An Innovative Proof-of-concept Approach to Identify Age-modulating Drugs Capable of Reversing Inflammation and Re-setting the Epigenetic Clock (Topical-RAPA)
Phase: EARLY_PHASE1
Conditions: Aging, Epigenetics, Inflammatory Mediators
Sponsor: The University of Texas Health Science Center at San Antonio

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Official Study Title: An innovative proof-of-concept approach to 
identify age-modulating drugs capable of reversing inflammation 
and re-setting the epigenetic clock (Topical-RAPA) 
 
 
NCT number: NCT04608448 
 
IRB Approval Date: 08-17-21 
 
Unique Protocol ID: HSC20200720H 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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An innovative proof-of-concept approach to identify age-modulating drugs 
capable of reversing inflammation and re-setting the epigenetic clock 
(Topical-RAPA) 
Study Chairman or Principal Investigator: 
Ellen Kraig, PhD 
University of Texas Health Science Center San Antonio 
Department of Cell Systems & Anatomy 
School of Medicine 
7703 Floyd Curl Drive, MC7762 
San Antonio, Texas 78229-3900 
210-367-3171 
 
kraig@uthscsa.edu 
 
 
Supported by: The National Institute on Aging 
 
Study Intervention Provided by: Grant Funds 
Sponsor of IND/IDE: 152906 Exempt per FDA 
Dean L Kellogg, Jr, MD, PhD 
UT Health Science Center 
Department of Medicine – Division of Geriatrics,  
Gerontology and Palliative Medicine 
7703 Floyd Curl, MC 7885 
San Antonio, TX 78229-3900 
210-617-5197 
kelloggd@uthscsa.edu 
 
Version 1.4  16 August 2021

Protocol Template, Version 3.0 
i 
TABLE OF CONTENTS 
TABLE OF CONTENTS .............................................................................................................. i 
Study Title .................................................................................................................................. iv 
Objectives .................................................................................................................................. iv 
Design and Outcomes ................................................................................................................ iv 
Interventions and Duration ........................................................................................................ iv 
Sample Size and Population....................................................................................................... iv 
STUDY TEAM ROSTER............................................................................................................. 5 
Principal Investigator:                Ellen Kraig, PhD ..................................................................... 5 
Co-Investigators:   Dean L. Kellogg, Jr, M.D., PH.D. ............................................................. 5 
1 
Study objectives ..................................................................................................................... 6 
1.1 
Primary Objective ........................................................................................................... 6 
1.2 
Secondary Objectives...................................................................................................... 6 
2 
BACKGROUND AND RATIONALE ................................................................................ 6 
2.1 
Background on Condition, Disease, or Other Primary Study Focus .............................. 6 
2.2 
Study Rationale ............................................................................................................... 7 
3 
STUDY DESIGN ................................................................................................................... 9 
4 
SELECTION AND ENROLLMENT OF PARTICIPANTS .......................................... 10 
4.1 
Inclusion Criteria .......................................................................................................... 10 
4.3 
Study Enrollment Procedures ....................................................................................... 11 
4.4 
Subject Recruitment and Retention .............................................................................. 12 
5 
STUDY INTERVENTION ................................................................................................. 12 
5.1 
Interventions, Administration, and Duration ................................................................ 12 
5.1.1.1 Rapamycin powder (from a supplier to Doyle’s pharmacy) ..................................... 13 
5.1.1.2 Petrolatum (Vaseline, vehicle only) .............................................................................. 14 
5.2 
Handling of Study Interventions ................................................................................... 15 
5.3 
Concomitant Interventions ............................................................................................ 15 
5.3.1 Allowed Interventions ............................................................................................... 15 
5.3.2 Prohibited Interventions ............................................................................................ 16 

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5.4 
Adherence Assessment ................................................................................................. 16 
6 
STUDY PROCEDURES .................................................................................................... 17 
6.1 
Schedule of Activities ................................................................................................... 17 
6.2 
Schedule of Evaluations ................................................................................................ 18 
7 
SAFETY ASSESSMENTS ................................................................................................. 20 
7.1 
Specification of Safety Parameters ............................................................................... 20 
7.2 
Adverse Events and Serious Adverse Events ............................................................... 21 
7.3 
Unanticipated Problems Involving Risk to Subjects or Others (UPIRSO)................... 21 
7.3.1 Adverse Event Reporting Period ........................................................................... 22 
7.3.2 Pre-existing Condition ............................................................................................ 22 
7.3.3 Abnormal Laboratory Values ................................................................................ 22 
7.3.4 Hospitalization, Prolonged Hospitalization or Surgery ...................................... 22 
7.4 
Recording of Adverse Events ....................................................................................... 22 
7.6 
 Reporting of Serious Adverse Events and Unanticipated Problems ............................ 23 
7.6.2 Investigator reporting: notifying the UTHSCSA IRB ............................................ 24 
Notifying the IRB and or FDA if SAE or UPIRSO .............................................................. 24 
7.7 
Medical Monitoring ...................................................................................................... 24 
7.8 
Investigator Reporting of Protocol Deviations/Violations ........................................... 24 
7.8.1 Deviations and violations may be identified in a number of ways including: ... 25 
7.8.2 Definitions of Protocol Deviations/Violations ....................................................... 25 
7.9 
Safety Monitoring ......................................................................................................... 25 
8 
INTERVENTION DISCONTINUATION........................................................................ 26 
9 
STATISTICAL CONSIDERATIONS .............................................................................. 26 
9.1 
General Design Issues ................................................................................................... 26 
9.2 
Sample Size and Randomization .................................................................................. 26 
9.2.1 Treatment Assignment Procedures ........................................................................... 27 
9.3 
Interim analyses and Stopping Rules ............................................................................ 27 
9.4 
Primary Objective ......................................................................................................... 27 
9.5 
Secondary Objectives.................................................................................................... 27 
9.6 
Data Analyses ............................................................................................................... 28 
10 DATA COLLECTION AND QUALITY ASSURANCE ................................................ 29 
10.1 
Data Integrity ................................................................................................................ 29 
10.2 
Data Management ......................................................................................................... 29 

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10.3 
Quality Assurance ......................................................................................................... 29 
10.3.1 
Training ............................................................................................................... 29 
10.3.2 
Quality Control Committee ............................................................................... 29 
10.3.3 
Study Monitoring Plan ....................................................................................... 29 
11 PARTICIPANT RIGHTS AND CONFIDENTIALITY ................................................. 30 
11.1 
Institutional Review Board (IRB) Review .................................................................... 30 
11.3 
Participant Confidentiality ............................................................................................ 30 
11.4 
Study Discontinuation ................................................................................................... 30 
12 ETHICAL CONSIDERATIONS ....................................................................................... 31 
13 COMMITTEES................................................................................................................... 31 
14 PUBLICATION OF RESEARCH FINDINGS ................................................................ 31 
15 REFERENCES .................................................................................................................... 32 
16 SUPPLEMENTS/APPENDICES ...................................................................................... 34 
 
 
 
 
 
 

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Study Title  
An innovative proof-of-concept approach to identify age-modulating drugs capable of 
reversing inflammation and re-setting the epigenetic clock (Topical-RAPA) 
Objectives  
AIM 1: Test whether epigenetic changes in skin are elicited by topical treatments with                  
RAPA 
AIM 2: Test whether baseline inflammation is affected by topical RAPA 
Design and Outcomes   
This is a double-blinded pilot treatment study, where each subject provides his/her own 
control to assess whether rapamycin will stop, slow, or reverse the “epigenetic clock” 
using DNA extracted from skin and the change in levels of inflammatory mediators 
measured in blister fluid in healthy subjects aged 65-95 years.  
Interventions and Duration  
Rapamycin 8% ointment will be applied topically, 0.25ml, to one of the participant’s 
forearms and matching placebo, 0.25ml, to the opposite forearm daily for a total of 6 
months. The total participant duration will include a consenting/screening visit (0) will 
last 60-90 minutes, followed by monthly visits (1-6) of up to 60 minutes, and followed by 
the final visit (7) of up to 4 hours. 
 
Sample Size and Population  
The study aims to recruit 75 subjects to ensure at least 40 completers, approximately half 
male and half female. 

