Open‐label, single‐arm, single‐center clinical study on the effectiveness and safety of a moisturizer containing tocotrienol‐rich composition in children with mild to moderate atopic dermatitis

Little is known about antioxidant efficacy of topical vitamin E on atopic dermatitis (AD) due to lack of controlled clinical studies.


| INTRODUC TI ON
Atopic dermatitis (AD) is the most common form of eczema characterized with chronic pruritic, relapsing form of skin inflammation, which is primarily localized to the flexural surfaces of body, face, eyelids, neck, external auditory meatus, and limb flexures.Globally, AD affects up to 3% adults and 20% of children, and it is associated with an increment of 2-to 3-folds in developing nations. 1 Despite the broad range of drugs (corticosteroids) available, patients are often dissatisfied with the treatments due to the risk associated with long-term usage. 2 Topicals such as moisturizers are the first-line therapy in AD due to lower risk of adverse effects than the systemic medications used.
Natural phenolic antioxidants such as vitamin E is an added lipophilic compound of skin care products due to its dermatological benefits.It was suggested that vitamin E plays a vital role in inhibiting lipid peroxidation and stabilizing lipid bilayers of stratum corneum.
Tocotrienols and tocopherols are the biologically active form of vitamin E, which are different in their aliphatic tails.The latter exhibited a superior antioxidant activity as it contains an isoprenoid instead of a phytyl side chain.The hydroxyl group on the chromanol ring acts to quench chain-carrying lipid peroxyl radicals on skin during oxidative stress.In fact, skin is a multi-layered tissue composed of continuously renewing keratinocytes at epidermis while underlying dermis is mainly made up of fibroblasts.In relation to this, vitamin E performs upregulation of gene expression of keratinocyte differentiation, indicating its therapeutic effect against AD. 3 On the other hand, the topical use of vitamin E has also raised concerns about allergic contact dermatitis (ACD) in AD patients.Contact allergy due to vitamin E and its derivatives, mainly the synthetic form, has been described in a few individual case reports. 4,5Although vitamin E has been described as the cause of the ACD in some sporadic cases, the incident rate is fairly low despite its widespread use. 4,6,7spite its emerging use in skincare formulations, little is known of its antioxidant efficacy on AD due to lack of controlled clinical studies.Thus, the aim of the present study is to access the effectiveness and safety of a topical moisturizer containing tocotrienol-rich composition (REMDI® Sensitive Intensive Moisturising Cream; RS) on 1 month-12 year children with mild to moderate AD in a period of 12 weeks.

| Study participants
Children (1 month-12 years old) were recruited through three routes.
First, patients who saw the trial advertisements on bulletins made available in UPM (specifically Pusat Kesihatan Universiti, Hospital Pengajar UPM's Family Medicine Clinic, Medical Specialist Clinic, or Pediatric Clinic) that contacted the sponsor voluntarily.Patients who saw the trial advertisements in social media, specifically Malaysia Eczema Support Community on Facebook, that contacted the sponsor voluntarily and patients who obtained words of mouth of the trial that contacted the sponsor voluntarily during the period of July 1, 2019-August 30, 2019.
Recruitment process were done from September 2019 to October 2019, followed by the study from November 2019 to February 2020 (4 weeks follow-up).The participants that completed the interventions were 30 patients due to three lost to follow-up and four participants withdrawn prematurely due to non-compliance.The sample size calculations were performed according to Cheon et al. 8 where the mean difference and standard deviation of the SCORAD index will be 10 and 13.576, respectively.Using 80% power and a 5% significance level, the required sample size will be corrected to at least 29 participants for this trial.product is formulated at a slightly acidic range; pH 5-5.5, which is the natural skin pH.

| Demographic
A total of 37 subjects were screened between September 2019 and October 2019 (Figure 1).Seven subjects were withdrawn prematurely due to non-compliance (N = 3) and lost to follow-up (N = 4).As shown in

| SCORAD and PO-SCORAD
As shown in Figure 2C,D, AD improved over the study period, with a decrease of SCORAD and PO-SCORAD scores from week-0 to week- i Excluded from analysis (n=0) Non-Randomized (n=37)

| Pruritus
A significant improvement of pruritus intensity was noted (p < 0.05), decreased from 6.77 ± 2.01 at week-0 to 3.63 ± 2.39 at week-12, with a relative reduction of 46.3% in 12 weeks' time as shown in Figure 2B.

| Skin color and erythema
As shown in Table 2, a* value of the patients decreased across the treatment periods significantly (p < 0.05), which indicated that the redness of the patients improved.Treatment with RS resulted in a progressive improvement of skin inflammation, as demonstrated by the change in the cumulative severity of erythema index.The erythema index of the AD patients showed a decreasing trend across the study period.As depicted in Figure 3A, the mean value of erythema index exhibited was 12.53 ± 2.24 at week-12 compared to baseline (15.17 ± 2.49) with a significant reduction of 17.4% (p < 0.05).

