The Efficacy and Safety of Topical Vitamin D and Supplementation In Acne Vulgaris The Study of VDR, IL-1β, IL-6, IL-10 and IL-17 Expression
Phase: PHASE4
Conditions: Acne Vulgaris, Vitamin D
Sponsor: Indonesia University

 
 
 
Research Protocol 
15 February 2023 
 
The Efficacy and Safety of Topical Vitamin D and Supplementation in 
Acne Vulgaris 
The Study of VDR, IL-1β, IL-6, IL-10 and IL-17 Expression 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Table of Contents 
 
1. INTRODUCTION ............................................................................................... 1 
2. STUDY OBJECTIVES ........................................................................................ 2 
3. STUDY DESIGN ............................................................................................... 2 
4. SUBJECT SELECTION AND WITHDRAWAL ....................................................... 3 
5. STUDY PROCEDURES ...................................................................................... 6 
6. SAMPLE SIZE .................................................................................................. 7 
7. ETHICAL COMMITTEES ................................................................................... 8 
 

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1 Introduction 
This document is a clinical trial protocol. This research will be conducted based on the 
standards of the Good Clinical Trial Method and regulations from the relevant institutions 
and ethics committees. 
1.1 Background 
Acne vulgaris (AV) is a chronic inflammatory disease with multifactorial causes in the skin's 
pilosebaceous follicular units, with clinical manifestations in the form of comedones,  
papules, pustules, nodes, and pseudocysts. The following factors are considered important 
for the etiology of AV: increased rate of sebum excretion, endocrinological factors such as 
androgens, abnormal keratinization of the follicular infundibulum, the proliferation of 
Cutibacterium acnes (C. acnes), and inflammation. Recent studies at the molecular and 
cellular levels have clarified how these factors interact and the role of the innate immune 
system. Inflammatory processes have been demonstrated in all types of lesions – preclinical 
microcomedones, comedones, inflammatory lesions, 'post inflammatory' erythema or 
hyperpigmentation, and scarring. Inflammation localized to the pilosebaceous can be 
considered a hallmark of acne and should be managed through several therapeutic routes. 
Clinicians tend to think that oral antibiotics should be used to treat inflammation in acne. 
However, this treatment are associated with resistance and low outcome due to its adverse 
events such as erythema, desquamation, and dry skin. There is evidence of the use and 
opportunity of vitamin D as a novelty treatment influencing the immune system. 25OHD and 
1,25(OH)2D are both catabolized by CYP24A1. 1,25(OH)2D is a ligand for the vitamin D 
receptor (VDR), a transcription factor that binds to sites in DNA called vitamin D response 
elements (VDRE). Thousands of these binding sites regulate hundreds of genes through 
several signaling pathways in different cell types, including their regulation in immune cells 
by toll-like receptors (TLRs), the primary signaling nucleus of C. acnes that interacts with 
the innate immune system, causing acute and chronic inflammation. 

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2 Study Objectives 
2.1 Primary Objective 
The primary objective of this study is to evaluate the efficacy and safety of combination 
topical vitamin D and supplementation as adjuvant therapy in acne vulgaris compared to 
placebo and topical vitamin D monotherapy. 
2.2 Secondary Objective(s) 
1. To assess vitamin D Receptor (VDR) expression on acne lesion and blood sample 
2. To assess the effect of combination topical vitamin D and supplementation on IL-1𝛽 
expression on acne lesion 
3. To assess the effect of combination topical vitamin D and supplementation on IL-6 
expression on acne lesion 
4. To assess the effect of combination topical vitamin D and supplementation on IL-10 
expression on acne lesion 
5. To assess the effect of combination topical vitamin D and supplementation on IL-17 
expression on acne lesion 
 
3 Study Design 
3.1 General Design 
It is a randomized double-blinded controlled trial to assess the efficacy and safety of 
combination topical vitamin D and supplementation as adjuvant therapy in acne vulgaris 
compared to placebo and topical vitamin D monotherapy. In week 8th, the patients will be 
observed to see if there is any recurrence of the lesion.  
 