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STUDY TEAM ROSTER  
 
 
Principal Investigator:               Ellen Kraig, PhD 
 
University of Texas Health Science Center San Antonio 
 
Department of Cell Systems & Anatomy 
 
School of Medicine 
 
7703 Floyd Curl Drive, MC7762 
 
San Antonio, Texas 78229-3900 
210-367-3171 
 
kraig@uthscsa.edu  
 
Co-Principal Investigator:        Dean L. Kellogg, Jr, M.D, Ph.D. 
UT Health Science Center 
Department of Medicine – Division of Geriatrics, 
Gerontology and Palliative Medicine 
   7703 Floyd Curl, MC 7885 
   San Antonio, TX 78229-3900 
                                                     210-617-5197  
                                                     kellogg@uthscsa.edu 
 
  
 
SITES: 
UT Health San Antonio 
 

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1 
STUDY OBJECTIVES 
1.1 Primary Objective 
Test whether epigenetic changes in skin are elicited by topical treatments with RAPA 
1.2 Secondary Objectives 
Test whether baseline inflammation is affected by topical RAPA 
 
2 
BACKGROUND AND RATIONALE  
2.1 Background on Condition, Disease, or Other Primary Study Focus 
The study is focused on the effects of mTOR antagonism specifically on elderly subjects. 
In order to identify drugs and/or interventions with the potential to delay the onset and progression 
of age-associated pathologies, preclinical studies have used animal models where extension of 
lifespan could be accurately assessed. Several outstanding candidate therapeutics have been 
identified over the last decade; these include mTOR inhibitors (like rapamycin), senolytic agents, 
acarbose, and others. However, translating anti-aging results from animal models into humans has 
been challenging. For example, measuring extension of lifespan would be impractical in humans, 
so it is necessary to identify and validate parameters that can serve as surrogate markers of healthy 
aging. Moreover, the efficacy of age-modulating drugs is often only demonstrable with older 
individuals where functional deficits are already evident. Thus, testing of potential anti-aging 
therapeutics should be done in a cohort of human subjects of an advanced age; issues of safe 
administration would be even more acute in this “at risk” population. To circumvent these 
limitations, we propose to develop and validate an innovative, minimally invasive, cost-effective 
assay in human subjects ranging from 65-95 years old. The putative anti-aging test drug will be 
applied topically to a discrete area of skin on the subject’s forearm while a “vehicle only” (control) 
is applied in parallel to the opposite arm. Thus, each subject will serve as his/her own control. The 
pharmacodynamic outcomes at the “test” and placebo sites will be compared in both males and 
females since the effects of age-modulating agents often differ between the sexes. Ascertaining 
the efficacy of topically-applied therapeutics requires robust biomarkers of aging that can be 
measured in skin, but will be generalizable to the intact organism. Towards this end, we have opted 
to use two independent parameters that are known to be modified with aging both locally (in 
individual tissues) and systemically: i) the DNA methylation pattern or “epigenetic clock” and  ii) 
biomarkers associated with inflammation. To validate the proposed technique, we have chosen a 
drug, rapamycin (RAPA), which has already been shown to modulate aging in rodents and which 
has been tested for safe systemic application in humans [1-4]. After a 6 month treatment phase, 
suction blisters will be generated at each site to allow collection of skin cells (blister flap) for use 
in the epigenomic analysis (Aim 1) and interstitial (blister) fluid for use in measuring cytokines 
and other inflammatory regulators (Aim 2). Once validated, our approach can be used to safely 
and efficiently screen the age-modulating potential of pharmacological agents in humans to 
identify those agents that warrant large, expensive human clinical trials with systemic agent 
administration. 
 
 

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2.2 Study Rationale 
Demonstrating age modulating benefits of drugs in long-lived humans is challenging. Although 
there are outstanding animal models used to identify candidate interventions which extend 
lifespan [2, 3, 13], an efficient and cost-effective approach to test which of these drugs can be 
used safely and with efficacy in an older human cohort is not available. Thus, we propose to 
develop and validate an innovative, relatively safe, minimally invasive approach for the initial 
evaluation of the potential efficacy of purported age-modulating agents in humans prior to 
moving forward with a large, costly human clinical trial. Our scientific premise is that putative 
age-modulating agents can be pre-screened for beneficial effects through topical application to 
the skin of humans. In this way, the drug effects will be limited to the small test area with 
minimal or no systemic delivery, thereby avoiding serious risks to the human subjects. As 
summarized in Figure 1, potential agents can be applied to different ‘experimental’ skin sites in 
the same person with simultaneous application of ‘control’ placebo treatments on opposite 
forearms. This approach will allow each subject to serve as his/her own control. Age-modulating 
efficacy of the tested agent(s) can be assessed from skin samples obtained from the experimental 
treated and placebo control sites. Skin samples can be easily obtained with suction blister or 
small punch biopsy techniques for subsequent testing [14-17]. Ascertaining the age modulating 
efficacy from these samples will require robust biomarkers of aging that can be measured in skin, 
but will be generalizable to the intact organism. Towards this end, we have opted to use two 
parameters that are known to be modified with aging both locally (in individual tissues) and 
systemically: i) the DNA methylation pattern or “epigenetic clock” and ii) biomarkers associated 
with inflammation.   
To validate the proposed pre-screening approach, we have chosen to initially test a single agent, 
rapamycin (RAPA), an mTOR pathway antagonist. RAPA has been shown to prolong lifespan in 
rodents. Improvements in cognition, cardiovascular function, and certain aspects of immunity 
were also demonstrated in animal models [7, 18-23].  Based on these promising results, we 
undertook a pilot study of orally-delivered RAPA in older (aged 70-95 yrs) human subjects as a 
potential anti-aging therapeutic. As described in detail [1], short-term (2 months) RAPA 
treatment was relatively well tolerated in these older individuals. In addition, we and others have 
established that RAPA can be efficaciously delivered transdermally [24, 25]. As shown in Figure 
2A, easily detectable levels of the drug are obtained locally (in the skin at the application site) 
within 12 hours of application while there was no detectable level of RAPA in the blood even 
after 7 days of topical application of 8% RAPA. Moreover, as shown in Figure 2B, 7 days of 8% 
RAPA led to improved endothelium-dependent vasodilation in older, but not younger persons, 
demonstrating that this treatment exerted pharmacological effects. In an "N of 1" study, 8 months 
of topical 8% RAPA treatment decreased the size of keloids (Kellogg, data not shown). Topical 
RAPA has also been tested clinically and shown to be efficacious in the treatment of psoriasis 
and tuberous sclerosis [24, 26, 27]. Thus, mTOR inhibition with RAPA should be an excellent 
model for development and validation of the proposed pre-screening protocol. The ‘long-term’ 
significance is that, once validated, our approach can be used to safely and efficiently screen the 
age-modulating potential of pharmacological agents in humans, in vivo, in a relatively short 
amount of time in order to identify those agents that warrant large, expensive human clinical 
trials with systemic agent administration. 

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b) Innovation: 
We will develop a new pre-screening approach that can be safely used for translational studies in 
humans. The proposed protocol, employing topical application, should both enhance our 
understanding of underlying mechanisms affected by aging and identify the most promising age-
modulating pharmacological therapies for future clinical trials with systemic administration. A 
priority for translating the pharmacological manipulation of aging from animal models into 
humans, especially in the geriatric age group, is subject safety. Since these test interventions will 
be given to essentially healthy persons to treat aging (a normal life process) rather than to 
mitigate an overt disease process, taking age-modulating drugs must entail no additional risks 
over those inherent in normal aging.  Furthermore, successful agents must act to prolong 
healthspan or at least delay the onset of age-related pathologies. Ideally, age modulating agents 
would ‘reset’ the biological clock and slow or reverse the increase in baseline inflammation; 
thereby rejuvenating treated individuals. The innovation in this proposal derives from the choice 
of biomarkers to be followed to assess efficacy of topical treatment. First, we propose to use two 
independent measures of aging such that simultaneous effects on both outcomes would be highly 
significant. Second, the “epigenetic clock” defined by Dr. Steve Horvath (a collaborator on this 
project), is an innovative tool for estimating chronological age of cells based on the DNA 
methylation at 391 CpG motifs. This test has been previously validated for both skin cells and 
blood cells, suggesting that the results from topical administration will be directly translatable to 
systemic dosing. Third, to test for the effect of the topically applied drug on baseline 
inflammation, we will use a multiplex Luminex array to measure levels of 29 human cytokines 
(both pro and anti-inflammatory). In addition, ELISAs will be performed on three analytes 
known to play critical roles in regulating inflammation: IL-6 (interleukin-6), soluble ICAM-1, 
and RAGE (receptor for advanced glycation end products). Importantly, by using RAPA as the 
initial test compound, we will be able to directly compare the results with topical application to 
results from human clinical trials with oral administration. The future implications and potential 
uses deriving from this “high risk, potentially high reward” R21 are vast.  
c) Approach: 
Rationale. The NIA Interventions Testing Program (ITP) was developed to assess the potential of 
drugs to extend lifespan in mice and has identified several promising candidates, including 
aspirin, rapamycin (RAPA), 17αEstradiol, acarbose, NDGA (nordihydroguaiaretic acid), and 
Protadim [28] Additional putative age-modulating compounds have been identified through other 
approaches; these include senolytics, NAD precursors, and Sirtuin activators, among others [2]. 

