Clinical trial NCT02937350
Conditions: Chronic Kidney Disease
Keywords: chronic kidney disease vitamin d catabolism
Sponsor: University of Washington
Start date: 2017-03-01; Completion date: 2021-01-01
Enrollment: 88 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The goal of this study is to better understand vitamin D catabolism and how it is affected by CKD and race.

Detailed description:
Specifically, the study team will evaluate the metabolic clearance of 25-hydroxyvitamin D3 in individuals with varying degrees of CKD and among participants who self-report race as Caucasian, African American or African. The long-term goal of this work is to enhance the clinical evaluation and treatment of impaired vitamin D metabolism.

Arms: arm 1: D6-25-hydroxyvitamin D3
Interventions: intervention 1: D6-25-hydroxyvitamin D3
Intervention descriptions: intervention 1: Intravenous administration of a deuterium-labeled 25(OH)D3 to evaluate the metabolic clearance of 25(OH)D3

Protocol documents:
Prot_SAP_000.pdf: 
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Clinical Research Protocol 
Clearance of 25-hydroxyvitamin D in chronic kidney disease 
 
Protocol Number: 
CLEAR-01 
Version: 
2.1 
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 
9 
1.1 
Overview of Clinical Studies ............................................................................................. 9 
2 
STUDY RATIONALE 
9 
2.1 
Risk / Benefit Assessment ................................................................................................. 11 
3 
STUDY OBJECTIVES 
11 
3.1 
Primary Objective .............................................................................................................. 11 
3.2 
Secondary Objectives ........................................................................................................ 11 
4 
STUDY DESIGN 
11 
5 
CRITERIA FOR EVALUATION 
12 
5.1 
Primary Efficacy Endpoint ................................................................................................ 12 
5.2 
Secondary Efficacy Endpoints ........................................................................................... 12 
5.3 
Safety Evaluations ............................................................................................................. 12 
6 
SUBJECT SELECTION 
13 
6.1 
Study Population ................................................................................................................ 13 
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 ......................................................................................................... 18 
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) ................................................................................ 20 
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) ................................................................. 21 
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 ................................................................................................... 25 
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 ...................................................................................... 26 
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 ................................................................................................................ 27 
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 ....................................................................................................... 28 
16.2 Institutional Review Boards and Independent Ethics Committees .................................... 28 
16.3 Informed Consent Form ..................................................................................................... 28 
16.4 Publications ....................................................................................................................... 29 
16.5 References ......................................................................................................................... 30 
 


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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 in chronic kidney disease 
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 
Patients with chronic kidney disease (CKD) are at high risk of 
adverse health outcomes, including progression to end stage 
renal disease, cardiovascular disease events, and death. 
Impaired vitamin D metabolism is commonly observed in 
CKD and may contribute to the morbidity of this disease.  In 
healthy individuals, the kidney produces the majority of 
circulating 1,25-dihydroxyvitamin D3 (1,25(OH)2D3, or 
calcitriol).  Calcitriol binds the vitamin D receptors in diverse 
target tissues and is known to modulate inflammation and 
fibrosis, suppress the renin-angiotensin system, and regulate 
proliferation and differentiation. In CKD, renal production of 
1,25(OH)2D3 is reduced, resulting in secondary 
hyperparathyroidism and dysregulation of other organs and 
tissues. Our preliminary data suggest that clearance of vitamin 
D metabolites is also reduced in CKD. 
The goal of this study is to better understand vitamin D 
catabolism and how it is affected by CKD and race. 
Specifically, we will evaluate the metabolic clearance of D6-
25-hydroxyvitamin D3 in individuals with varying degrees of 
CKD and among participants who self-report race as 
Caucasian, African American or African. The long-term goal 
of this work is to enhance the clinical evaluation and treatment 
of impaired vitamin D metabolism. 
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.  
Allocation: Non-Randomized 
Intervention Model: Single Group Assignment 
Masking: Open Label  
PRIMARY OBJECTIVE 
The goal of this study is to better understand vitamin D 
catabolism and how it is affected by CKD and race. 
Specifically, we will evaluate the metabolic clearance of 
25(OH)D3 in individuals with varying degrees of CKD and 
among participants who self-report their race as Caucasian, 
African American, or African. 


