A Phase 2, Exploratory Study to Investigate Safety and Efficacy of Doxycycline Monohydrate Hydrogel (NANODOX® HYDROGEL 1%) In Atopic Dermatitis
Phase: PHASE2
Conditions: Dermatitis, Atopic
Sponsor: University of Florida

IRB#201601657 
PI: Anna De Benedetto, MD 
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A Phase 2, Exploratory Study to Investigate Safety and Efficacy of Doxycycline 
Monohydrate Hydrogel (NANODOX®) 1% In Atopic Dermatitis 
 
 
Protocol Approval 
 
As authorized representative of the organizations involved with this study, we hereby 
give our dated signatures in scientific approval of the obligations and procedures set 
forth in this investigator initiated protocol as an instrument of proprietary information 
and instructions for the indicated research project. 
 
Authorization: 
 
 
 
Sponsor 
 
 
 
 
 
 
 
Date 
James Talton, PhD 
Alchem Laboratories, Inc 
 
 
 
Principal Investigator 
 
 
 
 
 
 
Date 
Anna De Benedetto, MD 
University of Florida College of Medicine 
Department of Dermatology 
 
Study Site: 
UF Health Department of Dermatology 
4037 NW 86th Terrace- 4th Floor 
Gainesville Florida 32606 
 
 
 
 
 
 
 
 
 
 
 
 

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IRB protocol -  
A Phase 2, Exploratory Study to Investigate Safety and Efficacy of Doxycycline 
Monohydrate Hydrogel (NANODOX®) 1% In Atopic Dermatitis 
Principal Investigator:  Anna De Benedetto, MD – Department of Dermatology, UF  
Phone Number: office (352) 294-4427; adebenedetto@ufl.edu 
Co-Investigators:  
- 
Gregory Schultz, PhD – Department of Obstetrics & Gynecology, UF 
Other research staff:   
Sanjuana Rodriquez, PA-C, Department of Dermatology, UF 
Kiran Motaparthi, MD – Assistant professor, Department of Dermatology, UF 
Jennifer Schoch, MD- Assistant Professor, Department of Dermatology, UF 
Kathryn Potter, MD- Resident in Training, Department of Dermatology, UF 
Michael Montuno, MD- Resident in Training, Department of Dermatology, UF 
Alexandra L. Owens , MD- Resident in Training, Department of Dermatology, UF 
Jennifer Nicole Harb, MD- Resident in Training, Department of Dermatology, UF 
Bahram Dideban, MD Resident in Training, Department of Family Medicine, UF 
 
Mary Bohannon, CCRA Research Coordinator mebohan@ufl.edu 
Lenise Thomas, LPN Research Nurse 
Gina Martorana, EPIC specialist, Department of Dermatology, UF 
Peter Nadeau, Biological Scientist III in Dr. De Benedetto’s lab, Department of dermatology, UF 
1. PURPOSE AND BACKGROUND 
Atopic Dermatitis (AD) is the most common inflammatory skin disease, affecting about 17% of 
children and 6% adults in the USAi, ii.  AD is characterized by skin barrier disruption, an aberrant 
adaptive immune response (i.e., Th2 polarized) to environmental allergens, susceptibility to 
cutaneous bacterial infections and intractable itchiii,iv. The intense pruritus and cutaneous 
infections contribute to the morbidity of AD and are major drivers of the reduced quality-of-life 
associated with this diseasev, vi. In the World Health Organization 2010 Global Burden of 
Disease survey, AD has ranked first among common skin diseasesvii. So far, AD treatments 
have targeted inflammation with the widespread use of topical and more intermittent use of 
systemic corticosteroids. In summary, despite its high prevalence, effects on quality-of-life and 
economic burden – there are few effective treatments for AD.  
 
Doxycycline are tetracycline antibiotics broadly used systemically to treat inflammatory-
dermatologic conditions. Several studies in human and animal models have shown doxycycline 
have anti-inflammatory and pro-healing properties, mainly by blocking tissue proteolytic activity.  
Doxycycline have been reported to nonselectively inhibit members of the metalloproteinases 
(MMP) superfamily [reviewed in viii, ix].  In addition to this direct inhibitory activity, doxycycline 
indirectly prevents tryptic kallikreins activation by MMPsx. Growing body of evidence suggests 
that the tetracycline might also directly downregulate Protease Activator receptor (PAR)-2 
expression and function, which was also found to play a role in induction of local inflammatory 

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mediatorsxi.  Importantly, the doxycycline antimicrobial activity could lead to reduced 
Staphylococcus infection/colonization in AD skin, a known trigger of AD flares.  
 
Alchem Laboratories Corp has recently acquired from Nanotherapeutics, Inc the first topical 
formulation of doxycycline: NanoDOX® Hydrogel, a novel small particle gel suspension of 
doxycycline 1%. The topical 1% doxycycline monohydrate hydrogel product is available for 
clinical testing through a FDA-cleared IND obtained in 2008 to treat chronic skin wound and a 
phase 2 study to evaluate safety and efficacy completed in 2011 at VA hospital in Gainesville 
("Safety Study of Topical Doxycycline Gel for Adult Diabetic Lower Extremity Ulcers", Protocol 
2008-DOX-NT/003, ClinicalTrials.gov Identifier: NCT00764361).  An amendment to the IND 
77520 has been filed with the FDA by Alchem Lab for the new IND indication for use in AD.  
 
