The Formulation #2LCRE.20 under evaluation as the Test Material (TM) in this study is comprised of the PAC, that includes ascorbates, tocopherols, tocotrienols, ubiquinone, enzymes and amino acids in an optimized DS, described in Table 1.

Table 1. List of PAC and DS ingredients in formulation #2LCRE.20.

Four TMs were used to complete the study described below. The Untreated Control consists of tissue samples that have not been treated with any substances. This TM was used as negative control to assess cytotoxicity. Triton X-100 is a non-ionic surfactant which disrupts cell membranes leading to cellular death is used as a positive control for cytotoxicity assessment. Cells exposed to Triton X-100 are not expected to survive, yielding a 100% death rate. A 0.9% Saline solution was used as negative control for the assessment of the gene expression changes in the 3-dimensional reconstructed skin tissue model. Its cytotoxicity potential was measured to ensure full cell viability during the gene expression study and isolate the TM’s cytotoxicity. This solution, considered neutral and inert to the skin cells, should induce very low to zero cytotoxicity or changes in gene expression. The results obtained with this solution serve as a comparison with the tested Formulation results. #2LCRE.20, also referred to as the Formulation, is described in Table 1. This is the main material to be assessed to determine the degree of change that will be induced after using the Formulation in the cytotoxicity, gene expression changes and in-vivo studies. Table 2 describes the approximate concentrations of actives in the PAC which are expected to provide most of the antioxidant activity of the TM. Table 3 expands further on the Test Materials design.

Table 2. List and concentrations of PAC ingredients in formulation #2LCRE.20.

Table 3. Test groups and result expectations.

The gene expression and cytotoxicity studies were performed on a commercially available 3-dimensional in vitro skin model (Mattek EFT-400) composed of epidermal keratinocytes and dermal fibroblasts as described in Figure 1. Tissues were equalized prior to individual inoculation with 15 uL of one of the four TM samples. For each treatment group, four tissue samples were included. Post distribution of TMs, the tissue samples were placed in an incubator at 37˚C with 5% CO2 and ~95% relative humidity for 24 hours. Following 24 hour incubation, each tissue sample surface was washed and retreated with a fresh TM sample. This process was performed a total of three times. Tissue samples were collected and gene expression was assessed after 72 hours exposure to the TMs utilising Genemarkers’ qPCR-based Standard Skin Panel containing 107 target genes (Appendix 1). After full incubation, tissue sample surfaces were washed, and each culture was placed in contact with a RNAlater solution ready for RNA isolation.

Figure 1. Gene Expression Process from inoculated tissue samples to raw data generation.

Following a qPCR process, statistical data analysis was performed using relative quantitation (RQ) method and converting any linear RQ values into linear fold-change values.

Controls:

High Control: 1% Triton X-100 was inoculated on the surface of two tissue samples as per process above.Low Control: Four untreated (UNT) tissue samples.

To assess the LDH activity (cytotoxicity) of the TM, the culture medium of each treated tissue sample was used and diluted 1:10 with Phosphate Buffered Saline (PBS). Every dilution was then combined with the LDH reaction mix at a ratio 1:1 and followed by a 20minute incubation at room temperature in the dark. A 1.0N HCl solution was used to stop the reaction of each dilution and absorbance was measured at 492 nm with a reference filter at 620nm. The LDH activity or cytotoxicity was calculated relative to the absorbance of the low control (0% cytotoxicity) and high control (100% cytotoxicity) following the below formula:

%Cytotoxicity = [(Test Media Value – Low Control)/(High Control – Low Control)] * 100.

Subjects

Patients were selected based on inclusion and exclusion criteria defined in Tables 4-6. Patients were directed not to use any other skincare products or undergo any form of aesthetic procedure before and during the study. Additionally, they were instructed to continue with their normal diet throughout the study. This study involved a retrospective review of previously treated patients. All patients signed an informed consent form approving their inclusion following an explanation of the study design and execution and agreed to the publication of results and images.

Table 4. Subjects’ inclusion criteria for selection.