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Although several of these agents have moved into human clinical trials, such efforts are limited 
due to: i) high costs; ii) the inability to use “lifespan” as an outcome measure in long-lived 
humans; and  iii) the reluctance to test drugs in an older “at risk” population. This last limitation 
is often circumvented either by using a younger subject cohort or by targeting individuals with 
serious illnesses; neither of these options is ideal. For example, some age-modulating drugs only 
show a treatment effect in older subjects where the loss of function with age changes the baseline 
allowing an effect to be detected (one such example is shown in Figure 2B where an effect of 
topical RAPA was seen only in older subjects). Similarly, studies of older subjects with cancer, 
autoimmunity, or another illness are compromised by associated co-morbidities and other drugs 
being taken to treat the primary illness. Thus, it is critically important to test putative age-
modulating drugs on an older human cohort that is otherwise clinically stable.   
The ‘gold standard’ for testing a drug for its ability to slow the ravages of aging would be a long-
term placebo-controlled human clinical trial where changes in lifespan and age-associated 
pathologies could be assessed. However, this is simply not feasible in humans and is even 
challenging in relatively long-lived non-human primate models. Thus, we propose to develop 
and validate an innovative, minimally invasive, safe, and cost-effective technique for initial 
translational testing of purported age-modulating agents in humans. Our approach utilizes topical 
application of the intervention being tested to the skin of consenting human subjects. In order to 
sample the treated skin regions, suction blisters will be generated as a source of skin cells 
(keratinocytes) and blister fluid to be used in determining efficacy of the putative anti-aging 
treatment. There are several significant advantages inherent in this new approach. Specifically, 
the topical administration of agents to small areas of skin in humans will obviate the greater risks 
of systemic drug administration. As the target area is relatively small, this approach will require 
significantly less drug than a systemic clinical trial at a much lower cost as well. Moreover, the 
risk to human participants will be minimized as the agent is only delivered into skin, not 
systemically. As our proposed approach is safer than systemic administration and less invasive, 
we anticipate that successful recruitment of volunteer subjects will be much easier which is 
particularly important when dealing with an older cohort. Lastly, multiple ‘experimental’ skin 
sites can be treated simultaneously such that each subject will serve as his/her own control, 
thereby increasing statistical power and validity of the trial.   
 
3 
STUDY DESIGN 
 
Overall study design.  
As shown diagrammatically in Figure 1, consenting subjects would apply the test compound to 
designated experimental and control sites on their forearms for the study period. As discussed 
below, we have opted for daily dosing over a 6-month period. At that point, suction blisters 

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would be generated at each of the sites; this will allow us to harvest both the skin flap and blister 
fluid. The efficacy of the potential age-modulating drug would then be assessed using two 
independent parameters: i) the potential to stop, slow, or reverse the “epigenetic clock” (Specific 
Aim 1) using DNA extracted from the skin and ii) changes in the levels of inflammatory 
mediators (Specific Aim 2) measured in the blister (interstitial) fluid. The topical approach to 
drug pre-screening is ideal for several reasons. First, skin is easily accessible and the applications 
can be handled by the subjects themselves. Second, human skin shows an invariant age-
dependent phenotype characterized by decreased epidermal thickness, changes in the 
vasculature, reduction in the number of melanocytes and dendritic (Langerhans) cells, and lower 
levels of specific collagen types. Third, it is relatively easy to access the treated skin and fluids 
therein using the already established suction blister protocol [15, 16]. Lastly, the cost associated 
with topical application is significantly lower than systemic dosing as one can obtain relatively 
high concentrations of the test agent when it’s being applied to a small area of skin; in this case, 
10 cm ²/site.   
 
4 
SELECTION AND ENROLLMENT OF PARTICIPANTS  
Participants will not be excluded based upon ethnicity or race. For the proposed studies, we will 
draw on the general population of San Antonio and surrounding area which is more than 50% 
Hispanic. We suspect that we will have approximately 50% (or slightly more) Hispanic subjects 
in our proposed study.  In our past studies, our volunteer population has been approximately 50% 
male and 50% female and we anticipate recruiting this equal sex distribution for the proposed 
trial.  All subjects will be in good health, non-smoking persons between the ages of 65 and 95.  
As outlined previously, all ethnicities, racial groups, and both sexes will be studied.  Subjects 
will be enrolled after confirmation of good health by history and physical exam, clinical 
laboratory tests (fasting glucose and lipid profile, metabolic profile, complete blood count, 
Hemoglobin A1C) and 12-lead ECG. The ethnicity of our past study populations generally 
reflected our local population: 47% Non-Hispanic White, 48% Hispanic, and 5% African 
American with an equal gender distribution.    
Vulnerable Populations:  No vulnerable populations will be involved in the proposed protocols. 
 
4.1 Inclusion Criteria  
Participants must meet all of the inclusion criteria to participate in this study: 
• 65-95 years of age. 
• 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). 
• All diseases or infirmities will be clinically stable whether managed by 
medications or not. 
• CLOX score of 10 or greater 

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• Women will be postmenopausal 
• Postmenopausal women taking hormone replacement will be included if they 
have been on a stable dose for ≥6 months 
• Participants will have been vaccinated for COVID-19 prior to beginning the 
treatment course 
• Participants will live within 20 miles of the UTHSA to facilitate home visits (or 
may choose to meet in a mutually convenient location within 20 miles of the 
UTHSA for all home visits) 
4.2   Exclusion Criteria  
All candidates meeting any of the exclusion criteria at baseline will be excluded from 
study participation: 
• Diabetes. 
• History of skin ulcers or poor wound healing, or keloid formers. 
• Smoking (current or within 5 years). 
• Current liver disease (acute or chronic). 
• Treatment for anticoagulation (coumadin or novel oral anticoagulant). 
• Treatment with drugs known to affect cytochrome P450 3A (diltiazem, 
erythromycin, etc. - due to role in rapamycin metabolism). 
• Treatment with an immunosuppressant (prednisone, etc.) within the 6 months. 
• History of recent (within 6 months) Myocardial Infarction or active Coronary 
Disease. 
• Hypersensitivity to rapamycin or petrolatum (ointment vehicle). 
• Tattoo or scar tissue on the forearm in the application area. 
  
4.3 
Study Enrollment Procedures  
Healthy human volunteers of normal cognitive function capable of consenting will be consented 
as study participants. Since the effectiveness of some age-modifying drugs (i.e., RAPA) can 
become more evident with age (Fig. 1B), we will recruit test subjects ranging from 65 to 95 years 
old. Subjects will be non-smokers and will be instructed to continue taking any currently 
prescribed medications. Female subjects will be postmenopausal. All ethnicities and races will be 
included. Prior to initiating treatment, potential subjects will be screened for exclusions, 
including serious co-morbidities, by medical history, clinical labs, and physical examination 
including EKGs. Both male and female subjects will be recruited. This is critically important as 
the age-modulating drugs often show greater efficacy in one sex. For example, RAPA was 
shown to extend lifespan to a greater extent in female mice than in males while acarbose showed 
the reverse [4, 29]. Thus, our goal is to recruit 75 subjects to ensure that there will be 40 
“completers”, approximately half female and half male. Towards this end, we will use the 
Subject Recruitment Call Center established and operated by the Research Core-2 (RC2) of the 
San Antonio OAIC (Pepper Center; Dr. Kellogg, RC2 co-leader). This center has been 
spectacularly successful in meeting subject recruitment goals for clinical trials of age modulating 
agents and has registered over 1800 persons who are willing to participate in future human trials. 

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Other instruments for subject recruitment may also be used. These include:  i) the national online registry 
at https://www.researchmatch.org,  ii) UT Health's Find-A-Study webpage at 
https://vpr.uthscsa.edu/findastudy/;  and  iii) the Barshop Clinical Trials webpage at  
https://barshopinstitute.uthscsa.edu/clinical-trials/; iv) ads may be placed in the San Antonio Express 
News or other local periodicals read by the target population, and v) ads may be placed on Facebook 
and/or other social media sites.   
Once subjects have been appropriately consented and cleared for participation, each individual 
will be given two Topi-CLICK® containers of ointment and complete instructions on the 
application protocol. Initially, we will test 1 concentration of the drug with a “vehicle only” 
(negative control, petrolatum) applied to the opposite arm. Importantly, since each subject will 
serve as his/her own control, no separate placebo group will be needed.   
4.4 
Subject Recruitment and Retention 
Subject recruitment will take place through the Subject Recruitment Call Center established and 
operated by the San Antonio Claude D. Pepper Older Americans Independence Center (SA 
OAIC) Research Core-2 (RC2, Dr. Kellogg Core Co-Leader).  RC2 developed the "Call Center 
and Participant Registry" for efficient subject recruitment.  The public is made aware of the Call 
Center through advertisements placed by the SA OAIC in local newspapers, websites, social 
media, or other online resources, on broadcast media, and through local presentations to civic 
groups.  The Center has one primary phone line that receives phone calls from potential 
participants.  Potential subjects are asked for permission to enter demographic and preliminary 
clinical data that are subsequently entered into a REDCap based registry. The Call Center is 
active 24 hours a day, 7 days a week, and is the main point of entry for participants for aging 
studies such as the present proposal.  RC2 staff determine each caller's study suitability based on 
the caller’s interest, initial screening information, and study eligibility criteria (which can vary 
among ongoing studies).  This subject database currently has records of 1811 persons who are 
willing trial participants.  The REDCap database of potential subjects will be queried for the 
proposed trial as the major source of recruitment, significantly reducing costs for external 
advertising. Enrolled subjects will be seen at in-person monthly Visits 1-6 in their homes (or at 
an alternate convenient site) and contacted bi-weekly by the study team to facilitate retention and 
to confirm treatment compliance. Visits 4 and 6 may be conducted by phone with those subjects who 
have demonstrated their strict compliance to protocol (in Visits 2 and 3) and have experienced no adverse 
effects.  
5 
STUDY INTERVENTION  
5.1 Interventions, Administration, and Duration  
The selection of topical application of 8% RAPA in petrolatum is based on our preliminary data.  
Our preliminary data indicate that topical 8% RAPA ointment: 
a) produces local effects (reduced EC50 for endothelium-dependent vasodilator response to 
methacholine in older, but not younger, persons; it also led to keloid regression); 
b) produces detectable intradermal RAPA levels (as measured with intradermal microdialysis);  
c) is without local or systemic adverse reactions; and  