Safety preclinical studies of NANODOX® were previously evaluated by Nanotherapeutics.  
Briefly, Franz diffusion cells were used to analyze the amount of doxycycline released out of a 
NanoDOX® Hydrogel or particle suspension across an exact area with respect to time.  These 
conditions may be used to mimic an exposed wound and the resulting drug release out of the 
NanoDOX® Hydrogel into the body. Overall, less than 1% w/w of the 15 mg total doxycycline 
available in the donor chamber in the gel (11.18µg, or 0.08%) as well as the suspension 
(29.88µg, or 0.21% w/w) diffused across the membrane into the receptor chamber. In a rat 
wound healing study, NanoDOX® Hydrogel was tested at doxycycline concentrations of 0.0% 
(placebo hydrogel), 0.3%, 1%, and 3% w/w as well as untreated control.  The results of the 
study demonstrated no significant differences between the histological scoring for all 
concentrations and the control.  All concentrations were judged to be non-irritants versus the 
control. In humans, NanoDOX® Hydrogel treatment was evaluated in a pilot, randomized, 
controlled trial of chronic, diabetic, lower-extremity foot ulcers.  All four chronic ulcers of diabetic 
patients treated daily with topical 1% NanoDOX® Hydrogel healed in the 20-week treatment 
period, whereas only one of three patients treated with vehicle healed (p=0.05). No adverse 
events were attributed to NanoDOX® Hydrogel treatment13. 
 
Aim of this protocol is to investigate the safety and clinical efficacy of 1% topical 
Doxycycline Monohydrate (NanoDOX® Hydrogel) in AD.  It is our hypothesis that daily 
application of NanoDOX® hydrogel blocking cutaneous proteases activity in AD subjects will 
reduce severity of the disease, by restoring skin barrier function and reducing skin driven 
inflammation. Importantly, we will also monitor the anti-microbial activity of this product on AD 
skin, as colonization with Staph aureus is typically associated with disease severity. This work 
will likely be the first step to developing new therapeutics targeting skin proteases activity.  
2. CHARACTERISTICS OF THE RESEARCH POPULATION 
Number of subjects We intend to have 15, non-pregnant, non-lactating women and men of any 
race and ethnicity, ages 18 to 65 yrs, who are generally healthy except for atopic dermatitis 
diagnosed by the US Consensus criteria complete this study. This may require enrolling up to 
30 subjects depending on retention rates and dropouts. Patients can have other allergic 
conditions such as asthma, allergic rhinitis, food allergies, etc. All AD subjects must have 

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moderate-severe AD as defined at screening by an Eczema Area and Severity Score (EASI) ≥ 
10. 
Gender of subjects We will be recruiting both male and female subjects  
Age of subjects We will be including subjects between the ages of 18 and 65 years. 
Racial and Ethnic Origin We will not have any restrictions based on racial or ethnic origin. We 
based our target research population racial and ethnic distribution on the data of the 2000 
United States Census for the Alachua Area.  
Vulnerable subjects We will not be targeting vulnerable subjects, however staff and students 
of UF and Shands may be enrolled as part of the general population with AD 
Inclusion Criteria 
- 
Male or female, 18 through 65 years of age, inclusive who are generally healthy 
except for active atopic dermatitis diagnosed by the following criteria.  
- 
Active Atopic Dermatitis: Subjects must have according to medical records, patient 
account or by medical exam of the investigator: 
 
Pruritus 
 
Eczema (acute, subacute, chronic) 
 
Chronic or relapsing history 
Most subjects will have (seen in most cases, adding support to the diagnosis): 
 
Early age at onset 
 
Atopy 
 
Personal and/or family history 
 
Xerosis 
Subjects may have (these clinical associations help to suggest the diagnosis of AD but 
are too nonspecific for defining or detecting AD for research or epidemiological studies): 
1. Atypical vascular responses (e.g., facial pallor, white dermographism, delayed 
blanch response) 
2. Keratosis pilaris/hyperlinear palms/ichthyosis 
3. Ocular/periorbital changes 
4. Other regional findings (e.g., perioral changes/periauricular lesions) 
5. Perifollicular accentuation/lichenification/prurigo lesions 
- 
Moderate to Severe AD: clinical score based on EASI ≥ 10 and/or IGA 1-2. 
- 
If receiving antihistamines, are on a stabilized dose, and expect to maintain this dose 
throughout the study 
- 
All female subjects of childbearing potential must have a negative pregnancy test at 
screening visit and must be on an acceptable methods of contraception from the 
Screening Visit continuously until 30 days after stopping study drug. 
 
 
 

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Exclusion Criteria: 
- 
As determined by the study doctor, a medical history that may interfere with study 
objectives (cancer, chronic illness) 
- 
Known allergy to tetracycline  
- 
Hand eczema only (no body involvement). 
- 
Subjects with a systemic infection requiring a course of systemic antibiotics or antivirals 
within the last 2 weeks 
- 
Unstable AD or any consistent requirement for systemic immune-modulant Rx (e.g. 
systemic steroids, phototherapy, Cyclosporine) 
- 
History of use of biologic therapy (including intravenous immunoglobulin) 
- 
Recent or anticipated concomitant use of systemic therapies that might alter the course 
of AD 
- 
Recent or current participation in another research study 
- 
Females who are breastfeeding, pregnant, or anticipate becoming pregnant during the 
study time frame  
- 
Subjects with a history of keloid formation  
- 
History of lidocaine, epinephrine or Novocain allergy 
- 
History of allergy to tape or other adhesive materials 
3. METHODS AND PROCEDURES  
 
 
 
 
 
