Clinical trial NCT05736224
Conditions: Skin Cancer
Sponsor: Yale University
Start date: 2023-05-15; Completion date: 2023-09-01
Enrollment: 30 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The primary objective of this study is to evaluate the effects of a novel sunscreen formulation by assessing the extent of ultraviolet radiation (UVR)-induced direct and indirect cellular and DNA damage to human skin, in the presence vs absence of the sunscreen, in a population of healthy adults with fair skin (Fitzpatrick Scale type I, II or III).

Detailed description:
Skin cancer is the most commonly diagnosed malignancy in the USA and ultraviolet radiation (UVR) exposure is the major environmental risk factor for skin cancer development. Currently available sunscreens utilize UVR filters that, while absorbing UVR energy, have been shown to induce ROS, resulting in oxidative DNA damage after UVR exposure. Organic sunscreen actives have also been shown to penetrate into the skin, raising direct toxicity, as well as irritant and photoallergic concerns. Further systemic absorption may result in additional health risks such as endocrine disruption. Novel sunscreens that more safely prevent both direct and indirect DNA damage are needed.

The study team have produced a bioadhesive nanoparticle (BNP) sunscreen designed to keep organic UVR filters from penetrating into the skin and have incorporated non-toxic natural products into this sunscreen to further safely boost UVR absorbing capacity and reduce oxidative, indirect DNA damage. This study will test the capacity of this sunscreen to prevent direct and indirect cellular and DNA damage in human skin exposed to UVR.

Arms: arm 1: Portion of skin covered by sunscreen. There are 3 different formulations of sunscreen (Formulation 1, Formulation 2, Formulation 3) arm 2: Portion of skin not covered by sunscreen.
Interventions: intervention 1: Sunscreen intervention 2: UV Light
Intervention descriptions: intervention 1: The sunscreen contains bioadhesive nanoparticles (BNP) encapsulating avobenzone and octocrylene plus the non-toxic natural products diosmin, ferulic acid, cytisine and trans-resveratrol. intervention 2: UV light to the correct sites, and the Multiport 610 solar simulator used to deliver 1 MED UVR to the appropriate subsites.

Protocol documents:
Prot_SAP_001.pdf: 
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CLINICAL STUDY PROTOCOL 
Interventional Drug or Biologic 
 
 
 
 
Assessing a Natural Product plus 
Bioadhesive Nanoparticle (BNP) 
Sunscreen 
Protocol Number 
200033876 
 
 
 
Protocol Version 
February 1, 2023 
Version 4 
PRINCIPAL INVESTIGATORS: 
Name: Michael Girardi, MD / Mark Salzman, PhD 
Department: Dermatology / Biomedical Engineering 
Telephone Number: 203 785 7432 
Email Address: girard@yale.edu 
 
 
IND Number: N/A 
IND Holder: N/A 
NCT Number: NCT05736224 
Study Phase (1, 2, 3, or 4-postmarketing): Phase 1 


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Include the IRB approved protocol version number and date for each revision of the protocol. All 
version history should remain in the table and never be deleted. The oldest IRB approved version of 
the protocol should be listed on the top row. The most recent IRB approved version should be listed 
on the bottom row. 
 
 
REVISION HISTORY: 
 
 
Revision # 
Version Date 
V1 
10/11/2022 
1 (V2) 
11/30/2022 
2 (V3) 
12/20/2022 
3 (V4) 
02/01/2023 
 
 
 
 


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Synopsis 
 
Primary Objective 
The primary objective of this study is to evaluate the effects of a novel sunscreen formulation 
by assessing the extent of UVR-induced direct and indirect cellular and DNA damage to human 
skin, in the presence vs absence of the sunscreen, in a population of healthy adults with fair 
skin (Fitzpatrick Scale type I, II or III). 
Secondary Objective (if applicable) 
N/A 
Study Duration 
9 months 
Study Design 
This is a phase 1, non-randomized pilot study designed to test the effectiveness of a sunscreen 
compared to a no treatment control. 
Number of Study Sites 
There will be one study site. Studies will be completed at the 4th floor of the Hunter Radiation 
Therapy (HRT – 4th floor) building, in the examination room of the YNHH Photopheresis Unit. 
Study Population Healthy Caucasian adults 
Healthy adult volunteers with fair skin. Individuals with fair skin are more susceptible to the 
UVR-induced DNA damage that we seek to prevent by development of a safer, more effective 
sunscreen, and thus will comprise the study population. 
Number of Participants 
30 
Primary Outcome Variables 
Biopsy tissue will be used to assess the effectiveness of the sunscreen by measuring the level 
of DNA and cellular damage in UVR-exposed vs unexposed, sunscreen treated vs untreated 
samples by measuring DNA mutation inducing cyclobutene pyrimidine dimers (CPD), DNA 
strand breaks (γH2AX), and cellular protein damage (3-nitrotyrosine) as biomarkers of 
response. 
Secondary and Exploratory Outcome Variables (if applicable) 
N/A 


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Explanation 
Abbreviation 
 
Explanation 
Glossary 
 
 
Abbreviations 
 
AVO 
avobenzone 
BNP 
bioadhesive nanoparticle 
BNP-A/O 
 bioadhesive nanoparticle encapsulating avobenzone and octocrylene 
CPD 
cyclobutane pyrimidine dimer 
DNA 
deoxyribonucleic acid 
FDA 
United States Food and Drug Administration 
gH2AX 
gamma (phosphorylated) histone H2AX 
GRASE 
generally regarded as safe and effective 
HPG 
hyperbranched polyglycerol 
hr 
hour 
MED 
minimal erythema dose 
mg 
milligram 
OCTR 
octocrylene 
PLA 
poly (lactic acid) 
PLA-HPG 
 poly (lactic acid)-hyperbranched polyglycerol 
PO 
padimate O 
PO/BNP 
bioadhesive nanoparticles encapsulating padimate O 
ROS 
reactive oxygen species 
SPF 
sun protection factor 
USP 
United States Pharmacopeia 
UVA 
ultraviolet A 
UVB 
ultraviolet B 
UVR 
ultraviolet radiation 
Glossary of Terms 
 


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Table of Contents 
Synopsis ................................................................................................................................... 3 
Primary Objective ................................................................. Error! Bookmark not defined. 
Secondary Objectives (if applicable) .................................... Error! Bookmark not defined. 
Study Duration ...................................................................... Error! Bookmark not defined. 
Study Design ........................................................................ Error! Bookmark not defined. 
Study Population .................................................................. Error! Bookmark not defined. 
Number of Participants ......................................................... Error! Bookmark not defined. 
Number of Study Sites .......................................................... Error! Bookmark not defined. 
Primary Outcome Variables .................................................. Error! Bookmark not defined. 
Secondary and Exploratory Outcome Variables (if applicable)Error! Bookmark not 
defined. 
Visit Schedule Table (Optional) ............................................ Error! Bookmark not defined. 
Study Flow Chart (optional) .................................................. Error! Bookmark not defined. 
Abbreviations ........................................................................................................................... 4 
Glossary of Terms .................................................................................................................... 4 
1 
Introduction ....................................................................................................................... 9 
1.1 
Introductory Statement .............................................................................................. 9 
2 
Background ..................................................................................................................... 10 
2.1.1 
Preclinical Experience ...................................................................................... 10 
2.1.2 
Clinical Experience ........................................................................................... 15 
2.2 
Background/prevalence of research topic ................................................................ 16 
3 
Rationale/Significance ..................................................................................................... 17 
3.1 
Problem Statement .................................................................................................. 17 
3.2 
Purpose of Study/Potential Impact ........................................................................... 17 
3.2.1 
Potential Risks .................................................................................................. 18 
3.2.2 
Potential Benefits .............................................................................................. 18 
4 
Study Objectives ............................................................................................................. 19 
4.1 
Hypothesis ............................................................................................................... 19 
4.2 
Primary Objective .................................................................................................... 19 
4.3 
Secondary Objectives (if applicable) ........................................................................ 19 
5 
Study Design .................................................................................................................. 20 


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5.1 
General Design Description ..................................................................................... 20 
5.1.1 
Study Date Range and Duration ....................................................................... 21 
5.1.2 
Number of Study Sites ...................................................................................... 21 
5.2 
Outcome Variables .................................................................................................. 21 
5.2.1 
Primary Outcome Variables .............................................................................. 22 
5.2.2 
Secondary and Exploratory Outcome Variables (if applicable) ........................ 22 
5.3 
Study Population ...................................................................................................... 22 
5.3.1 
Number of Participants ..................................................................................... 23 
5.3.2 
Eligibility Criteria/Vulnerable Populations ......................................................... 23 
6 
Methods .......................................................................................................................... 24 
6.1 
Treatment - Drug ..................................................................................................... 24 
6.1.1 
Identity of Investigational Product/New Drug .................................................... 24 
6.1.2 
Dosage, Admin, Schedule (if applicable) .......................................................... 25 
6.1.3 
Method of Assignment/Randomization (if applicable) ....................................... 26 
6.1.4 
Blinding and Procedures for Unblinding (if applicable) ..................................... 26 
6.1.5 
Packaging/Labelling .......................................................................................... 26 
6.1.6 
Storage Conditions ........................................................................................... 27 
6.1.7 
Concomitant therapy ........................................................................................ 27 
6.1.8 
Restrictions ....................................................................................................... 27 
6.2 
Assessments ........................................................................................................... 27 
6.2.1 
Efficacy ............................................................................................................. 27 
6.2.2 
Safety/Pregnancy-related policy ....................................................................... 27 
6.2.3 
Adverse Events Definition and Reporting ......................................................... 28 
6.2.4 
Pharmacokinetics (if applicable) ....................................................................... 30 
6.2.5 
Biomarkers (if applicable) ................................................................................. 30 
6.3 
Study Procedures .................................................................................................... 30 
6.3.1 
Study Schedule ................................................................................................ 31 
6.3.2 
Informed Consent ............................................................................................. 32 
6.3.3 
Screening ......................................................................................................... 32 
6.3.4 
Recruitment, Enrollment and Retention ............................................................ 33 
6.3.5 
On Study Visits ................................................................................................. 33 
6.3.6 
End of Study and Follow-up ............................................................................. 33 


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6.3.7 
Removal of subjects ......................................................................................... 33 
6.4 
Statistical Method .................................................................................................... 33 
6.4.1 
Statistical Design .............................................................................................. 33 
6.4.2 
Sample Size Considerations ............................................................................. 33 
6.5 
Planned Analyses .................................................................................................... 34 
6.5.1 
Primary Analyses .............................................................................................. 34 
6.5.2 
Secondary Objectives Analyses ....................................................................... 35 
6.5.3 
Safety/Pregnancy-related policy ....................................................................... 35 
6.5.4 
Analysis of Subject Characteristics ................................................................... 35 
6.5.5 
Interim Analysis (if applicable) .......................................................................... 35 
6.5.6 
Health economic evaluation ............................................................................. 35 
6.5.7 
Other ................................................................................................................ 35 
6.5.8 
Subsets and Covariates ................................................................................... 35 
6.5.9 
Handling of Missing Data .................................................................................. 35 
7 
Trial Administration ......................................................................................................... 36 
7.1 
Ethical Considerations: Informed Consent/Assent and HIPAA Authorization .......... 36 
7.2 
Institutional Review Board (IRB) Review ................................................................. 36 
7.3 
Subject Confidentiality ............................................................................................. 37 
7.4 
Deviations/Unanticipated Problems ......................................................................... 37 
7.5 
Data Collection ........................................................................................................ 37 
7.6 
Data Quality Assurance ........................................................................................... 37 
7.7 
Study Records ......................................................................................................... 37 
7.8 
Access to Source ..................................................................................................... 38 
7.9 
Data or Specimen Storage/Security ......................................................................... 38 
7.10 Retention of Records ............................................................................................... 38 
7.11 Study Monitoring ...................................................................................................... 38 
7.12 Data Safety Monitoring Plan .................................................................................... 38 
7.13 Study Modification ................................................................................................... 39 
7.14 Study Discontinuation .............................................................................................. 39 
7.15 Study Completion .................................................................................................... 40 
7.16 Conflict of Interest Policy ......................................................................................... 40 
7.17 Funding Source ....................................................................................................... 40 


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7.18 Publication Plan ....................................................................................................... 40 
8 
Appendices ....................................................................... Error! Bookmark not defined. 
9 
List of Tables .................................................................... Error! Bookmark not defined. 


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1 Introduction 
1.1 
Introductory Statement 
This document is a protocol for a human research study. The purpose of this protocol is to 
ensure that this study is to be conducted according to ICH GCP guidelines, and according to 
CFR 21 Part 312, other applicable government regulations and Institutional research policies 
and procedures. 
We have developed a novel bioadhesive nanoparticle technology that aims to make 
sunscreens safer and longer lasting by encapsulating UVR sunscreen active agents in non- 
toxic nanoparticles (1, 2). We have strong preclinical and clinical data demonstrating the 
potential for improved safety, long-duration of retention, and increased effectiveness of 
bioadhesive nanoparticles (BNPs) loaded with the over-the-counter, FDA GRASE chemical 
filters avobenzone (AVO) and octocrylene (OCTR). We have additionally identified four 
naturally occurring non-toxic products (diosmin, ferulic acid, cytisine, trans-resveratrol) that, 
in our preclinical studies, boost the UVR-absorbing capacity of our BNP sunscreen while 
also reducing the damaging effects of UVR-induced reactive oxygen species (ROS), to more 
efficiently prevent UVR-induced cellular and DNA damage. Herein, we propose to test the 
capacity of our combined natural product plus BNP sunscreen to prevent UV-induced 
cellular and DNA damage in human skin. 


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2 Background 
2.1.1 Preclinical Experience 
Nanoparticle encapsulation, used widely in the field of drug delivery, has demonstrated 
unique advantages including target specificity and toxicity reduction. Our initial preclinical 
studies demonstrated that the encapsulation of a model UVR filter, padimate O (PO), in 
bioadhesive nanoparticles (BNPs) prevents epidermal cellular exposure to UVR filters while 
enhancing UVR protection (1). We have performed additional studies demonstrating that the 
BNP platform can also enhance the broad-spectrum protection of UVR filters like 
avobenzone (AVO) and octocrylene (OCTR). The use of this technology confers bioadhesive 
characteristics that facilitate tight adherence to the stratum corneum without subsequent 
intra-epidermal or follicular penetration. As a result, our innovative sunscreen is not only 
persistently adherent and non-penetrant, but also highly protective against primary UVR 
damage and secondary ROS toxicity. 
 
