Conditions: Herpes Labialis Cold Sore
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 139
Healthy volunteers: no

Brief summary:
This study evaluates the safety and efficacy of SADBE in the prevention of recurrent herpes labialis in adults. Two-thirds of the participants will receive a SADBE solution, while the other third will receive only the vehicle as a placebo control. The solutions will be administered topically to the patient's arms. The study will compare a single-arm application versus a two-arm application versus two placebo doses on the arm.

Detailed description:

Arms:
arm 1: Patient will receive a topical 2% SADBE sensitization dose applied to the patient's upper arm. Three weeks after topical sensitization dose patient will receive topical placebo applied to the patient's upper arm. arm 2: Patient will receive a topical 2% SADBE sensitization dose applied to the patient's upper arm. A 0.5% SADBE intensification dose will be applied to the patient's upper arm 3 weeks after sensitization dose. arm 3: Patient will receive a topical placebo (vehicle-DMSO) dose applied to the patient's upper arm. A topical placebo (vehicle-DMSO) follow up dose will be applied to the patient's upper arm 3 weeks after the first placebo dose dose.

Interventions: intervention 1: SADBE intervention 2: Placebo
Intervention descriptions: intervention 1: Topical solution intervention 2: Topical solution

Protocol document:
Prot_SAP_000.pdf: 
 
1 
 
 
 
 
 
 
 
 
A PHASE 2, MULTI-SITE, RANDOMIZED, DOUBLE-BLIND, VEHICLE-
CONTROLLED STUDY OF THE EFFICACY AND SAFETY OF SQUARIC ACID 
DIBUTYL ESTER IN SUBJECTS WITH RECURRENT HERPES LABIALIS – 
SINGLE VERSUS TWO-DOSE ARM APPLICATION 
 
 
Detailed Protocol 
 
 
Amendment 5 
 
 
 
 
Investigator:   
 
Maria Alora-Palli, MD 
Clinical Unit for Research Trials in Skin (CURTIS) 
 
 
 
 
Massachusetts General Hospital 
 
 
 
 
50 Staniford St. (Suite 240) 
 
 
 
 
Boston, MA 02114 
 
 
 
Sponsor: 
 
 
Squarex, LLC 
 
Contacts: 
 
 
Hugh McTavish, Ph.D., J.D. 
Squarex, LLC 
7460 Pinehurst Road 
Pine Springs, MN 55115 
Tel. No. 651-492-0283 
hmctavish@squarex-pharma.com 
 
NCT Number:  
 
NCT02965781 
 
 
 
Version date: 16 March 2017  
 
 
 
 
 
 
 
 
 
 
 
 


 
2 
 
TABLE OF CONTENTS 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
   Page 
                                                                                                                             
I. OBJECTIVE 
 
....................................................................................................... 3 
 
II. BACKGROUND 
....................................................................................................... 3 
 
III.OUTCOMES 
 
....................................................................................................... 4 
 
IV.  SUBJECT SELECTION 
 
A, B 
Inclusion/Exclusion Criteria ........................................................................ 5 
 
C  
Randomization .............................................................................................. 5 
 
V.  SUBJECT ENROLLMENT 
 
A.  
Methods of enrollment ................................................................................. 6 
 
B.  
Informed consent .......................................................................................... 6 
 
VI. STUDY PROCEDURES ............................................................................................. 7 
 
 
VII. RISKS AND DISCOMFORTS .................................................................................... 10 
 
 
VIII. POTENTIAL BENEFITS 
 
A.   
Potential benefits to subjects ...................................................................... 10 
 
B.   
Potential benefits to society ........................................................................ 10 
 
IX. MONITORING AND QUALITY ASSURANCE 
 
A.   
Study Drug Management ............................................................................. 10 
 
B.   
Data and Safety monitoring ......................................................................... 11 
 
C.   
Data and Drug Handling ............................................................................... 11 
 
D.   
Site Monitoring .............................................................................................. 11 
 
E.        Sponsor Monitoring ...................................................................................... 11 
 
F.   
Adverse Event reporting .............................................................................. 12 
 
X. STATISTICAL METHODS ........................................................................................... 13 
 
XI. References 
 
....................................................................................................... 17 
 
Appendix A (Provider Assessment of Disease Severity)........................................... 18 
 
Appendix B (Patient Assessment of Disease Severity) ............................................. 19 
 
Appendix C (Short Diary) ............................................................................................... 20 
 
Appendix D (Study Schema) ......................................................................................... 21 


 
3 
 
I. Objectives 
Primary Objective: To compare the efficacy of SADBE versus placebo (DMSO) in  the 
treatment of herpes labialis 
 
Secondary Objective: To evaluate the safety and tolerability of SADBE versus placebo 
(DMSO) in the treatment of herpes labialis  
 