 
Study Design. We will perform a 6 weeks, pilot, investigator-initiated, single-center, open-label 
clinical study to determine the effect of NANODOX® 1% hydrogel in adult AD patients.  The 
study will be conducted in the UF Health Dermatology Department - Springhill.  
Subjects that voluntarily agreed to participate will be consented and enrolled in the study. 
Qualified subjects will be required to come to UF Dermatology Clinic for at least four visits, 
approximately 2 weeks apart, with an optional pre-screening (up to 28 days +/- 2 prior to V1) 
visit if washout is needed and/or to allow subjects to be enrolled in pre-established enrollment 
dates. Each visit will last approximately 30-60 minutes. 
Subjects will be asked to apply NanoDOX® Hydrogel 1% once daily at bedtime for total of four 
weeks (or until complete clearance) on the chosen target eczematous and adjoining clinically 
normal (perilesional) area (to cover an area of the size of an index card). NanoDOX® Hydrogel 
1% will be provided to the patient in single dose package containing 1.5 cc of hydrogel (equal to 
15mg Doxycycline) which is enough to cover a skin area of the size of an index card. Alchem 
will provide drug for the study at no cost. Of note, for systemic use doxycycline is used at 
concentration from 100 mg (antimicrobial) twice daily to 40 mg (anti-inflammatory) daily for 
periods ranging from 14 days to several months. We included non-lesional/perilesional skin to 
evaluate the safety (e.g. irritant or toxic effects) of the compound on atopic skin, as those 
subjects have a lower irritancy threshold as is compared to non-atopic subjectsxii.   Also, skin 
barrier impairment (e.g. increase water loss & increase pH) can be already detected in normal 

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appearing skin in AD subjects. It is our hypothesis that by normalizing the proteases activities, 
treatment with NanoDOX® Hydrogel will normalize skin barrier integrity.  
Subjects that are on any topical treatment (e.g. steroids, calcineurin antagonist) will be asked to 
discontinue the use of those medications only in the selected area for one week before starting 
the study (washout period). During the study, subjects will be allowed to stay on anti-histamine 
and any topical treatment (except for the treatment area) on a stabilized dose, and expect to 
maintain this dose throughout the study. In case of acute flare (e.g. intense itch, increase 
redness, pain or oozing) in the treatment area subjects will be allowed to use rescue medication 
(e.g. topical steroid or calcioneurin inhibitors that they used before the study) under the PI 
supervision. The need for rescue topical treatment will be monitored and recorded during the 
study and it will be used as an indication of the efficacy of the treatment. 
 
 
Screening visit (optional): 
1) Consenting and confirmation of inclusion & exclusion criteria 
2) Identification of the target area to be treated with NANODOX® and digital picture of the 
targeted lesion. The picture will not include subjects’ identifiers (e.g. facial features, tattoo, ect) 
3) possible washout period (topical only in the chosen area – treatment and biopsy sites) 
4) scheduling visit 1 
 
Visit 1 (Day 0):  
1) Consenting and confirmation of inclusion & exclusion criteria 
2) General History and Physical Exam (including review of concomitant medications) 
3) Urine Pregnancy test for woman of childbearing potential. Counseling on acceptable birth 
control measures to use throughout treatment period (28 days) and until 30 days after 
stopping study drug 
4) Questionnaire to clinically characterize AD  
5) Clinical score for atopic dermatitis  
6) Identification of the target area to be treated with NANODOX® and skin biopsy locations. 
Arms will be the preferable areas if lesions are present, otherwise trunk or lower extremity 
7) Digital pictures of the target area and area used for biopsy 
8) Non-invasive skin barrier Measurements: TransEpidermal Water Loss (TEWL). Optional: pH 
and Stratum Corneum (SC) hydration measurements may also be performed 
9) Repeated tape strippings (0, 5, 10, 15 tape-strippings with Cuderm™ tape) of non-lesional 
and lesional locations from treated area will be collected as part of the TEWL assessment 
and to assess for SC cohesion/integrity (rate of change in TEWL with repeated strippings). 

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Serial measurements of TEWL will be performed after tape stripping as previously 
describedxiii. This will also allow collecting proteins (eluded from the tape) for proteases 
activity assessment.  
10) Skin swab 
11) Optional Skin biopsy (5 mm punch) of a representative skin in the same anatomical area or 
symmetrical, avoiding the area that will be treated.  This biopsy may be performed on D28 at 
the same time as the post treatment biopsy. 
12) Venous blood draw to monitor AD biomarkers 
13) Dispense NANODOX® for two following weeks (14 +/- 2 monodose packages) 
- 
Subjects will be instructed to keep the NANODOX refrigerated at + 4C; subjects will be 
provided a cooler and ice packs keep the products refrigerated during transport.  
13) Provide instructions to complete at home diary.   
14) Provide date of Visit 2 and “morning of appointment” instructions 
 
Visit 2 (Day 14 +/-2):  
1) Review subject diary and address any questions, concerns, or missed doses 
2) Questionnaire to clinically characterize AD, review of medications and possible SE 
2) Clinical score for atopic dermatitis  
3) Digital pictures of the targeted area 
4) Non-invasive skin barrier Measurements: transepidermal water loss. Optional: pH and SC 
hydration measurements may also be performed. 
5) Repeated tape strippings (0, 5, 10, 15 tape-strippings with Cuderm™ tape)  
6) Dispense NANODOX® for two following weeks (14 +/-2 monodose packages) 
7) Suture removal at pre-treatment biopsy site, if applicable 
8) Review Diary instructions 
9) Provide date of Visit 3 and “morning of appointment” instructions 
 
In case of complete resolution of the treated area at V2, post treatment biopsy and swab and 
Venous blood draw will be performed at this visit (instead V3) and patient will only come back 
for 2 weeks post treatment. 
 
