Clinical trial NCT03253913
Conditions: Lymphangioleiomyomatosis
Sponsor: University of Cincinnati
Start date: 2018-03-31; Completion date: 2022-10-15
Enrollment: 25 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
RESULT is a phase II dose-escalating, open-label, safety and efficacy study to determine if there is a potential benefit of resveratrol in combination with sirolimus in patients with lymphangioleiomyomatosis (LAM). The primary study objective is to assess the change in serum vascular endothelial growth factor-D (VEGF-D) level after 24 weeks of treatment with a combination of resveratrol and sirolimus as compared to the VEGF-D level in patients on a stable dose of sirolimus alone. The secondary objectives of this study include an assessment of the safety and adverse effect profile of combined resveratrol and sirolimus in adult patients with LAM, and to determine the effect of treatment with a combination of resveratrol and sirolimus on changes in lung function and quality of life.

Detailed description:
Study Design RESULT was an open label, dose-escalation, phase 2 study with longitudinal repeated measures. In order to be eligible for trial inclusion, patients had to satisfy all of the following criteria: 1) confirmed diagnosis of LAM per the American Thoracic Society/Japanese Respiratory Society Clinical Practice Guidelines14, 2) age ≥18 years with signed informed consent, 3) on sirolimus for at least 20 weeks, and 4) VEGF-D stabilization as demonstrated by two stable values post initiation of sirolimus and drawn at least 12 weeks apart from each other. Key exclusion criteria included inability to provide consent, active listing for lung transplantation, enrolled in another interventional clinical trial, known allergy or hypersensitivity to resveratrol, and current or planned pregnancy in the next six months.

RESULT was a single site study conducted at the University of Cincinnati (UC). Study enrollment was open to LAM patients throughout the United States with travel reimbursement provided through the study. The study was reviewed and approved by UC's Institutional Review Board (IRB 2016-4904). An independent Data and Safety Monitoring Board (DSMB) was convened and met every six months to ensure the safety of study participants. All subjects provided written informed consent prior to the conduct of any study related activity.

Outcome Measures The primary objective of this study was to assess the change in serum VEGF-D value after 24 weeks of treatment with a combination of resveratrol and sirolimus, as compared to the historical VEGF-D values of patients prior to the study on sirolimus alone. Serial assessments of serum VEGF-D, pulmonary function tests (PFTs), treatment related adverse effects (AEs), complete blood count, liver and renal function tests, and HRQOL were performed at baseline and at 8, 16, and 24 weeks. In addition, sirolimus trough levels, measured 24 +/- 4 hours following the last dose, were assessed at baseline and at weeks 2, 4, 8, 12, 16, 18, 20 and 24. VEGF-D quantification was performed at the Translational Trial Development and Support Laboratory based at Cincinnati Children's Hospital Medical Center in a College of American Pathologists (CAP)/Clinical Laboratory Improvement Amendments (CLIA) approved manner. All other laboratory assessments were performed via Quest Diagnostics. HRQOL was measured by using four validated scales: St. George's Respiratory Questionnaire (SGRQ), University of California San Diego Shortness of Breath score (SDSOB), EuroQol visual analogue scale (EQVAS), and A Tool to Assess Quality of Life in LAM (ATAQ-LAM).

Study Drug Resveratrol used in this study was obtained from Evolva and manufactured by fermentation using genetically modified yeast in a process that results in trans-resveratrol with a purity of at least 98%, and packaged into capsules with each capsule containing 125mg resveratrol. The capsules were independently verified for content by mass spectrometry performed at UC and for sterility by microbial testing at Q Laboratories Inc., Cincinnati. Investigational new drug (IND) designation for studying Resveratrol in this trial was obtained from the United States Food and Drug Administration (FDA): IND 131722; IND Sponsor: N. Gupta.

Resveratrol was administered in a stepwise dose escalating fashion at 8-week intervals with assessment of VEGF-D, PFTs, AEs, and HRQOL prior to each dose escalation. The starting dose of resveratrol was 250mg once daily for the first 8 weeks, followed by 500mg daily from Weeks 8 - 16, and 500mg twice daily for the last 8 weeks (Weeks 16 - 24), with provisions for dose reduction if necessitated by AEs. Treatment related AEs were tabulated using the National Cancer Institute's Common Terminology Criteria for Adverse Events (CTCAE) version 4.

Arms: arm 1: Resveratrol 250mg daily for the first 8 weeks, followed by 250mg twice daily for the next 8 weeks, and then 500mg twice daily for the last 8 weeks.

Patients will be on a stable background therapy of sirolimus prior to enrolling and will continue on that regimen throughout the study.
Interventions: intervention 1: Sirolimus intervention 2: Resveratrol
Intervention descriptions: intervention 1: Patients will have been on a stable dose of sirolimus prior to enrolling and will continue that throughout the study. intervention 2: Escalating doses of resveratrol will be given throughout the study. Patients will start at 250mg daily for 8 weeks, followed by 500mg daily for 8 weeks, and 500mg BID for the last 8 weeks.

Protocol documents:
Prot_SAP_001.pdf: 
 
 
Official Title of the study:  Resveratrol and Sirolimus in Lymphangioleiomyomatosis Trial 
 
 
NCT number:  NCT03253913 
 
 
Date of the document:  July 31, 2018 


 
1 
MEDICAL IRB RESEARCH PROTOCOL 
________________________________________________________________ 
Protocol Synopsis 
Title 
Resveratrol and Sirolimus in LAM Trial 
Short Title 
The RESULT Trial 
Rationale 
Lymphangioleiomyomatosis (LAM) is a rare, progressive diffuse cystic lung 
disease characterized by the infiltration of lung parenchyma with abnormal smooth 
muscle cells, leading to progressive destruction of lung parenchyma. The origin of 
these smooth muscle cells is unknown, however, lung involvement happens as a 
result of metastatic spread of these smooth muscle cells via blood and lymphatics, 
thus characterizing LAM as a low-grade, destructive, metastasizing neoplasm. 
LAM is seen primarily in women with only a few reported cases in men. LAM can 
be seen in association with Tuberous Sclerosis Complex (TSC-LAM), or occur 
sporadically (S-LAM). Both TSC-LAM and S-LAM occur as a result of mutations 
in one of the two TSC genes: TSC1 or TSC2. Mutations in the TSC genes lead to 
constitutive activation of the mammalian target of rapamycin (mTOR) pathway, 
which drives cell proliferation and lymphangiogenesis.  
 
Sirolimus forms a complex with FKBP12, which binds to mTOR and blocks 
activation of downstream kinases, restoring homeostasis in cells with defective 
TSC function.  Sirolimus has been shown to shrink tumors in animal models of 
TSC deficiency. The Multicenter International LAM Efficacy of Sirolimus 
(MILES) Trial was a double blind, randomized, parallel group trial of one year of 
treatment with sirolimus versus placebo followed by one year of observation. 
While patients treated with placebo lost approximately 10% of their lung volume 
in the treatment phase, patients treated with sirolimus exhibited stabilization of 
their lung function, and had an improvement in their functional performance and 
quality of life. During the observation year, lung function decline resumed in the 
sirolimus group and paralleled that of the placebo group. The results from the 
MILES trial formed the basis for a recent FDA approval for sirolimus as a 
treatment for LAM.  
 
However, the resumption of lung function decline following drug stoppage 
suggests that the function of sirolimus is suppressive rather than curative. In 
addition, while the drug was well tolerated for the trial duration, the long-term 
safety and efficacy of sirolimus in LAM remains unclear. These limitations of 
sirolimus highlight the need to explore novel treatment opportunities for patients 
with LAM.  
 
Resveratrol (trans-3, 5, 4′-trihydroxystilbene) is a naturally occurring polyphenol 
found in grapes, berries, and red wine. Multiple studies have shown resveratrol to 


 
2 
have anticancer, anti-inflammatory, and anti-oxidant properties. Resveratrol has a 
complex mechanism of mTOR regulation. In addition, resveratrol inhibits the 
activity of Akt, AMPK and S6K1. Recent pre-clinical studies performed on TSC2 
null cells as well as murine models have demonstrated that a combination of 
resveratrol and sirolimus leads to downregulation of autophagy and promotes 
apoptosis in TSC2 null cells, decreases the metastatic capability of uterine 
leiomyoma-derived smooth muscle cells, and causes a significant reduction in the 
size and growth of TSC2 deficient xenograft tumors.  
 
The unmet need for a remission inducing (cytotoxic) as opposed to a suppressive 
(cytostatic) treatment option for patients with LAM, combined with strong pre-
clinical evidence demonstrating the efficacy of a combination of sirolimus and 
resveratrol form the basis for this dose-escalating, safety and efficacy clinical trial. 
Results from this trial will pave the way for pivotal phase III, randomized, 
controlled, efficacy studies of combined resveratrol and sirolimus in LAM.  
Clinical Phase 
II 
IND Sponsor 
Nishant Gupta, University of Cincinnati, Cincinnati OH 
IND Number  
131722  
Principal 
Investigator 
Nishant Gupta, University of Cincinnati, Cincinnati OH 
Participating 
Site(s) 
University of Cincinnati - Cincinnati, OH 
Accrual 
Objective 
20 evaluable subjects 
25 target enrollment 
Study 
Objectives 
Phase II dose-escalating, open-label, safety and efficacy study to determine if there 
is a potential benefit of resveratrol in combination with sirolimus in patients with 
LAM. Based on the results of this trial, additional phase III clinical development 
trials may be designed.  
Study Design  
Phase II: Open label, intention to treat study 
Study 
Duration 
24 weeks treatment  
Primary 
Objective 
Assess the change in serum VEGF-D level after 24 weeks of treatment with a 
combination of resveratrol and sirolimus as compared to the VEGF-D level in 
patients on a stable dose of sirolimus alone.  
Secondary 
Objectives 
1. Assess the safety and adverse effect profile of combined resveratrol and 
sirolimus in adult patients with LAM.  


 
3 
2. Determine the effect of treatment with a combination of resveratrol and 
sirolimus on changes in lung function and quality of life. 
3. Determine the feasibility and reliability of performing regular home 
spirometry in patients with LAM.  
Inclusion 
Criteria 
Subjects enrolled in the trial must meet all of the following criteria. 
1. Definitive diagnosis LAM based on the presence of characteristic cystic 
change on high-resolution computed tomography (HRCT) of the chest. The 
diagnosis must be confirmed by one of the following: 
A) Histopathological confirmation by biopsy (lung, abdominal mass, 
lymph node or kidney or cytology from thoracic or abdominal sources 
revealing HMB45+ staining of spindled/epithelioid cells)  
B) Compatible chest CT scan findings in the setting of tuberous sclerosis, 
angiomyolipomas (diagnosed by CT, MRI by the site radiologist or 
biopsy) or chylous pleural effusion (verified by tap)  
C) Chest CT scan findings compatible with LAM and a VEGF-D level ≥ 
800pg/ml. 
 
2. Age 18 years or greater. 
3. Signed and dated informed consent  
4. Currently on sirolimus for treatment of LAM for at least 20 weeks 
 
5. Evidence of disease stabilization on sirolimus as demonstrated by two 
stable values of serum VEGF-D post initiation of sirolimus drawn at least 
12 weeks apart from each other. For the purpose of this study, a variation in 
serum VEGF-D of less than or equal to 15% is considered stable. 
Exclusion 
Criteria  
Subjects who meet ANY of the following criteria are not eligible for enrollment as 
study participants:  
1. Known allergy or hypersensitivity to Resveratrol 
2. Inability to provide informed consent 
3. Active enrollment in other clinical drug trials for LAM 
4. Pregnant or plan to become pregnant in the next 6 months 
5. Breast feeding 
6. Inability to comply with pulmonary function tests or follow up visits 
7. Inadequate contraception 
8. Use of estrogen containing medications within the 30 days prior to 
randomization 
9. History of organ transplant 
10. Actively listed for lung transplantation 
11. Inability to comply with study procedures or attend scheduled study visits 
12. Any clinically significant medical disease (other than LAM) that is 
associated with an expected survival of less than 2 years, or likely to 
impact the ability of the patient to participate in the study in the opinion of 
the investigator, or impact the study efficacy or safety assessments.  


 
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Investigational 
Product 
Resveratrol at escalating doses starting at 250mg per day to a maximum of 
1000mg per day 
Study 
Procedures  
The primary objective of this trial is to evaluate the change in serum VEGF-D after 
24 weeks of combined resveratrol and sirolimus as compared to the baseline serum 
VEGF-D value on sirolimus alone. Secondary objectives include an assessment of 
the safety and tolerability of resveratrol in combination with sirolimus in patients 
with LAM, as well as the effect of combined resveratrol and sirolimus on lung 
function and quality of life. The frequency, severity and duration of all adverse 
events, regardless of cause, will be tabulated by body system and by severity using 
the NCI Common Terminology Criteria for Adverse Events v4.0. Subjects will 
have a medical history, physical examination, and laboratory measurements of 
complete blood count, renal and liver function tests, serum VEGF-D, and sirolimus 
levels drawn at every in-person visit. Pre and post bronchodilator spirometry 
measurements will be performed at each visit. St. George’s respiratory 
questionnaire (SGRQ), SD-SB, EuroQOL and ATAQ LAM will be used for 
quality of life assessment, and these questionnaires will be filled out at each in-
person study visit.  
Patients enrolling in RESULT will be trained on proper technique for home 
spirometry using an advanced vertical turbine based device that records and 
wirelessly transmits full flow volumes loops. Once a week, patients will perform at 
least three spirometry maneuvers that pass ATS/ERS acceptability criteria and 
choose the best value as their weekly FEV1 and FVC. All spirometric data will be 
reviewed by an expert pulmonary physiologist, who will judge the spirometry data 
and loops for intra-test acceptability and reproducibility as specified in the 
ATS/ERS guidelines, as well as provide near real time electronic feedback to 
patients regarding their technique. We will correlate spirometry values and trends 
(absolute as well as rate of decline) obtained from weekly home spirometry with 
the office-based spirometry values, as well as serum VEGF-D, and quality of life 
assessment scores. 
Data Analysis  
The analysis will be based on an intention to treat design. All subjects that receive 
study drug will be included in the safety and efficacy analysis set. The primary 
analysis will occur after the last enrolled participant has completed the 24-week 
visit. All analyses will be performed using SAS version 9.4. A p-value less than 
0.05 will be considered as statistically significant. Paired t-test will be performed 
to detect the difference of serum VEGF-D after 24 weeks of treatment with the 
serum VEGF-D prior to enrolment. The response variable VEGF-D will be 
measured repeatedly, at baseline and at weeks 8, 16, and 24. The change from 
baseline to the other study time points will indicate the treatment effect with 
resveratrol and sirolimus.  Initially, by using the paired t-test we will test whether 
these effects are statistically significant. Multivariate linear regression models will 
be used to assess the relationship between serum VEGF-D change and the 
covariates including patient’s clinical or demographic factors. A repeated measures 
ANOVA model and linear mixed models will be performed to compare the serum 
VEGF-D values at follow-up visits to the baseline taking into account the 


 
5 
correlation between repeated measurements within the same patient.  
 
Subjects will be evaluable for safety if they have received at least one dose of the 
study drug. The frequency, severity and duration of all adverse events, regardless 
of cause, will be recorded on the case report forms. The frequency and severity of 
adverse events will be calculated by patient, with each patient counted once using 
the most severe grade experienced. The duration of adverse events will be 
calculated by the number of days each event persisted. Adverse events will be 
tabulated by body system and by severity using the NCI Common Terminology 
Criteria for Adverse Events (CTCAE) v4.0, which can be obtained from the CTEP 
website: http://ctep.cancer.gov/reporting/ctc.html. Tables will be generated for all 
adverse events including those that are judged to be possibly, probably or 
definitely related to the drug. In order to assess the safety and adverse effect profile 
of resveratrol in LAM, we will assign a binary indicator variable to indicate a 
patient with or without adverse events. A multivariable logistic regression model 
will be used to assess of the effect of the covariates on the probability of having 
adverse events for patient with LAM. All interested factors will be adjusted in the 
logistic regression model. Model selection will be based on stepwise criterion 
when appropriate.  
 
Lung function parameters (FEV1 and FVC) will be measured at the baseline visit, 
and at every in-person visit. DLCO will be measured at the baseline visit and the 
last study visit. Similar analysis plan as VEGF-D will be employed for the lung 
function parameters. We will use the following questionnaire-based assessments to 
determine the quality of life in this trial: St. George’s Respiratory Questionnaire 
(SGRQ), EuroQOL scale, San Diego Shortness of Breath scale (SD-SOB), and A 
Tool to Assess Quality of Life in LAM (ATAQ-LAM). Written permission has 
been obtained to use all these questionnaires. The scores on each of these 
questionnaires will be calculated at baseline, and at every in-person visit. We will 
compare median values at baseline and 24 weeks with the Wilcoxon signed rank 
test for each of these scales. 
 
The spirometry values will be expressed as mean ± SD. All spirometry maneuvers 
will be checked for acceptability and reproducibility as specified in the ATS/ERS 
criteria by an expert pulmonary physiologist. The spirometry values obtained from 
maneuvers that do not satisfy the ATS/ERS criteria for acceptability will not be 
used in the primary analysis. If spirometric values meet the acceptability criteria 
but fail to satisfy the repeatability criteria, those values will be graded for quality, 
but will be included in the primary analysis. Overall compliance with weekly home 
spirometry will be recorded and reported as a percentage. Comparison of home 
spirometry and office-based spirometry values will be performed using the Bland-
Altman method at baseline, and at each time frame that patients get an office-based 
spirometry (i.e. every 8 weeks). Rate of change of FEV1 (slope) over time will be 
calculated by using all available values (without imputations) in a linear mixed 
effects model after considering the correlation of the observations within each 
subject, and the time between measurements will be treated as a fixed effect. In 


 
6 
order to analyze if the trends obtained from short-term follow up with frequent 
home spirometry correlate with overall disease trajectory, we will compare the rate 
of decline obtained after 8 weeks of home spirometry to the overall rate of decline 
obtained after 24 weeks of follow up. A similar analysis plan will be employed for 
serum VEGF-D, and other biomarkers. All statistical analyses for this study will be 
performed using SAS, version 9.4 (SAS Institute Cary NC). 
Sample Size  
The mean serum VEGF-D level at baseline was approximately 2,000pg/ml in both 
the MILES trial and the everolimus trial. In both studies, treatment with mTOR 
inhibitors resulted in an almost 50% reduction in the serum VEGF-D levels. Since 
patients in our study will be on sirolimus prior to enrolment, we estimate that the 
starting serum VEGF-D level in our study will be approximately 1,000pg/ml. The 
largest change between serial VEGF-D levels in the placebo group of MILES was 
42%. Thus, VEGF-D responders in MILES trial were conservatively defined as 
patients in whom serum VEGF-D decreased to a value greater than 42% from its 
baseline. Accordingly, we made our power calculations based on the ability to 
show a greater than or equal to 42% change in serum VEGF-D levels. Assuming 
the correlation between the baseline and subsequent VEGF-D levels is 0.7, a 
sample size of 20 subjects will provide 80% power to detect a mean VEGF-D 
difference from baseline to post-treatment of 420pg/ml or higher with the level of 
significant of 0.05 and a standard deviation of 800 for the difference. Allowing for 
a 20% dropout rate, we expect to recruit 25 patients for our study. Sample size 
calculations were done using SAS version 9.4. 
 