 
We successfully prepared nanoparticles using a 
biocompatible block copolymer called poly (lactic 
acid)-hyperbranched polyglycerol (PLA-HPG). These 
particles have a bioadhesive coating which is 
tunable for specific topical applications. Additionally, 
the properties of the coating allow the BNPs to be 
water-soluble while enabling the suspension of oil- 
based UVR filters, thereby simplifying the 
formulation process. We developed a method to load 
different bioactive agents into the 
particle (Fig. 1). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Fig. 2. Histology of dorsal mouse skin sections receiving different 
topical interventions three days after high dose UVB exposure 
(2160 J/m2) Topical interventions included (A-B) normal skin 
without UV exposure, (C-D) sunscreen, (E-F) PO/BNPs, (G-H) 
blank BNPs, (I-J) no protection. (K) Epidermal thickness and (L) 
percent area of keratin within the dorsal skin after receiving 
topical interventions and UV irradiation. The scale bar represents 
We demonstrated that a commercial 
UVR-filter, padimate O (PO), can be 
loaded into BNPs and retain a 
comparable or higher anti-UVR 
efficiency compared to commercial 
sunscreen. We compared a commercial 
sunscreen and PO loaded BNPs 
(PO/BNPs) on mouse skin and 
exposed the mice to a high dose of 
UVB. With the protection of PO/BNPs, 
the mouse skin showed no sign of 
sunburn by visual measurement and 
histopathologic staining (Fig. 2). On the 
molecular level, PO/BNPs exhibited a 
 
 
 
VERSITY IRB 2/21/2023 


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high efficiency in preventing the formation of cyclobutane pyrimidine dimers (CPD), a marker 
for sunburn, which is comparable to the control sunscreen (data not shown). In all 
experiments, we used a 20-fold lower dose of UVR-filter in PO/BNPs compared to the control 
sunscreen, indicating a higher anti-UVR efficiency of PO/BNPs than commercial sunscreen 
control. 
We showed that the PO/BNPs have no penetration into epidermis. We demonstrated that the 
BNPs themselves are non-penetrating; after applying the particles on pig skin, the BNPs 
showed substantially high retention, while no penetration of BNPs was observed within pig 
skin or within follicles (data not shown). When comparing the penetration of free PO and PO- 
encapsulated BNPs, we found that 6 hours after application on pig skin (a standard mimic for 
human skin), free PO led to a clear accumulation of the UVR filter in the skin, while the skin 
treated with BNP-based sunscreen showed no accumulation of PO (data not shown). In 
addition, the release of PO from BNP was minimal over a 24-hour period, minimizing the 
possible penetration risk of PO that has been released from the particles. We also 
demonstrated the non-penetrating property of the BNP-based sunscreen at the molecular 
level. Free UVR-filters present in the epidermis and dermis can produce reactive oxidation 
species and cause DNA double strand breaks. In our study, we applied both commercial 
sunscreen controls and PO/BNPs on mouse skin and treated with UVR irradiation. The 
samples protected by BNP-based sunscreen showed significantly fewer double strand breaks 
compared to the samples treated with the commercial sunscreen control, indicating less 
penetration into skin (data not shown). 
 
 
 
BNPs have low toxicity in vivo and can reduce the 
toxicity of reactive oxygen species (ROS) released 
by UVR filters. In an in vivo study aimed at testing 
the long-term toxicity of BNPs, the particles were 
applied to dorsal mouse skin every other day for 12 
days (six applications). The treated skins showed no 
evidence of cutaneous irritancy, toxicity, 
inflammation or damage to skin follicles (Fig. 3). 
Additionally, photo-induced chemical changes to 
UVR-filters composition can often produce toxic 
ROS that damage cells. We demonstrated the BNPs 
can prevent ROS-induced skin damage by 
containing the ROS inside the particles (data not 
shown). Finally, the BNP based sunscreen is long- 
lasting on the skin. We applied the BNPs on the skin 
of nude mice and demonstrated that the retention on mouse skin was stable over 24 hours 
and was water-resistant during this period, but can be readily removed by towel drying (Fig. 4). 


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n=322 
n=593 
 Assay 
n=219 
n=696 
 Screen 
n=118 
n=797 
 
 
 
 
 
n=20 
n=895 
 Screen 
n=13 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
We have further developed this platform by encapsulating UVR filters that can provide broad 
spectrum protection (Fig. 5) (2). In vitro testing demonstrates that a BNP sunscreen 
encapsulating a combination of avobenzone plus octocrylene (BNP-A/O) acts as a neutral 
density filter capable of absorbing both UVA and UVB wavelengths. This improved BNP 
formulation has a critical wavelength of 384nm, exceeding the broad spectrum requirements of 
the FDA monograph (critical wavelength = 370). 
 
 
In addition to addressing safety concerns regarding current sunscreen agents by engineering 
the novel BNP technology to enhance performance and prevent penetrance, we have also 
undertaken a high-throughput screen of naturally occurring compounds to identify safer 
sunscreen alternatives. 
High-throughput screening strategy: 
 
Two small molecule collections at the Yale Center for Molecular Discovery (MicroSource 
Pure Natural Products and NCI Natural Products Set) containing 915 natural products were 
sequentially screened for a) their ability to absorb UVA or UVB radiation, b) the degree of 
photostability they exhibit under solar simulator radiation, c) the extent to which they show 
minimal cytotoxicity when cultured in vitro with skin cells (keratinocytes, melanocytes), and 
 
13 
In Vitro Broad Spectrum Protection 
 
 
 
 
 
 
 
 
 
Fig. 5. In vitro broad spectrum protection testing 
shows uniform absorbance across UVA and UVB 
wavelengths. Critical wavelength measured: 384 nm. 
Absorbance (a.u.) 


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14 
R 
 
 
 
 
 
 
 
 
 
 
 
0 
0 
A. 
Diosmin 
Cytisine 
Ferulic Acid 
4 
 
 
4 
 
4 
 
 
 
3 
3 
3 
2 
2 
2 
1 
1 
1 
0 
0 
0 
300 
350 
 
40 
300 
350 
 
40 
300 
350 
 
400 
B. 
C. 
Figure 6. Characteristics of natural product sunscreen 
compounds. A. UVR absorbance pre- and post- solar 
simulator radiation exposure shows excellent 
photostability. B. in vitro culture of selected natural 
products with three skin cell lines (two keratinocyte lines 
and one melanocyte line) or vehicle control(0.1% DMSO 
in culture medium) demonstrates average negative % 
cytotoxicity – that is, cells cultured with each natural 
product remained healthier than cells cultured with the 
control. The fluorescent ROS probe dihydrorhodamine- 
123 was added to natural products and the mixture 
exposed to solar simulator radiation. Each natural product 
generated less ROS than the vehicle control. C. Natural 
product sunscreen formulated as 10% diosmin, 10% 
ferulic acid, 5% cytisine in FDA P2 cream vehicle was 
applied to the shaved dorsal bodywall skin of N=3 
C57Bl/6 mice once daily for 5 consecutive days. 
Throughout treatment, mice were examined daily, and no 
mice developed erythema, scale, fissuring, or erosions. 
Histologic findings were indistinguishable from that of the 
vehicle control group with normal appearing skin without 
inflammatory infiltrate. 
 
 
d) the extent to which they show minimal ROS generation when exposed to UVR. This 
resulted in selection of 13 natural compounds that were further scrutinized by additional in 
vitro testing as well as in vivo (mouse) irritant contact dermatitis testing. Of the 13 natural 
products resulting from the high-throughput screen, 9 were readily commercially available 
and passed confirmatory UV absorbance and photostability testing. These 9 then underwent 
in vitro SPF testing: 
 
Compound 
Average SPF (SD) 
Cytisine 
6.3 (0.1) 
Diosmin 
2.7 (0.2) 
Ferulic Acid 
3.1 (0.2) 
Gossypin 
1.7 (0.1) 
Isoliquiritigenin 
2.5 (0.3) 
Methylxanthoxylin 
2.1 (0.1) 
Rutin 
1.8 (0.0) 
Scopoletin 
1.6 (0.1) 
2/4-dihydroxychalcone 
2.2 (0.3) 
 
 
Three natural compounds, diosmin, ferulic acid, and cytisine, were ultimately chosen. Each 
displays excellent photostability (Fig 6A) and no in vitro cytotoxicity or ROS generation (Fig 
6B) was detected. When formulated as 10% diosmin, 10% ferulic acid, 5% cytosine in FDA 
P2 cream vehicle, this natural sunscreen gave an in vitro SPF = 48.6 and a high broad 
spectrum score, λcrit = 373, with no evidence of in vivo irritancy (Fig 6C). 
 
 
 
 
 
 
 
 
 
diosmin 
ferulic acid 
cytisine 
Cytotoxicity (% relative to vehicle control) 
-4.33% 
-9.67% 
-4.61% 
ROS generation (relative fluorescence units) 
-4670.84 
-3167.54 
-4441.45 
Absorbance 
Absorbance 
Absorbance 


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15 
Figure 7. Combined natural product BNP sunscreen is protective in vivo. 
C57Bl/6 mice underwent chronic solar simulated UVR exposures (1500 
J/m2 UVB + 2700 J/m2 UVA, 3x/wk, 10wks). 15 minutes before each 
exposure one group of mice was treated with the FDA monograph 
recommended amount (2 mg/cm2) of BNP-A/O containing 3% 
avobenzone and 9% octocrylene or with sunscreen consisting of 10% 
ferulic acid, 5% cytisine, 5% trans-resveratrol plus BNP-A/O. Four days 
following the final exposure, skin was harvested, epidermal sheets 
prepared and stained for quantification of mutant keratinocyte p53 
islands by confocal microscopy. Natural product BNP sunscreen reduced 
the total area occupied by p53 islands by 97.61%. 
 
 
 
 
 
Diosmin is a flavone glycoside found in many citrus fruits. It absorbs UVR primarily in the UVA 
spectrum. Orally administered diosmin has been used to treat blood vessel disorders and a 
variety of anti-inflammatory and antioxidant properties have been reported (3). One study also 
found that diosmin incorporated into a cream and applied to ex vivo human skin samples 
reduced ROS and CPD following UVB exposure (4). Cytisine is a UVB absorbing alkaloid 
found in some legumes. It has been marketed as a smoking cessation aid (TabexTM), and has 
also been suggested to have neuroprotective, anti-diabetic and anti-tumor effects (5). Ferulic 
acid is a phenolic acid commonly found in many plants. In addition to absorbing UVR 
(primarily UVB), it is an antioxidant, with excellent free-radical scavenging capacity. It is 
currently used in a variety of skin care formulations including for photoprotection, anti-aging 
and skin lightening (6). Trans-resveratrol is another plant derived polyphenolic antioxidant. 
Although not chosen as one of the final sunscreen candidates in our screen due to its 
relatively low UVR absorbing capacity, trans-resveratrol has been shown to have significant 
triplet-state energy quenching capacity (7). UVR-induced chemiexcitation generates high 
energy reactive oxygen and nitrogen species whose activity can be quenched by trans- 
resveratrol, thereby reducing subsequent indirect DNA damage. We have therefore included 
trans-resveratrol in some of our pre-clinical follow-up studies (below). 
We have tested BNP-A/O alone or in combination with select natural products in vivo (C57Bl/6 
mice) to assess their capacity to block the formation of DNA damaging cyclobutane pyrimidine 
dimers (CPD) immediately following UVR exposure and found that sunscreen comprising 
natural products diosmin, ferulic acid, cytisine and BNP-A/O successfully blocked 95.09% of 
the CPDs (not shown). We also performed a chronic UVR exposure experiment (3 
exposures/week for 10 weeks) in which sunscreen was applied to dorsal mouse skin before 
each UVR exposure (Fig 7). Such chronic UVR exposures cause expansion of p53-mutant 
keratinocyte clones (called “p53 islands”) that represent microscopic pre-cancerous lesions (8- 
10). The number of mutant p53 islands was reduced by 92.34% in the mice that received a 
sunscreen composed of natural products + BNP-A/O, and the area occupied by p53 islands 
was reduced by 97.61% in these same mice. Also, chronic application of this sunscreen 
caused no visible skin irritation (data not shown). Together, these data show that addition of 
select natural products to our BNP-A/O dramatically increases the ability of our sunscreen to 
block UVR-induced DNA damage. 
 


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Proposed doses and pharmacokinetics: 
The FDA monograph recommends applying 2 mg/cm2 formulated sunscreen. The FDA limits 
the amount of avobenzone (3%) and octocrylene (10%) that the formulation may contain. 
Our BNP-A/O will not exceed these limits and provide additional safety by preventing 
absorbance of the actives into the skin. 
Ferulic acid is used in medical cosmetology procedures (microneedling and non-needle 
mesotherapy, chemical peels, grooming treatments) at concentrations up to 12% (6). Based 
on our preclinical studies, we propose to use 10% ferulic acid, in a single topical dose equal 
to 0.9 mg. It has been estimated that ferulic acid intake through food consumption may 
reach 150-250 mg/day (11). The potential for ferulic acid toxicity in our study, using a dose of 
0.9 mg, is exceedingly low. “LD50s equal to 2445 mg/kg and 2113 mg/kg were calculated for 
male and female rats, respectively (Tada et al., 1999; Ou and Kwok, 2004), whereas an 
acute LD50 of 3200 mg/kg was calculated in mice (Wang and Ou-Yang, 2005). This low 
toxicity has been confirmed by numerous experimental studies.” (12-14). For reference, our 
dose of 0.9 mg applied to a 60 kg individual would equal 0.015 mg/kg, or applied to an 80 kg 
individual, would equal 0.011 mg/kg. After oral administration, ferulic acid plasma 
concentrations are maximal at 24 min and the amount of ingested ferulic acid that is 
absorbed has been estimated to range from 75% (15) to 92% (11). Following topical 
administration of ferulic acid, one study, using in vivo recovery from hairless mouse skin, 
reported 16.5 ± 1.92% was absorbed (16), In another study using in vitro human stratum 
corneum plus epidermis in a Franz cell system, only 2.62% of the applied ferulic acid 
penetrated through the epidermis (17). 
 
 
A clinical trial found that daily (oral) resveratrol at doses up to 5 g for 29 days was generally 
safe (18) and resveratrol (98% trans-resveratrol) has been used to improve lumbar bone 
mineral density at oral dosing of 75 mg twice daily for 12 months. Based on our preclinical 
studies, we propose to use a single topical dose of 0.45 mg (5% in formulated sunscreen) 
which we expect to be non-toxic based on the following studies:. 
“Low and high doses of resveratrol, up to 750 mg/kg/day for 3 months, were investigated in 
vivo in rabbits and rats. The authors concluded that resveratrol is well tolerated and non- 
toxic and has no effect on reproductive capacity in male or female rats and no embryo- fetal 
toxicity” (19). “The adverse effects in humans have been investigated in several studies after 
high-dose resveratrol intake, representing a total of 104 patients (including placebo). The 
highest doses were 5 g/70 kg for a single intake and 0.9 g/day for iterative administration, 
corresponding, respectively, to approximately 1/40 and 1/200 of the dose reported to cause 
nephrotoxicity and 1/4 and 1/20 of the highest dose reported to be safe in rats. No serious 


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adverse event was detected in any of these studies. Adverse events were mild and only 
lasted a few days.”(19). 
Pharmacokinetic studies in humans (20), reveal differences based on route of administration 
and dose, but generally agree that resveratrol is rapidly absorbed, with peak plasma 
concentrations 0.8 – 1.5 hr post-dose. There is significant metabolism of resveratrol to 
multiple forms, mostly excreted in urine (20). The oral absorption of resveratrol in humans is 
about 75% and is thought to occur mainly by transepithelial diffusion (21). Murakami et al., 
applied resveratrol orally or topically to mouse skin, and found that both oral and transdermal 
absorption levels were similar to that described for humans (22). Thus, we expect that 
approximately 75% of the applied trans-resveratrol will be absorbed. 
Cytisine has been used as a smoking cessation aid (e.g. TabexTM), where oral dosing of 1.5 
mg every 2 hr is suggested (23). We propose to use a single topical dose of 0.45 mg (5% in 
formulated sunscreen). We expect this dose to be non-toxic based on reports of clinical trials 
reviewed by Tzankova and Danchev (24). They report “The clinical trials and observations 
were carried out on more than 10,000 patients. Smoking cessation was achieved in 55 % - 
75 %. Cytisine administration showed no serious adverse reactions during the clinical trials.” 
(24). Toxicology from animal studies is presented in the following table, taken from 
PubChem (https://pubchem.ncbi.nlm.nih.gov/compound/Cytisine#section=Toxicity): 
 
