GP4G Diguanosine Tetraphosphate ISP Vincience Global Skin Research Center 1 655, Route du Pin Montard -B.P. 212-06904 Sophia Antipolis Cedex France - Tel: (33) -4 92 94 16 06 - Fax: (33) -4 92 94 16 00 e-mail : vincience_info@ispcorp.com
A Description B Energy & Cell Metabolism Activation C Epidermal & Dermal Regeneration D Protection 2
A Description B C D Presentation Composition Origin Properties 3
Presentation A GP4G is a cosmetic active ingredient extracted from the plankton artemia salina. GP4G is designed for energizing and regenerating skin care products. GP4G also offers a new way to increase cellular defenses against UV stress and shows multiple protective effects : Increases cellular protection against UV aggressions and DNA damage. Stimulates HSP70, proteins implicated in the fight against cellular stress, and increases skin defenses as well as resistance to UV- and heat-stress. Offers an anti-inflammatory action by decreasing the level of inflammatory cytokines such as IL-1a, IL-8 and TNFa. Cyst of Artemia salina with GP4G molecule 4
The GP4G Molecule A The originality of GP4G is based on its composition rich in nucleotide energetic messengers, and in particular, in the presence of an unusual nucleotide: Diguanosine tetraphosphate (GP 4 G) 5
Origin A 1. Artemia is a plankton that lives in hypermineral lakes since several millions of years. 2. When environmental conditions turn critical (stress), Artemia enters a state of dormancy, encapsulates and accumulates a vital molecule, the Diaguanosine Tetraphosphate (GP4G), a principal source of phosphate bonds with high energetic potential used for DNA protection as well as energy storage. 3. When environmental conditions turn favorable again, Artemia 'awakens', rehydrates and different metabolic activities take place again. Ultimately, GP 4 G is transformed into ATP. 4. GP4G provides Artemia with energy and protection for its survival and development. Life cycle of artemia salina plankton 1 Artemia Stress conditions 2 Dormancy Favorable conditions GP 4 G 3 Awakening 4 Development 6 ATP
GP4G Cell protector - Cell Revitalizer - Cell Energizer GP4G is an activator of G proteins Stimulus GP4G R Cell G GTP GP4G G G GTP GP4G AC ATP camp cell activation 7
Properties A Cell metabolism activation 1. GP4G triggers cellular second messengers: +40% camp content Epidermal and dermal regeneration 2. GP4G boosts epidermal synthesis: +68% keratin expression 3. GP4G boosts dermal synthesis: +54% collagen,+27% fibronectin DNA & HSP Protection 4. GP4G protects and repairs DNA: -45% D A damage 5. GP4G significantly induces HSP70 synthesis: +25% HSP 70 6. GP4G compensates for age related decrease in HSP70 7. GP4G decreases the synthesis of pro-inflammatory cytokines 8. GP4G increases skin defense and comfort during therapy with retinoids. 8
A B Energy & Cell Metabolism Activation C D A stable precursor of ATP Increases camp synthesis 9
Energy: GP4G is a stable precursor of ATP B µmoles/g protein 135 µg/ µµ/ 115 95 75 GP4G ATP+ GP4G ATP 55 ATP 35 0 1 2 3 4 Days GP4G When Artemia is awakened by favorable conditions, GP4G content declines rapidely while a sharp increase in ATP is observed. This is explained by a conversion of GP4G into ATP and a role of the GP4G in the awakening of the quiescent cells. 10
Skin efficacy tests GP4G increases camp synthesis B Cells: Human fibroblasts Dose: GP4G 2% Evaluation: immunoenzyme assay camp is a second messenger which activates enzymes of several metabolic pathways. camp content (fmol/well) 9 8 7 6 5 4 3 2 1 0 + 40% Control GP4G GP4G as an ATP precursor demonstrates energizing properties: it "boosts" campdependant metabolic pathways. 11
A B C Epidermal & Dermal Regeneration D Increases epidermal proteins Increases dermal proteins 12
Skin efficacy tests GP4G increases epidermal proteins C Cells: Human keratinocytes Dose: GP4G 1% Evaluation: immunoblotting (keratin) and immunofluorescence (filaggrin) Total keratins (pixels) 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 Keratin 25% 68% 10 h 24 h Control GP4G Filaggrin Control GP4G 1% GP4G demonstrates regenerating properties on epidermal cells. 13