 
 


 
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Table of Contents 
 
1. Participating Institutions/Investigators Table (contact information) ..................... 10 
2. Glossary of Abbreviations ................................................................................................. 11 
3. Background and Rationale ............................................................................................... 13 
3.1 Background: ................................................................................................................................ 13 
3.2 Role of Sirolimus in LAM ........................................................................................................ 13 
3.3 Serum Vascular Endothelial Growth Factor-D (VEGF-D) as a diagnostic, prognostic, 
and predictive biomarker of LAM ................................................................................................ 14 
3.4 Resveratrol and its role in treatment of LAM ..................................................................... 16 
3.5 
Role of home spirometry in pulmonary disorders .......................................................... 23 
3.6 
Rationale for the proposed study ........................................................................................ 24 
4. Study Objectives and Endpoints: .................................................................................... 25 
4.1 Primary Endpoint: ..................................................................................................................... 25 
4.2 Secondary Endpoints: ............................................................................................................... 25 
5. Study Design: ....................................................................................................................... 26 
5.1 Identification and Screening:................................................................................................... 26 
5.2 Inclusion Criteria: ...................................................................................................................... 26 
5.3 Exclusion Criteria: ..................................................................................................................... 26 
5.4 Recruitment: ............................................................................................................................... 27 
5.5 Study Drug Treatment: ............................................................................................................. 27 
5.6 Treatment Regimen: .................................................................................................................. 28 
5.7 Home Spirometry ....................................................................................................................... 28 
6. Study Procedures and Timelines ..................................................................................... 30 
6.1 Study and Timeline Overview ................................................................................................. 30 
6.2 Schedule of Events: .................................................................................................................... 31 
6.3 Study Visits .................................................................................................................................. 32 
6.4 Enrollment ................................................................................................................................... 35 
6.5 Patient retention and drug compliance ................................................................................. 35 
6.6 Consent for future contact ....................................................................................................... 36 
6.7 Consent for continuing data review of medical records .................................................... 36 
6.8 Consent for storage of blood samples for future use .......................................................... 36 
6.9 Consent for participation in the home spirometry substudy ............................................ 36 
7. Stopping rules ...................................................................................................................... 37 
7.1 Participant withdrawal criteria .............................................................................................. 37 
7.2 Criteria for terminating the study .......................................................................................... 37 
8. Study variables and measuring methods ....................................................................... 38 
8.1 Physical Exams: .......................................................................................................................... 38 
8.2 Pulmonary function testing ...................................................................................................... 38 
8.3 Questionnaires ............................................................................................................................ 38 
8.4 Biomarker Analyses ................................................................................................................... 39 
8.5 Home Spirometry ....................................................................................................................... 39 
9. Risks ....................................................................................................................................... 40 
9.1 Risks of the investigational product ....................................................................................... 40 
9.1.1 Pharmacokinetics of resveratrol in humans ................................................................................ 40 


 
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9.1.2 Safety Profile of Resveratrol in Humans ..................................................................................... 40 
9.2 Risks of study procedures ......................................................................................................... 42 
9.3 Adequacy of protection against risks ..................................................................................... 42 
9.4 Plans for assuring compliance with safety guidelines and regulations ........................... 43 
10. Benefits ................................................................................................................................ 44 
10.1 Benefits of the investigational agent ..................................................................................... 44 
10.2 Importance of the knowledge to be gained ......................................................................... 44 
10.3 Benefits of Study Procedures ................................................................................................. 44 
11. Drug and Drug Procurement: ........................................................................................ 45 
11.1 Resveratrol: ............................................................................................................................... 45 
11.2 Sirolimus: ................................................................................................................................... 46 
11.3 Accountability of Investigational Product .......................................................................... 47 
11.4 Assessment of compliance with the investigational product ........................................... 47 
12. Modification or discontinuation of the investigational product ............................. 48 
12.1 Modification of investigational product .............................................................................. 48 
12.2 Premature discontinuation of investigational product .................................................... 48 
12.3 Medication adjustments .......................................................................................................... 48 
12.3.1 Dose Delays ........................................................................................................................................ 48 
12.3.2 Resveratrol dose reduction ............................................................................................................. 49 
12.3.3 Sirolimus dose reduction ................................................................................................................ 49 
12.3.4 Concomitant medications ............................................................................................................... 50 
12.3.5 Prohibited medications .................................................................................................................... 50 
13. Procedures .......................................................................................................................... 51 
13.1 Stopping rules ........................................................................................................................... 51 
13.1.1 Study discontinuation ...................................................................................................................... 51 
13.1.2 Subject discontinuation ................................................................................................................... 51 
13.1.3 Study early stopping rules .............................................................................................................. 51 
13.1.4 Individual early stopping rules ..................................................................................................... 52 
13.2 Follow-up after early study termination ............................................................................. 52 
13.3 Participant replacement ......................................................................................................... 52 
14. Data and Safety Monitoring Plan ................................................................................. 53 
14.1 Study Oversight ........................................................................................................................ 53 
14.2 Adverse Events ......................................................................................................................... 53 
14.3 Definitions .................................................................................................................................. 53 
14.3.1 Adverse events ................................................................................................................................... 53 
14.3.2 Suspected adverse reaction and adverse reaction ................................................................... 53 
14.3.3 Adverse events associated with study procedures .................................................................. 54 
14.3.4 Serious Adverse Event (SAE) ....................................................................................................... 54 
14.3.5 Expected adverse events ................................................................................................................. 55 
14.3.6 Unexpected adverse event .............................................................................................................. 55 
14.4 Collecting, recording and managing adverse events ........................................................ 55 
14.4.1 Identifying adverse events .............................................................................................................. 55 
14.4.2 Recording AEs ................................................................................................................................... 55 
14.4.3 Recording SAEs ................................................................................................................................ 56 
14.5 Managing adverse events ........................................................................................................ 56 
14.6 Grading and Attribution ........................................................................................................ 57 
14.6.1 Grading Criteria ................................................................................................................................. 57 


 
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14.7 Definition of Attribution ......................................................................................................... 57 
14.8 SAE Reporting Criteria and Procedures ............................................................................ 59 
14.8.1 Unexpected, non-serious adverse events ................................................................................... 59 
14.8.2 Notifying the FDA ............................................................................................................................ 59 
14.8.3 Notifying the Data and Safety Monitoring Board .................................................................. 60 
14.8.4 Notifying the Institutional Review Board ................................................................................. 60 
14.9 Reporting Pregnancy ............................................................................................................... 60 
14.10 Non-serious adverse events (NSAEs) reporting .............................................................. 60 
14.10.1 Notifying the data and safety monitoring board ................................................................... 60 
14.10.2 Notifying the FDA .......................................................................................................................... 61 
14.10.3 Notifying the Institutional Review Board .............................................................................. 61 
14.10.4 Unanticipated Problems ................................................................................................................ 61 
14.11 Data Management .................................................................................................................. 61 
14.11.1 Registration ....................................................................................................................................... 61 
14.11.2 Data Entry ......................................................................................................................................... 62 
14.11.3 Data Quality and Monitoring Measures .................................................................................. 62 
14.12 Protocol Deviations ................................................................................................................ 63 
14.12.1 Protocol deviation Definition ..................................................................................................... 63 
14.12.2 Reporting Protocol Deviations ................................................................................................... 65 
15. Statistical considerations: ............................................................................................... 66 
16. Identification and access to source data: ..................................................................... 68 
16.1 Identifying source data ........................................................................................................... 68 
16.2 Updating source documentation ........................................................................................... 68 
16.3 Permitting access to source data ........................................................................................... 68 
16.4 Quality control and quality assurance ................................................................................. 68 
17. Ethical considerations and compliance with good clinical practice ...................... 69 
17.1 Statement of Compliance ........................................................................................................ 69 
17.2 Informed Consent .................................................................................................................... 69 
18. Privacy and Confidentiality ............................................................................................ 70 
19. Publications ........................................................................................................................ 71 
20. References: ......................................................................................................................... 72 
 
 
 


 
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1. Participating Institutions/Investigators Table (contact information) 
 
 
University of Cincinnati Principal Investigator:  Nishant Gupta, M.D. 
Contact: Nishant Gupta, M.D.  
Institution: University of Cincinnati 
Address: 231 Albert Sabin Way 
MSB Room 6053, ML 0564 
Cincinnati, OH 45267 
Phone: 513-558-4831 
Email: guptans@ucmail.uc.edu 
 
University of Cincinnati Sub-Investigator: Francis X. McCormack, M.D. 
Contact: Francis X. McCormack, M.D 
Institution: University of Cincinnati 
Address: 231 Albert Sabin Way 
MSB Room 6053, ML 0564 
Cincinnati, OH 45267 
Phone: 513-558-4831 
Email: mccormfx@ucmail.uc.edu 
 
University of Cincinnati Sub-Investigator: Adam Cole MD 
     Contact:     Adam Cole, M.D. 
     Institution: University of Cincinnati 
     Address:    231 Albert Sabin Way 
                       MSB Room 6053, ML 0564 
                       Cincinnati, OH 45267 
        Phone:    513-558-4831 
        Email:    colead@ucmail.uc.edu 
 
Key Person:  Marina K. Holz, PhD 
Contact: Marina K. Holz, PhD 
Institution: Yeshiva University 
Address: 245 Lexington Ave. 
New York, NY 10016  
Phone: 212-340-7838 
Email: mholz@yu.edu 
 
Biostatistician: Bin Zhang, PhD 
           Contact: Bin Zhang, PhD 
       Institution: Cincinnati Children’s Hospital Medical Center 
          Address: Division of Biostatistics and Epidemiology 
 
             3333 Burnet Avenue 
 
             Cincinnati, Ohio 45229-3026 
             Phone: 513-636-4200 
              Email: bin.zhang@cchmc.org 


 
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2. Glossary of Abbreviations 
 
AE 
Adverse event 
AEDAMS 
Adverse Event Data Management System 
AMPK 
5' AMP-activated protein kinase 
ATAQ-LAM 
A Tool to Assess Quality of Life in LAM 
ATS 
American Thoracic Society 
AUC 
Area under the curve 
BD 
Bronchodilators 
CAPA 
Corrective and Preventative Action 
CBC 
Complete blood count 
CFR 
Code of Federal Regulations 
cGCP 
Current Good Clinical Practice 
Cmax 
Peak plasma concentration 
CMP 
Comprehensive metabolic panel  
CRF 
Case report form  
CT 
Computerized tomography 
CTCAE 
Common Terminology Criteria for Adverse Events 
CYP 
Cytochrome P450 
DLCO 
Diffusion capacity for Carbon Monoxide 
DSMB 
Data Safety Monitoring Board 
DSMP 
Data Safety Monitoring Plan 
ERS 
European Respiratory Society 
eCRF 
 
Electronic Case Report Form 
Evaluable 
Patients 
Patients whose response to a treatment can be measured because 
enough information has been collected 
FDA 
Food and Drug Administration 
FEV1 
Forced expiratory volume in first second 


 
12 
FVC 
Forced vital capacity 
FPI 
Functional Performance Inventory 
GEE 
Generalized Estimating Equations 
HIPAA 
Health Insurance Portability and Accountability Act 
HRCT 
High-resolution computed tomography 
ICH 
International Conference on Harmonization 
IND 
Investigational New Drug 
IRB 
Institutional Review Board 
LAM 
Lymphangioleiomyomatosis 
MEF 
Mouse epithelial fibroblasts 
MILES 
Multicenter International LAM Efficacy of Sirolimus 
mTOR 
Mammalian Target of Rapamycin 
NCI 
National Cancer Institute 
NIH 
National Institutes of Health 
NSAES 
Non Serious Adverse Events 
OHRP 
Office for Human Research Protection 
PFT 
Pulmonary function test 
PI 
Principal Investigator 
SAE 
Serious Adverse Event 
SAR 
Suspected Adverse Reaction 
SD-SOB 
San Diego Shortness of Breath Scale 
SGRQ 
St. George’s Respiratory Questionnaire 
SUSAR 
Suspected, Unexpected Adverse Reaction 
TSC 
Tuberous Sclerosis Complex 
Tmax 
Median time to achieve peak plasma concentration 
TUNEL 
Terminal deoxynucleotidyl transferase dUTP nick end labeling 
VEGF-D 
Vascular Endothelial Growth Factor - D 


 
13 
3. Background and Rationale 
 
3.1 Background: 
 
Lymphangioleiomyomatosis (LAM) is a rare, progressive diffuse cystic lung disease 
characterized by the infiltration of lung parenchyma with abnormal smooth muscle cells, 
leading to progressive destruction of lung parenchyma (1). The origin of these smooth 
muscle cells is unknown, however, lung involvement happens as a result of metastatic 
spread of these smooth muscle cells via blood and lymphatics, thus characterizing LAM 
as a low-grade, destructive, metastasizing neoplasm (2, 3). LAM is seen primarily in 
women with only a few reported cases in men (1, 4). LAM can be seen in association 
with Tuberous Sclerosis Complex (TSC-LAM), or occur sporadically (S-LAM). Both 
TSC-LAM and S-LAM occur as a result of mutations in one of the two TSC genes: TSC1 
or TSC2 (5, 6). Mutations in the TSC genes lead to constitutive activation of the 
mammalian target of rapamycin (mTOR) pathway, which drives cell proliferation and 
lymphangiogenesis.  
 
3.2 Role of Sirolimus in LAM 
 
Sirolimus forms a complex with FKBP12, which binds to mTOR and blocks activation of 
downstream kinases, restoring homeostasis in cells with defective TSC function (7). 
Sirolimus has been shown to shrink tumors in animal models of TSC deficiency (8, 9). 
Based on these observations, mTOR inhibitors were studied as therapeutic agents in 
patients with LAM and TSC. Open label, phase 1-2 trials of sirolimus in patients with 
TSC, led to a reduction in the size of angiomyolipomas (10, 11). An improvement in lung 
function was also noted in a majority of patients on sirolimus (10). This formed the 
formed the rationale for conducting a larger clinical trial to evaluate the role of sirolimus 
in LAM.  
 
The Multicenter International LAM Efficacy of Sirolimus  (MILES) Trial was a double 
blind, randomized, parallel group trial of one 
year of treatment with sirolimus versus 
placebo followed by one year of observation 
(12). In the MILES trial, 89 patients with 
LAM 
and 
moderate 
lung 
impairment 
(defined as FEV1 less than 70% predicted) 
were randomized to treatment with sirolimus 
or placebo for 12 months followed by 12 
months 
of 
observation. 
The 
primary 
outcome for the MILES trial was the 
difference in the rate of change (slope) of 
FEV1. While patients treated with placebo 
lost approximately 10% of their lung volume 
in the treatment phase, patients treated with sirolimus exhibited stabilization of their lung 
function, and had an improvement in their functional performance and quality of life. 
Figure 1: Graph depicting the change in 
FEV1 in the MILES trial. 


 
14 
During the observation year, lung function decline resumed in the sirolimus group and 
paralleled that of the placebo group (Figure 1) (12).  
 
The resumption of lung function decline following drug stoppage suggests that the 
function of sirolimus is suppressive rather than remission inducing. In addition, while the 
drug was well tolerated for the trial duration, the long-term safety and efficacy of 
sirolimus in LAM remains unclear. These limitations of sirolimus highlight the critical 
need to explore novel treatment opportunities for patients with LAM.  
 
3.3 Serum Vascular Endothelial Growth Factor-D (VEGF-D) as a diagnostic, 
prognostic, and predictive biomarker of LAM 
Serum vascular endothelial growth factor-D (VEGF-D), a ligand for the lymphatic 
growth-factor receptor VEGFR-3/Flt-4, is a major angiogenic growth factor that induces 
formation of lymphatics and promotes the spread of malignant tumor cells (13). VEGF-D 
has been noted to be present at increased levels in the serum of LAM patients (13-15). 
While the exact source of origin of VEGF-D in LAM is not well established, it is 
believed to be derived from the LAM cells and promotes the metastatic spread of LAM 
cells (15). The role of serum VEGF-D as a diagnostic biomarker for LAM is now well 
established. Among patients with cystic lung disease on chest imaging, elevated serum 
VEGF-D greater than 800pg/ml is almost 100% specific for the diagnosis of LAM (14). 
Serum VEGF-D measurement is commercially available in a CAP/CLIA laboratory at 
CCHMC.  
 
In addition to its role as a diagnostic biomarker, serum VEGF-D is also a prognostic as 
well as a predictive biomarker. In the MILES trial, the baseline serum VEGF-D levels 
were similar in the placebo and treatment arms (12). Baseline serum VEGF-D levels 
correlated with multiple markers of disease severity, including pneumothorax, 
bronchodilator responsiveness and need for supplemental oxygen, and with physiologic 
parameters such as FVC (p=0.002), total lung capacity (TLC (p<0.0001)) and diffusion 
capacity for carbon monoxide ((DLCO) (p<0.0001)). Higher baseline serum VEGF-D 
levels were also associated with disease progression and treatment response. Each 1-unit 
increase in baseline log (VEGF-D) was associated with a baseline to 12-month change of 
-70 cc in the placebo group and +70cc in the sirolimus group (p<0.001). Placebo patients 
with baseline VEGF-D levels ≥600pg/ml were found to decline much faster than the 
placebo patients with VEGF-D <600pg/ml, and the ≥600pg/ml sirolimus group patients 
responded better than the sirolimus patients with VEGF-D <600pg/ml. While VEGF-D 
levels remained stable in the placebo arm, treatment with 12 months of sirolimus resulted 
in approximately 50% reduction in the mean VEGF-D levels. In the observation year, 
VEGF-D values remained stable in the placebo group, but increased again in the 
sirolimus group (Figure 2). Sirolimus induced reduction in serum VEGF-D was highly 
correlated with improvement in lung function. Improvement in baseline-to-12-month 
FEV1 occurred in 65% of VEGF-D responders (patients with a VEGF-D decrease ≥ 42% 
from its baseline value), as opposed to only 27% of VEGF-D non-responders (p=0.04) 
(Figure 3). These data indicate that VEGF-D is a clinically useful prognostic biomarker 
of disease progression and a predictive biomarker of treatment response (16). Similar 
findings have been replicated in another analysis (17). Similarly, reduction in serum 


 
15 
VEGF-D by approximately 50% was noticed after treatment with open-label everolimus 
in a recent trial (Figure 4) (18). These findings suggest that serum VEGF-D can serve as 
a biomarker to monitor the response to treatment in patients with LAM. Since LAM cells 
are the source of elevated VEGF-D, monitoring VEGF-D levels as treatment response 
can also serve as a surrogate for LAM cell burden and activity. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2: VEGF-D trends in the MILES trial. Serum VEGF-D stayed stable in the 
placebo group and decreased in the sirolimus group. VEGF-D levels increased again after 
stopping sirolimus.  
Figure 3: Baseline-to-12-month change in FEV1 in the placebo group (left), and sirolimus 
group (right) as stratified by VEGF-D responders and non-responders in the MILES trial.  