Cytisine peak plasma concentration is seen 1-2 hr following oral dosing and cytisine is 
eliminated unchanged, with 18% and 30% of the drug administered orally or intravenously, 
respectively, found in the urine after 24hr (5). In another study, Klocking et al., found the 
level of absorption to be 42% following oral administration (25). Internet searches did not 
identify any studies examining cytisine absorption following topical/skin application. 
Diosmin has been used to treat chronic venous insufficiency and hemorrhoid disease, with 
oral dosing of 1000 mg daily given in 2 divided doses. We propose to use a single topical 
dose of 0.9 mg (10% in formulated sunscreen), based on our preclinical studies. Diosmin 
(marketed as “Daflon”) has undergone extensive safety evaluation without evidence of 
toxicity in thousands of patients receiving systemically administered doses of 1000mg daily 
for 6 weeks to one year (26): “Overall analysis of mid-term and long-term trials: 2850 


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patients were treated with Daflon 500 mg at the dosage of two tablets per day for six weeks 
to one year.” “Regardless of the treatment duration, the clinical acceptability of Daflon 500 
mg was found to be good. The proportion of patients treated with Daflon 500 mg and 
developing a side effect (10%) was less than that in groups taking placebo (13.9%).” 
Following oral administration diosmin is hydrolyzed to diosmetin, which may then be 
absorbed, however the plasma concentration of diosmetin is low and variable (3), perhaps 
due to the low aqueous solubility of diosmin. Pharmacokinetic data on topically administered 
diosmin was not found, however because of the hydrophobic nature of diosmin, and the 
lipophilic nature of skin, we expect that at least a portion of the applied dose will penetrate 
the skin. 
Based on the single dose we propose to use, compared with doses reported above for other 
indications, we expect the natural products will not have toxic effects in this study. In 
addition, we have mixed these natural compounds together in various concentrations in vitro 
without any apparent chemical reaction (e.g. change in color, pH or temperature). We have 
also applied the natural products in combination (natural products combined alone or also 
along with BNP-avobenzone/octocrylene) topically to mouse skin, three times per week for 
10 weeks, without any apparent irritation, inflammation or cutaneous toxicity. 
Furthermore, many natural sunscreens include plant extracts that contain these compounds. 
For example, Hawaiian Tropic Sunscreen Powder contains papaya fruit extract and 
Passiflora extract which have been shown to contain diosmin, and mango extract and 
Psidium guajava extract which have been shown to contain ferulic acid. Resveratrol is also 
included in several sunscreens, for example, Paula’s Choice Essential Glow SPF30. Cytisine 
is derived from extracts of the plant family Fabaceae which includes lentils (Lens esculenta) 
and is included in some sunscreens, for example, Cay Skin Isle Glow SPF45, that uses Lens 
esculenta extract. 
 
 
In addition to using only low, non-toxic doses of natural products, and only FDA approved 
doses of the UV filters avobenzone and octocrylene, we aim to further increase the safety of 
this study by applying subject selection criterion that will eliminate any individuals with a 
history of skin cancer or history of any conditions that make you more sensitive to sunlight. 
The medical history obtained will emphasize the effects of sunlight on their skin, ascertain 
the general health of the individual, the individual's skin type (I, II, or III), whether the 
individual is taking medication (topical or systemic) that is known to produce abnormal 
sunlight responses, and whether the individual is subject to any abnormal responses to 
sunlight, such as a phototoxic or photoallergic response. In addition, women of child-bearing 
potential must have negative urine pregnancy test. 
 
 
We also specifically address whether diosmin, ferulic acid, cytisine, and trans-resveratrol 
meet the criteria for exemption from an IND: 


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We reviewed our protocol with Amy Hummel (YCCI, Associate Director fir UND/IDE 
Management) on November 2, 2022. Based on our exchange and response to questions, 
we believe that we are exempt from and IND. Our responses included that: 
The FDA sunscreen monograph allows us to proceed without an IND – we are using the 
actives allowed, under the max concentrations permitted. We are not modifying the actives 
(no covalent changes), just reformulation to improve performance and keep them on the 
surface of the skin. 
The BNP formulation utilizes PLA (which breaks down to lactic acid) and HPG (with breaks 
down to glycerol) on the surface of the skin. Yale Pharmacy has previously helped us set up 
GMP conditions for a previous pilot study we conducted and published with Mark Saltzman’s 
lab. 
A clinical investigation of a marketed drug is exempt from the IND requirements if all of the 
criteria for an exemption in § 312.2(b) are met: Are these met for diosmin, ferulic acid, 
cytisine, and trans-resveratrol? Yes: 
• The drug product is lawfully marketed in the United States. 
This is correct. Diosmin is marketed under several brand names in the US. For example, 
Diosmin capsules are available from PipingRock Health Products, Ronkonkoma, NY. 
Ferulic acid and resveratrol are included in many cosmetics marketed in the US. For 
example, The Ordinary Resveratrol 3% + Ferulic acid 3% antioxidant serum for daily skin 
protection. 
Cytisine is available as Tabex or Desmoxan, smoking cessation aids. 
• The investigation is not intended to be reported to FDA as a well-controlled study in 
support of a new indication and there is no intent to use it to support any other significant 
change in the labeling of the drug. 
This is correct. 
• In the case of a prescription drug, the investigation is not intended to support a significant 
change in the advertising for the drug. 
This is correct. This is not a prescription drug. 
• The investigation does not involve a route of administration, dose, patient population, or 
other factor that significantly increases the risk (or decreases the acceptability of the risk) 
associated with the use of the drug product (21 CFR 312.2(b)(1)(iii)). 
This is correct. Topical application is far safer than systemic administration. 
• The investigation is conducted in compliance with the requirements for review by an IRB 
(21 CFR part 56) and with the requirements for informed consent (21 CFR part 50). 
This is correct. 
• The investigation is conducted in compliance with the requirements of § 312.7 (i.e., the 
investigation is not intended to promote or commercialize the drug product). 


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This is correct. This is a pilot clinical study funded by the NCI to assess a novel strategy for 
melanoma prevention. 
 
 
2.1.2 Clinical Experience 
An interventional, nonrandomized trial performed in accordance with a protocol approved by 
the Yale Human Investigation Committee [HIC#: 1512016909], (N=10, ref. 2) was 
undertaken to determine the SPF of BNP-A/O (co-encapsulating 3% avobenzone and 9% 
octocrylene) in healthy volunteers with fair skin (Fitzpatrick skin type I and II), in comparison 
to FDA approved P2 formulation, which contains 7% padimate O and 3% oxybenzone and 
has an expected SPF of 16.3 (range 13.7-17.7). BNP-A/O performed comparably to the P2 
formulation (2). Skin irritation was not specifically studied in this trial, however no adverse 
reactions from photosensitivity or photo allergy were observed in subjects receiving BNP- 
A/O during the duration of the trial. 
Rational for proposed doses: 
The FDA monograph (21 CFR 201.327) recommends applying 2 mg/cm2 formulated 
sunscreen. The FDA limits the amount of avobenzone (3%) and octocrylene (10%) that the 
formulation may contain. Our BNP-A/O will not exceed these limits and provide additional 
safety by preventing absorbance of the actives into the skin. 
 
 
2.2 
Background/prevalence of research topic 
 
Nearly 90% of all skin cancers can be attributed to ultraviolet radiation (UVR) from the sun 
(27). UVR exposure leads to multiple adverse effects including cutaneous phototoxicity 
(sunburn), photo-aging, and carcinogenesis (27-31). Both UVA and UVB exposure markedly 
enhance the production of reactive oxidation species (ROS) that damage a variety of cellular 
components, including genomic DNA, and induce the secretion of immunosuppressive 
cytokines (32, 33). Conventional sunscreens, though protective against sunburn, have 
shown the capacity to raise the skin’s minimal erythema dose (MED), raising concerns due 
to their potential to penetrate the skin, enter the bloodstream, and bind hormone receptors. 
Thus, an innovative sunscreen that offers enhanced protection, while minimizing the 
potential toxicity induced by its active ingredients, is clearly advantageous over current 
formulations. 
 
Each year, there are more new cases of skin cancer than the combined incidence of breast, 
prostate, lung, and colon cancers; and incidence rates continue to rise (34). From 1992- 
2006, the treatment of non-melanoma skin cancers increased by nearly 77 percent (35). The 
diagnosis of melanoma is significantly more fatal; 2022 estimates indicate that one person 
dies of melanoma every hour (34). The economic impact of these high incidence rates also 
cannot be ignored—the annual cost of treating skin cancers in the United States is estimated 
at $8.1 B (36). Over the last decade, the average annual cost for skin cancer treatment has 
increased by more than 126%, compared to 25% for all other cancers (36). 
 
On average, a person’s lifetime melanoma risk increases by 80% if they have sustained five 
or more sunburns during youth (27, 37). Ambient UVR exposure also results in photo-aging; 
more than 90% of the visible changes in skin are caused by the sun (37). Epidemiologic 


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studies have proven that sunscreens play an important role in preventing UVR damage and 
reducing risk. Regular daily use of sunscreen reduces the risk of developing squamous cell 
carcinoma by 40 percent and the risk of developing melanoma by 50% (29, 30). People who 
use sunscreen daily also show 24% less skin aging than those who do not (37). 
 
Commercially available sunscreens have incorporated organic and inorganic UVR filters to 
provide protection, but their overall effectiveness in preventing skin cancer remains 
controversial (38-40). Both agents have shown the capacity to enhance ROS generation after 
UVR exposure, suggesting that even small quantities may contribute to cellular damage and 
ultimately carcinogenesis (38-40). Furthermore, transdermal penetration of these filters 
through the stratum corneum into epidermal cells, including keratinocytes and Langerhans 
cells, raises direct toxicity concerns. In addition, the potential for systemic absorption and 
deposition in adipose tissue may result in additional health risks such as endocrine disruption 
(38, 41). These adverse effects must be addressed in order to minimize skin cancer risk and 
minimize potential toxicity. 
 
Nanoparticle encapsulation, used widely in the field of drug delivery, has demonstrated unique 
advantages including target specificity and toxicity reduction. The incorporation of this 
technology will eliminate direct skin contact and subsequent epidermal penetration, and thus, 
provide unprecedented UVR protection and safety. Additionally, the properties of polymer- 
based nanoparticles may be tuned to offer increased substantivity and better equip individuals 
for prolonged sun exposure. Hence, we have engineered a safer sunscreen with enhanced 
UVR protection (1, 2). 
 
We have additionally utilized high-throughput screening to identify naturally occurring non- 
toxic products for use as additions to or alternatives for existing sunscreen actives. Natural 
products are compounds produced by living organisms (e.g. tropical plants, algae) that have 
evolved systems to respond to environmental stressors including UVR (42, 43). Indeed, 
harnessing the power of natural products for use as sun-protective agents is not a novel 
concept (44). Some natural compounds can directly interact with UVR to prevent direct DNA 
damage by absorbing UVR and thus acting as a sunscreen. For example, flavonoids, such as 
diosmin, contain an aromatic polyphenol backbone and may contain hydroxyl groups that 
confer excellent UVR absorbing capacity without concerns of paradoxically catalyzing 
photochemical reactions (45). Such compounds prevent direct formation of mutagenic 
cyclopyrimidine dimers (CPDs) in DNA. 
 
In addition to their UVR absorbing capacity, some natural products have also been shown to 
scavenge ROS, for example, ferulic acid and trans-resveratrol have excellent antioxidant 
properties (6, 7). This is critical for the reduction of indirect DNA damage. Chemiexcitation of 
electrons in skin chromophores such as melanin results in high-energy triplet states that are 
responsible for inducing so called “dark” CPDs, that is, indirect DNA damage that occurs after 
UVR exposure ends (46). Certain antioxidant natural compounds, including trans-resveratrol 
and ferulic acid, are able to quench triplet state energy and prevent dark CPD formation (7). 
Thus, the potential “triplet state quencher” capacity of certain natural compounds also makes 
them attractive candidates as a topical intervention before, during, and after UVR exposure to 
minimize the formation of dark CPDs. 
 
Our research, thus far, has demonstrated that the bioadhesive nanoparticle (BNP) sunscreen 
we have developed has demonstrated in an initital investigation superior skin protection from 
UVR exposure than commercial sunscreens. We have also identified non-toxic, naturally 
occurring plant-derived compounds that boost the effectiveness of our BNP sunscreen by both 
increasing the UVR-absorbing capacity and providing antioxidant, triplet state energy 
quenching capacity. These natural products combined with BNP sunscreen have the potential 


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to substantially reduce or eliminate both direct and indirect DNA damage caused by UVR 
exposure, reducing the risk of skin cancer development. 
 
 
 
3 Rationale/Significance 
3.1 
Problem Statement 
Skin cancer is the most commonly diagnosed malignancy in the USA and UVR exposure is 
the major environmental risk factor for skin cancer development. Currently available 
sunscreens utilize UVR filters that, while absorbing UVR energy, have been shown to induce 
ROS, resulting in oxidative DNA damage after UVR exposure. Organic sunscreen actives 
have also been shown to penetrate into the skin, raising direct toxicity, as well as irritant and 
photoallergic concerns. Further systemic absorption may result in additional health risks 
such as endocrine disruption. Novel sunscreens that more safely prevent both direct and 
indirect DNA damage are needed. 
3.2 
Purpose of Study/Potential Impact 
We have produced a bioadhesive nanoparticle (BNP) sunscreen designed to keep organic 
UVR filters from penetrating into the skin and have incorporated non-toxic natural products 
into this sunscreen to further safely boost UVR absorbing capacity and reduce oxidative, 
indirect DNA damage. In this study we propose to test the capacity of this sunscreen to 
prevent direct and indirect cellular and DNA damage in human skin exposed to UVR. 
 
 
3.2.1 Potential Risks 
UVR exposure: Ultraviolet radiation interacts with light absorbing molecules in different 
layers of the skin. UVB (280-320nm) is primarily absorbed in the epidermis and may be 
associated with direct DNA damage and immunosuppression, although narrowband UVB is 
used as a clinical therapy, and evidence of skin cancer risk is low. UVA (320-400nm) may 
penetrate into the dermis and is associated with oxidative cellular and DNA damage. 
UVR exposure for MED testing and subsequent sunscreen testing may result in erythema 
(redness). 
Biopsy: A total of 5 x 3mm punch biopsies will be taken by standard procedures. A skin 
biopsy is a generally safe procedure, with risks including: bleeding, bruising, and infection 
(all rare, <1%), and scarring (limited to <3mm per biopsy site). The resulting minimal 
erythema on UV exposed skin is not expected to increase risks i.e. over standard skin punch 
biopsy on non-exposed skin. The dose of UV used is too low and too superficial an exposure 
to adversely affect wound healing after a punch biopsy. Prior studies on much higher UV- 
exposed skin with much larger surgical wounds did not show compromised healing (47). 
Risk of agents acting in combination: based on the chemical structure of each agent, we do 
not have any reason to believe that there is any increased toxicity risk to using them in 
combination. We have mixed these compounds together in various concentrations in vitro 


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without any apparent chemical reaction (e.g. change in color, pH, or temperature). We have 
also applied the the natural products in combination (natural products combined alone or 
also along with BNP-avobenzone/octocrylene) topically to mouse skin (3 times/wk for 10 
wks) without any apparent irritation, inflammation, or cutaneous toxicity. 
 