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d) this concentration of RAPA ointment produces no detectable systemic RAPA levels.  
These preliminary results support our view that this treatment is safe for use in older humans. 
 
 
To validate the proposed technique, RAPA was chosen as our initial test agent; it has been found 
to extend lifespan in laboratory animals [4], be transdermally deliverable with topical application 
(see [24] and Fig. 2), improve epigenetic aging signatures in mice [30, 31], and restore some 
physiological functions in animal models when systemically delivered [7, 18-23, 32]. Moreover, 
we and others have shown that RAPA can be used with relative safety in older humans, even 
with systemic delivery [1, 5, 6]. In preliminary feasibility studies, RAPA ointments (1% or 8%) 
were applied to forearm skin sites and RAPA delivery into the skin interstitial space was 
assessed by placing an intradermal microdialysis probe at each site and then measuring the drug 
levels attained in the collected interstitial fluid by HPLC-mass spec. As shown in Figure 2A, 
significant RAPA levels (11.5±4.0 ng/ml) were seen with a 7 day course of the 8% RAPA 
ointment; while a 1% dose yielded much lower values. Simultaneous blood sampling after 1 
week of 8% RAPA dosing showed undetectable RAPA in the serum; thus, the treatment should 
be efficacious locally without having systemic consequences.   
    
5.1.1 Compounded Investigational Product 
 
5.1.1.1 Rapamycin powder (from a supplier to Doyle’s pharmacy) 
Molecular formula: C51H79NO13 
Synonyms: Rapamune, sirolimus 
PubChem Substance ID 57654583 
CAS No. 53123-88-9 
An FDA-approved macrocyclic triene antibiotic forms a complex with FKBP12 that binds to and 
inhibits the molecular target of rapamycin (mTOR). Rapamycin (RAPA) is a potent 
immunosuppressant and has anticancer activity. 

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i) Mechanism of Action 
The RAPA oral formulation developed by Pfizer (RAPAMUNE, sirolimus) inhibits T 
lymphocyte activation and proliferation that occurs in response to antigenic and cytokine 
(Interleukin [IL]-2, IL-4, and IL-15) stimulation by a mechanism that is distinct from that of 
other immuno-modulators.  In cells, sirolimus binds to the immunophilin, FK Binding Protein-12 
(FKBP-12). The sirolimus: FKBP-12 complex has no effect on calcineurin activity. This 
complex binds to and inhibits the activation of the mammalian Target Of Rapamycin (mTOR), a 
key regulatory kinase. This inhibition suppresses cytokine-driven T-cell proliferation, inhibiting 
the progression from the G1 to the S phase of the cell cycle. Dosage forms and strengths include: 
oral solution (60mg/60mL in amber glass bottle) and oral tablets (0.5, 1, and 2mg). 
ii) Safety 
From oral administration, serious allergic reactions identified by the manufacturer include: 
• swelling of face, eyes, or mouth 
• trouble breathing or wheezing 
• throat tightness 
• chest pain or tightness 
• feeling dizzy or faint 
• 
rash or peeling of skin 
In previous human studies cited earlier in this protocol, topical administration did not produce 
systemic or serious reactions. Potential Risks due to topical RAPA include local skin rash or 
irritation; the drug does not reach detectable levels systemically. Participants will be instructed to 
notify the research team and get help right away if any of the above symptoms of an allergic 
reaction occur. 
iii) Storage 
The powder is packaged in a glass bottle with instructions to keep container tightly closed in a 
dry and well-ventilated place. 
5.1.1.2 Petrolatum (Vaseline, vehicle only) 
i) Mechanism of Action 
Petrolatum is a pale yellow to yellow-colored, translucent, soft unctuous mass. It is odorless, 
tasteless, and not more than slightly fluorescent by daylight, even when melted. Petrolatum is an 
inert material with few incompatibilities. 
ii) Safety 
Petrolatum is mainly used in topical pharmaceutical formulations and is generally considered to 
be a nonirritant and nontoxic material. Animal studies, in mice, have shown petrolatum to be 
nontoxic and non-carcinogenic. Although petrolatum is generally nonirritant in humans 
following topical application, rare instances of allergic hypersensitivity reactions have been 
reported, as have cases of acne, in susceptible individuals following repeated use on facial skin. 
However, given the widespread use of petrolatum in topical products, there are few reports of 
irritant reactions. The allergic components of petrolatum appear to be polycyclic aromatic 
hydrocarbons present as impurities. The quantities of these materials found in petrolatum vary 
depending upon the source and degree of refining. Hypersensitivity appears to occur less with 
white petrolatum and it is often the preferred material for use in cosmetics and pharmaceuticals. 

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iii) Storage 
Petrolatum is an inherently stable material owing to the unreactive nature of its hydrocarbon 
components; most stability problems occur because of the presence of small quantities of 
impurities. On exposure to light, these impurities may be oxidized to discolor the petrolatum and 
produce an undesirable odor. Petrolatum should not be heated for extended periods above the 
temperature necessary to achieve complete fluidity (approximately 70°C/158°F). When heated 
to decomposition it emits acrid smoke and irritating fumes. 
Petrolatum may be sterilized by dry heat. Petrolatum should be stored in a well-closed container, 
protected from light, in a cool, dry place. 
iv) Regulatory Status 
GRAS listed. Accepted for use in certain food applications in many countries worldwide. 
Included in the FDA Inactive Ingredients Database (ophthalmic preparations; oral capsules and 
tablets; otic, topical, and transdermal preparations). 
5.1.2 Packaging 
Drug and placebo will be supplied by the compounding pharmacy in a metered dispenser called 
the Topi-CLICK® container. The opaque Topi-CLICK® dispenses 0.25 mL of ointment, 
protects the product from light exposure, and serves to conceal any subtle differences between 
placebo and investigational product. 
Drug will be labeled as an investigational drug as required by law: 
“Caution: New Drug — Limited by Federal (or United States) law to investigational use.”  
 
5.2 Handling of Study Interventions  
The 8% rapamycin and vehicle-only ointments will be compounded and purchased from Doyle’s 
Professional Pharmacy who have experience compounding rapamycin for topical application in 
human clinical trials.  All the rapamycin ointment and the placebo (petrolatum only) ointment 
will be provided to participants in Topi-CLICK® containers, a pump bottle for use in clinical 
trials that dispense 0.25ml per depression of the pump actuator.  Potency studies show that the 
8% rapamycin in petrolatum retains 96.8% potency after 60 days when stored at room 
temperature.  Dr. Kellogg holds a current IND (#144448) for topical rapamycin ointment used in 
another trial. The FDA determined that the current study of topical rapamycin was "exempt."   
Study drug will be shipped to and stored in a Research Pharmacy and dispensed to participants 
by the research pharmacy pharmacist. The study team will maintain records of drug 
accountability. 
NOTE: RAPA 8% ointment retains 96.8% potency after 60 days at room temperature, as 
documented in IND 144448 under Dr. Kellogg. 
 
5.3 Concomitant Interventions 
5.3.1 Allowed Interventions 
• 
For female postmenopausal participants, hormone replacement therapy use is 
permitted if the subject has been on a stable dose for at least 6 months. 

