HYDRANOV. A New Look at Hydration

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1 HYDRANOV A New Look at Hydration

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3 HYDRANOV A New Look at Hydration COSMOS EPIDERMIS SYNTHESIZES 4 TIMES MORE HYALURONIC ACID THAN THE DERMIS. Epidermal hyaluronic acid: a still overlooked potential. The rate of hyaluronic synthesis is higher in the epidermis than in the dermis. Since the dermis is much thicker than the epidermis, it contains 4 to 9 times more hyaluronic acid, but for equivalent tissue quantities, the epidermis synthesises 4 times more hyaluronic acid than the dermis (1). In the epidermis hyaluronic acid is localised in the intercellular space of the basal and spinous layers. In the same way as in the dermis the hygroscopic properties of hyaluronic acid are of great importance in hydrating the deep layers of the epidermis, but its function goes further than conventional hydration. It has been shown that the presence of high concentrations of hyaluronic acid in the intercellular space tends to renew desmosomes and tight junctions, and so reorganise the keratinocytes not only within the basal layer, but also throughout the process of differentiation as far as the stratum corneum (2). Hyaluronic acid acts therefore like an overall epidermal restructuring agent by initiating and maintaining constant cellular remodelling from the basal layer to the cornified layer. «HYALURONIC LIKE», HYDRANOV HAS HYGROSCOPIC PROPERTIES ALLOWING THE CAPTURE OF WATER MOLECULES AT THE SKIN SURFACE. «EPIDERMAL HYALURONIC ACTIVATOR», IT ALSO STIMULATES THE SYNTHESIS OF EPIDERMAL HYALURONIC ACID FOR AN OVERALL EPIDERMAL RESTRUCTING AND HYDRATING ACTION. HYDRANOV : High Tech Concentrate of Oligofurcellaran The oligofurcellaran is obtained by the depolymerisation of a sulfated furcellaran (galactose and anhydrous galactose) coming from the red alga Furcellaria lumbricalis, widespread in Scandinavian waters. Depolymerization is made using supercritical CO2 technology in anhydrous conditions. After depolymerization, the oligofurcellaran is rehydrated with an enriched sea salt solution. This final intake of marine nutrients, and mainly potassium, induces a double helicale reorganization of the polymer that gives it hygroscopic and gelling properties comparable to hyaluronic acid. The resulting oligofurcellaran has a molecular weight >200kDa. This depolymerisation method, water and acid free, complies with the policy of energetic resources managment developped by Codif R&N.

4 HYDRANOV CHRONOLOGICAL ACTION 1 «hyaluronic like» action at the surface of the skin 2 «hyaluronic activator» action : stimulates the synthesis of epidermal hyaluronic acid 3 Reinforcement of tight junctions New target : Claudin 1 4 Stimulation of ceramids synthesis 5 Reinforcement of NMF New target : Filaggrin HYALURONIC LIKE ACTION OF HYDRANOV INVITRO TEST The oligofurcellaran concentrated in Hydranov has high molecular weight. Therefore in the same way as hyaluronic acid it acts as a macromolecule with surface properties. Pure samples of Hydranov and hyaluronic acid are put in specific cells with a relative humidity that will vary from 0 to 95%. For each condition of relative humidity, the polymers capture water molecules to reach a maximum called «gain mass at sorption equilibrium». The value of gain mass reflects the ability of each polymer to trap water molecules. 120 mass gain % at equilibrium Hyaluronic Acid Hydranov % of relative humidity RESULT: Hydranov is able to trap atmospheric water molecules following a kinetic totally comparable to hyaluronic acid. The more atmosphere is saturated with water, the more Hydranov traps water molecules. For 95% of relative humidity, 86% mass gain is measured. HYDRANOV IS CAPABLE TO TRAP A QUANTITY OF WATER ALMOST EQUAL TO ITS INITIAL MASS AND THUS MAINTAINS A MOISTURE FILM ON THE SURFACE OF THE SKIN.