| Trans-epidermal water loss
The trans-epidermal water loss (TEWL) of the AD patients across the study week is depicted in Figure 3B.At week-0, the mean TEWL exhibited was 24.26 ± 11.33 g/m 2 h which further reduced to 12.21 ± 6.01 g/m 2 h at week-12 significantly (p < 0.05), with relative reduction of 49.7%.

| Questionnaire
The assessment of the overall satisfaction of RS was determined using a study-specific questionnaire administered to patients at week-12 (Table 3).The cream was found to be well tolerated after application.There was no adverse reaction observed in the form of redness, swelling, dryness, itching and burning sensation.The results showed that approximately 95% of patients perceived the study treatment (RS) as good or excellent in terms of overall satisfaction, and approximately 90% of patients regarded RS as better than commercial moisturizers used in the past.

| Steroid usage
As shown in Figure 4, there was a significant reduction in the frequency of hydrocortisone application from baseline (9.46 ± 4.70) to study end (4.25 ± 4.86) with a significant reduction of 55.07%(p < 0.05).

| DISCUSS ION
AD is strongly associated with keratinocyte damage and impaired skin barrier.It is widely accepted that reactive oxidants caused cellular damage to the epidermal keratinocytes via lipid peroxidation 10 and further trigger tissue inflammation through genes upregulation accounted for pro-inflammatory cytokines via the nuclear factor-κB pathway. 11Treatment with topical antioxidants provides a noninvasive alternative to repair the underlying mechanisms of cellular and molecular damage from oxidative-free radicals in AD patients.
Inflamed skin requires both hydrophilic and lipophilic antioxidants like vitamin C and E.
The lipophilic properties of vitamin E allows them to travel down to the deeper stratum corneum layer within the cell membranes through sebaceous gland secretions and protect them from oxidative damage.Vitamin E is delivered to the skin surface via sebum to protect the stratum corneum from the oxidative damage. 123-O-ethyl ascorbic acid which was used in this study is a potent chain-breaking agents with markedly affinity for biomembranes. 13It is absorbed easily through the skin than other ascorbic acid attributed to its lipophilic and hydrophilic properties.Thus,  factor for the formation of hydroxyproline and hydroxylysine. 16sults showed that the erythema index of the AD patients reduced across the treatment period which could be explained by the deactivation of free radicals by both vitamin E and vitamin C where they interact synergistically to provide antioxidant protection.Vitamin E quenches lipid peroxyl radicals by donating hydrogen from their phenolic group and becomes oxidized.The resulting tocopheroxyl and tocotrienoxyl radicals will be reduced by the redox cyclers (vitamin C) with lower redox potential to rejuvenate its activity. 17The synergistic relationship provided 4-fold protection against erythema induced by solar-simulated irradiation after topical application. 18This helps to alleviate the oxidative stress on the inflamed skin.Similarly, previous study revealed a significant improvement of melasma and pigmented contact dermatitis lesions using a combination of topical vitamins E and C, which prove to be superior to the single-treatment groups. 19pical applications of vitamin E permeate the dermis and epidermis. 20Despite the fact that topically applied dose vitamin E is unstable and will be destroyed on the skin after exposure to UV light, 21 nanotechnology could be used as an alternative to overcome to bioavailability problem of vitamin E. The incorporation of vitamin E nanoemulsion prepared using high pressure homogenizer was incorporated into the RS formulation; exhibited a mean droplet size of 150 nm was believed to improve the drug release profile and skin penetration attributed to the high interfacial area related to their nano-sized droplets.This is similar to the research by Akhtar et al. 22 in which β-cycloethosomes developed based on β-cycloamylose and carbomer 934P gel with nano-size of 228.33 ± 1.23 nm is able to reach the deeper skin layers.
All enrolled AD patients experienced skin dryness with higher TEWL at week-0.Across the treatment period, topical application of the treatment moisturizer containing the vitamin E increased the stratum corneum hydration and improved the water binding capacity.Generally, an average TEWL of a healthy skin lies between 0 and 15 g/m 2 h.Hence, it was evidenced that the enrolled AD patients exhibited higher TEWL associated with an impaired skin barrier function with more water lost to the ambient air.In average, the TEWL of all AD patients fell between the ranges of healthy skin TEWL at week-12.Hence, it is postulated that topical vitamin E could improve skin water-binding capacity.This is in line with the previous study by Gönüllü and co-workers, 23 which   Surface skin pH lies in the range of 4-6, inherently acidic in nature.It is a major factor in maintaining skin barrier integrity. 25evated surface skin pH is a hallmark of AD, rendering the skin more susceptible to irritation.It was evidenced that lesional skin on AD patients often exhibit 0.1-0.9pH units higher indicating a shallower pH gradient. 26Harsh alkaline detergents over the skin will alter the skin's acidic pH and dysregulates downstream enzyme activity and triggers AD.Both RS and BW (REMDII® Sensitive Calming Body Wash) are formulated with mild acidic pH range (5.0-5.5) and this could help to normalize the skin pH to its usual acidic range.Same result has been reported that low pH cleanser (pH 3.5) enhanced AD severity clinically in terms of Eczema Area and Severity Index, CDLQI, and pruritus index. 27pical corticosteroids such as hydrocortisone (1%) was allowed in this study for patients based on prescription of doctor.It is a mild topical corticosteroid used to treat swelling, redness and itchiness for skin inflammatory. 28However, the clinical usefulness of topical hydrocortisone is restricted with minimum usage in this study due to their propensity to induce dermal thinning.As shown in Figure 4,