II. Background 
Primary oral infection with the herpes simplex virus (HSV) typically occurs at a young age, is 
asymptomatic, and is not associated with significant morbidity. After primary oral infection, HSV 
may persist in a latent state in the trigeminal ganglion and later reactivate as the more common 
herpes labialis, or “cold sores.” Common triggers for reactivation are well known and include 
ultraviolet light, trauma, fatigue, stress, fever, inflammation, and menstruation. These lesions 
affect up to 45 percent of the U.S. population. They classically manifest as a well-localized cluster 
of small vesicles along the vermilion border of the lip or adjacent skin. The vesicles subsequently 
rupture, ulcerate, and crust within 24 to 48 hours. Spontaneous healing occurs over seven to 10 
days.  
In immunocompetent patients, herpes labialis usually is mild and self-limited. However, pain, 
swelling, and cosmetic concerns may prompt physician consultation. Orally administered antiviral 
agents, such as acyclovir (Zovirax) or valacyclovir (Valtrex), have a modest clinical benefit if 
initiated during the prodrome. Topical treatment with 1% penciclovir cream (Denavir) may reduce 
healing time and pain slightly, even if initiated after the prodrome. However, reduction in healing 
time with systemic or topical agents is modest. 
Squaric acid dibutyl ester (SADBE) is a topical immunotherapeutic agent used in the treatment of 
verruca vulgaris and alopecia areata. During a  recent FDA Compounding Advisory Committee 
Meeting, it was recommended that squaric acid dibutylester be included on the list of bulk drug 
substances allowed for use in compounding under section 503A of the Federal Food, Drug, and 
Cosmetic Act.  And SADBE has now been so listed under section 503A. 
 A study completed by Lee et al of 29 patients with recalcitrant warts demonstrated complete 
clearance in 69% of patients with application every 2-4 weeks. Silverberg et al showed a 
complete clearance of warts in 58% of patients (n=61) when SADBE was applied 3 times weekly.  
SADBE has also been used with some success in the treatment of alopecia areata. In a review of 
the literature, Rokhsar et al. noted a 50% to 60% success rate of SADBE in use for hair re-growth 
in this population. 
SADBE has been reported to cause eczema, lymphadenopathy, blistering, allergic contact 
dermatitis, skin hypopigmentation, a burning sensation after application, and systemic reactions 
including fever and arthralgias. A study completed by Oglio et al. of eight patients treated with 
SADBE for warts noted only mild and well tolerated side effects of erythema, desquamation, 
cutaneous edema, pruritus, burning, and pain.  
SADBE induces a delayed-type hypersensitivity response which in warts, is believed to induce 
the killing of virally infected cells by cytotoxic lymphocytes. This influx of lymphocytes into lesional 
tissue may also enhance the recognition and processing of viral antigens, leading to clonal 
expansion of effector cells. It is hoped that SADBE will offer subjects a safe and effective 


 
4 
 
therapeutic option to decrease the frequency and severity of future herpes labialis outbreaks 
through these mechanisms.  
A placebo-controlled clinical study completed at Massachusetts General Hospital showed that 
squaric acid prevented recurrence of herpetic lesions.  The effect of SADBE of delaying new 
herpes labialis outbreaks was highly significant (p<0.01) as compared to placebo.    (Throughout 
this document, where the term "squaric acid" is used, it refers to squaric acid dibutyl ester 
(SADBE).) 
 
III. Outcomes 
Primary Outcome Measure:  
1. No. of days until subject reported first new herpes labialis episode following 
sensitization dose (Dose A). 
   
Secondary Outcome Measures:  
1. Incidence of adverse events 
2. Number of days until subject reported first new herpes labialis episode following the 
intensification dose (Dose B). 
3. Number of days until subject reported first new herpes labialis episode beginning from 
21 days after the intensification dose (Dose B). 
4. Number of new herpes labialis episodes during the first 4 months following the 
sensitization dose (Dose A). 
5. Number of new herpes labialis episodes during the first 6 months following the 
sensitization dose (Dose A). 
6. Number of new herpes labialis eposides during the 12-month follow  up period following 
the sensitization dose (Dose A). 
7. Number of new herpes labialis episodes during the first 4 months following the 
intensification dose (Dose B). 
8. Number of new herpes labialis episodes during the first 6 months following the 
intensification dose (Dose B). 
9. Number of new herpes labialis eposides during the 12-month follow  up period following 
the intensification dose (Dose B). 
10. Number of days with herpes labialis during the first 4 months following the sensitization 
dose (Dose A). 
11. Number of days with herpes labialis during the first 6 months following the sensitization 
dose (Dose A). 
12. Number of days with herpes labialis during the 12-month follow-up period following the 
sensitization dose (Dose A).  
13. For only the subjects who are positive for IgG against HSV-2, number of days until a 
subject reports his or her first new herpes labialis episode following the sensitization 
dose (Dose A). 
14. For only the subjects who are positive for IgG against HSV-2, number of days until a 
subject reports his or her first new herpes labialis episode following the intensification 
dose (Dose B). 
15. Average duration of herpes labialis episodes during the 12-month follow up period 
following the sensitization dose (Dose A). 
16. Average duration of herpes labialis episodes during the follow up period following the 
intensification dose (Dose B). 
17. Change in anti-HSV-1 IgG levels between the screening visit and the 2-month visit. 
18. Change in lymphocyte counts between the screening visit and the 2-month visit. 
 
Each of the primary and secondary outcome measures will first be made by pooling the two drug 
treatment groups together and comparing the pooled drug treatment groups versus the placebo.  


 
5 
 
Secondarily, where appropriate, each of the two drug treatment groups will be separately 
compared to placebo and compared to each other. 
   
 
IV. Subject Selection 
 
A. Inclusion criteria  
 
1. Age ≥18 
2. Clinical diagnosis of herpes labialis, which may be made at the screening visit based 
on the patient's self-reported history of symptoms.  An active herpes labialis outbreak 
at the time of entry into the clinical trial will neither be required nor will be an exclusion 
criteria. 
3. Self report having four or more episodes of herpes labialis in the past 12 months.  
 
Subjects will NOT be told that four-or-more episodes in the previous 12 months is 
the entry criterion.  Subjects will be asked "How many separate episodes of cold sores 
have you had in the previous 12 months?"  They will be included if they give an answer 
of four or more and excluded if they give an answer of three or fewer.    
4. At least half of the subject's episodes of the previous 12 months should be vesicular in 
nature and at least half preceded by prodromal symptoms. 
 
