Activated Vitamin D for the Prevention and Treatment of Acute Kidney Injury (ACTIVATE-AKI)
Phase: PHASE2
Conditions: Critically Ill, Acute Kidney Injury
Sponsor: David Leaf

Title: ACTIVATE-AKI: Activated Vitamin D for the Prevention and Treatment of Acute Kidney 
Injury 
 
ClinicalTrials.gov Registration Number: NCT02962102 
 
Document Date: December 25, 2018 

 
BRIGHAM AND 
WOMEN’S HOSPITAL 
BRIGHAM HEALTH 
HARVARD 
MEDICAL SCHOOL 
 
 
 
ACTIVATE-AKI: 
Activated Vitamin D for the Prevention and Treatment of 
Acute Kidney Injury 
 
 
 
 
 
Detailed Protocol 
December 25, 2018 
 
 
 
 
 
 
 
 
Primary Investigators: 
 
David E. Leaf, MD, MMSc 
Assistant Professor of Medicine, Harvard Medical School 
Associate Physician, Division of Renal Medicine, Brigham and Women’s Hospital 
 
Sushrut S. Waikar, MD, MPH 
Associate Professor of Medicine, Harvard Medical School 
Constantine L. Hampers, MD Distinguished Chair in Renal Medicine  
Director of Translational Research and Ambulatory Services, Division of Renal Medicine, 
Brigham and Women's Hospital 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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TABLE OF CONTENTS 
 
ABBREVIATIONS…………………………………………………………………………………3 
TRIAL SUMMARY………………………………………………………………………………...4 
I. BACKGROUND AND SIGNIFICANCE……………………………………………………….5 
   A.  Historical background……………………………………………………………………....5 
   B.  Previous pre-clinical and clinical studies supporting the research…………………….6 
   C.  Justification of agents……………………………………………………………………....7 
   D.  Justification of dosing regimens…………………………………………………………...8 
   E.  Safety data on 25D (calcifediol)…………………………………………………………...11 
   F.  Safety data on 1,25D (calcitriol)…………………………………………………………..13 
   G.  Rationale and potential benefits to patients/society……………………………………14 
II. SPECIFIC AIMS……………………………………………………………………………….15 
III. SUBJECT SELECTION………………………………………………………………………16 
A. Inclusion and exclusion criteria…………………………………………………………...16 
B. Source of subjects………………………………………………………………………….16 
IV. SUBJECT ENROLLMENT…………………………………………………………………...17 
A. Methods of enrollment……………………………………………………………………..17 
B. Procedures for obtaining informed consent……………………………………………..17 
C. Treatment assignment and randomization………………………………………………18 
V. STUDY PROCEDURES………………………………………………………………………19 
   A.  Parameters to be measured……………………………………………………………….19 
   B.  Drugs to be used……………………………………………………………………………20 
   C.  Devices to be used…………………………………………………………………………21 
   D.  Procedures/surgical interventions………………………………………………………...21 
   E.  Data to be collected………………………………………………………………………...21 
   F.  Dosing modifications………………………………………………………………………..21 
   G. Adaptive trial design…………………………………………………………………………21 
VI. BIOSTATISTICAL ANALYSIS……………………………………………………………….22 
A. Specific data variables being collected for the study…………………………………...22 
B. Study end points…………………………………………………………………………....22 
C. Statistical methods………………………………………………………………………….22 
D. Power analysis………………………………………………………………………………22 
VII. RISKS AND DISCOMFORTS……………………………………………………………….21 
A. Complications of surgical and non-surgical procedures………………………………..23 
B. Drug side effects and toxicities…………………………………………………………....23 
C. Confidentiality……………………………………………………………………………….25 
D. Device complications/malfunctions……………………………………………………….25 
E. Psychosocial risks…………………………………………………………………………..25 
F. Radiation risks……………………………………………………………………………....25 
VIII. POTENTIAL BENEFITS………………………………………………………………….....26 
A. Potential benefits to participating individuals…………………………………………....26 
B. Potential benefits to society…………………………………………………………….....26 
IX. MONITORING AND QUALITY ASSURANCE……………………………………………..27 
A. Independent monitoring of source data……………………………………………….....27 
B. Safety monitoring……………………………………………………………………………27 
C. Outcomes monitoring……………………………………………………………………….27 
D. Adverse event reporting……………………………………………………………………27 
X. REFERENCES…………………………………………………………………………………28 

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ABBREVIATIONS 
 
1,25D = 1,25-dihydroxyvitamin D 
25D = 25-hydroxyvitamin D 
AKI = Acute Kidney Injury 
APACHE = Acute Physiology and Chronic Health Evaluation 
AUC = Area Under the Curve 
BWH = Brigham and Women’s Hospital 
CKD = Chronic Kidney Disease 
DSMB = Data safety and monitoring board 
HED = Human Equivalent Dose 
ICU = Intensive Care Unit 
ESRD = End Stage Renal Disease 
MCT Oil = Medium Chain Triglyceride Oil 
NIDDK = National Institute of Diabetes and Digestive Kidney Diseases 
NIH = National Institutes of Health 
PBMC = Peripheral Blood Mononuclear Cells 
PHRC = Partners Human Research Committee 
RCT = Randomized Controlled Trial 
RRT = Renal Replacement Therapy 
SCr = Serum creatinine 
SOFA = Sequential Organ Failure Assessment 
ULN = Upper limit of normal 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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TRIAL SUMMARY 
 
Title 
Activated Vitamin D for the Prevention and Treatment of Acute Kidney Injury (ACTIVATE-AKI) 
 
Objective 
To assess the efficacy of calcifediol (25-hydroxyvitamin D [25D]) and calcitriol (1,25-
dihydroxyvitamin D [1,25D]) in preventing and reducing the severity of acute kidney injury (AKI) 
in critically ill patients. 
 
Hypothesis 
Compared to placebo, treatment with calcifediol or calcitriol will reduce the occurrence and 
severity of renal injury, as determined by a composite of time-averaged daily serum creatinine 
(SCr) levels for 7 days, renal replacement therapy (RRT), and death. 
 
Study Design 
ACTIVATE-AKI is a 3-arm, randomized, double-blind, placebo-controlled, phase II trial (n=150) 
of vitamin D metabolites in critically ill patients at risk of severe AKI.  We will randomly assign 
participants in a 1:1:1 fashion to receive calcifediol, calcitriol, or placebo.  Study drugs will be 
administered on a daily basis for 5 days, and will be administered either orally or via 
naso/orogastric tube. 
 
Study Sites 
ACTIVATE-AKI is a single-center study that will recruit patients from intensive care units at 
Brigham and Women’s Hospital (BWH). 
 
Funding 
ACTIVATE-AKI is approved and funded by the National Institutes of Health (NIH) / National 
Institute of Diabetes and Digestive Kidney Diseases (NIDDK) grant K23DK106448 (to David 
Leaf, MD). 
 
Registration on clinicaltrials.gov 
NCT02962102 
 
 
 
 
 

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I. BACKGROUND AND SIGNIFICANCE 
 
A.  Historical background 
 
Overview of AKI 
Acute kidney injury (AKI) is associated with markedly increased hospital length of stay, 
mortality, and cost.1-3  Despite years of investigation, development of therapies for the 
prevention and treatment of AKI have been largely unsuccessful.  Therefore, investigation of 
novel therapeutic targets is needed. 
 
