In Vivo Methods to Prove Anti-Photoaging and Anti-Pollution Efficacy of Cosmetic Products

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1 In Vivo Methods to Prove Anti-Photoaging and Anti-Pollution Efficacy of Cosmetic Products Stephan Bielfeldt Anti-Aging Summit June 2016 Agenda Photoaging: The clinical picture What is the cause of photoaging? Skin aging by environmental pollution: A similar cause Cosmetic treatment for prevention and cure In vivo / ex vivo methods to assess efficacy Discussion of the methods Conclusion 1

2 Photoscore for Photoaging (Griffiths) 0 = no photodamage, 8 = severe photodamage From Han et al. Dermatol Clin 32 (2014) Age spots: Increased Length of Dermal Papillae International journal of cosmetic science 35.2 (2013):

3 The Cause: Reactive Oxigen Species (ROS) UV but also VIS and IR-A Produce Free Radicals in Skin Solar Elastosis: How is the Sun Causing Wrinkles? UV, VIS and IR form ROS in the skin MMP 1, MMP 3 and MMP 9 will be released Fibers, mainly collagen I und III are cleaved Subsequent repair of damaged tissue is incomplete A type of scar tissue is formed Consequences over years: Skin looses its elasticity and becomes wrinkled Chronicle exposure to UV-light triggers a misguided degradation response that nature created for wound healing 3

4 Solar Elastosis: How is the Sun Causing Wrinkles? Keratinocytes as well as Fibroblasts are Involved AP-1: Transcriptionsfactor; binding to DNA=>gene expression From Fisher et. al. Arch Dermatol 2002;138:1462 Environmental Pollution: Unhealthy when Inhaled But does it also Harm the Skin? 4

5 Skin Aging by Environmental Pollution is Similar to Photoaging! ROS components of pollution trigger the oxidization of skin proteins, skin lipids and further functional skin molecules This causes: Activation of matrix metalloproteinases (MMP s) => collagen cleavage Consumption of skin antioxidants (vitamin C, glutathione, ) Chronic effects are: Premature skin aging Formation of skin wrinkles Formation of pigmented spots Preventive Actions by Use of Cosmetics Sun screen filter actives are pivotal for prevention High protection from UVA and UVB wavelengths required Free radical scavengers/anti oxidants (vitamin C, E, carotenoids, polyphenols ) Inactivate reactive oxygen species (ROS) Improve the natural system of anti oxidants in the skin Supplement natural anti oxidants exhausted by UV-irradiation Not only UVB and UVA release ROS, but also VIS and IR-A! 5

6 Examples for Cosmetic Actives to Treat Photo Aged Skin Restoration of dermal matrix Topical Retinol (Skin metabolism forms the active retinoic acid) Retinol triggers the new formation of collagens Dose dependent skin irritation observed Systemic availability has to be avoided (Retinoic acid has a teratogenic potential) Reduction of pigmented spots and melasma Vitamin C, Kojic acid Arbutin Alpha-Hydroxy acids Hydrochinone is banned from cosmetics Measurement Methods Preventive effects Measurement of SPF and UVA protection (ISO standards) Measurement of antioxidant effects Quantification of collagen cleaving molecules (MMP 1 / TIMP 1) Curing effects Anti wrinkle measurement Measurement of dermal photo damage (22 MHz ultrasound) Measurement of pigmented spots and melasma 6

7 Measurement of Facial Wrinkles by Fringe Projection (AEVA-HE) 3D-Profile Image by AEVA-HE (Composition of 2 Side Views) Resolution in z-direction 2 µm 7

8 Evaluation Methods on Profile Images Extraction von 50 profile lines: Parameters: Line parameters: Ra, Rz, Area parameters: Algorithm for exact matching of profiles (before and after treatment) is crucial to detect small effects On 30 volunteers: Effects of approx. 5 % of wrinkle reduction can be detected as significant 22 MHz-Ultrasound Measurement on Photo Damaged Skin: Echo-poor Region due to Degraded Fibres in the Dermis Normal skin (outer forearm) Photoaging: Echo poor region below epidermal echo Parameter: Echo-density of the dermis 8