B. Exclusion criteria 
 
1. People that have had treatment with anti viral therapy within 2 weeks before 
sensitization dose.  
2. Pregnant or lactating females.  
3. Current or recurrent non-herpetic infection or any underlying condition that may 
predispose to infection or anyone who has been admitted to the hospital due to 
bacteremia, pneumonia or any other serious infection within the past 12 months. 
4. Therapy with glucocorticoid or immunosuppressants at time of recruitment or within 
past  4 weeks, except for inhaled corticosteroids for asthma or topical steroids in sites 
other than face.  
5. History of malignancy (except patients with surgically cured basal cell or squamous cell 
skin cancers) 
6. History of organ transplantation 
7. HIV-positive status determined by history at screening or known history of any other 
immunosuppressive disease. 
8. Severe co-morbidities (diabetes mellitus requiring insulin, CHF (EF<50% at baseline 
will be exclusionary) MI, CVA or TIA within 3 months of screening visit, unstable angina 
pectoris, oxygen-dependent severe pulmonary disease 
9. History of exposure to squaric acid or squaric acid dibutyl ester. 
10. Known hypersensitivity to DMSO 
11. Any condition judged by the investigator to cause this clinical trial to be detrimental to 
the patient. 
12. Subject is currently enrolled in another investigational device or drug trial(s), or subject 
has received other investigational agent(s) within 28 days of baseline visit. 
13. Previous or current participation in a clinical trial of SADBE to treat herpes labialis. 
14. Subject cannot be reliably expected to comprehend or satisfactorily assess a herpetic 
lesion. 
15. Subject has an abnormal skin condition (e.g., acne, eczema, rosacea, psoriasis, 
albinism, or chronic visiculo-bullous disorder) that occurs in the area ordinarily affected 
by herpes labialis. 
16. Subject has had a vaccine for either HSV-1 or HSV-2.   
 
 
C. Randomization 


 
6 
 
 
Randomization will be performed by a computer generated randomization table and will be 
blinded to study investigators and subjects. 
 
 
 
      Subjects will be randomized to one of three  groups on a double blind basis: 
1. Only sensitization dose. 
2. One treatment dose applied to the patient's upper arm 3 weeks after sensitization dose. 
3. Placebo.  Will receive DMSO alone (no SADBE) as placebo on a double blind basis.  
 
 
Group 
Sensitization 
(Dose A) 
3-weeks after 
sensitization 
(Dose B) 
1 
Arm (2%) 
Arm (placebo) 
2 
Arm (2%) 
Arm (0.5%)  
3 (Placebo) 
Arm (placebo) 
Arm (placebo) 
 
Subjects in groups 1 and 2 will receive 2% SADBE for sensitization.  Subjects in group 2 will 
receive 0.5% SADBE 3 weeks after the sensitization dose is applied.  All subjects will receive 
their dose at 0 weeks and at 3 weeks whether they have an active cold sore at that time or not.  
But the presence or absence of active cold sores at the time the doses are applied will be noted.     
 
In order to maintain blinding, the study staff applying study medication will not be involved in any 
of the subject assessments. 
 
V. Subject Enrollment 
 
Preliminary eligibility will be determined based on study staff interviews of interested subjects 
over the phone. Eligible subjects will then be scheduled for a screening visit. We plan to 
randomize and follow for at least 4 months a total of 94  subjects in the study.  To achieve this 
goal, we may have to randomize and enroll 120 subjects and screen up to 200 subjects. 
 
A. Methods of Enrollment 
 
 
 
All subjects who sign an informed consent form (ICF) and are screened will be documented 
on the enrollment log. A note will be made in the source documentation verifying that the 
subject has willingly signed the ICF prior to participation in any study procedures. All 
randomized subjects will receive a subject number to ensure their protected health 
information (PHI) and subject anonymity. Adult men and women of any race or and ethnic 
group may participate in this study. Women of childbearing potential must agree to use 
adequate birth control while in the study and for a period of one month after use of the study 
medication. No minors (i.e., <18 years of age) will be included in this study. 
 
B. Informed Consent 
 
The Investigator or sub-investigator will inform the potential study subject of all aspects of the 
study and answer all their questions. If the patient agrees to be a study subject, they will 
document their consent in writing by signing an ICF. If a subject needs more time to think 
about study participation, they will be given a copy of the consent and sign it upon return if 
they elect to participate. The investigator is responsible for using a consent form that has 
been approved by the IRB and is the most current version. If a new version of the consent 
form is approved by the IRB while a subject is in the treatment portion of the study, then the 
investigator or designee will inform the subject of the changes and, if the subject agrees to 
continue treatment, both the investigator and subject should sign the updated form. No 
minors will be included in this study. 
 


 
7 
 
VI. Study Procedures 
Screening Visit 
 
During the screening visit, the investigator will discuss with each subject the nature of the study, 
its requirements and its restrictions.  
 
The following will be performed:  
• 
Review of inclusion/exclusion criteria 
• 
Medical history and demographics 
• 
Physical examination (targeted skin examination) and vital signs 
• 
Diary distribution and explanation and explanation of the need for subjects to 
complete paper or electronic diaries during each day of the study during which they 
are experiencing a herpes labialis outbreak or redness, irritation, or rash at the drug 
application site. 
• 
Explanation of the need for subjects to take digital photographs with their own smart 
phones or digital cameras of affected areas of their face on each day of the study 
that they are experiencing a herpes labialis outbreak and to take photographs of the 
drug application site on each day of the study during which they are experiencing 
redness, rash, or irritation at the drug application site.   
• 
Photograph mouth and nose area of subject face below the eyes if subject is 
experiencing an outbreak in that area at the time of the clinic visit.   
• 
Blood collection for 
o 
CBC with Diff 
o 
HSV-1 and -2 Ab IgG 
• 
Swab herpetic lesion to collect sample* for  
o 
HSV-1 and -2 PCR* 
• 
Urine pregnancy test (for female participants of childbearing potential)  
• 
Review of concomitant medications 
• 
Adverse Events collection  
• 
Squaric acid sensitization on the right upper arm (application of Dose A, from a vial 
labeled xxx-A for Dose A to patient number xxx).   
* Viral specimen collection for HSV-1 and HSV-2 PCR testing will be conducted on the first study 
visit on which the subject has an HSV lesion. 
 
