- General Design This Phase 2 randomized clinical trial of high-dose vs. standard dose
Vitamin D (VitD) supplementation aims to prove feasibility of the intervention in a
diverse community-recruited cohort and to provide an estimate of the effect sizes of
treatment on cognitive change and brain MRI volumetric measures as the main outcomes. We
will study 180 elderly participants (approximately 60 with MCI, 60 with mild AD and 60
with No Cognitive Impairment) with longitudinal neuropsychological testing and brain MRI
scans over a 3 year study period. One-half of each diagnostic group will be randomized
to treatment with high-dose (4,000 IU p.o. daily) vitamin D3 (n=90) or to standard dose
vitamin D3 treatment (n=90 who will receive 600 IU vitamin D3 daily). The choice of the
low-dose amount of vitamin D3 was determined by the goal to meet the Institute of
Medicine (IOM) RDA recommendation of 800 IU/day for older adults (age >71y) from all
sources, including both diet and supplements. The estimated average dietary intake of
VitD by older adults is ~200 IU/day. Thus, the combined intake of VitD from diet and the
low-dose supplement in our study will be approximately the RDA (i.e. 800 IU/day). Blood
will be collected every 6 months (except month 30) for VitD levels along with other
This study aims to test if supplementation with high dose oral VitD (4,000 IU) will
successfully correct VitD insufficiency, compared to treatment with standard RDA dose
VitD (~800 IU total intake, as recommended by the IOM for those >age 71) in a diverse
community-based cohort with serum VitD levels <20 ng/ml at study entry. The primary aims
are to prove feasibility of the intervention, and the effectiveness of high-dose VitD in
correcting VitD insufficiency in this diverse old cohort. Our primary outcome will be
correction of VitD insufficiency in all subjects treated with 4,000 IU daily. Secondary
aims are to provide an estimate of the effect sizes of treatment on cognitive change
(executive function and global), and to gather preliminary data relevant to the evidence
for moving forward with a potential definitive Phase III study in elderly groups at risk
for dementia. Additional analyses will test if correction of VitD insufficiency
correlates with changes in key biomarkers measured in blood and urine. Longitudinal MRI
analyses will provide an estimate of the treatment effect size on brain atrophy rate. We
will also examine VitD receptor genotype polymorphisms and their impact on response to
2. Schedule of Visits Screening Visit (Visit 1). Elderly participants with a prior
diagnosis of either No Cognitive Impairment, MCI, or mild AD, will be screened for
potential study entry. Informed consent will be obtained before any study procedures are
conducted. The following assessments will be done at screen (before Visit 2): MOCA, GDS,
CDR, screening labs, then brain MRI for those who remain eligible. Blood and urine
screening lab results will be reviewed by the investigator or designee for assessment of
eligibility. Patients who do not meet all inclusion criteria or who meet any exclusion
criteria will be discontinued from the study. Up to 45 days are allowed for completion
of Visit 1 screening procedures, assessments, and evaluation of results from laboratory
tests and Brain MRI.
Baseline Visit (Visit 2). The treatment period is a double-blind phase beginning at
Visit 2, with treatment duration of 3 years from baseline to the final 42 month visit.
Patients who meet entry criteria will be enrolled and randomized to high-dose VitD 4,000
IU p.o. daily or standard dose VitD (600 IU daily). Randomization will be stratified by
diagnosis and race, such that 50% of each diagnostic subgroup will be randomized to the
high-dose treatment arm.
Assessments to be performed at the Baseline visits are: additional neuropsychological
tests including the SENAS and ADAS-Cog, the ECog which has sections completed by the
participant and the informant. Blood samples drawn at the Screening or Baseline visit
will be sent for measurements of parathyroid hormone (PTH) level and genotyping to
include polymorphisms of the VitD receptor for all enrolled subjects.
