Clinical trial NCT02874924
Conditions: Aging
Keywords: Geriatrics
Sponsor: The University of Texas Health Science Center at San Antonio
Start date: 2016-06-01; Completion date: 2018-09-01
Enrollment: 34 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The ability to mount an effective immune response declines with age, leaving the elderly increasingly susceptible to infectious diseases and cancer. Rapamycin, an FDA approved drug to prevent transplant rejection, increases the lifespan and healthspan of mice and ameliorates age-related declines in immune responsiveness, cancer survival, and cognition in laboratory animals. Investigators are conducting a translational trial to test whether rapamycin also improves life functions in humans focusing on elderly persons (aged 70-95).

Detailed description:
Inhibition of the mTOR pathway by rapamycin (RAPA), an immunosuppressive drug used as adjunct therapy in preventing solid organ allograft rejection, enhances longevity in mice. Importantly, RAPA was efficacious even when initiated in relatively old animals. Thus, it has been suggested that RAPA could be used therapeutically in humans to slow age-associated pathologies. Indeed, improvements in cognition, control of tumorigenesis, and enhancing certain aspects of immunity have been demonstrated in RAPA treated murine models. Moreover, long-term RAPA delivery in older mice is associated with changes in immune reactivity not evident in younger animals. Investigators propose to expand to a larger cohort of older humans to test the hypothesis that RAPA treatment, even in very old individuals, will result in simultaneous improvement in systems known to be negatively affected by aging. Investigators will focus on the immune system, cognition, and physical parameters of healthy aging, such as walking speed. Investigators will recruit healthy volunteers, aged 75-95 years, and randomize them to either RAPA or placebo, controlling for gender, ethnicity, and age. These groups will be used to address the following specific aims: Aim 1. Assess general parameters of immune health before and after RAPA treatment; these include serum inflammatory cytokines, PBMC subsets (naïve vs memory T cells, TREGS, etc.), and polyclonal T cell activation potential. Aim 2. Test the effects of RAPA treatment on responsiveness to a vaccine challenge; both B cell (antibody) and T cell responses will be assessed. Aim 3. Correlate immune function rejuvenation with cognitive and physical function measures in subjects treated with RAPA or placebo. Aim 4. Collect pilot data on effect of RAPA on cardiovascular function. Cognition will be assessed by three different testing tools (EXIT25, SLUMS, and TAPS). Physical performance will be measured by grip strength and 40 foot timed walks, parameters known to correlate with healthy aging. Measures of cardiovascular function (Substudy D) using MRI of the heart to evaluate diastolic function and brain MRI to analyze cerebral blood flow, with measures of pulse wave velocity and endothelial function using laser doppler flowmetry will be performed. In addition to scoring positive outcomes, investigators will assess whether there are adverse changes in clinical laboratory tests that could compromise the safe use of RAPA therapeutically in older individuals. The long-term goal is to assess whether RAPA is safe to use in an elderly population, while also being efficacious in slowing, or even reversing, the aging process.

Arms: arm 1: Rapamycin 1mg taken once daily for 8 weeks arm 2: Placebo taken once daily for 8 weeks arm 3: No placebo control; Rapamycin 1mg once daily for 8 weeks
Interventions: intervention 1: Rapamycin intervention 2: Placebo intervention 3: Rapamycin
Intervention descriptions: intervention 1: treatment intervention 2: control intervention 3: No placebo control in this substudy group

Protocol documents:
Prot_SAP_000.pdf: 
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1 
Protocol Template Form 
Item 1 UTHSCSA 
Tracking Number 
HSC20120304H 
Item 2 Abstract / 
Project Summary 
Provide a succinct and accurate description of the proposed research. State the purpose/aims. Describe 
concisely the research design and methods for achieving the stated goals. This section should be 
understandable to all members of the IRB, scientific and non-scientific.   
DO NOT EXCEED THE SPACE PROVIDED. 
Purpose/Objectives: Several drugs, including rapamycin (RAPA), an inhibitor of the mTOR pathway, and acarbose (ACA), a 
metabolic modulator, have been shown to extend the lifespan in laboratory rodents and also delay or reverse certain age-associated 
functional deficiencies.  However, the effects in older animals often differ from those in a younger cohort.  In order to determine 
whether these drugs will enhance healthy aging in older human subjects, a series of pilot studies is proposed where the underlying 
mechanisms and outcomes will be assessed with the focus being on physiological systems known to decline with aging.   
Research Design/Plan:  
Sub-study A Rapamycin Alone, or 
Sub-study B Acarbose followed by Rapamycin, or 
Sub-study C Acarbose Alone, or 
Sub-study D Rapamycin Alone – Cardiovascular Effects 
The effects of RAPA and/or ACA on cognitive, immune, physical, and other physiological systems known to be impacted with age 
will be investigated in a pilot study in a small number of elderly subjects.  In the sub-studies using RAPA, a control group, matched 
for age and ethnicity, will be given a placebo (Sub-studies A&B; Sub-study D RAPA/no placebo).  The study is designed such that a 
given subject will be enrolled in one of four sub-studies shown above as Sub-study A-D. 
Methods: Subjects who meet the inclusion criteria and choose to participate in one of the sub-studies will be consented and will 
then undergo a set of standard tests to obtain baseline data on their current health status.   
For Sub-study A Rapamycin Alone 
Subjects will receive Rapamycin or placebo for approximately two months.  We will assess many parameters to assess 
changes due to the drug (RAPA), particularly in physiological systems known to change with aging, and will also measure 
indicators of health to ensure safe delivery of RAPA in the elderly cohort.  Specifically, the tests to be run include the 
following:  immune response to challenge with the trivalent seasonal influenza vaccine effects on the gut microbiome, 
perception of quality of life and depression, grip strength, timed walk, cognitive assessment, blood chemistry,  immune and 
endocrine health, and other assessments of safe RAPA delivery. Some subjects may elect to participate in an optional 
imaging procedure to evaluate cerebral blood flow and metabolic rate of glucose using PET and MRI. 
For Sub-study B Acarbose followed by Rapamycin 
Subjects will receive ACA orally for approximately 2 months.  The battery of tests outlined above (except immunization) 
will be performed at three time points (pre-ACA, after 2 months on ACA, and 1 month post-ACA termination), to 
measure the effects of ACA on the gut microbiome, basic blood metabolic markers, blood cell distributions, physical 
function parameters (including walking speed and grip strength), cognitive abilities, depression, cytokine levels, and 
blood cell function.  ACA will be terminated and all subjects will be rested for approximately 1 month and will then enter 
the RAPA phase of study; they will be randomly assigned to either RAPA or placebo and will be maintained on the 
appropriate drug for a total of approximately 2 months.  During that time, we will assess response to vaccine challenges 
and to other physiological measures, including those listed above.  Subjects may also elect to participate in optional 
imaging procedures to evaluate cerebral blood flow and metabolic rate of glucose using PET and MRI. 
For Sub-study C Acarbose alone  
Effect of mTOR Inhibition and Other Metabolism Modulating 
Interventions on the Elderly: Immune, Cognitive, and Functional 
Consequences


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Subjects will be followed as in substudy B, but the tests will also be performed 1 month after termination of ACA. 
 
For Sub-study D Rapamycin Alone – Cardiovascular Effects 
 
Subjects will receive Rapamycin for approximately two months.  We will assess many parameters to assess changes due 
to the drug (RAPA), particularly in physiological systems known to change with aging, and will also measure indicators of 
health to ensure safe delivery of RAPA in the elderly cohort.  Specifically, the tests to be run include the following: aortic 
pulse wave velocity to assess aortic compliance and laser-Doppler flowmetry to measure endothelial function; 
perception of quality of life and depression; cognitive assessment; grip strength, timed walk, blood chemistry and 
immune health, and other assessments of safe RAPA delivery.  Subjects will also participate in cardiac and brain imaging 
procedures to evaluate cardiac function and cerebral blood flow using MRI. Subjects’ physical activity levels will be 
monitored by a wearable activity monitor throughout the study. 
 
 
 
Clinical Relevance: Our long-term goal is to define the beneficial and detrimental effects of mTOR inhibition and/or metabolic 
modulation in the elderly. 
 
 
 
Item 3  Background 
 
Describe past experimental 
and/or clinical findings 
leading to the formulation 
of your study.   
For research involving 
unapproved drugs, 
describe animal and 
human studies.   
For research that involves 
approved drugs or devices, 
describe the FDA approved 
uses of this drug/device in 
relation to your protocol. 
Background on rapamycin (RAPA) 
mTOR inhibition by RAPA was first shown to alter immune function in rodent models of 
induced autoimmunity.  It was speculated that RAPA inhibited T cell proliferative responses.  However, 
recent studies suggest that the drug’s effects are more complex, affecting both innate and adaptive 
immunity. The effects of mTOR inhibition on immune regulation and the control of T cell 
differentiation, migration, and expansion have been well studied. One of the most striking findings is 
an expansion of viral specific memory CD8 T cells. In addition, mTOR inhibition by RAPA appears to 
skew the CD4+ T cell compartment, with an expansion of suppressive regulatory (TREG) cells and a 
decrease in the pro-inflammatory TH1 and TH17 subsets. This is consistent with the reported efficacy of 
mTOR inhibition in suppressing autoimmunity in rodents and in humans (type I diabetes, multiple 
sclerosis, etc.).  Although mTOR inhibition by RAPA has been extensively tested in mice as a single 
agent, there are relatively few such studies in humans. Instead, most RAPA studies in humans have 
been conducted in solid-organ transplant patients taking multiple immunosuppressives (cyclosporine, 
tacrolimus, prednisone, etc) that complicate identifying mTOR inhibition by RAPA specific effects. 
Despite this complication, some surprising effects of mTOR have been reported. For example, 
although mTOR inhibition by RAPA is considered immunosuppressive, increased susceptibility to 
infection has not been found and immunity to certain pathogens, like CMV, may even be augmented 
by mTOR inhibition. Improved responses to other viral challenges have also been reported in 
transplant recipients.  
Similarly, no increases in neoplastic disorders have been reported with mTOR inhibition by 
RAPA, suggesting that tumor immunity is not further reduced and may even be improved by RAPA. 
Indeed, mTOR inhibition by RAPA appears to suppress or even regress skin cancer in humans. These 
results are consistent with the well-documented enhancement of viral-specific memory CD8+ T cell 
immunity in animals. Positive effects of mTOR inhibition by RAPA on autophagy, dendritic cells, and 
antigen processing also appear to enhance responses to intracellular bacteria. Where tested, humoral 
immunity is maintained during mTOR inhibition by RAPA, i.e., the addition of RAPA standard 
immunosuppressive therapy did not reduce the efficacy of influenza vaccination in solid organ 
transplant patients. Moreover, addition of RAPA correlated with improved responses in a lung 
transplant cohort and with broader antibody reactivity in a study of liver and kidney recipients. 