 
Risks will be minimized by: 
1. Using the minimal amount (1 MED) of UVR necessary to obtain the primary objective, and 
minimizing the area exposed to UVR by using the Multiport 601 solar simulator which limits 
UVR exposure to 8mm diameter (0.5 cm2) spots. 
2. Using FDA approved UVR filters encapsulated in nanoparticles (avobenzone and 
octocrylene) and formulating the sunscreen such that FDA approved doses are not 
exceeded (FDA OTC monograph specifies 3% avobenzone and 10% octocrylene). 
3. Using non-toxic natural products (ferulic acid, cytisine, diosmin, trans-resveratrol) and 
topical doses of these products that are below a) doses considered safe for repeated oral 
administration for other indications, or b) amounts used in existing cosmetic treatments 
4. Trained and experienced personnel will supervise subjects during every phase of 
participation. Personnel will be instructed to be aware of any warning signs of impending 
danger and will stop any procedure at that time. 
5. Using sterile skin biopsy techniques performed by a board-certified dermatologist, 
observing sites for 15 minutes after skin biopsy ensure adequate bleeding control, and 
provided post-biopsy care instructions (change bandage, wash site with soap and water 
daily, apply ointment and band aid daily; call for any redness, pain, swelling, or discharge; 
return in 7-10 days for suture removal). 
3.2.2 Potential Benefits 
The proposed study will enable us to scientifically assess and further develop our 
nanoparticle sunscreen technology combined with natural products for enhanced UVR 
protection. While direct benefits to participants are not expected, we believe that society at 
large would benefit from the translation of this technology for the development of safer more 
effective sunscreen which may minimize risk for skin cancer development. 


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4 Study Objectives 
4.1 
Hypothesis 
We hypothesize that our combined natural product plus BNP sunscreen will reduce both 
direct and indirect UVR-induced cellular and DNA damage to human skin by ≥90%. 
4.2 
Primary Objective 
The primary objective of this study is to evaluate the effectiveness of our novel sunscreen by 
assessing the extent of UVR-induced direct and indirect cellular and DNA damage to human 
skin, in the presence vs absence of the sunscreen, in a population of healthy adults with fair 
skin (Ftizpatrick Scale type I, II or III). 
4.3 
Secondary Objectives (if applicable) 
N/A 
4.4 
Exploratory Objectives (if applicable) 
N/A 


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fair-skinned individuals 
Physical examination of skin 
 
inherent MED 
Sunscreen application 
 
 
Visit #1: UVR exposure for 
Multiport 601 inherent MED determination 
test pad 
4 x 5.5 cm 
Sunscreen testing 
Multiport 601 solar simulator 
Liquid light guides individually 
control UVR exposure to the 
center of up to 6 sites. 
Exposed area = 0.8 cm diameter 
= 0.5 cm2. 
Visit #2: Sunscreen testing 
3 mm punch biopsy 
 
5 Study Design 
5.1 
General Design Description 
This is a phase 1 non-randomized pilot study designed to test the effectiveness of a 
sunscreen compared to a no treatment control. Sunscreen application and sample (biopsy) 
collection will not be blinded, but analysis of the samples will be conducted by laboratory 
staff blinded to the sample characteristics (to avoid bias). Data obtained from the sample 
analysis will be de-coded by the PI and used to determine sunscreen efficacy. 
 
 
The Multiport 601 solar simulator provides highly uniform UVR (290-400nm) and is fully 
compliant with ISO, FDA, JCIA and COLIPA spectral irradiance standards and all FDA 
sunscreen testing requirements (21 CFR 201.327). Calibrated probes monitor each 
exposure and specifically designed ports ensure directed exposure only to the 8mm 
diameter test sites identified by the blue test pad in the image above. The six black UV ports 
fit into the 6 openings in the blue patch. Only these six 0.5 cm2 spots of skin are exposed to 
UV light. The dose of UV going to each spot can be individually controlled. 


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The blue test pad is not used for sunscreen delivery; it is used to guide the UV light to six 
small defined spots (0.5 cm2 each). The sunscreen will be applied by rubbing the 
cream/emulsion onto the skin before placement of the blue pad. 
 
 
Visit #1: Subjects meeting eligibility requirements and providing written consent will undergo 
UVR exposure for inherent minimal erythema dose (MED) determination. An adhesive test 
pad will be placed on the upper arm to guide UV light delivery to six small defined spots (0.5 
cm2 each). No other skin is exposed to UV light. The doses used depend on the individuals 
Fitzpatrick Scale skin type: 
 
The Multiport 601 solar simulator will be used to deliver a 1.25x UVR dose series centered 
around the FDA Standard 1 MED dose for the participant’s individual Fitzpatrick Scale skin 
type. Examples of the UV dose given for different skin types is shown in the table below: 
 
Visit 1 
 
Test Site # 
(each site 
= 0.5 cm2) 
 
UV dose for MED testing (J/m2) 
 
 
 
 
 
Skin 
Type 
Skin Type 
Skin 
1 
2 
Type 3 
Corresponding time at the beach 
in France on the summer solstice 
(minutes) 
 
 
 
 
Skin 
Skin Type 
Skin 
Type 1 
2 
Type 3 
1 
0 
0 
0 
0 
0 
0 
2 
64 
128 
192 
5.91 
11.82 
17.72 
3 
80 
160 
240 
7.38 
14.77 
22.15 
4 
100 
200 
300 
9.23 
18.46 
27.69 
5 
125 
250 
375 
11.54 
23.08 
34.62 
6 
156 
313 
469 
14.42 
28.85 
43.27 


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The time needed to deliver the maximum UV light dose using our solar simulator ranges 
from approximately 30 seconds for skin type 1 to 90 seconds for skin type 3. 
 
 
 
Visit #2: 16-24 hr after Visit #1 the results of MED testing will be determined by visual 
inspection and recorded. 1 MED is defined as the smallest UVR dose that produces 
perceptible redness of the skin with clearly defined borders 16-24 hours after UVR exposure. 
The area used for MED determination on day 1 will be examined and the UV dose that 
produced the faintest perceptible redness will be identified. This dose = 1 MED for this 
individual, and this is the UV dose that will be used to test the sunscreen. 
The skin site to be used for sunscreen testing will then be delineated and sunscreen applied 
(by rubbing the cream/emulsion onto the skin with a gloved fingertip) to the appropriate 
subsites and allowed to dry for 15 min. An adhesive test pad will be placed over the 
delineated area to guide the UV light to the correct sites, and the Multiport 610 solar 
simulator used to deliver 1 MED UVR to the appropriate subsites. Based on our previous 
study, 1 MED ranged from 64 – 200 J/m2 for different individuals. The corresponding time at 
the beach on the summer solstice in France would be 5.91 – 18.46 minutes. 
Immediately following UVR exposure 3 x 3mm punch biopsies will be obtained by Dr. Girardi 
from the appropriate subsites of the test area: 
1. Untreated skin, No UVR 
2. Untreated skin + 1 MED UVR 
3. Sunscreen treated skin + 1 MED UVR 
Visit #3: 4 hr after the UVR exposure of visit #2, 2 additional 3 mm punch biopsies will be 
obtained by Dr. Girardi from the appropriate subsites of the test area: 
4. Untreated skin + 1 MED UVR 
5. Sunscreen treated skin + 1 MED UVR 
Visit #4: 7 – 10 days after biopsies for suture removal only. 
 
 
5.1.1 Study Date Range and Duration 
The expected length of the study is 9 months, beginning in November 2022. 
5.1.2 Number of Study Sites 
There will be one study site. Studies will be completed at the 4th floor of the Hunter Radiation 
Therapy (HRT) building, in the Examination Room of the YNHH Photopheresis Unit 
(Director: Dr. Michael Girardi). Physician Assistant Kacie Carlson, PA-C, has an office on 
this floor, and will serve as the study coordinator. Nursing staff are available and well- 
trained. Skin biopsies will be processed in Dr. Girardi’s laboratory (HRT618). 
5.2 
Outcome Variables 
Outcome data will be recorded for samples obtained at study visit #2 and #3. 


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Study visit #2: 
The results of MED testing will be determined by visual inspection and recorded. 
Samples (3 mm punch biopsies) will be obtained immediately after UVR exposure: 
1. Untreated skin with no UVR exposure 
2. Untreated skin exposed to 1 MED solar simulator UVR. 
3. Sunscreen treated skin exposed to 1 MED solar simulator UVR. 
Study visit #3: 
Samples (3 mm punch biopsies) will be obtained 4 hr after UVR exposure: 
4. Untreated skin exposed to 1 MED solar simulator UVR. 
5. Sunscreen treated skin exposed to 1 MED solar simulator UVR. 
The study endpoint for participants occurs following collection of 5 biopsy samples, 
described above. Biopsies will be analyzed to assess the primary objective (below). 
Study visit #4: Follow-up of biopsy sites will occur 7-10 days later with suture removal. 
 
5.2.1 Primary Outcome Variables 
Each biopsy tissue will be used to assess the effectiveness of the sunscreen by 
measuring the level of DNA and cellular damage in UVR-exposed vs unexposed and 
sunscreen treated vs untreated samples by the following 3 methods: 
1. DNA will be prepared and assayed by ELISA for quantification of CPDs. CPDs 
measured in samples obtained immediately after UVR exposure are indicative of 
direct DNA damage. CPDs will also be measured in samples obtained 4 hr after UVR 
exposure, at which time any increase in the CPD level, as compared to time 0, is 
indicative of so called “dark” CPDs that form in response to indirect oxidative DNA 
damage. 
2. Formalin fixed paraffin embedded skin will be stained with anti-gH2AX to identify 
DNA strand breaks. Indirect, oxidative DNA damage may result in DNA strand breaks 
that can be quantified by microscopic visualization of gH2AX, which builds up at the 
site of each strand break. 
3. Formalin fixed paraffin embedded skin will be stained with anti-3-nitrotyrosine to 
identify cellular damage. ROS and high energy triplet state species can result in 
nitration of tyrosine residues of cellular proteins. This type of damage can be 
quantified by microscopic visualization of 3-nitrotyrosine. 
All testing will be done by laboratory staff blinded to the sample characteristics. 
5.2.2 Secondary Outcome Variables (if applicable) 
N/A 
5.2.3 Exploratory Outcome Variables (if applicable) 
N/A 
5.3 
Study Population 
Individuals with fair skin are more susceptible to the UVR-induced DNA damage that we 
seek to prevent by development of a safer, more effective sunscreen, and thus will comprise 
the study population. 


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Healthy adult volunteers will be selected as follows: 
(1) Only fair-skin subjects with Fitzpatrick Scale skin types I, II, and III using the following 
guidelines shall be selected: 
Skin Type and Sunburn and Tanning History (Based on first 30 to 45 minutes sun exposure 
after a winter season of no sun exposure.) 
 
I- -Always burns easily; never tans (sensitive). 
II- -Always burns easily; tans minimally (sensitive). 
III- -Burns moderately; tans gradually (light brown) (normal). 
 
Do Not Include: 
IV- -Burns minimally; always tans well (moderate brown) (normal). 
V- -Rarely burns; tans profusely (dark brown) (insensitive). 
VI- -Never burns; deeply pigmented (insensitive). 
 
(2) 
(a) A medical history shall be obtained from all subjects with emphasis on the effects 
of sunlight on their skin. Ascertain the general health of the individual, the individual's skin 
type (I, II, or III), whether the individual is taking medication (topical or systemic) that is 
known to produce abnormal sunlight responses, and whether the individual is subject to any 
abnormal responses to sunlight, such as a phototoxic or photoallergic response. The subject 
should not have a history of any conditions that make you more sensitive to sunlight, or a 
history of skin cancer. 
(b) Test site inspection. The physical examination shall determine the presence of 
sunburn, suntan, scars, active dermal lesions, and uneven skin tones on the areas to be 
tested. The presence of nevi, blemishes, or moles will be acceptable if in the physician's 
judgment they will not interfere with the study results. 
5.3.1 Number of Participants 
We anticipate screening 40 individuals in order to enroll 30 individuals. 
5.3.2 Eligibility Criteria/Vulnerable Populations 
Eligibility will be determined by study coordinator, Kacie Carlson, PA-C. Dr. Michael Girardi, 
and Dr. Mark Salzman will not determine subject eligibility or consent subject due to conflicts 
of interest. If there are any questions regarding eligibility, Dr. Ian Odell will be consulted. 
INCLUSION CRITERIA: 
In order to be eligible to participate in this study, an individual must meet all of the following 
criteria: 
1. Provision of signed and dated informed consent form 
2. Stated willingness to comply with all study procedures and availability for the duration 
of the study 
3. Male or female, aged 18 years old or greater 
4. Women of child-bearing potential must have negative urine pregnancy test 
5. In good general health as evidenced by medical history 
6. Fair skinned with Fitzpatrick Scale skin types I, II or III using the following Skin Type 
and Sunburn and Tanning History (based on the first 30-45 minutes of sun exposure 
after a winter season of no sun exposure): 
a. I—always burns easily; never tans (sensitive) 
b. II—always burns easily; tans minimally (sensitive) 
c. III—burns moderately; tans gradually (light brown) (normal) 


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EXCLUSION CRITERIA: 
An individual who meets any of the following criteria will be excluded from participation in 
this study: 
 
1. Individuals with active or a history of dermatological disorders—psoriasis, rosacea, 
eczema, vitiligo, lupus, dermatomyositis, etc 
2. Individuals known to be subject to any abnormal responses to sunlight, such as 
phototoxic or photoallergic response. 
3. Current use of medication (topical or systemic) that is known to produce abnormal 
sunlight responses. 
4. History of skin cancer (such as basal cell carcinoma, squamous cell carcinoma, 
melanoma) 
5. Family history of melanoma 
6. Presence of sunburn, suntan, scars, active dermal lesions or uneven skin tone on the 
test site. 
7. Skin type falling under the Fitzpatrick Scale skin types IV, V or VI using the following 
Skin Type and Sunburn and Tanning History (based on the first 30-45 minutes of sun 
exposure after a winter season of no sun exposure): 
o IV—Burns minimally; always tans well (moderate brown) (normal) 
o V—Rarely burns; tans profusely (dark brown) (insensitive) 
8. Use of sunscreen within the last week on the test site area (such that UV filter 
penetration may confound results) 
9. Febrile illness within 48 hours. 
 