 
16 
  
 
 
Figure 4: Graphs depicting the trend of serum VEGF-D in LAM patients following 
treatment with everolimus (left) and sirolimus (right). Serum VEGF-D declined to 
approximately 50% of its baseline value following treatment with mTOR inhibitors. The 
everolimus study (left) had frequent, early blood draws which revealed that most of the 
reduction in serum VEGF-D occurs in the first 8 weeks following treatment initiation, 
after which it plateaus.  
3.4 Resveratrol and its role in treatment of LAM 
Resveratrol (trans-3, 5, 4′-trihydroxystilbene) is a naturally occurring polyphenol found 
in grapes, berries, and red wine (19). Multiple studies have shown Resveratrol to have 
anticancer, anti-inflammatory, and anti-oxidant properties (19-23).  
Resveratrol and mTOR regulation 
Resveratrol has a complex mechanism of mTOR regulation. Resveratrol inhibits 
mTORC1 phosphorylation in a dose-dependent manner by inhibiting phosphorylation at 
S2448 residue (19, 24). Resveratrol has also been shown to have multiple effects on 
mTORC2 signaling (25). Resveratrol is believed to regulate mTOR activity through 
modulation of 5' AMP-activated protein kinase (AMPK), a key molecule for energy 
sensing that negatively regulates mTOR activity. Resveratrol induced phosphorylation of 
AMPK causes decreased phosphorylation of mTORC1 and its downstream targets (26). 
Resveratrol has also been shown to inhibit Akt phosphorylation in a dose-dependent and 
time-dependent manner, thus leading to mTORC1 inhibition (27). Lastly, Resveratrol has 
been shown to promote association between mTOR and DEPTOR, a negative regulator 
of mTORC1 and mTORC2 that inhibits mTOR kinase activity (28). The inhibition of 
PI3, Akt and mTOR activity has also been postulated to be responsible for the pro-
apoptotic effects of Resveratrol (19).  
Resveratrol and its role in regulating autophagy 
Autophagy is an evolutionarily conserved process of recycling organelles or misfolded 
proteins that allows cells to maintain homeostasis under stress-induced conditions (29). 
Autophagy allows cancer cells to continue to survive by recycling intracellular 
components and persisting under low-nutrient or stress-induced conditions. The 
mTORC1 pathway is central to regulation of autophagy via its modulation of multiple 
autophagy-regulating factors such as Akt, AMPK, and S6K1. Resveratrol inhibits the 
activity of Akt, AMPK and S6K1, thus leading to autophagy down-regulation (19).  


 
17 
Sirolimus as a single agent may promote cell survival in LAM 
Results from the MILES trial show that sirolimus as a single agent can lead to 
stabilization of lung function decline in patients with LAM (12). However, lung function 
decline resumed at a rate similar to the placebo group after stopping sirolimus, suggesting 
a suppressive rather than curative effect of sirolimus in LAM. The inhibition of mTOR 
activity with sirolimus releases the negative feedback signaling from S6K to Akt. The 
resultant increased Akt activity prevents cancer cell death by protecting them from 
apoptosis. Additionally, TSC2–/– LAM cells are dependent on autophagy for survival, 
and, since mTORC1 is a major regulator of autophagy, inhibition of this pathway with 
rapamycin induces autophagy, paradoxically leading to cell survival (30).  
Combination of Resveratrol and Sirolimus treatment restores inhibition of Akt, 
while suppressing mTORC1 signaling 
In a recent study, wild type and TSC2 null mouse embryonic fibroblasts (MEFs) were 
treated with both Resveratrol and Sirolimus (30). Sirolimus effectively suppressed 
mTORC1 signaling in both cell types, as measured by markers of S6K1 activity. Due to 
inhibition of the negative feedback loop, Sirolimus treatment increased Akt signaling, as 
measured by phosphorylation of Akt and its target PRAS40. Resveratrol treatment alone 
suppressed basal Akt signaling and slightly decreased S6K1 pathway activation. 
Interestingly, Resveratrol was able to prevent rapamycin-induced reactivation of Akt 
signaling as well as phosphorylation of its downstream substrate, PRAS40, while 
simultaneously effectively suppressing mTORC1/S6K1 signaling (Figure 5). Similar 
results showing inhibition of Akt signaling and mTORC1 inactivation by a combination 
of Resveratrol and Sirolimus were replicated in uterine leiomyoma-derived smooth 
muscle cells, human LAM-derived TSC2-null 621–101 cells, and normal human 
bronchial cells (Images not shown) (30).   
 
Figure 5: The combination of Resveratrol and Sirolimus inhibits PI3/Akt and mTOR 
signaling pathways.  
Combination of sirolimus and resveratrol downregulates autophagy in TSC2-null 
cells  
Wild%type%MEFs%%%%%%%%%%%%%%%%%%%%%TSC2%Null%MEFs%


 
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The effect of the combination of sirolimus and resveratrol on autophagy induction was 
studied in wild type and TSC2 null MEFs (30). Levels of p62/SQSTM1 protein inversely 
correlate with autophagy induction. While resveratrol alone did not have an effect on p62 
levels, the combination treatment of sirolimus and resveratrol blocked autophagy 
induction in wild type and TSC2 null MEFs, and restored p62 levels back to baseline 
(Figure 6). Similar results showing a downregulation of autophagy were observed by 
treating human LAM-derived TSC2-null 621–101 cells with a combination of resveratrol 
and sirolimus (Images not shown) (30).  
 
Figure 6: Resveratrol prevents sirolimus induced induction of autophagy. Wild type 
mouse epithelial fibroblasts (MEFs) and TSC2 null fibroblasts were treated with 20nM 
Sirolimus and 100μM Resveratrol for 24 hours. Cells were lysed and protein levels of 
p62 and actin were detected by immunoblot.  
 
 
Apoptosis is induced in TSC2-null cells, but not in control cells  
Wild type and TSC2 null MEFs were treated with sirolimus and/or resveratrol for 24h 
and assayed for apoptosis induction by examining cleavage of 2 markers, PARP and 
Caspase 3. Resveratrol treatment, alone or in combination with sirolimus, was able to 
strongly increase cleavage of PARP and Caspase 3 specifically in TSC2 null MEFs and 
not in wild type MEFs (Figure 7). These findings of increased apoptosis by resveratrol in 
TSC2 null cells were replicated in uterine leiomyoma-derived smooth muscle cells, 
human LAM-derived TSC2-null 621–101 cells (Images not shown) (30).  


 
19 
 
Figure 7: Resveratrol, alone or in combination with sirolimus, leads to apoptosis in 
TSC2 null mouse epithelial fibroblasts (MEFs) and not in wild type MEFs. Wild type and 
TSC2 null MEFs were treated with 20 nM rapamycin and/or 100 μM resveratrol for 24 h. 
Cells were lysed and probed for PARP, cleaved-Caspase 3, and actin. The arrow indicates 
cleaved PARP isoform.  
 
 
Combination of sirolimus and resveratrol reduces the metastatic capacity of uterine 
leiomyoma-derived smooth muscle cells 
TSC2 null uterine leiomyoma derived smooth muscle cells (ELT3 cells), metastasize to 
the lungs following tail vein injection into mice replicating an in vivo model for LAM 
(31). After 24 h treatment, mice treated with the combination therapy of resveratrol and 
sirolimus had significantly fewer lung metastases as assayed by photon flux compared 
with control mice or mice treated with either sirolimus or resveratrol alone (Figure 8) 
(30).  
Wild%Type%MEFs%%%%%%%%%%%%%%%%%%TSC2%null%MEFs%


 
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Figure 8: Combination of sirolimus and resveratrol strongly reduces the survival of 
Tsc2-null cells in vivo. Mice were treated with vehicle, sirolimus, resveratrol, or 
sirolimus plus resveratrol. ELT3-luciferase-expressing cells were inoculated 
intravenously. Representative bioluminescent images of lung colonization at 1, 6, and 24 
hours post-cell injection are shown above depicting a significant decrease in pulmonary 
metastases following treatment with combination of resveratrol and sirolimus.  
 
 
 
 
 
 
 
 
!!!!!!!!!1!hour!!!!!!!!!!!!!!!!!!!!!!!!!!!!!6!hours!!!!!!!!!!!!!!!!!!!!!!!!!!!!!24!hours!


 
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Combination treatment with resveratrol and sirolimus causes a significant 
reduction of TSC2 deficient xenograft tumor size and growth 
Mice bearing ELT3-luciferase–expressing xenograft tumors were treated with sirolimus 
and resveratrol as single-agent treatments or in combination. There was no evidence of 
drug toxicity as evidenced by a lack of significant weight change among treatment 
groups. While sirolimus treatment alone showed a slight but significant reduction in 
tumor size in just 3 weeks of treatment, the combination of sirolimus and resveratrol 
caused  a statistically significant reduction in tumor size as compared with sirolimus 
treatment alone by as early as Week 2  of treatment, and this reduction in tumor size 
was maintained at 4 weeks after treatment (Figure 9) (32).  
 
Figure 9: Combination treatment with sirolimus and resveratrol reduces tumor size and 
inhibits growth. Female CB17-scid mice were inoculated with ELT3-luciferase cells 
subcutaneously. Mice were treated with vehicle, sirolimus, resveratrol, or a combination 
of sirolimus and resveratrol for 4 weeks. Bioluminescent intensity in xenograft tumors 
was recorded and quantified weekly. Vehicle- and resveratrol-treated tumors progressed 
faster than the tumors in sirolimus or sirolimus–resveratrol groups. Combination of 
sirolimus and resveratrol caused  a statistically significant reduction in tumor size as 
compared with sirolimus treatment alone.  
 


 
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The Combination of Sirolimus and Resveratrol Suppresses PI3K/Akt/ mTORC1 
Activation in the TSC2 deficient xenograft tumor  
Mice bearing ELT3-luciferase–expressing xenograft tumors were treated with sirolimus 
and resveratrol as single-agent treatments or in combination. The combination of 
sirolimus and resveratrol resulted in severe reduction of S6 phosphorylation and 
approximately 40% inhibition of 4EBP1 phosphorylation. Sirolimus alone resulted in 
mild reduction of S6 phosphorylation. Moreover, the combination of sirolimus and 
resveratrol caused a 45% inhibition of Akt phosphorylation at S473, with no effect on p-
ERK signaling, thus indicating the specificity of this treatment in inhibiting 
Akt/mTORC1 signaling (32). 
Combined Administration of Sirolimus and Resveratrol Reduces Proliferation and 
Induces Apoptosis within the TSC2 deficient xenograft tumor  
Mice bearing ELT3-luciferase–expressing xenograft tumors were treated with sirolimus 
and resveratrol as single-agent treatments or in combination for 4 weeks. Sirolimus and 
resveratrol combination treatment caused a 2-fold up-regulation of cleaved caspase-3, a 
marker of apoptosis. In addition, analysis of TUNEL staining of tumor sections showed 
that combination treatment increased the levels of apoptotic TUNEL-positive cells by 3-
fold, whereas single-agent treatment with either sirolimus or resveratrol had no effect on 
apoptosis (Figure 10). Taken together, these data indicate that a 4-week administration of 
the combination treatment of sirolimus and resveratrol inhibits mTORC1 signaling, 
inhibits Akt activation, and induces apoptosis (32).  
 
Figure 10: The combination of rapamycin and resveratrol induces apoptosis in xenograft 
tumors. TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling.  


 
23 
3.5 Role of home spirometry in pulmonary disorders 
In a recent study, 50 patients with idiopathic pulmonary fibrosis (IPF) performed daily 
home spirometry for a median of 279 days. Office-based spirometry was performed 6- 
and 12-months after enrollment. In this study, over 80% of the patients adhered to the 
routine of performing daily spirometry, daily spirometry values showed excellent 
correlation with office-based spirometry (Figure 11), and the rate of decline in FVC 
obtained after 3 months of home spirometry was highly predictive of patient outcomes 
and subsequent mortality (HR: 1.04, CI: 1.02–1.06, p<0.001) (Figure 12) (33). 
 
 
 
Figure 11: Bland-Altman plots demonstrating good agreement between office-based 
measurements of FEV1 (left) and FVC (right) with home-based measurements. There 
was a slight bias (0.20 liters) towards consistently higher readings obtained during office-
based measurements as opposed to home-based measurements 
 
 
 
 
 
Figure 12: Kaplan-Meier curve demonstrating that the trends obtained from home-based 
FVC monitoring are predictive of the overall survival in patients with IPF. Left: 
Increased mortality in patients with FVC decrease more than 5% from baseline after 3 
months of monitoring. Right: Increased mortality in patients with FVC decrease more 
than 10% from baseline after 12 months of monitoring. 
 


 
24 
 
Johannson et al. were able to achieve a compliance rate of 90% with once-weekly home 
spirometry in 24 patients with IPF, and demonstrated that weekly home spirometry is 
both reliable and feasible in this patient population. Addition of weekly home spirometry 
resulted in enhanced precision, and was estimated to reduce the sample size needed to 
study the efficacy of a new intervention by four-fold (34). In another recent study, 
patients with cystic fibrosis randomized to twice-weekly home spirometry measurements 
had a statistically significant shorter time to detection of disease-related exacerbations as 
opposed to usual care. However, the adherence to home spirometry in this study was 
suboptimal with 50% of the population consistently performing once-weekly testing, and 
only 19% of the population adhering to the twice-weekly regimen (35). Home spirometry 
has been shown to facilitate early detection of bronchiolitis obliterans following lung 
transplantation as compared to office-based spirometry (36, 37).  
 
 
3.6 Rationale for the proposed study 
Treatment with sirolimus alone has a suppressive rather than remission inducing effect. 
In addition, mTORC1 inhibition by sirolimus leads to upregulation of autophagy and 
paradoxically increases LAM cell survival. As explained in Section 3.4, the combination 
of resveratrol and sirolimus is effective in blocking autophagy and inducing apoptosis of 
TSC2–/– cells, as well as reducing their survival in vivo with minimal effect on normal, 
TSC2-expressing cells. These pre-clinical results form the rationale for investigating the 
combination of resveratrol and sirolimus as a treatment option for patients with LAM and 
TSC. Since there is no clinical data on the adverse effect profile and effective dosing of 
resveratrol in patients with LAM, we plan to conduct a phase II, dose escalating, safety 
and efficacy study of resveratrol in combination with sirolimus with plans for a multi-
center phase III study after successful completion of this project.   
Home spirometry has the potential to shorten the length of monitoring time needed to 
determine the disease trajectory. Reliable estimates of lung function typically require 
multiple measurements (3 or more) obtained over a prolonged monitoring period, usually 
12 months or more. We hypothesize that the trajectory of lung function decline and 
treatment response can be determined in a much shorter period of time using frequent 
home-based spirometric measurements. Our strategy of testing home spirometry in 
conjunction with RESULT will allow us to directly compare home spirometry to standard 
office-based spirometry in an ATS/ERS calibrated laboratory and with real time quality 
control for both home- and office-based pulmonary function studies. Our project will 
validate the use of home spirometry as a novel disease-monitoring tool for patients with 
LAM, and will help integrate home spirometry in clinical practice, and in future studies 
seeking to develop novel treatment approaches. 
 
 
 


 
25 
4. Study Objectives and Endpoints: 
 
4.1 Primary Endpoint: 
The primary objective of this study is to assess the change in serum vascular endothelial 
growth factor-D (VEGF-D) value after 24 weeks of treatment with a combination of 
resveratrol and sirolimus, as compared to the VEGF-D value on sirolimus alone.  
 
4.2 Secondary Endpoints: 
1. Safety and adverse effect profile of combined resveratrol and sirolimus in patients 
with LAM 
2. Mean change in FEV1 before and after treatment 
3. Mean change in FVC before and after treatment 
4. Difference in rate of change in FVC (slope) before and after treatment 
5. Difference in rate of change in FEV1 (slope) before and after treatment 
6. Mean change in diffusion capacity of carbon monoxide (DLCO) before and after 
treatment 
7. Difference in rate of change in DLCO (slope) before and after treatment 
8. Baseline to 24-week change in the score on St. George’s Respiratory 
Questionnaire (SGRQ) 
9. Baseline to 24-week change in the score on ATAQ-LAM (A tool to assess quality 
of life in LAM) 
10. Baseline to 24-week change in the score on SD-SOB score (San Diego Shortness 
of Breath Questionnaire) 
11. Baseline to 24-week change in the score on EuroQOL scale 
12. Overall compliance with weekly home spirometry. 
13. Correlation between home spirometry values (absolute as well as trends) with 
office-based spirometry values and trends.  
 
 
 
 


 
26 
5. Study Design: 
 
This is an open label, non-randomized, dose-escalation study conducted as a phase II 
safety and efficacy study. Patients will continue on their existing background therapy of 
sirolimus for treatment of LAM. 
 
5.1 Identification and Screening: 
 
Potential participants will be identified through the University of Cincinnati Medical 
Center LAM Clinic, which provides clinical care for patients with LAM. LAM patients 
may also learn of the study from the LAM Foundation web site or the study listing on 
ClinicalTrials.gov. Potential study participants will be screened for eligibility. Study staff 
expects to screen the required sample size of 25 patients over a 15-month time frame. 
Potential participants will be informed about the study, and following completion of the 
informed consent process, a screening evaluation will be performed. Informed consent 
will be obtained before any study procedures are performed. Informed consent for 
participation in this study may be obtained in-person or remotely via telephone.    
 
