A Prospective, Double-Blind, Cross-Over, Pilot Study to Assess Safety and Efficacy of Topical Sirolimus 2% in the Treatment of Plantar Blistering in Patients With Epidermolysis Bullous Simplex (EBS)
Phase: PHASE2
Conditions: Epidermolysis Bullosa Simplex, Epidermolysis Bullosa Simplex Kobner, Weber-Cockayne Syndrome
Sponsor: Stanford University

 
 
  
 
A Prospective, Double-Blind, Cross-Over, Pilot Study to Assess 
Safety and Efficacy of Topical Sirolimus 2% in the Treatment of 
Plantar Blistering in Patients With Epidermolysis Bullous Simplex 
(EBS) 
Study Protocol and Statistical Analysis Plan 
NCT02960997 
 April 7, 2017 
 

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Clinical Trial Protocol: 
 
A prospective, double-blind, cross-over pilot study to assess safety and 
efficacy of topical sirolimus 2% for the treatment of plantar blistering in 
patients with epidermolysis bullosa simplex 
 
Version 6 
 
Version Date: 07 April 2017 
 
Protocol Director: 
Joyce Teng, MD PhD 
Clinical Associate Professor, Stanford University School of Medicine, USA 
 
Sub-Investigator:  
Zachary Zinn, MD  
Fellow, Stanford University School of Medicine, USA 
 
Clinical Research Contact:  
Elidia Tafoya, MPH 
Clinical Research Coordinator, Stanford University School of Medicine, USA   
 
Clinical Research Contact:  
Monica Martin 
Clinical Research Coordinator, Stanford University School of Medicine, USA 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Table of Contents 
 
1.0 Protocol Summary 
 
2.0 Background and Rationale 
 
 2.1 Disease background 
 
 2.2 Sirolimus  
 
 2.3 Rationale 
 
3.0 Hypothesis 
 
4.0 Objectives and Scientific aims 
 
5.0 Criteria for Subject Eligibility 
 
5.1 Number of Participants 
 
5.2 Inclusion Criteria 
 
5.3 Exclusion Criteria 
 
5.4 Withdrawal from the Study 
 
6.0 Investigational Plan 
 
6.1 Study Design 
 
6.2 Recruitment process 
 
6.3 Compensation for participation 
 
6.4 Screening period 
 
6.5 Treatment period – Phase 1 
 
6.6 Wash-out period –  Phase 2 
 
6.7 Treatment period – Phase 3 
 

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6.8 Wash-out period – Phase 4 
 
6.9 Follow-up period 
 
7.0 Study treatments 
 
7.1 Investigational Product 
 
7.2 Treatment Assignment 
 
7.3 Blinding 
 
7.4 Prohibited Concomitant Medications 
 
8.0 Study Assessments  
 
8.1 Medical History 
   
8.2 Dermatological history 
 
8.3 Primary Outcome Measures 
 
8.4 Secondary Outcome Measures  
 
8.5 Safety Assessments 
 
8.6 Efficacy Assessments 
 
8.7 Adverse Events 
 
8.8 Pregnancy 
 
9.0 Data and Statistical Analysis 
 
9.1 Hypotheses 
 
9.2 Sample size calculation 
 
9.3 Treatment Comparisons 
 
 
10.0 Study Conduct Considerations 
 
10.1 Posting Information on Clinical Trials Registers 

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10.2 Regulatory and Ethical Considerations 
 
10.3 Records Storage 
 
10.4 Independent Data Monitoring Committee (IDMC) 
 
10.4 Private Health Information 
 
10.5 Privacy and Confidentiality  
 
10.6 Genomic Data Sharing 
 
 
11.0 References 
 
12.0 Figures and Tables 
 
Table 1. Topical Sirolimus Use in a Variety of Dermatological 
Conditions Off-Label  
 
Table 2. Schedule of Events 
 
Table 3. Trial Objectives and Outcomes 
 
Figure 1.  Schematic of the Proposed Study 
 
Figure 2. Method of defect measurement using Digital Wound 
Assessment System 
 
 
 
 
 
 
 
 
 
 
 
 
 

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1.0 Protocol Summary  
 
The purpose of this study is to investigate a targeted approach for the 
management of the plantar lesions of epidermolysis bullosa simplex (EBS). The 
drug to be tested is called sirolimus (Rapamycin), it is a mammalian Target of 
Rapamycin (mTOR) inhibitor to be used as a topical agent for treatment of the 
blistering/callus lesions on the soles of feet.  
 
Sirolimus 2% ointment, has been shown to reduce expression of keratin (KRT) 
6/16 and improve walking ability in a related condition affecting the feet, 
pachyonychia congenita, as well as being safe. Since sirolimus may have 
similar effects on the paired KRT 5/14 which are affected in EBS, we are 
conducting a prospective, double-blind, randomized, placebo-controlled, 
crossover study.  
 
Participants will be assigned to treat both feet with either topical sirolimus 2% 
ointment twice daily or placebo (vehicle-control) for 12 weeks followed by a 
washout period of four weeks, then retreatment to both feet with the switched 
intervention followed by a washout period of four weeks, and then a post-study 
phone call at 8 weeks thereafter.  
 
This study will exploit the naturally occurring transcriptional regulation of keratin 
sequences, the known gene aberration causing EBS, and assess the potential 
for mTOR pathway inhibition in treatment of EB patient’s plantar lesions. 
 
 
2.0 Background and Rationale 
 
2.1 Disease background 
 
Epidermolysis bullosa (EB) represents a spectrum of conditions characterized 
by blistering and mechanical fragility of the skin. Individuals with EB display 
tremendous clinical diversity, as the dermal-epidermal basement membrane 
zone contains a number of specialized adhesive structures that link the basal 
epidermis cytoskeleton to the papillary dermis. The molecular basis of EB has 
been linked to 18 genes, leading to the classification of 28 clinical (15 EBS, 8 
JEB, 14 DEB, 1 Kindler) 
EB subtypes and four major EB groups (Fine et al., 2014). With importance to 
this proposal, the EB simplex (EBS) subtype has been well-characterized 
genetically, and it is this characterization the authors plan to exploit with the goal 
of improving current state of the art. 
 