Subjects  randomized to one of the squaric acid groups will be given a sensitizing dose of 2.0% 
SADBE dissolved in DMSO applied to skin on a single spot (approximately 5 mm diameter) in the 
inner upper arm. The area will be encircled with petroleum jelly and Tegaderm™ dressing will be 
applied.  Subjects assigned to the placebo group will receive DMSO instead of 2.0% SADBE 
dissolved in DMSO. Subjects will be instructed to remove the dressing and rinse the area three 
hours after application of the sensitizing dose. 
 
Week 1 
The following procedures will be performed during this visit. 
• 
Physician assessment 
• 
Blood collection 
o 
CBC with Diff 
• 
Swab herpetic lesion to collect sample* for  
o 
HSV-1 and -2 PCR* 


 
8 
 
• 
Review of concomitant medications 
• 
Adverse events collection 
• 
Collection of diary and photographs 
• 
Photograph mouth and nose area of subject face below the eyes if subject is 
experiencing an outbreak in that area at the time of the clinic visit.   
• 
Photograph drug application site if subject is experiencing any redness, rash, or irritation 
at the drug application site at the time of the clinic visit. 
* Viral specimen collection for HSV-1 and HSV-2 PCR testing will be conducted on the first study visit 
on which the subject has an HSV lesion. 
 
Intensification Dose (Week 3)  
At 21 days (± 3 days) after the sensitization dose, the following procedures will be performed 
during this visit: 
• 
Physician assessment (Targeted Skin Examination) 
• 
Urine Pregnancy Test 
• 
Vital signs 
• 
Review of concomitant medications 
• 
Collection of any reported adverse events 
• 
Collection of photographs and diaries 
• 
Photograph mouth and nose area of subject face below the eyes if subject is 
experiencing an outbreak in that area at the time of the clinic visit.   
• 
Photograph drug application site if subject is experiencing any redness, rash, or irritation 
at the drug application site at the time of the clinic visit. 
• 
Squaric acid (or placebo) application on left upper arm (opposite arm from the 
sensitization dose) (Dose B).  This will involve application of Dose B, from a vial labeled 
xxx-B for Dose B to patient number xxx).  
• 
Swab herpetic lesion to collect sample* for  
o 
HSV-1 and -2 PCR* 
* Viral specimen collection for HSV-1 and HSV-2 PCR testing will be conducted on the first study visit 
on which the subject has an HSV lesion. 
 
 
Month 2   
Two months after the sensitization dose (Dose A), the following procedures will be performed: 
• 
Physician Assessment (Targeted Skin Examination) 
• 
Blood collection 
o 
CBC with Diff 
o 
HSV-1 and -2 Ab IgG 
• 
Swab herpetic lesion to collect sample* for  
o 
HSV-1 and -2 PCR* 
• 
Review of concomitant medications 
• 
Adverse events collection 
• 
Collection of diary and photographs 
• 
Photograph mouth and nose area of subject face below the eyes if subject is 
experiencing an outbreak in that area at the time of the clinic visit.   
• 
Photograph drug application site if subject is experiencing any redness, rash, or irritation 
at the drug application site at the time of the clinic visit. 
 


 
9 
 
* Viral specimen collection for HSV-1 and HSV-2 PCR testing will be conducted on the first study visit 
on which the subject has an HSV lesion. 
 
 
Months 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 
The following procedures will be performed during these visits: 
• 
Physician Assessment (Targeted Skin Examination) 
• 
Review of concomitant medications 
• 
Adverse events collection 
• 
Collection of diary and photographs 
• 
Photograph mouth and nose area of subject face below the eyes if subject is 
experiencing an outbreak in that area at the time of the clinic visit.   
• 
Photograph drug application site if subject is experiencing any redness, rash, or irritation 
at the drug application site at the time of the clinic visit. 
• 
Swab herpetic lesion to collect sample* for  
o 
HSV-1 and -2 PCR* 
* Viral specimen collection for HSV-1 and HSV-2 PCR testing will be conducted on the first study visit 
on which the subject has an HSV lesion. 
 
 
Photographs 
Digital photographs of the mouth and nose area of each patient will be taken by clinic staff at 
each clinic visit during which a patient has an outbreak occurring.  Eyes will be excluded from the 
photographs to protect patient privacy.   
 
In addition, patients will be asked to use their own smart phones or digital cameras to 
photograph herpes labialis lesions on or around the lips or nose once per day during each 
outbreak the that the patient experiences during the clinical trial.  The patients will also be asked 
to photograph the site of drug application when they experience any redness, rash, or irritation at 
that site.  Those photographs also should be taken once per day on any day they are 
experiencing rash, redness, or irritation at the drug application site. 
 
Patient Contacts and Monitoring 
At the initial visit, patients will be instructed to contact the investigators when they have a new 
outbreak. Patients will be sent text messages weekly asking whether they have had a new 
outbreak.  If they answer yes, they will be contacted by phone to schedule a visit for Provider 
Assessment and to ask the day the outbreak began. The clinic visit will only be scheduled if it is 
one of the first three outbreaks for the patient during the study.  If they answer no, it will be noted. 
If they do not reply to 4 consecutive weekly text messages, they will be contacted by phone.  If 
they cannot be reached for 3 consecutive months, they will be dropped from the study and if they 
had not reported any new herpes labialis outbreaks up to the time of the last response from them, 
it will be considered that they did not have a new outbreak up to the date of their last response or 
last clinic visit. 
 
Two to four days after each application of study drug to a subject, the subject will be contacted by 
telephone to ask about any erythema (redness), itching, or pain at the drug application site or 
other adverse event.  If the subject reports moderate or severe erythema or other adverse event 
they are concerned about, they will be asked to come in for a clinic visit for evaluation.  Patient 
responses will be recorded as data.  They will be asked to rate rash at the drug application site on 
a scale of 0 to 3, as no rash, mild rash, moderate rash, or severe rash, as in Question 7 of the 
Patient Assessment diary.  
 


 
10 
 
Patients will be asked to come in for a clinic visit during the first three separate herpes labialis 
outbreaks they have during the study.  If they come in for a visit during an outbreak at 7 weeks or 
more after the sensitization dose, the visit will be considered to be the next monthly visit of their 
monthly visits at 2-7 months, and the procedures specified for that visit will be performed.   
 