Skin efficacy tests GP4G increases dermal proteins C Total extracted cell proteins 6000 5000 4000 3000 2000 1000 0 Fibronectin +27% Control GP4G 1% Cells: Human fibroblasts Dose: GP4G 1% Evaluation: immunoblotting GP4G demonstrates regenerating properties on dermal proteins. Intensity (% of increase/control) 180 160 140 120 100 80 60 40 20 0 Collagen +54% Control GP4G 1% 14
A B C D Protection Heat Shock Proteins Protects & repairs DNA HSP & Protein Protection Reduces Pro-inflammatory Cytokines expression 15
The HSP or Heat Shock Proteins Heat shock proteins (HSP) or molecular chaperons, play an important role in protecting the cell from different types of stress. GP4G GP4G induces HSP70 which is important for many cell metabolic functions. HSP constitutive Native protein Protein denaturation Inductible HSP Protein folding STRESS STRESS 16
Skin efficacy tests GP4G protects and repairs DNA D Cells: Human keratinocytes Dose: GP4G 1% Evaluation: Tunel assay method Intensity of fluorecence (Pixels) 30 25 20 15 10 5 No UVB UVB 100mJ/cm 2 30% 40% 45% 0 Control No GP4G GP4G before GP4G after GP4G before & after DNA damage level DNA protection GP4G prevents and repairs DNA damages in cutaneous cells. 17
GP4G : DNA protection Low UVB doses D Culture: Human Fibroblasts Product application: GP4G 3% Application Time: 48h UVB Dose: 30 mj/cm 2 Evaluation: Comet Assay Irradiated cells Irradiated cells treated with GP4G The comet assay is a very sensitive method to examine D A damage and repair. D A damage is evaluated by the size of the tail of the comet, in relation to the head (undamaged D A). GP4G Under low doses of UVB, when cells were treated with GP4G, very little damage was seen, in contrast to untreated cells. 18
GP4G Increases HSP70 expression D Culture: Ex vivo Human skin Product application: GP4G 3% Evaluation: Immunofluorescence staining Skin treated with Placebo Skin treated with GP4G GP4G induces HSP70 expression in human skin, in stress-free manner. 19
D GP4G Increases HSP70 expression Culture: Human Fibroblasts Product application: GP4G 3% Application time: 24 h Evaluation: immunoblotting % of Expression 140 + 25% 120 100 80 60 40 20 0 Control 3% Inductylor GP4G GP4G significantly induces HSP70 synthesis. 20
GP4G Decreases inflammatory cytokines D pg IL-1 alpha/mg proteins 28 Control 3% Inductylor GP4G 24 20 Cell culture: HaCaT Cells Product application: GP4G 3% UVB Dose: 30 mj/cm 2 IL-1 dosage: Elisa assay 16 12 8 4 0 0 6 8 18 24 0 6 8 18 24 Hours GP4G decreases the synthesis of pro-inflammatory cytokines induced by UV, confirming that the increase in HSP is not due to an inflammation process. 21
GP4G Increases heat tolerance D Culture: Ex vivo human skin Product application: GP4G 3% Heat Stress: 47 C for 1 h, 37 C for 24 h Evaluation: H&E staining Skin treated with placebo at 47 C Skin treated with GP4G at 47 C GP4G increases the defense of skin exposed to heat stress. 22
GP4G compensates for age-related decrease in Hsp70 D Culture: Ex vivo human skin Application dose: GP4G 3% UVB Dose: 100 mj/cm 2 Evaluation: immunofluorescence Skin from 31 years old woman Skin from 53 years old woman Control skin UVB UVB + GP4G 23
GP4G helps restore the decrease in Hsp70 caused by retinoids D ATRA long term administration into the cells decreased their Hsp70 level. Inductylor 3% administration into ATRA-treated cells helps restore HSP70 content in these cells. Control skin After 5 days of ATRA treatment After 5 days of ATRA & GP4G treatment GP4G increases skin defense and comfort during therapy with retinoids. 24
A D E Cosmetic Applications 25
Cosmetic applications E Revitalizing or energizing skin care products Day creams, for DNA protection and repair Anti-photoaging cosmetic skin care Sun products to help DNA protection & repair Skin protection and defense against environmental stress (HSP) Co-active energetic boosters (cell signal enhancer) Recommended use level: 0.5-5% 26