10. Women with a positive urine pregnancy test 
 
 
6 Methods 
6.1 
Treatment 
6.1.1 Identity of Investigational Product 
The sunscreen to be tested will be administered topically. 
The sunscreen contains bioadhesive nanoparticles (BNP) encapsulating avobenzone and 
octocrylene plus the non-toxic natural products diosmin, ferulic acid, cytisine and trans- 
resveratrol. Each is described below: 
The BNP are composed of a biocompatible block copolymer called poly (lactic acid)- 
hyperbranched polyglycerol (PLA-HPG). Poly-lactic acid is a degradable polymer with a 
history of use in medicine, including many FDA-approved medical devices and drug delivery 
systems, stretching back to the 1970s. For example, PLA microspheres are currently 
approved as an injectable dermal filler for cosmetic purposes (Sculptra, injectable poly-(lactic 
acid) microspheres, FDA approval P030050, Aug 3 2004) and a bioabsorbable adhesion 
barrier (REPEL-CV®, FDA approval P070005, March 6, 2009). PLA has been demonstrated 
to be safe for human use. 
Our nanoparticles include a hyperbranched polyglycerol (HPG) surface modification that 
enables increased retention on the outer surface of the stratum corneum. This reduces 


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dermal penetration of the chemical filter, thereby increasing the safety of our sunscreen over 
currently approved over-the counter sunscreens. Because of this bioadhesion, there is little 
opportunity for the components of the nanoparticle to interact with cells, tissues and 
molecules deeper within the skin, or in the rest of the body. HPG is an inert polymer that is 
currently in advanced preclinical testing. Sitka Biopharma is testing nanoparticles of HPG 
and docetaxel for intravesicular treatment of bladder cancer, planning to initiate a phase 1 
clinical trial (http://www.sitkabiopharma.com). In addition, HPG has been tested in animals 
as an osmotic agent for use during peritoneal dialysis. In this application, large quantities of 
HPG are administered into the peritoneal cavity (HPG molecular weight 3 kDa; 2.5% - 15%). 
In this preclinical testing, exposure of the peritoneum to these large doses of HPG is found 
to produce less peritoneal injury than control solutions containing glucose as the osmotic 
agent (48). In addition, HPG appears to be less toxic than alternatives when used as a 
colloid in cold organ preservation solutions (49). Therefore, HPG appears to be inert, even 
when exposed to sensitive tissues deep within the body, such as those in the abdominal 
cavity and the endothelium of harvested organs. 
These nanoparticles will encapsulate only FDA-approved active ingredients and 
combinations as described in the monograph (21 CFR Part 352.1). The proposed human 
studies will use only approved or lower concentrations of UV filters avobenzone (AVO) and 
octocrylene (OCTR). We have conducted in vitro and in vivo studies with AVO and OCTR 
and padimate-O (PO), demonstrating improved safety and decreased penetration in animal 
models. 
We will manufacture the bioadhesive nanoparticles encapsulating avobenzone and 
octocrylene (BNP-A/O) under sterile conditions in Malone Engineering Center. We will 
ensure that the nanoparticles in solution (sterile distilled water) are prepared and handled in 
a sterile environment prior to application to human skin. 
USP grade (US Pharmacopoeia) natural products will be purchased. USP grade products 
meet or exceed the purity requirements of the US Pharmacopoeia and are suitable for food, 
drug, or medicinal use. USP and NF (National Formulary) are the official standards for all 
prescription and over-the-counter medicines, dietary supplements, excipients, and other 
healthcare products manufactured and sold in the US. A single lot of each natural product 
will be used for the entire study. The natural products will be handled in a sterile environment 
in Malone Engineering Center for formulation in vehicle (a standard vehicle approved for use 
in humans, such as Transcutol P or FDA P2 cream vehicle) prior to application to human 
skin. 
6.1.2 Dosage, Administration, Schedule 
Each participant will undergo topical UVR exposure one time for inherent minimal erythema 
dose (MED) determination. The Multiport 601 solar simulator will be used to deliver a 1.25x 
UVR dose series centered around the FDA Standard 1 MED dose for the participant’s 
individual Fitzpatrick Scale skin type. 
Following sunscreen application (below) each participant will undergo a single 1 MED UVR 
exposure, delivered by the Multiport 601 solar simulator, to test sunscreen efficacy. 


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The Multiport 601 solar simulator provides highly uniform UVR (290-400nm) and is fully 
compliant with ISO, FDA, JCIA and COLIPA spectral irradiance standards and all FDA 
sunscreen testing requirements (21 CFR 201.327). Calibrated probes monitor each 
exposure and specifically designed ports ensure directed exposure only to the 8mm 
diameter test sites. 
Formulated sunscreen will be applied topically, one time, on each participant, to a 1.5 x 3 cm 
area at the FDA monograph (21 CFR 201.327) recommended 2 mg formulated sunscreen 
per cm2. Thus, a total of 9 mg of formulated sunscreen will be applied to each participant. 
The formulated sunscreen will contain: 
3% avobenzone (9 mg x 3% = 0.27 mg avobenzone applied) 
9% octocrylene (9 mg x 9% = 0.81 mg octocrylene applied) 
10% diosmin (9 mg x 10% = 0.9 mg diosmin applied) 
10% ferulic acid (9mg x 10% = 0.9 mg ferulic acid applied) 
5% cytisine (9mg x 5% = 0.45 mg cytisine applied) 
5% trans-resveratrol (9mg x 5% = 0.45 mg trans-resveratrol applied) 
 
Sunscreen preparation: 
The sunscreen will be prepared under sterile conditions in Dr. Saltzman’s laboratory in the 
Department of Biomedical Engineering’s Malone Engineering Center (MEC). Over 2,000 sq. 
ft. of this space is solely committed to the laboratory efforts of Dr. Saltzman. This laboratory 
space includes a wide range of specialized equipment needed for bioengineering research. 
The sunscreen will be prepared following a written procedure with detailed record-keeping 
regarding raw ingredients, lot numbers, dates tested/used, measured amounts, and drug 
yields. All equipment and facilities used in manufacturing, such as biosafety cabinets and 
chemical hoods, will be clean and properly maintained. 
Over the course of the study, one lot of bioadhesive nanoparticle (BNP) sunscreen will be 
used. We will perform quality assurance testing prior to release. Non-adhesive nanoparticles 
will be stored at -80° C. Prior to use, these nanoparticles will be incubated in sodium 
periodate for conversion to the bioadhesive form and resuspended in water for use at a 
concentration of 250 mg/ml - 750 mg/ml BNP. We will ensure that the final concentration of 
the active ingredients do not exceed the limits set by the FDA monograph (3% avobenzone, 
10% octocrylene). Stability of these nanoparticles will be tested every month over the 
duration of this study to ensure quality of the product. 
Prior to use, USP grade natural products will be stored in the dark at room temperature. 
These products will be handled in a sterile environment in Malone Engineering Center for 
formulation in vehicle (a standard vehicle approved for use in humans, such as Transcutol P 
or FDA P2 cream vehicle) prior to application to human skin. Stability of the formulation will 
be tested every month over the duration of this study to ensure quality of the product. 
 
6.1.3 Method of Assignment/Randomization 


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N/A 
6.1.4 Blinding and Procedures for Unblinding 
Application of the sunscreen and collection of biopsies is not blinded, but all analysis of 
biopsies (measurement of CPDs, gammaH2AX, and 3-nitrotyrosine) will be done by 
laboratory staff blinded to the biopsy sample characteristics. 
6.1.5 Packaging/Labelling 
Nanoparticles used in the pilot study will be produced under sterile conditions in Dr. 
Saltzman’s laboratory in the Department of Biomedical Engineering’s Malone Engineering 
Center (MEC) as a single batch for quality control purposes and monitoring. Previous work 
has confirmed that these nanoparticles are stable beyond one year. Single lots of each USP 
grade natural product will also be used and are stable beyond one year. Formulated 
sunscreen will be produced under sterile conditions in Dr. Saltzman’s laboratory using a 
standard vehicle approved for use in humans, such as Transcutol P or FDA P2 cream 
vehicle. Formulated sunscreen will be aliquoted under sterile conditions into single use 
sealed aliquots properly labeled for use in this study. Delivery to the study personnel at HRT 
4 will be based upon participant enrollment. 
6.1.6 Storage Conditions 
Formulated sunscreen will be stored in the dark at 4°C prior to use. Following application of 
sunscreen on a participant, the remaining sunscreen will be disposed as per Yale OEHS 
guidelines in Dr. Girardi’s laboratory. 
6.1.7 Concomitant therapy 
Subjects will be excluded from the study if currently taking any photosensitizing drugs (see 
exclusion criteria). Litt's Drug Eruption & Reaction Manual, Shear, Neil H. 2022 
6.1.8 Restrictions 
There are no restrictions. Subjects will be given post-biopsy instructions not to remove the 
band aid for 24 hrs. 
6.2 
Assessments 
6.2.1 Efficacy 
Efficacy will be evaluated by analysis of the biopsy tissue: 
Each 3 mm punch biopsy will be bisected. Half will be snap frozen in liquid nitrogen 
and half will be fixed in 10% neutral buffered formalin. The tissue will be used to 
assess the effectiveness of the sunscreen by measuring the level of DNA and cellular 
damage in UVR-exposed vs unexposed and sunscreen treated vs untreated samples 
by the following 3 methods: 
1. DNA will be prepared and assayed by ELISA for quantification of CPDs. CPDs 
measured in samples obtained immediately after UVR exposure are indicative of 
direct DNA damage. CPDs will also be measured in samples obtained 4 hr after UVR 
exposure, at which time any increase in the CPD level, as compared to time 0, is 
indicative of so called “dark” CPDs that form in response to indirect oxidative DNA 
damage. 
2. Formalin fixed paraffin embedded skin will be stained with anti-gH2AX to identify 
DNA strand breaks. Indirect, oxidative DNA damage may result in DNA strand breaks 
that can be quantified by microscopic visualization of gH2AX, which builds up at the 
site of each strand break. 


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3. Formalin fixed paraffin embedded skin will be stained with anti-3-nitrotyrosine to 
identify cellular damage. ROS and high energy triplet state species can result in 
nitration of tyrosine residues of cellular proteins. This type of damage can be 
quantified by microscopic visualization of 3-nitrotyrosine. 
All testing will be done by laboratory staff blinded to the sample characteristics. 
 
 
6.2.2 Safety and Pregnancy-related policy 
The sunscreen will be prepared under sterile conditions in Dr. Saltzman’s laboratory in the 
Department of Biomedical Engineering’s Malone Engineering Center (MEC) following written 
procedures with detailed record-keeping regarding raw ingredients, lot numbers, dates 
tested/used, measured amounts, and yields. All equipment and facilities used in 
manufacturing, such as biosafety cabinets and chemical hoods, will be clean and properly 
maintained. Formulated sunscreen will be aliquoted under sterile conditions into single use 
sealed aliquots properly labeled for use in this study. Stability of the sunscreen will be tested 
every month over the duration of this study to ensure quality of the product. For quality 
control, we will conduct characterization studies to examine nanoparticle size, via dynamic 
light scattering, and in vitro UVR absorbance (for in vitro SPF and critical wavelength), via 
plate assays. Before proceeding, we will ensure that these parameters are consistent with 
those previously used in preclinical animal studies. 
Risks will be minimized by: 
1. Using the minimal amount (1 MED) of UVR necessary to obtain the primary objective, and 
minimizing the area exposed to UVR by using the Multiport 601 solar simulator which limits 
UVR exposure to 8mm diameter (0.5 cm2) spots. 
2. Using FDA approved UVR filters encapsulated in nanoparticles (avobenzone and 
octocrylene) and formulating the sunscreen such that FDA approved doses are not 
exceeded (FDA OTC monograph specifies 3% avobenzone and 10% octocrylene). 
3. Using non-toxic natural products (ferulic acid, cytisine, diosmin, trans-resveratrol) and 
topical doses of these products that are below a) doses considered safe for repeated oral 
administration for other indications, or b) amounts used in existing cosmetic treatments 
4. Trained and experienced personnel will supervise subjects during every phase of 
participation. Personnel will be instructed to be aware of any warning signs of impending 
danger and will stop any procedure at that time. 
5. Using sterile skin biopsy techniques performed by a board-certified dermatologist, 
observing sites for 15 minutes after skin biopsy ensure adequate bleeding control, and 
provided post-biopsy care instructions (change bandage, wash site with soap and water 
daily, apply ointment and band aid daily; call for any redness, pain, swelling, or discharge; 
return in 7-10 days for suture removal). 
Since we do not know the teratogenicity of the proposed natural products, women of child- 
bearing potential will undergo urine pregnancy testing prior to application of topical study 


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medications. If the test is negative, we will proceed with topical application study agents and 
UV exposure. 
 
 
6.2.3 Adverse Events Definition and Reporting 
Adverse events (skin irritancy, hypersensitivity, follicular occlusion effects) will be monitored 
for each participant from the time of the start of the study (first UVR exposure) through the 
end of intervention. 
Adverse events will be monitored by the personnel involved in the testing and administration 
of the study. 
Adverse events will be monitored immediately following UVR exposure (visit #1), 16-24 hr 
later (start of visit #2), 15 min following application of sunscreen (visit #2), immediately 
following 1 MED UVR exposure (visit #2), 4 hr post 1 MED UVR exposure (visit #3). 
Monitoring will include visual inspection and discussion with the participant. 
The Principal Investigator will be responsible for determining whether an AE is expected or 
unexpected. An AE will be considered unexpected if the nature, severity, or frequency of the 
event is not consistent with the risk information previously described for the study 
intervention. 
Adverse events will be attributed to the study procedures / design by the principal 
investigator Dr. Michael Girardi as definitely related, probably related, potentially related, 
unlikely to be related, or not related. 
Adverse events will be graded in severity according to the following scale using conditions 
noted during the study: 
• Mild: Awareness of sign or symptom, but easily tolerated. 
• Moderate: Discomfort enough to cause interference with usual activity. 
• Severe: Incapacitating with inability to work or do usual activity. 
• Not applicable: In some cases, an adverse event may be an “all or nothing” 
finding, which cannot be graded. 
Reporting: 
The principal investigator will report the following types of events to the IRB and all co- 
investigators listed on the protocol: 
Any incident, experience or outcome that meets ALL 3 of the following criteria: 
1. Is unexpected (in terms of nature, specificity, severity, or frequency) given (a) the research 
procedures described in the protocol-related documents, such as the IRB-approved protocol 
and informed consent document and (b) the characteristics of the subject population being 
studied; AND 
2. Is related or probably related to participation in the research (probably related means There 
is evidence to suggest a causal relationship, and the influence of other factors is unlikely); 


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AND 
3. Suggests that the research places subjects or others at greater risk of harm (including 
physical, psychological, economic, legal, or social harm) than was previously known or 
recognized. 
Unanticipated Problems Involving Risks to Subjects or Others (UPIRSOs) may be medical or 
non-medical in nature, and include – but are not limited to – serious, unexpected, and related 
adverse events and unanticipated adverse device effects. Please note that adverse events 
are reportable to the IRB as UPIRSOs only if they meet all 3 criteria listed above. 
These UPIRSOs/SAEs will be reported to the IRB in accordance with IRB Policy 710, using 
the appropriate forms found on the website. All related events involving risk but not meeting 
the prompt reporting requirements described in IRB Policy 710 should be reported to the IRB 
in summary form at the time of continuing review. If appropriate, such summary may be a 
simple brief statement that events have occurred at the expected frequency and level of 
severity as previously documented. In lieu of a summary of external events, a current DSMB 
report can be submitted for research studies that are subject to oversight by a DSMB (or 
other monitoring entity that is monitoring the study on behalf of an industry sponsor). 
The principal investigator, Michael Girardi, will conduct a review of all adverse events upon 
completion of every study subject. The principal investigator will evaluate the frequency and 
severity of the adverse events and determine if modifications to the protocol or consent form 
are required. 
6.2.4 Pharmacokinetics (if applicable) 
N/A 
6.2.5 Biomarkers (if applicable) 
N/A 
6.3 
Study Procedures 
All procedures are being performed exclusively for research purposes. 
Visit 1: Subjects will first visit the study center (HRT4) for screening, informed consent, and a 
physical examination with comprehensive medical history. If eligibility requirements are met, 
the participant will undergo initial evaluation and a test site area equivalent to the Multiport 
601 test patch will be delineated in indelible ink on the participant’s upper inner arm. This 
test site will be used for determination of the minimal erythema dose (MED). The minimal 
erythema dose (MED) is defined as the smallest UVR dose that produces perceptible 
redness of the skin with clearly defined borders 16-24 hours after UVR exposure. The 
Multiport 601 solar simulator will be used to deliver a series of six exposures (to the six 
subsites of the test pad) to untreated, unprotected skin to determine the subject's inherent 
MED. The doses selected shall be based on the Standard for the individuals Fitzpatrick 
Scale skin type and shall be a geometric series represented by (1.25n), wherein each 
exposure time interval is 25 percent greater than the previous time to maintain the same 
relative uncertainty (expressed as a constant percentage), independent of the subject's 
sensitivity to UVR radiation. This inherent MED (Unprotected Skin) shall be used to 