Visit 3 (Day 28 +/-2):  
1) Review subject diary and address any questions, concerns, or missed doses 
2)Questionnaire to clinically characterize AD, review of medications and possible SE 
3) Clinical score for atopic dermatitis  
4) Digital pictures of the targeted area 
5) Non-invasive skin barrier Measurements: transepidermal water loss. pH and SC hydration 
measurements may also be performed. 
6) Repeated tape strippings (0, 5, 10, 15 tape-strippings with Cuderm™ tape)  
7) Skin swab of treatment area 
8) Optional skin biopsy (5 mm punch) from treatment site and non-treatment site if not 
performed at Day 1 visit. 
9) Venous blood draw 
10) Provide date of Visit 4 and “morning of appointment” instructions 

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Visit 4 (Day 42 +/-5):  
1) Questionnaire to clinically characterize AD, review of medications and possible SE 
2) Clinical score for atopic dermatitis  
3) Take digital pictures of the targeted area 
4) Non-invasive skin barrier Measurements: transepidermal water loss. pH and SC hydration 
measurements may also be performed. 
5) Repeated tape strippings (0, 5, 10,15 tape-strippings with Cuderm™ tape)  
6) Suture removal at biopsy site(s) 
 
Study procedures & methods 
- 
Disease status assessment: 1) Investigator Global Assessment (IGA), which will 
provide information of the overall disease (four point scale: clear, mild, moderate, 
severe) and it is the score requested by FDA for clinical study and 2) Eczema Area and 
Severity Index (EASI), a well-accepted and validated scoring system for general disease 
activityxiv. It is the sum of the intensity scores (0 to 3) for four signs: erythema, induration, 
excoriation, lichenification and it is recorded for targeted treatment areas as well as four 
regions of the body (i.e., head, trunk, lower and upper extremities). Data will be reported 
as percent change in EASI score from baseline and each visit. Proportion of participants 
achieving EASI50 and EASI75 will be determined at each study timepoints. The 
presence or absence of skin irritation (e.g. macular/papular eruption), redness, atrophy 
and telangiectasia will be noted at each study visit.  
3) The pruritus intensity will be assessed using the 5-D Pruritus Scale.  The pruritus 
categorical scale is a 4-point scale commonly used in clinical studies of AD and has less 
of a “middling” effectxv The scale is rated as follows: 0: absence of pruritus; 1: mild, 
pruritus (occasional slight itching/scratching); 2: moderate pruritus (constant or 
intermittent itching/scratching that does not disturb sleep) and 3: severe pruritus 
(bothersome itching/scratching that disturbs sleep).  
- 
Digital pictures: A digital picture will be taken with a dedicated digital camera. The 
pictures will be labeled with the subject’s study ID and date. Pictures will not include 
identifying information (e.g. facial features, tattoo, etc). Pictures will be taken at a 
distance for easy identification of the anatomical area and closer to better capture 
clinical changes. Digital photographs will be used to record the location and monitor the 
size of target lesions.  Quantification of change in size will be obtained using Image J 
software.   
At conclusion of the study, two trained physicians/dermatologists will be asked to score 
clinical pictures (0-4 scale, clear, mild, moderate, severe) in a blind fashion (i.e., pictures 
will be de-identified and presented randomly/ not in chronologic order). This would allow 
to objectively confirm the clinical score taken during the study.   
- 
Skin swab: Skin swabs will be collected for microbiologic analysis to obtain a semi-
quantification of skin bacteria pool, including S. aureus spp. and to determine antibiotic 
sensitivity. Skin swabs are performed by swiping and rolling a sterile cotton-tipped 
applicator 4-5 times across the target area.  Swabs will be obtained from NanoDOX® 
Hydrogel sites pre and post treatment and may be repeated as needed to monitor 

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bacteria growth during study. The microbiology assay will be run by the Clinical 
microbiology laboratory at the University of Florida (Rocky Point Lab). 
- 
Optional Skin biopsy: One skin biopsy sample (5 mm ≈ pencil eraser size) will be 
obtained if possible within 4 cm from the treatment area or on the contralateral side. 
Second biopsy will be obtained from the treatment area post-treatment.  Biopsy will be 
performed after injecting a local anesthetic (Lidocaine +/- epinephrine) to minimize pain 
and bleeding. The biopsy will consist of removing a piece of skin with a sterile 5-mm skin 
biopsy punch. At the biopsy site one or two sutures will be placed to ensure better 
healing, minimize blood loss and to provide a more aesthetic scar formation. A pressure 
bandage will be applied to the biopsy site to minimize the risk of bleeding and infection. 
We will provide each subject with a wound care instruction sheet. 
Each biopsy will be used for analysis of proinflammatory cytokines (TSLP, IL33, TNFa, 
IL-1b, and IL-6) and epidermal barrier related protein (e.g. filaggrin, loricrin, claudin1, 
S100A7) as well as proteases activity (MMPs, elastase) that are typically upregulated in 
inflammatory skin. This will be done in collaboration with Dr. Gregory Schultz (Professor 
of Obstetrics/Gynecology & Ophthalmology at the University of Florida, co-PI in the 
study). It is our hypothesis that NanoDOX® Hydrogel by reducing the proteases activity 
in the epidermis will enhance the integrity of epidermal barrier structure.  Secondarily, 
we will evaluate changes in inflammatory (and pro-inflammatory) pathways downstream 
to proteases activity before and after treatment with NanoDOX® Hydrogel. We expect 
that normalization of molecular pattern parallels clinical improvement (and restore of skin 
barrier integrity). 
- 
Tape stripping for proteases activity measurements: the most superficial layers of 
the skin will be collected by tape stripping using D-squame (D-squame®, CuDerm, 
Dallas TX, USA – Figure 2). This is a well-established method used in clinical research 
to gently perturb the skin barrier. The first tape will be discarded to eliminate dirt and 
remnants of skin products. Each tape will be gently pressed against the skin with 
standardized pressure for 10 seconds (sec) using a standardized pressurizer (D500–D-
squame® pressure instrument, CuDerm, Dallas, TX, USA – Figure 2).  Non-lesional skin 
samples will be collected from the volar side of the forearm and lesional skin samples 
will be collected from an easily accessible area, such as arms or lower legs. 
All subjects will have 20 consecutive tapes collected from lesional skin and 20 
consecutive tapes from non-lesional skin. Once the tapes have been collected, as part of 
this study, we will test different methods to elute the skin proteins from the tapes. 
The corneocytes (cells in the most superficial layer of the skin) or their cellular contents 
that adhere to the discs must be released from the adhesive coating for subsequent 
analytical steps. So far several groups were able to use this non-invasive method to 
analyze for the presence of biomolecules, drugs and other cellular constituents. 
- 
Skin barrier measurements: Transepidermal water loss/TEWL, +/- pH and SC 
hydration measurements will be obtained on treatment area at a non-lesional site and on 
a site of inflammation. For each of the specific measurements, a probe will be gently 
applied on the skin surface for few (up to 30) seconds. We will perform all 
measurements in a climate-controlled examination room with temperatures ranging 20-
22°C and consistent humidity (preferably 40%).  The patient must be very comfortable in 