Overview of vitamin D metabolism 
Vitamin D is derived from dietary sources and sunlight-induced cutaneous synthesis, and is 
converted to 25-hydroxyvitamin D (25D) in the liver (Figure 1).4  25D is converted to its 
biologically active form, 1,25-
dihydroxyvitamin D (1,25D), in the 
kidneys and other tissues by 1-α 
hydroxylase.5  The conversion of 
25D to 1,25D is tightly regulated by 
two opposing hormones: 
parathyroid hormone (PTH), which 
stimulates 1-α hydroxylase, and 
fibroblast growth factor-23 
(FGF23), which inhibits 1-α 
hydroxylase.  FGF23 also 
stimulates 24-hydroxylase, which 
inactivates both 25D and 1,25D.6 
 
Non-classical actions of 1,25D 
In addition to maintaining calcium 
and phosphate homeostasis, a 
variety of non-classical effects of vitamin D metabolites are also appreciated.  1,25D binds to 
the intracellular vitamin D receptor (VDR), which is expressed nearly ubiquitously.7  This 
complex then translocates into the nucleus where it binds to specific DNA sequence elements in 
vitamin D-responsive genes, ultimately influencing the expression of over 200 target genes.8  
These target genes affect a variety of critical physiologic functions relevant to AKI, including 
immune/inflammatory pathways and vascular endothelial cell activation.9 
 
Interventional studies of vitamin D metabolites for AKI prevention in humans 
Despite promising data on vitamin D metabolites as therapeutic agents in animal models of AKI 
(summarized below), as well as epidemiologic studies from our group10 and others11-13 
suggesting that deficiencies in vitamin D metabolite levels contribute to mortality in critical 
illness, no interventional study has evaluated vitamin D metabolites as novel therapeutic agents 
for AKI prevention/treatment in humans.  We recently published the results of a pilot randomized 
controlled trial (RCT) evaluating the immunomodulatory effects of a single 2 µg IV dose of 
calcitriol (1,25D) in critically ill patients with severe sepsis.14  We found that calcitriol led to 
increased leukocyte mRNA expression of IL-10 and HO-1, which have important 
antiinflammatory effects and prevent AKI in animal models.15-19  The current study will expand 
on these findings by comprehensively evaluating the renoprotective effects of two vitamin D 
metabolites – calcifediol (25D) and calcitriol (1,25D) – in critically ill patients at risk of AKI. 
 
 

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B.  Previous pre-clinical and clinical studies supporting the proposed research 
 
Overview 
The “non-classical” effects of vitamin D metabolites on inflammation have been demonstrated in 
numerous in vitro studies.  Further, the beneficial effects of vitamin D metabolites on AKI 
prevention have been demonstrated in numerous animal models.  Finally, extensive safety data 
on vitamin D metabolites are available from clinical trials in humans. 
 
In vitro studies of 1,25D and inflammation 
In vitro studies have demonstrated potent antiinflammatory effects of 1,25D.  Treatment of T 
lymphocytes, monocytes, and other cells with 1,25D downregulates proinflammatory cytokines 
(e.g., IL-2, IL-6, TNF-α),20-25 upregulates antiinflammatory cytokines/enzymes (e.g., IL-10, heme 
oxygenase-1 [HO-1]), 26-31 and increases the frequency of antiinflammatory T regulatory cells 
(Tregs).32-34  Upregulation of IL-10, HO-1, and Tregs protect against AKI in animal models.15-19, 
35-37 
 
1,25D and AKI in animal models 
In rodents, 1,25D attenuates renal injury induced by a wide variety of insults/toxins including 
ischemia,38-40 gentamicin,41 cyclosporine,42 cisplatin,43 glomerulonephritis,44 and obstruction.45   
 
Clinical studies of vitamin D metabolites in non-critically ill patients 
Numerous RCTs have evaluated vitamin D metabolites as therapeutic agents across a wide 
variety of clinical settings, and are reviewed elsewhere as meta-analyses.46-49  While these 
studies have reported mixed findings with respect to efficacy, they have demonstrated a very 
favorable safety profile (discussed further below under sections on safety). 
 
Clinical studies of vitamin D metabolites in critically ill patients 
A recently published RCT evaluated the effects of a large (540,000 I.U.) oral dose of vitamin D 
versus placebo among 492 critically ill patients.  While the study found no effect on the primary 
outcome of hospital length of stay, a pre-specified subgroup analysis conducted in patients with 
severe vitamin D deficiency (defined as plasma 25D levels < 12 ng/ml) found a significant 
reduction in hospital mortality.50  Importantly, no change in mean total serum calcium levels was 
observed in the vitamin D versus placebo groups at the two follow-up time points (days 3 and 7) 
where it was tested, and only a single patient in the vitamin D group developed hypercalcemia, 
with a total serum calcium level of 12.0 mg/dl. 
 
Preliminary data from our pilot RCT of calcitriol 
We recently published the results of a pilot RCT of calcitriol (1,25D).14  We randomly assigned 
67 critically ill patients with severe sepsis to receive either a single 2 µg IV dose of calcitriol or 
placebo.  Although the primary endpoint of plasma cathelicidin levels was null, patients 
randomized to calcitriol had higher IL-10 (P=0.03) and HO-1 (P=0.004) leukocyte mRNA 
expression at 24h.  Additionally, we found a graded relation between change in 1,25D levels 
and change in IL-10 (P=0.004) and HO-1 (p<0.001) expression.  No hypercalcemia or other 
adverse events were observed. 
 
 
 
 
 
 
 

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C.  Justification of agents 
 
25D vs. 1,25D 
Despite promising data on the antiinflammatory and renoprotective effects of 1,25D in preclinical 
models, it is unknown whether administration of vitamin D metabolites to critically ill patients will 
have beneficial immunomodulatory effects, and whether these effects will translate into 
improved clinical outcomes.  Additionally, it is unknown whether 1,25D or its precursor, 25D, will 
have greater efficacy in humans.  While 1,25D is the metabolite used in most preclinical studies, 
it is unclear whether the concentrations of 1,25D used in vitro (1 to 100 nM in most studies) can 
be achieved in humans.  Additionally, although 1,25D is the “active” metabolite of vitamin D, it 
circulates in the plasma at ~1,000-fold lower concentrations than 25D.  Target cells such as 
monocytes express the 1-α-hydroxylase enzyme that converts 25D into 1,25D,51, 52 and are 
therefore capable of converting local 25D into 1,25D.  Indeed, the ability of circulating 1,25D to 
exert immunomodulatory effects in humans has been questioned,53 and some experts have 
advocated targeting 25D rather than 1,25D to facilitate local endogenous 1,25D production in 
target cells.53-56  The current 3-arm study will address these uncertainties by evaluating the 
effects of both calcifediol (25D) and calcitriol (1,25D) versus placebo. 
 
Overview of 25D 
Plasma 25D levels can be raised indirectly by administering its precursor, vitamin D, or directly 
by administering 25D itself (Figure 1).  A disadvantage of administering the precursor, vitamin 
D, is that it requires several days to saturate adipose tissue stores and raise plasma 25D levels 
to the low-normal range (30-35 ng/ml).55, 57  A recently published RCT evaluated the effects of a 
large (540,000 I.U.) enteral dose of vitamin D versus placebo among 492 critically ill patients, 
and found no effect on hospital length of stay, the primary outcome.50  In this study, only ~50% 
of the patients in the vitamin D arm actually achieved target plasma 25D levels >30 ng/ml by 
day 3.  In contrast, administration of oral 25D raises plasma 25D levels within hours.58  Thus, in 
the current study we will administer 25D. 
 
FDA status of 25D (calcifediol) 
Calderol.  25D was formerly available for use in patients as “Calderol”.  Calderol was approved 
by the FDA in 1980 for the treatment of hypocalcemia in patients undergoing chronic renal 
dialysis.  Calderol was withdrawn from the market in 2002 for commercial reasons which were 
not associated with safety or efficacy of the product. 
 