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determine the dose of UVR (equal to 1 MED) to be administered in subsequent sunscreen 
efficacy testing. The UVR will be administered by Research Personnel. The participant will 
be instructed to return to the clinic 16-24 hr post UVR exposure. 
Visit 2. 16-24 hr post UVR exposure, the participant will return to the clinic. The MED test 
area will be digitally photographed and recorded by Research Personnel. Dr. Girardi will 
read the MED, identifying the lowest dose of UVR that results in perceptible redness of the 
skin. This dose = 1 MED. 
A new test site area, near the MED test area, equivalent to the Multiport 601 test patch will 
be delineated in indelible ink on the subject’s arm. Six subsites corresponding to the 6 
openings of the test patch will also be delineated in indelible ink. The two subsites in the 
lower left corner of the total site (two adjacent subsites are equal to 1.5 x 3 cm = 4.5 cm2) will 
be treated with sunscreen: Research Personnel will apply the sunscreen in a thin film (2 mg/ 
cm2, as described by the FDA) to the delineated 4.5 cm2 area. The remainder of the area will 
remain untreated. Fifteen minutes after sunscreen application, the entire test site will be 
digitally photographed for visual record of irritation. The Multiport 601 solar simulator will be 
used to deliver a dose = 1 MED UVR to the two lower left (sunscreen treated) and two lower 
right (untreated) subsites of the test patch. The UVR will be administered by Research 
Personnel. The top two subsites will not receive UVR. 
Immediately following UVR exposure, 3 mm punch biopsies will be obtained by Dr. Girardi 
from the appropriate subsites of the test area: 
1. Untreated skin, No UVR 
2. Untreated skin + 1 MED UVR 
3. Sunscreen treated skin + 1 MED UVR 
Research Personnel will immediately bisect each biopsy, snap freeze half in liquid nitrogen, 
and fix half in 10% neutral buffered formalin. 
The participant will depart with instructions to return 4 hours after their UVR exposure. 
Visit #3. 4 hr after the UVR exposure of visit #2, the participant will return to the clinic. 3 mm 
punch biopsies will be obtained by Dr. Girardi from the appropriate subsites of the test area: 
4. Untreated skin + 1 MED UVR 
5. Sunscreen treated skin + 1 MED UVR 
Research Personnel will immediately bisect each biopsy, snap freeze half in liquid nitrogen, 
and fix half in 10% neutral buffered formalin. 
The participant will depart with instructions on post-biopsy wound care and contact 
information for study personnel, if there are any concerns about the biopsy sites 
The participant will be compensated at the conclusion of visit #3. 
6.3.1 Study Schedule 
Four total visits are expected. 


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Schedule of Events: 
Visit #1: 
Consent 
Medical history and physical examination 
UVR exposure for inherent MED determination 
Visit #2: 
Evaluate and record inherent MED 
Sunscreen application 
15 min wait 
UVR exposure = 1 MED for sunscreen efficacy testing 
3 x 3mm biopsies collected 
Visit #3: 
2 x 3mm biopsies collected 
Give instructions for biopsy site care 
Give study personnel contact information if any concerns about skin biopsy 
sites / healing 
Visit #4: 
Follow-up 7-10 days after biopsies for suture removal. 
 
6.3.2 Informed Consent 
The investigation will be conducted in compliance with the requirements for institutional 
(HIC) review and with the requirements for informed consent of the FDA regulations (21 CFR 
Part 50 and 21 CFR Part 56). 
All subjects will be of legal age such that parental or surrogate permission is not required. 
Enrollment is voluntary. Potential subjects interested in enrolling will initiate the process. 
Research personnel will confirm the subject’s consent during the eligibility screening. An 
informed consent form will be completed prior to the start of the before the research protocol. 
We stress that they are under no obligation to sign the consent form or participate in the 
study if they are uncomfortable with any aspects of it. We also stress that they will not be 
penalized if they decide not to participate. When the subject arrives for the consent process, 
we review the protocol with them and indicate that they should ask any questions or express 
concerns before signing the consent form. In addition, we tell the subjects that they are 
allowed to withdraw their consent at any time without prejudice and are advised of this fact 
prior to participating. We also inform the subjects that they may be disqualified based on 
information gathered on their first visit. 
Research personnel will verbally assess the subject’s capacity to provide consent. During 
the initial interview, the potential subject will be able to ask questions and express concerns 
about participation in conversation. If the subject cannot speak English well enough to 
understand the protocol, we do not allow them to continue the consent process. 
6.3.3 Screening 
Eligibility will be determined by personnel involved in the testing and administration of the 
study: Kacie Carlson, PA-C. Eligibility will be determined by study coordinator, Kacie 
Carlson, PA-C. Dr. Michael Girardi, and Dr. Mark Salzman will not determine subject 


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eligibility or consent subject due to conflicts of interest. If there are any questions regarding 
eligibility, Dr. Ian Odell will be consulted. 
Screening will include comprehensive medical history and a physical exam. 
(a) A medical history shall be obtained from all subjects with emphasis on the effects of 
sunlight on their skin. Ascertain the general health of the individual, the individual's 
skin type (I, II, or III), whether the individual is taking medication (topical or systemic) 
that is known to produce abnormal sunlight responses, and whether the individual is 
subject to any abnormal responses to sunlight, such as a phototoxic or photoallergic 
response. The subject should not have a history of any conditions that make you 
more sensitive to sunlight, or a history of skin cancer. The subject should not have 
applied sunscreen to the test area in the past week. 
(b) Test site inspection. The physical examination shall determine the presence of sunburn, 
suntan, scars, active dermal lesions, and uneven skin tones on the areas to be 
tested. The presence of nevi, blemishes, or moles will be acceptable if in the 
physician's judgment they will not interfere with the study results. 
(c) 
Women of child-bearing potential will be given a urine pregnancy test. If it is positive, 
they will not be enrolled. 
Subjects who fail screening due to application of sunscreen within the past week may be re- 
screened one week later. 
6.3.4 Enrollment 
Participants who have provided informed consent and who have been screened and meet 
eligibility criteria will be enrolled in the study by Kacie Carlson, PA-C. Eligibility will be 
determined by study coordinator, Kacie Carlson, PA-C. Dr. Michael Girardi, and Dr. Mark 
Salzman will not determine subject eligibility or consent subject due to conflicts of interest. If 
there are any questions regarding eligibility, Dr. Ian Odell will be consulted. 
On Study Visits 
• 
Visit #1, in order: consent, screen for eligibility, UVR exposure for MED determination 
• 
Visit #2, in order: read MED, apply sunscreen, UVR exposure = 1 MED, 3 biopsies 
taken 
• 
Visit #3: 2 additional biopsies taken 
• 
Visit #4: suture removal from biopsy sites 
Please refer to Schedule of Events (6.3.1) and procedures (6.3) above for more detail. 
6.3.5 End of Study and Follow-up 
At the end of the visit #3 participants will receive instructions for biopsy care, study staff 
contact information, and given an appointment to return in 7-10 days for suture removal. 
6.3.6 Removal of subjects 
Participants may withdraw voluntarily at any time for any reason. 
If a participant experiences an unexpected adverse event immediately following the UVR 
exposure for MED determination (visit 1), e.g. a photoallergic or phototoxic reaction, 
assessed by visual inspection and discussion with the participant, the event will be 
documented and the participant excluded from further study. 


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If a participant experiences an unexpected reaction to the sunscreen within 15 min of 
application (visit #2), as assessed by visual inspection and discussion with the participant, 
the event will be documented, and the participant will be excluded from further study. 
6.3.7 Statistical Design 
 
We will initially test the hypotheses using paired t-tests, e.g. comparing the CPDs within 
each subject, assuming a normal distribution. If needed, we will employ a non-parametric 
test. 
 
6.3.8 Sample Size Considerations 
 
To test the hypothesis that our sunscreen provides 90% protection over untreated skin, a 
subject with ≥90% difference within e.g., their immediate CPD values, will be considered a 
“success” and a less <90% difference will be a “failure.” We will summarize each patient’s 
outcome as binary-valued and analyze the data by binomial reference distribution. Analysis 
of the data as binomial facilitates a study of the power and sample size. The null hypothesis 
is an equal frequency of successes and failures, corresponding to a binomial parameter of 
p=0.5. We will assume two-tailed significance tests with (alpha) level 0.1, typical in such 
small clinical trials. The population p parameters that will exhibit 80% power for different 
sample sizes is p < 0.26 or p > 0.74 (25 subjects), p < 0.28 or p > 0.72 (30 subjects), and p < 
0.29 or p > 0.71 (35 subjects). Values were computed in R using the exact binomial 
distribution. We anticipate enrolling 30 subjects in the study. These calculations demonstrate 
only small changes in power for all sample sizes in the range illustrated here. 
 
6.4 
Planned Analyses 
6.4.1 Primary Objective Analysis 
1. Direct DNA damage assessed by the CPD level in biopsies obtained immediately 
following 1 MED UVR exposure. CPDs will be quantified by ELISA and we will compare the 
level of CPDs in untreated skin exposed to 1 MED UVR to the level of CPDs in sunscreen 
treated skin exposed to 1 MED UVR. We hypothesize that sunscreen will reduce UVR- 
induced CPDs by ≥90% within each participant. The level of CPDs in skin that was not 
exposed to UVR (negative control in ELISA) may be used to normalize the data between 
patients. Data will be analyzed by t-test, assuming a normal distribution. A non-parametric 
test will be used if needed. 
2. Indirect DNA damage will be assessed in two ways: 
a) by assessing the CPD level in untreated, UVR exposed skin immediately following 
UVR exposure (T0) vs 4 hr post UVR exposure. The CPD level at T=4hr minus the CPD 
level at T=0 defines “dark” CPDs that result from indirect DNA damage. This “dark” CPD 
level will be compared between untreated UVR exposed skin and sunscreen treated UVR 
exposed skin. We hypothesize that sunscreen will reduce “dark” CPDs by ≥90% within each 
participant. Data will be analyzed by t-test, assuming a normal distribution. A non-parametric 
test will be used if needed. 
b) by comparing the gH2AX level in untreated UVR exposed skin vs sunscreen 
treated UVR exposed skin using T0 biopsies and using T=4hr biopsies. gH2AX is detectable 
at sites of DNA strand breaks, which are associated with indirect DNA damage. gH2AX will 


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be measured by quantitative immunofluorescent staining with image analysis to determine 
the integrated density of gH2AX signal within the nuclear area (defined by DAPI stain). We 
hypothesize that sunscreen will reduce gH2AX signal by ≥90% within each participant. Data 
will be analyzed by t-test, assuming a normal distribution. A non-parametric test will be used 
if needed. 
3. Indirect cellular damage will be assessed by comparing the 3-nitrotyrosine level in 
untreated UVR exposed skin vs sunscreen treated UVR exposed skin using T0 biopsies and 
using T=4hr biopsies. 3-nitrotyrosine is detectable in proteins suffering indirect UVR 
damage, via ROS. 3-nitrotyrosine will be measured by quantitative immunofluorescent 
staining with image analysis to determine the integrated density of 3-nitrotyrosine signal. We 
hypothesize that sunscreen will reduce 3-nitrotyrosine signal by ≥90% within each 
participant. Data will be analyzed by t-test, assuming a normal distribution. A non-parametric 
test will be used if needed. 
6.4.2 Secondary Objectives Analyses 
N/A 
6.4.3 Exploratory Objectives Analyses (if applicable) 
N/A 
6.4.4 Safety 
Dermal irritation, allergic hypersensitivity, and follicular occlusion effects will be evaluated by 
Dr. Girardi. Specifically, clinical evaluation will assess evidence of erythema, edema, 
xerosis, desquamation, dermatitis, follicular occlusion on a 4-point scale as follows: 0=none; 
1=mild; 2=moderate; 3=severe. Subjective assessment of symptoms will be provided by 
each subject by verbal response to Dr. Girardi’s questioning regarding the following: burning, 
stinging, pruritus, tingling. Assessment will take place 15 min and 4 hr following application 
of sunscreen. 
6.4.5 Analysis of Subject Characteristics 
Healthy adult individuals with fair skin are more susceptible to the UVR-induced DNA 
damage that we seek to prevent by development of a safer, more effective sunscreen, and 
thus will comprise the study population. 
6.4.6 Interim Analysis (if applicable) 
N/A 
6.4.7 Health economic evaluation 
N/A 
6.4.8 Other 
No other analysis will be done. 
6.4.9 Subsets and Covariates 
N/A 
6.4.10 Handling of Missing Data 


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If either T=4hr biopsy is missing, only direct DNA damage will be assessed for that 
participant using the T0 biopsies and CPD quantification by ELISA. 
If a T0 biopsy is missing, but both T=4hr biopsies are available, only indirect DNA damage 
(by gH2AX) and indirect cellular damage (by 3-nitrotyrosine) will be assessed for that 
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7 Trial Administration 
7.1 
Ethical Considerations: Informed Consent/Assent and HIPAA Authorization 
This study will be conducted in accordance with the ethical principles that have their origin in 
the Declaration of Helsinki. 
There will be no deception; participants are informed of all drugs/procedures. 
Because the study does require considerable time and effort (3 timed visits and collection of 
5 biopsies), subjects who adhere strictly to the aforementioned research protocols will 
receive a one-time payment of $200 upon completion of the study. 
The subjects will provide private information on a medical history form** to indicate any 
reason to exclude them from the study. Only Research Personnel Michael Girardi, MD and 
Kacie Carlson, PA-C will have access to this information. Each subject will have a file that 
includes their medical history and consent form. These are kept in a locked file cabinet in the 
study coordinators office (HRT 4096). Only Michael Girardi, MD and Kacie Carlson, PA-C 
have access to the file cabinet. Each subject will be assigned a code number and all 
samples collected will be identified by this number. Laboratory staff performing sample 
testing will only be provided with the code numbers. Data resulting from testing will be stored 
on a computer and/or a laboratory notebook and will only be identified by sample code 
number. 
**Medical history to be collected from all subjects will focus on the 
• 
effects of sunlight on their skin to ascertain the individual's skin type (I, II, or III) 
• 
whether the individual is taking medication (topical or systemic) that is known to 
produce abnormal sunlight responses 
• 
whether the individual is subject to any abnormal responses to sunlight, such as 
a phototoxic or photoallergic response. 
• 
history of skin cancer to include squamous cell carcinoma, basal cell carcinoma 
or melanoma 
• 
history of skin disease (such as psoriasis, eczema, dermatomyositis, lupus or 
vitiligo) 
• 
family history of melanoma 
7.2 
Institutional Review Board (IRB) Review 
The protocol will be submitted to the IRB for review and approval. Approval of the protocol 
must be obtained before initiating any research activity. Any change to the protocol or study 
team will require an approved IRB amendment before implementation. The IRB will 
determine whether informed consent and HIPAA authorization are required. 
The IRB will conduct continuing review at intervals appropriate to the degree of risk, but not 
less than once per year. 
A study closure report will be submitted to the IRB after all research activities have been 
completed. 