Telephone Check-Ins at Months 1 and 30. Brief telephone calls will be made to all
enrolled participants at Month 1 & Month 30 to check on compliance with the study
Treatment Period Visits at Months 6 and 18 (Visits 3 & 5). Brief visits will be
performed at Month 6 and Month 18, with venipuncture for blood VitD level and the
ADAS-Cog. In addition, pill counts and study drug compliance will be reviewed and
Treatment Period Visits at Months 12, 24, 36 and 42 (Visits 4, 6, 8 & 9). These visits
will provide the main outcome measures for the trial. Assessments to be performed at
these visits are: Neuropsychological testing with the SENAS, MOCA and the ADAS-Cog.
Functional assessment instruments (collected from the participant and informant) are the
ECog and CDR. The GDS will also be collected. Blood samples will be drawn for
measurement of VitD level, and both plasma and serum will be stored for additional
studies. The final assessment visit at Month 42 (Visit 9) will take place on the day of
the patient's last VitD dose. A volumetric brain MRI study will also be obtained at this
Early Termination (ET) Visit (if applicable). For participants who do not complete this
42 month study, an early termination visit will be done whenever possible. For the early
termination visits, we plan to obtain all the measures usually collected at Month 42.
3. Protocol Procedures. Medical History, Physical, and Neurological Exam: A complete
medical history with review of systems, and recent medication history will be performed
at screening. All study patients will receive a comprehensive physical examination at
screening and annually. A detailed neurological exam will be performed either at
screening or baseline. Any deficits identified will be documented using a standardized
form. The physician also takes a functional history, and a psychiatric/behavioral
history. Participants will be closely monitored during the treatment period (from month
1 through 42) to assess side effects or adverse events.
Blood draws and Laboratory Tests. Blood samples will be collected into serum separator
tubes (to collect serum) containing EDTA. One EDTA tube will be sent directly to the UC
Davis Medical Center (UCDMC) clinical lab for a complete blood count and measurement of
hemoglobin A1C. A second EDTA tube will be centrifuged at 40C to collect plasma. The
buffy coat of the second tube will be collected and frozen for subsequent isolation of
genomic DNA. Serum and plasma samples will be aliquoted in volumes specific for each
assay, and frozen at -800C until analysis.
Laboratory Assays VitD (25-OHD) level will be performed at screen and every 6 months
after (serum/blood, except for the month 30 Telephone check-in visit). Clinical Labs
(screening): Comprehensive Metabolic Panel (CMP), CBC, UA, vitamin B12 at screen. PT/PTT
will be drawn for any participant with history of a coagulopathy or excessive bleeding.
If none of the above screening labs are exclusionary, we will add homocysteine, PTH,
high-sensitivity CRP, Interleukin-6 (IL-6), IL-10, TNF-alpha, Monocyte chemoattractant
protein-1 (MCP-1), blood and urine Isoprostane F 2- levels. Inflammatory markers:
annual CRP blood levels and cytokine panel (IL-6, IL-10, TNF-alpha, and MCP-1) will be
measured at Baseline and month 42 (or ET visit). Oxidative damage markers: Isoprostane F
2- levels in blood and urine will be measured at Baseline and month 42 (or ET visit).
Genetic analysis of the VitD receptor will be carried out for SNPs previously associated
with cognitive decline or AD: Apa1, Taq1, CdX, and Bsml. For safety purposes, the
following Labs will be done as needed: Urine & serum Calcium PRN renal stones, cultures
(e.g. urine, blood) and Microbiology tests if with high wbc count. We will repeat CMP
annually (months 12, 24, 36 & 42), and PTH level at month 42 (or ET visit).
Neuropsychological Testing. All subjects will undergo neuropsychological testing with
the MOCA at screening and at the 12, 24, 36 and 42 month visits. The Spanish English
Neuropsychological Assessment Scale (SENAS) will be administered at baseline and at the
12, 24, 36 and 42 month office visits. The ADAS-Cog will be administered at baseline and
every 6 months (except month 30).
Functional Assessments. All subjects will undergo functional assessments with the ECog
(informant based measure of daily function) and CDR at screen/baseline and at months 12,
24, 36, and 42.
Dietary Survey. Dietary data will be collected to assess overall intake of VitD from
food sources. Participants or their caregivers will be interviewed using a validated
Other Clinical Instruments/Assessments. All subjects will complete the Geriatric
Depression Scale (GDS) at screening and at the 12, 24 & 42 month assessments.