5 HYALURONIC ACTIVATOR ACTION OF HYDRANOV Given the size of the oligofurcellaran and the low probability that it can penetrate into the epidermis, the effect of Hydranov has been evaluated by topical application on human skin explants or human reconstructed epidermis. HYDRANOV INCREASES THE EXPRESSION OF HYALURONAN SYNTHASE 3 EXVIVO TEST Hyaluronan synthase 3 is an enzyme involved in hyaluronic acid synthesis. Reconstructed human epidermis Topical application of 1% Hydranov for 72 hours. Gene expression analysis using RTqPCR. RESULT: +64% HYALURONAN SYNTHASE 3 expression % compared to untreated control Control +64% 1% Hydranov HYDRANOV INCREASES THE SYNTHESIS OF EPIDERMAL HYALURONIC ACID EXVIVO TEST Human skin explants Topical application of 1.5% Hydranov for 9 days. Quantification of epidermal hyaluronic acid synthesis using immunolabelling (fuchsia colour). RESULT: +211% HYALURONIC ACID X3 : TOTAL SURFACE AREA OCCUPIED BY HYALURONIC ACID p<0.01 Student test Untreated explant Explant treated with 1.5% Hydranov INCREASE IN EPIDERMAL HYALURONIC ACID SYNTHESIS WILL PROVIDE A BETTER HYDRATION OF THE DEEPER LAYERS OF THE EPIDERMIS BUT ALSO STIMULATE THE RENEWAL OF TIGHT JUNCTIONS IN THE MIDDLE AND UPPER LAYERS.

6 RESTRUCTURING ACTION OF HYDRANOV : CLAUDIN 1 ET CERAMIDS The function of tight junctions is to block circulation of fluids between cells and to ensure in this way that there is a tight seal between two tissue compartments. They were identified in human skin explants in the medium layers (granular and subbasal layers) (3). The main factor contributing to the formation of these tight junctions is a protein called Claudin 1. In 2002, Furuse and al. showed that mice which did not express Claudin 1 died of massive TransEpidermal Water Loss due to a deficient barrier function in the granular layer (4). Tight junction: protein complex Lipid bilayer Claudine Tight junction Claudin 1 HYDRANOV STIMULATES THE SYNTHESIS OF CLAUDIN 1 EXVIVO TEST Human skin explants Topical application of 1.5% Hydranov for 9 days. Quantification of Claudin 1 synthesis using immunolabelling. RESULT: +45% CLAUDIN 1 expression % versus untreated control Control +45% 1.5% Hydranov HYDRANOV STIMULATES THE SYNTHESIS OF CERAMIDS EXVIVO TEST Human skin explants Topical application of 1.5% Hydranov for 9 days. Quantification of Ceramids synthesis using immunolabelling. Untreated explant RESULT: : +56% CERAMIDS p<0.01 Student test Explant treated with 1.5% Hydranov INCREASE IN THE SYNTHESIS OF CLAUDIN 1 AND CERAMIDS HELPS TO STRENGTHEN THE SEALING OF THE EPIDERMIS. THE HYDRATION RATE PREVIOUSLY INCREASED VIA EPIDERMAL HYALURONIC ACID IN THE DEEPER LAYERS IS PRESERVED.

7 NMF BOOSTER EFFECT OF HYDRANOV Numerous studies report the essential role of Filaggrin which, when it is broken down in the stratum corneum, adds hygroscopic amino acids (AA) to the NMF (Natural Moisturizing Factors). An absence of filaggrin expression leads to an almost complete lack of stratum corneum (ichtyosis) and major skin dehydration (xerosis) (5). Filaggrin is broken down in the stratum corneum by Caspase 14 which is itself matured under the control of a serine protease called kallikreinrelated peptidase 7 (KLK7). Activation of KLK7 depends on a second kallikrein coded by the gene KLK5 Procaspase 14 Filaggrin maturation KLK7 KLK5 activation degradation Caspase 14 Hygroscopic AA NMF HYDRANOV STIMULATES THE SYNTHESIS OF FILAGGRIN EXVIVO TEST Reconstructed human epidermis Topical application of 1% Hydranov for 72 hours. Gene expression analysis using RTqPCR. RESULT: +43% FILAGGRIN expression % versus untreated control % 0 Control 1% Hydranov HYDRANOV FAVOURS FILAGGRIN DEGRADATION EXVIVO TEST Reconstructed human epidermis Topical application of 1% Hydranov for 24 hours (KLK5 and KLK7 analysis) and 72 hours (caspase 14 analysis). Analysis of genes expression using RTqPCR. Visualization of Caspase 14 using immunolabelling (fuchsia colour) in human skin explant treated with 1.5% Hydranov. Visualization of Caspase 14 in untreated explant RESULT: +60% KLK5 / +39% KLK7 +65% CASPASE 14 Visualization of Caspase 14 in explant treated with 1.5% Hydranov BY FAVOURING FILAGGRIN BREAKDOWN, HYDRANOV BOOSTS NMF AND REINFORCES THE HYDRATION OF CORNIFIED LAYER. THIS LATEST PROPERTY COMPLETES HYDRANOV ACTION: A GLOBAL HYDRATION FROM THE BASAL LAYER TO THE STRATUM CORNEUM.

