proderm Institute for Applied Dermatological Research GmbH States of Mobility of Water Molecules Measured by in Vivo Confocal Raman Spectroscopy

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1 proderm Institute for Applied Dermatological Research GmbH States of Mobility of Water Molecules Measured by in Vivo Confocal Raman Spectroscopy Presented at Sepawa 2017

2 About the Raman Effect Chandrasekhara Venkata Raman was an indian physicist. One of his major achievements was the experimental discovery of inelastic scattering of light which since then is known as 'Raman Scattering' or 'Raman Effect'. For this work he was awarded with the nobel prize for physics in 1930.

3 About the Raman Effect Raman Spectroscopy is a technology that allows to quantify specific molecules in different skin levels on the basis of the 'Raman Effect'. This effect describes the process when monochromatic light interacts with molecular vibrations: energy is transfered to the molecular cloud (green arrow) and then returned on a different wavelength, meaning less energy (red arrow). This shift is called 'Raman Effect'.

4 About the Raman Effect This picture shows a complex biomolecule. Due to the complex structure and the many different vibrational conditions resulting shifts in wavelength are unique for each existing molecule. This is what is called the specific 'Fingerprint'.

5 About the Raman Effect This picture above illustrates the confocal technology which enables to capture data in different layers in the depth of the skin. By detecting the Raman Scattering in a defined depth, profiles related to e.g. the skin thickness can be obtained. Left picture: Monochromatic light is irradiated into the skin. The technology enables the determination of specific molecules in different depths of the skin and the detection of spectra in each of the depths.

6 Basic Raman Spectra - Water Measuring the amount of water in the skin has always been and still is an important topic for the cosmetic industry. The spectra measured in the stratum corneum is illustrated by the blue line, which actually shows one peak for keratin and one for water. The proportion of those peaks would result in the concentration of water in specific skin levels. The picture on the left illustrates the concentration of water in specific skin depths. Within the stratum corneum a water gradient is visible. This gradient resembles the skin barrier funtion. The calculated interface in between stratum corneum and epidermis (the stratum corneum thickness) can also be seen. ww.

7 proderm Case Study on Body Cream containing Urea Test product Test area Subjects Application Measurements Body cream containing urea Legs 12 for Raman Spectroscopy 2 x daily for 21 days Before and after after 21 days of application Stratum Corneum Thickness Among other parameters the thickness of the Stratum Corneum was measured. The according results illustrate that the product had no significant impact on the Stratum Corneum thickness (comparison between baseline values and values on day 21).

8 proderm Case Study on Body Cream containing Urea Another parameter measured was the penetration of urea into the skin. Our specific Raman Spectrometer features a dabase of the fingerprints for the molecules present in different skin depths. These can therefore be detected and measured (i.e. Urea). Additional (unknown) molecules can be added into the database and with that also be detected and measured (i.e. Active Ingredients). Urea Penetration

9 proderm Case Study on Body Cream containing Urea Urea Penetration The picture above illustrates an example of a penetration curve showing the amount of compound in different skin depths. Alternatively the amount of compound over time could also be investigated.

10 proderm Case Study on Body Cream containing Urea Before Urea Penetration 21 days after application Urea is an important natural moisturizning factor and with that is responsible for bounding water in the upper skin levels. Dry skin can often be connected with a lack of urea. The results show a significant increase of urea in different skin levels.

11 proderm Case Study on Body Cream containing Urea Water molecules can have different states of mobility. One distinguishes in between (i) bound water, (ii) water which is intermediately mobile and (iii) water which is most mobile. This differentiation is important since only bound water (either bound or intermediately mobile) impacts the long-term skin moisturization level. It is therefore the aim of many cosmetic products to transform mobile water into intermediately mobile or bound water. Water Mobility

12 proderm Case Study on Body Cream containing Urea Before Water Mobility at Baseline and at 21 days post application 21 days after application This is an overview of the different states of water mobility as measured at baseline and at day 21 post application. The results indicate that the product applied improved the amount of intermediately mobile water while reducing the amount of mobile water at the same time.

13 proderm Case Study on Body Cream containing Urea The difference to baseline is illustrated in the graph above. It could be demonstrated that the product transfered mobile water into bounded / intermediately water and with that increased long-term skin moisturization. Water Mobility Differences to Baseline

14 Results Raman Spectroscopy is a unique method for the determination of water in different skin depths 'Water Mobility' is a new and important parameter for many manufacturers of cosmetic products since only bound water impacts the level of long-term skin moisturization After analyzing the Raman spectra the fraction of mobile water compared to the fraction of intermediately mobile water and of bound water had decreased, while the total amount of water was comparable for treated and untreated test areas In vivo confocal Raman spectroscopy can be used to swiftly measure the mobility of water at different depths in the SC with high precision. In addition to the classical water measurements this innovative method provides data to distinguish between different states of mobility of water in the skin. It could be shown that while the total amount of water in the SC and its barrier function stayed constant, the mobility of water can even shift to stronger bound water after applying a suitable cosmetic product.

15 proderm is an international leading CRO offering claim support, tolerability and safety testing with a strong focus on topical applications. Being headquartered in Schenefeld/Hamburg, the institute today has over 100 staff members including over 20 scientists and certified specialists in dermatology, ophthalmology, dentistry, pediatrics and gynaecology. proderm established a very good reputation as being a high-quality provider of clinical trial services on the basis of outstanding technical equipment and extraordinary know-how. proderm operates as a full-service CRO and conducts a total number of around 600 studies per year. The institutes state-of-the art facilitiy comprises more than m² with dedicated laboratories such as: investigational rooms, Hair Care, Sun Care, Sensory Laboratory, Unit for Standardized and Reproducible Photography etc. In a regulatory perspective, the institute offers clinical studies according to ICH-GCP and testing in an environment approximating ICH-GCP guidelines. Product wise proderm offers contract research for Pharmaceuticals, Medical Devices, Cosmetics, Food Supplements and Consumer Goods. Ever since 2001, proderm is certified according to quality management system ISO The institute has worked for more than 400 clients worldwide. proderm Institute for Applied Dermatological Research Kiebitzweg Schenefeld/Hamburg Germany phone: fax: proderm.de