Clinical trial NCT02906566
Conditions: Intrinsic Aging of Skin
Sponsor: Stanford University
Start date: 2015-11-01; Completion date: 2018-03-09
Enrollment: 110 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
There has been increasing evidence for different rates of natural aging in humans and one of the best organs to study human aging is skin. Studies have demonstrated anti-aging effects of topical agents (such as creams, gels, lotions, or ointments) and one of them, retinol or vitamin A, was shown to decrease fine wrinkling in skin of older individuals. Additionally, studies of retinol in humans have largely occurred in white populations and so this study aims to focus on skin aging in individuals of East Asian descent as they represent a majority of the world population. This study aims to better characterize the molecular basis of rejuvenation effects and to potentially discover new topical agents with similar and/or more effective preservation of skin youthfulness.

Detailed description:

Arms: arm 1: Active and Placebo. Participants received retinol lotion on one arm and placebo to match on the other arm. arm 2: Participants in the group will give tissue sample only for comparison.
Interventions: intervention 1: Retinol intervention 2: Placebo
Intervention descriptions: intervention 1: Retinol in the form of vitamin A lotion intervention 2: Placebo of Retinol

Protocol documents:
Prot_SAP_000.pdf: 
 
Discovery of Effects of Retinol on Human Skin Aging in 
Individuals of East Asian Descent 
Protocol and Statistical Analysis Plan 
NCT02906566 
October 12, 2016 
 
Anne Lynn S. Change, MD, Principal Investigator 
Stanford University School of Medicine 
Redwood City, California, 94063 


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Title of Proposal: 
 
 
 
 
Discovery of Rejuvenation Effects of Vitamin A (Retinol) Lotion  
on Naturally Aged Skin in an East Asian Cohort 
 
 
 
 
 
Version 101216 
 
 
 
 
 
 
 
 
 
Principal Investigator: 
 
Anne Lynn S. Chang, MD 
Stanford University School of Medicine 
Assistant Professor, Department of Dermatology 
450 Broadway Street
 
Redwood City, CA 94063 
 
 
 
 
 
 


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Introduction 
 
 
Increasing evidence from multiple organ systems suggests that there are different 
rates of natural aging in humans. One of the best organs to study human aging is the skin, 
due to its amenability to clinical inspection and serial biopsy. Recently, genome-wide 
analysis in an Ashkenazi population has provided the evidence that skin can age at 
different rates and that youthful appearing skin is associated with different gene variants 
(Chang et al., 2014). The first studies to demonstrate anti-aging effects of a topical agent 
were published in 1988 in which topical tretinoin was used to improve the appearance in 
photo-aged skin (Weiss et al., 1988). Subsequently, topical retinol (vitamin A) lotion was 
shown in a placebo-controlled study to decrease fine wrinkling in photo-protected skin in 
older individuals residing in nursing homes (Kafi et al., 2007).  
 
Recent advances in deep sequencing technology have enabled the unbiased and 
detailed analysis of gene expression changes in the skin (Chang et al., 2013). 
Furthermore, a type of deep sequencing technique called 3’-end sequencing has 
demonstrated the ability to identify changes in a class of transcripts called long non-
coding RNAs (lnc RNAs) that are now understood to be a previously unrecognized way to 
regulate gene expression. Here, we propose to utilize this state-of –the-art technology to 
discover, in unbiased fashion, the rejuvenation effects of the most widely used class of 
topical anti-aging agents, retinoids. In this study, we will focus on topical retinol, as it is an 
over-the-counter agent that has minimal side effects. 
Furthermore, studies on retinol to date in humans have occurred largely in white 
population even though individuals of Asian descent represent the majority of the world 
population. The potential effects of retinol on individuals of Asian descent are 
uncharacterized and represent an untapped potential to explore differences in aging 
across ethnic groups. 
While prior studies have indicated an increase in glycosaminoglycans (GAGs) and 
collagen after topical retinol usage (Kafi et al., 2007), the true breadth of anti-aging effects 
including the mechanisms by which these effects are controlled has not been explored. It 
is possible that topical retinol has additional effects on major pathways of skin aging, such 
as NFkB related genes (Chang et al., 2013) as well as previously unknown aging 
pathways. This study aims to discover the molecular basis of rejuvenation effects of 
retinol so that new topical agents can be identified that might mimic aspects of the retinol 
rejuvenation effect. We aim to do this in a Han Chinese cohort, as this is the most 
populous ethnic group among the East Asian population. Given the similarity in haplotype 
of Han Chinese with Korean and Japanese populations, these latter groups will be 
included as well. 
 