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Significance It has been suggested that mTOR inhibition by RAPA may slow the progression of aging 
even if treatment were initiated relatively late in life.  However, concerns about RAPA-induced 
immune alterations remain as there are no human studies that directly assess cognitive, immune, and 
physiological effects in elderly individuals who are not being treated with other immunosuppressive or 
anti-proliferative drugs. If successful, this study will provide insights and critical pilot data to facilitate 
future grant applications to support a large-scale clinical study. 
PRELIMINARY DATA 
Thus far in our studies, no unanticipated clinically significant effects have occurred. Importantly, the 
oral glucose tolerance tests (OGTTs) revealed no RAPA-induced diabetogenic effects.  Similarly, no 
changes were noted in any other clinical laboratory, immune, cognitive, physical performance, or self-
perceived health status measures. Based on these substudy findings, RAPA can be used safely in older 
persons who are otherwise healthy. 
 
Effects of mTOR inhibition by RAPA on humoral immunity  An age-associated decrease in humoral 
(antibody) responses has been reported for many different antigens in both humans and mice.  As we 
published, this loss of immune competence is particularly acute when the antigen has never before 
been encountered by the responding individual.  To assess whether mTOR inhibition by RAPA could 
reverse the age-associated loss of immune competence, mice were fed diets containing RAPA or 
control without RAPA for 7 months; they were then immunized with bacteria.  The mice were bled 2 
weeks later and ELISAs performed to measure anti-bacterial antibody titers.  As shown in Figure 1, 
RAPA had no effect on antibody titers elicited in young mice.  However, in older animals, where the 
immune responses were expectedly lower, some RAPA treated mice showed higher responses. These 
data provide support for our hypothesis that mTOR inhibition will improve vaccine efficacy in the 
elderly. 
 
 
Effects of mTOR inhibition by RAPA on parameters of immune health in mice:  Since inhibitors of 
mTOR are pleiotropic, affecting many immune parameters, we opted to focus on biomarkers that are 
also implicated in aging.  In order to ask whether RAPA reverses or exacerbates phenotypes associated 
with immunosenescence, studies have already been undertaken in mice through the auspices of the 
NIA-funded GO grant (RC2 AG 036613-01; A. Richardson, PI) by Drs. Curiel and Kraig.  These data are 
summarized in Table 1.  Of particular interest are markers (like baseline inflammation) which increase 
in response to one condition (aging) while decreasing in response to the other (RAPA).  The ratio of 
naïve to memory T cells is also affected in opposite directions by aging and RAPA.  These murine data 
provide the rationale for parameters to be studied in Aim III.   
 
Immune parameter 
Effect of aging 
Effect of RAPA 
Expected outcome 
INNATE IMMUNITY 
ELISA antibody reactivity
OD at 1/125 serum dilution
0
0.2
0.4
0.6
YOUNG
OLD
*
NS
Control   RAPA       Control  RAPA
FIG 1. RAPA enhances the antibody response to 
bacterial challenge, but only in old mice.  Young (2 
month) and old (20 month) mice were placed on diets 
containing encapsulated rapa (RAPA,    ) or control 
(capsular material,   ) for 7 months and were then 
immunized with bacteria.  The mice were bled 2 weeks 
post challenge and ELISAs were performed to measure 
anti-bacterial antibody titers. [Data from Benavides & Kraig]
ELISA antibody reactivity
OD at 1/125 serum dilution
0
0.2
0.4
0.6
YOUNG
OLD
*
NS
Control   RAPA       Control  RAPA
FIG 1. RAPA enhances the antibody response to 
bacterial challenge, but only in old mice.  Young (2 
month) and old (20 month) mice were placed on diets 
containing encapsulated rapa (RAPA,    ) or control 
(capsular material,   ) for 7 months and were then 
immunized with bacteria.  The mice were bled 2 weeks 
post challenge and ELISAs were performed to measure 
anti-bacterial antibody titers. [Data from Benavides & Kraig]


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Baseline inflammation 
 
 
Could reverse age-
associated inflammation 
ADAPTIVE IMMUNITY 
Naive humoral 
response 
 
Varies with age & 
antigen  
Has not been tested in 
elderly humans 
Ratio of naïve/memory 
T cells 
 
 
Could enhance responses 
in elderly 
T cell activation by 
antigen 
 
No change seen  
 
TREG cells 
Mixed results 
No major change 
No rapa-induced immune 
suppression 
TH1 and TH17 
TH1  and TH17  
increased changes in 
the same direction 
for both 
 Th1 will reduce 
inflammation  
 Th17 good for tumor 
immune surveillance 
PD-1 (negative 
signaling) 
mice; humans 
unclear 
 
Would improve beneficial T 
cell functions 
TABLE 1.  Summary of immune parameters impacted by aging and/or RAPA; as studied in the Curiel and Kraig 
labs 
 
Discoveries typically begin at the bench with basic research and are then translated to the 
clinical level.  A major unanswered question is whether mTOR inhibition by RAPA would be harmful or 
beneficial in humans. Studies of a variety of physiological functions in rodents have found no evidence 
for negative effects of mTOR inhibition by RAPA or related rapalogs agents, and in a few cases, e.g., 
grip strength and cognition, mTOR inhibition by RAPA may have actually improved function.  
 
 
The age-related decline in physical function by loss of muscle strength negatively impacts 
health outcomes in older people by increasing the risk of falls, hospitalization, disability, morbidity, 
and mortality all of which carry significant health-care cost. An association between general health 
status and muscle function in relation to muscle mass and has been noted in healthy older adults. 
Interventions that improve muscle function provide benefits to both physical and emotional well-
being of older individuals. Can mTOR inhibition by RAPA do the same? Furthermore, mTOR inhibition 
has shown promise against memory impairment and β-amyloid deposition in transgenic mouse 
models of Alzheimer’s disease.  Can mTOR inhibition by RAPA or rapalogs do the same in humans?  
It was recently reported that 16 weeks of RAPA treatment in symptomatic AD mice increased 
cerebral blood flow (CBF) through nitric oxide (NO) dependent mechanisms. RAPA treatment also 
restored brain vascular density, reduced cerebral amyloid angiopathy and microhemorrhages, 
decreased amyloid burden, and improved cognitive function. Based on these findings, RAPA appears 
to be a promising agent to ameliorate cognitive dysfunction in elderly humans.  
As noted above, pre-clinical studies in animals show that RAPA has beneficial effects on 
lifespan and health span. More importantly, RAPA increases CBF in laboratory animals and improves 
cognitive dysfunction in AD animal models. Despite these findings, the potential for adverse reactions 
to RAPA has limited investigations in healthy human subjects such that our current knowledge 
remains incomplete, especially for clinical application in older humans. Most knowledge of mTOR 
antagonist use in humans is derived from studies of patients with serious chronic medical problems 
such as solid organ transplant patients or cancer patients who also receive multiple agents that 
confounding results. We have found oral RAPA to be safe in humans who are in the ‘oldest old’ (over 
85) age group; a group with the greatest burden of age-related disease. If RAPA ameliorates age-
related disease, this age group is likely to benefit more than younger age groups. We will administer 
RAPA to healthy elderly (70-95 year old) persons who in good health, with all medical conditions well 
controlled and clinically stable.  
While it will not be possible to directly evaluate the long-term anti-aging (lifespan, 
healthspan, physical performance) actions of RAPA because subjects will be treated for 4 months 
(Substudy D is 2-3 months), we have designed this study as an initial step to generate information 
concerning potential beneficial and adverse effects of RAPA. We have preliminary data that suggests 


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physical performance (walking speed) and cognition are improved in the ‘oldest old’ with RAPA. We 
propose to expand our studies of oral RAPA treatment to a larger group of healthy older adults and to 
explore the vascular effects of RAPA on brain and peripheral vascular beds. The findings from this 
study will provide important preliminary data for a dedicated clinical trial of RAPA in older subjects. 
 
Background on acarbose (ACA) 
The NIH-funded Interventions Testing Program (ITP) was developed to assess the potential of 
drugs, delivered as dietary supplements, to extend lifespan in mice. In 2009, the ITP reported that 
rapamycin (RAPA) was efficacious in enhancing longevity, even if treatment was initiated late in life. 
Since RAPA is an immune modulator whose effects can vary with age, we undertook a preliminary 
clinical trial in healthy elderly human subjects. Our early pilot data suggested improvements in 
cognition, physical function, and immunity [Kellogg, Kraig, Chiodo, Curiel]. However, given scientific 
concerns about the potential immunosuppressive side effects of RAPA, we were intrigued by a recent 
publication in which the ITP showed that another drug, acarbose (ACA), was even more efficacious at 
extending lifespan, at least in male mice. ACA is an FDA–approved a-glucosidase inhibitor with a long 
history of clinical use in management of diabetes with an excellent safety profile that is far superior to 
RAPA.  
To investigate ACA as a potential anti-aging therapy for use in humans, we propose a pilot 
study of ACA in elderly subjects. Our goals are to: i) obtain preliminary data to better estimate power 
for a larger trial, ii) establish a safe dosing strategy for ACA in non-diabetic elderly humans, iii) 
determine whether positive ACA outcomes seen in diabetics (i.e., improved endothelial function) are 
also seen in older non-diabetic subjects, iv) assess whether ACA can reverse age-associated deficits in 
cognition, physical function, and immunity, and v) determine whether ACA alters the gut microbiome 
as such changes could impact longevity and immune competence. For ACA, such a translation into 
geriatric medicine would be relatively straight-forward as it is FDA approved with a low incidence of 
side effects.   
 