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the room in which they are being tested to ensure that perspiration due to stress or 
acclimatization does not alter the TEWL. TEWL will be measured at baseline and after 
tapestripping. SC hydration and surface pH will be assessed on non-lesional and 
lesional skin using the Corneometer® CM825 and Skin-pH-Meter® PH 905, respectively 
(Courage and Khazaka Electronic GmbH, Kohn GE – Figure 1). TEWL will be assessed 
using the AquaFlux (Biox, London UK). These measurements should provide data to 
determine if the treatment has an effect on skin barrier integrity. 
- 
Venous blood sample: (4 ml) will be obtained to evaluate biomarkers of disease 
severity. 4mLs will be collected into a redtop serum blood collection tube and allowed to 
clot at room temperature for minimum of 30 minutes. Blood will be spanned down and 
serum pipetted into Eppendorf tubes to be stored in -80°C. Tubes will be labeled with 
study subject ID, data and Visit number, not subject identifying information Tubes will be 
stored in Dr. De Benedetto’s laboratory for measurement of Th2 biomarkers (markers of 
disease severity), including serum total IgE and TARC/CCL17. 
 
4. RISK, BENEFITS and PROTECTION AGAINST RISKS 
 
Risks Associated with Digital Picture 
There are no known risks for the digital picture. The image will not include identifying features 
and will be labeled with study subject ID and date. Pictures will be taken with a dedicated 
camera, stored on a secure server and transferred into RedCap database. 
Risks Associated with Barrier Assessments 
There are no known risks for the barrier assessments. 
Risks Associated with Tape Stripping 
The risks associated with tape stripping, theoretically, include the rare possibility of an allergic 
reaction to the tape and infection.  Since the tape is removed immediately after application, the 
risk of reaction is extremely low.  We will exclude study subjects with known allergy to tapes or 
adhesive material. In previous and ongoing studies involving tape stripping, it has been noted 
that a very mild erythema may develop immediately after a series of tape strippings on one 
localized area of skin, presumably due to the mild mechanical disturbance.  The erythema is 
expected to resolve within 12 hours without sequelae.  The risk of skin infection is extremely low 
since only superficial skin layers are removed.  Subjects will be provided with wound care 
information and a department phone number to contact should they have any concerns.  
Risks of Phlebotomy 
The risks of having blood drawn include some pain when the needle goes in, and a small risk of 
bruising and/or infection at that site. Some people will get lightheaded, nauseous, or faint. 4mLs 
is approximately 1 teaspoon of blood and is not likely to cause lightheadedness, however 
subjects will be observed after blood draw to be sure they are comfortable with deambulation. 
Subjects will be encouraged to drink plenty of water prior to the blood draw. Phlebotomy will be 
performed at the antecubital fossa to minimize pain and bruising. Subjects will be seated during 

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the blood draw to minimize dizziness and any risk of falling. Pressure will be applied to the site 
after the draw and a band-aid will be applied to minimize the risk of bleeding and infection. 
 
 
Risks of Punch Biopsy 
The biopsies will be no larger than 5 mm in diameter (about a pencil eraser head). A 
nonabsorbable suture (1-2 stiches) will be placed in each site for hemostasis and enhanced 
wound healing. A small scar will remain at the site. The scar will be smaller than the initial 
biopsy but may also be hypo- or hyperpigmented. Sutures will be removed in 14 (+/- 2) days. 
The risks of this procedure include adverse reaction to anesthesia (lidocaine), pain, bleeding, 
infection and hypertrophic scars or keloids. Patients with a history of Novocain or lidocaine 
allergy or a history of keloid formation will not be allowed to participate in order to minimize the 
risk of adverse reactions. Biopsy will be performed after injecting a local anesthetic to minimize 
pain. A pressure bandage will be applied to the biopsy site to minimize the risk of bleeding and 
infection. Written wound care instruction will be provided to the subjects after the procedure. In 
case of increased redness, pain or oozing of the sites subjects will be instructed to call the 
dermatology department if appropriate subjects will be asked to come to clinic in the next 24-48 
hours for wound check. 
 
Risks of NANODOXY® 1% hydrogel 
Subjects may experience local irritation including redness, pain, stinging or burning sensation in 
the treatment area. Systemic doxycycline can enhance sensibility to the sun light. It is possible 
that topical application might temporarily enhance risk of sunburn in the treated site. To 
minimize risk of sunburn we will instruct the subjects to apply the topical at bedtime and rinse off 
in the morning. In case of sun exposure subjects should use appropriate sun protection (SPF 
>30 or clothing). Some patients may experience some irritation from the NANODOX® hydrogel 
especially in areas of open skin from scratching. Some patients may experience increased 
dryness from the hydrogel formulation.  All patients will be advised to moisturize in the morning 
and use gentle body wash to minimize these potential side effect.  
 
