BARNET ACTIVE INTELLIGENT MOISTURIZATION AESTHIGEL. Aesthetic Moisturizer acting from the bottom to the surface in every layer of the skin

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1 BARNET ACTIVE INTELLIGENT MOISTURIZATION AESTHIGEL Aesthetic Moisturizer acting from the bottom to the surface in every layer of the skin The information contained in this technical bulletin is, to the best of our knowledge, true and accurate. No warranty, expressed or implied is made or intended. The use should be based upon the customer s own investigations and appraisal. No recommendation should be construed as an inducement to use a material in infringement of patents or applicable government regulations. May 2015

2 CONCEPT: AESTHIGEL IS A MOISTURIZER FROM THE BASAL LAYER TO THE SURFACE EPIDERMAL DEPTH EPIDERMAL HYDRATION SKIN SURFACE Hyaluronic Like Action CORNIFIED LAYER +43% Filaggrin / +60% Caspase % / 65% KLK 7 & KLK 5 MEDIUM LAYERS +45% Claudin 1 (tight Junction) +56% Ceramides DEEPER LAYERS +211% Epidermal Hyaluronic Acid AESTHIGEL STRATEGY 2

3 AESTHIGEL S RESTRUCTURING EFFECT Untreated explant Explant treated with AesthiGel Application: Topical application of 1.5% AesthiGel for one week on human skin explants. Explants treated with AesthiGel have an homogenous and smoothed surface with only a few desquaming cells. 3

4 WHY DOES AESTHIGEL IMPROVE THE SKIN S SURFACE? The following tests demonstrate AesthiGel s efficacy: Hygroscopic effect: water retention at the surface of the skin Genomics: study of the upregulation of genes involved in the improvement of the hydro lipidic barrier Ex vivo: Confirmation that AesthiGel increases moisturizing molecules in the skin and the bridging of the cells 4

5 FIELD TO FACE AesthiGel is a hydrolyzed furcellaran obtained from the depolymerization of a sulfated carrageen extracted from the red algae Furcellaria lumbricalisc, also known as Black Carrageen. AesthiGel is red brown in color but when it is illuminated from the rear, appears to have a dark brown color. It becomes blackish when dried. Furcellaria lumbricalis grows on hard bottoms in Scandinavian waters and it is often the dominant alga in those areas. 5

6 AESTHIGEL AND FURCELLARAN FURCELLARIA LUMBRILICALIS Active components in AesthiGel Water extraction and drying POWDER OF SULFATED CARRAGEEN (FURCELLARAN) Subcritical CO2 extraction POWDER OF HYDROLYZED FURCELLARAN Polysaccharide obtained via an original depolymerization process seen to the right Solubilization (water+sea salts +phenoxyethanol) AesthiGel In solution 6

7 HYALURONIC LIKE ACTION HYDROSCOPY COMPARATIVE STUDY Subjects: Samples: 5 volunteers 100% AesthiGel and 100% Hyaluronic Acid (HA) Application: Put in cells with a relative humidity that will vary from 0 to 95% Measurement: For each condition of relative humidity, the polymers capture water molecules to reach a maximum called gain mass sorption equilibrium. The value of gain mass reflects the ability of each polymer to trap water molecules. The higher the humidity, the more the polysaccharide quenches water. 7

8 HYALURONIC LIKE ACTION HYDROSCOPY COMPARATIVE STUDY Results AesthiGel is able to trap atmospheric water molecules following a kinetic comparable to that of hyaluronic acid. AesthiGel is able to trap nearly its own weight in water, and to maintain a moisture film on the surface of the skin. Hyaluronic Acid Aesthigel Relative Mass Gain % of Relative Humidity AesthiGel is HA like. 8

9 AESTHIGEL vs. HA IN VIVO TEST Subjects: 30 volunteers Samples: solution containing 1.5% AesthiGel (equivalent to 0.009% Furcellaran) solution containing 0.025% Hyaluronic Acid (MW = 1.3 to 1.8 MD) Application: 15 volunteers apply AesthiGel solution 15 volunteers apply Hyaluronic Acid Applied twice daily for 2 weeks Skin hydration level measured using corneometer HA is a high molecular weight: 2,000,000 D AesthiGel is 200,000 D 9

10 AESTHIGEL VS. HYALURONIC ACID AesthiGel increases the skin hydration level 4 hours after only a single application. 12 Variation % of skin hydration level versus T p<0.05 student test 4H 6H 24H AesthiGel Hyaluronic Acid Its hydrating action is faster and more efficient than Hyaluronic Acid. 10