Rayaldee.  25D is currently FDA-approved as “Rayaldee” for the treatment of vitamin D 
deficiency in chronic kidney disease stage 3 or 4 patients with secondary hyperparathyroidism. 
 
Overview of 1,25D 
The rationale for evaluating 1,25D as a therapeutic agent in AKI is three-fold: 1) the 
renoprotective effects of vitamin D metabolites in animal models of AKI were demonstrated 
exclusively with 1,25D; 2) there may be impaired renal59 and extra-renal60 conversion of 25D to 
1,25D in critical illness due to elevated FGF2361 or other mechanisms; 3) our preliminary data 
from a pilot RCT in critically ill patients with sepsis demonstrate that a single 2 µg IV dose of 
1,25D increases leukocyte mRNA expression of IL-10 and HO-1,14 which are important 
antiinflammatory proteins that prevent AKI in animal models. 
 
FDA status of 1,25D (calcitriol) 
1,25D is available for use in patients as calcitriol, and is currently FDA-approved for the 
treatment of hypocalcemia in patients undergoing chronic renal dialysis. 
 
 

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D.  Justification of dosing regimens 
 
Overview of dosing regimens 
Dosing regimens are shown in Figure 2.  Participants assigned to 25D will be administered a 
liquid formulation of calcifediol, either orally or via naso/orogastric tube, at a dose of 400µg on 
day 0 (the day of enrollment) and 200µg on days 1, 2, 3, and 4.  Participants assigned to 1,25D 
will be administered a liquid formulation of calcitriol, either orally or via naso/orogastric tube, at a 
dose of 4µg daily on days 0, 1, 2, 3, and 4.  Participants assigned to placebo will be 
administered medium chain triglyceride (MCT) oil. 
 
 
 
Rationale for dose of 25D 
The dosing regimen of calcifediol in the current study is intended to achieve plasma 25D levels 
> 50 ng/ml throughout the majority of the first 7 days of the study.  While optimal plasma 25D 
levels in critically ill patients are unknown, epidemiologic studies in this patient population have 
consistently shown that lower levels are associated with adverse clinical outcomes.11-13, 62  A 
study conducted in healthy volunteers found that raising plasma 25D levels to 55 ng/ml with 
administration of oral vitamin D resulted in a significant increase in the frequency of regulatory T 
cells (Tregs),63 which have an important protective role in AKI.64  Additionally, a recently 
published RCT conducted in critically ill ventilated patients found that raising 25D levels to 55 
ng/ml resulted in decreased hospital length of stay.65 
 
Rationale for dose of 1,25D (data from in vitro studies) 
Most in vitro studies demonstrating immunomodulatory effects of calcitriol used concentrations 
ranging between 1 to 100 nmol/L.8, 31, 33, 66-68  In our recently completed pilot RCT evaluating the 
immunomodulatory effects of a single 2 µg intravenous dose of calcitriol among 67 critically ill 
patients, maximum mean (SD) plasma 1,25D levels were 0.2 (0.1) nmol/L,14 substantially lower 
than concentrations used in vitro, which may account for the largely negative findings in the 
study.  Thus, higher doses of calcitriol – which are nonetheless still substantially lower than 
doses that have been used safely in prior humans studies (discussed further in section F below) 
– are planned in the current study.  Additionally, repeated dosing is planned (Figure 2) to 
account for the short half-life of calcitriol. 
 
 

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Rationale for dose of 1,25D (data from animal studies) 
 
Animal models of AKI prevention with calcitriol 
▪ 
Xu et al69 evaluated the effects of calcitriol pretreatment on renal oxidative stress 
in an LPS model of sepsis-induced AKI in mice.  Mice were pretreated with three 
oral doses of calcitriol at a dose of 25 µg/kg at 1, 24, and 48 hours before LPS 
injection.  Calcitriol treatment compared to placebo resulted in significant 
attenuation of LPS-induced renal oxidative stress determined by markers of lipid 
peroxidation, renal inducible nitric oxide synthase expression, and other 
measures.  The human equivalent dose (HED) of calcitriol is shown below in 
Table 1. 
 
▪ 
Kapil et al70 subjected rats to 40 minutes of bilateral renal ischemia-reperfusion 
injury (IRI).  Prior to IRI, calcitriol was administered intraperitoneally on a daily 
basis for 7 days at the following doses: 0.25 µg/kg, 0.5 µg/kg, and 1 µg/kg.  
Treatment with each of these calcitriol dosing regimens compared to control-
treated animals significantly attenuated the effect of IRI on kidney function, with 
the maximum beneficial effect observed in the group that received calcitriol 0.5 
µg/kg.  The HED of calcitriol in shown below in Table 1. 
 
▪ 
Sezgin et al40 subjected rats to a combination of right-sided nephrectomy plus left 
sided IRI.  Rats were pretreated with calcitriol 0.5 µg/kg intraperitoneally on a 
daily basis for 7 days proir to renal IRI.  Treatment with calcitriol compared to 
control-treated animals had a significant beneficial effect on biochemical 
parameters such as serum creatinine and blood urea nitrogen, as well as renal 
tissue expression of glutathione, superoxide dismutase, and nitric oxide activity.  
The HED of calcitriol in shown below in Table 1. 
 
Animal models of autoimmune disease prevention/treatment with calcitriol 
 
▪ 
Mathieu et al71 evaluated the ability of calcitriol to prevent autoimmune diabetes 
in non-obese diabetic (NOD) mice.  NOD mice received calcitriol 5 µg/kg 
intraperitoneally every other day beginning at the age of 21 days and ending at 
the age of 200 days or on the day diabetes was diagnosed.  Calcitriol compared 
to placebo resulted in significant prevention of diabetes.  The HED of calcitriol in 
shown below in Table 1. 
 
▪ 
Branisteanu et al72 evaluated the efficacy of calcitriol in preventing autoimmune 
encephalomyelitis in mice.  Calcitriol at a dose of 5 µg/kg intraperitoneally every 
2 days was effective at preventing paralysis in 70% of treated mice.  The HED of 
calcitriol in shown below in Table 1. 
 
 
 
 

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Reference 
Animal Model 
Calcitriol 
Dose 
HED 
HED in a 100 kg 
individual 
Xu et al69 
Sepsis-induced AKI in mice 
25 µg/kg 
2.0 µg/kg 
203 µg 
Kapil et al70 
Renal IRI in rats 
0.5 µg/kg 
0.1 µg/kg 
8.1 µg 
Sezgin et al40 
Unilateral nephrectomy + renal IRI in rats 
0.5 µg/kg 
0.1 µg/kg 
8.1 µg 
Mathieu et al71 
Autoimmune diabetes in NOD mice 
5 µg/kg 
0.4 µg/kg 
40.7 µg 
Branisteanu et al72 
Autoimmune encephalomyelitis in mice 
5 µg/kg 
0.4 µg/kg 
40.7 µg 
 
 
 
 
 
 
 
 
 
Average calcitriol 
dose = 60.1 µg 
 
 
 
 
 
Median calcitriol 
dose = 40.7 µg 
 
Table 1.  Summary of animal studies in which calcitriol exerted protective 
immunomodulatory effects.  HED is calculated by applying a conversion factor (6.2-to-1 for 
rats-to-humans; 12.3-to-1 for mice-to-humans) to account for differences in metabolism between 
rodents versus humans.  Abbreviations: AKI, acute kidney injury; HED, human equivalent dose; 
IRI, ischemia reperfusion injury; NOD, non-obese diabetic. 
 