3.2 Settings and Conduct  
This study was performed in Central Jakarta, Jakarta Capital Special Region, Indonesia. 
Patients were assigned into three groups. The first group was given the combination of topical 
vitamin D and supplementation also standard treatment with topical adapalene. In 

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comparison, the second group was given topical vitamin D monotherapy and standard 
treatment with topical adapalene. Last group was given only the standard treatment with 
topical adapalene as control group. 
3.3 Primary Study Endpoints 
- 
Clinical symptoms  
Changes in counts of inflammation and non-inflammation lesions (assessed in week 8th) 
- 
Changes in VDR, IL-1β, IL-6, IL-10 and IL-17 expression on acne lesion (Time frame: 
change of expression from baseline to week 8th) 
3.4 Primary Safety Endpoints 
Adverse events will be assessed during study periods. Any adverse events including 
local/skin inflammation caused by either oral or topical vitamin D will also be monitored by 
dermatologist in charge.  
4 Subject Selection and Withdrawal 
4.1 Inclusion Criteria 
a. Men and women diagnosed with moderate or severe acne vulgaris 
b. Aged over 18-50 years. 
c. During the study, were willing not to use skin care products either in oral and/or 
topical form on the face, as well as other treatments outside of standard AV treatment. 
d. Willing to take part in the examination and treatment in accordance with the provisions 
of the study and follow the control time schedule in accordance with the predetermined 
research plan also willing to sign the informed consent form. 
4.2 Exclusion Criteria 
Patients will be excluded if 
a. Women who are pregnant and breastfeeding. 
b. Have history using topical antibiotics in the past 2 weeks. 

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c. Have history using topical corticosteroids in the past 2 weeks. 
d. Have history taking vitamin D supplements in the last 1 month. 
e. Have history taking oral antibiotics in the last 1 month. 
f. Have history using oral corticosteroid use in the last 1 month. 
g. Have history using oral and topical retinoid use in the last 3 months. 
h. Have history using topical BPO in the last 1 month. 
i. Using hormonal contraception for women. 
j. Have history of drug allergies or skin disorders due to side effects of first-line therapy 
drugs for moderate/severe acne vulgaris. 
k. Impaired liver and kidney function. 
4.3 Early Withdrawal of Subjects 
The protocol treatment will be discontinued if patients  
a. 
There are severe side effects in the subject in the form of gastrointestinal disorders, 
severe liver function disorders, and severe irritation, edema or bullous lesions at the site 
of topical drug application. 
b. 
Subjects decided to withdraw from the study before the second week. 
c. 
Subjects did not come for 2 consecutive follow-ups. The tolerance limit for follow-up 
is D+3 from the specified examination date. 
d. 
Subject confirmed positive for COVID-19 
e. 
Subjects did not follow established research protocols. 
4.4 
Sample Estimate 
The sample size in this study can be calculated based on the ratio of two proportions, using 
the following formula: 
𝑁1 = 𝑁2 = (Zα√2PQ + Zβ√P1 Q1 + P2 Q2)2
(P1 −P2 )2
 
 
N1 = Control group sample size 

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N2 = N3 = The number of samples of the test group 
α = Type 1 error, set at 0.10 (90% confidence level) with a one-way hypothesis, so 
 
Z α = 1,64 
 
β = if it is set at 20%, then the power of this study is 1- β = 80%, from the table so 
that Z β = 0.84 
P2= The proportion of treatment effectiveness in the control group obtained from the 
previous research was 16 = 0.70 
 
Q2 = 1 – P2 = 1 - 0,70 = 0,30 
P1 - P2 = The minimum difference in the proportion of treatment effectiveness that is 
considered significant is set at 0.25. 
 