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5.3.2  Prohibited Interventions 
• Systemic steroid or immunosuppressant therapy 
• Body lotions, astringents, or other skin applications including cosmetics and 
especially sunscreen to the targeted skin area(s) 
• Other antifungal, antibiotic, or topical preparations 
 
5.3.3 Drug interactions 
In previous human studies cited earlier in this protocol, topical administration did not produce 
systemic or serious reactions. Participants will be instructed to notify the research team and get 
help right away if any of the above symptoms of an allergic reaction occur. 
Drug interactions with systemic use include: Inducers of CYP3A4 and P-gp may decrease 
sirolimus concentrations whereas inhibitors of CYP3A4 and P-gp may increase sirolimus 
concentrations. Avoid concomitant use of sirolimus with strong inducers (e.g., rifampin, 
rifabutin) and strong inhibitors (e.g., ketoconazole, voriconazole, itraconazole, erythromycin, 
telithromycin, clarithromycin) of CYP3A4 and P-gp. Avoiding consumption of grapefruit juice 
is also cited. 
 
5.4 Adherence Assessment  
Subjects will be contacted monthly either in person or by phone (Visits 2-7) to verify compliance. In 
addition, subjects will be called approximately 14 days after Visit 1 to ensure that they have been 
following the application directions and have no questions or potential adverse events. Subjects also 
receive a drug diary to help record administrations and any notable effects, which supports reporting 
the participant’s experiences at phone follow-ups and clinic visits. 
Each Topi-CLICK® container will be weighed before being given out and at collection (after 2 
months) to quantify compliance. 
 
 
 
 
 
 
 
 
 
 

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6 
STUDY PROCEDURES 
6.1 
Schedule of Activities 
 
VISIT # (Month #) 
V0 
V1 (0) 
V2 (1) 
V3 (2) 
V4 (3) 
V5 (4) 
V6 (5) 
V7 (6) 
Visit goal 
Consent/ 
Screen 
Pre-
drug 
RAPA/ 
Placebo 
RAPA/ 
Placebo 
RAPA/ 
Placebo 
RAPA/ 
Placebo 
RAPA/ 
Placebo 
RAPA/ 
Placebo 
SCREENING PROCEDURES 
Consent & 
CLOX1=10+ 
X 
 
 
 
 
 
 
 
RAPA/CONTROL OINTMENTS 
60 day supplies of 
study drug 
 
X 
 
X 
 
X 
 
 
SAFETY / ADVERSE EVENTS / METABOLIC 9  
Physical Exam 
X 
 
 
 
 
 
 
X 
ECG/EKG 
X 
 
 
 
 
 
 
 
Self-Reported AEs 
 
 
X 
X 
X 
X 
X 
X 
CBC  
X 
 
 
 
 
 
 
 
Metabolic profile, 
fasting blood, 
sugar/lipids, HbA1c 
X 
 
 
 
 
 
 
 
RAPA blood levels 
 
 
 
 
 
 
 
X 
SUCTION BLISTER COLLECTIONS 
Blister fluid collection 
for inflammatory 
mediators 
 
 
 
 
 
 
 
X 
Blister roof collection 
for DNA methylation  
analysis 
 
 
 
 
 
 
 
X 

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6.2 Schedule of Evaluations  
6.2.1 Visit 0 (60 to 90 minutes) 
 6.2.1.1 Consenting Procedure 
Before any screening procedure is performed, informed consent must be obtained. Eligible 
subjects will be asked to come to the research area to review and sign the consent form. Staff 
conducting the consent process adhere to local standard operating procedure (SOP) for consent 
administration. When scheduling consent appointments, staff will ask participants to arrive 
fasting. 
Participants will be asked at each visit whether they would like to continue participation in this 
study.  
 
6.2.1.2 Screening /Baseline 
Following consent and signature, participants will undergo minimal cognitive screen using the 
CLOX1 test to ensure consent is valid. The CLOX test is a clinical tool widely used in screening 
for cognitive disorders and dementia, which involves a clock drawing task designed to elicit 
executive impairment and discriminate it from non-executive failure.   Study staff will: 
•  Obtain vital signs, height and weight, lab work (fasting) to ensure safety and rule out 
systemic infection (complete blood count [CBC], comprehensive metabolic panel [CMP], 
lipid panel, HbA1C content) 
• Electrocardiogram (ECG/EKG)  
•  Document medical history, concomitant medication review, physical examination, and  
•  Perform evaluation of eligibility criteria listed in Sections 4.1 – 4.2 above. 
 
Additional baseline activities and data collection to include: 
• Current and recent medications within past 6 months 
• Verification of application sites of study drug, RAPA/placebo 
 
6.2.2 Visit 1 (Less than 60 minutes) 
• Verify participant is still willing to participate in study 
• Explain how to apply medication and possible side effects to monitor.  
Use of the study drug 
Each ointment will be applied to a 10 cm² area of skin on the forearm with randomization 
among subjects of which side (left vs. right) receives RAPA vs. placebo to eliminate any 
positional bias. Importantly, each subject will be his/her own control. To permit double-
blinding while avoiding treatment confusion, dispensers will be color coded and subjects 
will have the skin sites similarly colored using surgical markers. The initial application 
will be done for subject education; thereafter, the subjects will apply the compounds daily 
and refresh the pen outlines as needed. Fresh Topi-CLICK® containers will be given to 
each subject on a bi-monthly basis.  

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After ointment application, subjects will cover the site with hypo-allergenic bandage   to 
prevent ointment removal or soiling of clothing. Subjects will be instructed to avoid 
cross-contamination between forearm sites by washing hands between applications and 
using new disposable finger cots (glove-like, but cover only the finger) with each application. 
Subjects will be contacted/visited monthly to verify compliance. Topi-CLICK® containers 
will be weighed before they are given to participants and at collection (after two months) 
to quantify compliance and the amounts used. 
• Dispense study drug, two color coded Topi-CLICK® containers.  
• 
Phone calls will be made to follow up with subjects regarding adherence or AEs approximately 
14 days after Visit 1.  
 
6.2.3 Visit 2 (Less than 60 minutes) 
• Verify participant is still willing to participate in study 
 
6.2.4 Visit 3-6 (Each less than 60 minutes) 
• Verify participant is still willing to participate in study 
• Study drug will be dispensed at Visit #3 and #5 (60-day supply) 
• 
For subjects who have demonstrated outstanding compliance and have experienced no adverse 
events, Visits 4 and/or 6 may be replaced by phone calls. 
 
6.2.5 Visit 7 (up to 4 hours)  
• Verify participant is still willing to participate in study 
• Physical exam 
• Self-reported AEs 
• Rapamycin blood levels 
• Suction blister fluid collection 
• Suction blister skin flap collection 
Process for induction of a suction blister 
Blisters are induced at the sites of rapamycin/placebo administration on the subjects by means of 
a negative pressure cutaneous suction method.  The Negative Pressure Instrument System is a 
commercially available self-contained instrument package that combines all the necessary 
elements to successfully create suction blisters on a patient's skin. The blisters are created 
through the use of suction chambers that are attached to the patient's skin. The instrument 
console contains the power source, vacuum pump, temperature controls and all related controls 
to operate multiple suction chambers. The suction chambers are connected to the console by a 
flexible connection. Each of the chambers is controlled by a preset temperature control to 
provide an optimal skin warming temperature. Both chambers share an adjustable common 
vacuum source that affects all chambers equally. 
1) Assemble the suction device. 
2) Disinfect the orifice plate and the skin of the volunteer using alcohol swabs. 

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3) Attach the suction device within the treatment area on each forearm, by loosely securing 
the chamber with the provided straps. The chamber will adhere to the skin and maintain 
its position once negative pressure is applied. 
4) Turn the suction device on and adjust the pressure to -20kPa (150mmHg) 
5) After 30 minutes, increase the negative pressure to -25kPa (187.5 mm Hg), and then to -
30kPa (225 mmHg) after 60 minutes. Keep the pressure at -30kPa (225 mm Hg) until a 
blister is fully formed. The blister induction phase ranges from 60-180 minutes. 
6) Once the blister is fully formed, release the pressure, and carefully remove the suction 
chamber whilst maintaining integrity of the blister. 
7) Note the time taken for blister induction. 
 
Harvesting the suction blister fluid and blister roof 
1) Using a sterile 1-3 ml syringe with a 23G-26G needle, insert the needle into the 
top/lateral side of the blister roof and slowly aspirate the fluid, avoiding touching the 
floor of the cavity. Withdraw as much fluid as possible, and transfer to a sterile 
microfuge tube. 
2) Remove the blister roof using a sterile scalpel or sterile surgical scissors and store at -
80°C for subsequent nucleic acid isolation. 
3) Apply an antibiotic ointment (such as Neosporin) and a band aid to the blister, as long as 
the subject does not have any allergic reaction to the antibiotic ointment. The blister 
should heal within a week, no anesthesia is required for this procedure. 
 