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Other study events (e.g. data breaches, protocol deviations) will be submitted per Yale’s IRB 
policies. 
7.3 
Subject Confidentiality 
Subject confidentiality is held in strict trust by the research team. Confidentiality of all 
information in the study will be maintained by identifying subjects by unique code numbers. 
No subjects are identified by name in any of the published literature and only by code in data 
storage areas, to which access is limited to Research Personnel. The individual subject files 
are the only place where names are noted and linked to their unique code number, and these 
files are kept in a locked file cabinet in the office of the PI. Access to this file cabinet is limited 
to Michael Girardi and Kacie Carlson. To date, we have never had a violation of confidentiality. 
The Yale Human Investigation Committee may inspect all study records. 
7.4 
Deviations/Unanticipated Problems 
If the study team becomes aware of an anticipated problem (e.g. data breach, protocol 
deviation), the event will be reported to the IRB by PI Michael Girardi. 
7.5 
Data Collection 
At visit #1 the subjects will provide private information on a medical history form to indicate 
any reason to exclude them from the study. Only Research Personnel Michael Girardi, MD 
and Kacie Carlson, PA-C will have access to this information. Each subject will have a file 
that includes their medical history and consent form. These are kept in a locked file cabinet 
in the study coordinators office (HRT4096) office. Only Michael Girardi, MD and Kacie 
Carlson, PA-C will have access to the file cabinet. Each subject will be assigned a code 
number and all samples collected will be identified only by this number. Laboratory staff 
performing sample testing will only be provided with the deidentified code numbers. Data 
resulting from sample testing will be stored on a computer and/or in a laboratory notebook 
and will only be identified by sample code number. 
7.6 
Data Quality Assurance 
Dr. Girardi will be responsible for monitoring the data, assuring protocol compliance, and 
conducting the safety reviews at the specified frequency, which must be conducted at a 
minimum of every 6 months (including when reapproval of the protocol is sought). During 
the review process, the principal investigator (monitor) will evaluate whether the study 
should continue unchanged, require modification/amendment, or close to enrollment. 
Due to Conflict of Interests declared by Dr. Michael Girardi, Dr. Mark Salzman and Julia 
Lewis, PhD, Dr. Jeffrey Gehlhausen, Assistant Professor of Dermatology – YUSM, will 
provide and independent review of the raw data and data analysis. 
A single medical history form will be used throughout the study. 
All sample testing will be performed in Dr. Girardi’s laboratory by staff who have been trained 
in all test procedures and who will follow a single standardized testing protocol. 
7.7 
Study Records 


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The study protocol, consent forms, subject medical records and code numbers, and data 
generated from subject samples will be considered study records. 
7.8 
Access to Source Documents 
The subject’s medical history form will be used to determine study eligibility. Only Research 
Personnel Michael Girardi and Kacie Carlson will have access to these forms / documents. 
7.9 
Data or Specimen Storage/Security 
De-identified specimens will be stored in Dr. Girardi’s locked research laboratory (HRT 618). 
Data collected from specimen testing will be stored on a computer and/or a laboratory 
notebook and will be identified only by the specimen code number. 
7.10 Retention of Records 
Study documents should be retained for a minimum of 2 years after the last approval of a 
marketing application in an International Conference on Harmonisation (ICH) region and 
until there are no pending or contemplated marketing applications in an ICH region or until at 
least 2 years have elapsed since the formal discontinuation of clinical development of the 
study intervention. These documents should be retained for a longer period, however, if 
required by local regulations. No records will be destroyed without the written consent of the 
sponsor, if applicable. It is the responsibility of the sponsor to inform the investigator when 
these documents no longer need to be retained. 
Study documents will be maintained for a minimum of 3 years, as required by the funding 
organization (NIH). 
7.11 Study Monitoring 
Dr. Girardi and Kacie Carlson, PA-C will be responsible for monitoring the data, assuring 
protocol compliance, and conducting the safety reviews. Monitoring will occur throughout 
the entire study, after each patient completes the study visits. Modifications will be made if 
needed based on any issues that may arise. The IRB will be notified of any modifications. 
7.12 Data Safety Monitoring Plan 
Dr. Girardi will be responsible for monitoring the data, assuring protocol compliance, and 
conducting the safety reviews at the specified frequency, which must be conducted at a 
minimum of every 6 months (including when reapproval of the protocol is sought). During 
the review process, the principal investigator (monitor) will evaluate whether the study 
should continue unchanged, require modification/amendment, or close to enrollment. 
Due to Conflict of Interests declared by Dr. Michael Girardi, Dr. Mark Salzman and Julia 
Lewis, PhD, Dr. Jeffrey Gehlhausen, Assistant Professor of Dermatology – YUSM, will 
provide and independent review of the raw data and data analysis. 
Either the principal investigator, or the Yale HIC shall have the authority to stop or suspend 
the study or require modifications. 
The risks associated with the current study are deemed greater than minimal for the following 
reasons: 
(1) pre-clinical testing showed no evidence of skin irritancy or hypersensitivity for the natural 
products or BNP-sunscreen, with or without UV exposure; (2) our BNP technology does not 


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require covalent modifications to encapsulate the UV filter, and instead physically entraps 
these agents with excipients, such as PLA, which are generally recognized as safe (GRAS); 
(3) per the FDA OTC Monograph that details the active ingredients and concentrations 
permissible without need for additional approval (Code for Federal Regulations-Part 352), 
we will adhere to these guidelines and formulate the active ingredients accordingly, for which 
avobenzone and octocrylene have been approved for use at up to 3% and 10%, 
respectively. 4) Since we do not know the teratogenicity of the proposed natural products, 
women of child-bearing potential will undergo urine pregnancy testing prior to application of 
topical study medications. If the test is negative, we will proceed with topical application 
study agents and UV exposure. 
This protocol presents greater than minimal risks to the subjects and Unanticipated 
Problems Involving Risks to Subjects or Others (UPIRSOs), including adverse events, are 
not anticipated. In the unlikely event that such events occur, Reportable Events (which are 
events that are serious or life-threatening and unanticipated (or anticipated but occurring 
with a greater frequency than expected) and possibly, probably, or definitely related) or 
Unanticipated Problems Involving Risks to Subjects or Others that may require a temporary 
or permanent interruption of study activities will be reported immediately (if possible), 
followed by a written report within 5 calendar days of the Principal Investigator becoming 
aware of the event to the IRB (using the appropriate forms from the website) and any 
appropriate funding and regulatory agencies. The investigator will apprise fellow 
investigators and study personnel of all UPIRSOs and adverse events that occur during the 
conduct of this research project through regular study meetings, and via email as they are 
reviewed by the principal investigator. 
The protocol’s research monitor(s), e.g., Data and Safety Monitoring Boards, study 
sponsors, funding and regulatory agencies, and regulatory and decision-making bodies will 
be informed of adverse events within 5 days of the event becoming known to the principal 
investigator. 
Although we have assessed the proposed study as one of greater than minimal risk, the 
potential exists for anticipated and/or unanticipated adverse events, serious or otherwise, to 
occur since it is not possible to predict with certainty the absolute risk in any given individual 
or in advance of first-hand experience with the proposed study methods. Therefore, we 
provide a plan for monitoring the data and safety of the proposed study as follows: 
 
 
Attribution of Adverse Events: 
Adverse events will be monitored for each subject participating in the study and attributed to 
the study procedures / design by the principal investigator (Insert Investigator Name) 
according to the following categories: 
a.) Definite: Adverse event is clearly related to investigational procedures(s)/agent(s). 
b.) Probable: Adverse event is likely related to investigational procedures(s)/agent(s). 
c.) Possible: Adverse event may be related to investigational procedures(s)/agent(s). 


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d.) Unlikely: Adverse event is likely not to be related to the investigational 
procedures(s)/agent(s). 
e.) Unrelated: Adverse event is clearly not related to investigational procedures(s)/agent(s). 
Plan for Grading Adverse Events: 
The following scale will be used in grading the severity of adverse events noted during the 
study: 
1. Mild adverse event 
2. Moderate adverse event 
3. Severe 
Plan for Determining Seriousness of Adverse Events: 
Serious Adverse Events: 
In addition to grading the adverse event, the PI will determine whether the adverse event 
meets the criteria for a Serious Adverse Event (SAE). An adverse event is considered 
serious if it results in any of the following outcomes: 
1. Death; 
2. A life-threatening experience in-patient hospitalization or prolongation of existing 
hospitalization; 
3. A persistent or significant disability or incapacity; 
4. A congenital anomaly or birth defect; OR 
5. Any other adverse event that, based upon appropriate medical judgment, may 
jeopardize the subject’s health and may require medical or surgical intervention to 
prevent one of the other outcomes listed in this definition. 
An adverse event may be graded as severe but still not meet the criteria for a Serious 
Adverse Event. Similarly, an adverse event may be graded as moderate but still meet the 
criteria for an SAE. It is important for the PI to consider the grade of the event as well as its 
“seriousness” when determining whether reporting to the IRB is necessary. 
Plan for reporting UPIRSOs (including Adverse Events) to the IRB 
The principal investigator will report the following types of events to the IRB: 
Any incident, experience or outcome that meets ALL 3 of the following criteria: 
1. Is unexpected (in terms of nature, specificity, severity, or frequency) given (a) the 
research procedures described in the protocol-related documents, such as the IRB- 
approved protocol and informed consent document and (b) the characteristics of 
the subject population being studied; AND 
2. Is related or possibly related to participation in the research (possibly related 
means there is a reasonable possibility that the incident, experience, or outcome 
may have been caused by the procedures involved in the research); AND 
3. Suggests that the research places subjects or others at greater risk of harm 
(including physical, psychological, economic, legal, or social harm) than was 
previously known or recognized. 


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Unanticipated Problems Involving Risks to Subjects or Others (UPIRSOs) may be 
medical or non-medical in nature, and include – but are not limited to – serious, 
unexpected, and related adverse events and unanticipated adverse device effects. Please 
note that adverse events are reportable to the IRB as UPIRSOs only if they meet all 3 
criteria listed above. 
These UPIRSOs/SAEs will be reported to the IRB in accordance with IRB Policy 710, using 
the appropriate forms found on the website. All related events involving risk but not meeting 
the prompt reporting requirements described in IRB Policy 710 should be reported to the IRB 
in summary form at the time of continuing review. If appropriate, such summary may be a 
simple brief statement that events have occurred at the expected frequency and level of 
severity as previously documented. In lieu of a summary of external events, a current DSMB 
report can be submitted for research studies that are subject to oversight by a DSMB (or 
other monitoring entity that is monitoring the study on behalf of an industry sponsor). 
Plan for reporting adverse events to co-investigators on the study, as appropriate the 
protocol’s research monitor(s), e.g., industrial sponsor, Yale Cancer Center Data and 
Safety Monitoring Committee (DSMC), Protocol Review Committee (PRC), DSMBs, 
study sponsors, funding and regulatory agencies, and regulatory and decision- 
making bodies. 
For the current study, the following individuals, funding, and/or regulatory agencies will be 
notified (choose those that apply): 
X All Co-Investigators listed on the protocol. 
 
X Yale IRB 
 
The principal investigator Michael Girardi, MD will conduct a review of all adverse events 
upon completion of every study subject. The principal investigator will evaluate the frequency 
and severity of the adverse events and determine if modifications to the protocol or consent 
form are required. 
7.13 Study Modification 
Dr. Girardi will be responsible for monitoring the data, assuring protocol compliance, and 
conducting the safety reviews at the specified frequency, which must be conducted at a 
minimum of every 6 months (including when reapproval of the protocol is sought). During 
the review process, the principal investigator will evaluate whether the study should continue 
unchanged, require modification/amendment, or close to enrollment. 
All proposed modifications will be submitted to the IRB as protocol modifications and 
changes will be implemented upon IRB approval. 
7.14 Study Discontinuation 
If the study is prematurely terminated or suspended, the Principal Investigator (PI) will 
promptly inform study participants and the Institutional Review Board (IRB). Circumstances 
regarding the termination or suspension will be provided. Study participants will be 
contacted, as applicable, and be informed of changes to study visit schedule. 


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Circumstances that may warrant termination or suspension include, but are not limited to: 
• 
Determination of unexpected, significant, or unacceptable risk to participants 
• 
Demonstration of efficacy that would warrant stopping 
• 
Insufficient compliance to protocol requirements 
• 
Data that are not sufficiently complete and/or evaluable 
• 
Determination that the primary endpoint has been met 
• 
Determination of futility 
7.15 Study Completion 
The study will be complete when subjects have been enrolled and data analyzed. The IRB 
will be notified when subjects are enrolled, and data analyzed. At this time, the study will be 
closed. 
7.16 Conflict of Interest Policy 
The independence of this study from any actual or perceived influence, such as by the 
pharmaceutical industry, is critical. Therefore, any actual conflict of interest of persons who 
have a role in the design, conduct, analysis, publication, or any aspect of this trial will be 
disclosed and managed. Furthermore, persons who have a perceived conflict of interest will 
be required to have such conflicts managed in a way that is appropriate to their participation 
in the trial. The study leadership in conjunction with the appropriate conflict of interest review 
committee has established policies and procedures for all study group members to disclose 
all conflicts of interest and will establish a mechanism for the management of all reported 
dualities of interest. 
All investigators will follow the applicable conflict of interest policies. And, as stated above, 
due to Conflict of Interests declared by Dr. Michael Girardi, Dr. Mark Salzman and Julia 
Lewis, PhD, Dr. Jeffrey Gehlhausen, Assistant Professor of Dermatology – YUSM, will 
provide and independent review of the raw data and data analysis. 
 
 
7.17 Funding Source 
This study is funded by the Yale SPORE in Skin Cancer, NCI P50CA121974. 
7.18 Publication Plan 
Published manuscripts generated from this work will be made available to the public free of 
charge, per NIHPA guidelines and we will follow the Final NIH Statement on Sharing 
Research Data (Notice NOT-OD-03-032) issued February 26, 2003. Dr. Girardi holds 
primary responsibility for publishing the study results. 


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8 Bibliography 
 
 
1. 
Deng Y, Ediriwickrema A, Yang F, Lewis J, Girardi M, Saltzman WM. A sunblock 
based on bioadhesive nanoparticles. Nat Mater. 2015;14(12):1278-85. Epub 2015/09/29. 
doi: 10.1038/nmat4422. PubMed PMID: 26413985; PubMed Central PMCID: 
PMCPMC4654636. 
2. 
Suh HW, Lewis J, Fong L, Ramseier JY, Carlson K, Peng ZH, et al. Biodegradable 
bioadhesive nanoparticle incorporation of broad-spectrum organic sunscreen agents. Bioeng 
Transl Med. 2019;4(1):129-40. Epub 2019/01/27. doi: 10.1002/btm2.10092. PubMed PMID: 
30680324; PubMed Central PMCID: PMCPMC6336670. 
3. 
Gerges SH, Wahdan SA, Elsherbiny DA, El-Demerdash E. Pharmacology of 
Diosmin, a Citrus Flavone Glycoside: An Updated Review. Eur J Drug Metab 
Pharmacokinet. 2022;47(1):1-18. Epub 2021/10/24. doi: 10.1007/s13318-021-00731-y. 
PubMed PMID: 34687440. 
4. 
Boisnic S, Branchet MC, Gouhier-Kodas C, Verriere F, Jabbour V. Anti-inflammatory 
and antiradical effects of a 2% diosmin cream in a human skin organ culture as model. J 
Cosmet Dermatol. 2018;17(5):848-54. Epub 2018/09/12. doi: 10.1111/jocd.12778. PubMed 
PMID: 30203575. 
5. 
Gotti C, Clementi F. Cytisine and cytisine derivatives. More than smoking cessation 
aids. Pharmacol Res. 2021;170:105700. Epub 2021/06/05. doi: 10.1016/j.phrs.2021.105700. 
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Mancuso C, Santangelo R. Ferulic acid: pharmacological and toxicological aspects. 
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ICF_000.pdf: 
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COMPOUND AUTHORIZATION AND CONSENT FOR PARTICIPATION IN A RESEARCH 
STUDY
YALE UNIVERSITY
YALE UNIVERSITY SCHOOL OF MEDICINE
YALE-NEW HAVEN HOSPITAL
Study Title: Assessing a Natural Product plus Bioadhesive Nanoparticle (BNP) 
Sunscreen 
Principal Investigator (the person who is responsible for this research):   Michael Girardi, 
MD, LMP 5040
Phone Number: 203 785 7432 
Research Study Summary:

We are asking you to join a research study.