 
 
 
 
 

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E.  Safety data on 25D (calcifediol) 
Overview 
The only known toxicity of vitamin D metabolites, including calcifediol (25D) and calcitriol 
(1,25D), is hypercalcemia.  The dosing regimens of calcifediol planned in the current study are 
very unlikely to produce hypercalcemia because they are lower than doses that have been used 
safely in prior studies in both animals and humans. 
 
Animal studies 
In mice, doses of 25D required to induce hypercalcemia are 1mg/kg orally on a daily basis for 5 
weeks.73  In contrast, the highest dose of calcifediol in the current proposal is 400 µg, or 0.004 
mg/kg in a 100 kg adult.  Thus, we propose a dosing regimen that is approximately 200-fold 
lower than doses which induce hypercalcemia in mice.  Further, we propose a much shorter 
duration of administration (5 doses over 5 days, in contrast to daily dosing for 5 weeks). 
 
Human studies 
In a study conducted in 27 healthy adult volunteers, oral calcifediol was administered at doses 
of 1.5 µg/kg, 5 µg/kg, and 10 µg/kg of body weight.  Thus, a 70 kg individual assigned to the 
highest group would have received a 700 µg dose of calcifediol.  No hypercalcemia nor any 
other adverse effects were observed.58  In the same study, a single 1,000 µg IV dose of 
calcifediol was administered to two healthy volunteers and did not result in hypercalcemia or 
any other adverse events.58 
 
In a separate study conducted in 12 healthy adult volunteers, oral calcifediol was administered 
at 10 µg/kg of body weight.  No hypercalcemia nor any other adverse effects were observed.  
Mean plasma calcium levels rose only minimally from 9.5 mg/dl to 9.7 mg/dl at 24 hours.74 
 
In a study conduced in elderly nursing home patients, 22 patients were assigned to received a 
single dose of calcifediol 10 µg/kg of body weight.  No hypercalcemia nor any other adverse 
effects were observed.  No significant differences were observed in serum calcium levels in 
calcifediol-treated versus control patients.75 
 
In a more recent study conducted in 29 subjects with stage 3 or 4 chronic kidney disease, 
subjects were randomly assigned to receive a single dose of oral calcifediol 450 µg, a single 
dose of oral calcifediol 900 µg, or an intravenous dose of calcifediol 448 µg.  No significant 
increases in serum calcium were observed in any treatment group during the post-dose 
period.76 
 
The safety of repeated (daily) dosing of oral calcifediol in humans has been demonstrated in 
multiple smaller studies77-79 as well as in two phase III RCTs, which included a total of 429 
subjects.80  In the latter studies, patients received 30 or 60 µg of 25D on a daily basis for 26 
weeks.  Subjects assigned to 25D had an inconsequential increase in mean serum total calcium 
levels (0.2 mg/dl versus 0.1 mg/dl in the placebo group), and the percentage of patients who 
experienced at least one treatment-emergent adverse event was similar in the 25D versus 
placebo groups.80  Only six subjects (2%) in the calcifediol group required dose reductions for 
protocol-defined hypercalcemia (2 consecutive serum calcium values > 10.3 mg/dl). 
 
The current study will administer a total of five enteral doses of calcifediol over 5 days.  The 
rationale for this approach is that 25D levels are known to decline by ~50% of peak levels by 24 
hours.58, 74  To minimize the potential for a stepwise increase in plasma 25D levels with repeated 
dosing, the first dose will be 400 µg, and subsequent doses will be 200 µg (Figure 2).   

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Summary 
Plasma levels of 25D considered “toxic” (i.e. associated with hypercalcemia) have been 
reported to be in excess of 214 ng/ml.81 82  Based on the known pharmacokinetics of calcifediol 
from the studies above, plasma 25D levels are very unlikely to ever approach this limit. 
 
 
 
 

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F.  Safety data on 1,25D (calcitriol) 
 
Overview 
The only known toxicity of vitamin D metabolites, including calcifediol and calcitriol, is 
hypercalcemia.  The dosing regimens of calcitriol planned in the current study are very unlikely 
to produce hypercalcemia because they are lower than doses that have been used safely in 
previous studies in humans. 
 
Human studies of oral calcitriol 
In a phase I dose-escalation pharmacokinetics study of oral calcitriol in patients with advanced 
malignancy, patients received calcitriol for 3 consecutive days each week.  The highest dosing 
level of calcitriol administered was 38 µg.  At this dose, the average increase in serum calcium 
was 0.5 mg/dl, and no dose-limiting toxicity occurred.83 
 
In a phase II study of high dose oral calcitriol, 39 patients with prostate cancer were randomly 
assigned to receive calcitriol 0.5 µg/kg (e.g., 35 µg in a 70 kg adult) once per week for 4 weeks 
or placebo.  No patients developed hypercalcemia, and no significant changes in serum calcium 
were detected.  The median and range of serum total calcium levels at baseline was 9.3 (8.5 to 
10.4) mg/dl, compared with 9.3 (8.6 to 10.1) mg/dl on day 3, 9.4 (8.7 to 10.2) mg/dl on day 10, 
and 9.3 (7.8 to 10.0) mg/dl on day 25.  No significant differences in adverse events were 
reported between calcitriol vs. placebo-treated patients.84 
 
In a phase I pharmacokinetics study of an oral calcitriol formulation, DN-101, 38 patients 
received a single-dose administration of 15, 30, 60, 75, 90, 105, 135, or 165 µg of DN-101.  No 
hypercalcemia was observed at doses less than 60 µg, and the hypercalcemia that developed 
at higher doses (in 13.2% of patients) was grade 1 (minor and transient) in all cases.85 
 
Human studies of intravenous calcitriol 
In a phase I dose-escalation pharmacokinetics study of IV calcitriol in patients with advanced 
solid tumors, no patients developed hypercalcemia at weekly IV doses up to 15 µg.  At higher 
weekly IV doses of 74 µg, only one of seven patients developed clinically relevant (grade 2 or 
higher) hypercalcemia, defined as serum calcium > 11.5 mg/dl.86 
 
In a phase I pharmacokinetics study of IV calcitriol in combination with oral dexamethasone and 
gefitinib in patients with advanced solid tumors, 20 patients received IV calcitriol on weeks 1, 3, 
and weekly thereafter.  The starting dose of calcitriol was 57 µg and escalation occurred in 
cohorts of three patients until the maximum tolerated dose was defined.  Escalations included 
74, 96, 125, and 163 µg doses.  Doses of 125 µg were associated with grade 2 hypercalcemia 
(serum calcium 11.5-12.5 mg/dl) and doses of 163 µg were associated with two cases of grade 
3 hypercalcemia (serum calcium 12.5-13.5).  However, doses ranging from 57 to 96 µg did not 
produce any clinically relevant (grade 2 or higher) hypercalcemia (defined as serum calcium > 
11.5 mg/dl).87 
 
Summary 
The dosing regimen of calcitriol planned in the current study (4 µg daily for 5 days) represents a 
dose far lower than dosing regimens that have been used safely in prior studies, and thus is 
very unlikely to cause hypercalcemia or other adverse events. 
 
 
 

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G.  Rationale behind the proposed research, and potential benefits to patients/society 
 
Despite compelling data from the pre-clinical and epidemiologic studies discussed above, it 
remains unknown whether administration of vitamin D metabolites (25D or 1,25D) to critically ill 
patients will have beneficial effects on acute kidney injury (AKI) or other clinically relevant 
outcomes.  Finding an effect of vitamin D metabolites on AKI would represent a major advance 
in the field, especially since currently there are no drugs or interventions that reliably prevent or 
treat AKI in humans.  Finding a beneficial effect of vitamin D metabolites on AKI in the current 
study would provide preliminary data to justify a larger interventional study of vitamin D 
metabolites among critically ill patients, which would be powered to detect hard clinical end 
points such as need for renal replacement therapy and death.  Finally, the current study will 
answer important questions about vitamin D metabolism in critically ill patients, which will inform 
future interventional studies of vitamin D metabolites in this and other clinical settings. 
 