7 
SAFETY ASSESSMENTS  
Participant safety will be monitored once an individual is enrolled in the study. Dr. Kellogg will 
be responsible for ensuring the first-hand monitoring of the data and safety of study participants 
on a monthly basis. He will be assisted by other members of the study staff. 
7.1 Specification of Safety Parameters 
Protection against risk is paramount in our research and in the operation of our laboratory. The 
risks of the proposed studies are extremely small.  This is by design and reflects the history and 
philosophy of the laboratory.  All of our proposed studies are reviewed and approved by our IRB 
prior to any studies in the clinic or laboratory.  All volunteers will be in good health, as 
documented by a medical history and physical examination performed by overseeing clinical 
staff. Volunteers will be queried using minimally invasive methods.  Antibiotic ointment and 
gauze are used to cover and protect the area of suction blister formation.  In the unlikely event of 
an adverse effect, appropriate medical procedures will be undertaken.  There are no known 
social, legal, or psychological risks.  At the time of collection, all data will be de-identified and 
stored that way so that there is no risk to the privacy of individuals or confidentiality of data.  
Data are stored in electronic form behind institutional electronic firewalls, or in physically 
secured laboratory facilities that are patrolled by armed police forces.  These procedures have 
adequately protected our data for decades and we believe that they will continue to be highly 
effective in the future.  To minimize infection risk at suction blister sites, all subjects will have 

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Version 1.2   21 
the sites treated with topical antibiotic ointment and covered with a bandage or gauze.  They will 
be provided with antibiotic ointment and bandages for use at home until the sites have healed.  
Any participants with an allergy to RAPA and persons with such sensitivity are excluded. Risks 
are minimized by the monitoring procedures we use, the selection of methods, and by the 
monitoring of adverse events and by using minimally invasive harvesting techniques for fluid 
aspiration and blister roof excision. 
7.2 Adverse Events and Serious Adverse Events  
Adverse Event (AE): In general, AE is used very broadly and encompasses physical and 
psychological harms and includes:   
 
 
Any untoward or unfavorable medical occurrence in a human subject, including any abnormal 
sign (for example, abnormal physical exam or laboratory finding), symptom, or disease, 
temporally associated with the subject’s participation in the research, whether or not it is 
considered related to the subject’s participation in the research. 
 
Intercurrent illnesses or injuries should be regarded as adverse events.  Abnormal results of diagnostic 
procedures are considered to be adverse events if the abnormality: 
• 
results in study withdrawal 
• 
is associated with a serious adverse event 
• 
is associated with clinical signs or symptoms 
• 
leads to additional treatment or to further diagnostic tests 
• 
is considered by the investigator to be of clinical significance 
Serious Adverse Event (SAE): Any adverse event that: 
• 
Results in death 
• 
Is life threatening, or places the participant at immediate risk of death from the event 
as it occurred 
• 
Requires or prolongs hospitalization 
• 
Causes persistent or significant disability or incapacity 
• 
Results in congenital anomalies or birth defects 
• 
Is another condition which investigators judge to represent significant hazards 
 
7.3 
Unanticipated Problems Involving Risk to Subjects or Others (UPIRSO) 
Any incident, experience, or outcome that meets all of the following criteria:  
• 
Unexpected in nature, severity, or frequency (i.e. not described in study-related documents such 
as the IRB-approved protocol or consent form, the investigators brochure, etc.) 
• 
Related or possibly related to participation in the research (i.e. possibly related means there is a 
reasonable possibility that the incident experience, or outcome may have been caused by the procedures 
involved in the research) 
• 
Suggests that the research places subjects or others at greater risk of harm (including physical, 
psychological, economic, or social harm). 
 

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7.3.1 Adverse Event Reporting Period 
The study period during which adverse events must be reported is normally defined as the period 
from the initiation of any study procedures to the end of the study treatment follow-up. For this 
study, the study treatment follow-up is defined as 30 days following the last administration of 
study treatment. 
7.3.2 Pre-existing Condition 
A preexisting condition is one that is present at the start of the study. A pre-existing condition should be 
recorded as an adverse event if the frequency, intensity, or the character of the condition worsens during 
the study period. 
 
At screening, any clinically significant abnormality should be recorded as a preexisting condition. At the 
end of the study, any new clinically significant findings/abnormalities that meet the definition of an 
adverse event must also be recorded and documented as an adverse event. 
7.3.3 Abnormal Laboratory Values 
A clinical laboratory abnormality should be documented as an adverse event if any one of the following 
conditions is met:  
• 
The laboratory abnormality is not otherwise refuted by a repeat test to confirm the abnormality 
• 
The abnormality suggests a disease and/or organ toxicity 
The abnormality is of a clinically significant degree requiring active management; e.g. change of dose, 
discontinuation of the drug, more frequent follow-up assessments, further diagnostic investigation, etc. 
 
7.3.4 Hospitalization, Prolonged Hospitalization or Surgery 
Any adverse event that results in hospitalization or prolonged hospitalization should be documented and 
reported as a SAE unless specifically instructed otherwise in this protocol. Any condition responsible for 
surgery should be documented as an AE if the condition meets the criteria for and adverse event.  
 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery are reported as an AE in the 
following circumstances: 
• 
Hospitalization or prolonged hospitalization for diagnostic or elective surgical procedures for a 
preexisting condition (e.g., colonoscopy, SCI annual evaluation, respite care).  
• 
Surgery should not be reported as an outcome of an adverse event if the purpose of the surgery was 
elective or diagnostic and the outcome was uneventful. 
• 
Hospitalization or prolonged hospitalization required to allow efficacy measurement for the study. 
• 
Hospitalization or prolonged hospitalization for therapy of the target disease of the study, unless it 
is a worsening or increase in frequency of hospital admissions as judged by the clinical investigator. 
 
7.4 
Recording of Adverse Events 
At each contact with the subject, the investigator or study staff will seek information about adverse events 
by specific questioning and, if appropriate, by examination.  Information on all AEs will be recorded 
immediately in the source documentation. Site staff will record AEs using the appropriate data collection 
form in REDCap, which will be exported as an AE Log (See Section 9.3) for periodic review, at least 
monthly and ad hoc, depending on severity and expected/unexpected nature of the event. 
 

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The clinical course of each event will be followed until resolution, stabilization, or until it has been 
determined that the study treatment or participation is not the cause. SAEs that are still ongoing at the end 
of the study period will be followed up to determine the final outcome. Any SAE that occurs after the study 
period and is considered to be possibly related to the study treatment or study participation will be recorded 
and reported per Institutional policy and according to FDA requirements. 
7.6 
 Reporting of Serious Adverse Events and Unanticipated Problems 
 
SAEs and UPIRSOs will be reported per local IRB policy and procedure. 
 
Each subject is evaluated for any adverse events (AE). Any event that is reported to either the 
principal investigator or designated research staff by either the subject or medical staff caring for 
the subject and which meets the criteria will be documented. Any AE reported as serious will 
necessitate an adverse event report, which will be submitted to the IRB and to the Pepper Center 
DSMB.   
Unanticipated risks to subjects or others (UPIRSO) that are a result of study participation are 
promptly reported to the IRB and, if deemed appropriate, to the Pepper Center DSMB. The 
report will include a description of the event, when and how it was reported, as well as any 
official chart records or documentation to corroborate the event or the reporting of the event. All 
adverse events will be graded as mild, moderate, or severe. All adverse events will be 
summarized annually and submitted to the IRB. Any action resulting in a temporary or 
permanent suspension of this study (e.g. local site IRB actions) will be reported to FDA or drug 
manufacturer per IRB stipulations.   
7.6.1 Investigator Reporting: notifying DSMB 
Any study-related SAE-UPIRSO, must be reported to the Principal Investigator by telephone 
within 24 hours of the event. To report such events, a Serious Adverse Event (SAE) form must 
be completed and submitted within 24 hours.  The investigator will keep a copy of this SAE form 
on file at the study site.   
 
Report SAEs by email and facsimile to: 
 
Dean L. Kellogg, Jr, MD, PhD 
Professor, Medicine-Geriatrics 
Email: kelloggd@uthscsa.edu 
210-617-5132 fax      210-235-3681 page
 
 
Within the following 48 hours, the Principal Investigator provides further information on the 
SAE or the UPIRSO in the form of a written narrative. This should include a copy of the 
completed SAE form, and any other diagnostic information that will assist the understanding of 
the event.  
 
 
 

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7.6.2 Investigator reporting: notifying the UTHSCSA IRB 
 
Notifying the IRB and or FDA if SAE or UPIRSO 
 
• Within 7 calendar days 
Any study event that is: 
– associated with the use of the study drug 
– unexpected,  
– fatal or life-threatening, and  
 
• Within 15 calendar days 
Any study event that is: 
– associated with the use of the study drug, 
– unexpected, and 
– serious, but not fatal or life-threatening 
-or- 
– a previous adverse event that was not initially deemed reportable but is later found to fit 
the criteria for reporting (reporting within 15 calendar days from when event was 
deemed reportable). 
Any finding from tests in laboratory animals that:  
– suggests a significant risk for human subjects including reports of mutagenicity, 
teratogenicity, or carcinogenicity. 
 