 
5.2 Inclusion Criteria: 
Subjects enrolled in the trial must meet all of the following criteria. 
1. Definitive diagnosis LAM based on the presence of characteristic cystic change 
on high-resolution computed tomography (HRCT) of the chest. The diagnosis 
must be confirmed by one of the following: 
A) Histopathological confirmation by biopsy (lung, abdominal mass, lymph node 
or kidney or cytology from thoracic or abdominal sources revealing HMB45+ 
staining of spindled/epithelioid cells)  
B) Compatible chest CT scan findings in the setting of tuberous sclerosis, 
angiomyolipomas (diagnosed by CT, MRI by the site radiologist or biopsy) or 
chylous pleural effusion (verified by tap)  
C) Chest CT scan findings compatible with LAM and a VEGF-D level ≥ 
800pg/ml. 
2. Age 18 years or greater 
3. Signed and dated informed consent  
4. Currently on sirolimus for treatment of LAM for at least 20 weeks  
5. Evidence of disease stabilization on sirolimus as demonstrated by two stable 
values of serum VEGF-D post initiation of sirolimus drawn at least 12 weeks 
apart from each other. For the purpose of this study, a variation in serum VEGF-D 
of less than or equal to 15% is considered stable. 
 
5.3 Exclusion Criteria:  
Subjects who meet any of the following criteria are not eligible for enrollment as study 
participants:  


 
27 
1. Known allergy or hypersensitivity to resveratrol 
2. Inability to provide informed consent 
3. Active enrollment in other clinical drug trials for LAM 
4. Pregnant or plan to become pregnant in the next 6 months 
5. Breast feeding 
6. Inability to comply with pulmonary function tests or follow up visits 
7. Inadequate contraception 
8. Use of estrogen containing medications within the 30 days prior to randomization 
9. History of organ transplant 
10. Actively listed for lung transplantation 
11. Inability to comply with study procedures or attend scheduled study visits 
12. Any clinically significant medical disease (other than LAM) that is associated 
with an expected survival of less than 2 years, or likely to impact the ability of the 
patient to participate in the study in the opinion of the investigator, or impact the 
study efficacy or safety assessments.  
13. Concomitant use of resveratrol with drugs with a narrow therapeutic range that 
are CYP3A, CYP1A2, CYP2D6, or CYP2C9 substrates (for example, 
theophylline, tizanidine, warfarin, phenytoin, alfentanil, dihydroergotamine, 
ergotamine, fentanyl, pimozide, quinidine, cyclosporine, tacrolimus, thioridazine) 
will be prohibited from Day 1 until the end of study.  
 
 
5.4 Recruitment: 
 
Patients will be recruited from The University of Cincinnati Medical Center LAM Clinic, 
and referred by physicians and LAM support groups such as the LAM Foundation, and 
the Tuberous Sclerosis Alliance. IRB approved recruitment materials may be posted in 
public places such as LAM clinics, The LAM Foundation, the Tuberous Sclerosis 
Alliance, LAM patient and/or physician events, conferences and websites.  The study will 
be posted on clinicaltrials.gov.  
 
Potential study participants will be contacted by the study staff to determine interest in 
study participation. Contact may be made by mail, electronic mail, telephone or in 
person. Interested individuals will receive study information, including a cover letter and 
a consent form. Informed consent for study participation may be obtained in-person or 
over the phone.  
 
5.5 Study Drug Treatment: 
 
Patients who meet eligibility criteria, provide informed consent and are enrolled in the 
study will receive instructions regarding resveratrol dosage, route and administration. A 
8-week supply of resveratrol 250mg daily will be provided at visit 1. Patients will be 
reminded about the side effects of resveratrol, and will be given a drug information sheet. 
Patients will be instructed to take the study medication at the same time every day. 
Patients will be carefully monitored monthly at subsequent visits for adverse effects 


 
28 
related to resveratrol. Common side effects of resveratrol include nausea, vomiting, 
diarrhea, flatulence, abdominal discomfort, and fatigue. Patients will be instructed to 
contact the study staff should they experience any of these side effects. Resveratrol doses 
may be held or reduced in the event of unacceptable side effects.  
5.6 Treatment Regimen: 
 
We have chosen an intra-patient dose escalation strategy because there is no previous 
data from human studies to demonstrate the safety of resveratrol in combination with 
sirolimus, in patients with LAM. Patients will begin resveratrol at a starting dose of 
250mg daily, and increase the dose to 500mg daily after 8 weeks, followed by dose 
escalation to 1000mg daily in two divided doses (500 mg twice a day) at week 16 of the 
study. If no adverse effects are seen, patients should reach a dose of 1000mg per day at 
week 16 of the study, and then continue with this dose for the rest of the study duration. 
If patients do not tolerate a certain dose of resveratrol, the dose will be reduced to the last 
tolerated strength. Patients will remain on the last well-tolerated dose of resveratrol for 
the entire study duration. Patients will remain on their current dose of sirolimus without 
interruption, as clinically indicated. Patients who are unable to tolerate the lowest dose of 
resveratrol will be withdrawn from the study.  
 
5.7 Home Spirometry  
 
We will provide patients with a home spirometer (GoSpiro, Monitored Therapeutics Inc.) 
at the beginning of the study This device was chosen after a careful survey of 9 different 
home spirometers, based on its ability to assess acceptability and reproducibility of 
maneuvers and fully comply with the ATS/ERS standards of a spirometer (38). FEV1 and 
FVC will be obtained once weekly from patients agreeing to participate in the home 
spirometry sub study. Detailed instructions regarding the proper use of the home 
spirometer will be provided at the time of enrollment, along with access to detailed 
instruction manuals for future use. Once a week, patients will perform at least three 
spirometric measurements at approximately the same time, and record the best FEV1 and 
FVC values as their weekly results. Patients will also be asked to complete a brief survey 
to rate their experience with the home spirometry every week. These results, along with 
the associated flow-volume and volume-time loops will then be transmitted electronically 
to the study coordinator. The spirometric values as well as the loops will be reviewed in 
near real time along with direct feedback to the subject on the performance and trouble 
shooting of any issues by an expert pulmonary physiologist (Dr. Adam Cole and/or Dr. 
Nishant Gupta). The spirometry data and loops will be judged for their acceptability and 
reproducibility as specified in the ATS/ERS guidelines (38). A maneuver will have to 
satisfy both the satisfactory start as well as end of test criteria in order for it to be 
considered acceptable. Difference between the largest recorded FEV1/FVC and the next 
largest FEV1/FVC less than 150ml will determine satisfactory intra-test reproducibility. 
Inter-test reproducibility will be assessed by comparing the FEV1 values obtained during 
the first 4 weeks of home spirometry measurements, with a target between-test variation 
of less than 5%. Overall compliance to weekly home spirometry will be recorded with 


 
29 
success defined as greater than 80% compliance with weekly measurements throughout 
the 24-week study duration.  
 
 


 
30 
6. Study Procedures and Timelines 
 
6.1 Study and Timeline Overview 
 
Patients will be identified by the medical and research staff at the LAM clinic at the 
University of Cincinnati Medical Center. Study staff will screen up to 60 patients over 15 
months with a goal of enrolling 25 participants. Patients who meet eligibility criteria, 
provide informed consent and are enrolled in the study will receive instructions regarding 
resveratrol dosage, route and administration. An 8-week supply of resveratrol will be 
provided at visit 1. Patients will begin resveratrol at a starting dose of 250mg daily, and 
increase the dose in two increments to a maximum dose of 1000mg daily. If patients do 
not tolerate a certain dose of resveratrol, the dose will be reduced to the last tolerated 
strength. Patients will remain on the last well-tolerated dose of resveratrol for the entire 
study duration. Patients who are unable to tolerate the lowest dose of resveratrol will be 
withdrawn from study. 
 
The patients will be given a copy of the informed consent form. Patients will be given 
ample time to read and discuss the consent form with the study staff. The study staff will 
then answer any remaining questions asked by the patients. The patients will be offered 
the option of signing the consent form. Those who agree to participate will undergo the 
remainder of screening visit.  
 
Prior to starting study drug, pre/post bronchodilator spirometry, diffusion capacity of 
carbon monoxide (DLCO), and serum VEGF-D will be obtained to establish baseline 
values prior to study drug initiation. The best values for FEV1 and FVC obtained during 
the spirometry maneuvers will be used. Patients will be followed with serial spirometry 
and VEGF-D levels collected at the initial visit, followed by week 8, week 16 and week 
24 visits. DLCO will also be obtained at the 24-week visit in addition to spirometry.  
 
For patients who agree to the home spirometry study, we will teach them on the proper 
technique to perform, record and transmit home spirometry, and provide them with a 
home spirometer. The patients will perform their first test during the initial visit and we 
will use those values to compare the home spirometer values with the office-based 
spirometry values. After that patients will be instructed to perform home spirometry once 
a week as detailed in Section 5.7.  
 
Section 6.2 provides a summary of the study timeline and procedures. 
 
 
 
 
 
 
 
 
 


 
31 
6.2 Schedule of Events: 
 
Event 
Screening  
Visit* 
Initial 
Visit 
Week 
2  
Week 
4 
Week 
8 
Week 
12 
Week 
16  
Week 
18 
Week 
20 
Week 
24 
Visit number 
0 
or 
Phone 
1 
Lab 
check 
Phone 
2 
Phone 
3 
Lab 
check  
Phone 
4 
Informed 
consent  
X 
 
 
 
 
 
 
 
 
 
History and 
physical 
 
X 
 
 
X 
 
X 
 
 
X 
CBC, CMP 
 
X 
 
 
X 
 
X 
 
 
X 
Lipid panel  
 
X 
 
 
X 
 
 
 
 
X 
Serum 
VEGF-D 
X 
X 
 
 
X 
 
X 
 
 
X 
Sirolimus 
level  
 
X 
X*** 
X*** 
X 
X*** 
X 
X*** 
X*** 
X 
Urine 
Pregnancy 
Test** 
 
X 
 
 
X 
 
X 
 
 
 
Saved serum 
and plasma 
 
X 
 
 
X 
 
X 
 
 
X 
Spirometry 
post BD 
 
X 
 
 
X 
 
X 
 
 
X 
Diffusion 
capacity 
 
X 
 
 
 
 
 
 
 
X 
Home 
spirometry 
dispensed  
 
X 
****  
 
 
 
 
 
 
 
 
Review 
concomitant 
meds 
 
X 
 
X 
X 
X 
X 
 
X 
X 
Review 
adverse 
events 
 
 
 
X 
X 
X 
X 
 
X 
X 
SGRQ  
 
X 
 
 
X 
 
X 
 
 
X 
ATAQ-
LAM 
 
X 
 
 
X 
 
X 
 
 
X 
EuroQOL 
 
X 
 
 
X 
 
X 
 
 
X 
SD-SOB 
 
X 
 
 
X 
 
X 
 
 
X 
 
Window for all visits after the initial visit: ±7 days, except Weeks 2 and 18 where the 
window is ±3 days 
 
* Screening visit can be the initial visit if a patient meets all inclusion/exclusion criteria. 
Informed consent for study inclusion can be obtained remotely over the phone rather than 
in-person. VEGF-D levels to confirm eligibility for trial participation can be drawn 
remotely rather than an in-person visit.  
 
** Only required for women of childbearing potential. 
 
*** Can be drawn remotely. This is a trough level to be obtained 24 ± 4 hours after the 
last sirolimus dose.  


 
32 
**** FEV1 and FVC will be obtained once weekly from patients agreeing to participate 
in the home spirometry sub study. Detailed instructions regarding the proper use of the 
home spirometer will be provided at the time of enrollment, along with access to detailed 
instruction manuals for future use. Once a week, patients will perform at least three 
spirometric measurements at approximately the same time, and record the best FEV1 and 
FVC values as their weekly results. These results, along with the associated flow-volume 
and volume-time loops will then be transmitted electronically to the study coordinator. 
 
ATAQ-LAM: A Tool to Assess Quality of Life in LAM, BD: Bronchodilators, CBC: 
complete blood count, CMP: comprehensive metabolic panel, SGRQ: St. George’s 
Respiratory Questionnaire, SD-SOB: San Diego Shortness of Breath scale, VEGF-D: 
Vascular Endothelial Growth Factor-D. 
 
 
6.3 Study Visits 
 
Screening/Initial Visit 
 
At the screening visit, the consent form will be presented to the potential patient. If the 
patient is competent to understand the risks, agrees to participate and provides informed 
consent and HIPAA release, the patient will undergo screening. Patients will also be 
informed about the home spirometry portion of this study, and given the option to opt-in 
or opt-out of the home spirometry evaluation. Patients may enroll in the clinical trial 
without agreeing to participate in the home spirometry portion. During this visit, we will 
review the inclusion/exclusion criteria, obtain a urine pregnancy test for women of 
childbearing potential (defined as any fertility status other than natural or surgical 
menopause or post hysterectomy), and check a serum VEGF-D level (if two previous 
stable values post initiation of sirolimus are not drawn). The screening visit and VEGF-D 
testing can be accomplished either remotely or in-person. If patients meet the study 
inclusion criteria, the screening visit can be their initial study visit. We anticipate that in 
most patients the screening visit will be the initial study visit, and the following 
additional steps will be completed during the visit: 
 
•  Review current medications.  
•  Review medical history and conduct a physical examination.  
•  Perform spirometry with appropriate safety precautions. All spirometry tests  
 during all visits will be performed after supervised administration of   
 bronchodilator in the pulmonary function laboratory.  
•  Perform diffusion capacity (DLCO) testing. DLCO will be performed in a     
 supervised setting in the pulmonary function laboratory. 
•  Obtain clinical labs including: complete blood count (CBC), comprehensive 
 metabolic panel (CMP), lipid panel, serum VEGF-D, sirolimus level, urine   
 pregnancy test, and urinalysis.  
•  Quality of life, dyspnea and fatigue assessments 
• 
Dispense resveratrol tablets for the next 8 week supply. 


 
33 
• 
Provide a drug diary to document drug compliance. Patients will bring the drug 
diary at each subsequent visit 
• 
If the patient has consented obtain a blood sample to be stored on-site for 
potential future use 
• 
If the patient has consented, instruct them on the proper technique to perform 
home spirometry, provide them with a home unit, and obtain the first set of 
FEV1 and FVC values. Patients will also be taught on how to record and transmit 
electronically the home spirometry values on a weekly basis throughout the 24-
week trial duration.  
 
 
Lab Check (Week 2 ± 3 days) 
During this study, the patient will have a sirolimus level checked for safety monitoring. 
This level can be drawn remotely.  
 
Phone Visit (Week 4 ± 7 days) 
During this visit a study coordinator will contact the patient and review the interval 
history in detail. A report of any adverse events will be assembled in detail, along with a 
review of their concurrent medications. Any concerns brought up during this phone call 
will be reviewed with the investigator. In addition, sirolimus level for safety monitoring 
will be drawn at this visit. The sirolimus level can be drawn remotely.  
 
Visit 2 (Week 8 ± 7 days) 
During this visit the following will be performed:  
 
• Obtain a urine pregnancy test for women of childbearing potential  
• Obtain urinalysis 
• Review current medications 
• Review the interval medical history, and perform a physical examination 
• Perform spirometry after supervised administration of bronchodilator in the 
pulmonary function laboratory 
• Quality of life, dyspnea and fatigue assessments 
• Obtain blood for CBC, CMP, lipid panel, VEGF-D measurement and 
sirolimus level; if the patient has consented obtain an additional sample to be 
stored on-site for potential future use  
• Dispense resveratrol tablets for the next 8 weeks 
• Review drug diary and any reported adverse events 
• Arrange for inter-visit phone call conduct 
 
Phone Visit (Week 12 ± 7 days) 
During this visit a study coordinator will contact the patient and review the interval 
history in detail. A report of any adverse events will be assembled in detail, along with a 
review of their concurrent medications. Any concerns brought up during this phone call 
will be reviewed with the investigator. In addition, sirolimus level for safety monitoring 
will be drawn at this visit. The sirolimus level can be drawn remotely. 
 


 
34 
 
Visit 3 (Week 16 ± 7 days) 
During this visit, the following will be performed: 
• Obtain a urine pregnancy test for women of childbearing potential 
• Obtain urinalysis 
• Review current medications 
• Review the interval medical history, and perform a physical examination 
• Perform spirometry after supervised administration of bronchodilator in the 
pulmonary function laboratory 
• Quality of life, dyspnea and fatigue assessments 
• Obtain blood for CBC, CMP, VEGF-D measurement and sirolimus level; if 
the patient has consented obtain an additional sample to be stored on-site for 
potential future use  
• Dispense resveratrol tablets for the next 12 weeks  
• Review drug diary and any reported adverse events 
 
Lab Check (Week 18 ± 3 days) 
During this study, the patient will have a sirolimus level checked for safety monitoring. 
This level can be drawn remotely.  
 
Phone Visit (Week 20 ± 7 days) 
During this visit a study coordinator will contact the patient and review the interval 
history in detail. A report of any adverse events will be assembled in detail, along with a 
review of their concurrent medications. Any concerns brought up during this phone call 
will be reviewed with the investigator. In addition, sirolimus level for safety monitoring 
will be drawn at this visit. The sirolimus level can be drawn remotely. 
 
Visit 4 (Week 24 ± 7 days) 
During this visit, the following will be performed: 
• Obtain a urine pregnancy test for women of childbearing potential 
• Obtain a urinalysis 
• Review current medications 
• Review the interval medical history, and perform a physical examination 
• Perform spirometry after supervised administration of bronchodilator in the 
pulmonary function laboratory.   
• Perform DLCO in the pulmonary function laboratory 
• Quality of life, dyspnea and fatigue assessments 
• Obtain blood for CBC, CMP, lipid panel, VEGF-D measurement and 
sirolimus level; if the patient has consented obtain an additional sample to be 
stored on-site for potential future use  
• Review drug diary and any reported adverse events 
• Advise women of childbearing potential that they should continue to use their 
method of birth control for at least 4 weeks after the study ends  


 
35 
• Remind patients they will be contacted via phone in 4 weeks as a follow up 
• Remind patients to follow up with their regular physicians for routine care 
 
If at visit 4, it is determined there has been clinical benefit, the patient and her regular 
physician will decide how to proceed after the conclusion of the clinical study.  
 
End of study telephone call (Week 28 ± 7 days) 
An end of study phone call will be conducted at 4 weeks ± 7 days after the last study 
visit. The purpose of the call is to follow up on any open or unresolved adverse events.  
 