Application and Directions for Use. Each subject is his/her own control.

Table 5. Formulation application instructions.

Kinetics

Parameters tested and the time points are described below:

Table 6. Kinetics known as in-vivo tests parameters and check points.

In-VivoCase Study

This assessment was performed on 7 patients using products within a personalized pre-established skincare ritual. The TM was introduced into patients existing skincare routine for a period of 4 weeks. Visia images (Visia, Canfield Scientific, NJ, USA) were taken on clean skin prior to commencement of the study then again after 4 weeks of introducing #2LCRE.20 into each subject’s skincare routine. To measure and observe physiological changes, the following Visia-defined parameters were captured: Overview, Spots, Brown Spots, UV Spots and Red Spots. Detailed data are reported for one single representative case. Further details on the rest of the cohort are available in Appendix 2.

Cytotoxicity and gene expression studies were performed on the Test Groups described in Table 3. Increased LDH activity is interpreted as an indication of cell damage or cell death. The positive control, Triton X-100 is the reference for full cytotoxic LDH activity. Levels of cytotoxicity were assessed after 72 hours of treatment and were compared to the untreated, negative control.

The cytotoxicity level of the #2LCRE.20 was minimal and comparable to the 0.9% Saline solution results as shown in Figure 2. The LDH activity threshold allowing to conclude that the TM is affecting the gene expression results is set at 50% cytotoxicity.

Of the 107 genes tested (Appendix 1), approximately 50 presented with significant changes in genetic expression. Each gene change is reported to influence structural and functional activity within skin cells (anti-inflammation, DNA repair, survival, apoptosis, antioxidation etc). A selection of 6 genetic markers was curated and reported above as their functions and mechanism of action are most relevant to this study as displayed in Table 7.

Note. The * (p ≤ 0.05) symbol designates statistical significance after performing unpaired t-test.

Figure 2. Relative % LDH activity (cytotoxicity) after 72 hours of treatment.

Table 7. Gene expression linear fold change 72 hours after TM application.

Each patient’s skin was assessed at Day 0, 28 and 56 under dermatologist supervision, thus representing its own control.

Each patient was provided with the TM to apply every morning, a self-assessment questionnaire and a daily log to report any changes and unusual or adverse occurrences.

The cohort recruited is detailed below in Table 8:

Table 8. In-vivo test demographics (total number of subjects n = 21).

Cutaneous Compatibility

This assessment was performed by a dermatologist. One patient experienced some clinical signs that were not attributed to use of the product and was therefore classified as not relevant. A second patient reported a functional sign of skin tightness during the trial period which was classified as relevant to use of the product (Table 9).

Table 9. Clinical and reported signs of non-acceptability.

Cutaneous Hydration

Cutaneous hydration was measured with a corneometer [13], and results were compared before and after using the TM (Figure 3). 95% of the patients experienced an increase in hydration of 40% after applying #2LCRE.20 daily for 28days and continuing using it for 56 days, 78% of the patients experienced increased hydration. An average of 22% elevation in epidermal moisture levels was recorded after 56 days of continual use.

Subjective Questionnaire

Each patient was requested to complete a questionnaire related to perceived efficacy of the Formulation based on various criteria after 28 and 56 days. Affirmations were proposed and subjects were asked to rate their degree of agreement with the statement on a four-point scale from agree to disagree. Where patients agreed or strongly agreed, the results were considered positive. Twenty-one patients were considered for this assessment therefore 1 patient represents 4.8% of the total result. To evaluate the significance of the answers, a 95% confidence interval (CI) was determined using the Wilson Methodology and compared to the lower limit of significance (Figure 4).

Overall, most patients observed and reported a global improvement in the appearance and feel of their skin including hydration, firmness, radiance, evenness of skin tone, suppleness and softness.

Figure 3. Variations of cutaneous hydration rate measured with a corneometer (in arbitrary units) after using the Formulation for up to 56 days compared to baseline. A significant increase in hydration is interpreted as enhanced moisturization attributable to use of the TM, while no change of hydration status indicates a non-drying effect.