 
 
 

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II. SPECIFIC AIMS 
 
We will enroll 150 critically ill patients at risk of severe AKI into a 3-arm, double-blind, 
randomized study of calcifediol (25D), calcitriol (1,25D), or placebo to test the following: 
 
Primary Objective – Prevention of AKI incidence/severity 
Test whether administration of calcifediol (25D) or calcitriol (1,25D), compared to placebo, will 
decrease the incidence/severity of AKI among ICU patients, assessed by daily measurement of 
SCr over 7 days. 
 
Secondary Objectives – Urinary Injury and Immune/Inflammatory Biomarkers  
Test the effects of calcifediol (25D) or calcitriol (1,25D), compared to placebo, on urinary injury 
biomarkers (NGAL and KIM-1) and key immune/inflammatory markers assessed by monocyte 
mRNA expression and flow cytometry. 
 
Tertiary Objectives – Clinical Outcomes (exploratory) 
Test the effects of calcifediol (25D) or calcitriol (1,25D), compared to placebo, on ICU- and 
hospital length of stay, mortality, duration of mechanical ventilation, and other clinical outcomes. 
 
 
 
 

Version date 12/25/18;  Page 16 
 
III. SUBJECT SELECTION 
 
A.  Inclusion and Exclusion Criteria 
 
Inclusion Criteria 
Exclusion Criteria 
• Age ≥ 18 
• Admitted to the ICU within 48h prior to enrollment 
• Likely to remain in the ICU (alive) for ≥72h 
• Naso/orogastric tube or ability to swallow 
• High risk of severe AKI* 
• Total serum calcium > 9.0 mg/dl or phosphate > 6.0 mg/dL 
within previous 48h 
• Currently receiving oral calcium supplementation 
• Ingestion of vitamin D3 >1,000 IU/day or any 25D or 1,25D 
during the previous 7 days 
• AKI stage 2 or 3 (based on SCr and/or UOP criteria) 
• History of transplantation or chronically (>7days) receiving 
immunosuppressive medications (not including glucocorticoid 
steroids at a dose ≤ prednisone 20 mg/day) 
• Neutropenia (<1000 PMNs/mm3) in the previous 48h 
• Primary parathyroid disease (active), granulomatous disease 
(active), or symptomatic nephrolithiasis in the previous 3 
months 
• Receiving cytochrome P450 inhibitors† 
• CKD stage V or ESRD 
• Hgb < 7 g/dL 
• GI malabsorption 
• Prisoner 
• Pregnant or breastfeeding 
• Enrolled in a conflicting study, including any study with a 
research blood draw 
 
Table 2.  Inclusion and Exclusion Criteria.  *Patients are considered to be at high risk of 
severe AKI if they have either: AKI stage 1, defined as an increase in SCr ≥ 0.3 mg/d within the 
previous 48 hours, ≥ 50% within the previous 7 days, or urine output ≤ 0.5 ml/kg/h x 6 hours); or 
if they have an AKI risk score ≥6 – see Table 3.  †Patients will be excluded if they are receiving 
any of the following medications: ketoconazole, atazanavir, clarithromycin, indinavir, 
itraconazole, nefazodone, nelfinavir, ritonavir, saquinavir, telithromycin, or voriconazole, since 
these drugs may inhibit enzymes involved in vitamin D metabolism. 
 
 
Risk Factor 
Points 
Chronic 
 
Age>70y 
Hypertension 
Diabetes Mellitus 
1 
1 
2 
Chronic Liver Disease (cirrhosis, NAFLD, AIH, or chronic viral hepatitis) 
2 
   Congestive Heart Failure (NYHA class 3 or 4) 
   Chronic Kidney Disease (baseline eGFR<60 ml/min/1.73m2) 
2 
3 
Acute 
 
   Blood pH (venous or arterial) ≤ 7.30 
2 
Sepsis 
2 
Mechanical ventilation expected to last > 24 hours 
2 
Hypotension/shock, defined as MAP<70 mm Hg or use of pressors/inotropes 
2 
 
Table 3.  AKI Risk Prediction Score 
 
B.  Source of Subjects 
Subjects will be enrolled from the following intensive care units (ICUs) at Brigham and Women’s 
Hospital (BWH):  medical ICU (MICU), cardiac care unit (CCU), thoracic surgery ICU (TICU), 
surgical ICU (SICU), and neurology/neurosurgical ICU (NICU/NSICU). 
 

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IV. SUBJECT ENROLLMENT 
 
A.  Methods of enrollment 
Patients from each of the ICUs listed above will be screened for eligibility, using electronic 
medical records, based on the inclusion/exclusion criteria listed above.  Women of child-bearing 
potential will not be included without documentation of a negative pregnancy test, unless they 
have previously undergone hysterectomy or bilateral oophorectomy.  Women of child bearing 
potential will be defined as any woman who has begun menstruation and not yet reached 
menopause.  Menopause will be defined as any woman over the age of 45 who has not had a 
menstrual period for at least 12 months.  For those patients who appear eligible based on 
electronic medical records, study investigators will confirm eligibility with the patient’s nurse and 
attending physician of record. 
 
B.  Procedures for obtaining informed consent 
Once permission for study enrollment has been obtained from the patient’s attending physician, 
study investigators will request that a member of the patient’s primary treating team (e.g., the 
intern, resident, fellow, attending, or nurse caring for the patient) ascertain the willingness of the 
patient to discuss the study with investigator David Leaf, M.D., investigator Sushrut Waikar, 
M.D, or investigator Finnian McCausland, M.D.  Patients eligible and willing will then be 
approached by Dr. Leaf, Dr. Waikar, or Dr. McCausland to explain the rationale of the study, 
answer any questions, and ask for written informed consent to participate.  Only physician-
investigators will obtain informed consent.  Subjects will be given up to 6 hours to consider 
participation given the time-sensitive nature of the study.  The 6-hour period will begin once the 
patient or surrogate has received the consent form. 
 
For patients deemed to lack decisional capacity by the primary treating team, study 
investigators will follow identical procedures as above with the patient’s surrogate.  The Partners 
Human Research Committee (PHRC) preferred order of surrogates will be followed, and the 
investigator will document the relationship of the surrogate to the subject in the research record.  
The order of surrogates will be: 
 
i) court appointed guardian with specific authority to consent to participation in research 
or authority to make health care decisions for a class of diagnostic and therapeutic 
decisions inclusive of the proposed research; 
ii) health care proxy/person with durable power of attorney with specific authority for 
making health care decisions inclusive of the proposed research; or 
iii) spouse, adult child, or other close family member who knows the subject well and has 
been involved in their care. 
 
For patients deemed to lack decisional capacity who are enrolled by surrogate consent, assent 
of subjects will be required for participation in the research unless the subject is incapable of 
providing assent due to his/her medical condition (such as intubation/sedation).  For patients 
deemed to lack decisional capacity who are enrolled by surrogate consent and who 
subsequently regain capacity during the study, investigators will explain the study (including 
updating them on what has happened so far as a result of being in the study), answer any 
questions, and ask for written informed consent to continue to participate.  Alternatively, patients 
will have the option of withdrawing from the study, in which case all data and biological samples 
collected will be destroyed. 
 