The purpose of this research study is to evaluate the effects of a new sunscreen 
formulation.

Study procedures will include: Eligibility screening, consent, physical examination of 
skin, application of sunscreen, ultraviolet exposure of skin, five skin biopsies

Four visits are required.

These visits will take 1-1/2 to 2 hours total. 

There are some risks from participating in this study. Skin irritation, sunburn to exposed 
areas of skin, bleeding, skin infection and small scar at skin biopsy sites.  

The study may have no benefits to you. Participation in this study will enable the 
development of a safer, more protective UV sunscreen that may benefit individuals 
across multiple communities.

Taking part in this study is your choice. You can choose to take part, or you can choose not 
to take part in this study.  You can also change your mind at any time.  Whatever choice you 
make, you will not lose access to your medical care or give up any legal rights or benefits. 

If you are interested in learning more about the study, please continue reading, or have 
someone read to you, the rest of this document. Take as much time as you need before you 
make your decision. Ask the study staff questions about anything you do not understand. 
Once you understand the study, we will ask you if you wish to participate; if so, you will have 
to sign this form.
Why is this study being offered to me?
We are asking you to take part in a research study because your skin type is sensitive to UV 
exposure.  We would like you to help us test the effectiveness of a new natural product 
bioadhesive sunscreen.  This new sunscreen has a special coating that forms a small capsule 
around the sunscreen filters to keep the sunscreen on the outside of your skin.  Current 
sunscreen products are absorbed into your skin. We are looking for 30 participants to be part of 
this research study. 
Who is paying for the study?
This study is funded by the Yale SPORE in Skin Cancer Grant from the National Cancer 
Institute (NCI).
Who is providing other support for the study?
No other support is being provided.  
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What is the study about? 
The purpose of this study is to test a new natural product bioadhesive nanoparticle sunscreen 
that we have developed.  We hope to create a safer, non-toxic, longer lasting sunscreen that will 
help prevent damage to human skin when it is exposed to the sun.  
What are you asking me to do and how long will it take? 
If you agree to take part in this study, this is what will happen: 
Before you can enroll in the study, we will need to confirm your eligibility.  To be eligible, you 
must be at least 18 years old, and you cannot have a history of skin cancer (such as basal cell 
carcinoma, squamous cell carcinoma, or melanoma); and you cannot have a history of skin 
disease (such as psoriasis, eczema, lupus, dermatomyositis or vitiligo).  You cannot have a 
family history of melanoma.  If you are a woman of childbearing potential, pregnancy testing will 
be required before you may be enrolled in the study.
In addition, your skin type must be I, II, or II on the following scale:

Skin Type Characterization and Sunburn/Tanning History:
o
Based on first 30 to 45 minutes sun exposure after a winter season of no sun 
exposure, under which category would you characterize yourself:
I--Always burns easily; never tans (sensitive).
II--Always burns easily; tans minimally (sensitive).
III--Burns moderately; tans gradually (light brown) (normal).
IV--Burns minimally; always tans well (moderate brown) (normal).
V--Rarely burns; tans profusely (dark brown) (insensitive).
VI--Never burns; deeply pigmented (insensitive).

You will also be asked to provide a brief medical history with emphasis on the effects of 
sunlight on your skin. To be eligible, you cannot be using any medications that are 
known to make you more sensitive to sunlight. 

We will perform a test site inspection. The physical examination shall determine the 
presence of sunburn, suntan, scars, active dermal lesions, and uneven skin tones on the 
areas of the back to be tested. The presence of nevi, blemishes, or moles will be 
acceptable if they will not interfere with the study results. 
If you are deemed eligible and choose to voluntarily participate in this study, you will be asked to 
complete the following:
Visit 1: Participants will first visit the study site for screening, informed consent, and a physical 
examination with comprehensive medical history. If eligibility requirements are met, the research 
personnel will identify one area of your skin for the evaluation of your minimal erythema dose 
(MED). (MED is the shortest exposure to ultraviolet radiation that produces reddening of the 
skin within 1 to 6 hours and disappears in 24 hours.)  This area will then be exposed to 
ultraviolet light that is similar to the sun’s rays.  You will be asked to return to the study center 
the next day, approximately 16 to 24 hours later, for more testing and skin biopsies.  If you are a 
woman of childbearing potential, a urine pregnancy test will be given before you proceed in the 
study.
Visit 2: Upon return the next day, your MED will be determined. The research personnel will 
then identify a 4 x 5.5 cm area on your inner upper arm that will be used for the testing of the 
sunscreen. Within this small area, four smaller areas will be identified.  Two the areas will have 
the test sunscreen applied to them.  The other two areas will not receive any sunscreen.  After 
the sunscreen has been applied, you will be asked to rest for 15 minutes while the sunscreen 
absorbs into your skin.  After the 15 minutes have passed, all four areas will then be exposed to 
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the ultraviolet light.  The time your skin is exposed to the ultraviolet light will depend on the 
results of your MED testing from Day 1 / Visit 1.  Immediately after the ultraviolet exposure is 
completed, Dr. Michael Girardi will take three small (3 mm) skin biopsies from your skin.  After 
these skin biopsies are completed, then you may leave the clinic area.  You will need to return 
in 4 hours.    
Visit 3: You will be instructed to return 4 hours after the ultraviolet exposure has occurred for 
two more small (3 mm) skin biopsies.  After the skin biopsies are completed, you will receive 
instructions on how to take care of the skin biopsies.  You will be given contact information for 
study personnel to call if you have any issues or questions about the biopsy sites.  You will be 
given a date and time to return in 7 to 10 days for suture removal.  
Visit 4:  Suture removal (7 -10 days after Visit 3).
What are the risks and discomforts of participating? 
As a participant of this study, you should be aware of the following foreseeable discomforts and 
inconveniences.

Allergic reaction to the sunblock ingredients.  This might cause a red itchy rash.

Sunburn for unprotected control and some test sites after UV irradiation for SPF testing.

Some of the complications associated with punch biopsy include local bleeding and 
bruising, pain, infection, allergic reaction to the numbing medicine used in the procedure, 
or damage to the structures beneath the skin site (such as an artery or a nerve). Your 
doctor will take care to reduce the likelihood of these rare problems

To obtain additional information about the investigational sunscreen in the event of an 
emergency situation, please call Michael Girardi, MD and Kacie Carlson, PA-C at 203-
785-7432 (Monday – Friday 9 am – 5 pm), 203 785 4445 (after hours / weekends).
How will I know about new risks or important information about the study? 
We will tell you if we learn any new information that could change your mind about taking part in 
this study. 
How can the study possibly benefit me?
You may or may not benefit from your participation in this study.  But, you will be helping in the 
development of a safer sunscreen which will potentially decrease risk of skin cancers in the 
future.  
How can the study possibly benefit other people?
Participation in this study may enable the development of a safer, more protective UV 
sunscreen that may benefit individuals across multiple communities.
Are there any costs to participation? 
If you take part in this study, you will not have to pay for any services, supplies, study 
procedures, or care that are provided for this research only (they are NOT part of your routine 
medical care).  However, there may be additional costs to you.  These can include costs of 
transportation and your time to come to the study visits. 
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Will I be paid for participation? 
You will be paid for taking part in this study.  You will receive $200 upon completion of the 
study. This includes the initial visit, visit 2, 3 and 4 (as outlined above).  You must complete the 
entire study to receive the $200. You are responsible for paying state, federal, or other taxes for 
the payments you receive for being in this study.  Taxes are not withheld from your payments
What are my choices if I decide not to take part in this study?
Because this investigation does not constitute treatment-based research, alternatives to 
participation would include not participating.
How will you keep my data safe and private?
We will keep information we collect about you confidential. We will share it with others if you 
agree to it or when we have to do it because U.S. or State law requires it. For example, we will 
tell somebody if you we learn that you are hurting a child or an older person.
When we publish the results of the research or talk about it in conferences, we will not use your 
name. If we want to use your name, we would ask you for your permission.  
We will also share information about you with other researchers for future research, but we will 
not use your name or other identifiers. We will not ask you for any additional permission.
Your information and skin biopsies collected as part of the research, even if identifiers are 
removed, will not be used or distributed for future research studies.
We understand that information about you obtained in connection with your health is personal, 
and we are committed to protecting the privacy of that information.  If you decide to be in this 
study, the researcher will get information that identifies you and your personal health 
information.  This may include information that might directly identify you, such as your name.  
This information will be de-identified at the earliest reasonable time after we receive it, meaning 
we will replace your identifying information with a code that does not directly identify you. The 
principal investigator will keep a link that identifies you to your coded information, and this link 
will be kept secure and available only to the PI or selected members of the research team. Any 
information that can identify you will remain confidential.  Your responses/data will be de-
identified upon collection. Post-processed research data will be stored on a password-protected 
computer. The research team will only give this coded information to others to carry out this 
research study. The link to your personal information will be kept for 3 years after which time the 
link will be destroyed and the data will become anonymous. The data will be kept in this 
anonymous form until it is destroyed. 
What Information Will You Collect About Me in this Study?
The information we are asking to use and share is called “Protected Health Information.” It is 
protected by a federal law called the Privacy Rule of the Health Insurance Portability and 
Accountability Act (HIPAA). In general, we cannot use or share your health information for 
research without your permission. If you want, we can give you more information about the Privacy 
Rule. Also, if you have any questions about the Privacy Rule and your rights, you can speak to 
Yale Privacy Officer at 203-432-5919.
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The specific information about you and your health that we will collect, use, and share includes:
• Research study records
• Medical and laboratory records of only those services provided in connection with this 
Study.  
• The entire research record
• Records about phone calls made as part of this research
• Records about your study visits
• Information obtained during this research regarding

Physical exams of your skin

Medications you are taking

History of skin diseases

Personal and family history of skin cancer

Records about any study drug (sunscreen) you received

pregnancy test (females of child-bearing potential only)
How will you use and share my information?
We will use your information to conduct the study described in this consent form. 
We may share your information with:
•
The U.S. Department of Health and Human Services (DHHS) agencies
•
Representatives from Yale University, the Yale Human Research Protection Program and 
the Institutional Review Board (the committee that reviews, approves, and monitors 
research on human participants), who are responsible for ensuring research compliance.  
These individuals are required to keep all information confidential. 
•
The U.S. Food and Drug Administration (FDA) This is done so that the FDA can review 
information about new sunscreen involved in this research.  The information may also be 
used to meet the reporting requirements of drug regulatory agencies.  
•
The study sponsor or manufacturer of study drug/device
•
Drug regulatory agencies in other countries
•
Governmental agencies to whom certain diseases (reportable diseases) must be reported
•
Health care providers who provide services to you in connection with this study.
•
Laboratories and other individuals and organizations that analyze your health information 
in connection with this study, according to the study plan.
•
Principal Investigator of the study, Dr. Michael Girardi
•
Co-Investigators, Dr. Mark Saltzman, Study Coordinator and Members of the Research 
Team.  
•
Study Coordinator and Members of the Research Team 
•
Data and Safety Monitoring Boards and others authorized to monitor the conduct of the 
Study 
o
Dr. Jeffrey Gehlhausen
We will do our best to make sure your information stays private. But, if we share information with 
people who do not have to follow the Privacy Rule, your information will no longer be protected 
by the Privacy Rule. Let us know if you have questions about this. However, to better protect your 
health information, agreements are in place with these individuals and/or companies that require 
that they keep your information confidential.
Why must I sign this document?
By signing this form, you will allow researchers to use and disclose your information described 
above for this research study. This is to ensure that the information related to this research is 
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available to all parties who may need it for research purposes. You always have the right to review 
and copy your health information in your medical record. 
What if I change my mind?
The authorization to use and disclose your health information collected during your participation 
in this study will never expire.  However, you may withdraw or take away your permission at any 
time. You may withdraw your permission by telling the study staff or by writing to Michael Girardi, 
MD at the Yale University, P.O. Box 208059, New Haven, CT 06520.
If you withdraw your permission, you will not be able to stay in this study but the care you get from 
your doctor outside this study will not change.  No new health information identifying you will be 
gathered after the date you withdraw. Information that has already been collected may still be 
used and given to others until the end of the research study to ensure the integrity of the study 
and/or study oversight.  
Who will pay for treatment if I am injured or become ill due to participation in the study? 
If you are injured while on study, seek treatment and contact the study doctor as soon as you 
are able.  
Yale School of Medicine does not provide funds for the treatment of research-related injury.  If 
you are injured as a result of your participation in this study, treatment will be provided.  You or 
your insurance carrier will be expected to pay the costs of this treatment.  No additional financial 
compensation for injury or lost wages is available.
What if I want to refuse or end participation before the study is over? 
Taking part in this study is your choice. You can choose to take part, or you can choose not to 
take part in this study.  You also can change your mind at any time.  Whatever choice you 
make, you will not lose access to your medical care or give up any legal rights or benefits. 
Not participating or withdrawing later will not harm your relationship with your own doctors or 
with this institution.  
To withdraw from the study, you can call a member of the research team at any time and tell 
them that you no longer want to take part.  
The researchers may withdraw you from participating in the research if necessary. Conditions 
under which a subject may be withdrawn might include subject non-compliance.
What will happen with my data if I stop participating?
When you withdraw from the study, no new health information identifying you will be gathered 
after that date.  Information that has already been gathered may still be used and given to others 
until the end of the research study, as necessary to ensure the integrity of the study and/or study 
oversight.  
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Who should I contact if I have questions? 
Please feel free to ask about anything you don't understand. 
If you have questions later or if you have a research-related problem, you can call the Principal 
Investigator at 203 785 7432 
If you have questions about your rights as a research participant, or you have complaints about 
this research, you call the Yale Institutional Review Boards at (203) 785-4688 or email 
hrpp@yale.edu.
Conflict of Interest Declared by Investigators on this Study:
You should be aware that Dr. Girardi, principal investigator, Mark Salzman, PhD and Julia 
Lewis, PhD - co -investigators for this study, are named as co-inventors on a patent application 
covering the use of natural products in sunscreen, which is being tested in this protocol.  They 
may have a potential financial interest in this research if it leads to the development of products 
that will be commercialized.  If this happens, there is no plan to share any financial gain with 
you. You may speak with them at any time should you have questions regarding these 
investigator interests.
Authorization and Permission
Your signature below indicates that you have read this consent document and that you agree to 
be in this study.  
We will give you a copy of this form.
Participant Printed Name
Participant Signature
Date
Person Obtaining Consent Printed 
Name
Person Obtaining Consent Signature
Date
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