EBS refers to a group of rare inherited disorders caused by blister formation 
within the epidermis. The clinical features of all forms of EBS manifest with skin 

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blistering. Palmoplantar-keratoderma, nail dystrophy and shedding, alopecia 
and oral involvement can be seen. Excruciating pain and severe impairment in 
quality of life in EBS patients has been reported (Fine at el., Clin Exp Dermatol 
2004). Causes of early mortality in EBS patients include infections and protein 
loss leading to malnutrition, fluid and electrolyte imbalances, and anemia. 
Patient care has been limited to supportive measures, including wound care, 
nutritional support, and antibiotics. In 1991, two groups published in Cell and 
Science independently revealing the first keratin disorder with a genetic basis in 
humans, EBS. EBS is usually caused by missense mutations in the genes 
encoding keratin 5 and 14 (KRT5 and KRT14, respectively) leading to a 
dominant negative effect on the function of normal keratins (Lane et al., J Pathol 
2004), resulting in keratin cytoskeleton dysfunction. The prevalence is estimated 
to be 6 to 30 per 1 million live births. 
 
2.2 Sirolimus  
 
Sirolimus binds to immunophilin FK506 binding protein-12 and creates a 
complex that inhibits the activation of mTORC1 that leads to decreased 
translational initiation. Consequently, sirolimus has shown to decrease overall 
protein synthesis by 20% and cause cell cycle arrest in G1 phase. (Castedo et 
al., 2001) Other effects of sirolimus include apoptosis, inhibition of T-cell 
activation and reduced expression of specific proteins involved in angiogenesis 
and lymphangiogenesis, specifically hypoxia inducible factor-1a (HIF-1a) and 
vascular endothelial growth factor (VEGF). (Guba et. al 2002)  
 
Systemic use of sirolimus is associated with side effects that include 
opportunistic infection, mucositis, hypercholesterolemia, decreased renal 
function, elevation in liver transaminases, thrombocytopenia, neutropenia, and 
delayed wound healing. Minor adverse cutaneous events of systemic therapy 
include acne like eruptions, edema, and nail disorders. mTOR inhibitors in 
topical preparations show promise as an ideal delivery system to minimize 
systemic adverse effects in the treatment of dermatological disease.  
 
Topical formulations of sirolimus are being used off-label in a variety of 
dermatological diseases (see Table 1) (Fogel et al, 2015). Formulations are 
mostly crushed sirolimus from tablet compounded with petrolatum to form an 
ointment, with a few studies utilizing the commercially available oral sirolimus 
solution (1mg/ml). Most of the topical formulations were found to have no local 
adverse effects, with irritation being more common using the aqueous solution. 
In all cases the systemic level of sirolimus was less than 2ng/ml that is below 
the standard serum levels required for immunosuppression (5-15 ng/ml). 
Treatment duration and frequency varied according to condition.  
 
2.3 Rationale 
 
The development of new treatment modalities is critical in improving disease 
state and the quality of life for EBS patients. Disease classification has changed 
from descriptive to a detailed pathogenesis over the past several decades, and 

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now scientists are drawn to ask the question, “How can we approach this 
disease in a targeted fashion?” 
 
Therapeutic approaches targeting the deleterious effects of abnormal and 
abundant keratins are emerging.  Topical sirolimus 2% is one potential 
medication yet to be utilized in EBS patients. It has been shown to selectively 
block 5’oligopyrimidine (TOP) mRNA translation thereby altering mRNAs 5’TOP 
motifs (Raught et al., 2001). Published sequence data suggest KRT5 mRNA, 
but not KRT14 mRNA, contain the 5’TOP motif (Hickerson et al., 2009) and thus 
are viable drug targets of mTOR for the treatment of EBS variants. 
 
 
3.0 Hypothesis  
 
Our hypothesis is that the mTOR inhibitor sirolimus will down regulate the 
translation of defective KRT5 protein, therefore ameliorating the severity of EBS 
in patients affected. Additionally, altering the mTOR pathway may directly affect 
pain and itch (R Kasper, May 2015, unpublished correspondence). Itch and pain 
are two of the three top symptoms that matter to patients living with EB 
(Challenges & Unmet Needs in EB, Jemima Mellerio, EB2015 Conference, 
Atlanta, GA), both of which are secondary outcome measures in this study.   
 
4.0 Objectives and Scientific aims 
 
Aim 1: To assess the safety of topical sirolimus 2% use in the treatment of plantar 
lesions in epidermolysis bullosa simplex (EBS).   
Aim 2: To assess the efficacy of topical sirolimus 2% to improve the clinical 
severity of lesional skin, including pain and itch symptoms. 
Aim 3: To assess improvement in walking ability.  
 
 
5.0 Criteria for Subject Eligibility  
 
5.1 Number of Participants 
 
We aim to enroll a total of 8-10 patients in this study at Stanford University. 
 
  
5.2 Inclusion criteria 
 
● 
Diagnosis of EBS 
● 
Minimum EBDASI feet activity score of 2/10 
● 
Healthy male or non-pregnant female 
● 
Age –4 years old or older 

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● 
Ability to complete 7 study visits, each for approximately 30-60 minutes, 
including 3 scheduled phone calls within a 40-week period 
● 
Anticipated life expectancy ≥52 weeks 
● 
Males and females of childbearing potential should be using an effective 
means of contraception. 
● 
Laboratory values within the range of normal unless the PI feels they are not 
clinically relevant. 
 
5.3 Exclusion criteria 
• 
Allergy to sirolimus or components of the vehicle ointment 
• 
Pregnancy, breast feeding 
• 
Prior history of liver disease 
• 
Serious known concurrent medical illness or infection, which could 
potentially present a safety risk and/or prevent compliance with the 
requirements of the treatment program. 
• 
Known immunodeficiency virus or syndrome including those with: 
o Acquired Immunodeficiency Syndrome (AIDS) 
o Human Immunodeficiency Virus (HIV) 
o Hepatitis B 
 
• 
Prior history of grafting surgeries or other surgeries in the dermatologic 
treatment area  
• 
History of significant condition in the dermatologic treatment area such as 
trauma or non-healing chronic wound, which could impair evaluation for the 
treatment of EBS. 
• 
Use of other investigational drugs within 30 days of the screening visit 
and/or has not recovered from any side effects of prior investigational drugs 
or procedure in the affected area (e.g., a biopsy).  
• 
Use of acitretin within the last 1 month 
• 
Use of Roaccutane within last 3 months 
• 
Botox injections to the feet within the last 6 months. 
• 
Participant is planning extra physical activities within the next 3 months.  
• 
Amputated foot 
 
5.4 Withdrawal from the study 
 
Participants must discontinue investigational medicinal product (IMP) and be 
withdrawn from the study for the following reasons: 
 
• 
Participant withdraws consent 
• 
Investigator request e.g. non-compliance with visits 
• 
Participant meets any of the above exclusion criteria 
 
 
6.0 Study Design and Intervention 

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6.1 Study Design 
 
This study is a prospective, double-blind, randomized, placebo-controlled 
crossover study at Stanford University School of Medicine, USA.  It includes a 6-
week screening period, followed by a 4 Phase Pilot Clinical Trial (see Table 2).  
 