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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.  
NUMBER OF SUBJECTS 
Up to 120 
SUBJECT SELECTION 
CRITERIA 
Inclusion Criteria:  
• Age ≥ 18 years 
• Self-reported race Caucasian, African American, or 
African  
• Serum total 25(OH)D 10-50 ng/mL 
• Chronic kidney disease status: 
Estimated GFR ≥60 mL/min/1.73m2 (N=40) 
Estimated GFR <60 mL/min/1.73m2, not treated with 
dialysis (N=40) 
End stage renal disease treated with hemodialysis (N=40) 
 
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 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 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 
ensure reliable detection of circulating deuterated 25(OH)D3, 
without administering a dose that might alter the underlying 


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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 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, within 3 
months (wash-out allowed) 
Prohibited (within last 4 weeks, 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 
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. Formal interim analyses are 
not planned.  
STATISTICS 
Primary Analysis Plan 
We will examine the distribution of D6-25(OH)D3 clearance 
by CKD status (3 categories) and race (2 categories) using box 
plots and summary statistics. We will test associations of CKD 
status and race with D6-25(OH)D3 clearance (continuous 
outcome variable) using linear regression. Models will be 
adjusted for age, gender, diabetes status, body size, baseline 
25(OH)D concentration, and other covariates strongly related 
to 25(OH)D3 clearance. We will test whether associations of 
CKD status with clearance are modified by race by including a 
CKD x race interaction term.   
Rationale for Number of 
Subjects 
Our power calculations conservatively assume an interaction 
between CKD status and race, for which each cell in the 3x2 
recruitment grid would require individual comparison. With 
this approach, we estimate that 17 people in each of two 
groups will provide 80% power to detect a difference in D6-
25(OH)D3 half-life of 2 days, assuming a standard deviation 
of 2 days. We will recruit 20 people per cell to allow for 
multivariable adjustment. 
 


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1 
BACKGROUND  
Among people with chronic kidney disease (CKD), impaired vitamin D metabolism may 
contribute to cardiovascular disease, progression to end stage renal disease, and premature 
death.1 In healthy individuals, the kidney produces the majority of circulating 1,25-
dihydroxyvitamin D3 (1,25(OH)2D3, calcitriol), which binds to the vitamin D receptors in 
diverse target tissues.  Activation of Vitamin D3 signaling pathways is known to modulate 
inflammation and fibrosis, suppress the renin-angiotensin system, and attenuate secondary 
hyperparathyroidism.  The evaluation and treatment of impaired vitamin D metabolism is a 
major focus of current clinical care for CKD patients however, clinical decision making is 
limited by the lack of an effective measure of functional 1,25(OH)2D3 deficiency.  25-
hydroxyvitamin D3 (25(OH)D3) is a relatively inactive substrate form of vitamin D; circulating 
1,25(OH)2D3 concentration is tightly regulated and poorly reflects tissue levels; and circulating 
parathyroid hormone reflects functional 1,25(OH)2D3 deficiency at only one of many relevant 
biological sites.  
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 
Patients with chronic kidney disease (CKD) are at high risk of adverse health outcomes, 
including progression to end stage renal disease, cardiovascular disease events, and death.8 
Impaired vitamin D metabolism is commonly observed in CKD and may contribute to the 
morbidity of this disease.1  In healthy individuals, the kidney produces the majority of circulating 
1,25-dihydroxyvitamin D3 (1,25(OH)2D3, or calcitriol).  Calcitrol binds the vitamin D receptors 
in diverse target tissues, and is known to modulate inflammation and fibrosis, suppress the renin-
angiotensin system, and regulate proliferation and differentiation. In CKD, renal production of 
1,25(OH)2D3 is reduced, resulting in secondary hyperparathyroidism and dysregulation of other 
organs and tissues. Our preliminary data suggest that clearance of vitamin D metabolites is also 
reduced in CKD. 
Concentrations of vitamin D metabolites in blood and tissues must necessarily represent a 
balance of production and catabolism. While basic mechanisms of vitamin D catabolism have 
been defined,9 the clinical evaluation of vitamin D catabolism has received remarkably little 
attention. In health, CYP24A1 is the major enzyme responsible for catabolism of both 25(OH)D3 
and 1,25(OH)2D3.1,9-15 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.16-18 As a result, CYP24A1 expression is 
commonly used as a marker of tissue 1,25(OH)2D3 activity in basic science.19-35 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 
CYP24A1.9 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)36,37 
and has a circulating half-life of approximately 7 days.38 Circulating 24,25(OH)2D3 appears to 


