V. 14 A NEW COLLAGEN STABILISING TETRAPEPTIDE

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1 V. 14 A NEW COLLAGEN STABILISING TETRAPEPTIDE

2 EXTRACELLULAR MATRIX (ECM) The extracellular matrix (ECM) consists of three major types of macromolecules: Fibres Fibres: responsible for the ability of each tissue to resist tension, compression, extensibility and torsion Ex: Collagen and Elastin Glycoproteins Glycoproteins: provide linkage between matrix components and cells Proteoglycans: a diverse group of soluble macromolecules that have both structural and metabolic roles

3 PROTEOGLYCANS The basic structure of all proteoglycans includes a core protein and at least one, but frequently more (up to tens or hundreds) carbohydrate chains, so called glycosaminoglycans They act as tissue organisers, influence cell growth and maturation of specialised tissues, play aroleasbiological filters and modulate growth-factor activities Protein core GlycosAminoGlycans

4 PROTEOGLYCANS SMALL LEUCINE-RICH PROTEOGLYCANS (SLRPs) belong to a family of secreted proteoglycans (about a dozen) that feature regions rich in leucine along the protein core SMALL LEUCINE-RICH PROTEOGLYCANS (SLRPs) are characterised by their functional roles in the orderly assembly of extracellular matrixes, modulation of growth factor activity, and regulation of cell growth properties Examples: Decorin, versican, biglycan, fibromodulin, lumican

5 DECORIN STRUCTURE Decorin has a horseshoe-like structure where the ß-sheets form the inner concave surface and the α-helices make up the outer convex face Decorin Collagen The inner concave surface of decorin is of suitable size to accommodate a single triple helix of collagen

6 COLLAGEN FIBRILLOGENESIS After being secreted into the extracellular space, collagen molecules assemble into ordered polymers called collagen fibrils, in a process called fibrillogenesis RERECM

7 DECORIN Decorin binds to surface of collagen fibrils, induces a delayed fibril assembly and uniformises fibril diameter Fibrils are stabilised and fibrillogenesis is orientated Helps to establish and maintain tissue shape

8 DECORIN FUNCTIONS Decorin controls fibril dimensions by preventing lateral fusion of collagen molecules, uniformity of fibril diameter and the regular spacing of fibrils Irregular fibrils Fibrils with uniform diameter Decorin governs fibril growth and influences higher order matrix assembly, giving gsuppleness and strength to the skin

9 DECORIN STRUCTURE Decorin is associated with collagen fibrils at specific binding sites via the protein core Collagen binding sites on decorin Two tetrapeptide sequences have been identified as the specifically binding sites of decorin to collagen fibrils

10 SKIN AGING Dramatic changes occur in skin as a function of age, including changes in morphology, physiology and mechanical properties A catabolic fragment of decorin is abundant in mature skin. This truncated form of decorin lacks regions of decorin proved to be important for interaction with collagen The appearance of this catabolic product may have significant effect on skin elasticity and morphologic differences between collagen fibres of young and mature skin

11 DECORINYL Anew tetrapeptide, which is amimic from the sequence of the specifically binding sites of decorin DECORINYL has proved regulate collagen fibril growth to regulate fibrillogenesis and Improves structure and suppleness of the skin

12 EFFICACY TESTS IN VITRO: COLLAGEN FIBRILLOGENESIS ASSAY SKIN MODEL- TRANSMISSION ELECTRON MICROSCOPY (TEM) EX VIVO: HISTOCHEMICAL STUDY OF HUMAN SKIN BIOPSIES IN VIVO: MEASUREMENT OF SKIN BIOMECHANICAL PROPERTIES: SKIN MEASUREMENT OF SKIN BIOMECHANICAL PROPERTIES: SKIN SUPPLENESS

13 IN VITRO REGULATION OF COLLAGEN FIBRILLOGENESIS Type I Collagen from calf skin (Sigma) were treated with DECORINYL peptide at concentrations of 0.001%, 0.01%, 0.05% and 0.1% The process of fibrillogenesis was measured by turbidity readings All tested concentrations of DECORINYL peptide present significant regulation activity on fibrillogenesis respect to control, in a dose- dependent manner

14 IN VITRO SKIN MODEL - TEM Tissues from a tridimensional i human skin model EFT-200 (MatTek Corporation) are treated with DECORINYL peptide 0.01% Non treated tissues are used as controls Skin sections are observed by Transmission Electron Microscopy (TEM) Control (untreated skin) Skin treated with DECORINYL peptide

15 IN VITRO SKIN MODEL - TEM The diameter of collagen fibres is measured and statistically analysed using the One Way ANOVA analysis Control Standard deviation (nm) Control -34.7% (of standard deviation) DECORINYL The standard deviation is 34.7% lower after the treatment with DECORINYL peptide p 0.01%. The analysis of variances shows that the fibres treated with DECORINYL peptide are more uniform, due to its low variability Frequency Frequency Range: nm n: Diameter (nm) DECORINYL Range: nm n: Diameter (nm)

16 EX VIVO HISTOCHEMICAL STUDY OF HUMAN SKIN BIOPSIES Skin biopsies of 3 patients were evaluated before and after a two-month treatment with a cosmetic formulation containing 0.01% DECORINYL peptide. The collagen fibril diameter was measured from Transmission Electron Micrographs After a two-month treatment, collagen fibrils in all patients show a decrease in the standard deviation of the collagen fibril diameter 0 months 2 months Transmission electron micrographs of dermal collagen from skin biopsies of Patient 1 of r (nm) St tandard Deviation collag gen fibril diameter 14 Patient 1 12 Patient 2 10 Patient MONTHS 2 MONTHS 0 MONTHS 2 MONTHS 0 MONTHS 2 MONTHS The average decrease of the standard deviation of collagen fibril diameter was 9.63%

17 EX VIVO HISTOCHEMICAL STUDY OF HUMAN SKIN BIOPSIES Analysis of frequency and distribution of collagen fibril diameter reveals that the range and distribution is different before and after treatment with DECORINYL formulation Frequen ncy Patient 1 0 MONTHS 2 MONTHS Diameter (nm) The reduction of the standard deviation in all patients implies a decrease in the variability of the collagen fibril diameters and a high uniformity of collagen fibrils after the treatment with the peptide

18 IN VIVO TEST Our in vivo test was performed on 22 female volunteers, aged 40 to 58. A cream containing 5% DECORINYL was applied daily on the face (temple) during 28 days Another group of 21 female volunteers was treated with a placebo cream Variations on skin suppleness were measured with a MPA 580 Variations on skin suppleness were measured with a MPA 580 Cutometer, at time 0 and after 28 days

19 IN VIVO TEST After 28 days, the cream containing DECORINYL induced a 54% increase in skin suppleness Variations on skin suppleness (%) T+28 days Placebo Placebo DECORINYL DECORINYL At the end of the test, 95% of the volunteers had increased their skin suppleness No significant increase was observed for the placebo cream

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