 
Objective:  
 
To discover new pathways of rejuvenation using topical vitamin A application on naturally 
aged in East Asian women.  
 
Specific Aims: 


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Specific Aim 1: To discover molecular pathways of skin rejuvenation by identification of 
gene expression profiles after topical vitamin A in East Asian women 
 
Specific Aim 2: To identify changes in wrinkling and biometrics associated with skin 
rejuvenation after topical vitamin A application in East Asian women 
 
Specific Aim 3: To identify type and severity of adverse events associated with topical 
retinol usage  
 
 
Materials and Methods 
 
Formulation of topical retinol per Kafi et al. by combining a 41% retinol solution in 55% 
polysorbate 20 in sufficient proportions with fragrance-free Norwegian Formula Neutrogena 
Body Moisturizer (Ortho-Neutrogena Co, Los Angeles, Calif ) to yield a 0.4% retinol lotion. 
The vehicle was similarly prepared with 55% polysorbate 20 solution in Norwegian Formula 
Neutrogena Body Moisturizer. There were no discernable differences in color, odor, 
or consistency between the active lotion with retinol and the placebo lotion control. A stability 
study using high-performance liquid chromatography will be performed to demonstrate that 
more than 90% of 0.4% retinol remained in the moisturizer 3 months after preparation.  
Both 0.4% retinol lotion and its vehicle were placed in dark glass containers covered with foil 
to eliminate transmission of UV radiation and stored at 4°C. 
 
After Stanford Human Subjects Panel approval and written informed consent, participants will 
be screened for eligibility as follows:  
 
Inclusion Criteria  
Able to provide written informed consent 
Older group: Age between 50 and 75 years 
Young group: Age less than or equal to 25 years 
Female  
Fine wrinkling score at baseline =7 or greater if in older group 
All 4 grandparents of Han Chinese, Korean or Japanese descent 
Willing and able to apply topical agents to inner arms 
Uncontrolled medical problems including diabetes, active chemotherapy 
Body mass index within normal or overweight range  
No history of weight loss of >20 lbs. within past 5 years 
 
Exclusion Criteria 
Skin condition in the areas of skin biopsy that would obscure results of analysis 
Topical creams or treatment to arms 2 weeks prior to study baseline visit, including 
sunscreens, topical moisturizers, sunless tanners 
Individuals with known hypersensitivity to retinoid class of agents 
Prior anti-aging treatments to arms including retinol, microdermabrasion within 2 weeks of 
baseline visit 


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Prior laser therapy or surgical procedure to arms 
Prior radiation or other trauma (extensive burns or abrasions) to arm skin 
Hormone-based therapy within 4 weeks of enrollment 
 
Screening Visit (VISIT 1—older group only) 
Only individuals fulfilling eligibility criteria will be enrolled. 
As part of the screening process, clinical histories, physical examinations including vital 
signs, and photography of sun exposed and non sun-exposed arm will be performed. 
Cutometry, Raman spectroscopy (Biophotonic scanning) and transepidermal water loss will 
be measured.  
 
Enrollment Visit (VISIT 2—subset of 30 participants from the older group above will be 
invited for this visit) 
Upon enrollment into the study, patients will be instructed on proper usage topical retinol 
versus vehicle placebo. The placebo versus treatment arm will be clearly labeled on the 
subject’s diary to minimize subject error.  Participants will apply treatment to one arm and 
placebo to the other arm based on computer generated randomized assignment of left or 
right for the retinol. Participants are blinded to the treatment. Participants will be given a diary 
to record adherence to study procedures. Participants will be instructed to discontinue study 
if there is any itching or burning sensation that is moderate or severe and to contact study 
personnel immediately should this occur. 
 
Week 12 visit (VISIT 3—the 30 participants from VISIT 2 (older group) only) 
Adverse events and concomitant medication changes will be assessed and recorded. Study 
diary will be reviewed for adherence to study regimen. Photographs, cutometry and TEWL 
measurements will be taken. Two 4 mm skin biopsies will be taken at this time, one from the 
side with the retinol exposure and one from the side without retinol exposure after injection of 
a small amount of lidocaine anesthetic. Skin biopsies will be bisected and placed in either 
formalin for hematoxylin and eosin staining and mucin staining (for GAG), or placed into RNA 
later for subsequent gene expression analysis by 3’-end sequencing as described in Chang 
et al., 2013. Tissue will also be sent for microRNA analysis. 
 