11 AESTHIGEL VS. HYALURONIC ACID Day after day, AesthiGel increases skin hydration levels. It is still more efficient than hyaluronic acid. Treatment with hyaluronic acid needs more than two weeks to reach the hydration level provided by one day of treatment with AesthiGel. 20 Variation % of skin hydration level versus T p<0.05 student test J1 J7 J14 AesthiGel Hyaluronic Acid AesthiGel is better than HA. 11

12 AESTHIGEL: GENE EXPRESSION Goal: To Identify the Genes Activated by AesthiGel to anticipate which molecules can be produced Cell Model: Reconstructed Human Epidermis (RHE) Sample: AesthiGel at 1% (w/w) Application: One topical application at T0 and measurement after 24 hours Or Four topical applications at T0, T24H, T48H, and T72H Analysis: Analysis of gene expression by RT gpcr (quantitative approach) Expressed in % of gene expression in treated epidermis in comparison with untreated epidermis. 12

13 AESTHIGEL: IMMUNO STAINING Goal: To confirm in explants in culture that AesthiGel increases the production of key molecules for moisturization Cell Model: Human skin explant Sample: AesthiGel at 1.5% (w/w) Multiple Applications: D1, D2, D3, D6 and D9 of the active versus control Analysis: Analysis of the intensity of staining (specific immuno labeling) Expressed in % of staining vs. non treated explants 13

14 HYDRATION MECHANISM Hydrating strategies are usually focused on two mechanisms: Improvement of NMF in the stratum corneum to boost hydration level Improvement of cellular cohesion and lipidic cement to avoid water loss Both mechanisms target the superficial layers of the epidermis (cornified layer and granular layer). However, water gradient is not limited to these upper layers, but to the whole epidermis. 14

15 MARKERS OF HYDRATION Hydration strategies developed by AesthiGel: Holistic action on the whole epidermis: from the basal layer to the cornified layer A key but ignored target: epidermal Hyaluronic Acid Increase of Hyaluronic Acid in the dermis Increase of Ceramide Increase of Filaggrin Increase of binding molecules: Claudin 1 15

16 AESTHIGEL AND HA SYNTHESIS HAS3 an enzyme involved in epidermal Hyaluronic Acid synthesis AesthiGel, tested at 1%, regulated the expression of the gene coding for Hyaluronan Synthase 3 (HAS3), the protein involved in Hyaluronic Acid synthesis in epidermis. AesthiGel increased the expression of HAS3 by 64%. This effect is in favor of stimulating Hyaluronic Acid synthesis by epidermal keratinocytes. It could add to the hydrating effect of AesthiGel. Relative Expression (% of Control) 70% 60% 50% 40% 30% 20% 10% 0% 64% 23% 24H 72H SHORT TERM EFFECT LONG TERM EFFECT 16

17 AESTHIGEL 1.5% AND HA SYNTHESIS (EX VIVO) 9 DAYS Cell Model: Human skin explant Sample: AesthiGel at 1.5% Applications: topical application for 9 days Untreated Explant Analysis: Quantification of epidermal Hyaluronic Acid synthesis using immuno labeling. HA is highlighted in pink purple. After 9 days, a significant increase in epidermal Hyaluronic Acid synthesis was observed (+211%**) in the epidermis treated with 1.5% AesthiGel. **p<0.01 Student Test The total surface occupied by Hyaluronic Acid in the epidermis is increased by a factor of 3 after treatment with AesthiGel. epidermis dermis 17

18 AESTHIGEL 1.5% AND HA SYNTHESIS (EX VIVO) 9 DAYS % Intensity of HA staining (%) % Untreated with with 1.5% 1.5% Aesthinet Aesthigel 5 0 Epidermis Dermis With AesthiGel the skin makes HA. 18

19 AESTHIGEL AND HYALURONIC ACID Stratum corneum EPIDERMIS Granular layer Spinous layer Basal layer DERMIS 1 2 At the surface: (1) HA like In the skin: (2) Helps to increase HA 19

20 AESTHIGEL AND CERAMIDE PRODUCTION Effect of AesthiGel on the Expression of Genes Involved in Ceramide Synthesis AesthiGel, tested at 1%, regulates the expression of UGCG and GBA (enzymes involved in ceramide synthesis). AesthiGel increased the expression of GB by 31% in 24 hours and increased the expression of UGCG by 89% in 72 hours; it favors the strengthening of the epidermal barrier. Relative expression (% of the control) % +31% GBA UGCG SHORT TERM EFFECT LONG TERM EFFECT 20

21 AESTHIGEL 1.5% AND CERAMIDE PRODUCTION Effect of AesthiGel on the Expression of Genes Involved in Ceramide Synthesis in 9 Days Untreated explant Explant treated with Aesthigel AesthiGel at 1.5% applied topically on human skin explants Increased ceramide synthesis of +56% in the epidermis. Ceramides are the mortar of the bricks in the barrier to retain water. 21