As for any antibiotic, there might be a risk of inducing antibiotic resistance. To minimize the risk 
the drug will be used only until the lesion is active/visible. Also we will be monitoring for bacteria 
growth during the study to determine possible change in bacteria contamination as well as 
antibiotic resistance. 
  
Preclinical studies have shown that over time (12-24 hours) the color of leftover products in the 
package might change from a white transparent tone to a more yellow-brown color. Change in 
color has not been associated with change in efficacy. To minimize risk of skin discoloration we 
will advise the subjects to rinse off the site in the morning.  
 
Subjects with sensitivity to doxycycline might develop an allergic drug reaction, which could vary 
from local irritation to more severe systemic reaction. Subjects with sensitivity/allergy to 

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tetracycline will not be enrolled in the study.  
 
In case of any abnormal reaction we will instruct the subjects to call the dermatology office and 
stop using NANODOX® until discussing symptoms with study team.  
 
Risks to children or pets are not known and subjects will be instructed to keep the products out 
of reach from children and pets. Because this is an investigational medication/formulation some 
side effect may not yet be known. Any adverse reaction will be accounted. 
 
Based on preliminary data, we do not anticipate any complications associated with the use of 
topical doxycycline.  
 
Common skin reactions with taking oral 
doxycycline include:  
• 
Redness  
• 
Swelling  
• 
Blistering rash  
 
Common allergic reactions with taking oral 
doxycycline include:  
• 
Hives  
• 
Shortness of breath  
• 
Swelling of the face, lip, tongue, or     
         throat  
 
Common systemic reactions with taking oral 
doxycycline include:  
• 
Headache  
• 
Dizziness  
• 
Fever  
• 
Chills  
• 
Rash  
• 
Nausea  
• 
Vomiting  
• 
Diarrhea  
• 
Thrush  
• 
Vaginitis  
If a subject experiences an unexpected reaction to the investigational product, it should be 
recorded as an AE or SAE.  
 
The study agent (NanoDOX™ Hydrogel) consists of doxycycline gel 1% w/w.  The 1% w/w 
doxycycline formulation of NanoDOX™ Hydrogel contains:  1% w/w doxycycline monohydrate in 
carboxymethylcellulose (CMC), a hydrogel.  CMC functions as the vehicle for the doxycycline 
active ingredient and assists in providing a moist wound environment.  The primary function of 
doxycycline in NanoDOX™ Hydrogel is to promote wound healing by harnessing doxycycline's 
inhibition of matrix metalloproteinase (MMPs) and TNF-a converting enzyme (TACE).  This 
property of doxycycline is hypothesized as the mechanism of action of NanoDOX™ Hydrogel for 
enhanced wound-healing. 
 
Alchem will prepare, package and label the investigative material in easy-to-open 1.5 gram 
disposable packets.  Each packet of doxycycline gel will have a two-part tear-off label.  The gel 
should be refrigerated between applications.  
 
Bench Testing  
Systemic absorption of the doxycycline component in NanoDOX® Hydrogel through the skin 
was evaluated by Nanotherapeutics.  Briefly, Franz diffusion cells were used to analyze the 
amount of doxycycline released out of a NanoDOX® Hydrogel or particle suspension across an 
exact area with respect to time.  These conditions may be used to mimic an exposed wound 
and the resulting drug release out of the NanoDOX® Hydrogel into the body. Overall, less than 
1% w/w of the 15 mg total doxycycline available in the donor chamber in the gel (11.18µg, or 
0.08%) as well as the suspension (29.88µg, or 0.21% w/w) diffused across the membrane into 
the receptor chamber. 

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Animal Testing  
In a rat wound healing study, NanoDOX® Hydrogel was tested at doxycycline concentrations of 
0.0% (placebo hydrogel), 0.3%, 1.0%, and 3.0% w/w as well as untreated control. The results of 
the study are:  
a) No significant differences between the histological scoring for all concentrations and the 
control; and,  
b) All concentrations were judged to be non-irritants versus the control.  
 
Details relating to additional in vivo and in vitro nonclinical pharmacology and nonclinical 
toxicology studies of NanoDOX® Hydrogel can be found in the Investigator’s Brochure. 
Reproductive Risks 
The effects of topical doxycycline on an unborn fetus or nursing (breast feeding) infant are 
unknown. It is possible that use of NANODOX® hydrogel may be associated with unanticipated 
risks to a pregnancy or fetus. Women of child bearing potential will be counseled to use 
acceptable birth control methods. Women will not be allowed to participate in this study if they 
are pregnant, become pregnant, or are nursing an infant. 
 
Property damage risks 
Objects in contact with NANODOX® may have discoloration (e.g. bedding, towels, clothes). 
Subjects are advised to be careful handling the opened NANODOX® packages and to rinse the 
excess of product from their skin if the observe change in color to minimize the likelihood of 
stain on towels, rugs or other clothing.  
General Risks 
When participating in any research study there is a possibility for invasion of privacy or breach 
of confidentiality.  
Adverse Event Reporting 
An adverse event (AE) is any observation whether or not considered to be product related, that 
is unfavorable and unintended and that occurs after any use of an investigational medicinal 
product. 
Any observation of abnormality will be documented on the Adverse event form.  The 
Investigator will document the date of occurrence, severity, resolution, and will assess the 
relationship of the adverse event to the study drug by assigning a causality assessment. 
Results from laboratory evaluations which are outside of the laboratory reference range will 
constitute an adverse event only if the Investigator determines that the result is clinically 
significant.  
Severity will be assessed by the following system:  
• 
Mild: easily tolerated, causing minimal discomfort  
• 
Moderate: sufficiently discomforting to interfere with every day activities  
• 
Severe: prevents normal every day activities 

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Serious Adverse Events 
FDA definition: A serious adverse event results in death, is life-threatening, requires inpatient 
hospitalization or prolongation of existing hospitalization, results in persistent or significant 
disability / incapacity, results in a congenital anomaly / birth defect. Events fitting these 
descriptions will be considered adverse events if they occur after treatment with the study drug. 
As soon as reasonably possible, subjects will contact the Investigator to report any unfavorable 
events. The Investigator will assess the case, determine the need for treatment, and consult 
with Alchem Laboratory, Inc, particularly in the occurrence of: 
 
A serious adverse event. 
 