Our current understanding of ACA’s effects primarily derives from patient-based trials in 
diabetics that report reductions in weight, improved cardiovascular function, and other health 
benefits. Only a few trials of ACA have been done in non-diabetics and these report beneficial weight 
reductions. In elderly diabetics (average age ≈70) ACA was found to be safe and moderately effective 
in reducing hyper-glycemia.  These elderly patients experienced ACA side effects similar to younger 
groups: primarily flatulence.  We propose to assess ACA safety and potential anti-aging benefits in 
elderly non-diabetics. 
Our early preliminary data demonstrate that RAPA at 1 mg/day is well tolerated by elderly 
humans and hypothesized adverse events (diabetes, immunosuppression, etc.) have not been 
observed. While these results indicate that RAPA use in healthy elderly persons is not problematic, we 
have found suggestions of improved cognitive, immune, and physical function. This leads us to seek 
objectively measurable outcomes likely to show RAPA benefit. We have concluded that the 
cardiovascular parameters of aortic compliance and endothelial function answer our quest. 
SubStudy D - Background and Significance – Rapamycin Alone – Cardiovascular Effects 
RAPA as a modulator of age-related cardiovascular dysfunction. Aging is associated with reduced 
compliance of large elastic arteries and endothelial dysfunction due to reduced nitric oxide (NO) 
bioavailability. Recent work by Lesniewski, et al (10) showed that 6-8 weeks of RAPA treatment 
improved compliance in elastic vessels and NO-dependent endothelial function in old (30mo) mice. 
RAPA treatment improved aortic compliance in old mice through decreasing collagen and advanced 
glycosylation end-products (AGEs) in the aorta without changes in intima-media thickness or aortic 
elastin content while improved endothelial function was effected by improved NO bioavailability 
through decreased arterial oxidative stress. Their results are consistent with our unpublished findings 
that topical RAPA decreases collagen levels in keloids and improves NO-dependent endothelial 
function in older humans. The foregoing results suggest that increased mTOR activation may underlie 
reductions in large elastic artery compliance, structural adaptations in the vessel wall, and endothelial 
dysfunction due to vascular oxidative stress with advancing age. The finding that RAPA treatment 
reversed these deleterious cardiovascular changes suggests that such treatment holds promise for the 


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treatment of large arterial as well as arteriolar aging and thus the potential prevention of age-
associated CVD in humans. We will explore the role of mTOR in these age-associated vascular 
dysfunctions, by examining the impact of mTOR antagonism with RAPA on aortic PWV and NO-
dependent endothelial function in cutaneous arterioles in elderly humans. 
With aging, systemic inflammation increases and likely contributes to the development of 
age-associated pathologies including cardiovascular diseases (CVDs) (3, 7, 14). CVDs are primarily 
diseases of the arterial system and are associated with reduced large artery compliance and reduced 
endothelial function. Recent work in old (30mo) mice showed that both large vessel compliance and 
nitric oxide (NO) dependent endothelial function improved with 6-8 weeks of RAPA treatment (10). 
Subjects will undergo imaging procedures to evaluate cardiac function and cerebral blood flow using 
MRI. 
To test whether the same RAPA effects occur in elderly humans (age 70-95), we propose to 
add noninvasive measurements of the following cardiovascular and physical activity variables::  
• 
Aim D-1 aortic pulse wave velocity (an index of large vessel compliance) 
• 
Aim D-2) heat induced arteriolar vasodilation (an index of NO-dependent endothelial 
function) 
• 
Aim D-3 cardiac and brain MRI to measure diastolic function and cerebral blood flow  
• 
Aim D-4 physical activity monitoring (an index of changes in activity, sleep, and calorie 
expenditure) Importantly, this would be beneficial through validation of the CV measures in 
human subjects. 
 
 
 
 
 
 


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Item 4 
Purpose and 
rationale 
Insert purpose, 
objectives and 
research 
questions/hypotheses 
here. 
 If you cut and paste 
from another 
document, make sure 
the excerpted 
material answers the 
question 
Purpose and Aims for Sub-study A & B – Rapamycin  
Rapamycin (RAPA), an inhibitor of the mTOR pathway, is used clinically as an immunosuppressive drug in 
preventing transplant rejection and with drug eluting stents to limit coronary restenosis.  When tested in 
mice, RAPA was shown to extend lifespan, even if treatment was initiated relatively late in life. The drug 
was shown to delay, but not alter, the range of illnesses at death and also attenuated the age-related 
decline of spontaneous activity suggesting that it increased healthspan. Rapamycin (RAPA) extends lifespan 
in laboratory animals and has been shown to delay the onset of several age-related diseases; however, 
clinical administration of RAPA has been reported to effect hyperglycemia and potentially new onset 
diabetes in human solid organ transplant patients.  Given that the human population has a high prevalence 
of obesity and age-related insulin resistance, extrapolation of this observation to the human population in 
general has raised concerns as to whether RAPA administration in healthy humans could lead to diabetes.  
Human clinical studies that report RAPA-induced hyperglycemia have focused on transplant patients with 
pre-existing conditions with multiple medical problems who receive complex pharmacological 
immunosuppressive treatments. No long-term studies of the effects of RAPA in non-transplant patients 
who are otherwise healthy have been done, although in our experience RAPA treatment of up to 4 months 
does not lead to hyperglycemia in healthy older persons age 80-95. Given that it is unclear whether the 
previously mentioned transplantation-associated hyperglycemic risks of RAPA are directly to RAPA per se 
or confounding factors in transplant patients, we propose to add oral glucose tolerance tests (OGTT) to our 
ongoing RAPA trial in healthy older persons to clarify this issue. Our long-term goal is to define the 
beneficial and detrimental effects of mTOR inhibition in the elderly. Using subjects (70-95 years old) 
available through the Geriatric Evaluation and Management Clinic (GEM) at the STVHCS (South Texas 
Veterans Health Care System), we are uniquely positioned to assess the effects of mTOR inhibition in vivo 
on health status, vaccine efficacy, and on parameters of immune health. We hypothesize that RAPA will 
improve immunity by reducing the immune dysfunction associated with increasing age. Expected outcomes 
are decreased inflammation and enhanced vaccine-mediated immunity.   
Aim I. 
Assess mTOR inhibition safety in elderly human subjects, 70-95 years of age.   
Prior human studies of mTOR inhibition by RAPA have focused on subjects with underlying health issues 
(transplant recipients, patients with cancer or autoimmunity, etc.) so RAPA was often used in combination 
with other immunosuppressive or anti-proliferative drugs.  Thus, we propose to assess whether RAPA can 
be safely delivered to an otherwise healthy, but elderly test population.  Towards this end, subjects will be 
placed on mTOR inhibition by RAPA (Rapamune, Wyeth/Pfizer) and an equal number on placebo.  Their 
health status will be assessed by: i) physical examination, ii) clinical blood work  iii) weekly self-reporting of 
complications to the clinical nurse, and iv) use of both a depression scale and the SF36 questionnaire.  
Participants will remain on drug therapy (either Rapa or placebo) for the entire ~2 months of the RAPA 
study. 
Aim II.  Assess the effects of mTOR inhibition on vaccine efficacy in the elderly.   
Humoral immune responses decline with advancing age in humans as evidenced by decreased vaccine 
efficacy and increased susceptibility to infection. Thus, to determine whether mTOR inhibition by RAPA 
alters the age-associated loss in immune competence, study participants will be immunized with the 
seasonal flu vaccine (subjects will not have had prior exposure to the composition used).  Both humoral 
(antibody titer) and cellular (T cell proliferation) responses will be assessed. In addition, the subjects will 
receive a second immunization to test effects on memory B (antibody) and T cell responses.   
Aim III.  Determine whether mTOR inhibition can improve general immune features in the aged 
mTOR inhibition’s effects on immune parameters characteristically altered by aging will be assessed.  These 
include: i) serum cytokine levels (IL-6, TGF-β, etc.) as indicators of increased inflammation, ii) the naive to 
memory T cell ratio that decreases with age due to thymus involution and memory expansion; and iii) T cell 
functional subsets as assessed by fluorescence-activated cell sorting (FACS) for cell surface markers and 
cytokine measures. 
Aim IV.  Determine whether mTOR inhibition can improve muscle strength and cognitive function  
Measurements of handgrip strength, timed walking, and cognition will be assessed. The objective of this 
sub-study is to assess the short-term effect of rapamycin in healthy age 70-95 subjects on parameters that 
assess physical function and allow us to assess whether this agent has anti-aging, health-span promoting 
effects in humans.  Our limited goal would be to test the hypothesis that mTOR inhibition improves muscle 
strength and cognition, consistent with improving health span.  It was reported that 16 weeks of RAPA 


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treatment in symptomatic AD mice increased cerebral blood flow (CBF); RAPA also restored brain vascular 
density, reduced cerebral amyloid angiopathy and microhemorrhages, decreased amyloid burden, and 
improved cognitive function. Based on these findings, RAPA appears to be a promising agent to ameliorate 
cognitive dysfunction in elderly humans.  
Aim V. Assess the effects of RAPA on the gut microbiome.  The microbiota is known to change with aging 
and this likely contributes to increased inflammation in older individuals. RAPA also likely alters the 
microbiome, potentially reversing the aberrant effects of age; this will be examined before and after RAPA 
administration. 
 
Purpose and Aims for Sub-study B & C – Acarbose  
Treatment with acarbose (ACA), an α-glucosidase inhibitor, was shown to extend median lifespan by 22% in 
male mice. Since ACA is FDA-approved for use in humans and has been extensively employed for the 
management of diabetic symptoms, largely without adverse outcomes, it has been posited that acarbose 
could be efficacious as a safe anti-aging therapeutic. However, the effects of ACA treatment could be 
differentially affected by age, so it is imperative to test ACA directly in older individuals (aged 70-95 years 
old). Thus, we propose to perform a small pilot study assessing the effects of ACA on systems known to be 
impacted by aging (endothelial function, cognition, muscular, and immune). In addition, since oral ACA is 
likely to impact the gut microbiome with downstream effects on inflammation and other aging phenotypes, 
the microbiota will be assessed in these elderly subjects before, during, and after treatment. This pilot study 
will directly test our hypothesis that ACA treatment could be used in elderly humans to slow the progression 
of increased inflammation and age-associated pathologies due, at least in part, to changes in the gut 
microbiome.  
 