22 AESTHIGEL AND NMF Expression of Genes Involved in Production of NMF Components NMF (Natural Moisturizing Factor) KALLIKREIN RELATED PEPTIDASE 5 (KLK5) = Serine protease expressed by the epidermis with the ability to activate KLK7 KALLIKREIN RELATED PEPTIDASE 7 (KLK&) = Protease with the ability to regulate CASP14 maturation * CASPASE 14 (CASP14) = Protein which plays a role in the hydration of the skin by activating the maturation of filaggrin and its hydrolysis in essential amino acids to NMF FILAGGRIN (FLG) = Essential protein degrading in free amino acids, giving an important constituent to NMF *Yamamoto, et al. Maturation mechanism of Caspase 14,

23 SHORT TERM EFFECT OF AESTHIGEL AND NMF Effect of AesthiGel on the Expression of Genes Involved in the Production of NMP Components AesthiGel, tested at 1%, regulates the expression of genes involved in the formation of filaggrin like KLK 5, KLK 7, Caspase 14 and Filaggrin genes AesthiGel 24H (1%) 70% Relative Expression (%) 60% 50% 40% 30% 20% 60% 39% 10% 0% KLK 5 KLK 7 SHORT TERM EFFECT AesthiGel increased the expression of KLK 5 by 60% and the expression of KLK 7 by 39% after only one application (24H) 23

24 LONG TERM EFFECT OF AESTHIGEL AND NMF Effect of AesthiGel on the Expression of Genes Involved in the Production of NMP Components AesthiGel, tested at 1%, regulates the expression of genes involved in the formation of filaggrin like KLK 5, KLK 7, Caspase 14 and Filaggrin genes 70% 65% AesthiGel 72H (1%) Relative Expression (%) 60% 50% 40% 30% 20% 10% 43% 0% CASP14 LONG TERM EFFECT FLG AesthiGel increased caspase 14 (CASP14) expression by 65% and filaggrin (FLG) expression by 43% after 3 successive applications (72h). 24

25 AESTHIGEL 1.5% AND NMF PRODUCTION Effect of AesthiGel on the Synthesis of Caspase 14 Involved in the Production of NMF Components in 9 Days Cell Model: Human Skin Explants Sample: Application: Topical Analysis: 1.5% AesthiGel Visualization of Caspase 14 synthesis using immuno labeling (pink purple) Stratum corneum Expression area of Caspase 14 Untreated explant The expression of Caspase 14 was increased by +65% in the epidermis treated with 1.5% AesthiGel Explant treated with Aesthigel 25

26 AESTHIGEL AND TIGHT JUNCTION Effect of AesthiGel on the Expression of Claudin 1, a Protein Involved in Tight Junction to Limit Water Loss The function of tight junctions is to block water circulation in intercellular spaces and to ensure the seal between two tissue compartments. This kind of junction has been identified in human skin explants into the granular layer [2]. The main component of the tight junction is the protein Claudin 1. In 2002, Furuse, et al. showed that mice that did not express Claudin 1 died of a massive TEWL due to a deficient barrier funtion of the granular layer [3]. Role of Claudin 1 in the formation of tight junctions Paracellular pathway Tight junction protein complex Tight junctions Basolateral side [2] Adherens Junctions, Desmosomes and Tight Junctions in Epidermal Barrier Function Johanna M. Brandner, Marek Haftek and Carien M. Niessen. The Open Dermatology Journal, 2010, 4, [3] Claudin based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin 1 deficient mic. Furuse M, Hata M, Lipid bilayer Claudin 26

27 AESTHIGEL AND TIGHT JUNCTION AesthiGel, tested at 1%, regulated the expression of the gene coding for Claudin 1, a protein present in all layers of the epidermis. AesthiGel increased the expression of Claudin 1 by 78%. +78% Relative Expression (vs. Control) Claudin 1 Control with with 1% 1% Aesthinet AesthiGel Better cohesion = water retention 27

28 AESTHIGEL 1.5% AND TIGHT JUNCTION Untreated explant Cell Model: Human Skin Explants Sample: 1.5% AesthiGel Application: Topical Analysis: Quantification of Claudin 1 synthesis using immuno labeling. Claudin 1 is highlighted in pink purple. Explant treated with Hydranov A significant increase in Claudin 1 synthesis was observed (+45%*) in the epidermis treated with 1.5% AesthiGel. *p<0.1 Student Test 28

29 AESTHIGEL AND EPIDERMIS TIGHTENING MEDIUM LAYERS +45% Claudin 1 (Tight Junctions) MEDIUM LAYERS +56% Ceramides 1 2 Increase in the synthesis of Claudin 1 (1) and ceramides (2) help to tighten the epidermis. The hydration rate previously increased via epidermal Hyaluronic Acid in the deeper layers is preserved. 29