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come from both renal and non-renal sources.15,22,39-41 In the kidney proximal tubule, CYP24A1 
transcription is induced by FGF-23 and suppressed by PTH.17,42 
To study vitamin D catabolism in humans, we developed a novel high-throughput mass 
spectrometry assay for 24,25(OH)2D3, measured concurrently with 25(OH)D3, 25(OH)D2, 
1,25(OH)2D3, and 1,25(OH)2D2.43 We applied this assay to the serum of 278 participants in the 
Seattle Kidney Study (SKS), a single-center cohort study of pre-dialysis CKD .36 Serum 
25(OH)D3 did not vary by eGFR, but mean serum concentrations of 24,25(OH)2D3 were lower 
with lower eGFR: 3.6, 3.2, 2.6, 2.6, and 1.7 ng/mL for eGFR ≥60, 45-59, 30-44, 15-29, and <15 
mL/min/1.73m2, respectively (p<0.001). These data are consistent with smaller CKD studies that 
observed low circulating concentrations of 24,25(OH)2D3 measured by immunoassay6,44-48 and 
with one study suggesting substantially reduced metabolic clearance of 25(OH)D in ESRD.49 In 
SKS, serum 24,25(OH)2D3 correlated moderately with 25(OH)D (r2=0.56) but not with 
1,25(OH)2D (r2<0.01). 
In addition to 24-hydroxylation, 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 the liver by CYP3A450 and SULT2A1 (unpublished results). As 
with CYP24A1, CYP3A4 expression is induced by 1,25(OH)2D3.51 In a preliminary analysis of 
plasma from 20 prevalent hemodialysis patients and 14 normal controls, we found that the 
relationships of plasma 4β,25(OH)2D3 and 25(OH)D3-3-sulfate with plasma 25(OH)D3 were 
distinctly different for the two populations. Specifically, slopes were less steep in dialysis 
patients, with lower metabolite concentrations at any given concentration of 25(OH)D3. This 
parallels the relationship we previously observed for 24,25(OH)2D3 and suggests that hepatic 
25(OH)D3 metabolism is also reduced in CKD. In addition, plasma 25(OH)D3-3-sulfate 
concentrations were comparable in magnitude to plasma 25(OH)D3 concentrations, suggesting 
that sulfation may be a quantitatively important unrecognized route of vitamin D catabolism. 
Because 25(OH)D3-3-sulfate is probably eliminated by the kidney, the degree to which 
25(OH)D3 sulfation is reduced in CKD may be underestimated by steady state plasma 
25(OH)D3-3-sulfate concentrations. 
Vitamin D metabolism also differs substantially by race.52 The circulating concentration of 
25(OH) D is substantially lower in people who define their race as African American or African  
compared to Caucasian populations, presumably due to differences in skin pigmentation and 
cutaneous cholecalciferol synthesis. Interestingly, the circulating concentrations of 1,25(OH)2D3 
do not vary by race, although there are noted differences in bone density between the two 
populations. Data from our cohort suggest associations between low 25(OH)D levels and 
coronary heart disease among Caucasians, but not in African American or African  populations. 
This data suggests that previously unrecognized differences in vitamin D catabolism may 
contribute to racial differences in disease susceptibility. 
The goal of this study is to better understand vitamin D catabolism and how it is affected by 
CKD and race. Specifically, we will evaluate the metabolic clearance of 25-hydroxyvitamin D3 
in individuals with varying degrees of CKD and among participants who self-report their race as  
Caucasian, African American, or African . The long-term goal of this work is to enhance the 
clinical evaluation and treatment of impaired vitamin D metabolism. 