Figure 4. Patient self-assessment after using the Formulation for 28 and 56days. * “effect bonne mine” meaning healthy glow.

Out of the 7 patients participating in this study (Table 10), one subject was selected to be presented in this report based on their results. The patient is of Type III on the Fitzpatrick scale and has a combination skin.

Prior to starting the study, a skin therapist assessed all subjects skin conditions and concerns as well as goals and current skincare ritual.

Pre-assessment results for all patients are presented in Table 10; baseline and final Visia images after 4 weeks of Formulation usage are presented in Figure 5 and 6 for the selected participant (#1).

Visia (Canfield Scientific, NJ) Skin Analysis imaging deploys a non-invasive measure of skin changes using cross polarized and UV light to reveal superficial, epidermal and dermal changes in skin tone and texture. The Visia images and measurements reported in Figure 5, Figure 6 and in Figure 7 for the selected participant #1 provide results for 8 different skin markers including an improvement percentage score and visible assessment. An increase in score results indicate an improvement of the specific skin marker.

The first parameter, Spots, typically identifies brown or red skin lesions including freckles, acne scars, hyper-pigmentation and vascular lesions. The before and after scores are very close, from 82% to 84%. The second parameter examined is the appearance of Pores. They are identified by their circular shape and their size being much smaller than a spot. The reduction in pore size score in this subject’s skin increased by 34%. Wrinkles are defined as folds or creases in the skin which are known to be associated with a decline in skin elasticity and are often considered to be a result of excessive sun exposure over time. The baseline results for this patient were 89% and increased to 99% over a 4-week period of using the Formulation. The Skin Texture parameter, assessed through Visia imaging is essentially an analysis of skin smoothness. It measures skin colour and smoothness by identifying gradients in colour from the surrounding skin

Table 10. Pre-selected patients pre-assessment performed by a Skin Therapist including skin type, conditions and goals.

The patient signed the informed consent form.

Figure 5. Selected patient #1 visible improvements in the appearance of wrinkles, texture, brown spots and pores after 4 weeks (below) of using the Formulation. A score (%) increase from baseline (above) to end results, indicates an improvement in skin appearance.

tone, as well as peaks (shown in yellow) and valleys (shown in blue) on the skin surface that indicate variations in the surface texture [Canfield Visia]. This patient has seen her skin texture improve from 79% to 94% after 4 weeks of using #2LCRE.20. The assessment of Porphyrins investigates the bacterial excretions that can settle in pores, potentially leading to acne. Canfield Visia imagery measures the fluorescence of UV light, manifesting as circular white spots and appear in yellow on the images. The porphyrin score remained somewhat similar post usage of the Formulation. UV Spots is a parameter directly linked to melanin coagulation below the skin surface and is considered to be a result of sun damage. They are generally invisible under normal lighting conditions but the selective absorption of the UV light by epidermal melanin enhances its display and detection by VISIA. In this study, there was not a significant improvement in the appearance of UV Spots. Red Areas were examined in this study to identify improvement in a variety of potential skin conditions that could be exacerbated by sun damage (inflammation, rosacea etc.). This patient experienced a 6% decrease in Red Spots. The last parameter assessed is Brown Spots or skin lesions that could be considered as hyperpigmentation, freckles, lentigines or melasma. These conditions occur due to an excess of melanin which is produced by the melanocytes in the bottom layer of the epidermis in response to solar exposure over time. This patient experienced a 20% increase in the improvement score in Brown Spots.

The patient signed the informed consent form.

Figure 6. Selected patient #1 overall visible improvements in skin tone, radiance and pigmentation before incorporating the Formulation (left) and after 4 weeks (right). Visual assessment from baseline (left) to end results (right), indicates an improvement in skin appearance.

Figure 7. Selected patient #1 skin markers measurements at baseline and after 4 weeks of using the Formulation.

The Visia images presented in Figure 6 highlight the improved skin tone and radiance overall after 4weeks of incorporating the TM in the skincare regimen as well as a diffused pigmentation around the eye area.