Aim VI.  Develop a safe protocol for testing ACA in elderly non-diabetic human subjects.  Subjects will be 
recruited from the VA Geriatrics Clinics and placed on a regimen of ACA.  Safety of treatment will be 
assessed by i) physical examination, ii) clinical blood work, iii) self-reporting, and iv) SF36 questionnaires. 
 
Aim VII. Ask whether ACA can ameliorate age-associated functional deficits. Participants will be tested 
prior to ACA initiation, during treatment, and post drug withdrawal for the following parameters: physical 
function (grip strength and walking speed), cognition (EXIT, SLUMS, TAPS), and immunity (cytokine 
profiles, TREGS numbers, the CD4/CD8 T cell ratio, and naïve/memory T cell ratios).  
 
Aim VIII. Assess the effects of ACA on the gut microbiome.  The microbiota is known to change with aging 
and this likely contributes to increased inflammation in older individuals. ACA also likely alters the 
microbiome, potentially reversing the aberrant effects of age; this will be examined longitudinally. 
 
Purpose and Aims for Sub-study D – Cardiovascular Function with Rapamycin 
With aging, systemic inflammation increases and likely contributes to the development of age-associated 
pathologies including cardiovascular diseases (CVDs) {Franceschi, 2014 #2611;Howcroft, 2013 
#2610;Michaud, 2013 #2612}. CVDs are primarily diseases of the arterial system and are associated with 
reduced large artery compliance and reduced endothelial function. Recent work in old (30mo) mice showed 
that both large vessel compliance and nitric oxide (NO) dependent endothelial function improved with 6-8 
weeks of RAPA treatment {Lesniewski, 2017 #2605}. RAPA has also been shown to attenuate the age-related 
decline of spontaneous activity suggesting that it increased healthspan {Miller, 2011 #1350}. Similar effects 
on activity were reported in dogs treated with RAPA {Urfer, 2017 #2617}.  
Subjects will be recruited from the VA Geriatrics Clinics, posted study flyers in community-based private 
practice offices or senior centers, UT Health Find A Study website, and or newspaper advertisements. 
Interested candidates will be directed to contact the Barshop Clinical Research Call Center at (210) 450-0020. 
Potential candidates may be contacted by phone from the study team for phone screening after verbal 
consent is provided. Once the study team establishes contact with a potential candidate who then  
understands the basics of the study and if desired, interested subjects may receive a copy of the informed 
consent form by US Postal Service for review prior to the screening appointment. (Email to VA subjects is 
prohibited.) 
Aim D-1. Test whether RAPA increases aortic compliance in elderly subjects. Aortic pulse wave velocity (PWV) 
will be measured before and during 8 weeks of RAPA administration. Applanation tonometry will be used to 


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record peripheral waveforms from the carotid and femoral arteries simultaneously with electrocardiogram 
(ECG) recording to measure PWV  Typically carotid and femoral recordings are performed on the right side 
of the body, but if necessary may be done on the left.  
Aim D-2 Test whether RAPA improves endothelial function in elderly subjects. NO-dependent endothelial 
function will be measured by monitoring the cutaneous arteriolar vasodilation with laser-Doppler flowmetry 
(LDF) in response to local warming of the skin from 34°C to 39°C and 42°C. Measurements will be made 
before, during, and at the end of 8 weeks of RAPA (Table 2). 
Aim D-3. Examine whether mTOR inhibition improves diastolic heart function and increases cerebral blood 
flow in humans.  
Does mTOR inhibition improve diastolic function and/or increase cerebral blood flow in 70-95 year olds? 
This portion of the project will explore whether mTOR inhibition with RAPA increases cerebral blood flow 
(CBF) in mouse models of Alzheimer’s Disease (AD) and improves cognition (1). Also, rapamycin has been 
shown to improve diastolic heart function in aging animal models {Chiao, 2016 #2623;Urfer, 2017 #2617}. 
Our preliminary data in ‘oldest old’ humans (age 89-93) and a published pilot study (1) suggest similar 
cognitive effects in humans. We will examine these RAPA effects in older humans (age 70-95). Based on the 
foregoing observations, we hypothesize that RAPA effects on heart function, the CBF as well as cognitive 
and physical function are beneficial. We will test our hypothesis in 70-95 year old humans treated with 
RAPA for 8 weeks by addressing whether systemic RAPA increases CBF in elderly humans. MRI will be used 
to measure EF, SV, LV volumes, peak LV filling rate, and the E/A wave ratios of flow through the mitral 
valve. Cardiac data will be normalized to BSA.  CBF also will be measured by MRI. Measurements will be 
made before RAPA is administered, as well as after completion of RAPA administration. Each subject will be 
his own control in this design. 
 
Aim D-4. Examine whether mTOR inhibition increases levels of activity in humans. 
In previous mouse studies, rodents (males) treated with RAPA demonstrated increased physical activity. 
We hypothesize that RAPA will increase activity levels in humans. Activity data will be collected using a 
Garmin Vivofit3. We will examine effects on overall activity (time, distance, step count, calories burned, 
total hours of sleep, periods of movement, and periods of restful sleep) {O’Brien, 2015 #2716;Schrack, 2016 
#2715;Šimůnek, 2016 #2714} 
 
 
Literature Cited 
 
1. Boutouyrie P, Briet M, Collin C, Vermeersch S, and Pannier B. Assessment of pulse wave velocity. Artery 
Research 3: 3-8, 2009. 
2. Chiao, Y.A., Kolwicz, S.C., Basisty, N., Gagnidze, A., Zhang, J., Gu, H., Djukovic, D., Beyer, R.P., Raftery, 
D., MacCoss, M., Tian, R., Rabinovitch, P.S., 2016. Rapamycin transiently induces mitochondrial 
remodeling to reprogram energy metabolism in old hearts. Aging 8, 314-326. 
3. Choi PJ, Brunt VE, Fujii N, and Minson CT. New approach to measure cutaneous microvascular function: 
an improved test of NO-mediated vasodilation by thermal hyperemia. J Appl Physiol 117: 277-283, 
2014. 
4. Franceschi C, and Campisi J. Potential Contribution to Age-Associated Diseases. J Gernontol A Biol Sci 
Med Sci Sci  69 (Suppl 1): S4-S9, 2014. 
5. Harrison DE, Strong R, Sharp ZD, Nelson JD, Astle CM, Flurkey K, Nadon NL, Wilkinson JE, Frenkel K, 
Carter CS, Pahor M, Javors MJ, Fernandez E, and Miller RA. Rapamycin fed late in life extends lifespan in 
genetically heterogeneous mice. Nature 460: 392-395, 2009. 
6. Holowatz LA, Thompson-Torgerson C, and Kenney WL. Aging and the control of human skin blood flow. 
Frontiers in Bioscience 15: 718-739, 2010. 
7. Holowatz LA, Thompson-Torgerson CS, and Kenney WL. Altered mechanisms of vasodilation in aged 
human skin. Ex Sports Sci Rev 35: 119-125, 2007. 
8. Howcroft TK, Campisi J, Louis GB, Smith MT, Wise B, Wyss-Coray T, Augustine AD, McElhaney JE, 
Kohanski R, and Sierra F. The role of inflammation in age-related disease. Aging 5: 84-93, 2013. 
9. Keating R, Hertz T, Wehenkel M, Harris TL, Edwards BA, McClaren JL, Brown SA, Surman S, Wilson ZS, 
Bradley P, Hurwitz J, Chi H, Doherty PC, Thomas PG, and McGargill MA. mTOR modulates the antibody 
response to provide cross protective immunity to lethal influenza infections. Nature Immunology 14: 
1266–1276, 2013. 


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10. Kellogg DL, Jr., Johnson JM, Kenney WL, Pergola PE, and Kosiba WA. Mechanisms of control of skin 
blood flow during prolonged exercise in humans. Am J Physiol Heart Circ Physiol 265: H562-H568, 1993. 
11. Lesniewski LA, Seals DR, Walker AE, Henson GD, Blimline MW, Trott DW, Bosshardt GC, LaRocca TJ, 
Lawson BR, Zigler MC, and Donato AJ. Dietary rapamycin supplementation reverses age-related 
vascular dysfunction and oxidative stress, while modulating nutrient-sensing, cell cycle, and senescence 
pathways. Aging Cell 16: 17-26, 2017. 
12. Lin A-L, Zheng W, Halloran JJ, Burbank RR, Hussong SA, Hart MJ, Javors M, Shih Y-YI, Muir E, Fonseca RS, 
Strong R, Richardson AG, Lechleiter JD, Fox PT, and Galvan V. Chronic rapamycin restores brain vascular 
integrity and function through NO synthase activation and improves memory in symptomatic mice 
modeling Alzheimer’s disease. J Cerebral Blood Flow & Metab 33: 1412-1421, 2013. 
13. Livi CB, Hardman RL, Christy BA, Dodds SG, Jones D, Williams C, Strong R, Bokov A, Javors MA, Ikeno Y, 
Hubbard G, Hasty P, and Sharp ZD. Rapamycin extends life span of Rb1+/- mice by inhibiting 
neuroendocrine tumors. Aging 5: 100-110, 2013. 
14. Majumder S, Caccamo A, Medina DX, Benavides AD, Javors MA, Kraig E, Strong R, Richardson A, and 
Oddo S. Lifelong rapamycin administration ameliorates age-dependent cognitive deficits by reducing IL-
1beta and enhancing NMDA signaling. Aging cell 11: 326-335, 2012. 
15. Michaud M, Balardy L, Moulis G, Gaudin C, Peyrot C, Vellas B, Cesari M, and Nourhashemi F. 
Proinflammatory cytokines, aging, and age-related diseases. J Am Med Dir Assoc 14: 877-882, 2013. 
16. Miller, R.A., Harrison, D.E., Astle, C.M., Baur, J.A., Boyd, A.R., de Cabo, R., Fernandez, E., Flurkey, K., 
Javors, M.J., Nelson, J.F., Orihuela, C.J., Pletcher, S., Sharp, Z.D., Sinclair, D., Starnes, J.W., Wilkinson, 
J.E., Nadon, N.L., Strong, R., 2011. Rapamycin but not resveratrol or simvastatin, extends life span of 
genetically heterogenous mice. J. Gerontol. A Biol. Sci. Med. Sci. 66, 191-201. 
17. Minson CT, Holowatz LA, Wong BJ, Kenney WL, and Wilkins BW. Decreased nitric oxide- and axon 
reflex-mediated cutaneous vasodilation with age during local heating. J Appl Physiol 93: 1644-1649, 
2002. 
18. O’Brien, T., Troutman-Jordan, M., Hathaway, D., Armstrong, S., Moore, M., 2015. Acceptability of 
wristband activity trackers among community dwelling older adults. Geriatric Nursing 36, S21-S25. 
19. Schrack, J.A., Cooper, R., Koster, A., Shiroma, E.J., Murabito, J.M., Rejeski, W.J., Ferrucci, L., Harris4, 
T.B., 2016. Assessing Daily Physical Activity in Older Adults: Unraveling the Complexity of Monitors, 
Measures, and Methods. J Gerontol A Biol Sci Med Sci 71, 1039–1048. 
20. Šimůnek, A., Dygrýn, J., Gába, A., Jakubec, L., Stelzer, J., Chmelík, F., 2016. Validity of Garmin Vívofit and 
Polar Loop for measuring daily step counts in free-living conditions in adults. Acta Gymnica. 46, 129–
135. 
21. Urfer, S.R., Kaeberlein, T.L., Mailheau, S., Bergman, P.J., Creevy, K.E., Promislow, D.E.L., Kaeberlein, M., 
2017. A randomized controlled trial to establish effects of short-term rapamycin treatment in 24 
middle-aged companion dogs. GeroScience 39, 117–127. 
 