8 COMPARATIVE HYDRATING PROPERTIES OF HYDRANOV AND HYALURONIC ACID INVIVO TEST 15 volunteers apply a solution containing 1.5% HYDRANOV (equivalent to 0.009% pure oligofurcellaran) 15 volunteers apply a solution containing 0,025% pure HYALURONIC ACID (% recommended by the supplier) of high molecular weight: >200kDa. Twice daily applications for 2 weeks on forearms Measure of skin hydration level using corneometer COMPARATIVE EFFICACY AFTER A SINGLE APPLICATION Hydranov hydrates the skin with an immediate and continuous efficacy during the 24 hours following its application. Its hydrating action is faster and higher than thus of Hyaluronic acid. HYDRATION INCREASE INDUCED BY HYDRANOV IS 3 TIMES HIGHER THAN THUS TRIGGERED BY HYALURONIC ACID. p<0.05 Student test Hydration variation versus T0 (%) Hydranov Hyaluronic acid 4H 6H 24H COMPARATIVE EFFICACY AFTER 2 WEEKS Hydranov has a cumulative hydrating effect. Day after day, skin hydration is increased. AFTER A 2 WEEKS TREATMENT, THE HYDRATION PROVIDED BY HYALURONIC ACID IS STILL LOWER THAN THUS PROVIDED BY HYDRANOV AFTER ONLY 24H. p<0.05 Student test Hydration variation versus T0 (%) Hydranov Hyaluronic acid D1 D7 D14

9 VISUALIZATION OF THE RESTRUCTURING ACTION OF HYDRANOV EXVIVO TEST Human skin explants. Topical application of 1.5% Hydranov for 1 week. Staining, rinsing, drying and observation of the surface using Scanning Electron Microscopy. Untreated explant Explant treated with Hydranov THE EXPLANT TREATED WITH HYDRANOV HAS AN HOMOGENEOUS AND SMOOTHED SURFACE WITH ONLY FEW DESQUAMING CELLS. THIS OBSERVATION ILLUSTRATES THE RESTRUCTURING ACTION OF HYDRANOV ON THE WHOLE EPIDERMIS, FROM THE BASAL LAYER TO THE CORNIFIED LAYER. CONCLUSION : HYDRANOV targets a molecule with outstanding potential and little known: the epidermal hyaluronic acid. Hydranov reinvents hydration by targeting new molecules with unexploited potential. By increasing the synthesis of epidermal hyaluronic acid, Hydranov promotes not only hydration of the deepest layers of the epidermis, but also renewal of tight junctions to reorganise the keratinocytes in all the layers of the epidermis. In the corneal layer, Hydranov activates metabolism of filaggrin to enrich NMF with hygroscopic amino acids while on the surface, in the same way as hyaluronic acid, it captures water molecules to maintain a hydrating film. Hydranov is a HiTech concentrate of oligofurcellaran, faster and 3 times more efficient than hyaluronic acid to hydrate and restructure the skin. Marine plant origin Hydrosoluble active Invitro test exvivo test Invivo test COSMOS Cosmos Validation ingredient

