submission to FDA and 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. 
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. 

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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. 
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.  

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25. 
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Swami S, Krishnan AV, Wang JY, Jensen K, Horst R, Albertelli MA, Feldman D. 
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Protocol Template © CFFT TDN   Effective: 12 SEP 2007 
APPENDIX 1. SCHEDULE OF STUDY PROCEDURES 
Procedure 
VISIT 0  
SCREENING 
VISIT 1  
RE-SCREENING 
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 
X 
 
 
 
 
 
 
 
 
 
Focused Medical History 
X 
X 
 
 
 
 
 
 
 
 
 
Physical Exam and Vital Signs 
 
X 
X 
 
 
 
 
 
 
 
 
Medications Inventory 
X 
X 
X 
 
 
 
 
 
 
 
X 
Blood Samples* 
 
X 
X 
X  
X 
X 
X 
X 
X 
X 
X 
24-hour urine collections 
 
 
X 
X 
 
 
 
 
 
 
 
DNA collection 
 
 
X 
 
 
 
 
 
 
 
 
Prescribe vitamin D3 
X 
 
 
 
 
 
 
 
 
 
 
Study Drug Administration 
 
 
X 
 
 
 
 
 
 
 
 
*Blood Draws Detailed in Appendix 2. 

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Protocol Template © CFFT TDN   Effective: 12 SEP 2007 
APPENDIX 2. DRAFT BLOOD DRAW TYPES AND VOLUMES 
Blood sample 
VISIT 1 
SCREENING 
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 
TOTAL VOLUME 
(ML) 
Clinical lab: CBC  
(3 mL EDTA-plasma 
[purple top]) 
X 
 
 
 
 
 
 
 
 
 
3 
Clinical lab: chemistries  
(4 mL serum [lime green 
top, PST]) 
X 
X 
 
 
X 
 
 
 
 
 
12 
Clinical lab: total 
25(OH)D 
(4 mL serum [lime green 
top, PST]) 
X 
 
 
 
 
 
 
 
 
 
4 
Clinical lab: PTH* 
 
X 
 
 
X 
 
 
 
 
 
0* 
Vitamin D metabolites 
(10 mL serum [red top]) 
 
X** 
X 
X 
X 
X 
X  
X 
X 
X 
114 
Stored blood 
(10 mL EDTA-plasma 
[purple top]) 
 
X 
 
 
 
 
 
 
 
 
10 
DNA*** 
 
X 
 
 
 
 
 
 
 
 
0** 
Total volume (mL) 
11 
48 
10 
10 
14 
10 
10 
10 
10 
10 
143 
Notes: * PTH measured with chemistries, requires no additional volume; ** 14 mL serum prior to injection of D6-25(OH)D3 plus two 10 mL serum 
samples after injection of D6-25(OH)D3; ***DNA extracted from EDTA plasma vacutainer, requires no additional volume