 
 
 
      
 
 
Item 5 
Study Population(s) Being 
Recruited 
In your recruitment plan, how 
many different populations of 
prospective subjects do you plan to 
target? Provide number: 1 
 
 
Identify the criteria for inclusion: 
 
 
Identify the criteria for exclusion: 


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11 
e.g., a population can be individuals 
with type 2 diabetes controlled with 
diet and/or a population of healthy 
controls.  Or a population can be 
individuals attending an education 
program, etc. 
 
List each different population on a 
separate row and provide a short 
descriptive label:   
(e.g., normal-healthy, diabetics, 
parents, children, etc.) 
 
To add rows use copy & paste 
normal, healthy 70-95 years old; 
Veterans and non-veterans 
(All sub-studies) 
 
In Sub-study D we will enroll males 
only, 70-95 years old, veterans and 
non-veterans. 
 
• 
Since this is a small pilot study, we have 
opted to limit subject heterogeneity by 
focusing on 70-95 years of age.   
• 
Subjects will be in good health with all 
chronic diseases (hypertension, 
coronary artery disease, etc.) clinically 
stable. Selected subjects will be in good 
health (Per the World Health 
Organization good health will be 
defined as complete physical, mental, 
and social well-being and not merely 
the absence of disease or infirmity.   
• 
For our purposes all diseases or infirmi-
ties will be clinically stable whether 
managed by medications or not.   
• 
All ethnicities will be included. 
• 
For cardiac and brain imaging by MRI, a 
pre-MRI screening questionnaire will be 
used to assess MRI safety and 
neurological health. 
 
Exclusion criteria include: 
• 
Received current season influenza 
vaccine (exclusion for substudy A, B, C*;  
*NOT used in SubStudy D) 
• 
Diabetes, (*Substudy D - with A1c ≥6.5 
or if  treated with medication affecting 
glucose homeostasis History of skin 
ulcers or poor wound healing,  
• 
Smoking,  
• 
Liver disease,  
• 
Coumadin anti-coagulation,  
• 
treatment with drugs known to affect 
cytochrome P450 3A (diltiazem, 
erythromycin, etc) due to its role in 
RAPA metabolism 
• 
Treatment (>30days of therapy or long 
term) with a systemic 
immunosuppressant (prednisone, etc.) 
within the last year.   
• 
Patients with history of recent (within 6 
months) Myocardial Infarction or active 
Coronary Disease. 
• 
Patients with history of recent (within 6 
months) intestinal disorders 
• 
Exclusion criteria for MRI scan: known 
claustrophobia, metal implants in soft 
tissue of the body including 
pacemakers, aneurysm clips, ferrous 
metal fragments not anchored to bone 
(bullets, BBs, shrapnel, metal shavings), 
implanted medication pumps, and oral-
facial metal appliances that are 
permanently secured but may result in 
low image quality. Participants may also 
be excluded for history of severe head 
trauma, brain injury, brain surgery, 
inflammation of the brain, or history of 
seizures. 
 
 
 
 


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Item 6 
Research Plan / Description of the Research Methods a.  Provide a comprehensive narrative describing the research methods.   
Provide the plan for data analysis (include as applicable the sample size calculation). 
 
 
Step-by-Step Methods:  
Methods for Sub-study A & B Rapamycin  
mTOR inhibition: Subjects will be randomized to mTOR inhibition by RAPA or placebo in equal sized groups. Those 
receiving the drug will be started on 1 mg per day with no loading dose.  Dosage will be adjusted for a target whole 
blood concentration of 10-15 ng/ml and maintained there until the study’s conclusion. RAPA blood levels will be 
determined by mass spec. The initial trough level will be drawn one week after initiation of therapy. When a 
maintenance dose is adjusted, the new dose will be maintained for 7 days before additional adjustments are made 
based on concentration monitoring. Reported adverse reactions to RAPA and related agents when used as solitary 
agents include mucositis, pneumonitis, skin rash, hyperlipidemia, hyperglycemia, thrombocytopenia, and anemia.  
Clinical assessment: Subject health will be confirmed initially by history and physical exam, normal laboratory tests 
(fasting lipid profile, metabolic profile, CBC, and CK), and 12-lead EKG. Oral glucose tolerance test (OGTT): Baseline 
(fasting) samples for determination of glucose, insulin, and free fatty acid (FFA) concentrations will be drawn at -30, -15 
and -0 min (these basal values are averaged).  At time zero, each subject will ingest 75 g of glucose.  Glucose, insulin 
and FFA are determined every 30 min for 2 h following glucose ingestion. The Matsuda index of insulin sensitivity will 
be calculated from OGTT results, as described (Matsuda and DeFronzo, 1999). At each visit, patients will receive a 
standard history, physical examination, blood chemistry assessment, and be queried in detail for RAPA adverse 
reactions including infections. A questionnaire (SF-36) designed to assess the benefits of health care interventions and 
the Geriatric Depression Scale questionnaire will allow us to characterize any adverse or beneficial effects. Between 
visits, subjects will contacted weekly by the Clinical Nurse to ensure that no complications have arisen. Blood test for CK 
will be monitored. 
If subjects have previously undergone all or part of screening labs/tests within the past 4 months, the data may be 
accepted for eligibility determination without screening labs or procedures having to be repeated. 
Aim II.  Assess the effects of mTOR inhibition on vaccine efficacy in the elderly.   
Rationale:  One serious consequence of immune senescence is the age-associated loss of responsiveness to immune 
challenges. Where examined in humans, mTOR inhibition by RAPA did not appear to decrease vaccine efficacy, but this 
is difficult to conclude given that these subjects were on combination therapies for serious underlying health issues 
(recent transplant or cancer).  Thus, using the cohort described in Aim I, we have a unique opportunity to ask whether 
mTOR inhibition by RAPA will enhance immunity in otherwise clinically stable elderly individuals. 
 
Immunization:  We chose a vaccine that is commonly given to the elderly and was previously reported to show small 
improvements in a study of mTOR inhibition in adults performed by Novartis (Mannick, et al.).  The study will be timed 
such that none of our subjects will have already been immunized with that season’s trivalent influenza vaccine although 
it is likely that they will have been previously immunized with other flu variants and/or previously had flu infections.  
We will compare pre-immune and post-immune data to control for the heterogeneity in the population.  Typically, the 
seasonal flu vaccine contains three different influenza viruses determined each year by the CDC based on variants 
predicted to dominate in the US during.  A flu shot is considered standard of care for individuals in this age group and 
the composition used will be the one recommended by the VA; it is approved for humans. Standard adult dosing of 
Trivalent influenza vaccine will be initiated 1½ months after RAPA treatment begins and it will be given only once, as 
recommended.  
Outcome measures and expected results:  Subjects will return to the clinic for blood collection as indicated on Figure 2; 
both humoral (antibody titer) and cellular (T cell proliferation) responses to influenza antigens will be assessed.  Effects 
of mTOR inhibition by RAPA on B cell responses:  Following each immunization (at day 7 and 14), 5 ml of blood will be 
collected and serum prepared. The titer of anti-flu virus antibodies in these specimens will be determined by ELISAs 
using standardized kits (Alpha Diagnostics Inc).  If our hypothesis were correct, the primary responses would be low in 
these elderly subjects with higher levels in individuals on RAPA.  
Effects of mTOR inhibition by RAPA on influenza virus-specific T cells:  Age associated deficiencies are also seen for 
cellular (T-cell mediated) responses. This is likely due to a loss of naïve T cell production (a consequence of thymus 
involution), coupled with an increase in memory T cells due to antigenic exposure. Thus, it is also critical to ask whether 
RAPA will compensate for losses in T cell immunity with age.  Towards this end, 25 ml blood will be collected 7 days 


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after each immunization. Peripheral blood mononuclear cells (PBMCs) will be harvested and stimulated in vitro in 
triplicate with serial dilutions of recombinant flu viral antigens or with control stimuli [PHA or anti-CD3/CD28 (positive 
controls) and vehicle (negative control)]. Proliferation and cytokine production in response to the test stimulus are 
measured to determine both the extent of T cell activation and the nature of the T cell response (i.e. TH1, TH2, TH17).  
The effects of mTOR inhibition on T cell maturation in response to an immune challenge have not been rigorously 
investigated and have never been tested in elderly humans. If our hypothesis were correct, RAPA would enhance 
antigen-specific T cell proliferation while suppressing overall proliferation to PHA or anti-CD3/CD28 stimuli. 
 