Brain MRI Procedure & Analysis. Structural Brain MRI will be acquired at Screening and
at study end (42 months, or at time of ET visit) for all enrolled participants. Subjects
will be scanned at the UC Davis Imaging Research Center using a 3 Tesla Siemens Tim Trio
machine and ADNI-compatible pulse sequences.
MRI acquisition and analysis. MRI acquisition will include T1-weighted, fast T2,
fluid-attenuated inversion recovery (FLAIR), and diffusion tensor imaging (DTI). MRI
data will be analyzed for quantification of regional brain, gray, white and CSF volumes.
All subjects will have baseline and longitudinal quantification of regional WMH, DTI and
gray matter measures.
4. Statistical Analysis Plan (SAP), Hypothesis Testing & Statistical Considerations.
A brief description of hypothesis testing strategies and power calculations are provided
separately by aim below. All power calculations assumed a two-sided test and alpha=0.05
unless otherwise specified.
Aim 1: To compare the effectiveness of high-dose versus standard-dose oral VitD in
correcting low VitD status in a diverse community-based cohort.
Hypothesis 1.1: Rate of correction of VitD insufficiency is higher after high-dose
versus standard dose oral VitD supplementation.
Analytic Plan. The primary analyses for this aim will involve two parts: 1) a
descriptive summary of the percentage of individuals randomized to each group whose VitD
levels reach and continue in the adequate range (>20 ng/ml); and 2) a comparison of the
percentage of individuals whose levels reach the adequate range between the high-dose
and standard dose groups. We will compute the percentage of individuals who attain
adequate VitD levels by six months, the percentage who maintain these levels from months
6 through 42, and 95% confidence intervals. A stratified exact test of proportions will
be used to compare percentages between groups. Secondary analyses will characterize the
trajectory of VitD levels in the two groups over time using mixed effects models to
assess patterns of change, and in particular, whether levels plateau or continue to
Power calculations. With 76-90 individuals per group, we will have over 80% power to
detect a difference in percentage that reach or maintain adequate levels between groups
assuming the percentage in the high-dose group is at least 90% and the percentage in the
standard-dose group is no more than 72%.
Aim 2: To assess the effect of high-dose vs. standard-dose VitD on altering cognitive
trajectories, and gather preliminary data relevant to the design of a potential Phase
III trial in elderly at-risk groups.
Hypothesis 2.1: High dose VitD supplementation is associated with less cognitive decline
(better cognitive trajectory) in executive function over 3 years than in those
receiving the standard dose.
Rationale. Published data from our observational study showed a significant difference
in rates of decline between those with insufficient or deficient serum VitD levels and
those whose levels are in the adequate range. If high-dose VitD supplementation results
in a slowing of cognitive decline, similar to that seen between the decline in the
inadequate or deficient group and the adequate group, in addition to evidence of
effectiveness (Aim 1) and biological mechanism (Aim 3), such a result would support
moving on to a Phase III clinical trial for a definitive demonstration of clinical
efficacy of high-dose VitD.
Analytic Plan. The primary outcome for this aim is the SENAS executive function score,
assessed annually over the course of the trial. A secondary outcome is the ADAS-Cog
(global cognition). The analytic strategy is the same for each outcome and will utilize
an intent-to-treat approach in which everyone randomized to the high dose group will be
compared to those randomized to the standard dose regardless of adherence to treatment;
secondary as-treated analyses will utilize continuous serum VitD levels as a measure of
exposure to VitD to assess a "dose-response" relationship. Mixed effects regression
models, similar to those described above for the secondary analyses for VitD
trajectories will be used to assess differences in rates of decline. Models will further
include age, education, ethnicity, ApoE4 status, BMI, and season of blood draw as
covariates. Secondary analyses will compare those with adequate serum VitD levels by six
months (who sustain levels throughout the treatment period) to those with continued VitD
insufficiency. Further secondary analyses will utilize the continuous serum VitD levels
as time-varying variables in the models to assess how levels over time are associated
with level and change in cognitive function.