10 FORMULATION GUIDELINE HYDRARESTRUCTURING FLUID This fluid is formulated with HYDRANOV: hydrarestructuring ingredient, HYDROFILTRAT MENYANTHES G: dermorestructuring active and a moisturizing ingredient: PHEOHYDRANE. Phase Raw material / commercial name INCI name % NIKKOMULESE LC (1) Behenyl Alcohol & Stearyl Alcohol & PEG20 Phytosterol & Cetyl Alcohol & Phytosterols & Glyceryl Stearate & Hydrogenated Lecithin & Caprylic/Capric Triglyceride 5 CRODAMOL GTCC / MIGLYOL 812 / WAGLINOL (2) Caprylic/Capric Triglyceride 3 A SILICON (DIMETHICONE (100CS) (3) Dimethicone 0.5 PHENOXYETHANOL (4) Phenoxyethanol 0.8 ARLAMOL HD (5) Isohexadecane 4 LANOL 99 (6) Isononyl Isononanoate 5 B B DEMINERALIZED WATER Aqua AVICEL PC 611 (7) Microcrystalline Cellulose & Cellulose Gum 1 DIPROPYLENE GLYCOL (8) Dipropylene Glycol 3 KELTROL CGSFT (9) Xanthan Gum 0.5 B ELESTAB CPN (10) Chlorphenesin 0.27 HYDRANOV P (11) Aqua & Phenoxyethanol & Sodium Carrageenan & Maris Sal 1.5 C PHEOHYDRANE P (11) Aqua & Hydrolyzed Algin & Maris Aqua & Phenoxyethanol & Chlorella Vulgaris Extract 1 HYDROFILTRAT MENYANTHES G (11) Glycerin & Aqua & Menyanthes Trifoliata Extract 2 FRAGRANCE FLOWER AND FRUIT (12) Fragrance 0.1 (1) IMCD, (2) Croda, (3) Quimdis, (4) Laserson, (5) Quimasso, (6) Seppic, (7) FMC BioPolymer, (8) Brenntag, (9) CP Kelco, (10) Cognis, (11) CODIF Recherche et Nature, (12) Expressions parfumées Product Aspect: White Glitter Fluid cream Product Properties: ph = 6.54 ± 0.5 Operating Method: Heat A at 75 C Heat the phase B at 75 C under emulsifier 2000 rpm during 10 minutes. Add the mixture B under emulsifier 1500 rpm during 5 minutes. Add the mixture B under emulsifier 1500 rpm during 5 minutes. Add A into B+B +B under emulsifier 2500 rpm, then let shake in these conditions during 10 minutes. Around 2530 C, add the phase C.

11 BIBLIOGRAPHIC REFERENCES (1) Tammi M, Saamanen AM : Degradation of Newly Synthesized High Molecular Mass Hyaluronan in the Epidermal and Dermal Compartments of Human Skin in Organ Culture. J Invest Dermatol 97: , 1991 (2) Tammi R, Tammi M: Hyluronan in the Epidermis (3) 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, 1420 (4) Claudinbased tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin1deficient mice. Furuse M, Hata M, Furuse K, et al. J Cell Biol 2002; 156: (5) Filaggrin in the frontline: role in skin barrier function and disease. Sandilands A, Sutherland C, et al. J Cell Science, 2009, 122,

12 HYDRANOV - A NEW LOOK AT HYDRATION Cosmetic activities «hyaluronic like» action: traps water molecules at the surface of the skin «hyaluronic activator» action : stimulates the synthesis of epidermal hyaluronic acid Reinforces tight junctions via Claudin 1 Increases ceramids synthesis Reinforces the NMF via Filaggrin Hydrating efficacy faster and higher than hyaluronic acid Restructuring action INCI name Hydranov P: Water (and) Phenoxyethanol (and) Sodium Carrageenan (and) Sea salt Hydranov PA : Water (and) Phenethyl alcohol (and) Sodium Carrageenan (and) Sea salt COSMOS Recommended % of use: Hydranov P : 1.5% Hydranov PA : 1.5% Technical department : infotech@codifrechercheetnature.com Commercial department : commercial@codifrechercheetnature.com 70, rue du Commandant l Herminier CS SaintMalo cedex FRANCE Tel : Fax : CODIF Recherche & Nature is committed to sustainable development. All our wastewater is processed and purified using filtrating gardens. A succession of airtight horizontal and vertical filters is used to depollute wastewater by the roots of plant species chosen for their draining properties. Iris, water mint, flowering rush, willows etc now form part of the image of the brand which is recognized for its commitment to the preservation of natural resources. RPDOC133GB