 
Aim III.  Determine whether mTOR inhibition can improve general immune features in the aged 
 
Rationale  The effects of mTOR inhibition are pleiotropic and the consequences of treatment may be different in young 
immunologically-competent individuals than in elderly subjects with age-associated immune dysfunctions (Fig. 1). Thus, 
we will examine several parameters of immune health in the mTOR inhibition by RAPA and placebo controlled subjects 
to assess whether the drug has mitigated the effects of aging. Importantly, we will be able to correlate improved 
humoral responses (Aim II) with changes in specific immune parameters measure in Aim III. 
 
Immune parameters assessed and expected results  We will focus on parameters known to change with age (Table 1) 
using blood collected after approximately 1, 6, and 8 weeks on RAPA or placebo, the following will be assessed and 
compared to levels determined prior to initiation of the treatment:  i) Serum cytokine levels; Luminex-based assays will 
be used; these allow simultaneous measurement of 28 different cytokines. It is expected that RAPA will have the 
beneficial effect of decreasing the levels of pro-inflammatory cytokines (TGF-β, IL-12, IL-6, TNF-α).  ii) Naive T cells are 
lost with aging due to thymus involution and a lifetime of clonal expansion of memory T cells. In mice, RAPA appears to 
protect these naive T cells which could then allow for a healthier immune response to “new” antigens in older 
individuals.  To assess whether the same is seen in humans, PBMCs will be analyzed by FACS to assess the proportion of 
memory vs. naive T cells both before and after RAPA treatment.  iii) T cell functional subsets will be assessed by FACS 
for cell surface markers and intracellular cytokines. Although expectations are less clear here, it will be important to 
assess whether RAPA has effects on human T cell development that are analogous to those seen in rodents.  Moreover, 
these measurements have not previously been ascertained for human subjects in this advanced age group.  
 
Aim IV.  Determine whether mTOR inhibition can improve muscle strength and cognitive function  
The following tests will be performed to assess RAPA affects on physiological parameters: 
1) Grip strength (3 consecutive measurements by grip-strength dynamometer): participants will be seated in a chair 
with the forearm at a 90° elbow bend and 3 consecutive grip strengths will be completed with a standard grip strength 
dynamometer (in Kg) for the dominant hand.  Results will be averaged for analysis.  
2) Timed 40-foot walk: each participant will perform 3 walks (timed with a stopwatch) at their preferred walking speed 
over a measured 40-foot path.  Results will be averaged for analysis.  
Statistical comparisons will be made between measurements made before and after, as well as both within and 
between mTOR inhibited and placebo groups to test our hypothesis.  
Cognitive assessment: All subjects will undergo cognitive assessments within one week of their recruitment (before 
treatment) and at the end of treatment (approximately 2 months after entering the RAPA phase of the study).  The 
cognitive tests will be selected from a battery of the following validated tests – EXIT, SLUMS, and/or TAPS – with care 
taken to avoid use of ones employed in the ACA phase (as appropriate) to limit the effects of “learning/memory”.   
Results of the foregoing (grip strength, timed walk, and cognitive assessment scores) will be compared between groups.  
 
Aim V. Assess the effects of RAPA on the gut microbiome.   
Rationale Composition of the gut microbiota is known to change with age and this most likely contributes to increases 
in inflammation seen in older individuals. Given its broad effects on metabolic regulation and its oral delivery route, 
RAPA is likely to alter the gut microbiome and may thereby elicit changes in the host immune system (addressed in 
Aims II and III).  In order to address this possibility, we will collect fecal specimens from the RAPA and placebo treated 
subjects at 4 time points, prior to RAPA treatment and after 1, 6, and 8 weeks on the drug.  The gut microbiome will be 
characterized initially from the pre-treatment and 8 week treatment samples.  The other fecal specimens will be stored 
for future analysis of the kinetics of interesting changes observed.   


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Approach The advent of next generation DNA sequencing has revolutionized efforts to characterize the endogenous 
microbiome. An oral medication, like RAPA, will likely target the gut microbiome. Thus, subjects will self-collect feces in 
sterile containers and deliver it to the clinic at their regular visits. DNA isolation will be performed by the Genomics 
Core facility on a fee-for-service basis and the samples will be delivered to the Sequencing Core Facility for analysis. The 
bacterial rDNA in each fecal sample will be amplified using primers from conserved regions of the 16S ribosomal RNA 
genes. Amplified DNAs are then used to generate a library which is subsequently subjected to deep (next gen) 
sequencing.  
 
 
Sub-study B & C Acarbose 
ACA delivery:  After baseline data are collected, ACA treatment will be initiated in all subjects.  For the first week, the dose 
will be 50mg once daily at supper-time. This will be increased to 50mg twice daily (breakfast and supper) during the 
second week and 50mg with each meal (breakfast, lunch, supper) during the third week. By the fourth week, the full dose 
of 100mg with each meal will be taken. Subjects will be instructed to take their pills at the beginning of a meal. 
Clinical assessment:  Subject health will be confirmed initially by history and physical exam, normal laboratory tests 
(fasting lipid profile, fasting blood sugar, complete metabolic profile, CBC), and 12-lead EKG. Oral glucose tolerance test 
(OGTT): Baseline (fasting) samples for determination of glucose, insulin, and free fatty acid (FFA) concentrations will be 
drawn at -30, -15, and 0 min (these basal values are averaged).  At time zero, each subject will ingest 75 g of glucose.  
Glucose, insulin and FFA are determined every 30 min for 2 h following glucose ingestion. The Matsuda index of insulin 
sensitivity will be calculated from OGTT results, as described (Matsuda and DeFronzo, 1999).At each visit, patients will 
receive a standard history and physical examination and be queried for any adverse reactions. A questionnaire (SF-36) 
will be administered before and after ACA treatment to assess patient perceived health effects and the Geriatric 
Depression Scoring system will be used to assess any changes in this parameter.  Between visits, subjects will be 
contacted weekly to ensure that no complications have arisen. If the highest dose were not well tolerated by a given 
subject, we would then drop down to 50mg ACA three times a day, but they would remain in the study.  At this lower 
dose, gastrointestinal issues have been reported in fewer than 6% of subjects. 
 
If subjects have previously undergone all or part of screening labs/tests within the past several months, the data may 
be accepted for eligibility determination without screening labs or procedures having to be repeated. 
Subjects that have completed taking placebo in SubStudy A and pass other necessary screening evaluations may be 
enrolled to SubStudy C. 
 
Approach Subjects will be studied at 3 time points: pretreatment, after 2 months on ACA, and 1 month after 
termination of the drug. Thus, each subject will serve as his own control and no placebo group is needed for the ACA 
phase.   
Physical function will be assessed using two independent assays of parameters known to decline with age, handgrip 
strength and timed 40-foot walk. Performance measures will be done longitudinally at each clinic visit to offer the 
greatest potential to detect changes due to ACA treatment and subsequent withdrawal.  
1) Grip strength (3 consecutive measurements by grip-strength dynamometer): participants will be seated in a chair 
with the forearm at a 90° elbow bend and 3 consecutive grip strengths will be completed with a standard grip strength 
dynamometer (in Kg) for the dominant hand.  Results will be averaged for analysis.  
2) Timed 40-foot walk: each participant will perform 3 walks (timed with a stopwatch) at their preferred walking speed 
over a measured 40-foot path.  Results will be averaged for analysis.  
Cognitive function will be assessed within one week of subject recruitment (before treatment), and at the end of 
treatment (2 months). These tests will be performed only twice to avoid learning effects that could confound the 
results. Three independent tests will be employed: EXIT (includes letter fluency) (13), SLUMS (St. Louis University 
Mental Status exam, which includes a memory test, digit span, and animal fluency) (14), GDS (Geriatr Depression Scale) 
and TAPS (Texas Assessment of Processing Speed; a digit/symbol coding test available in alternate forms to eliminate 
learning effects) (15).  If significant improvement were seen with one (or more) of the cognitive measures, that test (or 
those tests) would be repeated at the end of the withdrawal period to assess longevity of the response.  In addition, we 
will assess depression using the Geriatric Depression Score questionnaire. 


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CLOX Assessment: clock-drawing tests (CDT) as a screen for cognitive impairment at screening. 
Immune function Blood will be collected from each subject before treatment and after two months on ACA.  General 
parameters of immune health that are known to change with age will be assessed, as described below: 
1) Serum cytokine levels will be determined using Luminex-based assays that allow simultaneous measurement of 27 
different cytokines. It is known that aging is associated with an increase in the levels of pro-inflammatory cytokines (IL-
12, IL-6, TNF-α), which contributes to the loss of immune regulation with age and increased susceptibility to certain 
pathogens.  ACA reversal of this effect would be highly significant. 
2) TREGs have been shown by us and others to increase with age. These cells function to suppress ongoing immune 
responses and their presence in older individuals correlates with diminished immunity (19). To address whether ACA 
can reverse this increase in TREGs, PBMCs (peripheral blood mononuclear cells) will be analyzed by FACS to assess the 
proportion and number of CD4+CD25+foxp3+ cells (TREGs). Future experiments (beyond this pilot grant) would then be 
needed to examine TREG function and expression profiles.  
3) CD4/CD8 T cell ratio is a parameter known to change with aging and HIV infection. Using flow cytometry of PBMCs, 
the ratio of CD4+ T cells / CD8+ T cells will be determined before, during, and after ACA. 
4) Naive T cell numbers decline with age due to thymus involution and a lifetime of clonal expansion of memory T cells. 
This contributes to the loss of immune competence leading to decreased efficacy of vaccines and increased 
susceptibility to infection in the elderly. The loss of naïve T cells among PBMCs will be analyzed by FACS at the 5 time 
points (before, during and after treatment) for both CD4+ helper T cell (TH) and naïve CD8+ cytotoxic T cell (Tc) subsets. 
ACA induced recovery of naïve T cells would be significant. 
Expected Results Statistical comparisons will be made between the pre-treatment time point and the post-treatment 
point (2 months on ACA). It is our hope that the ACA-induced extension of lifespan in mice will be echoed by 
improvements in all of the parameters measured which were selected due to their known deficiencies with aging. This 
would suggest simultaneous effects on three systems: immune, neuromuscular, and cognitive.  
 