6.4 Enrollment 
Enrollment in the study will continue until the enrollment target of 25 patients is reached. 
This research study will be explained in lay terms to each potential research participant. 
The potential participant will sign an informed consent form before undergoing any 
screening study procedures.  Participants who are deemed eligible for the study (see 
sections 6.1 through 6.3) at each of the study sites will be enrolled and assigned a unique 
participant ID number. 
 
6.5 Patient retention and drug compliance 
Patient retention will be encouraged in several ways. Extensive contact information will 
be captured for each patient, at the time of study enrollment. This will include home, 
work, and cellular telephone numbers and that of an emergency contact or nearest family 
member. Prior to each study visit, the study staff will contact the patient to remind her 
about the upcoming study visit. A check for the reimbursement of expenses up to $500 
will be issued after each study visit.  
The study staff and physician will explain the importance of compliance with the study 
protocol at each patient contact. If a patient fails to comply with a study visit, the study 
staff will contact her by telephone. If the study staff is unsuccessful in reaching the 
patient, a follow up letter will be sent by overnight carrier, on two consecutive weeks, 
seeking follow-up.  
Compliance with the protocol and drug administration will be strongly emphasized. Pill 
counts will be performed at visits and episodes of medication noncompliance will be 
recorded, including the reason for noncompliance. Patients will receive a calendar to 
document medication and any side effects experienced that day. If a patient wishes to 
withdrawal from the treatment phase of the study or has a serious adverse event (SAE) 
(whether related to study drugs or not), patient follow-up assessments will continue for 
safety monitoring and to avoid problems associated with missing data. The inclusion of 
such follow-up data will allow for analysis by intention-to-treat.  
If a patient is withdrawn from study drug exposure for any reason, the patient will be 
strongly encouraged to continue with and complete the remainder of the study 
assessments, as scheduled.  


 
36 
 
6.6 Consent for future contact 
During the consent process, patients will have the option to provide permission for future 
contact. This permission allows for patient contact information, including their full name, 
address and telephone number, to be retained. The permission for future contact allows 
for possible patient re-contact regarding future IRB-approved studies. If the patient 
denies permission, no future contact will be made.  
 
6.7 Consent for continuing data review of medical records 
During the consent process, patients will have the option to provide permission for future 
data review of their medical records. This permission allows for study staff to contact the 
patient (or the patient’s caregiver(s)) to obtain medical records and other information for 
a period of up to two years after the patient has withdrawn from or completed the study. 
The purpose of this data review is to capture information including but not limited to 
clinical, radiographic, pathologic, laboratory and pulmonary function data, after the 
patient has exited the study. The rationale for future data review is that there may be long 
term effects of study treatment that are unknown to the investigators at this time.   
 
6.8 Consent for storage of blood samples for future use 
Patients will be asked to consent to the storage of leftover blood and serum samples for 
future use related to LAM. Patients can refuse without impacting their care or ability to 
participate in this study.  
 
6.9 Consent for participation in the home spirometry substudy 
Patients will be asked to consent for participation in the home spirometry evaluation, 
being done in conjunction with the clinical trial. Patients can refuse to participate in the 
home spirometry evaluation without impacting their care or ability to participate in this 
study.  
 
 
 
 


 
37 
7. Stopping rules 
7.1 Participant withdrawal criteria 
Participants may be terminated early from the study for the following reasons: 
a. The participant elects to withdraw consent from all future study activities, including 
follow-up. 
b. The participant is “lost to follow-up” (i.e., no further follow-up is possible because 
attempts to reestablish contact with the participant have failed). 
c. The participant dies.  
d. The participant develops a medical condition or is started on new medication(s) not 
previously mentioned in the list of prohibited medications that, in the opinion of the 
investigator, may pose additional risks from participation in the study, may interfere with 
the participant’s ability to comply with study requirements or that may impact the quality 
of the data obtained from the study. 
e. The participant is listed for transplant and is required to stop sirolimus therapy. 
f. In the opinion of the investigator, it is not in the participant’s best medical interest to 
continue to participate in the study.   
g. The participant meets any of the individual stopping rules as delineated in section 13.1. 
 
7.2 Criteria for terminating the study 
Up to 25 patients will be enrolled in this study. The maximum duration of patient 
participation is up to 28 weeks, including a follow-up phone contact 4 weeks after the last 
study visit. In the event of any death, or Grade IV Serious adverse event (SAE) 
attributable to the study agent and unexpected, enrollment will be suspended, pending 
Data Safety Monitoring Board (DSMB) review.  
 
 
 


 
38 
8. Study variables and measuring methods 
 
8.1 Physical Exams: 
Exams will be performed in a routine manner, consistent with the standard of care. 
 
8.2 Pulmonary function testing 
Pre and post bronchodilator spirometry and DLCO measurement will be performed in a 
standardized manner according to American Thoracic Society/European Respiratory 
Society criteria for acceptability and repeatability (38). The study site will attempt to 
have the same respiratory therapist administer testing to patients for consistency in the 
study. All PFTs obtained as part of the study will be performed post bronchodilator only, 
regardless of whether bronchodilator responsiveness is demonstrated. All spirometry data 
will be assessed for quality by spirometry standardization expert and study investigators, 
Dr. Francis X. McCormack, Dr. Adam Cole, and Dr. Nishant Gupta.  
 
8.3 Questionnaires 
St. George’s Respiratory Questionnaire (SGRQ) 
The SGRQ is a disease-specific instrument designed to measure impact on overall health, 
daily life, and perceived well-being. It has been developed to be used by patients with 
fixed or reversible airway obstruction. It has been shown to correlate well with 
physiologic parameters and to show changes with time in patients with LAM (39). The 
administration is performed by face to face or telephone interview; completion takes 
approximately 10 minutes. The questionnaire consists of 76 items divided into 3 
domains: symptoms (frequency and severity); activity (activities that cause or are limited 
by breathlessness); and impacts (social functioning, psychological disturbances resulting 
from airways disease).  
A Tool to Assess Quality of Life in LAM (ATAQ-LAM) 
ATAQ-LAM is a disease-specific instrument designed to assess health related quality of 
life in patients with LAM. The questionnaire consists of 32 items divided into 4 domains: 
exertional dyspnea, cough, fatigue, and emotional well-being.  
The EuroQOL visual analogue scale for quality of life 
The EuroQOL visual-analogue scale measures self-reported ratings of health status. 
Scores range from 0 to100, with lower scores indicating worse functioning. Patients 
receiving sirolimus in the MILES trial had an increase in their scores on the EuroQOL 
scale as compared to the placebo group (12).  
 


 
39 
San Diego Shortness of Breath Questionnaire 
The San Diego shortness of breath questionnaire measures the quality of life impairment 
secondary to dyspnea resulting from pulmonary disorders. It is a 24-item measure that 
assesses self-reported shortness of breath while performing a variety of activities of daily 
living (40).  
 
8.4 Biomarker Analyses 
VEGF-D biomarker analysis will be conducted on blood samples collected from each 
study patient at scheduled times. Assays will be performed by a central laboratory in 
Cincinnati. Serum VEGF-D has been shown to be elevated in patients with LAM and is a 
useful diagnostic, prognostic, as well as predictive biomarker (13, 14, 16).  
 
8.5 Home Spirometry  
FEV1 and FVC will be obtained once weekly from patients agreeing to participate in the 
home spirometry sub study. Detailed instructions regarding the proper use of the home 
spirometer will be provided at the time of enrollment, along with access to detailed 
instruction manuals for future use. Once a week, patients will perform at least three 
spirometric measurements at approximately the same time, and record the best FEV1 and 
FVC values as their weekly results. These results, along with the associated flow-volume 
and volume-time loops will then be transmitted electronically to the study coordinator. 
The spirometric values as well as the loops will be reviewed in near real time along with 
direct feedback to the subject on the performance and trouble shooting of any issues by 
an expert pulmonary physiologist (Dr. Adam Cole and/or Dr. Nishant Gupta).  
 


 
40 
9. Risks 
9.1 Risks of the investigational product 
9.1.1 Pharmacokinetics of resveratrol in humans 
Absorption: In general, resveratrol is rapidly absorbed following oral administration. 
After oral administration, median time to achieve peak plasma concentration (Tmax) is 
approximately 30 minutes for low-dose resveratrol (41), and approximately 60-90 
minutes for high-dose resveratrol (42, 43). Absorption of resveratrol is affected by food 
intake. Peak plasma concentration of resveratrol (Cmax) occurred 3 hours after a standard 
breakfast and 5 hours after consuming a high-fat meal (44). Co-administration of alcohol 
does not seem to affect resveratrol bioavailability (44).  
 
Distribution: Because of its lipophilic nature, resveratrol has a high volume of 
distribution. Volumes of distribution ranging between 730 liters to 22000 liters have been 
described after ingesting resveratrol (44, 45). 
 
Metabolism: Resveratrol undergoes extensive metabolism after human consumption. 
With small doses of resveratrol (up to 50mg per day), glucuronides are the main 
metabolites present in plasma (46). Monosulfates are the major metabolites when taking 
higher doses (> 250 mg per day) of resveratrol (47). It is possible that the resveratrol 
metabolites could act as a reserve for the parent molecule and, thus, contribute to 
resveratrol’s pharmacological effects (48). 
 
Elimination: The half-life of resveratrol varies depending upon the form of the drug. The 
half-life for micronized resveratrol is approximately 1 hour (49), whereas the half-life for 
nonmicronized resveratrol is approximately 8 hours (42). Resveratrol and its metabolites 
are mainly excreted through feces and urine (41). Fecal excretion is the major route of 
elimination for resveratrol and its metabolites accounting for greater than 70% of the 
excretion, with urinary elimination accounting for the remaining portions (41, 50).  
9.1.2 Safety Profile of Resveratrol in Humans 
In general, resveratrol is a well-tolerated drug with a robust safety profile. No adverse 
effects related to resveratrol were reported in clinical studies evaluating doses up to 1 
gram of resveratrol per day (50-55). Resveratrol is well tolerated even at higher doses, 
however there is an increase in the gastrointestinal side effects at doses exceeding 1 gram 
per day. Howells et al. conducted a phase I study evaluating the safety profile of a 
micronized oral formulation of resveratrol in patients with liver metastases of colorectal 
cancer. Six patients received 5 grams per day of resveratrol for a total of 14 days. 
Resveratrol was well tolerated, in general, with the most common adverse effects being 
mild nausea and diarrhea. Other adverse effects included chills, lethargy, rash, peripheral 
neuropathy, skin irritation and vascular flushing, which resolved without sequelae (49). 
Using the same dose and formulation in 24 patients with multiple myeloma, Popat et al. 
noticed a higher rate of development of adverse effects. The most commonly reported 
adverse events were: nausea (79%), diarrhea (71%), vomiting (54%), fatigue (46%) and 
anemia (38%). 54% of patients reported grade 3 adverse events; most commonly 21% 
hematological (anemia and thrombocytopenia), 21% renal failure, 13% nausea and 13% 
infections (56).  


 
41 
In a phase I study, 10 healthy volunteers were given 4 different strengths of resveratrol 
(0.5 grams, 1 gram, 2.5 grams and 5 grams per day). Resveratrol was well tolerated at all 
doses, with no serious adverse effect noted. Minor adverse events were seen in 57% of 
the patients, and were self-limited resolving 2-4 days post administration (45). The same 
study was repeated again yielding similar results. There were no serious adverse events 
related to resveratrol. However, subjects taking the higher dose of resveratrol (2.5 grams 
and 5 grams per day) had an increased incidence of gastrointestinal symptoms, including 
nausea, flatulence, abdominal discomfort and diarrhea. Most of these events were mild 
(severity grade 1, NCI CTCAE v.4.0), although 4 participants on the 2.5 gram and 5 gram 
doses presented with nausea and/or diarrhea of moderate severity (grade 2). The 
gastrointestinal side effects commenced after 2-4 days of the intervention and occurred 
half to one hour after resveratrol ingestion (42). Other studies have reported similar 
findings with an increase in the incidence of gastrointestinal side effects at resveratrol 
doses higher than 1 gram per day, with most side effects being mild and spontaneously 
resolving after a brief drug holiday (41, 44, 47, 57, 58). Due to the increased incidence of 
gastrointestinal side effects related to resveratrol at high doses (42), and the increased 
incidence of renal failure noted in one study (56), we have decided to limit the highest 
dose in our study at 1 gram per day.   
Adverse reaction 
Incidence (%) 
Gastrointestinal disorders  
 
Nausea  
10 - 15 
Vomiting  
8 - 12 
Diarrhea  
20 – 30 
Flatulence 
5 – 10 
Abdominal discomfort 
5 - 10 
Nervous system disorders 
 
Headache 
< 1 
Peripheral neuropathy 
< 1 
General disorders 
 
Fatigue 
< 5 
Chills  
< 1  
Musculoskeletal disorders 
 
Myalgias and muscle cramps 
< 1 
Skin and subcutaneous tissue disorders  
Rash  
< 1 
Skin discoloration/irritation 
< 1 
Acne 
< 1 
Flushing  
< 1 
Hematologic disorders 
 
Anemia 
2 - 4 
Thrombocytopenia 
2 - 4 
Renal disorders 
 
Acute renal failure  
0 – 4% 
Laboratory abnormalities 
 
Elevated bilirubin 
< 1 


 
42 
Table 1: Adverse reactions associated with the use of resveratrol (41, 42, 44, 45, 47, 49, 
56-58). Percentages for the incidence of a particular adverse event are estimated after 
combining the patient numbers in clinical studies evaluating high dose resveratrol.  
 
9.2 Risks of study procedures 
Questionnaires: Participants will complete questionnaires about their health and basic 
information about demographics will be obtained. These procedures represent no more 
than minimal risk. Care will be taken to ensure confidentiality. Research records will be 
held in locked cabinets or locked storage rooms when staff members are not in 
attendance. All transmission of data to the investigators’ laboratory for analysis will 
contain only subject’s coded identification. Subjects’ names or any other personal 
identifying information will not be used. Publication of results will involve aggregate 
data only so that individual participants cannot be identified. On occasion, it may be 
necessary, for legal reasons, or for good clinical practice, for third parties such as the 
IRB, to review medical records that are identified by name, but this should be an 
uncommon occurrence, and every effort will be made by the investigators to provide 
confidentiality. 
Blood draws: The risks of blood draws include bleeding, bruising, discomfort, fainting 
and lightheadedness infection or pain at the needle site. All research personnel drawing 
blood are trained in the procedure and are familiar with proper infection control protocol.  
Pulmonary function tests: In general, PFTs involve minimum risks, which will be 
reviewed with the subject prior to performing the testing. In general, these tests are well 
tolerated but occasionally some people may experience temporary tiredness, mild chest 
tightening, and coughing. Short acting bronchodilators such as albuterol will be available 
and will be administered if needed. Subjects with LAM are routinely evaluated with 
breathing tests as a matter of clinical care.  
 
9.3 Adequacy of protection against risks 
Each participant in this study will be informed of the intent of the study and asked to sign 
an IRB approved informed consent form. The consent form will fully describe the 
procedures, risks, alternatives, and potential benefits of the study. Consent will be 
obtained from eligible participants by a physician investigator or by the study research 
coordinator. The participants will be given the opportunity to refuse to participate in the 
study, under the assurance that such refusal will in no way affect their regular treatment. 
All questions asked by study subject will be answered to the best of the knowledge of the 
investigator and the study staff. Time will be given for the patient to read the consent 
form and ask questions prior to performing any study procedures. A copy of the signed 
and dated consent form will be given to subject prior to leaving the clinic. There are no 
alternatives in this study other than not to participate.  Potential risks and benefits will be 
thoroughly explained to the subjects participating and they are free to withdraw from 
these studies at any point in time prior to or during the course of these investigations. 


 
43 
All information, data, etc., collected as a result of these studies is considered confidential 
and will be released only as required by law. Information published as a result of this 
study will be protected completely preserving the anonymity of the participants.  Per 
HIPAA guidelines, research records will be stored in a locked room identified 
specifically as a patient record room. All transmitting of data is done by coded names and 
does not identify any types of names (first or last). Results of any procedures or tests will 
also be de-identified so as to hide the true identity of patient's names or any other 
identifiers. On occasion, it may be necessary, for legal reasons, or for good clinical 
practice, for third parties such as the IRB, FDA, or DSMB members to review medical 
records that are identified by name. This however would be on special occasions and 
would not be a common occurrence. Every effort in all areas of care will be made to keep 
the patient's information as confidential as possible. As noted above, we will minimize 
risks to human subjects by creating and maintaining a standard of excellence such as we 
had with our previous clinical trials in our sites.  Such standard includes active 
interactions between faculty and staff on a weekly formal basis, and as needed otherwise, 
careful attention to training of staff in procedures and their possible complications, and a 
philosophy of ‘patient first’ in all of our dealings. Specifically, we use only trained 
personnel to perform procedures. The plan to handle adverse events is described in detail 
in the section of data and safety monitoring plan.  
 
9.4 Plans for assuring compliance with safety guidelines and regulations 
All clinical staff will be required to participate in training offered through their respective 
Institutional Review Boards. Training is also required in Health Insurance Portability and 
Accountability Act (HIPAA) in order to have access to study subject records.  
International Conference of Harmonization (ICH) Good Clinical Practice Guidelines 
(GCPs) are utilized daily in the clinical research practice and are reviewed periodically.  
The clinical research teams will have twice a month conference calls to provide an 
opportunity for staff to review and discuss the activities of the study. All clinical and 
laboratory staff are required to take courses handling blood borne pathogens on a yearly 
basis as well as training in laboratory safety, handling of biohazards, storage and shipping 
of clinical and laboratory specimens including dangerous goods.   
 
 


 
44 
10. Benefits 
10.1 Benefits of the investigational agent 
As with other studies investigating novel treatment for LAM, there may or may not be 
any benefits for the subject participating in the study. However, participation in this study 
will provide additional data on the safety and effectiveness of resveratrol for the 
treatment of LAM. Some participants in this study may benefit from the general 
evaluation of their disease from a qualified specialist for the duration of their 
participation in the trial. The participants may benefit from the physical exams, 
pulmonary function tests, and other study procedures. The information learned from this 
research study may benefit other subjects with LAM in the future. Subjects who 
participate in the study will receive all study medications at no charge, will have all 
testing at no cost to them or their insurance carrier (and the results of those tests will be 
made available to their primary care physician).   
 
10.2 Importance of the knowledge to be gained 
In general, the knowledge gained from this study may benefit many subjects with LAM. 
The information obtained may improve the scientific community's understanding of the 
management of LAM. Specifically, the study will identify the safety and efficacy of the 
combination of resveratrol and sirolimus in LAM and thus may fundamentally alter the 
current clinical approach to patients with LAM. 
 