An unusual frequency of non-serious adverse events. 
 
Death, even if this does not appear to be related to therapy with the study drug.  
The Investigator will notify the sponsor (Alchem Laboratory, Inc and CTSI) and the UF 
Institutional Review Board within approximately 3 working days if a serious adverse event 
occurs.  
Causality assessments 
 
Probable 
              All of the following apply: 
 
Reasonable association in time between the administration of the study drug and 
onset and duration of the reported event. 
 
Description of the clinical phenomenon should be consistent with, or at least 
plausible, given the known pharmacology and toxicology of the product. 
 
There should be no other equally plausible explanation(s) of the case. In particular, 
concurrent use of other products (and possible interactions) or concurrent disease 
should be taken into account in the assessment. 
Possible 
 
The causality is one (of other) possible and plausible causes for the described 
event, but the data do not meet the criteria for inclusion in Category A. 
Unknown 
 
An association with the study drug cannot be discounted, but other factors prevent 
a conclusion from being drawn. 
Unlikely 
 
Sufficient information exists to establish beyond reasonable doubt that the product 
was not likely to be the cause of the event. 
Classification of adverse events 
At the conclusion of the study, Medicinal Dictionary for Drug Regulatory Authorities (MeDDRA) 
terms will be assigned to all adverse events and will be included in the final study report.  
Stopping Criteria 
If more than 5 patients report a greater than moderate local side effect (irritation, worsening of 
rash, etc) or if the clinical dermatological score in the target lesion area as assessed by the 
clinician has worsened by more than 50% from baseline in more than 5 patients, then all 

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patients will stop medication until further review and changes made to the protocol.  If any 
unanticipated serious adverse effects are noted all subjects will be notified and changes will be 
made to future protocols and submitted for approval. 
5. DATA ANALYSIS AND DATA MONITORING 
Primary Outcomes:  
1. Assessment of safety and tolerability of NanoDOX® Hydrogel in AD subjects on lesional and 
non-lesional (clinically normal appearing) skin, including pain and dermatologic/skin toxicity 
(incidence, severity, frequency, duration and causality) in lesional skin at target site 
(NanoDOX® Hydrogel). 2. Improvements rate of IGA and EASI score in lesional skin treated 
with NanoDOX® Hydrogel before and after treatment. We will quantify clinical improvement as: 
IGA (0-4) fold improvement from baseline; percent of subjects reaching EASI50 (50% 
improvement over baseline) or EASI75 (75% improvement over baseline) at each time point 
post treatment (V2 and V4). 3. Objective Clinical improvement based on digital photography 
scoring (0-4 scale and size of active lesion), measured as fold improvement over baseline at 
each time point (V2 and V4). 
Secondary Outcomes:  
1. Improvements of itch score (0-5 scale) in lesional skin, before and after treatment at each 
time point. 2. Will evaluate Staphylococcal Aureus rate of colonization (positive or negative), 
difference in number of growth (0 to 3+++) and antibiotic sensitivity pre and post treatment.  
Doxycycline could have an effect on AD lesions just by reducing the Staphylococcal Aureus 
colonization. By integrating these findings with clinical outcomes, drug penetration and 
molecular changes we will be able to start to address this question. 3. Improvement of barrier 
function in non-lesional (normal appearing) and lesional skin pre and post treatment (V1 to V3). 
We will expect that clinical improvement parallels with improvement of barrier function, such as 
reduced TEWL, increased stratum corneum hydration and reduced pH in active treatment 
areas. On the other hand, worsening of the measurement will suggest damaging effect of the 
treatment on the skin, which could be indication of subclinical irritation induced by the treatment 
and/or vehicle. 
The purpose of this study is a proof of concept.  We are adopting a similar strategy to Phase II 
cancer trials that seek a clinically significant response.   Using exact binomial methods, we shall 
obtain point and interval estimates for the fraction of subjects who respond as (a) At least EASI 
50% by visit 2 or 4 and (b) At least EASI 75% by visit 2 or 4. We shall also tabulate the IGA 
score from baseline to visit 4.    We would consider 6 or more responses (a) in 15 subjects as 
promising.  The probability of declaring a promising result when the true response rate is 60% or 
higher is over 96% (power). 
 
Side effects and baseline characteristics will be tabulated qualitatively.  The blinded pre-vs post 
evaluation will be analyzed for IGA scores by the Wilcoxon sign-rank test.    
 
Data Storage and Confidentiality 

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All data from the clinical trial will be securely stored using the REDCap. We have already 
obtained funding to work with CTSI to build the REDCap database for the study. 
Only the minimal necessary data is being collected.  This includes: Name, contact information, 
age, sex, MRN, history of allergies, dermatologic diagnosis, history of other medical conditions, 
current medications and clinical scores.  This information will be stored in REDcap.   
Samples will be assigned unique identifiers and stored separately from the key linking the 
sample to the identifiers.  The electronic key will be stored in REDcap file, accessible only to 
study personnel.  Any paper documents containing identifiers will be kept in a binder in a locked 
cabinet accessible only to study personnel.   
 