Power calculations. Because this Phase II trial will assess preliminary evidence of
efficacy, the type I error rate (alpha) will be set at 0.05 and power will be based on a
one-tailed test as is standard for Phase II trials to assess whether rates of decline
are slower in the high-dose group than in the standard-dose group. Using estimates of
between and within-person variability in change in executive function from preliminary
data, with 76 individuals (completers) per group, we will have 80% power to detect a
difference in the rate of executive function decline, based on the effects seen in our
observational study. We allow for up to a 15.5% drop out rate with dropout uncorrelated
to missing data.
Aim 3 (exploratory): To compare the effects of high-dose VitD versus standard-dose VitD
on key brain, blood and urine biomarkers relevant to healthy brain aging.
Hypothesis 3.1: High dose VitD supplementation is associated with less brain atrophy
(total gray + white matter volume) and less loss of hippocampal volume over 3 years
than in those on standard dose VitD.
Hypothesis 3.2: Blood inflammatory markers are lower in persons on high-dose VitD
supplementation compared to persons on standard dose VitD.
Hypothesis 3.3: Blood and urine markers of oxidative stress are lower in persons on
high-dose VitD supplementation compared to persons on standard dose VitD.
Rationale. MRI, blood and urine markers provide information on the potential biological
mechanisms of VitD supplementation, and in particular, correction of VitD insufficiency.
These exploratory hypotheses will provide further evidence of the benefit of VitD
supplementation in at risk elderly individuals.
Analytic Plan The analysis strategy will be similar for each of these hypotheses. MRI
and measures from blood (e.g. CRP, IL-6, IL-10, TNF-alpha), and urine (e.g. isoprostane)
will be acquired at baseline and at month 42 (or the ET). The primary outcome will be
the value from the final visit. Because this is a randomized trial, it is expected that
the baseline means will be similar in the two groups, so that a primary comparison of
the measure at the final visit provides an assessment of the impact of high- vs standard
dose VitD supplementation. Linear regression, using the baseline measure as a covariate
will be used. Other covariates, including age, and ApoE4 status will be included in the
models. To account for differences in the timing of the final visit (since some may be
terminal visits prior to the end of the trial), time since baseline will be included.
Model assumptions of normality of the residuals, constant variance, and linearity will
be checked and transformations or nonlinear models will be used if needed. Secondary
analyses will compare those individuals who have sustained corrected VitD levels versus
those who remain insufficient. Hypotheses will be supported if the average levels at the
terminal visit are improved in the high-dose group relative to the standard dose group.
Power calculations. With 152 individuals, we will have 80% power to detect a difference
between groups as small as 0.46 standard deviations (SD). Using data from the
Alzheimer's Disease Neuroimaging Initiative, brain atrophy and hippocampal atrophy over
3 years between Normal and MCI individuals ranged from 0.53-0.69 SD. Therefore, we
will have sufficient power to detect a slowing in atrophy rates comparable to an
improvement of the MCI subjects to the rate of cognitively normal individuals through
Additional exploratory analyses. Additional analyses will examine the effects of
high-dose VitD on measures of mood (GDS), using the GDS as the outcome in mixed effects
regression models. Because response to VitD supplementation may differ by VitD receptor
genotype, the association between genotype and cognitive decline and brain atrophy will
be explored by adding genotype to the models used for Aim 2 and Hypothesis 3.1. For
planning of the Phase III clinical trial, differences in effects by diagnosis will be
explored. We will biobank serum and plasma making these materials available for further
analyses which could include metabolomics and further genetic characterization.
5. Safety analyses. The Data Safety Monitoring Board for this study will review Adverse
Events and all safety data collected every six months, commencing with the first patient
enrolled. The DSMB will be particularly concerned with falls and fractures, renal
stones, hepatic/renal insufficiency, and achieving extremely high levels of VitD.
Frequencies of these events will be recorded and compared between the high- and standard
dose groups. Overall, we expect the tolerability of VitD supplementation to be excellent
in this population, but will test if safety and aspects of health are superior/inferior
in either of the treatment arms.