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CORRELATIVES AND COMPLEMENTARY EXPERIMENTAL MODELS FOR THE DEVELOPMENT OF ANTIAGEING DERMATOCOSMETIC PRODUCT Authors: Laura Olariu, Brindusa Dumitriu BIOTEHNOS, Research & Development, Otopeni, Romania. Dermatocosmetic ingredients testing on standardized cellular cultures INTRODUCTION: Despite the exponential develop of cosmetics with pharmaceutical activity, appropriates methods to quantify the effects are not yet established. The efficacy claim is a widespread effect for many skin care products, but usually a few test were done to scientific prove this action. Development of new concepts regarding anti-ageing active ingredients Performing the right “in vitro” and “in vivo” screening to prove the efficacy Scientific basis for cosmetic products claims INNOVATIVE ALGORITHM FOR Innovation of dermatocosmetic products Research of cellular physiological mechanisms Development of cellular investigative techniques Comparative analysis and correlation with non-invasiveness tissue methods New solutions for antiageing topical product investigations DEFINITION: “in vitro” and “in vivo” screening techniques comprise an assembly of instrumental methods, comparative and correlative, oriented to prove: the claimed effect and the specific target action of an ingredient. Efficacy checking of intermediate or final formulations Innovation in clinical dermatology Technological process innovation / Rising performances for tested products RECONSTITUTED HUMAN SKIN Classical methods of diagnosis and testing Innovation in dermatocosmetic, anti-ageing tests Cell cultures: “IN VITRO” METHODS TO PROVE THE “ANTIAGEING “ EFFECT OF DERMATOCOSMETIC INGREDIENTS: IMPLEMENTATION OF “IN VITRO” AND “IN VIVO” METHODS FOR SCREENING TECHNIQUES AND PRODUCT DEVELOPMENT STANDARDISED CELL LINES: dermal human fibroblasts (ex. HS27), normal human keratinocytes (ex. Immortalised cell line HaCat) Relevant cellular parameters: CITOTOXICITY “IN VIVO” , NON-INVASIVE TECHNIQUES TO PROVE THE “ANTIAGEING “ EFFECT OF DERMATOCOSMETIC PRODUCTS: Microscopic evaluation Studies on healthy volunteers using NON-INVASIVE METHODS: (selection) MTT/MTS reduction SKIN ELASTICITY MTS is a tetrazolium salt which is transformed in a formazan type product by dehydrogenase enzymes found in metabolically active cells. The quantity of formazan product as measured by 490nm absorbance is directly proportional to the number of living cells in culture LDH release Lactat dehidrogenase release in the extracellular medium is a potent indicator of citotoxic agent action CUTOMETER After treatment: R4= 0.113; R3= 0.389; Fo= 0.040 The mechanical parameters R4 R3 and F0 (area) provided by Cutometer measurements are most indicative of human skin fatigue. COLAGEN DEPOSITS VISUALISATION Citotoxicity curve for a dermatocosmetic ingredient Riss, T. and Moravec, R.A. (2004) Assay Drug Dev. Technol. 2, 51–62. DERMASCAN The age-related decrease of skin elasticity results in larger fatigue of adult skin than young skin after applying multiple stress at one and the same anatomic region. The non-invasive method applied can be useful for objective and quantitative investigation of age-related changes in skin fatigue and evaluation of the effects of cosmetic and antiaging topical products. CELL PROLIFERATION Collagen deposits visualization by high frequency ultrasound scanners - (ex. DERMASCAN Cortex Technology) Succesive generation proliferation for a dermatocosmetic ingredient CFSE (carboxy fluorescein diacetat succin imidil ester ) is a cell permeant fluorescein-based dye which covalenty attaches to cytoplasmic components of cells, resulting in uniform bright fluorescence. Upon cell division, the dye is distributed equally between daughter cells, allowing the resolution of up to eight cycles of cell division by flow cytometry. ANTI-WRINKLE ROUGHNESS CC_5 ZILE FIBRO_G9_G09.fcs 1024 1000 Gate 2 512 218 250 256 109 0 0 0 0 250 500 750 1000 0 256 512 768 1024 0 256 512 768 1024 PE-A ageing treatment SMOOTHNESS Gate 2 512 SKIN VISIOMETER / After anti- 256 PE-W PE-A PI staining of nuclear DNA show a histogram representation of G0/G1, S and G2/M cell cycle phases Count PE-A 500 768 326 Before anti- VISIOSCAN CC_5 ZILE FIBRO_G10_G10.fcs 1024 435 768 750 Count D N A synthesis: Cell cycle progression visualized by flow cytometry CC_5 ZILE FIBRO_G10_G10.fcs PE-A CC_5 ZILE FIBRO_G9_G09.fcs ageing treatment 0 0 256 512 768 1024 PE-W Cell cycle sequentiation for a dermatocosmetic ingredient BA Bach, WA Knappe, MG Edinger – Am. J.Pathol. 