Although written consent from surrogates in person will be obtained whenever possible as the 
preferred means of consent for patients who are unable to consent, for patients who do not 

Version date 12/25/18;  Page 18 
 
have a surrogate available to provide written consent in person we will ask a member of the 
patient’s primary treatment team (e.g., doctor or nurse) to contact the surrogate (using the same 
PHRC preferred order described above) by telephone to inform them of the study and to 
ascertain their willingness to speak with one of the M.D. study investigators.  The primary team 
member making the phone call will verify the surrogate’s name and relationship to the patient, 
and will be provided with a printed script containing bullet points for the call (script provided as 
separate attachment). 
 
For surrogates expressing interest in hearing about the study, physician-investigator David Leaf, 
M.D., Sushrut Waikar, M.D., or Finnian McCausland, M.D., will first describe an overview of the 
study and answer any questions.  If the surrogate is interested in hearing more about the study, 
the physician-investigator will ascertain whether the surrogate has plans of coming to the 
hospital in the next few hours, and will therefore be available to provide in person written 
consent.  If the surrogate states that he/she is planning on coming to the hospital in the next few 
hours, the physician-investigator will complete the consent process in person.  If the surrogate 
states that he/she is not planning on coming to the hospital in the next few hours, the physician-
investigator will send the consent form to the surrogate electronically, either by secure email or 
facsimile.  After confirming that the surrogate has a copy of the consent form in front of them, 
the physician-investigator will summarize the required elements of informed consent, review the 
consent form with the surrogate, answer any additional questions, and ask for written consent.  
If the surrogate agrees, the physician-investigator will ask the surrogate to sign and date the 
consent form and to send it back electronically by secure email or facsimile.  Given the time-
sensitive nature of the study, the surrogate will be asked to provide written consent as soon as 
possible and no longer than 6 hours from the time they receive the consent form. 
 
Non-English speaking patients will be eligible for inclusion.  We will use official Partners 
interpreters and the PHRC approved ‘short form’ consent document in the native language of 
the patient and/or their surrogate. 
 
Using a checklist, we will document the following items of the informed consent process for 
each patient: 
 
▪ 
If consent was from subject or surrogate 
▪ 
If surrogate, in-person or on the phone 
▪ 
If surrogate, relationship to subject 
▪ 
If surrogate, could subject assent, and if not, why not? 
▪ 
Was an interpreter required and short form used? 
▪ 
If subject regains capacity to consent a later time;  if so, document whether the subject 
provides written consent to continue to participate or not 
▪ 
Copy of consent form provided to subject or surrogate/ 
▪ 
Copy of consent form uploaded into Epic? 
▪ 
Research study associated to the patient in Epic? 
 
C.  Treatment assignment and randomization 
Participants will be randomly assigned, in a 1:1:1 fashion, to receive calcifediol, calcitriol, or 
placebo.  Randomization will be stratified by the presence or absence of AKI on enrollment, 
defined by an increase in serum Cr ≥ 0.3 mg/dl within the previous 48 hours, ≥ 50% within the 
previous 7 days, or urine output ≤ 0.5 ml/kg/h x 6 consecutive hours during any 6-hour block in 
the previous 24 hours.  Randomization will be performed by the BWH research pharmacy using 
a computer generated sequence and permuted blocks. 
 
 

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V. STUDY PROCEDURES 
 
A.  Parameters to be measured 
 
Clinical data 
Upon enrollment in the study, the following demographic variables will be recorded:  age, 
gender, race, ethnicity, comorbidities, active medications, active medical issues, reason for ICU 
admission, date of ICU/hospital admission, ICU location (e.g., MICU, CCU), and severity of 
illness at the time of study enrollment, as measured by APACHE II and SOFA scores. 
 
In addition, the following clinical outcomes data will be recorded: 
 
-ICU-, hospital-, and 28-day mortality 
-ICU and hospital length of stay 
-Incidence/severity of new/worsening AKI (defined by KDIGO guidelines)88 within 1 week of 
enrollment 
-Duration of mechanical ventilation 
-Duration of vasopressor support 
-Daily Sequential Organ Failure Assessment (SOFA) scores for the first 7 days of the study 
-Physiologic data including heart rate, blood pressure, oxygenation, and urine output during the 
first 7 days of the study 
-Hypocalcemia requiring I.V. calcium replacement during the first 7 days of the study 
-Total serum calcium levels on a daily basis for the first 7 days of the study 
 
28-day mortality 
For patients discharged from the hospital prior to day 28, investigator David Leaf, M.D. will 
ascertain 28-day survival by calling the patient and/or their surrogate 28 days after enrollment 
as a courtesy follow-up to thank them for participating and to answer any questions they may 
have.  The duration of the phone call is expected to last less than 2 minutes. 
 
Blood samples 
We will obtain blood samples on days 0, 1, 2, 3, 4, and 5.  A summary of the volumes of blood 
that we will obtain at each time point is shown in Figure 3.  On days 0 and 2 we will collect 
18mL of blood, which will allow us to isolate peripheral blood mononuclear cells (PBMCs) and 
store them in liquid nitrogen for comprehensive analyses in the future, including flow cytometry 
and RT-PCR.  18mL of blood will ensure that we have a sufficient number of PBMCs to perform 
all of the planned analyses.  Specifically, each mL of blood has approximately 106 PBMCs, and 
therefore 18mL of blood has approximately 18 x 106 PBMCs.  We will aliquot and store the 
PBMCs as follows: aliquot #1 will have 10 x 106 PBMCs, and will be used for RT-PCR analyses 
on monocytes.  Aliquots #2 and #3 will have 4 x 106 PBMCs each, and will be used for flow 
cytometry.  For each of these analyses, the number of PBMCs being collected and archived 
represents the minimum amount necessary for the intended analyses. 
 
In addition to the 18mL of blood on days 0 and 2 for PBMC isolation, we will also collect 2.5mL 
blood on days 0, 1, and 2 into PAXgene RNA tubes, which will allow us to evaluate leukocyte 
gene expression profiles.  Finally, on days 1, 3, 4, and 5, we will collect 9mL of blood and store 
the plasma and DNA.  9mL of blood contains approximately 4.5mL of plasma (approximately 
50% of whole blood is plasma), which will allow us to archive nine 500uL plasma aliquots at 
each time point.  Collection of nine plasma aliquots at each time point will allow us to test the 
multiple hypotheses and measurements described below. 
 

Version date 12/25/18;  Page 20 
 
Thus, the total volume of blood that will be drawn over the course of the entire study, including 
PBMCs, DNA, RNA, and plasma, will be 79.5mL.  We will not enroll patients who are already 
enrolled in competing studies in which blood is being drawn, and we will not enroll patients with 
a hemoglobin level < 7 g/dl (Table 2).  In most cases, we anticipate that blood samples will be 
obtained from central venous or arterial catheters by the patient’s nurse.  For patients who do 
not have a central venous or arterial catheter, blood will be obtained by venipuncture by a 
hospital phlebotomist, drawn concurrently with clinical blood samples whenever possible. 
 
 
 
 
The primary measurements that are anticipated to be performed on the plasma obtained from 
these collections will include: 25D, 1,25D, vitamin D-binding protein, heme oxygenase-1, 
interleukin-6, interleukin-10, thrombomodulin, parathyroid hormone, fibroblast growth factor-23, 
and cystatin C. 
 