30 AESTHIGEL AND BARRIER EFFECT Effect of AesthiGel on Gene Expression of Proteins Involved in the Keratinocytes Adhesion (Desmosomes) Proteins involved in adhesion between 2 keratinocytes: Catenin alpha 1 (CTNNA1) protein that associates with the cytoplasmic domain of a variety of cadherins. Association to catenins to cadherins produces a complex which is connected to the network of actin filaments, important for cell adhesion. Desmoglein 1 (DSG1) transmembrane glycoprotein component of desmosomes (cell cell junctions) Desmoplakin (DSP) essential component of desmosomes that anchors intermediate filaments to desmosomal plaques. Epiplakin 1 (EPPK1) essential protein for the barrier function of the skin and the integrity Proteins involved in adhesion of keratinocyte to epidermal dermal junction (basal membrane): Integrin, alpha 2 (ITGA2) laminin, collagen, fibronectin and E cadherin receptor protein, responsible for the strength and the organization of the extracellular matrix newly synthesized 30

31 AESTHIGEL AND EPIDERMAL STRENGTH Effect of AesthiGel on Gene Expression of Proteins Involved in the Keratinocytes Adhesion (Desmosomes) Relative expression (in % of control) 70% 60% 50% 40% 30% 20% 10% 0% AesthiGel 24H (1%) 61% 51% 33% 27% CTNNA1 DSG1 DSP EPPK1 Relative expression (in % of control) 70% 60% 50% 40% 30% 20% 10% 0% AesthiGel - 72H (1%) 65% AesthiGel increases the expression of CTNNA1, DSG1, DSP and EPPK1 by respectively 61%, 27%, 33% and 51% after 1 application (24h). All these protein are located into suprabasal layers of the epidermis. SHORT TERM EFFECT AesthiGel increases by 69% the expression of ITGA after 3 applications (72h). This integrin is located deeper into epidermis, at the level of basal cells. LONG TERM EFFECT Better cohesion = water retention 31

32 AESTHIGEL AND STRATUM CORNEUM STRENGTH Effect of AesthiGel on Gene Expression of Proteins Involved in Corneocyte Formation, Adhesion and Integrity Corneodesmosin (CDSN) adhesion protein of the extracellular portion of corneodesmosomes, involved in the cohesion of corneocytes Calmodulin_like 5 (CALML5) forming part of the family of calcium binding protein, a key enzyme in the terminal differentiation of keratinocytes Cornuline (CRNN) protein which plays a role in epidermal differentiation Small proline rich protein 2A (SPRR2A) protein important for the barrier function of the epidermis Loricrine (LOR) protein which plays an important role in the structure of corneocytes in cornified envelope and junctions between corneocytes formation 32

33 AESTHIGEL AND STRATUM CORNEUM STRENGTH Relative expression (in % of control) 70% 60% 50% 40% 30% 20% 10% 0% AesthiGel 24H (1%) CDSN 65% AesthiGel increases by 41% the expression of CDSN after one application (24H). This protein is located at the superficial layer of the epidermis. SHORT TERM EFFECT Relative expression (in % of control) 120% 100% 80% 60% 40% 20% 0% 65% AesthiGel 72H (1%) 98% 48% 69% DEFB4 RNASE SPRR2A LOR AesthiGel increases the expression of CALM5, CRNN, SPRR2A and LOR by respectively 86%, 54%, 48% and 69% after 3 applications (72H). These proteins are located deeper. LONG TERM EFFECT 33

34 SUMMARY AesthiGel stimulates the expression of epidermal genes involved in: Filaggrin activation (KLK5, KLK7, Caspase 14, Pro Filaggrin): more NMF Tight Junction (Claudin 1): less water loss Envelop production (loricrin, CALML5): healthy corneocytes Keratinocytes adhesion to the Dermal epidermal Junction: (ITGA) Keratinocytes adhesion (CNDS): stronger bridge (Desmosomes) Corneocytes adhesion (CNDS): stronger bridge (Corneodesmosomes) Lipids synthesis (GBA, UGCG) Epidermal Hyaluronic Acid synthesis (HAS3): water holding EX VIVO: AesthiGel at 1.5% increased ceramide synthesis by 56% and Hyaluronic Acid by 211% IN VIVO: Faster and more efficient than Hyaluronic Acid to hydrate the skin after a single application but also after 2 weeks of daily applications 34

35 AESTHIGEL Primary Use: INCI Name: REACH: Canada DSL: China Registration: Moisturizer Suggested Use Level: 1% 3% Water (and) Sodium Carrageenan (and) Sea Salt All components are pre registered or exempt. All components are listed or sold in commerce All components are listed on the Inventory of Existing Cosmetic Ingredients in China (IECIC). 35