P1 = P2 + 0,20 = 0,70 + 0,25 = 0,95 
Q1 = 1 – P1 = 1 – 0,95 = 0,05 
P = 
P1+P2
2
= 0,825 
Q = 1 – P = 1- 0,825 = 0,175 
Obtained results: 
𝑁1 = 𝑁2 = (1,64√2 x 0,825 x 0,175 +  0,84 √0,95 x 0,05 + 0,70 x 0,30)2
(0,25)2
 
= 27,35 
= 28 
 
Assuming that the difference in the proportion of moderate/severe acne vulgaris 
improvement between the group with standard therapy + oral vitamin D and topical vitamin 
D and the control group was only 25%, so the sample size for each group was 28 people. 
Anticipation of drop out by 20%: 
n’= 
𝑛
(1−𝑓) 
n’ = 
28
(1−0,2) =  35 ≈ n1 = n2 = n3 = 35 people. 
 

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Information: 
n’1 = n’2 = n’3   
= the size of the research sample for each group 
n’ 
 
 
= sample size after correction with anticipation of DO 
n  
 
 
= sample size based on previous estimates 
f  
 
 
= prediction of the percentage of sample drop out 
 
The sample size after being corrected with anticipation of dropping out became n'1 = 
n'2 = n'3 = 35 people. 
 
5. Study Procedures 
Treatment phase: 
 
The selected subjects were divided into three groups through a process of randomization and 
sample stratification per group. The first group (A) will receive a combination of oral vitamin 
D 2000 IU 1x1 and topical 7-Dehydrocholesterol 5000 mcg 2x1, the second group (B) will 
receive an oral placebo and topical 7-Dehydrocholesterol 5000 mcg 2x1 and the third group 
(C) will receive an oral placebo and basic ingredient placebo topical vitamin D similar to the 
ointment to be tested. All subjects were still given 0.1% adapalene cream as a standard topical 
treatment for AV. The three groups will receive intervention for 8 weeks (56 days). AV lesion 
extraction will be performed, peripheral venous blood sampling will be performed to examine 
serum 25(OH)D and serum VDR. Examination of serum VDR and tissue VDR were using 
flowcytometry. Examination of inflammatory cytokines IL-1β, IL-6, IL-10, and IL-17 in 
tissues were using the Luminex Kit. Random allocation is done by block randomization 
technique using a computer program (Randlist®). Capsule drugs will be recalculated and 
topical drugs will be weighed when the subject returns for follow-up. Research drugs when 
given to subjects or returned to researchers were included in sealed envelopes. Each subject 
received a diary sheet which included a statement column to drink/not drink/apply/not apply 
and have/no side effects and their type, which had to be filled in every day according to the 
diary attached. 
 

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Arms and intervention  
Arm 
Intervention/treatment 
Experimental: combination topical vitamin 
D and supplementation 
Daily supplementation (1x/day, in the 
morning) and topical application (2x/day, in 
the morning and in the afternoon, minimum 
duration 4 hours on the skin) for 56 days 
Drug: combination vitamin D 2000 IU 1x1 
and topical 7-Dehydrocholesterol 5000 mcg 
2x1 
Active Comparator: oral placebo and 
topical vitamin D 
Daily supplementation (1x/day, in the 
morning) and topical application (2x/day, in 
the morning and in the afternoon, minimum 
duration 4 hours on the skin) for 56 days 
Drug: 
oral 
placebo 
and 
topical 
7-
Dehydrocholesterol 5000 mcg 2x1 
Placebo Comparator: 
Daily supplementation (1x/day, in the 
morning) and topical application (2x/day, in 
the morning and in the afternoon, minimum 
duration 4 hours on the skin) for 56 days 
Drug: oral placebo and basic ingredient 
placebo topical vitamin D similar to the 
ointment 
 
6 Sample Size 
6.1 Target sample size 
105 patients (35/arm) 
 
7 Ethical Committees 
- 
Name of ethics committee 
 
: Ethics committee of the Faculty of Medicine, 
University 
of 
Indonesia 
– 
Cipto 
Mangunkusumo Hospital  

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  Medical Sciences  
- 
City  
 
 
 
 
: Central Jakarta 
- 
Province  
 
 
 
: Jakarta Capital Special Region 
- 
Country 
 
 
 
 
: Indonesia 
- 
Approval date 
 
 
 
: September, 5th 2022 
- 
Ethics committee reference number 
: KET-922/UN2.F1/ETIK.PPM.00.02/2022