Aim VII.  Assess the effects of ACA on the gut microbiome.   
Rationale Composition of the gut microbiota is known to change with age and this most likely contributes to increases 
in inflammation seen in older individuals. Given its function in gastrointestinal metabolism, ACA is also likely to alter the 
gut microbiome; this may contribute to the beneficial effects of ACA on diabetic symptoms since it is known that 
changes in the resident microbes can impact both immune and endocrine functions  Although there were no searchable 
publications reporting characterization of the effects of ACA on the gut microbiome, there is a 2012 listing on 
ClinicalTrials.gov, entitled “Efficacy of acarbose on intestinal microbiome and incretins of Type 2 diabetes” from Dr. 
Ning, Shanghai Jiao Tong University School of Medicine; no results have been posted to-date. Also, an abstract from the 
University of Alabama, Birmingham (Dr. Brewer) reports on gut microbiome changes in mice treated with ACA 
[http://www.uab.edu/medicine/ camac/images/5-Abstract.pdf]. Our hypothesis is that ACA will result in improvement 
of functions known to decline with age (immune, endothelial, cognitive, and neuromuscular).  We further speculate 
that changes to the microbiome induced by ACA may play a role in mediating these pleiotropic effects. In order to 
address this possibility, we will collect fecal specimens from the ACA cohort and characterize the gut microbiome at the 
2 time points prescribed above [pretreatment and after 2 months on ACA].   
Approach The advent of next generation DNA sequencing has revolutionized efforts to characterize the endogenous 
microbiome. In fact, these genomic-based technologies have revealed many new bacterial species that were missed 
using microbiologic and serologic approaches. The endogenous microbiota plays a critically important role in generating 
an efficacious immune system and a healthy functioning gut. Of relevance to this proposal, the microbiome also 
regulates susceptibility to obesity and diabetes.  An oral medication, like ACA, will likely target the gut microbiome. 
Thus, subjects will self-collect feces in sterile containers and deliver it to the clinic at their regular visits. DNA isolation 
will be performed by the Genomics Core facility on a fee-for-service basis and the samples will be delivered to the 
Sequencing Core Facility for analysis. The bacterial rDNA in each fecal sample will be amplified using primers from 
conserved regions of the 16S ribosomal RNA genes. Amplified DNAs are then used to generate a library which is 
subsequently subjected to deep (next gen) sequencing.  
Expected Results For humans, there have been many reports documenting the bacterial species that dominate in older 
vs. young individuals (24).  Given the ages of our subjects, we would expect to see a profile more similar to the “old” 
profile. It would be most exciting if ACA were to alter the microbiome, making it more “youthful”.  
 


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Sub-study D Rapamycin Alone – Cardiovascular Effects 
For Aims D-1, D-2, D-3, and D-4: 
Intervention with RAPA will begin at the end of Visit 2 once all screening and baseline measurements have been 
collected. Subjects will self-administer RAPA once daily for a period of 8 weeks (56 doses) and there will be no 
placebo group in this Sub-study. We will screen 12 MALE subjects to complete 6. No females will be enrolled in this 
pilot substudy. 
Clinical assessment: See also Sub-study A (except without OGTT). In substudies A, B and C, no detrimental diabetogenic 
effects of RAPA were found, i.e. OGTT, FBS, HbAic were unaltered by RAPA. 
If subjects have previously undergone all or part of screening labs/tests within the past 4 months, the data may be 
accepted for eligibility determination without screening labs or procedures having to be repeated. 
Physical function will be assessed using two independent assays of parameters known to decline with age, handgrip 
strength and timed 40-foot walk. Performance measures will be done longitudinally at Visits 2, 3, and 4 to offer the 
greatest potential to detect changes due to RAPA treatment.  
1) Grip strength (3 consecutive measurements by grip-strength dynamometer): participants will be seated in a chair 
with the forearm at a 90° elbow bend and 3 consecutive grip strengths will be completed with a standard grip strength 
dynamometer (in Kg) for the dominant hand.  Results will be averaged for analysis.  
2) Timed 40-foot walk: each participant will perform 3 walks (timed with a stopwatch) at their preferred walking speed 
over a measured 40-foot path.  Results will be averaged for analysis.  
3) Activity will be continuously monitored in participants by wearing a Garmin Vivofit3 Activity Monitor. This battery 
operated, waterproof activity monitor will be worn continuously over the period of enrollment to record participant 
activity (steps per day, periods of sleep). The Garmin Vivofit and similar devices have been used successfully in other 
studies and  have been found to be acceptable to older volunteer subjects {O’Brien, 2015 #2716;Schrack, 2016 
#2715;Šimůnek, 2016 #2714}. 
Immune function will be assessed pre-treatment and at the end of treatment (Visits 1 and 4). We will perform tests for 
serum cytokine profiles, flow cytometry with T cell subsets, and T cell activation potential. 
Cognitive function will be assessed before treatment at Visit 2, and at the end of treatment (2 months, Visit 4). These 
tests will be performed only twice to avoid learning effects that could confound the results. Three independent tests 
will be employed: EXIT (includes letter fluency) (13), SLUMS (St. Louis University Mental Status exam, which includes a 
memory test, digit span, and animal fluency) (14), GDS (Geriatric Depression Scale) and TAPS (Texas Assessment of 
Processing Speed; a digit/symbol coding test available in alternate forms to eliminate learning effects) (15).  If 
significant improvement were seen with one (or more) of the cognitive measures, that test (or those tests) would be 
repeated at the end of the withdrawal period to assess longevity of the response.  In addition, we will assess depression 
using the Geriatric Depression Score questionnaire. 
Aim D-1. Test whether RAPA treatment increases aortic compliance in elderly subjects. 
Approach.  PWV will be measured before, during, and at the end of RAPA treatment as per Table 2. Measurement of 
aortic PWV is the simplest way to assess arterial compliance non-invasively in humans and reflects the ability of the 
arterial system to cope with systolic ejection volume. It also implies that the compliant arterial system’s major function 
is to relay cardiac contraction during diastole. Indeed, cardiac contraction lasts only one third of a cardiac cycle of the 
time. Due to the distension of the compliant large arteries during systole, after the end of cardiac contraction, the 
compliant large arteries return to their initial dimension and thereby help maintain blood flow. This phenomenon is of 
key physiological importance as diastolic flow in arteries may represent more than half of cardiac output. Large artery 
compliance represents the dynamic force opposing ejection (also called impedance): if high, it helps in ejection, the 
lower the compliance, diastolic facilitation of ejection is lessened. Many methods have been proposed to quantify this 
phenomenon.  The simplest non-invasive approach to measure arterial compliance is PWV, i.e., the transit time 
measurement of the pulse wave from the aortic valve to the terminus of the aorta (1). We will use the SphygmoCor 
system, which has been used in successful large clinical trials and population surveys. This device uses a large band 
piezoelectric probe and allows the arterial pulse recording in succession (carotid then femoral), both signals being 
synchronized with the same time basis (ECG R wave) (1). We have such a device available and are experienced with its 
use in humans. To measure PWV, the subject will rest in supine position for at least 5-10 min to reach the physiological 
baseline conditions, the room will be maintained at a standardized temperature, and the blood pressure will be 


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17 
accurately measured within minutes from the PWV recording times. Serial longitudinal measurements are performed 
as summarized in Table 2. All PWVs will be done in fasting condition. Overall, the reproducibility of PWV measurements 
is good (CV in the range of 8-12%) (1).  
Expected Results-There are many reports documenting that the PWV is greater in older vs. young humans (1). Given 
the ages of our subjects, we expect to see a pattern of initially high PWV before RAPA treatment that would be reduced 
with RAPA. In the placebo group, we expect that their initially high PWV will be unaltered or increase during the study.  
Aim D-2. Test whether RAPA treatment improves endothelial function in elderly subjects.   
Approach. It is well known that microvascular endothelial dysfunction precedes dysfunction in conduit arteries and is 
predominantly due to reduced NO bioavailability in both aging and disease states. Choi, et al (2), have developed 
microvascular function test that reflects primarily NO-dependent vascular function and is thus of significant clinical utility. 
This test uses rapid local heating of forearm skin from 34°C to 39°C to produce a hyperemic vasodilation (an increase in 
cutaneous vascular conductance (CVC=LDF/MAP)) that is 80% dependent on NO. This is the most robust test of NO-
dependent dilation that can be performed non-invasively in humans, as the multifold increase in conductance from 34C 
to 39°C is almost entirely effected by NO. Due to the noninvasiveness and ease of performing the test, we will use rapid 
local heating to 39°C to assess NO-dependent dilation and thus microvascular function. Rapid local heating to 39°C elicits 
a plateau which reaches 50% of maximal CVC as effected by local warming to 42°C, which allows for assessment of 
interventions that may alter maximal vasodilatory mechanisms that are NO-independent. Upon arrival in the laboratory, 
subjects will be placed in a semi-supine position in a dialysis chair and be instrumented on their right forearm for laser-
Doppler flowmetry measurements of skin blood flow (SkBF) with a LDF probe equipped with a local heating unit and 
thermocouple. A Finapres cuff placed on one finger for blood pressure measurements (MAP). Local skin temperature 
(Tloc) at the LDF site will be held at 34°C for approximately 5-10 minutes to establish a stable baseline measurement. 
Tloc will be then increased at a rate of 0.1°C/sec to 39°C and then held constant. After LDF has reached a stable plateau 
(30–40 min) at Tloc=39°C, Tloc will be raised to 42°C for about 30 min to maximally vasodilate the skin (9, 15, 17). For 
data analysis, CVC will be calculated. Averages of maximal absolute values of CVC (in mV) with Tloc=34, 39, and 42°C will 
be calculated and compared both within and between subjects by repeated measures ANOVA. All of the needed 
equipment is available in Dr. Kellogg’s lab.  
Expected Results. It is well known that NO-dependent vasodilation is reduced in older vs. young individuals (5, 6). We 
expect that CVC responses to skin warming to 39°C will increase both during and after RAPA when compared to pre-RAPA 
responses.  
 
**Depending on availability of subjects, shared lab equipment, and personnel resources, the vascular function 
assessments for baseline measurements of PWV and or LDF may be scheduled and performed at Visit 1 OR Visit 2 (see 
Table 2). This allows flexibility for the subject and facilitates protocol compliance for the research team. 
 