Screening: Up to 6 weeks  
Phase 1: 12-week treatment period  
Phase 2: 4-week wash-out period 
Phase 3: 12-week treatment period 
Phase 4: 4- week wash out period 
Follow-up: 8 weeks after end-of-trial visit.  
 
This research study is expected to take approximately 40 weeks. There will be 2 
different parts of the study which the subject may receive for initial treatment 
period either (a) placebo (no drug) or (b) study drug for 12 weeks, during their 
enrollment in the 40-week study. After the initial 12-week period, there will be a 
washout period consisting of 4 weeks, before starting secondary treatment. 
During the secondary treatment period, a switch will occur and subject will 
receive either the (a) placebo (no drug) or (b) study drug depending on which 
treatment was received initially. At some time point during the 40-week study, at 
least both feet will receive study drug. There will also be a post-treatment visit at 
week 16 and week 32, with a post study phone call at Week 40. The end of 
treatment period will occur at Week 12 and Week 28, with the final in-person 
study visit occurring at week 32. Structured phone calls made by a physician will 
occur at Week 2, Week 14, Week 18, and Week 40. 
 
The EBDASI feet score needs to be within 20% of baseline to progress to 
Phase 3. If subject’s score does not meet this criteria the Phase 2 wash-out can 
be extended to 6 weeks.   
 
Treatments are switched after Phase 2.  
 
In-person study visits:  
 Week 0 (screening/baseline) 
 Week 4 
 Week 12 
 Week 16 
 Week 20 
 Week 28 
 Week 32 
 
Structured physician phone-call will be scheduled at: 
 Week 2 
 Week 14 
 Week 18 
 Week 40 

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6.2 Recruitment Process 
 
EBS is a rare orphan disease and Stanford is one of several multidisciplinary 
clinics in the world that focuses on the treatment of EB.  With a prevalence of 6-
10 per million, there are roughly 3,200 patients in the United States.  This study 
aims to enroll a total of 8-10 patients at Lucile Packard Children's Hospital 
Stanford. We cooperate with national and international networks of families, 
researchers, and physicians who care for children and adults with EB and plan 
to use these groups to recruit subjects for this study. In addition we will use our 
email listserv (ebcarenetwork@lists.Stanford.edu) to inform our national 
communities that we are recruiting for this study. Patients have previously 
signed Consent Forms that have been approved by the Stanford ethics 
committee to be involved in these databases and contacted. Over 200 people 
are contained within these databases, 24 of which have EB with known EBS 
genetic KRT 5/14 mutation.  We will access these databases to gain participant 
names, addresses, phone numbers and email addresses that we will use to 
invite them to participate in the study. Preference in enrolment will be given to 
participants in proximity to the study site and those with genetic KRT 5/14 
mutations. Genetic testing is not a requirement for EBS diagnoses nor study 
enrollment. The mechanism of disease in EBS is well known as an autosomal 
dominant defect in keratin 5 or keratin 14. We will not be genotyping, the cost of 
genotyping is high at $3-4k per subject. If genotyping is already known, this 
information will be utilized. Our recruitment methods will not involve material 
inducements.  A telephone recruitment form has been submitted with this IRB 
request. Study personnel will not receive incentives for recruiting study 
participants or for any other purpose directly related to the study. 
 
 
6.3 Compensation for participation 
 
There will be no compensation for participation in this study but travel expenses 
may be reimbursed e.g. by using IPTAAS (Isolated Patients Travel and 
Accommodation Assistance Scheme). 
 
6.4 Screening/Baseline Period 
 
Before any study-specific interventions are performed, the appropriate written 
informed consent must be obtained for the trial and a separate consent for skin 
biopsy, which is an optional additional procedure.  
 
To assess subject eligibility, all screening assessments as follows should be 
completed: eligibility criteria, medical history and physical exam, weight and 
height, demography, vital signs, concomitant medications, Epidermolysis 
Bullosa Diseases Activity Score (EBDASI), Quality of Life in Epidermolysis 
Bullosa (QOLEB),  collection of blood for: metabolic panel, complete blood cell 
count with differential, urinalysis, lipid profile.  

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The participant will also be initiated in how to use the Fitbit and completion of 
daily diaries and the various different questionnaires to be used in the trial.   
 
6.5 Treatment Period – Phase 1 
 
Participants who meet all eligibility criteria will be randomized 1:1 to receive 
either topical sirolimus 2% or placebo ointment. The on-site Registered Nurse 
will teach the participant how to apply the ointment to both feet and will 
administer the first dose of IMP at week 0. Subsequent twice daily application of 
IMP to both feet will be performed by the participant, this will continue for 12 
weeks.  
 
6.6 Wash-Out Period – Phase 2 
 
After completion of Phase 1 the participant will stop applying the IMP to the feet 
for a period of 4 weeks at which point their baseline activity will be assessed. If 
their EBDASI feet score is within 20% of baseline they can proceed to Phase 3.  
 
6.7 Treatment Period – Phase 3 
 
Participants who received topical sirolimus 2% during phase 1 will switch to 
placebo ointment and vice versa. Twice daily application of IMP will be 
continued by the participant for a further 12 weeks. The trial will remain double-
blinded throughout. 
 
6.8 Wash-Out Period – Phase 4 
 
After completion of Phase 3 participants will enter a further 4-week wash-out 
period where they will stop applying the IMP to their feet. At the end of Phase 4 
they will attend for their final end-of-trial physician visit. 
 