9 Dermatoscope to Assess Pigmented Spots or Melasma Example: Dermatoscopic Assessment and Image Analysis to Measure Efficacy on Melasma Baseline before treatment After 8 weeks of treatment Melasma-area Normal pigmentation area 9

10 Ex Vivo Measurement of IR-A Protection Ratio MMP-1 / TIMP-1 as a marker (on skin biopsies) From Schröder et al. Journal of Investigative Dermatology (2008) 128, TIMP-1 = Tissue Inhibitor of Metallo-Proteinases - 1 In Vivo Detection of Carotenoids as Markers of the Status of Skin Own Antioxidant System after UV - Irradiation From Lademann et al. Skin Pharmacol Physiol 2011;24:

11 Ex Vivo Detection of Antioxidant Potential (AP) by Electron Spin Resonance From Herrling et al. (2006). Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 63(4), In Vivo Assessment of Lipid Peroxidation: Triggered by Tobacco Smoke (Pollution Model) or UVA irradiation (Photoaging Model) Non invasive, human ex vivo Cigarette smoke is used in vivo as the model pollutant Assessment of peroxidation of human sebum or skin barrier lipids Target molecules of lipid peroxidation: Squalene monohydroperoxide (Sebum) and Malondialdehyde (Sebum or Barrier lipids) Both UVA-light as well as cigarette smoke are suitable to trigger lipid peroxidation and investigate product efficacy 11

12 Example: In Vivo Measurement of Antioxidant Effects Anti Pollution efficacy (Cigarette Smoke Model) Instant Protection Long term Protection Subjects 10 Parameters Test areas for barrier lipids Test area for sebum lipids Test products Test procedure Quantification of Squalene Monohydroperoxide (SQQOH), Malondialdehyde (MDA) [LC/MS determination] 5 testing areas on the volar forearm 5 testing areas on the upper back 3 cosmetic products, positive control (untreated area exposed to smoke), negative control (untreated area not exposed to smoke) Day 1: Product application, e.g. 15 min. waiting time, 15 min. exposure to pollution, sampling Day 1, 2, 3, 4: Product application After h, on day 5: 15 min. exposure to pollution, sampling Product Application on the Back 12

13 Smoke Suction Device A pump produces negative pressure and transports the smoke to the skin Smoke Application on the Back 13

14 Sampling Method to Harvest Lipids from Skin Swab method with a cocktail solution Analysis by HPLC-MS SQOOH analysis MDA analysis 14

15 Example Study Results (Squalene monohydroperoxide) SQOOH found in test samples mean and SD [ng/mg SQOOH] Product A Product B Positiv control Negativ control Product A Product B Positiv control Negativ control Test product Example Study Results (Malondialdehyde) 90.0 MDA found in test samples mean and SD [ng/mg MDA] Product A Product B Positiv control Negativ control Product A Product B Positiv control Negativ control Test product 15

16 Discussion of Test Methods: Preventing Effects In vivo methods to assess protection from sun light SPF, UVA-PF: Well standardized ISO methods Yet no standard-methods for protection from VIS and IR-A available Biopsy method available to detect MMP s after IR-A irradiation Would it work also for VIS? Less invasive approach possible? (e.g. on suction blister fluid) Methods to assess antioxidant effects: In vivo and ex vivo methods available (established in single laboratories) ESR methods available: In vivo is possible but difficult Skin own antioxidants (carotenoids) as marker molecules available Endpoints of skin lipids peroxidation as marker molecules available Endpoints of protein oxidation appreciated Discussion of Test Methods Curing Effects Methods to quantify skin wrinkles, pigmented spots or parameters of elastosis are well established, but In general cosmetic products show only weak effects even after long treatment times Studies require high volunteer numbers (n > 30) Test duration of 3 and even 6 month are typical Volunteer compliance is crucial 16

17 Conclusion Premature skin aging is caused by sun light but also by environmental pollution Further efforts to improve the efficacy of cosmetics are required A wide range of methods is available to proof antiphotoaging and anti-pollution efficacy of cosmetics Development of new actives and new test methods have to go hand in hand 17

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