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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. 
3 
STUDY OBJECTIVES 
3.1 
Primary Objective 
The goal of this study is to better understand vitamin D catabolism and how it is affected by 
CKD and race. Specifically, we will evaluate the metabolic clearance of 25(OH)D3 across stages 
of CKD and among participants who self-report race as Caucasian,  African American or 
African. The long-term goal of this work is to enhance the clinical evaluation and treatment of 
impaired vitamin D metabolism. 
3.2 
Secondary Objectives 
• To explore the biochemical pathways through which 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 potential biomarkers of 25(OH)D3 
4 
STUDY DESIGN 
This is a cross-sectional, observational study that uses the intravenous administration of stable 
isotope-labeled D6-25(OH)D3 to evaluate the metabolic clearance of 25(OH)D3. It is a single-
dose, open-label PK study of 25(OH)D3. 


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We will recruit a total of up to 120 study subjects, with recruitment stratified by CKD status and 
race. 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.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 
• 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: 


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• 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 
study. 
6 
SUBJECT SELECTION 
6.1 
Study Population 
Recruitment for this study will be stratified by CKD status and race (Table 6.1). Study 
participants will be recruited from the Seattle Kidney Study (an established, active cohort study 
of people with CKD) and other Kidney Research Institute cohorts and trial, Nephrology clinics 
that refer to the Kidney Research Institute, the Healthy Kidney Study (a cohort study of people 
without known kidney disease, conducted in parallel to the Seattle Kidney Study), primary care 
clinics within the University of Washington system, dialysis units within the Northwest Kidney 
Centers and Puget Sound Kidney Centers, and the University of Washington Kidney Research 
Institute participant registry. 
  
Table 6.1 Target recruitment by chronic kidney disease status. 
 
Self-reported race 
 
Caucasian African American or African  
End state renal disease treated 
with hemodialysis 
20 
20 


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Estimated GFR <60 
ml/min/1.73m2, not treated with 
dialysis 
20 
20 
Estimated GFR ≥60 
ml/min/1.73m2 
20 
20 
 
6.2 
Inclusion Criteria 
We will study only adults (Table 6.2). In addition to eligibility criteria stratified by CKD status 
and race, we will limit to a range of total serum 25(OH)D. 
 
Table 6.2. Participant Eligibility – Inclusion criteria 
Target chronic kidney disease status as described in table 6.1 
Race as described in table 6.1 
Age ≥ 18 years 
Serum 25(OH)D concentration 10 – 50ng/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 vitamin D3 or vitamin D2 supplements (e.g. ergocalciferol or cholecalciferol > 400 
IU/day) within 3 months 
• 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) 
 
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. If 
excluding for ergocalciferol or cholecalciferol supplementation >400 IU/day proves to be a 
major impediment to successful recruiting, we will consider liberalizing this exclusion criterion 
to >800 IU/day or >1000 IU/day. 
 
Table 6.3. Patient Eligibility – Exclusion criteria 


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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 
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 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 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 
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 
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volume. A typical dose would be 32 µg (1.6 mL) for an 85 kg individual. We will monitor 
achieved D6-25(OH) D3 concentrations throughout the course of the study. If our initial 
approach fails to achieve peak D6-25(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 µg (5 mL) D6-25(OH) D3 for any participant. 
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 
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 
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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. 
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 at 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 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) 
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. 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. 


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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. 
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 
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. 
• 
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. 
• 
Collect urine for measurement of albumin and creatinine (except for hemodialysis patients). 
• 
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 


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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 
• 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:   
• Perform dual energy x-ray absorptiometry (DEXA) to measure adiposity.   
• Administer D6-25(OH) D3 intravenously.  
• 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: 
• 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. 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.  
10.5 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.  
• Collect Accelerometer 


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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. 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  
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. 
 


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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. 
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 


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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. 
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. 


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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 
• 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).   