6.9 Follow-up Period 
 
Week 32-40 will be the follow-up period with a structured physician phone 
consultation at Week 40.  Participants who withdraw from treatment will also 
enter the follow-up period. 
 
 
7.0 Study Treatment 
 
7.1 Investigational Product 
 
Topical sirolimus 2% and placebo ointment will be made and supplied by PCCA 
compounding pharmacy. Placebo ointment will consist of vehicle only – i.e. all 
the same components as the topical sirolimus 2% ointment minus the active 

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ingredient. Both will have the same appearance, texture and odor and will be in 
a matching container or tube.   
 
Tubes will contain vehicle or sirolimus 2% ointment in a 60 gram supply, the 
amount of topical for twice daily application over 2 weeks. We expect 360 grams 
for each phase of the treatment, 720 grams total with 360 grams intervention 
and 360 grams vehicle. Vehicle is an ointment plasticized base contained no 
water that provides at least 6 months sirolimus stability (Chemistry RX, Lars 
Brichta April 8 communication; PCCA plasticized base information provided in 
Section 16). 'A kit' describes two components in a plastic bag or cardboard box: 
(1) vehicle (placebo) or intervention (sirolimus 2%) ointment in 60 gram tubes 
and (2) our "Study Drug Application Kit Instructions' (provided in Section 16). 
 
Contents of the label will be in accordance with all applicable regulatory 
requirements.   
 
Study Drug Storage:  The IP will be stored at room temperature in a dry place, 
away from direct sunlight.  
 
 
 
At each physician visit the participant will be re-supplied with sufficient IMP until 
their next visit. The containers will also be weighed to monitor how much IMP is 
being used.  
 
Patients will be instructed to wear gloves and change gloves between 
applications. 
 
 
7.2 Treatment Assignment 
 
Participants who provide informed consent and undergo screening assessments 
receive a unique participant number. This assignment of participant numbers 
occurs at the screening and baseline visit and serves to identify data in an 
anonymous fashion. Participant eligibility will be established at the conclusion of 
the baseline evaluations (prior to randomization and first dose of IMP at Week 
0). Participants who meet all entry criteria will then be randomized in a 1:1 
allocation to receive either topical sirolimus 2% or placebo ointment according to 
a computer-generated randomization list.  
 
The participants and the investigator staff (except the unblinded pharmacist) will 
be blinded to the treatment assignment. Participants who withdraw from the 
study following informed consent, but before randomization to study treatment, 
will be classified as screen failures.  
 
7.4 Blinding 
 
This is a double-blind study. All study staff (which includes the investigator, sub-

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investigators, other site staff) and participants will be blinded to the treatment 
allocation throughout the trial.  
 
The blinded study treatment assignments for individual participants will be 
maintained by a Central System used by the study pharmacy. Emergency 
unblinding will be available via the central system in a case where the identity of 
the IMP must be known in order to treat an emergent adverse experience for an 
individual patient. 
 
If the treatment blind is broken for a participant in the treatment period, the 
participant in question will be withdrawn from the treatment period and moved 
into the follow-up period. 
 
During the study, investigators will not receive central laboratory data that have 
the potential to unblind a subject’s treatment assignment.  
 
7.5 Prohibited concomitant medications 
 
Participants may take other medications for treatment of the symptoms 
associated with EBS, so long as they do not fall into any of the prohibited 
medications category (see exclusion criteria). 
 
 
8.0 Study Assessments  
 
Table 2 shows the schedule of assessments for the study. The investigators 
(medically qualified) are to conduct all study assessments. The investigator or 
designee must obtain written informed consent prior to performing any of the 
study information that will be recorded in source documents. 
 
If a visit cannot be scheduled on the appropriate date, the visit should be re-
scheduled as close as possible to the planned date, ideally within 3 days. 
 
The study participants and caregivers will be given specific instructions about 
gentle skin care and avoid possible irritants on the treated area during the entire 
study. Patients will also be instructed to inspect the plantar surface daily to 
ensure no trauma. 
 
8.1 Medical history and examination 
 
A complete medical history will be taken at the Screening Visit. Information from 
the medical history is important to establish the baseline condition of the 
participant and will impact the safety monitoring assessments during the study. 
Any significant medical conditions affecting the participant in the past 5 years 
should be recorded. The history should include the following: 
 

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• 
A thorough review of systems, including any past or current conditions 
• 
Prior surgical procedures 
• 
Full medication history 
• 
Prior immunosuppressive therapies, including type, number, and duration 
• 
Allergies and significant allergic reactions 
• 
Significant infections, or history of recurrent infection, including urinary and 
respiratory tract infections 
• 
Smoking history (current or previous smoker, number of packs per day 
smoked) 
• 
Full physical examination will be performed by an experienced medical 
practitioner.  
 
8.2 Epidermolysis bullosa simplex history 
 
Details of the participant’s EBS history will be collected (e.g. dates of symptom 
onset, diagnosis) and recorded. These data are critical for establishing subject 
eligibility, as well as establishing a baseline for subsequent clinical 
assessments.  
 
8.3 Primary outcome measures 
 
1. Foot function utilizing the validated Foot Health Status Questionnaire 
(FHSQ) as a change from baseline to the end of each treatment. 
2. Safety and tolerability of topical sirolimus 2% ointment (serum trough 
sirolimus levels, monitoring for adverse events). 
 
8.4 Secondary Outcome Measures 
 
1. Plantar defect size measurements using 3D photography (% change in total 
defect area) from baseline to the end of each treatment.  
2. EB Disease Activity and Scarring Index (EBDASI) feet activity scores as a 
change from baseline to the end of each treatment 
3. Quality of life assessment in EB (QOLEB) as a change from baseline to the 
end of each treatment. 
4. Itch severity measured using 5-D Pruritus Scale as a change from baseline 
to the end of each treatment. 
5. Participants will wear a FitBit® for the duration of the study to assess 
number of steps walked per day as a change from baseline to the end of 
each treatment. 
6. Changes in foot plantar pressure measurements before and after treatment 
using the Podotech Elftman Foot Scanner from baseline to the end of each 
treatment. 
7. Molecular biology study of skin biopsies assayed for mTOR pathway 
inhibition (e.g. determination of phosphoprotein inhibition included ribosomal 
protein S6, S6 kinase and/or eIF-4E binding protein) as a change from 
baseline to the end of each treatment.  
 