MRI Studies of Cardiac function and Cerebral Blood flow 
 
Aim D-3. Examine whether mTOR inhibition improves diastolic function and increases cerebral blood flow in humans.  
We will explore whether mTOR inhibition with RAPA improves cardiac diastolic function and cerebral blood flow (CBF).  
Diastolic function and CBF will be measured by MRI pre- and post-treatment with RAPA at Visits 2 and 4. 
 
Expected Results-There are reports that the cardiac function and CBF are improved in animal models by RAPA 
treatment {Chiao, 2016 #2623;Urfer, 2017 #2617}. Given the ages of our subjects, we expect to see a pattern of initially 
impaired diastolic function and CBF before RAPA treatment that would be improved with RAPA.  
Aim D-4. Examine whether mTOR inhibition increases physical activity in humans. 
Expected Results-RAPA treatment increases physical activity in animal models. {Miller, 2011 #1350;Urfer, 2017 #2617} . 
We anticipate that similar activity increases will be found in humans with RAPA administration. 
Pilot data will be collected on at least 6 subjects to analyze whether treatment with RAPA increases physical activity. 
Using a Garmin Vivofit3 Activity Monitor [Garmin, Inc.] provided by the study to each participant. we will examine 
whether RAPA effects overall activity by measuring time, distance, step count, calories burned, total hours of sleep, 
periods of movement, and periods of restful sleep. Subjects will wear activity monitors continuously for approximately 
2 weeks from consent to baseline visit at which we will download pre-intervention activity levels. Subjects continue 
wearing bracelet-style activity monitors throughout the study duration and upon returning to the clinic (4 weeks and 8 
weeks of RAPA treatment), provide data downloads at each of the Visits 3 and 4. Participants will keep the activity 


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18 
tracking devices after the study is complete as a thank-you for their participation {O’Brien, 2015 #2716;Schrack, 2016 
#2715;Šimůnek, 2016 #2714}. 
Data Analysis Plan:  
This is a pilot study designed to produce the data necessary for future larger studies.  No data are currently available to 
guide sample size calculations. 
 
OVERALL EXPERIMENTAL DESIGN   The collection of samples and performance of procedures for Substudies A, B and C will typically 
be performed as indicated below**.  Also, V1 may be combined with V0 if subject arrived fasting.  
The collection of samples and performance of procedures for Substudy D will typically be performed as indicated in Table 2 on the 
following page.. However, at the discretion of the PI, some collections may be done at a visit before or after the one shown.   
Table 1. Substudy A Rapamycin Only study visits 0, 1, 7-10; Substudy B ACA+RAPA study visits 0-10; Substudy C ACA Only study visits 0- 6 (as 
summarized in table below).  TABLE 1 DOES NOT APPLY TO SUBSTUDY D** 
 
 
Visit number 
V0 
V1 
V2 
V3 
1V4 
1.5V5 
2V6 
3V7 
4V8 
5V9 
6V10 
Visit goal 
Consent 
Screeni
ng 
Pre-ACA 
ACA – 1 
month 
ACA – 6-
7 wks 
ACA 
ends 
Post 
ACA2 
month 
termina
tion 
7 day 
RAPA 
6 wks 
RAPA 
Safety 
Imm 
Study Phase 
A,B,C 
A,B,C 
B,C 
B,C 
B,C  
B,C  
B,C  
A,B 
A,B 
A,B 
A,B 
Week number (of phase) 
(time variable between visits 3 
and 4) 
 
 
0 
4 
6-7 
48 
12 
0 (for 
RAPA) 
1 
6 
8 
Timing (days) – range 
 
 
 
 
 
 
25-35 
days 
after V2 
38-45 
days 
after V2 
50-70 
days 
after V2 
25-35 
days 
after V5 
25-35 
days 
after 
visit 2 
6-9 days 
after V7 
40-45 
days 
after V7 
13-15 
days 
after 
V9 
PROCEDURES 
 
 
 
 
 
 
 
 
 
 
 
Consent form 
X 
 
 
X 
 
 
 
 
 
 
 
EKG 
 
X 
 
X 
 
 
 
 
 
 
 
Physical exam & history;  
Safety measures: AE’s and 
problems 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Clinical blood tests, include: 
 
 
 
X 
 
 
X 
X 
X 
X 
X 
  Complete blood count  
 
X 
# 
 
 
X 
X 
# 
X 
X 
X 
  Metabolic profile 
 
X 
# 
 
 
X 
X 
# 
X 
X 
X 
  Fasting blood sugar; HbA1c 
 
X 
# 
 
 
X 
X 
# 
X 
X 
X 
  Fasting lipid profile 
 
X 
# 
 
 
X 
X 
# 
X 
X 
X 
Urinalysis 
 
 
 
 
 
 
 
x 
 
 
x 
OGTT 
 
X 
 
 
X 
 
X 
 
x 
X 
 
Acarbose treatment begins 
 
 
X 
 
X 
 
 
 
 
 
 
Acarbose treatment ends 
 
 
 
 
 
X 
X 
 
 
 
 
RAPA or placebo begins 
 
 
 
 
 
 
 
X 
X 
 
 
SF-36 questionnaire 
 
 
X 
 
X 
X 
X 
X 
X 
 
X 
RAPA levels 
 
 
 
 
 
 
 
 
X 
X 
X 
Blood draw for lab, includes: 
 
 
 
X 
 
 
X 
X 
X 
 
X 
  Cytokine profiles 
 
 
X 
X 
 
X 
X 
X 
X 
X 
X 
  Blood cell subsets by flow 
 
 
X 
X 
 
X 
X 
X 
X 
X 
X 
  T cell subsets  
 
 
X 
X 
 
X 
X 
X 
X 
X 
X 
  T cell proliferation to mitogen 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
Collect feces 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
Constipation Assessment 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Immunizations: flu vaccine 
 
 
 
 
 
 
 
 
 
X 
X 
T cell prolif to flu antigens 
 
 
 
 
 
 
 
 
X 
X 
X 
Antibody to flu antigens 
 
 
 
 
 
 
 
 
X 
X 
X 
Hand grip and 40 ft walk test* 
 
 
X 
X 
 
X 
X 
X 
X 
X 
X 
Cognitive tests: EXIT/SLUMS 
and/or TAPS* 
 
 
X 
X 
 
X 
X 
X 
X 
 
X 
GDS (Geriatr Depression Scale)* 
 
 
X 
X 
 
X 
X 
X 
X 
 
X 
CLOX Assessment (cognitive 
assessment)* 
 
X 
 
X 
 
 
 
 
 
 
 
PET/MRI 
 
 
 
 
 
 
 
O 
O 
 
O 


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Notes:        X=scheduled procedure; O=optional procedure (if funding found and subject consents) *May be done 
over Visit 0 and Visit 1;  #=procedure performed if > 2 weeks from prior measure at V1 
 
 
Table 2. VISIT SCHEDULE: Sub-study D. Rapamycin Alone – Cardiovascular Effects 
 
VISIT NUMBER 
V1 
V2 
FU1 
V3 
FU2 
V4 
Visit goal 
Consent/ 
screen 
(fasting) 
Pre-drug 
(not 
fasting) 
 Phone 
follow up* 
4 wks 
RAPA 
Phone  
follow up* 
8 wks 
RAPA 
Week number  
 
0 
1 
4 
 
8 
PROCEDURES 
2-2.5h 
3-3.5 
 
2.5-3 
 
4 
  Consent form and CLOX>10 
 
 
 
 
 
 
SAFETY/AEs/METABOLIC 
 
 
 
 
 
 
  Physical exam 
 
 
 
 
 
 
  ECG 
 
 
 
 
 
 
  Self 
reported 
Adverse 
Events (AEs) 
 
 
 
 
 
 
  CBC (blood cell counts) 
 
 
 
 
 
 
  Fasting blood sugar/CMP 
 
 
 
 
 
 
  Fasting lipids 
 
 
 
 
 
 
  HbA1c 
 
 
 
 
 
 
  Urinalysis 
 
 
 
 
 
 
IMMUNE FUNCTION 
 
 
 
 
 
 
  Serum for cytokine profiles  
 
 
 
 
 
 
  Flow 
cytometry: 
T 
cell 
subsets 
 
 
 
 
 
 
  T cell activation potential 
 
 
 
 
 
 
  RAPAMYCIN LEVEL 
 
 
 
 
 
 
Meal/snack 
 
 
 
 
 
 
COGNITIVE FUNCTION 
 
 
 
 
 
 
  Geriatric depression scale 
 
 
 
 
 
 
  EXIT, SLUMS, TAPS 
 
 
 
 
 
 
  VR-36 QOL 
 
 
 
 
 
 
PHYSICAL FUNCTION 
 
 
 
 
 
 
Hand grip and 40 foot walk 
 
 
 
 
 
 
Garmin VivoFit3 Activity 
Monitor 
DISPENSE 
BRING* 
 
BRING* 
 
BRING*  
DISPENSE STUDY MED 
 
 
 
 
 
 
VASCULAR FUNCTION 
 
 
 
 
 
 
  Aortic Pulse Wave Velocity 
(PWV) 
** 
** 
 
 
 
 
  Arteriolar Endothelial 
Function (LDF) 
** 
** 
 
 
 
 
CARDIAC/CEREBRAL 
BLOOD FLOW FUNCTION 
 
 
 
 
 
 
  MRI 
 
 
 
 
 
 
      Schedule MRI -  Date 
 
 
 
 
 
 
 
 
*Telephone follow up of subjects to assess tolerability and or adverse reactions to RAPA will be conducted at least once between V2 and 
V3. If tolerating well after 1-2 weeks of RAPA treatment without side effects, no further calls may be necessary. If side effects are reported at any 
time during RAPA administration, the investigator may choose to modify dosing, if appropriate, and follow by phone on a weekly basis until 
resolved. 
 
 
BRING* – At Visits 2, 3 and 4, subjects will bring their Garmin VivoFit3 Activity Monitor so that activity data can be downloaded to a 
protected server with adequate storage and backup. 
 


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20 
 
**Depending on availability of subjects, shared lab equipment and personnel resources, the vascular function assessments for baseline 
measurements may be scheduled and performed at Visit 1 OR Visit 2. This allows flexibility and promotes protocol compliance. 
 
Item 7 
Risks Section. Risks are described in detail in the Step 2-Institutional Form.