 
VII. Risks and discomforts 
 
Possible side effects of Squaric Acid include: 
 
1. Localized erythema 
2. Increasing lesional inflammation. 
3. Pruritis  
4. Contact dermatitis 
5. Lymphadenopathy 
6. Vitiligo or leukoderma  
7. Generalized allergic reaction 
8. Blistering 
9. Burning sensation with application 
10. Fever 
11. Arthralgias 
 
VIII. Potential Benefits 
A. Potential benefits to participating individuals 
 
Subjects may or may not benefit from participating in the study. Their skin condition may get 
better, stay the same, or get worse.  
 
B. Potential benefits to society 
 
Information gathered from this study may help other people in the future with herpes labialis. 
 
IX. Monitoring and Quality Assurance 
 
Written informed consent will be obtained from each subject at the initiation of the screening visit.  
In all cases, the consent will be witnessed by an appropriate health care professional. A copy of 
the signed consent form will be given to the subject to keep. All efforts will be made to insure the 
privacy rights of the study subject. No written or oral communication will be made about any 
patient with anyone other than the patient, unless the patient so requests.  Medical information 
obtained from the study may become part of the patient’s permanent clinic or hospital record, 
subject to the confidentiality and privacy regulations of the clinic or hospital. 
 
A. Study Drug Management 
 
Study medication will be provided by Squarex and shipped directly to the study site clinic. 
 
All study medications will be retained in secure and restricted access storage by the Investigator’s 
designee for the duration of the study. All study medication will be stored in a freezer (-20°C ± 
5°C, or -13°F to +5°F).  Each vial of study medication is labeled with a subject number. Each vial 
will be transferred to room temperature (23°C ±5°C) to thaw one business day before the clinic 
visit at which it is scheduled to be used.  If the clinic visit for that subject is postponed for some 
reason, the vial will be stored at room temperature (23°C ±5°C) for up to 3 months after thawing.  


 
11 
 
Controlled access to study medications will be maintained until Squarex, Inc. has completed final 
drug accountability and provided instructions for drug return and destruction. 
 
 
 
B. Data and Safety Monitoring Plan 
 
This study is considered to be moderate risk to human subjects as the study drug is not FDA 
approved, but is commonly used for treatment of warts and alopecia areata. SADBE is included 
on the "List of Bulk Drug Substances That Can Be Used To Compound Drug Products in 
Accordance With Section 503A of the Federal Food, Drug, and Cosmetic Act." SADBE is being 
proposed for the 503A Bulks List, to appear in § 216.23(a) of Title 21 of the CFR. 
  
A Data and Safety Monitor (DSM) will be appointed, who will be Dr. Thomas Horn, MD, a board-
certified dermatologist and Chief Medical Officer of Squarex.. The DSM will  advise the IND 
sponsor regarding both the safety of current and potential subjects, as well as trial validity and 
merit. The study blind will not be broken unless deemed necessary for the safety and welfare of 
the study subjects. If the blind needs to be broken, the Principal Investigator will not be informed 
unless deemed necessary for the safety and welfare of the study subjects.The FDA will be 
informed of all recommendations made by the DSM to the IND sponsor regarding safety, 
regardless of whether the definition for serious adverse event is met. 
 
C. Data and Drug Handling Guidelines 
 
Data will be collected on case report forms and drug accountability logs and will be complete and 
accurate based on available source documentation.  Corrections of data will be made in a 
manner that allows Squarex to track changes according to FDA regulations. The investigator will 
respond to inquiries regarding data errors, inconsistencies, and missing data in a timely fashion.   
 
The study site will keep all study records, including source docs, CRFs, signed ICFs, regulatory 
papers, patient logs, drug accountability logs, etc. until Squarex determines that they can be 
returned or destroyed.  The investigator will follow the procedures outlined in the protocol and 
discuss any deviations with Squarex.   
 
D. Site Monitoring of Source Data 
 
The principal investigator and members of the study staff not directly involved in clinical 
assessments or clinical study procedures will monitor the study. All data relevant to the 
assessments outlined in this protocol will be recorded in the case report form (CRF) and the 
subject’s sourcebook. 
 
The original case report form for each subject will be audited to source documents 
(inpatient/outpatient medical records, clinical assessment profiles) at the study site by the study 
monitor. In many cases, the case report form may serve as the source document.  The study staff 
monitor will review the progress of the study to ensure proper study conduct and accurate data 
collections through ongoing reviews of Case Report Forms, clinical records, and administrative 
documents. Reviews will be made at least once a month.   
  
 
E. Sponsor Monitoring of Study Data 
 
Representatives of Squarex will also monitor this study’s data regularly via scheduled monitoring 
visits. Monitoring procedures include pre-study preparations, site initiation visit, interim monitoring 
visits, and study close-out preparations and visits. During sponsor’s visits, the Investigator will 


 
12 
 
provide Squarex’s monitors with access to all protocol regulatory documents, all medical data 
collected on the participating subjects, screening logs, enrollment logs, drug accountability logs 
and the drug supply, case report forms, dermatological efficacy assessments, photographs of 
lesions and sites of drug application, all questionnaires, patient diaries, signed informed consent 
forms, and any other information that Squarex may consider to be necessary to evaluate the 
efficacy of the investigational product and patient safety.   Both the monitors and study staff will 
review the accuracy and completeness of case report form entries, all log entries, source 
documents, and informed consent documents.    
 
F. Adverse event reporting guidelines 
 
Definitions 
 
Adverse Event (AE) is any untoward medical occurrence associated with the use of the study 
drug in a subject, whether or not considered drug related.. An AE can therefore be any 
unfavorable and unintended sign, symptom, or disease temporally associated with the use of an 
investigational product, whether or not related to the investigational product.  
 