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8.5 Safety Assessments 
 
Safety and tolerability of the medication will be assessed at each visit through 
recording of adverse events, serious adverse events, evaluation of systemic 
absorption through measurement of trough sirolimus levels in serum, and 
additional safety laboratory tests listed below will also be performed.  
 
Investigator assessment of local skin reaction (including increased erythema, 
edema, weeping/exudates, flaking/scaling/dryness, scabbing/crusting, and 
erosion/ulceration) will be assessed independently of other adverse effects. 
 
Serum sirolimus levels 
Trough serum sirolimus levels are a frequently run outpatient laboratory ELISA 
assay and will be performed at Stanford University at specific study visits from 
baseline. 
 
Other Safety Laboratory Assessments 
The clinical safety laboratory parameters will be assessed at each study visit. 
Laboratory tests may be repeated more often if clinically indicated.  
 
All out-of-normal range laboratory values must be evaluated by the Investigator 
or subinvestigator as to whether they are clinically significant (i.e., require 
medical intervention or have clinical signs and/or symptoms). Abnormal clinical 
laboratory parameters that are considered clinically significant by the 
Investigator will be recorded on the Adverse Event (AE) Case Report Form 
(CRF) except Baseline visit. Clinically significant laboratory abnormalities noted 
from the Baseline visit will be recorded as medical history. The AE should be 
reported as a clinical diagnosis when possible. Laboratory tests will be 
performed at Stanford Children’s Health clinics.  
 
Urine Pregnancy Tests (UPT) will be performed at each scheduled visit on 
female subjects of childbearing potential starting at Baseline. If a UPT results in 
a positive test, the subject must be discontinued. 
 
Sample management (collection, storage, shipping, etc…) will be done 
according to Stanford Clinical Laboratories standards and policies.  
 
Clinical safety laboratories will include:  
• Full blood count (CBC, Metabolic Panel, Liver function tests) 
• Lipid panel profile 
• Urinalysis 
• Sirolimus trough (at specific study visits) 
 
These will be performed at each scheduled study visit from screening/baseline 
visit (7 total), except for sirolimus trough. 
 
Adverse Events (AE) and Serious Adverse Events (SAEs) 

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1. AE frequency, severity, and relationship to IMP. 
2. Frequency of withdrawals due to treatment-related AEs. 
3. Frequency of serious adverse events (SAEs) 
 
8.6 Efficacy Assessments 
Clinical assessments of the extent and severity of plantar lesions will be 
performed throughout the study.  
 
Foot Health Status Questionnaire (FHSQ) 
The purpose of this questionnaire is primarily to measure foot health related to 
foot function and quality of life. There are 19 questions regarding foot health and 
its impact with 4 subscales: foot pain, foot function, footwear and foot health. It 
has been shown to have excellent validity and ability to detect change in clinical 
trials and has demonstrated good reliability (Riskowski et al., 2011). 
 
Epidermolysis Bullosa Disease Activity Score (EBDASI) 
The EBDASI is a validated scoring system that objectively quantifies the 
severity of EB affecting the entire body. It has been designed to evaluate the 
response to new therapies for the treatment of EB. It was developed and 
validated many time over in the literature (Loh et al., 2014) 
 
EBDASI feet score 
Is a subsection of the EBDASI and objectively quantifies activity and damage 
levels relating to the feet only. Activity is graded according to size and number 
of lesions (blisters/erosions) in specific anatomical area. Damage is graded 
according to the presence/absence of the following signs: erythema, 
pigmentation, poikiloderma, atrophy, hyperkeratosis, scarring and milia.  
 
Method of defect assessment 
Tracing of affected wounds will be performed using Visitrak Digital Wound 
Assessment System. This is a portable device that provides accurate, 
reproducible data for tracking wound progress (See Figure 2). 
 
Quality of life in Epidermolysis Bullosa (QOLEB)  
QOLEB is a Quality of Life Questionnaire specifically designed for people with 
EB. The QOLEB can be used to identify everyday life occurrences negatively 
affected by EB. It assesses change in quality of life over time, an important 
measure when assessing the success of new treatments for EB (Frew et al., 
2009). 
 
Plantar Pressure Measurements 
The Podotech Elftman foot pressure scanner uses 1600 electronic sensors 
incorporated into a thin mat to measure pressures at various points on the soles 
of the feet. This analysis can be performed while standing to get a static 
analysis or when walking to get a dynamic analysis. Peak pressures can be 
calculated and can correlate with areas of hyperkeratosis and changes in 
loading due to pain. A before and after assessment of participants feet using this 
device will give us insight into how hyperkeratosis has changed as well as 

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changes in loading and pain.  This technology is extensively investigated in 
rheumatoid arthritis patients (Van der leeden et al 2008). 
 
Fitbit Pedometer Mobility Measurements 
Each enrolled study subject will receive a FitBit®/pedometer during their 
participation in the study to track their activity levels. This purchase will be made 
using our SPARK grant funding and be provided to subjects at no cost. Will add 
the information below to the consent and in Section 16.  
 
The FitBit / pedometer will sync stats wirelessly to a deidentified premium 
account at www.fitbit.com/zip. An Excel form of the data will be downloaded on 
health protected computers every two weeks. FitBit pedometers have been 
demonstrated in the literature as reliable and accurate (Storm FA et al. Step 
Detection and Activity Recognition Accuracy of Seven Physical Activity Monitors. 
PLoS ONE, 2015). 
 
Erythema score of soles of feet 
An erythema score of a specified location on the soles of the feet will be 
obtained through use of Mexameter® MX 18 to detect any change in erythema 
at each study visit. This will be an objective measure of local skin irritation at the 
treatment site. This is an easy, quick and economical tool to measure erythema 
by reflectance. The instrument is worldwide established and used in many 
scientific studies. 
 
Skin biopsies assayed for mTOR pathway inhibition 
The skin biopsies are optional and will be assayed for transcriptional factors of 
mTOR pathway inhibition using Western blot / immunohistochemical methods 
(Transderm laboratory, Santa Cruz USA) e.g. determination of phosphoprotein 
inhibition included ribosomal protein S6, S6 kinase and/or eIF-4E binding 
protein. Tissue samples will be transferred to Transderm laboratory deidentified 
and without identifiable personal health information. A separate consent, and if 
applicable, assent form(s) will be used. 
 