10.3 Benefits of Study Procedures 
None. 
 
 
 


 
45 
11. Drug and Drug Procurement: 
 
11.1 Resveratrol: 
 
Resveratrol (3,5,4'-trihydroxystilbene) is a polyphenolic phytoalexin. It is a stilbenoid, a 
derivate of stilbene, and is produced in plants with the help of the enzyme stilbene 
synthase. It exists as two structural isomers: cis-(Z) and trans-(E). The molecular formula 
for resveratrol is C14H12O3 and its molecular weight is 228.24 g/mol. The structural 
formula of resveratrol is: 
 
 
 
Resveratrol used in this study is manufactured by fermentation using genetically 
modified yeast in a process that results in trans-resveratrol with a purity of at least 98%. 
The product is free from DNA, proteins and cells from the production host. Resveratrol is 
available in the form of an off white powder that is highly lipophilic, and almost 
insoluble in water.  
 
Resveratrol is available as white nº0 opaque capsules containing 125mg/capsule of 
resveratrol. The excipients present in the capsules are cellulose and magnesium stearate. 
Resveratrol capsules come in white plastic containers containing 200 capsules per 
container.  Containers are to be stored at room temperature for a maximum of 2 years 
after encapsulation.  
 


 
46 
11.2 Sirolimus: 
Sirolimus is an immunosuppressive agent. Sirolimus is a macrocyclic lactone produced 
by Streptomyces hygroscopicus. The chemical name of sirolimus (also known as 
rapamycin) is (3S,6R ,7E,9R ,10R,12R ,14S,15E ,17E,19E ,21S,23S ,26R,27R ,34aS)- 
9,10,12,13,14,21,22,23,24,25,26,27,32,33,34, 
34a-hexadecahydro-9,27-dihydroxy-3- 
[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy- 
6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4] 
oxaazacyclohentriacontine-1,5,11,28,29 (4H,6H ,31H)-pentone. Its molecular formula is 
C51H79NO13 and its molecular weight is 914.2. The structural formula of sirolimus is:  
 
 
 
 
Sirolimus is a white to off-white powder and is insoluble in water, but freely soluble in 
benzyl alcohol, chloroform, acetone, and acetonitrile.  
 


 
47 
Sirolimus is available for administration as an oral solution containing 1 mg/mL 
sirolimus. Sirolimus is also available as a white, triangular-shaped tablet containing 1 mg 
sirolimus, and as a yellow to beige triangular-shaped tablet containing 2 mg sirolimus.  
The inactive ingredients in sirolimus Oral Solution are Phosal 50 PG® 
(phosphatidylcholine, propylene glycol, mono- and di-glycerides, ethanol, soy fatty acids, 
and ascorbyl palmitate) and polysorbate 80. Rapamune Oral Solution contains 1.5% - 
2.5% ethanol.  
 
The inactive ingredients in sirolimus tablets include sucrose, lactose, polyethylene glycol 
8000, calcium sulfate, microcrystalline cellulose, pharmaceutical glaze, talc, titanium 
dioxide, magnesium stearate, povidone, poloxamer 188, polyethylene glycol 20,000, 
glyceryl monooleate, carnauba wax, and other ingredients. The 2 mg dosage strength also 
contains iron oxide yellow 10 and iron oxide brown 70.  
 
Patients will remain on sirolimus and continue to obtain those agents in the manner 
already established. Sirolimus has approval from the FDA for the treatment of LAM. 
 
11.3 Accountability of Investigational Product 
 
Under Title 21 of the Code of Federal Regulations (21CFR §312.62) the investigator will 
maintain adequate records of the disposition of the investigational product(s)/intervention 
material(s), including the date and quantity of the drug received, to whom the drug was 
dispensed (participant-by-participant accounting), and a detailed accounting of any 
investigational product(s)/intervention material(s) accidentally or deliberately destroyed. 
Records for receipt, storage, use, and disposition will be maintained by the study site.  A 
dispensing log will be kept current for each participant. This log will contain the 
identification of each participant and the date and quantity of investigational 
product(s)/intervention material(s) dispensed. All records regarding the disposition of the 
investigational product will be available for inspection by the DSMB, FDA, or IRB.  
 
11.4 Assessment of compliance with the investigational product 
 
Drug adherence will be assessed by pill counts. Pill counts will not be done in the 
presence of participants to mitigate “dumping” behavior, and therefore allow more 
accurate assessment of drug use. Adherence will be reinforced using standardized non-
judgmental questioning and tailored behavioral strategies.  
 
 
 


 
48 
12. Modification or discontinuation of the investigational product 
 
12.1 Modification of investigational product 
 
The product will not be modified during the course of the study by the investigator, 
pharmacy or subject.   
 
12.2 Premature discontinuation of investigational product 
 
Refer to sections 12.3 and 13.1.  
 
12.3 Medication adjustments  
12.3.1 Dose Delays 
For patients who are unable to tolerate the protocol-specified dosing schedule, dose 
adjustments are permitted in order to keep the patient on study drug. If administration of 
sirolimus must be interrupted because of unacceptable toxicity, drug dosing will be 
interrupted or modified according to rules described in Section 12.3.3. Sirolimus therapy 
may also be interrupted to accommodate surgical procedures or other invasive therapies 
at the discretion of treating clinicians. Major Events are non-treatment-related grade 3 
and 4 pulmonary and non-pulmonary toxicities. Treatment should be delayed for major 
events if resveratrol may further complicate the non-treatment related event. If a major 
event requires a delay of treatment, treatment must be delayed until toxicity is resolved (≤ 
Grade 1 or ≤ Baseline). For treatment-related toxicities and major events, if toxicity is not 
resolved in ≤ 4 weeks, patient will be taken off the treatment that is most likely to be 
related to the toxicity. While this most likely will result in withdrawal from study drug 
exposure at the investigator’s discretion patients may continue on single agent therapy, if 
toxicities are ≤ grade 2 and there is some evidence of clinical benefit to the patient.  
Toxicity 
Dose reduce first 
Dose reduce second 
Nausea 
Resveratrol 
Sirolimus 
Diarrhea 
Resveratrol 
Sirolimus 
Abdominal pain 
Resveratrol 
Sirolimus 
Infections 
Sirolimus 
Resveratrol 
Headache 
Resveratrol 
Sirolimus 
Table 2: Approach to overlapping toxicities of sirolimus and resveratrol. The above table 
is a suggestion. Final decision on the order of dose reduction will be at the discretion of 
the investigator.  


 
49 
12.3.2 Resveratrol dose reduction 
Any AE of ≥ Grade 3 and attributed as possibly, probably or definitely related to 
resveratrol will result in the dose being held until the AE has resolved to ≤ grade 1 or 
baseline, while sirolimus dosing may continue uninterrupted. If the AE resolves, 
reinstitution of treatment can occur but at a reduced dose as described in Table 3. If the 
AE recurs at the reduced dose, treatment will be held until the AE has resolved to ≤ grade 
1 and when resolved treatment can be reinstituted at the next lower dose level. No more 
than 2 dose reductions are allowed during the maintenance cycles. Participants who are 
unable to tolerate the lowest dosage of resveratrol will be withdrawn from study drug 
exposure.  
Toxicities that may be attributable to resveratrol include nausea, vomiting, abdominal 
pain, diarrhea, headache, and fatigue. If any of these AEs occur at grade ≤ 2, resveratrol 
may be continued and the AE managed with supportive care. For any AE with a grade ≥ 
3, the rules outlined in Sections 14.2 – 14.10 apply for holding of dose, dose reduction, 
removal from study and reporting requirements.  
Dose  
Reduce to  
250 mg daily 
Hold or withdrawal from resveratrol 
exposure  
500 mg daily 
250 mg daily 
1000 mg daily 
500 mg daily 
Table 3: Dose reduction schema for resveratrol 
12.3.3 Sirolimus dose reduction 
Known sirolimus toxicities will not be attributed to resveratrol but will only be attributed 
to sirolimus and may result in sirolimus dose modifications, at the discretion of the 
treating physician. Sirolimus trough levels will be checked at 2-, 4-, 8-, 12-, 16-, 18-, 20-, 
and 24- week intervals following study drug initiation. Sirolimus dose will be reduced if 
the serum trough level increases to a value greater than 10ng/ml. If sirolimus dose is 
reduced based on a high trough value, the trough level will be repeated 2 weeks after 
dose reduction. This process will be repeated until a sirolimus trough value less than 
10ng/ml is achieved. The exact dose reduction scheme for sirolimus will be left at the 
discretion of the treating physician. Sirolimus toxicity monitoring labs will be performed 
as clinically indicated at the discretion of the physician. For severe/non-resolving 
toxicities, the treating physician may decide to stop sirolimus. Resveratrol may be 
continued as a single agent after sirolimus has been stopped. All reasonable efforts will 
be made to avoid sirolimus dose changes throughout the study duration, as long as it does 
not impact clinical patient care. A notification letter will be sent to the patient’s primary 
pulmonologist about the patient’s participation in the study and urging them to avoid 
sirolimus dose changes throughout the study duration, unless absolutely clinically 
indicated. 


 
50 
12.3.4 Concomitant medications 
All concomitant medications including prescription and over-the-counter preparations 
taken during the course of the study will be documented on a CRF including start and 
stop dates, dose, and indication for use.  
12.3.5 Prohibited medications 
Participants will not be allowed to receive any concomitant medication(s) that may 
interfere in the interpretation of data or the subject’s participation in the study in the 
Investigator’s opinion.  
Metabolism of sirolimus occurs in the liver and the small intestine by the CYP3A4 family 
of enzymes. Thus, concomitant administration of sirolimus with other CYP3A substrates 
or inducers can alter the oral bioavailability of sirolimus (59). Although not a strong 
modulator of CYP activity, resveratrol has been reported to inhibit the activity of 
CYP3A4 in vitro and in vivo (60). Thus, co-administration of sirolimus and resveratrol 
has the potential to increase the blood levels of sirolimus or lead to an increase in the side 
effects of sirolimus. We will periodically monitor the sirolimus level during the study, as 
well as monitor closely for any adverse effects related to the study medications.  
Because of the potential interaction with other drugs involving CYP3A4 metabolic 
pathways, strong inhibitors and strong inducers of CYP3A4 will not be allowed to be 
used concomitantly with the study medication. A washout period of 14 days will be 
required after discontinuation of the below listed medications before beginning the study 
medication. A list of the prohibited medications is provided in Table 4 below: 
List of prohibited medications for this study 
Strong CYP inducers 
Barbiturates 
Carbamazepine 
Phenytoin 
Rifampin, Rifabutin 
St. John’s Wort 
Strong CYP inhibitors 
Ketoconazole 
Voriconazole 
Itraconazole 
Nefazodone 
Clarithromycin 
Erythromycin 
Telithromycin 
Gemfibrozil 
Grapefruit juice 
Table 4: List of prohibited medications for this study 
Concomitant use of resveratrol with drugs with a narrow therapeutic range that are 
CYP3A, CYP1A2, CYP2D6, or CYP2C9 substrates (for example, theophylline, 


 
51 
tizanidine, warfarin, phenytoin, alfentanil, dihydroergotamine, ergotamine, fentanyl, 
pimozide, quinidine, cyclosporine, tacrolimus, thioridazine) will be prohibited from Day 
1 until the end of study.  
13. Procedures 
13.1 Stopping rules 
13.1.1 Study discontinuation 
The DSMB, FDA, and IRB have the authority to stop or suspend this trial at any time. 
This study may be suspended or closed if: 
• Early stopping rules have been met 
• The study objectives have been met 
• The study chair/investigators believe it is not safe for the study to continue 
• The DSMB suspends or closes the trial 
• The FDA suspends or closes the trial 
13.1.2 Subject discontinuation 
An intent-to-treat approach will be used. All data acquired prior to termination for the 
reasons outlined below will be included in the primary analysis unless patient withdraws 
consent. Every effort will be made to conduct a final study visit with the participant and 
participants will be followed clinically until, if applicable, all adverse events attributable 
to study drug resolve. Participants may be terminated early from the study for the 
following reasons: 
• Withdrawal of consent 
• Withdrawal by the participant 
• Withdrawal by the investigator 
• Intercurrent illness or event that precludes further visits to the study site or ability 
to evaluate disease (e.g. mental status change, large pleural effusion etc.) 
13.1.3 Study early stopping rules 
Study enrollment will be suspended pending expedited review of all pertinent data by the 
IRB and the DSMB if any one of the following occurs: 
• Death from any cause in any subject believed to be related to study drug 
• Multiple medically similar serious adverse events related to study medication 
• Events that, in the opinion of the Principal Investigator contraindicate further 
dosing of additional subjects 
If one of the above-listed stopping rules are met, a prompt cumulative review of safety 
data and of the circumstances of the event(s) in question will be conducted to determine 
whether study entry should be resumed, whether the protocol will be modified, or 
whether the study will be discontinued permanently. Review and approval by the DSMB 
is required for resumption of the study in the event the study is interrupted because of one 
of the above-listed events. 


 
52 
 
 
13.1.4 Individual early stopping rules 
A study participant will be terminated from the study if any of the following occurs 
during the study: 
• Recurrent or persistent resveratrol-related toxicity unresponsive to treatment in 
spite of dose adjustments. 
• Pregnancy or failure to comply with adequate contraception 
• The participant refuses to continue in the study  
• Continued non-compliance 
• The investigator determines that the participant should be withdrawn from the 
study for reasons not listed above 
A subject will be considered evaluable if they receive treatment for at least 8 weeks and 
the relevant information collected during this period. Subjects who are withdrawn after 
less than 8 weeks of treatment will be considered as pre-mature discontinuation, and will 
be replaced by another participant.  
 
Every effort will be made to obtain endpoint evaluations for subjects who withdraw or 
who discontinue prematurely from the study. The same evaluations performed at final 
visit will be completed at the time of termination. If withdrawal is due to inability to 
tolerate investigational drug or other untoward event related to investigational drug, the 
withdrawal will be noted in data analysis as treatment failure.  
 
13.2 Follow-up after early study termination 
 
Participants who are prematurely withdrawn from study drug exposure due to adverse 
events will be followed to monitor safety for a minimum of 30 days or until resolution or 
stabilization of the disqualifying event or until the Principal Investigator determines that 
further follow-up is not needed. 
 
Subjects considered “lost-to-follow-up” are those enrolled in the study, but, for whatever 
reason, refuse to return to complete the study per the protocol. Reasonable efforts will be 
made to contact any subject lost to follow-up during the course of the study in order to 
complete assessments and retrieve any outstanding data, study drugs or clinical supplies. 
The investigator must make at least three documented attempts to have the patient return 
for study withdrawal evaluations.  
 
13.3 Participant replacement 
 
Participants with early termination from this study will be replaced if they are deemed 
non-evaluable.  


 
53 
14. Data and Safety Monitoring Plan 
The study protocol will be reviewed and approved by the DSMB and University of 
Cincinnati IRB prior to study initiation.  Participant enrollment may only begin with IRB 
approved consent forms. This is an interventional exploratory phase II study. 
14.1 Study Oversight 
The Principal Investigator has primary oversight responsibility of this clinical trial. The 
DSMB has oversight responsibility of the Data Safety Monitoring Plan (DSMP) for this 
clinical trial. The DSMB will review accrual, patterns and frequencies of all adverse 
events, and protocol compliance every 6 months. The DSMB makes recommendations to 
the PI regarding the continuation status of the protocol. 
The site Principal Investigator and their research team (co-Investigators, research nurses, 
clinical trial coordinators, and data managers) are responsible for identifying adverse 
events. Aggregate report - detailed by severity, attribution (expected or unexpected), and 
relationship to the study drug/study procedures – will be available for site review.  
14.2 Adverse Events 
This section defines the types of adverse events and outlines the procedures for 
appropriately collecting, grading, recording, and reporting them.  Information in this 
section complies with ICH Guideline E2A: Clinical Safety Data Management: 
Definitions and Standards for Expedited Reporting and ICH E6: Guideline for Good 
Clinical Practice, and applies the standards set forth in the National Cancer Institute 
(NCI), Common Terminology Criteria for Adverse Events version V 4.0.  These criteria 
have been reviewed by the study investigators and have been determined appropriate for 
this study population. 
14.3 Definitions 
14.3.1 Adverse events 
An adverse event (AE) is any occurrence or worsening of an undesirable or unintended 
sign, symptom, laboratory finding, or disease that is experienced during participation in 
the trial.  An AE can therefore be any unfavorable and unintended sign (including an 
abnormal laboratory finding), symptom, or disease temporarily associated with the use of 
a medicinal (investigational) Study Agent(s) or a study procedure, whether or not related 
to the medicinal (investigational) Study Agent(s) or study procedure.  Any medical 
condition that is present at the time that the subject is screened will be considered as 
baseline and not recorded as an AE.  However, if the condition deteriorates or changes in 
severity at any time during the study it will be recorded and reported as an AE.   
14.3.2 Suspected adverse reaction and adverse reaction 
Suspected adverse reaction (SAR) means any adverse event for which there is a 
reasonable possibility that the drug caused the adverse event. For the purposes of safety 
reporting, ‘reasonable possibility’ means there is evidence to suggest a causal relationship 
between the drug and the adverse event. A suspected adverse reaction implies a lesser 
degree of certainty about causality than adverse reaction, which means any adverse event 
caused by a drug. 


 
54 
An adverse reaction (AR) means any adverse event caused by a drug.  Adverse reactions 
are a subset of all suspected adverse reactions for which there are reasons to conclude 
that the drug caused the event. 
14.3.3 Adverse events associated with study procedures 
The following clinical situations will be considered to be outside of the normal range of 
findings and will be recorded as Adverse Events. These situations do not limit the 
principal investigator from reporting any other events, associated or not with these 
procedures, from being recorded and reported as AEs.   
Blood draws 
• Fainting/vasovagal events 
• Bruising at puncture site greater than 2 cm in diameter 
• Bleeding from puncture site lasting more than 5 minutes 
• Swelling at puncture site larger than 2 cm 
Pulmonary function testing 
• Wheezing or bronchoconstriction requiring treatment with bronchodilators within 
30 minutes from the procedure 
• Coughing requiring treatment with bronchodilators within 30 minutes from the 
procedure 
Adverse events related to study medication will be tabulated by body system and by 
severity using the NCI CTCAE v4.0.  
14.3.4 Serious Adverse Event (SAE) 
An AE or SAR is considered “serious” if it results in any of the following outcomes (21 
CFR 312.32(a): 
• Death: A death that occurs during the study or that comes to the attention of the 
investigator during the protocol-defined follow-up.  
• A life-threatening event: An adverse event or suspected adverse reaction is 
considered “life-threatening” if, in the opinion of the investigator, its occurrence 
places the subject at immediate risk of death. It does not include an adverse event 
or suspected adverse reaction that, had it occurred in a more severe form, might 
have caused death. 
• An inpatient hospitalization or prolongation of existing hospitalization. 
• Persistent or significant disability/incapacity or substantial disruption of the 
ability to conduct normal life functions. 
• An important medical event that may not result in death, be life threatening, or 
require hospitalization may be considered an SAE when, in the opinion of the 
investigator, it may jeopardize the participant and may require medical or surgical 
intervention to prevent one of the outcomes listed above. 
• Congenital anomaly or birth defect. 