 
Data Monitoring 
 
The study will be monitored in compliance with the relevant parts of 21 CFR and according to 
the ICH GCP Guidelines. 
 
A Monitor will visit the Study Center on a pre-arranged schedule to monitor adherence to the 
protocol and to applicable FDA regulations; and the maintenance of adequate and accurate 
clinical records.  A Case Report Form will be completed for every subject that was enrolled to 
participate in the Study.  The Case Report Form (CRF) will be reviewed in detail, for which the 
Monitor will have access to subject study records, laboratory data, and other source 
documentation.   
 
Case Report Forms will be completed as information becomes available or within five days of a 
Study Visit. 
 
Case Report Forms will be reviewed in detail by the Monitor who will make a decision as to their 
acceptability.  If errors or omissions are found in the course of a data audit, or if clarification of 
data is required, the Case Report Form(s) in question will be corrected by the Investigator or 
his/her designee.  Data Resolution or Query Forms may be generated on omissions or 
clarifications, to be completed, signed and dated, and maintained as a part of the CRF. 
 
The Principal Investigator or a Sub-investigator will sign and date the indicated places of the 
Case Report Form.  This signature will indicate that thorough inspection of the data therein has 
been made and will thereby certify the contents of the form.   
 
The procedures outlined in the protocol and case report forms will be carefully reviewed by the 
Sponsor, Investigator and staff prior to Study Initiation to ensure appropriate interpretation and 
implementation.  No deviations from the protocol shall be made except in emergency situations 
where alternative treatment is necessary for the protection, proper care and well being of 
subjects. 
 

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Amendments will be submitted to the IRB for their review and approval prior to implementation.  
When an amendment to a protocol substantially alters the study design or increases potential 
risk to the study subject, the Informed Consent form will be revised and if applicable, subject’s 
consent to continue participation will again be obtained. 
 
5. SUBJECTS IDENTIFICATION, RECRUITMENT AND CONSENT 
Method of subject identification and recruitment 
Dr. Anna De Benedetto will identify potential subjects from the CTSI consent2share cohort, UF 
dermatology clinics, the IRB-approved Dermatology Research database (IRB201600411) or 
from advertisements (UF website, fliers and newspaper advertisements) directed at the general 
population. In the case of clinical subjects, Dr. De Benedetto will provide the subject with a 
contact phone number for the study coordinator who is approved to enroll and consent subjects. 
While Dr. De Benedetto will be available for questions, consent might be obtained by other 
study personnel for subjects she sees in her clinical practice. It will be made clear to the 
potential subjects that their participation is voluntary and their care at the Department of 
Dermatology will not be affected by their decision to participate in this study. 
 
Process of consent  
Dr. De Benedetto or other team members (e.g. coordinator, nurse, co-investigator) will be 
meeting directly with the subjects in a private suite. The consent form will be discussed with the 
potential subject. Time allotted to consenting will be dependent on the subjects’ needs, 
questions, and comprehension of the information in the consent form.  Each subject will be 
encouraged to read the consent form on his/her own and ask any specific questions. At the time 
of consent, subjects will be offered the option to provide permission to be contacted in the future 
regarding this and other dermatologic studies.  Those who provide permission for future contact 
will be added to the IRB201600411 approved database. 
Subject capacity  
We will not be recruiting subjects incapable of giving informed consent. 
Subject/representative comprehension  
The subject’s capacity to comprehend the information in the consent form will be assessed by 
asking questions about the content and encouraging the subject to describe briefly the study 
procedures. 
Documentation of consent  
The consent will be documented by signing the consent form by both the study subject and the 
person obtaining consent. A copy of the consent form will be provided to the subject. 
Costs to the subject  
Procedures performed during the study will be at no cost to the subject. 
Payment for participation  
Subjects that agree to participate in the study will receive compensation for their time:  $25.00 

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for the screening visit.  Each study visit will be $45.00 and an additional $45.00 for the optional 
biopsy. The Final visit (Follow up Visit) will also be $25.00. For a total of up to $275.00 including 
the 2 biopsies or $185.00 without the biopsy if all visits are completed.  UF VISA cards will be 
used for the reimbursement. Payments will be issued to VISA card within 7 business days of 
completed study visit. 
 
Potential benefits to the subjects  
There are no benefits for the individual participant. The investigational medication may 
temporally improve or worsen the lesion in the target area of application. The nature of this pilot 
study does not promise benefit for the individual. We hope that this research will lead to a better 
understanding of atopic dermatitis pathogenesis and pave the way for development of topical 
medications that aim to repair skin barrier and skin driven inflammation.  
 
 
Figure 1: Multi probe System MPA5 – Courage & Khazaka (Germany) and Aquaflux (Biox, 
London UK).  
 
 
 
Figure 2: D500–D-squame® pressure instrument and D-squame® - CuDerm (Dallas, TX) 
 
 
                                                          
i NEJM 2008 Apr 3;358(14):1483-94 
ii JAAD 2002 Mar;46(3):361-70 

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iii JID 2009 Aug;129(8):1892-908 
iv J Clin Invest. 2012 Feb;122(2):440-7 
v J Eur Acad Dermatol Venereol 2013;27:239-42 
vi BJD 2006; 154(4):719-25 
vii Lancet 2012;380:2197 
 
viii Wounds, 15:315-323, 2003 
ix Biol Res Nurs. 2010 Apr;11(4):336-44 
x JID 2012;132:1435 
xi J. Periodontology 2015;2:10 
xii Arch Dermatol. 1994 Nov;130(11):1402-7 
xiii Br J Dermatol, 2011. 165(2): p. 329-34 
xiv Br J Dermatol. 2015 Aug;173(2):316-7. 
xv Allergy, 2006. 61(3): p. 375-81.