5-D Pruritus Scale 
People with EBS can experience the aggravating symptom of itch.  This 
validated tool will be utilized to assess the effect (if any) topical sirolimus has on 
this symptom (Elman et al., 2010) 
 
8.7 Adverse Events (AE) and Serious Adverse Events (SAEs) 
The investigator is responsible for detecting, documenting, and reporting events 
that meet the definition of an AE or SAE. AEs will be collected from the start of 
study treatment through the protocol-specified follow-up period. 
 
Definition of an AE: Any untoward medical occurrence in a subject or clinical 
investigation subject, temporally associated with the use of a medicinal product, 
whether or not considered related to the medicinal product. 
Note: An AE can therefore be any unfavorable and unintended sign (including 
an abnormal laboratory finding), symptom, or disease (new or exacerbated) 

 Page 18 of 30
temporally associated with the use of a medicinal product. This also includes 
failure to produce expected benefits (i.e. lack of efficacy), abuse, or misuse. 
 
A serious adverse event (SAE) is any untoward medical occurrence that: 
 
a. Results in death 
b. Is life-threatening 
c. Requires hospitalization or prolongation of existing hospitalization 
d. Results in disability/incapacity, or 
e. Results in a congenital anomaly/birth defect 
 
8.8 Pregnancy 
 
Women of childbearing potential must have a negative urine pregnancy test 
within 48 hours prior to starting treatment with study drug. Females of non-
childbearing potential are defined as meeting at least one of the following 
criteria: (1) over the age of 60 years (2) amenorrheic for at least 2 years, (3) 
have had a hysterectomy and/or bilateral oophorectomy. All other females 
(including those with tubal ligation) will be considered to be of childbearing 
potential. At visits 2-12 urine pregnancy testing need only be performed if the 
subject has missed a menstrual cycle. The pregnancy must be followed up to 
determine outcome (including premature termination) and status 
of mother and child. 
 
9.0 Data and Statistical Analysis 
 
9.1  Hypotheses 
The null hypotheses is that there is no difference in efficacy between topical 
sirolimus 2% and placebo ointment in improving the clinical 
severity/function/pain scores of the plantar lesions in EBS. The alternative 
hypothesis is that topical sirolimus 2% is superior to placebo ointment.  
 
9.2  Sample size calculation 
Recruitment of 8 subjects needed to detect an average 30% improvement of 
foot function score between topical sirolimus 2% and placebo with standard 
deviation of 40%, with power >0.8 and two-sided significance level of 5% 
(Stanford Statistician Group, USA). 
 
9.3  Treatment comparisons 
The primary comparison of interest is between placebo and topical sirolimus 2% 
on foot function using the validated Foot Health Status Questionnaire. 
 
Secondary comparisons of interest will be made between placebo and  
topical sirolimus 2% on the secondary outcome measures. 
 
 

 Page 19 of 30
10.0 Study conduct considerations 
 
10.1 Posting information on Clinical Trials Registers 
Study information from this protocol will be posted on publicly available clinical 
trial registers before enrollment of participants begins. (clinicaltrials.gov) 
 
10.2  Regulatory and ethical considerations 
The study will be conducted in accordance with ICH Good Clinical Practice, and 
the ethical principles that are outlined in the Declaration of Helsinki 2008. 
 
10.3  Records storage 
Following closure of the study, the investigator will maintain all site study 
records in a safe and secure off-site location for duration of 15 years. The 
records will be easily accessible when needed and will be available for review 
by regulatory bodies.  
 
All of the records will be maintained in a format other than hard copy (e.g. 
scanned,electronic). 
 
10.4  Independent data monitoring committee (IDMC) 
This will consist of the following: 
- 
, MD PhD 
- 
 MB MD FRCP 
- 
, MD PhD 
- 
, MD 
 
10.5  Protected Health Information 
During screening, patient's initials must be used for tracking purposes. If the 
subject enrolls in the study, then name and medical number will be necessary 
as each subject will have a medical chart during the study. Demographic 
information such as date of birth, phone numbers, and address will be used for 
communication purposes. Names and medical numbers will be required for lab 
and other necessary testing, clinic appointments, etc. This includes office visits 
for the study, medical history and medication history, blood test results, and 
processing of laboratory samples including tests. However, with exception of 
initials and subject number, no identifiers are included on survey instruments 
and questionnaires.  
 
A complete list of health information and identifiers include:  
Diagnosis and Procedures by visit  
Clinical notes  
List of ICD 9 diagnosis  
Lab Test Results  
Pathology reports  
Radiology report  
Pharmacy data/Medication list  
Gender  

 Page 20 of 30
Age at Diagnosis  
MRN  
Names  
Contact information (phone number, address, email address)   
Date of Birth 
Medical History 
 
10.6 Privacy and Confidentiality  
Documents pertaining to the study and any other information received will be 
kept strictly confidential. The data may be reviewed by representatives of 
regulatory authorities only for the purpose of verifying procedures. Other than 
this, disclosure of data will only occur if it is required by law.  
 
Physical documents will be kept within folders, which will be locked within the 
Department of Dermatology, St George Hospital. Electronic data will be kept on 
locked departmental computers and stored in password-protected files. Only 
study staff will have access. Records pertaining to the study will be kept for 15 
years in a secure location away from the study site. After this amount of time 
has transpired the records will be disposed of securely.  
 
No identifying information will be included in publications or presentations of the 
results without prior written consent from the participant. 
 
10.7   Genomic Data Sharing Plan1 
 
The data that will be shared: 
I will share phenotypic and genotypic data associated with the collected 
samples by depositing these data at Database of Genotypes and Phenotypes 
(dbGaP), which is an NIH-funded repository.  Additional data documentation 
and de-identified data will be deposited for sharing along with phenotypic data, 
which includes demographics, family history of Epidermolysis bullosa simplex 
disease, and diagnosis, consistent with applicable laws and regulations.  I will 
comply with the NIH GWAS Policy and the funding Institute and Centers existing 
policies on sharing data on Epidermolysis bullosa simplex disease genetics to 
include secondary analysis of data resulting from a genome wide association 
study through the repository.  Meta-analysis data and associated phenotypic 
data, along with data content, format, and organization, will be available at 
Database of Genotypes and Phenotypes (dbGaP).  Submitted data will conform 
to relevant data and terminology standards. 
 