Additional reporting requirements 
 
Post marketing surveillance and adverse events may be submitted on FDA Form 3500A or in a 
narrative format. The contact information for submitting safety reports is noted below: 
 
Food and Drug Administration 
Center for Drug Evaluation and Research 
Division of Dermatology and Dental Products (DDDP) 
5901-B Ammendale Road 
Beltsville, MD 20705-1266 
Phone: (301) 796-2290 
Fax: (301) 796-9712  
 
7.7 
Medical Monitoring 
The Investigator and or Co-PI will review the safety and progress of this study on a monthly 
basis or when needed if SAE or SAE-UPIRSO occurs.   
7.8 Investigator Reporting of Protocol Deviations/Violations 
Departures during the conduct of a research study constitute a protocol deviation, violation or 
exception and as such must be reported to the UTHSCSA IRB. 
 

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Tracking and reporting of protocol deviations and violations to the IRB is the responsibility of 
the PI. To determine whether deviations or violations require prompt reporting or other action, 
refer to the IRB document entitled “Decision Tree – Evaluating Departures” on the IRB website. 
Failure to report departures from the protocol according to IRB policy may constitute possible 
non-compliance, which will require a Prompt Report Form and possible FDA reporting by IRB. 
 
7.8.1 
Deviations and violations may be identified in a number of ways including:  
• 
A report by an individual can be made directly to the IRB Office.  
• 
The IRB may learn of event through its continuing review of ongoing research.  
• 
Compliance reviews (audits) conducted by the Office of Regulatory Affairs and 
Compliance or one of the HSC affiliated institutional compliance offices.  
• 
A report by an individual can be made directly to the Office of Regulatory Affairs 
and Compliance (Hotline) or one of the HSC affiliated institutional compliance 
offices.  
• 
A report by another committee, department, institution, or official.  
• 
An audit or report from the study sponsor or sponsor’s monitoring entity. 
 
7.8.2 
Definitions of Protocol Deviations/Violations  
• 
Protocol deviations – such as out of window visit, missed lab, usually recognized 
after the fact, etc. 
• 
Protocol violations – enrolling an ineligible participant, using wrong consent version, 
willful act of not following protocol 
• 
Emergency 
violations 
Refer 
to 
UTHSCSA 
IRB 
Policy 
website: 
https://research.uthscsa.edu/irb/policy/deviations for more information 
  
7.9 Safety Monitoring 
Data Safety Monitoring Plan (DSMP):  Our studies are designed to explore both the responses 
to topical RAPA on DNA methylation (the Skin Epigenetic Clock) and the altered mechanisms 
involved in inflammation.  Dr. Kellogg will be responsible for ensuring the first-hand 
monitoring of the data and safety of study participants on a weekly and monthly basis. He will 
be assisted by other members of the study staff. A REDCap database will be designed to include 
an inclusion/exclusion checklist that is reviewed and signed by Dr. Kellogg for each subject 
enrolled before study medications are ordered by the investigators.  
 
The San Antonio Claude D. Pepper Older Americans Independence Center Data Safety 
Monitoring Board (DSMB) will be responsible for reviewing clinical trial data from the 
proposed study on an ongoing basis to ensure the safety of study subjects. This DSMB was 
created by the SA OAIC under the auspices of the SA OAIC Research Core 2 (RC2) to ensure 
subject safety and data integrity for studies involving older persons Board members are 
independent, with no vested interest in this study. The DSMB will assign a staff member to 
conduct quarterly assessments for data quality control/assurance on collected data, which is 
reviewed on an annual basis by the Pepper Center Regulatory Coordinator and PI at the time of 

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Version 1.2   26 
preparing continuing review documentation for IRB submission.  This study will be reviewed 
by the Pepper Center (DSMB) at least annually. The Pepper Center DSMB meets 3-4 times a 
year, by teleconference call, to review study progress and participants’ safety. 
8 
INTERVENTION DISCONTINUATION  
Should a subject develop a rash during study intervention, RAPA medication will be stopped and 
their participation will cease. 
In the unlikely event that a study-related death or SAE occurs, the decision to stop the trial, 
either temporarily or permanently, will be the collaborative responsibility of the Pepper Center 
DSMB and the Principal Investigators. 
9 
STATISTICAL CONSIDERATIONS  
9.1 General Design Issues  
The intent of this pilot project is to validate the novel approach of examining age-modulating 
effects of pharmacological agents in humans, in vivo, through local transdermal drug application 
thus avoiding the risks of systemic drug administration.  We propose to study at least 40 older 
persons, age 65-95.  We will study at least 20 women and 20 men.  The test agent for the project 
will be 8% rapamycin (RAPA) ointment with petrolatum-only ointment as a control agent.  
Ointment treatments will be for 6 months.  The duration derives in part from our unpublished 
observation that 8 months of topical 8% RAPA ointment caused significant regression of an 
established keloid in a 63 year old person.  At present, there are no other in vivo data relevant to 
power analyses for our proposed project; however, recently published in vitro work by our 
collaborator, Dr. Horvath, suggests that our approach may be viable in vivo.  In that study, 
Horvath and colleagues examined the in vitro effects of rapamycin on keratinocytes obtained 
from 3 different persons and found that rapamycin retarded keratinocyte aging independently 
from its effects on replicative senescence, proliferation rate or frequency, and differentiation 
(Horvath, Lu et al. 2019).  They also found that rapamycin inhibited cellular senescence, as well 
as epigenetic aging.  Our proposed trial will recapitulate the foregoing in vitro keratinocyte study 
design with substitution of in vivo treatment of keratinocytes in humans, in lieu of in vitro cell 
culture approach. Instead of samples from 3 different persons, we will study 50 different persons, 
half women and half men.  Our study uses a "within-subject" design where each person will be 
his/her own control and thus increase statistical power.  In our pilot trial, we are interested in 
precise estimates of feasibility and acceptability, as well as outcome variability that will aid in 
the planning of future sufficiently powered efficacy trials.  
  
9.2 Sample Size and Randomization 
A sample size of 40 persons with experimental (RAPA) and control samples from each person 
will allow us to be relatively precise in our conclusions regarding feasibility outcomes.  

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9.2.1 Treatment Assignment Procedures 
Participants are both the active comparator and control arm for the study with one forearm 
assigned as the active site and the other the control site. Randomization codes will be assigned to 
each subject for active study drug and placebo. Each participant receives color-coded Topi-
CLICK® containers of the study drug, which will be assigned to the same color-coded patch on 
the corresponding forearm. 
  
9.3 Interim analyses and Stopping Rules 
The Sponsor, Dr. Kellogg, has the authority to stop patient participation, suspend accrual or stop 
the study in its entirety at any time for safety. (in particular any rash or skin reaction will stop 
that subject's experiment.)  Dr. Kellogg will report all events (internal / external) to the 
UTHSCSA Pepper Center DSMB and IRB per the UPIRSO reporting criteria.  UPIRSOs being 
submitted promptly and non-UPIRSOs reported at minimum annually.    
As described above, if an individual event or accumulation of events should occur that warrant 
stopping the study, this will be done by Dr. Kellogg and DSMB.  Dr. Kellogg and DSMB will 
review safety summary reports and they will be reported to the IRB with progress reports or 
more promptly if they meet UPIRSO criteria. 
  
9.4 Primary Objective 
Test whether epigenetic changes in skin are elicited by topical treatments with RAPA 
 
Methylation of cytosine residues in DNA, particularly when found in CpG islands, is often 
associated with silencing of downstream genes. Dr. Horvath, a collaborator on this project, has 
identified a set of 391 DNA methylation marks that change with aging and can be used to assess 
the age of an individual [34, 35]. His novel “epigenetic clock” has been used to predict “time to 
death” and parameters of healthy aging. To determine the effects of topical RAPA on the 
epigenetic clock, DNA will be extracted from the blister skin flaps (keratinocytes) taken after 
treatment from the two sites on each subject using Qiagen DNeasy kits. The genomic DNA will 
be sent to the University of Minnesota Genomics Center for analysis on Infinium Methylation 
EPIC arrays. The data will be analyzed by Dr. Horvath; he will compare the profiles seen with 
RAPA to the profiles seen at the placebo site for each individual focusing on the methylation 
marks that comprise the aging clock. 
 