This is a 12 2 . 4 |
weeks, prospective, open-label clinical study conducted in the Clinical Research Ward of Universiti Putra Malaysia Teaching Hospital.The study was conducted from November 2019 to February 2020.Permission for the human subject study was granted by the Ethics Committee For Research Involving Human Subject, JKEUPM-2019-274 (NMMR-19-1588-49234).In order to keep topical treatments consistent, participants were instructed to exclusively use the moisturizer (REMDII® Sensitive Intensive Moisturising Cream, RS) and body wash (REMDII® Sensitive Calming Body Wash) given and were abstained from using other soap-based products and commercial moisturizer during the whole study period.In accordance with this, participants were advised to apply the study product (RS) thrice daily on the affected AD lesion sites for 12 consecutive weeks.To ensure their compliance, each participant was requested to return the bottles at every visit to assess the amount of applied moisturizer.Mild potency corticosteroids such as hydrocortisone 1% cream was used for the first 2 weeks concurrently with the study product.Thereafter, the use is only limited to flare up.The utilization of corticosteroids were recorded in the provided diary.Outcome measure Physician based severity measurement, namely investigator global assessment (IGA), and Scoring Atopic Dermatitis Score (SCORAD) were used.Patient's directed severity measurement was assessed using Patient-Oriented Scoring Atopic Dermatitis Score (PO-SCORAD).Patients' quality of life improvement were measured using Children Dermatology Life Quality Index (CDLQI) or Infant Dermatitis Quality of Life (IDQOL).We objectively assessed the skin color and trans-epidermal water loss (TEWL) using a skin analyzer (Dermalab Series SkinLab Combo).These outcome measures were assessed at five different time points, namely baseline (week-0, week-2, week-4, week-8 and week-12).Toward the end of the study, subjects were asked to assess the satisfaction and opinion of the efficacy of the cream in comparison to other cream used in the past.

2. 4 . 2 |
PO-SCORADPatient-oriented SCORAD (PO-SCORAD) is a self-assessment scale for atopic patients for evaluating severity of AD lesions, intensity of itch, and sleep disturbance.9It involves the patient/parents directly in the monitoring of chronic AD, allowing them to evaluate the AD based on subjective and objective criteria derived mainly from the SCORAD.2.4.3 | IDQOL and CDLQIInfants' Dermatitis Quality of Life Index (IDQOL) and Children's Dermatology Life Quality Index (CDLQI) were used to assess the quality of life (QOL) of patients.IDQOL questionnaire is designed for use in infants with AD below the age of 4 years while CDLQI questionnaire is designed to measure the impact of AD on the lives of children above 4 years.2.4.4 | Measurement of trans-epidermal water lossTEWL of the test sites using TEWL probe in duplicates, expressed as grams of water per square meter per hour (g/m 2 h).It is based on the diffusion of water through the skin due to the water vapor pressure gradient between the skin surface and the ambient air.Prior to measurement, all patients were allowed to equilibrate in a closed environment with a constant temperature (20°C ± 2°C) and humidity (45%-55% RH).The same skin areas were recorded and assessed each time.2.4.5 | Measurement of skin color and erythemaSkin analyzer (Dermalab Series SkinLab Combo) is used to measure the skin color and erythema intensity of the test sites using color probe in duplicate, indicating L*, a*, b* result where L* refers to color luminosity, a* refers the color range from green (−) to red (+), and b* refers the color range from blue (−) to yellow (+).Erythema is expressed in arbitrary units.The same areas were recorded and assessed each time.Erythema is one of the skin parameters evaluated using non-invasive procedure.Red color of the skin indicated the sign of inflammation and acute stage of AD.Hence, changes in a* value from green (−) to red (+) was correlated to erythema intensity.The higher the a* value, the intense the erythema intensity.2.4.6 | Pruritus Pruritus intensity is determined by the 10-point visual analogue scales (VAS), where 0 indicates none itching and 10 indicates the worst itching.This was found in SCORAD.2.4.7 | Statistical analysis All experiments were conducted in triplicates and the experimental data were analyzed based on one-way analysis of variance (ANOVA) using Minitab Statistical Software Release 16 (Minitab Inc.).The differences between the means were considered significantly different at p < 0.05, as determined by Tukey's test.