Urine 
We will collect 30mL of urine at each of the above time points.  In the majority of cases, urine 
will be collected from foley catheters placed for routine clinical care of ICU patients.  Foley 
catheters will not be placed for research purposes.  If a patient does not have a foley catheter in 
place for routine clinical care, urine will be collected using a bed pan, commode, or direct 
urination into a specimen cup.  Anticipated tests that will be performed on the urine samples 
include neutrophil gelatinase-associated lipocalin (NGAL), interleukin 18, and kidney injury 
molecule-1 (KIM-1). 
 
 
B.  Drugs to be used 
We will administer calcifediol, calcitriol, or placebo.  The dosing regimens are provided in Figure 
2.  The rationale for these agents, justification of dosing regimens, and safety data are provided 
in sections 1C-F.  Patients assigned to the placebo arm will receive medium chain triglyceride 
(MCT) oil, which has an identical appearance, color, consistency, and odor as calcifediol and 
calcitriol, both of which are also compounded in MCT oil. 
 

Version date 12/25/18;  Page 21 
 
Double-dummy design 
Although calcifediol, calcitriol, and placebo will be identical to each other in appearance, the 
volumes administered will differ.  For example, the concentration of calcitriol is 1 µg/ml, and 
therefore each 4µg dose will be administered in 4 ml.  In contrast, the concentration of 
calcifediol is 100µg/ml.  Since the first dose of calcifediol is 400µg, the first dose will also be 
administered in 4ml, identical to the calcifediol arm.  However, calcifediol will be administered at 
a dose of 200µg on days 1, 2, 3, and 4 (Figure 2), which will be in 2ml.  Therefore, to maintain 
blinding, we will use a double-dummy approach for drug administration on days 1, 2, 3, and 4 as 
follows: 
 
Calcifediol Arm 
Calcitriol Arm 
Placebo Arm 
Calcifediol 2ml 
Placebo 4ml 
Calcitriol 4ml 
Placebo 2ml 
Placebo 4ml 
Placebo 2ml 
 
C.  Devices to be used 
None 
 
D.  Procedures/surgical interventions 
None 
 
E.  Data to be collected 
The specific data variables that will be collected are provided in section V-A. 
 
F.  Dosing modifications 
All patients enrolled will have total serum calcium levels monitored on a daily basis as part of 
routine clinical care in the ICU.  All subsequent doses of study drug will be held for any patient 
who develops a total serum calcium level > 10.0 mg/dl after enrollment.  Additionally, if at any 
time during the course of the study >10% of participants in either of the active treatment arms 
develops hypercalcemia, defined as per the BWH hospital laboratory as a total serum calcium 
level > 10.7 mg/dl, the dosing regimen for future participants assigned to that arm will be 
reduced by 25%.  For the calcifediol arm, this 25% dose reduction would result in a new dosing 
regimen of 300 µg on day 0 and 150 µg on days 1, 2, 3, and 4.  For the calcitriol arm, this 25% 
dose reduction would result in a new dosing regimen of 3 µg daily for 5 days.  Unblinded safety 
data on hypercalcemia will be evaluated by the data and safety monitoring board (DSMB). 
 
G.  Adaptive Trial Design 
After enrollment of the first 18 patients (6 patients in each of the three arms), we will stop 
enrollment to measure plasma 25D levels in the patients who received calcifediol to ensure that 
mean plasma 25D levels on days 1, 2, 3, 4, and 5 are > 50 ng/ml.  If mean plasma 25D levels 
on days 1, 2, 3, 4, and 5 are < 50 ng/ml and the mean increase in serum total calcium levels 
from day 0 to day 5 is < 0.5 mg/dl, we will increase the dosing regimen for the calcifediol arm for 
the remaining study participants as follows: 
 
Mean plasma 25D level on days 1 through 5 
Calcifediol dose adjustment for remaining participants 
40-50 ng/ml 
500 µg on day 0 and 250 µg on days 1, 2, 3, and 4 
<40 ng/ml 
600 µg on day 0 and 300 µg on days 1, 2, 3, and 4 
 
 
 

Version date 12/25/18;  Page 22 
 
VI.  BIOSTATISTICAL ANALYSIS 
 
A.  Specific data variables being collected for the study 
The specific data variables that will be collected are provided in section V-A. 
 
B.  Study end points 
Primary end point.  The primary end point is a composite of renal injury, RRT, and death.  
Renal injury will be assessed by time-averaged (area under the curve [AUC]) levels of daily SCr 
for the first 7 days following enrollment.  This end point has been used in previous clinical trials 
conducted in AKI,89 and these measurements will be obtained through routine clinical testing.  
To account for missing data, we will standardize the AUC of daily SCr levels relative to the 
number of days in which SCr data are available.  RRT and death are important confounders of 
SCr levels, as well as important end points by themselves, and will therefore be considered 
along with the AUC of SCr levels as part of the primary composite end point.  RRT will be 
defined as receipt of any form of dialysis within the first 7 days of enrollment.  Death will be 
defined as all-cause mortality within the first 7 days of enrollment. 
 
To evaluate the primary composite end point, which includes both continuous (SCr levels) and 
dichotomous (RRT and death) variables, we will use a rank-based procedure, as prior studies 
have done,90 as follows: 
 
-Patients who die within 7 days of enrollment will be assigned the highest (worst) rank, with no 
difference in rank assigned to patients who die at different times within the first 7 days. 
-Patients who survive >7 days but who require RRT within 7 days of enrollment will be assigned 
the second highest rank. 
-All other patients will be assigned a rank based on the AUC of their SCr, as described above. 
 
Secondary end points.  Secondary end points will include new or worsening AKI (defined by 
KDIGO guidelines);91 kidney function on hospital discharge (assessed by SCr); hospital- and 
ICU length of stay; and hospital-, ICU-, and 28-day mortality.  We will also evaluate the AUC of 
urinary injury biomarkers, including KIM-1, NGAL, and IL-18, during the first 5 days using a 
similar global rank score strategy as described above.  Finally, we will evaluate organ-failure 
free days (assessed by daily SOFA scores) during the first 7 days following enrollment.  For the 
cardiovascular component of the SOFA score, we will used a modified scoring system that has 
improved accuracy in predicting outcomes,92 and for the renal component we will assign a score 
of “4” (the highest score) to any participant who requires RRT. 
 
C.  Statistical methods 
To evaluate the primary end point, we will compare the ranks between treatment groups versus 
the placebo group with the use of the Wilcoxon Rank Sum test.  To evaluate secondary end 
points, we will compare means and medians between each of the active treatment groups 
versus the placebo group using parametric (e.g., t-test) and non-parametric (e.g., Wilcoxon 
Rank Sum) tests, as appropriate.  To account for two active treatment groups, we will consider p 
values <0.025 to be significant for the primary end point.  For secondary end points, all of which 
will be considered exploratory, we will consider p values <0.05 to be significant. 
 
D.  Power analysis 
150 participants (N=50 per group) will provide >80% power to detect a 0.62 standard devation 
difference in the global rank score in each of the active treatment groups vs. placebo, with a 
two-sided α of 2.5%. 
 