Serious Adverse Event (SAE) is any untoward medical occurrence that: 
• 
Results in death, 
• 
Is life-threatening, 
• 
Requires inpatient hospitalization or prolongation of existing hospitalization, 
• 
Results in persistent or significant disability or incapacity, 
• 
Is a congenital anomaly/birth defect, or 
• 
Is another medically important condition 
 
 
Reporting and Documenting Adverse Events 
 
All untoward medical occurrences that occur after the subject signs a consent form should be 
documented as an AE. The Investigator should ensure that all events that occur during the study 
period are recorded. All AEs should be followed until resolution or until, in the Investigator’s 
judgment, they are chronic and stable. If an emergency situation should occur, appropriate 
medical measures should be taken to stabilize the subject. 
 
Documentation of AEs includes: date and time of onset and resolution of AE, intensity, frequency, 
seriousness, related interventions and outcome. The Investigator must also evaluate the 
probability of a causal relationship of the AE to the study treatment as being: “definite, probable, 
possible, unlikely, or unrelated.” Intensity of adverse events will be graded as mild, moderate, or 
severe according to the following criteria: 
 
Mild: symptoms that are easily tolerated and transient in nature with minimal or no impairment of 
normal activity 
Moderate: symptoms that are poorly tolerated, are sustained, and interfere with normal activity 
Severe: symptoms that are incapacitating and render the subject unable to work or participate in 
many or all usual activities 
 
All SAEs will be reported to the IRB according to the IRB’s requirements. They will also be 
reported to the study sponsor and FDA according to regulatory guidelines. 
 
 
Study Discontinuation 
 
At any time after enrollment, a subject may be discontinued. Reasons for discontinuation of a 
subject from the study will include, but may not be limited to, the following: 


 
13 
 
• 
Subject is found to be intolerant to a required study procedure at any time point 
• 
Subject experiences a serious adverse experience at any time point.  
• 
Subject develops an inter-current illness that would, in the judgment of the investigator, 
affect assessments of clinical status to a significant degree.  
• 
Subject begins a medication that, in the judgment of the investigator, may affect 
assessments of clinical status to a significant degree. 
• 
Subject becomes pregnant while participating in the study 
• 
Subject enrolls in another investigational study 
• 
Subject requests to withdraw from the study 
• 
The study sponsor decides to suspend or terminate the study. 
 
 
X. STATISTICAL METHODS 
 
Statistical analyses will be performed using SAS software. Default estimation methods in version 
9.4 of SAS are always used unless an alternative is specified below. In general, data summaries 
will include the mean, standard deviation, median, minimum and maximum values for continuous 
data; the median, 25th and 75th percentiles, minimum and maximum values for time-to-event 
endpoints; and the number and percentage of patients in each category for categorical data. 
Pointwise 95% confidence intervals (CI) will also be estimated for the mean (continuous data), 
median (time-to-event endpoints) or percentage of patients (categorical data). 
 
All primary statistical comparisons of 2% SADBE to placebo (i.e., regardless of whether the 
comparison uses a primary or secondary endpoint) will combine data from patients randomized to 
treatment group 1 or 2 for testing against data from patients randomized to treatment group 3. 
Secondary analyses of primary and secondary endpoints will be conducted to assess differences 
between individual treatment groups. 
 
No interim data analyses are planned. (However, blinded review of safety data may be performed 
if needed to ensure patient safety.) 
 
Randomization 
 
Random assignment of patients to three treatment groups will be on a 1:1:1 ratio. Thus the ratio 
of patients assigned to one of the two 2% SADBE treatment groups will be 2:1 versus placebo. 
 
Sample Size 
 
The sample size of 94 patients was selected for this study so that a two-sided log-rank at the 5% 
level of significance would have 90% power under the following assumptions: 
 
• 
Median times to first new herpes labialis episode of 90 and 40 days for patients treated 
with 2% SADBE and placebo, respectively 
• 
2:1 allocation of patients to the two 2% SADBE treatment groups versus placebo 
• 
Patient accrual over a period of 1 year and a total study duration of 18 months 


 
14 
 
• 
Patients may be lost to follow-up (and censored according to the rules specified below) 
according to an exponential distribution with a median of 9 months 
 
The method for controlling the overall two-sided 5% level of significance against the inflationary 
effects of testing multiple efficacy endpoints is discussed below. 
 
Primary Endpoint Analysis 
 
The primary endpoint is the number of days until a patient reports his or her first new herpes 
labialis episode following the sensitization dose (Dose A). The date of the sensitization dose is 
defined as Day 1. Therefore, any herpes labialis episodes occurring prior to the sensitization 
dose, including any episodes between treatment group randomization and the day prior to the 
sensitization dose, will not be counted when assessing the primary endpoint. Data from patients 
in treatment groups 1 and 2 will be combined (since patient in both groups receive 2% SADBE for 
sensitization) and compared against data from patients in treatment group 3 (placebo). The 
nonparametric log-rank test will be used to test the treatment effect (2% SADBE versus placebo) 
at the two-sided, 5% level of significance. Patients withdrawn from the study for any reason prior 
to experiencing a first new episode of herpes labialis will have their primary endpoint censored on 
the last date of active study participation. Cumulative time-to-endpoint distributions (“survival 
curves”) will be estimated by the Kaplan-Meier method. 
 
Secondary Endpoint Analyses 
 
Without taking appropriate measures, testing multiple efficacy parameters inflates the overall 
significance level since the probability of making a type-1 error in at least one from a set of 
hypothesis tests is greater than the probability of making a type-1 error in any single test. The 
hierarchical closed test procedure [Reference #s 5 & 6] will be used to maintain the overall 5% 
significance level. The order of efficacy endpoints for the hierarchical closed test procedure is: 
 
1. Number of days until a subject reports his or her first new herpes labialis episode 
following the sensitization dose (Dose A) (primary endpoint) 
2. Number of days until subject reported first new herpes labialis episode following the 
intensification dose (Dose B). 
3. Number of days until subject reported first new herpes labialis episode beginning from 21 
days after the intensification dose (Dose B). 
4. Number of new herpes labialis episodes during the first 4 months following the 
sensitization dose (Dose A). 
5. Number of new herpes labialis episodes during the first 6 months following the 
sensitization dose (Dose A). 
6. Number of new herpes labialis eposides during the 12-month follow  up period following 
the sensitization dose (Dose A). 
7. Number of new herpes labialis episodes during the first 4 months following the 
intensification dose (Dose B). 
8. Number of new herpes labialis episodes during the first 6 months following the 
intensification dose (Dose B). 
9. Number of new herpes labialis eposides during the 12-month follow  up period following 
the intensification dose (Dose B). 