Who will have access to the data: 
I agree that data will be deposited and made available through Database of 
Genotypes and Phenotypes (dbGaP), which is an NIH-funded repository, and 
that these data will be shared with investigators working under an institution with 
a Federal Wide Assurance (FWA) and could be used for secondary study 
purposes.  I agree that the names and Institutions of persons either given or 
                                                        
 

 Page 21 of 30
denied access to the data, and the bases for such decisions will be summarized 
in the annual progress report.  Meta-analysis data and associated phenotypic 
data, along with data content, format, and organization, will be made available 
to investigators through Database of Genotypes and Phenotypes (dbGaP). 
 
Where will the data be available: 
I agree to deposit and maintain the phenotypic data, and secondary analysis of 
data (if any) at Database of Genotypes and Phenotypes (dbGaP), which is an 
NIH-funded repository.  The repository has data access policies and procedures 
consistent with NIH data sharing policies. 
 
When will the data be shared: 
I agree to deposit genetic outcome data into Database of Genotypes and 
Phenotypes (dbGaP) repository as soon as possible but no later than within one 
year of the completion of the funded project period for the parent award or upon 
acceptance of the data for publication, or public disclosure of a submitted patent 
application, whichever is earlier. 
 
How will researchers locate and access the data: 
I agree that I will identify where the data will be available and how to access the 
data in any publications and presentations that I author or co-author about these 
data, as well as acknowledge the repository and funding source in any 
publications and presentations.  As I will be using Database of Genotypes and 
Phenotypes (dbGaP), which is an NIH-funded repository, this repository has 
policies and procedures in place that will provide data access to qualified 
researchers, fully consistent with NIH data sharing policies and applicable laws 
and regulations 
 
NOTE:  NIH is currently updating their guidance on data sharing plans, which 
will be available soon. For questions, contact the NIH Office of Extramural 
Research (OER) at Sharing@nih.gov or your RPM. 
 
 
 
 
 
 
 
 
 
 

 Page 22 of 30
11.0 References  
 
Fine, Jo-David, et al. "Inherited epidermolysis bullosa: updated recommendations on 
diagnosis and classification." Journal of the American Academy of Dermatology 70.6 
(2014): 1103-1126. 
 
Fine, J‐D., et al. "Assessment of mobility, activities and pain in different subtypes of 
epidermolysis bullosa." Clinical and experimental dermatology 29.2 (2004): 122-127. 
 
Lane, E. B., and W. H. I. McLean. "Keratins and skin disorders." The Journal of 
pathology 204.4 (2004): 355-366. 
 
Castedo, M., K. F. Ferri, and G. Kroemer. "Mammalian target of rapamycin (mTOR): 
pro-and anti-apoptotic." Cell death and differentiation 9.2 (2002): 99-100. 
 
Guba, Markus, et al. "Rapamycin inhibits primary and metastatic tumor growth by 
antiangiogenesis: involvement of vascular endothelial growth factor." Nature 
medicine 8.2 (2002): 128-135. 
 
Fogel, Alexander L., Sharleen Hill, and Joyce MC Teng. "Advances in the therapeutic 
use of mammalian target of rapamycin (mTOR) inhibitors in dermatology." Journal of 
the American Academy of Dermatology 72.5 (2015): 879-889. 
 
Raught, Brian, Anne-Claude Gingras, and Nahum Sonenberg. "The target of rapamycin 
(TOR) proteins." Proceedings of the National Academy of Sciences of the United 
States of America 98.13 (2001): 7037. 
 
Hickerson, Robyn P., et al. "Rapamycin selectively inhibits expression of an inducible 
keratin (K6a) in human keratinocytes and improves symptoms in pachyonychia 
congenita patients." Journal of dermatological science 56.2 (2009): 82-88. 
 
Roger Kaspar PhD TransDerm Inc www.clinicaltrials.gov: Topical Sirolimus for the 
Treatment of Pachyonychia Congenita (PC) 
 
 Riskowski, Jody L., Thomas J. Hagedorn, and Marian T. Hannan. "Measures of foot 
function, foot health, and foot pain: American Academy of Orthopedic Surgeons Lower 
Limb Outcomes Assessment: Foot and Ankle Module (AAOS‐FAM), Bristol Foot Score 
(BFS), Revised Foot Function Index (FFI‐R), Foot Health Status Questionnaire (FHSQ), 
Manchester Foot Pain and Disability Index (MFPDI), Podiatric Health Questionnaire 
(PHQ), and Rowan Foot Pain Assessment (ROFPAQ)." Arthritis care & 
research 63.S11 (2011): S229-S239. 
 
Loh, Clement CH, et al. "Development, reliability, and validity of a novel Epidermolysis 
Bullosa Disease Activity and Scarring Index (EBDASI)."Journal of the American 
Academy of Dermatology 70.1 (2014): 89-97. 
 
Venugopal, Supriya S., et al. "A phase II randomized vehicle-controlled trial of 
intradermal allogeneic fibroblasts for recessive dystrophic epidermolysis 
bullosa." Journal of the American Academy of Dermatology 69.6 (2013): 898-908. 
 

 Page 23 of 30
Frew, J. W., et al. "Quality of life evaluation in epidermolysis bullosa (EB) through the 
development of the QOLEB questionnaire: an EB‐specific quality of life 
instrument." British Journal of Dermatology 161.6 (2009): 1323-1330. 
 
Elman, S., et al. "The 5‐D itch scale: a new measure of pruritus." British Journal of 
Dermatology 162.3 (2010): 587-593. 
 