VISIT 1—age<25 years group only 
As part of the screening process, clinical histories, physical examinations including vital 
signs, and photography of face, sun exposed and non sun-exposed arm skin will be 
performed. Cutometry, Raman spectroscopy (non invasive Biophotonic scanning) and 
transepidermal water loss will be measured. Photographs, cutometry and TEWL 
measurements will be taken. A 4 mm skin biopsies will be taken from the inner arm skin after 
injection of a small amount of lidocaine anesthetic. Skin biopsies will be bisected and placed 
in either formalin for hematoxylin and eosin staining and mucin staining (for GAG), or placed 
into RNA later for subsequent gene expression analysis by 3’-end sequencing as described 
in Chang et al., 2013. Tissue will also be sent for microRNA analysis. 
 
Statistical Considerations 
 
This study is powered using data from Kafi et al. for the primary clinical endpoint of fine 


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wrinkling score as measured by semi-quantitative scale of 0 to 9. A sample size of 36 
provides a power level of 0.80 in detecting a difference of 1.00 unit on mean value before 
and after treatment for the fine wrinkling scale, with a type I error rate of 0.05 for a 2-tailed 
hypothesis, assuming a standard deviation of differences to be 1.8. Assuming a dropout rate 
of 10%, the sample size needed is 28 subjects.  
 
 
Anticipated Results 
 
Results of photographs will be analyzed in blinded fashion by a board certified dermatologist 
for skin aging parameters including fine wrinkling (tertiary endpoint), dyspigmentation, and 
surface texture with results graded on a 9 point Likert scale per Kafi et al. We anticipate that 
there will be statistically significant differences in mean score of these parameters between 
pre- and post- treatment groups for both vitamin A and placebo. 
 
Results of histology will be evaluated in blinded fashion by a dermatopathologist. Parameters 
of skin aging will include measurement of epidermal thickness, and density of collagen and 
elastin. Results from the pre- and post- treatments groups for topical vitamin A versus 
placebo will be compared. 
 
Results of the cutometry and TEWL measurements will be grouped and mean and standard 
deviations for the older patients before and after topical vitamin A treatment will be calculated 
and compared to placebo. We anticipate there will be statistically significant differences in the 
mean scores in cutometry and TEWL measurements between treated and placebo groups. 
 
Whole transcriptome libraries will be created from the samples stored in RNA later and sent 
for 3’-end sequencing at Stanford Functional Genomics Core Facility after de-identification. 
We have utilized this Core facility for other projects in the past with high quality results. 
Results will be analyzed by our bioinformatician using the methods outlined in Chang ALS et 
al., 2013. Specifically, comparisons will be made in skin from older participants between the 
pre and post treatment compared to placebo. All results of interest from 3’-end sequencing 
will be confirmed by RT-qPCR. 
 
We anticipate that the adverse event rate (deemed related to study agent by investigator) for 
participants is less than 10% and may include mild (grade 1) itching or burning.  
 
Significance 
 
This study will be the first broad based, unbiased investigation into the molecular basis of the 
anti-aging effects of topical vitamin A. This study is a high quality, well-controlled study using 
state-of-the art technology of 3-Seq and microRNA analysis. Our research group has 
established the workflow from previous larger studies in skin aging (unpublished data) and 
can now leverage this experience for maximal efficiency and positive results. Specifically, we 
would be able to compare our results to inner arm skin from females younger than 30 years 
whose gene expression profiles are already contained in our database. 
 


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This study will provide novel candidate gene pathways as well as confirm suspected/known 
genes important for skin rejuvenation in humans. This information will aid in discovery of new 
topical agents that might have rejuvenative capacity due to their ability to mimic aspects of 
the vitamin A gene expression changes. 
 
References 
 
Chang ALS, Atzmon G, Bergman A, et al. (2014) Identification of genes promoting skin 
youthfulness by genome-wide association study. J Invest Dermatol 134 (3): 651-657. 
 
Chang ALS, Bitter PH, Qu K, et al. (2013) Rejuvenation of Gene Expression Pattern of Aged 
Human Skin by Broadband Light Treatment: A Pilot Study. J Invest Dermatol 133 (2): 394-
402. 
 
Kafi R, Kwak HR, Schumacher WE, et al. (2007) Improvement of naturally aged skin with 
vitamin A (retinol). Arch Dermatol 143: 666-12. 
 
Weiss JS, Ellis CN, Headington JT, et al. (1988) Topical tretinoin for photoaged skin: a 
double-blind vehicle-controlled study. JAMA 259:527-532.