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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 
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 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 D6-25(OH) D3clearance by CKD status (3 categories) and 
race (2 categories) using box plots and summary statistics. We will explore the distribution of 
D6-25(OH) D3clearance by key covariates (age, gender, diabetes status, 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 D6-25(OH) D3clearance, including 
multivariable modeling to determine independent associations. We will test associations of CKD 
status and race with D6-25(OH) D3clearance (continuous outcome variable) using linear 
regression. Models will be adjusted for age, gender, diabetes status, vitamin D supplement use 
(within eligibility criteria), body size, baseline 25(OH)D concentration, and other covariates 
strongly related to D6-25(OH) D3 clearance. We will test whether associations of CKD status 
with clearance are modified by race by including a CKD x race interaction term. If we find 
evidence of interaction, we will analyze each CKD x race category separately. Absent 


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interaction, we will test associations of each exposure in parallel (adjusted for each other) to 
maximize precision and power. 
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 
Our power calculations conservatively assume an interaction between CKD status and race, for 
which each cell in the 3x2 recruitment grid would require individual comparison. With this 
approach, we estimate that 17 people in each of two groups will provide 80% power to detect a 
difference in D6-25(OH) D3half-life of 2 days, assuming a standard deviation of 2 days. We will 
recruit 20 people per cell to allow for multivariable adjustment. 
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. 


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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 
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). 


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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. 
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.  


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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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16.5 References 
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Compston JE, Merrett AL, Ledger JE, Creamer B. Faecal tritium excretion after intravenous administration 
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Davies M, Heys SE, Selby PL, Berry JL, Mawer EB. Increased catabolism of 25-hydroxyvitamin D in 
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Batchelor AJ, Compston JE. Reduced plasma half-life of radio-labelled 25-hydroxyvitamin D3 in subjects 
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Gray RW, Weber HP, Dominguez JH, Lemann J. The metabolism of vitamin D3 and 25-hydroxyvitamin 
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Beckman MJ, Tadikonda P, Werner E, Prahl J, Yamada S, DeLuca HF. Human 25-hydroxyvitamin D3-24-
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Knutson JC, DeLuca HF. 25-Hydroxyvitamin D3-24-hydroxylase. Subcellular location and properties. 
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Annalora AJ, Goodin DB, Hong WX, Zhang Q, Johnson EF, Stout CD. Crystal structure of CYP24A1, a 
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15. 
Kumar R, Schaefer J, Grande JP, Roche PC. Immunolocalization of calcitriol receptor, 24-hydroxylase 
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Schuster I. Cytochromes P450 are essential players in the vitamin D signaling system. Biochim Biophys 
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17. 
Shinki T, Jin CH, Nishimura A, Nagai Y, Ohyama Y, Noshiro M, Okuda K, Suda T. Parathyroid hormone 
inhibits 25-hydroxyvitamin D3-24-hydroxylase mRNA expression stimulated by 1 alpha,25-dihydroxyvitamin D3 in 
rat kidney but not in intestine. J Biol Chem. 1992;267(19):13757-62. PubMed PMID: 1618870. 