 
15 
 
10. Number of days with herpes labialis during the first 4 months following the sensitization 
dose (Dose A). 
11. Number of days with herpes labialis during the first 6 months following the sensitization 
dose (Dose A). 
12. Number of days with herpes labialis during the 12-month follow-up period following the 
sensitization dose (Dose A).  
13. For only the subjects who are positive for IgG against HSV-2, number of days until a 
subject reports his or her first new herpes labialis episode following the sensitization dose 
(Dose A).  
14. For only the subjects who are positive for IgG against HSV-2, number of days until a 
subject reports his or her first new herpes labialis episode following the intensification 
dose (Dose B). 
15. Average duration of herpes labialis episodes during the 12-month follow up period 
following the sensitization dose (Dose A). 
16. Average duration of herpes labialis episodes during the follow up period following the 
intensification dose (Dose B). 
 
Safety and pharmacodynamic endpoints are not included in the hierarchical closed test 
procedure. 
 
Each efficacy endpoint will be tested at a two-sided 5% significance level using the statistical 
procedures described below. Days until a patient reports his or her first new herpes labialis 
episode following the sensitization dose, the primary efficacy endpoint, will be declared 
statistically significant if its test p-value is ≤0.05. A secondary efficacy endpoint will be declared 
statistically significant only if its test p-value is ≤0.05 and all efficacy endpoints preceding it in the 
hierarchy are statistically significant. In other words, declaration of statistical significance for 
efficacy endpoints will advance in order down the hierarchy only until a p-value >0.05 is found. 
 
Only the p-value from an endpoint’s primary analysis will be used for deciding statistical 
significance. Supportive (sensitivity) analyses used to demonstrate the robustness of primary 
results to slight modifications to how endpoints are defined and/or analyzed will not be considered 
for the purpose of determining primary significance. 
 
The log-rank test (as described above for the primary endpoint) will be used to test secondary 
time-to-event endpoints. Secondary endpoints that are total or average durations of specific 
events will be tested using nonparametric Wilcoxon rank sum tests. 
 
Safety Analyses 
 
Reported adverse event (AE) terms will be mapped to MedDRA preferred terminology. All 
reported events will appear in AE listings, however only treatment-emergent adverse events will 
be summarized. A treatment-emergent adverse event (TEAE) is an AE that starts or increases in 
severity any time after the first application of any study drug up to 30 days following the last 
application of any study drug. 
 


 
16 
 
A high-level safety summary will display the numbers of patients within each treatment group and 
overall who experience one or more AEs in each of the following categories: 
 
• 
All TEAEs regardless of severity or presumed relationship to study drug 
• 
TEAEs judged related to study drug 
• 
Treatment-emergent serious adverse events (SAEs) 
• 
TEAEs leading to a delay in the application of study drug 
• 
TEAEs leading to discontinuation of study drug 
• 
TEAEs leading to withdrawal from the study 
 
The base summary of TEAEs will show within– and between–treatment-arm incidence rates for 
each MedDRA primary system organ class and/or preferred term by highest reported severity 
grade and overall. A separate summary will be produced each of the AE subsets listed above. 
Additional AE summaries may be produced using safety data from subsets of patients (e.g., 
patient subsets according to age, sex or race). 
 


 
17 
 
References 
 
1. Lee AN, Mallory SB. Contact immunotherapy with squaric acid dibutylester for the treatment 
of recalcitrant warts. J Am Acad Dermatol 1999;41(4): 595-599. 
2. Rokshar CK, Shupak JL, Vafai JJ, Washenik K. Efficacy of topical sensitizers in the treatment 
of alopecia areata. J Am Acad Dermatol 1998; 39(5)751-761 
3. Silverberg NB, Lim JK, Paller AS, Mancini AJ. Squaric acid for immunotherapy for warts in 
children. J Am Acad Dermatol 2000;42:803-808. 
4. Millican EA, Conley JA, Sheinbein D. Cutaneous lymphoid hyperplasia related to squaric acid 
dibutyl ester. J Am Acad Dermatol 2011; 65(1): 230-231. 
5. Yue L. Testing Multiple Secondary Endpoints in Confirmatory Comparative Studies – A 
Regulatory Perspective. Presented at: 2nd Annual AdvaMed Statistical Issues for Medical 
Devices and Diagnostics; April 29, 2009; Bethesda, MD. Available at: 
http://www.advamedmtli.org/download/File/Statistics_April_2009/Yue_lilly_pres.pdf. 
Accessed February 5, 2015. 
6. Koch GG, Gansky SA. Statistical Considerations for Multiplicity in Confirmatory Protocols. 
Drug Information J. 1996;30:523-533. Available at: 
http://ferran.torres.name/download/shared/general/Multiplicity%20dj302612.pdf. Accessed 
February 5, 2015. 


 
18 
 
Appendix A.   
 