3. 2
| IGA IGA showed a significant reduction at week-12 compared to the baseline (p < 0.05) with an overall improvement rate of 63.4% at the end of the treatment period as indicated in Figure 2A.At week-2, the average values of IGA (1.67 ± 0.66) showed a statistically significant reduction (p < 0.05), compared to baseline (2.37 ± 0.49).
direct topical application of these antioxidants is expected to facilitate this protection.Vitamin C (3-O-ethyl ascorbic acid) was shown to protect cells against UV-induced oxidative insult by scavenging free radicals and reactive oxygen species in vitro.14Besides, vitamin E (α-tocopherol) at 20 and 40 μM was shown to improve barrier function by means of reduced TEWL compared to controls in vivo.153-O-ethyl ascorbic acid is a stable l-ascorbic acid derivative with an ethyl group at third carbon position (C3).This biologically active form of vitamin C is a potent antioxidant and can protect the cells from oxidative damage by scavenging free radicals and reactive oxygen species.Besides, it helps in skin renewal by stimulating collagen biosynthesis, being co- Atopic dermatitis severity measured by IGA scores across the treatment period.(B) Pruritus scores according to physician's assessment on a visual analogue scale.(C) PO-SCORAD scores according to patients' assessment across the treatment period.(D) SCORAD scores according to physician's assessment across the treatment period.(E) IDQOL/CDLQI scores according physician's assessment across the treatment period.CDLQI, Children's Dermatology Life Quality Index; IDQOL, Infant's Dermatitis Quality of Life Index; IGA, investigator global assessment; PO-SCORAD, Patient-Oriented Scoring Atopic Dermatitis index.TA B L E 2 Mean values of skin color (L*a*b*) of the patients across the study period.

F I G U R E 3 HOW
(A) Erythema measured using skin analyzer on the patient's affected area.(B) Trans-epidermal water loss measured using skin analyzer on the patient's affected area.et al.indicated that tocopherols containing emulsion exhibited higher skin conductance than calcium ascorbate and ascorbic acid containing emulsion, suggesting that vitamin E exhibited better skin hydration compared to vitamin C. Another study suggested that topical vitamin E enhanced the hygroscopicity and water holding capacity of the stratum corneum by enhancing the ceramide synthesis.24It induced the differentiation of epidermal keratinocytes by increasing Ca 2+ uptake and stimulating gene expression of ceramide synthetase (serine palmitoyltransferase) and led to increment of ceramide content in stratum corneum.This results in improved skin barrier moisture-retention.
there was a significant reduction in the frequency of hydrocortisone application from baseline (9.46 ± 4.70) to study end (4.25 ± 4.86) with a significant reduction of 55.07%(p < 0.05).Similar result has been highlighted whereby medium-potency topical corticosteroids when used in combination with emollients were found to minimize the relapsing risk for children with moderate to severe AD.29 The limitations of the study includes of the lifestyle factors of the patients; which may lead to imprecise results and the small sample size (N = 30).Besides, it is a non-randomized, single arm and open labeled study which may reduce the reality of the data.5 | CON CLUS IONMost of the AD patients in this study observed an improvement in clinical symptoms, with reduction of pruritus intensity measured through SCORAD index; PO-SCORAD; IDQOL/CDLQI.IGA scores indicated no adverse events.The overall improvement in terms of TEWL and erythema index also makes the tocotrienol enriched moisturizer used within the study a safe and effective moisturizer for management of AD in young children.
1.96 + 0.84) 2 × 13.576 2 ÷ 10 2 = 28.932.All informed consent was signed and obtained from the parent or guardian before starting the study.Eligible participants included those who are diagnosed with mild (IGA score = 2) to moderate (IGA score = 3) AD; at least 5% of body surface areas affected during the inclusion time; with a carer-reported pruritus score based on VAS of at least 4 and above; without other chronic health conditions such as asthma or allergic rhinitis; were free of dermatological disorders other than AD that would interfere with the assessment of treat-
Demographics of the study population.
TA B L E 1