9.5 Secondary Objectives 
Test whether baseline inflammation is affected by topical RAPA  
 
In order to examine the effect of topical RAPA (or any future test drug) on the inflammatory 
milieu in skin, Luminex-based assays will be performed on the interstitial fluid collected from 
suction blisters at the end of the 6 month treatment period. These allow simultaneous 

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measurement of ≥29 different human cytokines/chemokines from as little as 30µl. It is 
anticipated that pro-inflammatory cytokines (i.e., IL-12, IL-6, TNF-α) would be elevated in this 
older population, so a decrease in their levels upon treatment with RAPA (but not the vehicle) 
would support an anti-aging potential. Since inflammation underlies many age-associated 
pathologies, this finding would have broad implications. For this reason, we will also perform 
sensitive ELISAs for three analytes known to play a functional role in regulating age-associated 
inflammatory phenotypes. These will include: i) interleukin-6 (IL-6); although present in the 
Luminex panel, IL-6 detection often requires a more sensitive ELISA [1]; ii) sICAM-1 (soluble 
intercellular adhesion molecule-1, a biomarker of vascular endothelial cell activation/damage) 
[36] and iii) RAGE (receptor for advanced glycation end products) [37, 38]. Validated ELISA 
kits are commercially available for these three analytes and have been tested by us with sera 
from the subjects in the pilot human trial of oral RAPA (Fig. 4). Sufficient blister fluid will be 
recovered in order to perform all of the tests outlined including: i) RAPA levels by HPLC-mass 
spec; ii) Luminex multiplex cytokine arrays; and iii) ELISAs for IL-6, RAGE, and sICAM-1. 
Remaining fluid will be aliquoted and frozen at -20°C. 
9.6 Data Analyses 
As pilot studies are preparatory investigations that provide specific information necessary for 
planning subsequent definitive trials. A major purpose of the present proposal is to perfect the 
study design, measures, procedures, recruitment criteria, and operational strategies for use in 
larger and/or longer subsequent trials with rapamycin and/or other purported anti-aging agents. 
 Our project will provide the means to evaluate the technical aspects of our novel approach of 
testing topical treatments with simultaneous untreated controls within individual subjects.  This 
pilot project will serve as a platform to generate preliminary data and foster new methodological 
development. Our pilot 'proof of concept' study is designed so that the information gained can be 
put to optimal use in future studies that build on scientific evidence of the efficacy and reduced 
costs of our approach.  
Our proposed 'proof of concept' trial will contribute to the development and design of future 
(larger and/or longer) studies by: 
– 
Refining the research hypotheses 
– 
Identifying barriers to successful study completion 
– 
Evaluating acceptability of methods and instruments to participants 
– 
Estimating the time required for study participation 
– 
Providing estimates of missing data and dropout 
– 
Estimating rates and variability in outcomes for future studies 
– 
Testing mechanistic efficacy/ ‘proof of concept’ for future trials 
Regarding analyses of inflammatory mediators:  
Assay results will be analyzed by 2 factor ANOVA (RAPA vs Placebo as within factor and sex 
as second factor); power calculations based on our prior studies with systemically administered 

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rapamycin indicate that N=9 may be needed for those inflammatory mediators we previously 
found to decrease significantly with systemic RAPA (ICAM-1 and RAGE).  Calculations 
indicate that as many as 141 samples may be needed for others (IL-6) that we have not found to 
differ significantly with systemic RAPA. These estimates are based on unpaired comparisons 
between subjects who received systemic RAPA or placebo for 8 weeks. The design for the 
proposed trial permits each subject to serve as his/her own control and thus should reduce these 
estimates. 
 
10 
DATA COLLECTION AND QUALITY ASSURANCE 
10.1 Data Integrity 
Data integrity will be assessed by review of the written/computer (REDCap) documents used to 
record all collected data on a weekly basis. The representational faithfulness (composed of four 
essential qualities or core attributes: completeness, currency/timeliness, accuracy/correctness and 
validity/authorization data) of all data to the true state of the measurement that the information 
represents will be the responsibility of Dr. Kraig, Dr. Kellogg and study coordinator. 
 
10.2 Data Management  
Data integrity will be assessed by review of the written/computer documents used to record all 
collected data on a weekly basis. The representational faithfulness (composed of four essential 
qualities or core attributes: completeness, currency/timeliness, accuracy/correctness and 
validity/authorization data) of all data to the true state of the measurement that the information 
represents will be the responsibility of the Principal Investigators and the study coordinator. 
10.3 Quality Assurance  
10.3.1 
Training 
All study personnel interacting with human subjects or with access to PHI will 
complete all human subjects training to include Good Clinical Practice training.  
 
10.3.2 Quality Control Committee  
 We will use the Regulatory Office and Data Safety Monitoring Board operated by 
RC2 to ensure subject safety and that all regulatory aspects of human trials are met. 
   
10.3.3 Study Monitoring Plan 
 The Principal Investigators will ensure that the designated regulatory coordinator or 
other quality assurance reviewer is given access to all the above noted study-related 
documents and study related facilities (e.g. pharmacy, diagnostic laboratory, etc.), and 
has adequate space to conduct study monitoring visits as assigned. 
 
10.3.4 Monitoring and Auditing 
The Principal Investigators will permit study-related monitoring, audits, and 
inspections by the IRB, the funding sponsor, the OAIC Pepper Center Data Safety 

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and Monitoring Board (DSMB), government regulatory bodies, and University 
compliance groups of all study related documents (e.g. source documents, regulatory 
documents, data collection instruments, study data etc.). The investigators will 
ensure the capability for inspections of applicable study-related facilities (e.g. 
pharmacy, diagnostic laboratory, etc.). 
Participation as an investigator in this study implies acceptance of potential 
inspection by government regulatory authorities and applicable University 
compliance and quality assurance offices. 
11 
PARTICIPANT RIGHTS AND CONFIDENTIALITY  
11.1 Institutional Review Board (IRB) Review 
This protocol and the informed consent document and any subsequent modifications will be 
reviewed and approved by the IRB responsible for oversight of the study. The IRB responsible 
for review of this study will be the University of Texas Health Science Center San Antonio 
Institutional Review Board (UTHSA IRB). 
11.2 Informed Consent Forms 
Consent forms will be IRB approved and the participant will be asked to read and review the 
document. The investigator will explain the research study to the participant and answer any 
questions that may arise. A verbal explanation will be provided in terms suited to the 
participant’s comprehension of the purposes, procedures, and potential risks of the study and of 
their rights as research participants.  Participants will be informed that participation is voluntary 
and that they may withdraw from the study at any time, without prejudice, and that the quality of 
their medical care will not be adversely affected if they decline to participate in this study. 
Participants will have the opportunity to carefully review the written consent form and ask 
questions prior to signing. The participants will be given a copy of the ICF so that they may 
discuss the study with their family or surrogates or think about it prior to agreeing to participate. 
The informed consent process will be conducted and documented in the source document 
(including the date), and the form signed, before the participant undergoes any study-specific 
procedures. A copy of the signed informed consent document will be given to the participants for 
their records.  
11.3 Participant Confidentiality  
Any data, specimens, forms, reports, video recordings, and other records that leave the site will 
be identified only by a participant identification number (Participant ID, PID) to maintain 
confidentiality.  All records will be kept in a locked file cabinet.  All computer entry and 
networking programs will be done using PIDs only. Information will not be released without 
written permission of the participant, except as necessary for monitoring by IRB, the FDA, the 
NIA, and the OHRP. 
11.4 Study Discontinuation  
The study may be discontinued at any time by the IRB, the NIA, the OHRP, the FDA, or other 
government agencies as part of their duties to ensure that research participants are protected.  

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12 
ETHICAL CONSIDERATIONS 
 This study is to be conducted according to US and international standards of Good Clinical 
Practice (FDA Title 21 part 312) applicable government regulations and Institutional research 
policies and procedures. 
This protocol and any amendments will be submitted to the Institutional Review Board (IRB), in 
agreement with local legal prescriptions, for formal approval of the study conduct. The decision 
of the IRB concerning the conduct of the study will be made in writing to the investigator and a 
copy of this decision will be provided to the funding sponsor before commencement of this 
study.   
All subjects for this study will be provided a consent form describing this study and providing 
sufficient information for subjects to make an informed decision about their participation in this 
study.  
 
13 
COMMITTEES 
 The San Antonio Claude D. Pepper Older Americans Independence Center Data Safety 
Monitoring Board (DSMB) will be responsible for reviewing clinical trial data from the 
proposed study on an ongoing basis to ensure the safety of study subjects and validity and 
integrity of the data.   
14 
PUBLICATION OF RESEARCH FINDINGS 
Publication authorship will be determined by the relative contributions of the PIs. It is likely that 
the first paper from this work will demonstrate validity of the topical age-modulating assay using 
rapamycin as the test drug. In this case, as in our previous paper (1), Dr. Kraig would likely 
handle much of the bench data analysis and would be first author, while Dr. Kellogg would have 
overseen all clinical aspects and comprehensive data analysis, so he would be the appropriate 
senior author. However, if the epigenomic data were used more extensively in a second paper, 
then Dr. Horvath might be first and the two PIs would have shared senior authorship. We make it 
a priority to discuss authorship openly before writing the paper(s).     
 
Dissemination Plan 
This trial has been registered in ClinicalTrials.gov; Identifier NCT04608448.  All consent 
documents for the trial will include a specific statement concerning ClinicalTrials.gov. Results 
will be submitted to ClinicalTrials.gov as required.  UT Health San Antonio has an internal 
policy that ensures that clinical trials registration and results reporting occur in compliance with 
policy requirements. Open access publication of results will also be made as significant results 
are obtained. 

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15 
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16 
SUPPLEMENTS/APPENDICES