 
55 
Regardless of the relationship of the adverse event to the study, the event will be reported 
per Section 14.3.4 as an SAE if it meets any of the above definitions.  
14.3.5 Expected adverse events 
This section lists those adverse events regarded as expected for resveratrol: 
Gastrointestinal: Nausea, vomiting, diarrhea, abdominal discomfort, flatulence 
General: Fatigue 
Hematological: Anemia, thrombocytopenia 
Renal: Acute kidney injury 
Liver: Increased serum bilirubin 
Infectious: Increased risk of infections 
 
14.3.6 Unexpected adverse event 
An adverse event or suspected adverse reaction is considered “unexpected” when its 
nature, severity or frequency is not consistent with the information that is provided with 
the information in the protocol.  
14.4 Collecting, recording and managing adverse events 
14.4.1 Identifying adverse events 
Any adverse event that occurs from the moment the subject has signed the consent form 
will be recorded and is reportable. Collection time for AEs and SAEs will begin after the 
screening visit when the consent form is signed and will continue until the end of the 
treatment. 
Adverse events may be discovered through any of these methods: 
• Observing the participant 
• Questioning the participant, with standardized questions/procedures. 
• Receiving an unsolicited complaint from the participant. 
• An abnormal value or result from a clinical or laboratory evaluation can also 
indicate an adverse event when determined to be clinically significant by the 
Principal Investigator.  
14.4.2 Recording AEs 
Throughout the study all identified adverse events (serious and non-serious) will be 
recorded on all appropriate source document and adverse event case report forms 
regardless of their severity or relation to the study.  A complete description of all adverse 
events will include event description, time of onset, investigator assessment of severity, 
relationship to study agent or procedures, time of resolution/stabilization of the event, 
expectedness, determination of whether the AE qualifies as a SAE, and action taken.  A 
change in the severity of the AE will also be documented. Assessment of severity and 
relationship will be documented on the source documents or the on the CRF.  
 


 
56 
14.4.3 Recording SAEs 
Serious adverse events will be recorded on the serious adverse event case report form and 
will include a narrative of the event signed and dated by the Principal Investigator.  
Adverse event reporting timeline 
• Within 24 hours (of learning of the event), investigators must report any 
reportable Serious Adverse Event (SAE) that: 
o Is considered life-threatening/disabling or results in death of subject 
-OR- 
o Is unexpected/unanticipated 
• Investigators must report all other reportable SAEs within 5 working days (of 
learning of the event). 
• All other (suspected) reportable AEs must be reported to the DSMB and the IRB 
within 20 working days of the notification of the event or of the investigators 
becoming aware of the event.  
 
14.5 Managing adverse events 
The study investigator must apply his or her clinical judgment as to whether an AE is of 
sufficient severity to require that the participant immediately be removed from further 
receiving the study agent under the protocol. The investigator must institute any 
necessary medical therapy to protect a participant from any immediate dangers. 
An adverse event will be followed until any of the following takes place:  a) it is 
resolved, b) participant is stable, c) a minimum of 30 days after participant is terminated 
from the study and the Principal Investigator determines that follow-up is no longer 
needed. 
If an abnormal value or result from a clinical or laboratory evaluation is determined to be 
an AE, then the evaluation that produced the value or result can be repeated until the 
value or result returns to normal, or the result can be explained, or the usual standard of 
care does not require further follow-up, and the participant’s safety is not at risk.   
Non-serious expected adverse events will be submitted to the DSMB in a timely fashion. 
The events will be presented in tabular form and given to the DSMB on a bi-annual basis. 
Study investigators are also required to fulfill all reporting requirements of their local 
institutions. 
 
 


 
57 
14.6 Grading and Attribution 
14.6.1 Grading Criteria 
In addition to determining whether an adverse event fulfills criteria for a serious adverse 
event or not, the severity of adverse events experienced by study participants will be 
graded according to the criteria set forth in the National Cancer Institute’s Common 
Terminology Criteria for Adverse Events Version V4.0.  This document (referred to 
herein as the NCI-CTCAE manual) provides a common language to describe levels of 
severity, to analyze and interpret data, and to articulate the clinical significance of all 
adverse events.  All adverse events whether or not listed in the NCI-CTCAE will be 
graded on a scale from 1 to 5 according to the following standards in the NCI-CTCAE 
manual (A semi-colon indicates ‘or’ within the description of the grade): 
Grade 1 = Mild; asymptomatic or mild symptoms; clinical or diagnostic observations 
only; intervention not indicated.  
Grade 2 = Moderate; minimal, local or noninvasive intervention indicated; limiting age-
appropriate instrumental ADLs (preparing meals, shopping for groceries or clothes, using 
the telephone, managing money, etc). 
Grade 3 = Severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; limiting self care 
ADLs (bathing, dressing and undressing, feeding self, using the toilet, taking 
medications, and not bedridden).  
Grade 4 = Life-threatening consequences; or urgent intervention indicated. 
Grade 5 = Death related to AE. 
 
14.7 Definition of Attribution 
The attribution, of an adverse event to the study will be determined by the Principal 
Investigator or designated physician co/sub-investigator. The Principal Investigator or 
designee will record the determination of attribution on the appropriate adverse event or 
serious adverse event form.  The attribution of an adverse event to the investigational 
drug or to a study procedure will be determined using the descriptors in the following 
table. For the purpose of this study, in addition to all study medications, the following 
procedures will be considered when determining attribution: 
 
 
 
 


 
58 
Attribution of adverse events 
Code 
Descriptor 
Definition (guidelines) 
UNRELATED CATEGORY  
1 
Unrelated 
The adverse event is clearly not related to study.  The event is 
completely related to an etiology other than the study product or 
study intervention (the alternative etiology must be documented in 
the study subject’s medical record) 
2 
Unlikely 
The adverse event is doubtfully related to study and likely to be 
related to factors other than study product or study intervention. 
RELATED CATEGORIES 
3 
Possible 
The adverse event may be related to study.  There is an association 
between the event and the administration of study product and there 
is a plausible mechanism for the event to be related to the study 
product; there may be also an alternative etiology, such as 
characteristics of the subject’s clinical status and/or underlying 
disease 
4 
Probable 
The adverse event is likely related to study.  There is (1) an 
association between the event and the administration of study 
product or study intervention, (2) a plausible mechanism for the 
event to be related to the study product, and (3) the event could not 
be reasonably explained by known characteristics of the subject’s 
clinical status and or an alternative etiology is not apparent 
5 
Definite 
The adverse event is clearly related to study.  There is (1) an 
association between the event and the administration of the study 
product or study intervention, (2) a plausible mechanism for the 
event to be related to the related to the study product, and (3) causes 
other than the study product have been ruled out and/or the event re-
appeared on re-exposure to the study product 
 
(For additional information and a printable version of the NCI-CTCAE manual, consult 
the NCI-CTCAE website: http://ctep.cancer.gov/reporting/ctc.html) will be consulted.) 
In a clinical trial, the study product\intervention will always be suspect when attributing 
an AE and the “unrelated” attribution will be used only when there is an undisputable or 
likely alternative explanation for the AE.  


 
59 
14.8 SAE Reporting Criteria and Procedures 
The Principal Investigator will be notified by the study staff as soon as a study staff 
member becomes aware of the SAE.  In the absence of the Principal Investigator, a 
physician sub-investigator will be notified.   
The Principal Investigator determines causality (definitely not related, probably not 
related, possibly related, probably related, definitely related) of the adverse event. The 
DSMB Chair may request further information if necessary and possibly request changes 
to the protocol or consent form as a consequence of the adverse event. A back-up 
notification system will be instituted so that any delays in review by the Chair beyond a 
specified period of time are forwarded to a secondary reviewer.  
14.8.1 Unexpected, non-serious adverse events 
An unexpected, non-serious adverse event that is of Grade 3 severity or higher and study 
related will be recorded and reported under the serious adverse event reporting procedure 
(i.e. within 24 hours).  
14.8.2 Notifying the FDA 
The IND sponsor (The PI) is responsible for FDA safety submissions as follows: The 
IND Sponsor for this study will handle all regulatory communications with the Food and 
Drug Administration and will be responsible to update the IND to include this trial, file 
all regulatory related issues including any MEDWATCH reports which will be reviewed 
and approved by the PI and Steering Committee. The IND sponsor will be responsible for 
submission of yearly reports and final report for this study. All DSMB recommendations 
will be communicated to the IND sponsor in a timely fashion.  
 
The following process for reporting a serious adverse event ensures compliance with the 
ICH guidelines, 21CFR §312.32.  
 
• Expedited safety report to the FDA applies if the adverse event is classified as one 
of the following: 
 
o Serious and unexpected suspected adverse reaction (SUSAR) (Sections 
14.3.4, 14.3.6, and 14.7) (i.e. serious, unexpected, and related) 
 
Or 
 
o Aggregate analysis of adverse events that suggest a causal relationship to 
the study medications 
 
Or 
 
o Any findings from clinical, epidemiological, pooled analysis of data 
pooled across multiple studies, published or unpublished scientific papers 
or any findings from animal or in vitro testing that would result in a 
safety-related change in the protocol, informed consent, investigator 


 
60 
brochure or other aspects of the overall conduct of the trial will be 
reported. 
 
Expedited Safety Reports must be reported by the IND Sponsor to the FDA within 15 
calendar days; fatal or immediately life-threatening serious, unexpected, suspected 
adverse reactions must be reported within 7 calendar days. SAEs that do not strictly fit 
the above criteria may be reported to the FDA in an expedited manner if the IND Sponsor 
chose to do so.  Each 7-day report must be followed up by a 15-day report. 
 
 
The following types of SAEs will be reported in the IND Annual Report: 
• 
Serious, expected, suspected adverse reactions  
• 
Serious but not a suspected adverse reaction  
 
For standard reporting, the IND Sponsor or designee will file the IND Annual Report. All 
adverse events (not just those requiring 24 hour reporting) will be reported in the Annual 
IND Report. 
 
14.8.3 Notifying the Data and Safety Monitoring Board 
The PI will submit all safety reports on an ongoing basis to the DSMB.  Individual or 
clusters of SAEs may be reported expeditiously to the DSMB upon determination.  
14.8.4 Notifying the Institutional Review Board 
The Principal Investigator will ensure the timely dissemination of SAE information, 
including expedited reports, to the IRB in accordance with IRB regulations and 
guidelines.  
14.9 Reporting Pregnancy 
A research subject will be terminated from the study due to pregnancy. The principal 
investigator will be informed immediately of any pregnancy and will report all 
pregnancies within 24 hours of becoming aware of the event to DSMB utilizing the SAE 
report form.  This report is for tracking purposes only.  All pregnancies that are identified 
during the study will be followed to conclusion and the outcome of each will be reported.  
The site principal investigator will discuss with the participant and/or the treating 
physician the known possible risks of the investigational product(s) on the fetus.  
Monitoring of the participant will continue until the conclusion of the pregnancy, and a 
follow-up SAE report form detailing the outcome of the pregnancy will be submitted to 
the DSMB. 
14.10 Non-serious adverse events (NSAEs) reporting 
14.10.1 Notifying the data and safety monitoring board 
The DSMB will be notified of non-serious adverse events during their regularly 
scheduled meetings. The events will be presented to the Board in tabular format.  


 
61 
14.10.2 Notifying the FDA 
The IND sponsor (the Study Chair) will file all adverse events per 21 CFR 312.32, 
whether expedited or part of the IND Annual Report.  The IND sponsor will be 
responsible for compiling the IND Annual Report. 
14.10.3 Notifying the Institutional Review Board 
The Principal Investigator will ensure the timely dissemination of AE information to the 
IRB in accordance with applicable regulations and guidelines.  
14.10.4 Unanticipated Problems 
The Office for Human Research Protections (OHRP) considers unanticipated problems 
involving risks to subjects or others to include, in general, any incident, experience, or 
outcome that meets all of the following criteria: 
Unexpected in terms of nature, severity, or frequency given (a) the research procedures 
that are described in the protocol-related documents, such as the IRB-approved research 
protocol and informed consent document; and (b) the characteristics of the subject 
population being studied; 
Related or possibly related to participation in the research (“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); and 
Suggests that the research places subjects or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or 
recognized. 
Incidents or events that meet the OHRP criteria for unanticipated problems will be 
reported to the DSMB and IRB, in the following time frames: 
1. Unanticipated problems that are serious adverse events should be reported to the IRB 
within 1 week of the investigator becoming aware of the event. 
2. Any other unanticipated problem should be reported to the IRB according to the IRB’s 
reporting policies. 
14.11 Data Management 
All study data will be collected via systems created by using RedCap (an online data 
management software) and will comply with all applicable guidelines regarding patient 
confidentiality and data integrity.  
14.11.1 Registration 
Registration of participants on this protocol will employ an interactive data system in 
which the clinical site (University of Cincinnati) will attest to the participant’s eligibility 
as per protocol criteria and obtain appropriate informed consent. IRB approval for the 
protocol must be obtained before accrual can occur from the clinical site. 
Each participant enrolled will be assigned a local identifier by the enrollment site.  This 


 
62 
number can be a combination of the site identifier (location code) and a serial accession 
number.  Only the registering site will have access to the linkage between this number 
and the personal identifier of the subject.   
14.11.2 Data Entry 
Data collection for this study will be accomplished with a combination of paper as well 
as online electronic case report forms.  Using encrypted communication links, on-line 
forms will be developed that contain the requisite data fields.  
14.11.3 Data Quality and Monitoring Measures 
As much as possible data quality is assessed at the data entry point using intelligent on-
line data entry via visual basic designed screen forms. Data element constraints, whether 
independent range and/or format limitations or ‘relative’ referential integrity limitations, 
can be enforced by all methods employed for data input. QA reports assess data quality 
post-data entry. As we note, data quality begins with the design of the data collection 
forms and procedures and incorporates reasonable checks to minimize transcription and 
omission errors.  Of the more important quality assurance measures are the internal 
validity checks for reasonableness and consistency.  
The DSMB will review data quality on an ongoing basis. A risk-based monitoring plan 
will be developed to support the study. The purposes of study monitoring are to ensure 
the safety and welfare of study participants and to confirm the accuracy of study data.  
An onsite monitoring visit will occur once during the conduct of the trial, with the option 
of making a second visit if determined by Principal Investigator to be needed, to address 
enrollment, or protocol deviation issues.  The Monitoring Plan will detail the frequency 
and level of intensity of on-site monitoring visits.  In general, the study will be monitored 
for all participants at a level of 100% of study data gathered for inclusion and exclusion 
criteria, informed consent procedures, adverse events and unanticipated problems.   The 
study monitor(s) will collaborate with the Principal Investigator to conduct remedial site 
training to ensure compliance with GCP and all regulations.    
During scheduled interim remote monitoring visits, the clinical monitors will verify that 
the protocol is being followed and that data are being collected according to protocol 
requirements.  The clinical monitor(s) will review the Study Regulatory File to determine 
that all required documentation is being collected and that the IRB approval for the site is 
current.  The monitor will then verify that each participant has signed the correct version 
of the informed consent document, and that this document is filed in the participant’s 
source documents.  At each visit, the monitor will perform an audit of the source 
documents in the subject’s binder by checking them against the database.  Adverse event 
documentation will be checked for completeness and accuracy.  At the study closeout, the 
monitor(s) will confirm that all data have been reviewed, all source documents have been 
verified, and all required documents are present in the Study Regulatory File.   
Protocol deviations and unanticipated problems will be reviewed by the Principal 
Investigator, and the regulatory and monitoring staff to determine the need for corrective 
and preventative action (CAPA).  In the event that corrective and/or preventative action is 


 
63 
warranted, a root cause analysis will be performed and a CAPA will be completed as per 
institutional policy.  The Principal Investigator is responsible for communicating the 
CAPA plan to appropriate staff (i.e. Site Investigator, study team) and obtaining 
appropriate signatures to address the issues at hand.  The Principal Investigator will work 
with regulatory and monitoring staff to insure that the CAPA is followed and is effective 
in resolving the issue(s).  Protocol deviations and unanticipated problems will be reported 
to the IRB as per institutional policy.   
 
The table below describes the variables to be reviewed during monitoring visits. 
Variable 
% of Records 
Reviewed 
Informed consents 
100% 
Eligibility criteria for all screened subjects 
100% 
Adverse events 
100% 
Protocol adherence 
20% of active subjects 
Verification of REDCap with source documents 
20% of active subjects 
Central study files-inclusion of all applicable 
documents 
100% 
Protocol deviations/violations 
100% 
 
14.12 Protocol Deviations 
The practices below are consistent with Good Clinical Practice (GCP ICH E6) Sections: 
• Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3 
• Quality Assurance and Quality Control, section 5.1.1 
• Noncompliance sections 5.20.1, and 5.20.2 
14.12.1 Protocol deviation Definition 
14.12.1.1 Protocol Deviation - Any change, divergence, or departure from the study 
design or procedures of a research protocol that affects the subject's rights, safety, or 
well-being and/or the completeness, accuracy and reliability of the study data constitutes 
a protocol violation. Changes or alterations in the conduct of the trial which do not have a 
major impact on the subject's rights, safety or well-being, or the completeness, accuracy 
and reliability of the study data are considered non-major protocol deviations. Site 
Principal Investigator is responsible for reporting protocol deviations to the IRB using the 
standard reporting form. As a result of deviations, corrective actions are to be developed 


 
64 
by the site and implemented promptly. 
14.12.1.2 Major Protocol Deviation (Protocol Violation) - A protocol violation is a 
deviation from the IRB approved protocol that may affect the subject's rights, safety, or 
well-being and/or the completeness, accuracy and reliability of the study data.  
If the deviation meets any of the following criteria, it is considered a major protocol 
deviation.  However, the lists of deviation examples that follow are not exhaustive.  
• The deviation has harmed or posed a significant or substantive risk of harm to the 
research subject. 
Examples: 
1. A research subject received the wrong treatment or incorrect dose. 
2. A research subject met withdrawal criteria during the study but was not 
withdrawn 
3. A research subject received an excluded concomitant medication. 
 