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18. 
Akeno N, Saikatsu S, Kawane T, Horiuchi N. Mouse vitamin D-24-hydroxylase: molecular cloning, tissue 
distribution, and transcriptional regulation by 1alpha,25-dihydroxyvitamin D3. Endocrinology. 1997;138(6):2233-
40. PubMed PMID: 9165006. 
19. 
Tomon M, Tenenhouse HS, Jones G. Expression of 25-hydroxyvitamin D3-24-hydroxylase activity in 
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20. 
Goff JP, Reinhardt TA, Engstrom GW, Horst RL. Effect of dietary calcium or phosphorus restriction and 
1,25-dihydroxyvitamin D administration on rat intestinal 24-hydroxylase. Endocrinology. 1992;131(1):101-4. Epub 
1992/07/01. PubMed PMID: 1611988. 
21. 
Akeno N, Saikatsu S, Kawane T, Horiuchi N. Mouse vitamin D-24-hydroxylase: molecular cloning, tissue 
distribution, and transcriptional regulation by 1alpha,25-dihydroxyvitamin D3. Endocrinology. 1997;138(6):2233-
40. Epub 1997/06/01. PubMed PMID: 9165006. 
22. 
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. 
23. 
Flanagan JN, Zheng S, Chiang KC, Kittaka A, Sakaki T, Nakabayashi S, Zhao X, Spanjaard RA, Persons 
KS, Mathieu JS, Holick MF, Chen TC. Evaluation of 19-nor-2alpha-(3-hydroxypropyl)-1alpha,25-dihydroxyvitamin 
D3 as a therapeutic agent for androgen-dependent prostate cancer. Anticancer research. 2009;29(9):3547-53. Epub 
2009/08/12. PubMed PMID: 19667147. 
24. 
Stubbs JR, Idiculla A, Slusser J, Menard R, Quarles LD. Cholecalciferol supplementation alters calcitriol-
responsive monocyte proteins and decreases inflammatory cytokines in ESRD. Journal of the American Society of 
Nephrology : JASN. 2010;21(2):353-61. Epub 2009/12/17. doi: 10.1681/ASN.2009040451. PubMed PMID: 
20007751; PubMed Central PMCID: PMC2834546. 
25. 
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antiviral agent suppressing hepatitis C virus in human hepatocytes. Hepatology. 2011;54(5):1570-9. Epub 
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26. 
Koszewski NJ, Horst RL, Goff JP. Importance of apical membrane delivery of 1,25-dihydroxyvitamin D3 
to vitamin D-responsive gene expression in the colon. Am J Physiol Gastrointest Liver Physiol. 2012. Epub 
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27. 
Goff JP, Koszewski NJ, Haynes JS, Horst RL. Targeted delivery of vitamin D to the colon using beta-
glucuronides of vitamin D: therapeutic effects in a murine model of inflammatory bowel disease. Am J Physiol 
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28. 
Bacchetta J, Sea JL, Chun RF, Lisse TS, Wesseling-Perry K, Gales B, Adams JS, Salusky IB, Hewison M. 
FGF23 inhibits extra-renal synthesis of 1,25-dihydroxyvitamin D in human monocytes. Journal of bone and mineral 
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29. 
Swami S, Krishnan AV, Wang JY, Jensen K, Horst R, Albertelli MA, Feldman D. Dietary vitamin D(3) 
and 1,25-dihydroxyvitamin D(3) (calcitriol) exhibit equivalent anticancer activity in mouse xenograft models of 
breast and prostate cancer. Endocrinology. 2012;153(6):2576-87. Epub 2012/03/29. doi: 10.1210/en.2011-1600. 
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cytokines in response to respiratory syncytial virus infection of calves with high or low circulating 25-
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Alimirah F, Peng X, Yuan L, Mehta RR, von Knethen A, Choubey D, Mehta RG. Crosstalk between the 
peroxisome proliferator-activated receptor gamma (PPARgamma) and the vitamin D receptor (VDR) in human 
breast cancer cells: PPARgamma binds to VDR and inhibits 1alpha,25-dihydroxyvitamin D(3) mediated 


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transactivation. Exp Cell Res. 2012;318(19):2490-7. Epub 2012/08/14. doi: 10.1016/j.yexcr.2012.07.020. PubMed 
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34. 
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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. Epub 2012/06/01. doi: 
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37. 
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B, Brunzell JD. Circulating vitamin D metabolites and kidney disease in type 1 diabetes. The Journal of clinical 
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48. 
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49. 
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50. 
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: 
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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; 
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54. 
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. 
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55. 
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. 
56. 
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. 
57. 
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 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. 


Protocol Number 
Confidential 
 
Version 2.0 
Version Date: 9/3/15  
Page 34 of 35 
Protocol Template © CFFT TDN   Effective: 12 SEP 2007 
APPENDIX 1. SCHEDULE OF STUDY PROCEDURES 
Procedure 
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 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
Focused Medical 
History 
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 
 
 
 
 
 
DXA 
 
X 
 
 
 
 
 
 
 
 
Study Drug 
Administration 
 
X 
 
 
 
 
 
 
 
 
*Blood Draws Detailed in Appendix 2. 


Protocol Number 
Confidential 
 
Version 2.0 
Version Date: 9/3/15  
Page 35 of 35 
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