Storm FA et al. Step Detection and Activity Recognition Accuracy of Seven Physical 
Activity Monitors. PLoS ONE, 2015 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 Page 24 of 30
 
12.0 Tables 
 
Table 1: Review of topical sirolimus use in dermatological disease 
(Fogel et al., 2015) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Schedule of Events:  
 
Study period 
Phase 1b 
Screening/Baseline 
Phase 
1b 
Phase 1b Phase 1b Phase 2c Phase 3d Phase 3d 
Phase 
3d 
Phase 
3d 
Phase 
4e 
Follow-upf 
 
Visit Number 
1 
� 
g 
2 
 
 
(EOT)
a  
 
3    
�
g        
WASH-OUT 
4 
� 
g 
5 
  (EOT)
a 
  
6 
7 
� 
g 
 
Study Week 
Week 0 
Week 2 Week 4 Week 12 
 
Week 16 Week 18 Week 20 Week 28 Week 32 Week 40 
 
Informed Consent/Assent (If applicable) 
X 
 
 
 
 
 
 
 
 
 
  
 
Epidermolysis Bullosa Genotyping (Optional) 
X 
 
 
 
 
 
 
 
 
 
 
 
Verify eligibility criteria 
X 
 
 
 
 
 
 
 
 
 
 
 
Vital signs 
X 
  
X 
X 
  
X 
  
X 
X 
X 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
 
 
 
Adverse events 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Serious Adverse events 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Concomitant medications 
X 
  
X 
X 
  
X 
  
X 
X 
X 
  
 
Mexameter erythema score  
X 
  
X 
X 
  
X 
  
X 
X 
X 
  
 
Medical Exam 
X 
  
  
X 
  
  
  
  
X 
  
  
 
Foot Health Status Questionnaire 
X 
  
X 
X 
  
X 
  
X 
X 
X 
  
 
Defect area measurement using Visitrak 
X 
  
X 
X 
  
X 
  
X 
X 
X 
  
 
2D photography of feet 
X 
  
X 
X 
  
X 
  
X 
X 
X 
  
 
EBDASI feet score 
X 
  
X 
X 
  
X 
  
X 
X 
X 
  
 
EBDASI total score 
X 
  
  
X 
  
X 
  
  
X 
  
  
 
QOLEB 
X 
  
  
X 
  
X 
  
  
X 
  
  
 
5-D Pruritus Scale 
X 
  
  
X 
  
X 
  
  
X 
X 
  
 
Plantar pressure measurement 
X 
  
  
X 
  
X 
  
  
X 
X 
  
 
Study drug collection/weighing/accountability 
X 
  
X 
X 
  
X 
  
X 
X 
  
  
 
Supervise study drug self-administrationI 
X 
  
X 
  
  
X 
  
X 
  
  
  
 
Fitbit  (daily) 
X 
X 
X 
X 
X 
X 
  
X 
X 
X 
X 
Page 24 of 27 
 

 
 
Collection of blood for: 
  
  
  
  
  
  
  
  
  
  
  
 
Urine Pregnancy Testh 
X 
  
X 
X 
  
X 
  
X 
X 
X 
  
 
Visit Number 
1 
� 
g 
2 
(EOT)
a 
 
3    
�
g        
WASH-OUT 
4 
� 
g 
5 
  (EOT)
a 
  
6 
7 
� 
g 
 
Study Week 
Week 0 
Week 2 Week 4 Week 12 
 
Week 16 Week 18 Week 20 Week 28 Week 32 Week 40 
 
 
Sirolimus trough level 
  
  
X 
X 
  
  
  
X 
X 
  X** 
  
 
Urinalysis 
X 
  
 
X 
  
X 
  
 
X 
X 
  
 
CBC 
X 
  
X 
X 
  
X 
  
X 
X 
X 
  
 
Metabolic panel 
X 
  
X 
X 
  
X 
  
X 
X 
X 
  
 
Lipid profile 
X 
  
X 
X 
  
X 
  
X 
X 
X 
  
 
Skin biopsy (optional) 
X 
  
  
X 
  
X 
  
  
X 
  
  
 
Structured phone-call 
  
X 
  
  
X 
  
X 
  
  
  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
If a washout period is needed prior to baseline, subject has up to 6 weeks before starting treatment. 
a. EOT= End of Treatment  
 
b. After screening, subject will be randomized and enter Phase 1, the initial treatment application period. Subject will receive study drug or vehicle during this 12-week treatment period.  
 
c. When Phase 1 is complete, a wash-out period will occur, which will last 4 weeks. 
 
 
 
 
 
 
 
  
 
d. Phase 3 is the secondary treatment application period. The subject will receive either study drug or vehicle, switching from treatment initially used in Phase 1 during this secondary 12-week 
treatment period. Phase 3 may be delayed if subject has not reached 20% baseline EBDASI activity / Visitrak 2cm diameter. 
 
e. Phase 4 will be a 4 week follow-up period after study treatment has concluded. 
 
  
 
f. A scheduled phone call will occur at Week 40, after post treatment period at Phase 3. 
 
 
 
 
 
 
  
 
g. Structured physician phone-call will be scheduled 
 
 
 
 
 
 
 
 
 
  
 
h. Eligible female subjects of childbearing potential  
 
 
 
 
 
 
 
 
 
  
 
i. Application occurs at the site on visit days 
 
 
 
 
 
 
 
 
 
 
  

 
Table 3: Trial Objectives and Outcome Measures 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Page 25 of 27 

 
Figure 1. Schematic of the proposed study 
 
 
Recruitment and consent  
(N=8-10) 
Randomization to topical sirolimus 
2% or placebo ointment 
Secondary application of topical 
sirolimus, 2% ointment on both 
feet, twice daily for 12 weeks 
Initial application of topical 
sirolimus, 2% ointment on both feet, 
twice daily  for 12 weeks 
4-week wash-out period  
(No ointments are applied to feet) 
Interventions are then switched 
Secondary application of 
placebo ointment on both feet, 
twice daily for 12 weeks 
Follow-up phone call  
12 weeks after last visit 
Initial application of placebo 
ointment  on both feet, twice 
daily for 12 weeks 
 
PHASE 1  
PHASE 2 
Initial a
ointmen
da
of topical 
nt on both f
12 weeks
Page 26 of 27 

 
 
Figure 2.  Method of defect measurement using  
Visitrak Digital Wound Assessment System 
 
Tracing of affected areas (hyperkeratosis or wounds) 
 
 
 
A. Selection of paired chronic wounds in a patient with RDEB.  
B. Wound size measurements made by 2-D tracing method.  
C. RDEB wound ulcers improved after vehicle injections at baseline, with a 
maximum response at 3 months (Venugopal et al., JAAD 2013). The 
same visitrak method will be used to trace affected areas in EBS.  
 
Page 27 of 27