• The deviation compromises the scientific integrity of the data collected for the 
study. 
Examples: 
1. A research subject was enrolled but does not meet the protocol's eligibility 
criteria. 
2. Changing the protocol without prior IRB approval, except for modification to 
ensure the safety of study participants. 
3. Inadvertent loss of samples or data. 
 
• The deviation is a willful or knowing breach of human subject protection 
regulations, policies, or procedures on the part of the investigator(s). 
Examples: 
1. Working under an expired professional license or certification. 
2. Failure to follow federal and/or local regulations. 
3. Repeated minor deviations. 
 
• The deviation is inconsistent with the NIH Human Research Protection Program’s 
research, medical, and ethical principles.  
Examples: 
1. A breach of confidentiality. 
2. Inadequate or improper informed consent procedure. 


 
65 
14.12.1.3 Non-Major Protocol Deviation - A non-major protocol deviation is any 
change, divergence, or departure from the study design or procedures of a research 
protocol that has not been approved by the IRB and which DOES NOT have a major 
impact on the subject's rights, safety or well-being, or the completeness, accuracy and 
reliability of the study data.  
 
14.12.2 Reporting Protocol Deviations 
Deviations from the protocol are not allowed. It is the responsibility of the study site to 
use continuous vigilance to identify and report any protocol deviations. Upon 
determination that a protocol deviation has occurred, the study staff will a) notify the 
Principal Investigator, b) notify Project Manager and c) complete the Protocol Deviation 
form.  The Principal Investigator will complete and sign the Protocol Deviation form and 
submit it to the IRB, per IRB regulations.  Major protocol deviations will be reported to 
the DSMB at the regular DSMB reporting interval. The IND sponsor (PI) will also be 
responsible for notifying the FDA. 
 
 
 


 
66 
15. Statistical considerations: 
Sample size determination: As mentioned previously, the mean serum VEGF-D level 
prior to treatment was approximately 2,000pg/ml in both the MILES trial (12) as well as 
the everolimus trial (18). In both studies, treatment with mTOR inhibitors resulted in an 
almost 50% reduction in the serum VEGF-D levels (12, 18). Since patients in our study 
will be on sirolimus prior to enrolment, we estimate that the starting serum VEGF-D 
level in our study will be approximately 1,000pg/ml. The largest change between serial 
VEGF-D levels in the placebo group of MILES was 42%. Thus, VEGF-D responders in 
MILES trial were conservatively defined as patients in whom serum VEGF-D decreased 
to a value greater than 42% from its baseline (16). Accordingly, we made our power 
calculations based on the ability to show a greater than or equal to 42% change in serum 
VEGF-D levels. Assuming the correlation between the baseline and subsequent VEGF-D 
levels is 0.7, a sample size of 20 subjects will provide 80% power to detect a mean 
VEGF-D difference from baseline to post-treatment of 420pg/ml or higher with the level 
of significant of 0.05 and a standard deviation of 800 for the difference. Allowing for a 
20% dropout rate, we expect to recruit 25 patients for our study. Sample size calculations 
were done using SAS version 9.4. 
 
Data Analysis: The analysis will be based on an intention to treat design. All subjects that 
receive study drug will be included in the safety and efficacy analysis set. The primary 
analysis will occur after the last enrolled participant has completed the 24-week visit. All 
analyses will be performed using SAS version 9.4. A p-value less than 0.05 will be 
considered as statistically significant. 
Specific Aim #1: To determine if combined resveratrol and sirolimus leads to reduction 
in serum VEGF-D compared to VEGF-D reduction on sirolimus alone. Paired t-test will 
be performed to detect the difference of serum VEGF-D after 24 weeks of treatment with 
the serum VEGF-D prior to enrolment. The response variable VEGF-D will be measured 
repeatedly, at baseline and at weeks 8, 16, and 24. The change from baseline to the other 
study time points will indicate the treatment effect with resveratrol and sirolimus.  
Initially, by using the paired t-test we will test whether these effects are statistically 
significant. Multivariate linear regression models will be used to assess the relationship 
between serum VEGF-D change and the covariates including patient’s clinical or 
demographic factors. Linear mixed models will be performed to detect the correlation 
between serum VEGF-D and the covariates taking into account the correlation between 
repeated measurements within the same patient.  A repeated measures ANOVA model 
and linear mixed models will be performed to compare the serum VEGF-D at follow-up 
visits to the baseline taking into account the correlation between repeated measurements 
within the same patient.  
 
Specific Aim #2: To determine the safety and adverse effect profile of combined 
resveratrol and sirolimus in LAM. Subjects will be evaluable for safety if they have 
received at least one dose of the study drug. The frequency, severity and duration of all 
adverse events, regardless of cause, will be recorded on the case report forms. The 
frequency and severity of adverse events will be calculated by patient, with each patient 
counted once using the most severe grade experienced. The duration of adverse events 


 
67 
will be calculated by the number of days each event persisted. Adverse events will be 
tabulated by body system and by severity using the NCI Common Terminology Criteria 
for Adverse Events (CTCAE) v4.0, which can be obtained from the CTEP website: 
http://ctep.cancer.gov/reporting/ctc.html. Tables will be generated for all adverse events 
including those that are judged to be possibly, probably or definitely related to the drug. 
In order to assess the safety and adverse effect profile of resveratrol in LAM, we will 
assign a binary indicator variable to indicate a patient with or without adverse events. A 
multivariable logistic regression model will be used to assess of the effect of the 
covariates on the probability of having adverse events for patient with LAM. All 
interested factors will be adjusted in the logistic regression model. Model selection will 
be based on stepwise criterion when appropriate.  
Specific Aim #3: To determine the effect of combined resveratrol and sirolimus on lung 
function and quality of life parameters in patients with LAM. Lung function parameters 
(FEV1 and FVC) will be measured at the baseline visit, and at every in-person visit. 
DLCO will be measured at the baseline visit and the last study visit. Similar analysis plan 
as VEGF-D will be employed for the lung function parameters. We will use the following 
questionnaire-based assessments to determine the quality of life in this trial: St. George’s 
Respiratory Questionnaire (SGRQ), EuroQOL scale, San Diego Shortness of Breath scale 
(SD-SOB), and A Tool to Assess Quality of Life in LAM (ATAQ-LAM). Written 
permission has been obtained to use all these questionnaires. The scores on each of these 
questionnaires will be calculated at baseline, and at every in-person visit. We will 
compare median values at baseline and 24 weeks with the Wilcoxon signed rank test for 
each of these scales.  
Home spirometry evaluation: The spirometry values will be expressed as mean ± SD. 
All spirometry maneuvers will be checked for acceptability and reproducibility as 
specified in the ATS/ERS criteria (38) by an expert pulmonary physiologist. The 
spirometry values obtained from maneuvers that do not satisfy the ATS/ERS criteria for 
acceptability will not used in the primary analysis. If spirometric values meet the 
acceptability criteria but fail to satisfy the repeatability criteria, those values will be 
graded for quality, but will be used in the primary analysis. Overall compliance with 
weekly home spirometry will be recorded and reported as a percentage. Comparison of 
home spirometry and office-based spirometry values will be performed using the Bland-
Altman method at baseline, and at each time frame that patients get an office-based 
spirometry (i.e. every 8 weeks). Rate of change of FEV1 (slope) over time will be 
calculated by using all available values (without imputations) in a linear mixed effects 
model after considering the correlation of the observations within each subject, and the 
time between measurements will be treated as a fixed effect. In order to analyze if the 
trends obtained from short-term follow up with frequent home spirometry correlate with 
overall disease trajectory, we will compare the rate of decline obtained after 8 weeks of 
home spirometry to the overall rate of decline obtained after 24 weeks of follow up. A 
similar analysis plan will be employed for serum VEGF-D, and other biomarkers. All 
statistical analyses for this study will be performed using SAS, version 9.4 (SAS Institute 
Cary NC).  
 


 
68 
16. Identification and access to source data: 
16.1 Identifying source data 
The site PI will keep accurate records to ensure that the conduct of the study is fully 
documented.  Data derived from source documents will be transferred to protocol-
specific CRFs.  The results of all clinical and clinical laboratory evaluations will be 
maintained in the participant’s medical records or hospital database and the data will be 
transferred to clinical CRFs, as applicable. 
16.2 Updating source documentation 
Documents describing the safety profile of an investigational product, such as the 
investigator’s brochure, will be amended as needed by the investigational product 
manufacturer to ensure that the description of safety information adequately reflects any 
new clinical findings.  
The site PI will provide the IRB with the most up-to-date versions of the above 
documents as soon as the PI becomes aware of any changes.  For investigational product, 
the PI will confirm that there are no changes to the Investigator Brochure every 3 months. 
16.3 Permitting access to source data 
The investigational site participating in this study (University of Cincinnati) will maintain 
the highest degree of confidentiality for the clinical and research information obtained 
from the participants in this clinical trial.  Medical and research records will be 
maintained at the study site in the strictest confidence.  However, as a part of the quality 
assurance and legal responsibilities of an investigator, the investigational site will permit 
the study monitor, authorized representatives of the IRB, the FDA, and health authorities 
to examine (and when required by applicable law, to copy) clinical records for the 
purpose of quality assurance reviews, audits, and evaluations of the study safety and 
progress.  Unless required by the laws that permit copying of records, only the coded 
identity associated with documents or with other participant data may be copied (and all 
personally identifying information will be removed).  Authorized representatives as noted 
above are bound to maintain the strict confidentiality of medical and research information 
that is linked to identify individuals.   
16.4 Quality control and quality assurance 
The PI will keep accurate records to ensure that the conduct of the study is fully 
documented.  The investigator will ensure that all CRFs and participant study files are 
legible and complete for every participant. 
The PI, through the use of the DSMB, will be responsible for the regular review of the 
conduct of the trial, for verifying adherence to the protocol, and for confirming the 
completeness, consistency, and accuracy of all documented data and accuracy of source 
documentation verification.   
When the CRFs are complete, they will be reviewed and signed by the PI.  All 
discrepancies identified by the site monitor will be reviewed, and any resulting queries 
will be resolved with the site PIs and the CRFs will be amended as needed. 


 
69 
17. Ethical considerations and compliance with good clinical practice 
17.1 Statement of Compliance 
This study was designed to ensure the protection of subjects according to the ethical 
principles of the Declaration of Helsinki and amendments concerning medical research in 
human subjects. This clinical study will be conducted using current good clinical practice 
(cGCP), as delineated in ICH, Guidance for Industry: E6 Good Clinical Practice 
Consolidated Guidance, and according to the criteria specified in this study protocol.  
Before study initiation, the protocol, the informed consent documents, and the CRFs will 
be reviewed and approved by the DSMB, IRB, FDA, as well as any other appropriate 
health authorities. Any amendments to the protocol or to the consent materials will also 
be approved by the appropriate bodies listed above prior to implementation. 
17.2 Informed Consent 
Each subject will be provided with oral and written information describing the nature and 
duration of the study. Written consent must be obtained prior to any study procedures. 
Prospective participant must be given ample opportunity to review the informed consent 
and inquire about the results of the study. All participants must read, sign, and date a 
consent form prior to study participation.  Subject will enter the date of the consent. The 
original, signed consent form will be retained with the study center’s records, and each 
subject will receive a copy. The informed consent form will provide information about 
the study to a prospective participant to allow for an informed decision about 
participation in the study.  We anticipate all, or at least the vast majority, of the study 
participants to speak and understand English. In case a participant doesn't speak or 
understand English well, we will use the smart form and an interpreter to obtain informed 
consent.  
The informed consent form will be revised and receive IRB approval if and when 
important new safety information is available, or whenever any new information becomes 
available that may affect participation in the trial. 
A copy of the informed consent form will be given to a prospective participant for 
review.  The Principal Investigator or an approved designee will discuss the consent with 
the prospective participant and answer questions.  The prospective participant will be told 
that being in the trial is voluntary and that he or she may withdraw from the study at any 
time, for any reason. The site will document details of the informed consent process 
within the study records. 
 
 


 
70 
18. Privacy and Confidentiality 
A participant’s privacy and confidentiality will be respected throughout the study.  Each 
participant will be assigned a sequential identification number and these numbers rather 
than names will be used during collection, storage, and reporting of participant 
information. 
Data collection for this study will be accomplished with a combination of paper as well 
as online electronic case report forms.  Using encrypted communication links, on-line 
forms will be developed that contain the requisite data fields.  Data will also come from 
EPIC notes written as part of the research encounters, as well as field data collected using 
specific case report forms designed for this study. Field data will be entered into the 
eCRF in RedCap. Electronic data collected in the field will be protected from theft using 
whole disk encryption. Paper data will be stored in the locked secure office of study staff 
according to institution policies and procedures.  During the study, data will be hosted at 
University of Cincinnati’s secure Data Center. Upon completion of the study, private data 
will be destroyed by certified system administrators per UC policy. 
 
 


 
71 
19. Publications 
Publication of any data from this study must be carried out in accordance with the clinical 
study agreements. The results of the study will be reported in clinicaltrials.gov per 
current NIH regulations.  
 
 


 
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20. References: 
 
1. 
Gupta N, Vassallo R, Wikenheiser-Brokamp KA, McCormack FX. Diffuse Cystic 
Lung Disease. Part I. Am J Respir Crit Care Med. 2015;191(12):1354-66. 
2. 
McCormack FX, Travis WD, Colby TV, Henske EP, Moss J. 
Lymphangioleiomyomatosis: calling it what it is: a low-grade, destructive, metastasizing 
neoplasm. Am J Respir Crit Care Med. 2012;186(12):1210-2. 
3. 
Gupta N, Han MK, McCormack FX. Regional Sparing in an Oligemic Lung 
Segment Supports Hematogenous Spread as a Pathogenic Mechanism in 
Lymphangioleiomyomatosis. Ann Am Thorac Soc. 2015;12(8):1247-8. 
4. 
Schiavina M, Di Scioscio V, Contini P, Cavazza A, Fabiani A, Barberis M, et al. 
Pulmonary lymphangioleiomyomatosis in a karyotypically normal man without tuberous 
sclerosis complex. Am J Respir Crit Care Med. 2007;176(1):96-8. 
5. 
McCormack FX. Lymphangioleiomyomatosis: a clinical update. Chest. 
2008;133(2):507-16. 
6. 
Henske EP, McCormack FX. Lymphangioleiomyomatosis - a wolf in sheep's 
clothing. J Clin Invest. 2012;122(11):3807-16. 
7. 
Sengupta S, Peterson TR, Sabatini DM. Regulation of the mTOR complex 1 
pathway by nutrients, growth factors, and stress. Mol Cell. 2010;40(2):310-22. 
8. 
Kwiatkowski DJ, Zhang H, Bandura JL, Heiberger KM, Glogauer M, el-
Hashemite N, et al. A mouse model of TSC1 reveals sex-dependent lethality from liver 
hemangiomas, and up-regulation of p70S6 kinase activity in Tsc1 null cells. Hum Mol 
Genet. 2002;11(5):525-34. 
9. 
Lee L, Sudentas P, Donohue B, Asrican K, Worku A, Walker V, et al. Efficacy of 
a rapamycin analog (CCI-779) and IFN-gamma in tuberous sclerosis mouse models. 
Genes Chromosomes Cancer. 2005;42(3):213-27. 
10. 
Bissler JJ, McCormack FX, Young LR, Elwing JM, Chuck G, Leonard JM, et al. 
Sirolimus for angiomyolipoma in tuberous sclerosis complex or 
lymphangioleiomyomatosis. N Engl J Med. 2008;358(2):140-51. 
11. 
Davies DM, Johnson SR, Tattersfield AE, Kingswood JC, Cox JA, McCartney 
DL, et al. Sirolimus therapy in tuberous sclerosis or sporadic lymphangioleiomyomatosis. 
N Engl J Med. 2008;358(2):200-3. 
12. 
McCormack FX, Inoue Y, Moss J, Singer LG, Strange C, Nakata K, et al. 
Efficacy and safety of sirolimus in lymphangioleiomyomatosis. N Engl J Med. 
2011;364(17):1595-606. 
13. 
Young LR, Inoue Y, McCormack FX. Diagnostic potential of serum VEGF-D for 
lymphangioleiomyomatosis. N Engl J Med. 2008;358(2):199-200. 
14. 
Young LR, Vandyke R, Gulleman PM, Inoue Y, Brown KK, Schmidt LS, et al. 
Serum vascular endothelial growth factor-D prospectively distinguishes 
lymphangioleiomyomatosis from other diseases. Chest. 2010;138(3):674-81. 
15. 
Seyama K, Kumasaka T, Souma S, Sato T, Kurihara M, Mitani K, et al. Vascular 
endothelial growth factor-D is increased in serum of patients with 
lymphangioleiomyomatosis. Lymphat Res Biol. 2006;4(3):143-52. 
16. 
Young L, Lee HS, Inoue Y, Moss J, Singer LG, Strange C, et al. Serum VEGF-D 
a concentration as a biomarker of lymphangioleiomyomatosis severity and treatment 
response: a prospective analysis of the Multicenter International 


 
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Lymphangioleiomyomatosis Efficacy of Sirolimus (MILES) trial. Lancet Respir Med. 
2013;1(6):445-52. 
17. 
Radzikowska E, Jagus P, Sobiecka M, Chorostowska-Wynimko J, Wiatr E, Kus J, 
et al. Correlation of serum vascular endothelial growth factor-D concentration with 
clinical presentation and course of lymphangioleiomyomatosis. Respir Med. 2015. 
18. 
Goldberg HJ, Harari S, Cottin V, Rosas IO, Peters E, Biswal S, et al. Everolimus 
for the treatment of lymphangioleiomyomatosis: a phase II study. Eur Respir J. 2015. 
19. 
Alayev A, Berger SM, Holz MK. Resveratrol as a novel treatment for diseases 
with mTOR pathway hyperactivation. Ann N Y Acad Sci. 2015;1348(1):116-23. 
20. 
Lin JK, Tsai SH. Chemoprevention of cancer and cardiovascular disease by 
resveratrol. Proc Natl Sci Counc Repub China B. 1999;23(3):99-106. 
21. 
Tsai SH, Lin-Shiau SY, Lin JK. Suppression of nitric oxide synthase and the 
down-regulation of the activation of NFkappaB in macrophages by resveratrol. Br J 
Pharmacol. 1999;126(3):673-80. 
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