1991; 96:615-627 TGF-β ACTIVATION, SOLUBLE PROTEIN RESPONSIBLE FOR COLLAGEN HOMEOSTASIS COLAGEN METABOLISM L.Rhein – Ageing Skin: Current and future Therapeutic strategies –Allured Bussiness Media -2010 MATRIX METALOPROTEINASES activation ←MMP-9 (92kDa) ←MMP-2 (72kDa) The MMPs are members of the unique family of proteolytic enzyme which contain a zinc ion at their active sites and can degrade native collagens and other ECM components. Application: Materials: •Human TGF beta1 Single Plex Flex Set (BD CBA) •Human Soluble Protein Master Buffer Kit (BD CBA) Method :beads - based flow cytometry assay for the quantization of soluble proteins (TGF- β) Decreased cell function in aged and photodamaged skin appears to be due to abnormalities in the signaling pathways that regulate matrix production. In human dermal fibroblasts, TGF-β not only functions as primary stimulus for collagen synthesis, but also reduced collagen degradation by inhibiting MMP-1 expression. Sandwich Complex sample bead with specific antibodies Detection antibodies , PE stained Compounds designed in Biotehnos Laboratories: • Dermo – Oz – and Dermo – O – phytocompounds from Callendula officinalis • Rise the fibroblasts proliferation, but activate the metalloproteinases from extracellular matrix; significant effects on tissue repair and photoageing treatment. • Dermo – U – phytocompound from Salvia officinalis • “Estrogen-like” effect on fibroblasts, quickly acts on proliferation stimulation and collagen synthesis; • Dermo – ET - biocomplex from Trifolium Pratense • “Estrogen-like” effect on fibroblasts, quickly acts on cellular turn-over (augment D N A synthesis and proliferation) Gelatin-based zymography of the culture medium of HS27 cells treated with COSMETIC INGREDIENTS. CONCLUSIONS: Hee-Jae Cha, Soo-Kyung Bae, Ho-Young Lee, Ok-Hee Lee, Cancer Research 56, 2281-2284, May 15, 1996 COLAGEN synthesis Absorbance 550nm 1.2 Collagen contains a unique aminoacid in its structure: Hydroxyproline. Hyp level is strong correlated with the total collagen synthetised. (1mg of collagen contain 0.0122mg Hyp) Before treatment: R4= 0.217; R3= 0.642; Fo= 0.057 1 0.8 0.6 y = 0,272x + 0,1514 R² = 0,9956 0.4 Collagen is one of the more important structural proteins in the body being of particular importance in connective tissues by providing their durability. As such, knowledge of at least the amonunt of collagen in a particular tissue is essential for the complete understanding of the structural and mechanical proprities of that tissue. Fields and Dunn have suggest that it is the structural proteins, collagen primarily, which are responsible for the tissue’s echographic visualizability . 0.2 •All anti-ageing skin care products have pharmaceutically effect, including the enhanced wound healing effects, acting at cellular / molecular level, so scientific testing methodologies are strictly recommended •A cosmetic may be characterized in the light of increasing knowledge of its effect on normal or damaged skin • “in vitro” tests provide a relevant and complex quantification regarding anti-ageing ingredients effect at cellular/molecular level – a necessary preliminary report about product efficacy •“In vivo” relevance of “in vitro” tests are strong connected with the efficacy of future anti-ageing skin care products •The regulatory agencies should protect the public from unproven claims and unsafe materials, imposing scientific test for cosmetic effects •The implementation of a skin functional parameters scanning in respect of the active ingredients action will have a deep impact in: • • • the quality of final product formulation a better treatment scheme for dermatological dysfunctions rising performances for the new skin care anti-ageing ingredients 0 -1.5 -0.5 0.5 1.5 2.5 Unknown analyte Concentration of hydroxyproline added (µg/ml) concentration at… 3.5 G. Kesava Reddy and Chukuka S. Enwemeka, Clinical Biochemistry, Vol.29, No. 3. 225-229, 1996 Experimental developement and inovation activities in dermatocosmetics are done with financial help from POS CCE ID 383 SMIS CSNR 6009 CTR 107/2010 This report does not necessarily represent the official position of the European Union or the Romanian Government Project title: POS CCE ID 383 SMIS CSNR 6009 CTR 107/2010 - DERMOLAB “International standards implementation for the organisation of a dermato-cosmetic research and testing core” Material editor: SC BIOTEHNOS SA Date of publication: 11.05.2011