Version date 12/25/18;  Page 23 
 
VII.  RISKS AND DISCOMFORTS 
 
A.  Complications of surgical and non-surgical procedures 
Not applicable 
 
B.  Drug side effects and toxicities 
The only known toxicity of vitamin D metabolites, including calcifediol and calcitriol, is 
hypercalcemia.81 82  Despite an excellent safety profile, transient hypercalcemia has been 
reported with calcitriol and, to a much lesser extent, with calcifediol.  The risks of hypercalcemia 
in the current study are low, since the planned doses of calcifediol and calcitriol are lower than 
doses that have been used safely in prior studies in humans (discussed in detail in sections 1E 
and 1F).  Additionally, the following precautions will further minimize risk to patients: 
 
1) Limiting our study to patients with serum total calcium levels ≤ 9.0 mg/dl.  Most prior 
clinical studies of calcifediol and calcitriol (discussed in sections 1E and 1F) were conducted in 
patients who were generally normocalcemic on enrollment.  In contrast, most ICU patients have 
serum calcium levels in the low-normal range, and many patients (55.2% in one study)93 are 
frankly hypocalcemic on arrival to the ICU.  In the current study, we will exclude patients with 
serum total calcium levels > 9.0 mg/dl on enrollment (Table 2).  By limiting enrollment to 
patients with serum total calcium levels ≤ 9.0 mg/dl, a substantial barrier exists for serum 
calcium levels to rise into the hypercalcemic range (>10.7 mg/dl) and an even larger barrier for 
clinically relevant hypercalcemia (>11.5 mg/dl). 
 
2) Limited number of doses of study drug.  In most prior clinical studies, calcifediol and 
calcitriol were administered on a regular (daily) basis for weeks to months.  The current study 
will administer a total of only 5 doses of calcifediol or calcitriol drug over 5 days. 
 
3) Close monitoring of total serum calcium levels.  All patients enrolled will have total serum 
calcium levels monitored on a daily basis.  Subsequent doses of study drug will be held in any 
patient with a serum calcium > 10.0 mg/dl. 
 
4) Exclusion of patients with primary parathyroid disease or granulomatous disease.  
These conditions may be associated with upregulation of 1-α hydroxylase, which could increase 
the conversion of 25D to 1,25D and increase the risk of developing hypercalcemia.  Additionally, 
we will exclude patients with symptomatic nephrolithiasis in the preceding 3 months, and those 
receiving oral calcium supplementation (Table 2). 
 
5) Exclusion of patients receiving cytochrome P450 inhibitors.  We will exclude patients 
receiving any of the following medications, since these drugs may inhibit enzymes involved in 
vitamin D metabolism and therefore could increase the risk of hypercalcemia: ketoconazole, 
atazanavir, clarithromycin, indinavir, itraconazole, nefazodone, nelfinavir, ritonavir, saquinavir, 
telithromycin, or voriconazole (Table 2). 
 
6) Highly monitored setting.  Patients will be in the highly-monitored setting of an ICU at a 
tertiary care academic hospital.  Accordingly, if hypercalcemia or other adverse events occur 
they are likely to be appreciated immediately and appropriate resources will be readily available. 
 
7) Pre-specified dose modification.  As described in section V-F, if at any time during the 
course of the study >10% of participants in either of the active treatment arms develops 
hypercalcemia, defined by the BWH hospital laboratory as a total serum calcium level > 10.7 
mg/dl, the dosing regimen for future participants assigned to that arm will be reduced by 25%.   

Version date 12/25/18;  Page 24 
 
 
8) DSMB.  A DSMB will meet every 6 months to review unblinded data on all adverse events, 
with an emphasis on serum total calcium levels, and will submit a report to the IRB after each 
meeting.  Additionally, data safety monitoring will be reviewed by investigators David E. Leaf, 
MD, and Sushrut S. Waikar, MD, on a weekly basis. 
 
 
 
 
 

Version date 12/25/18;  Page 25 
 
C.  Confidentiality 
Loss of confidential patient information is a potential risk in any clinical trial.  To minimize this 
risk we will collect our database only on partners-protected computers; we will not include any 
protected health information in our primary database or in any study documents; the key linking 
the patient’s study ID# with their name and medical record number will be kept in a separate 
folder on a partners-protected computer.  The key linking the treatment group with the study ID 
will be kept on a separate password-protected computer by the BWH research pharmacy.  
Biological specimen tubes containing plasma and urine will be tagged with study ID#’s only and 
will not include any protected health information.  Specimens will be stored in locked freezers at 
Brigham and Women’s hospital. 
 
Storing of genetic samples 
Genetic samples will be stored in locked freezers at Brigham and Women’s Hospital and kept 
for a maximum of 25 years.  The samples will be labeled with a study ID only;  no protected 
health information will be shared with collaborators outside of Partners.  Genetic data will not be 
placed onto publicly available repositories such as the National Institutes of Health (NIH) central 
repository. 
 
D.  Device complications/malfunctions 
Not applicable  
 
E.  Psychosocial risks 
Not applicable  
 
F.  Radiation risks 
Not applicable  
 
 

Version date 12/25/18;  Page 26 
 
VIII.  POTENTIAL BENEFITS 
 
A.  Potential benefits to participating individuals 
Individuals receiving calcifediol or calcitriol may derive benefit through the drug’s actions on 
immune/inflammatory pathways that are directly involved in the pathogenesis of AKI.  Our 
hypothesis that these pharmacologic agents may prevent/attenuate AKI in critically ill patients is 
supported by ample data from preclinical studies and preliminary data in humans (discussed in 
detail in section I). 
 
B.  Potential benefits to society 
Potential benefits to society include elucidation of the physiology of vitamin D metabolism in 
critically ill patients.  Moreover, this study will help answer important questions about 
mechanisms responsible for the association between vitamin D deficiency and adverse 
outcomes, including AKI, in critically ill patients.  Understanding whether the 
immune/inflammatory response can be enhanced by calcifediol or calcitriol will inform the 
research community whether a larger study of either agent, powered to evaluate hard end 
points such as mortality, is justified. 
 
 
 
 

Version date 12/25/18;  Page 27 
 
IX.  MONITORING AND QUALITY ASSURANCE 
 
A.  Independent monitoring of source data 
Data collection will be performed by study investigators in a blinded fashion.  The patient 
assignment key linking study ID# with treatment group assignment will be maintained securely 
by the BWH research pharmacy throughout the data collection period. 
 
B.  Safety monitoring 
We have assembled a DSMB consisting of Dr. Gearoid McMahon, a faculty member in the 
BWH Division of Nephrology, and Dr. Benjamin Raby, a faculty member in the BWH Division of 
Pulmonary and Critical Care Medicine.  Drs. McMahon and Raby both have experience 
providing care to critically ill patients and also have expertise in patient-oriented research, 
clinical trials, and biostatistics.  The DSMB will meet every 6 months to review unblinded data 
on all adverse events, with an emphasis on serum total calcium levels, and will submit a report 
to the IRB after each meeting.  The DSMB will be responsible for determining if the research 
should be altered or stopped. 
 
Additionally, the PI (Dr. David Leaf) will review daily serum creatinine values for the first 7 days 
in real-time for each patient enrolled, and will take appropriate action as needed for any patient 
who develops hypercalcemia. 
 
C.  Outcomes monitoring 
Not applicable.  The primary end point is a biomarker, SCr, and will not be assessed until study 
completion. 
 
D.  Adverse event reporting 
All adverse events and unanticipated problems involving risks to subjects and others will be 
reported to the PHRC in accordance with PHRC adverse event and unanticipated problems 
reporting guidelines: 
 
http://navigator.partners.org/ClinicalResearch/Reporting_Unanticipated_Problems_including_Ad
verse_Events.pdf 
 
We will report any adverse events to the PHRC within 5 working days/7 calendar days of the 
date the investigator first becomes aware of the problem.  Other adverse events and minor 
protocol deviations will be reported during continuing review. 
 
Additionally, in accordance with IND regulations, we will report all adverse events to the FDA in 
an IND safety report as soon as possible, but no later than 15 calendar days after the primary 
investigator (David Leaf, MD) becomes aware of the safety information and determines that the 
information qualifies for reporting: 
 
http://www.fda.gov/downloads/Drugs/.../Guidances/UCM227351.pdf 
 
 
 

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X.  REFERENCES 
 
1. 
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