Subject #: 
 
 
Month          Day              Year     
Subject Initials:  
 
 
Visit Date:  
   
   
 
 
 
Provider Assessment of Disease Severity 
 
1. Are there HSV lesions present (please circle):     Yes               No 
 
2. Location of lesions (please choose from the following):    
 
 
 
 
3.  Redness of lesions (circle)  
0 = no erythema  
1 = faint pink, 
2 = red 
3 = violaceous. 
 
4.  Nature of lesion (circle all that apply) 
a. prodrome (patient reports tingling but no visible lesion) 
b. erythema 
c. papule 
d. vesicle 
e. erosion or ulcer  
f. crust  
g. normal skin 
 
5.  Please assess for local irritation from therapy. Please circle the choice that most accurately 
describes the treatment response: 
0 = no rash, 
1 = mild rash 
2 = moderate rash 
3 = severe rash in need of treatment with topical hydrocortisone 
 
 
 
 
________________________________________________ 
_______________ 
Signature 
 
 
 
 
 
 
Date 


 
19 
 
 
Appendix B 
Subject #: 
 
 
Month          Day              Year     
Subject Initials:  
 
 
Date:  
   
   
 
 
 
 
 
 
 
 
 
Patient Assessment of Disease Severity: please encircle your answers. 
 
1. Do you have any HSV lesions present:     Yes               No 
 
2. Location of lesions (please label the following):    
 
 
 
3.  Redness:  
0 = no redness  
1 = faint pink 
2 = red 
3 = purple 
 
4. Pain: 
0 = no pain 
1 = mild pain 
2 = moderate pain 
3 = severe pain 
 
5. Tingling: 
0 = no tingling 
1 = mild tingling 
2 = moderate tingling 
3 = severe tingling 
 
6.  Nature of lesion (check all that apply): 
a. prodrome (tingling, no visible rash) 
b.  erythema (redness)  
c. papule (raised skin) 
d. vesicle (fluid-filled raised skin) 
e. erosion or ulcer (open sore)  
f. crust (dry sore)  
g. normal skin 
 
 
7. Please assess for local irritation from therapy; 
0 = no rash, 
1 = mild rash 
2 = moderate rash 
3 = severe rash 
 
 
 
 
 
 
Signature: _______________________ 


 
20 
 
 
Appendix C. Short Diary 
 
Subject No. 
 
 
 
Month:  
Year:  
 
 
Date 
Do you have any cold sores (HSV lesions) present? 
Photos Taken 
 
Initials 
                       (If yes, please complete detailed sheet) 
 
 
 
1              
Yes               No  
 
 
 
Yes               No            ________ 
2  
 
Yes               No  
 
 
 
Yes               No            ________ 
3 
 
Yes               No  
 
 
 
Yes               No              ________ 
4 
 
Yes               No  
 
 
 
Yes               No              ________ 
5 
 
Yes               No  
 
 
 
Yes               No              ________ 
6 
 
Yes               No  
 
 
 
Yes               No              ________ 
7 
 
Yes               No  
 
 
 
Yes               No             ________  
8 
 
Yes               No  
 
 
 
Yes               No             ________ 
9 
 
Yes               No  
 
 
 
Yes               No             ________ 
10 
 
Yes               No  
 
 
 
Yes               No             ________ 
11 
 
Yes               No  
 
 
 
Yes               No             ________ 
12 
 
Yes               No  
 
 
 
Yes               No             ________ 
13 
 
Yes               No  
 
 
 
Yes               No             ________ 
14 
 
Yes               No  
 
 
 
Yes               No             ________ 
15 
 
Yes               No  
 
 
 
Yes               No             ________ 
16 
 
Yes               No  
 
 
 
Yes               No             ________ 
17 
 
Yes               No  
 
 
 
Yes               No             ________ 
18 
 
Yes               No  
 
 
 
Yes               No             ________ 
19 
 
Yes               No  
 
 
 
Yes               No             ________ 
20 
 
Yes               No  
 
 
 
Yes               No             ________ 
21 
 
Yes               No  
 
 
 
Yes               No            ________  
22 
 
Yes               No  
 
 
 
Yes               No            ________ 
23 
 
Yes               No  
 
 
 
Yes               No            ________ 
24 
 
Yes               No  
 
 
 
Yes               No            ________ 
25 
 
Yes               No  
 
 
 
Yes               No            ________ 
26 
 
Yes               No  
 
 
 
Yes               No            ________ 
27 
 
Yes               No  
 
 
 
Yes               No           ________  
28 
 
Yes               No  
 
 
 
Yes               No           ________ 
29 
 
Yes               No  
 
 
 
Yes               No           ________ 
30 
 
Yes               No  
 
 
 
Yes               No           ________ 
31 
 
Yes               No  
 
 
 
Yes               No           ________ 
 
 


 
21 
 
Appendix D. Study Schema 
 
  
Screening 
and Sensitization 
dose (Dose A) 
application  
Week 1 
Intensification 
Dose (Dose B) 
(Week 3)  
Month 2 
Month 3 
Monthly 
visits at 
months 4-
12 
+ 2 d 
+2 W 
+1 W 
+ 2 W 
+ 2 W 
Informed Consent 
X 
 
  
  
  
  
Inclusion/Exclusion 
X 
 
  
  
  
  
History/Demographics 
X 
 
  
  
  
  
Physician Assessments (Targeted Skin Examination) 
X 
X 
X  
X 
X 
X 
Urine Pregnancy Test 
X 
 
 X 
  
  
  
Vital Signs 
X 
 
 X 
  
  
  
Photography 
X 
X 
X 
X 
X 
X 
Diary distribution and explanation / explanation of taking self 
photographs. 
X 
 
 
 
 
 
Photograph Collection and Diary review 
X 
X 
X 
X 
X 
Squaric acid application 
X 
 
X  
  
  
  
Blood Collection (Processing, storing, shipping, dry ice)  
X 
X 
 
 X 
  
 
CBC with Differential 
X 
X 
 
 X 
  
 
HSV-1 and -2 Ab IgG 
X 
 
 
 X 
  
 
Viral specimen collection* 
X  
HSV-1 and -2 PCR* 
 X 
Concomitant medications/Adverse event collection 
X 
X 
X 
X 
X 
X 
* Viral specimen collection and HSV-1 and -2 PCR in the specimen collection will be conducted on the first study visit on which the patient has an HSV lesion.