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SEPAWA 2011 - Gematria Test Lab

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58 <strong>SEPAWA</strong> Congress 12-14 October, Fulda, Germany<br />

The Radical Status:<br />

a new method for the determination<br />

of the barrier function of skin<br />

Katinka Jung, Marietta Seifert, Thomas Herrling<br />

<strong>Gematria</strong> <strong>Test</strong> <strong>Lab</strong> GmbH, Berlin, Germany<br />

Wolfgang Pittermann, Bettina Blume<br />

Simred GmbH, Großburgwedel, Germany<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

1


Introduction – skin irritants and skin protection<br />

SUN CHEMICALS DETERGENTS ACIDS/ALCALINE<br />

• Several chemical substances / physical influences may damage<br />

the skin.<br />

• Due to the different irritants, the universal protectant does not exist.<br />

• The skin protection should not only isolate the skin‘s surface, but<br />

should be based on a synergistic interaction between the barrier<br />

and the reservoir function of the skin and the protectant.<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

2


How to measure skin irritation and skin protection?<br />

20 % of the occupational diseases are skin diseases caused by<br />

chemicals (BG Chemie 2009)<br />

Due to the variety of noxa and the variety of individual<br />

sensitivities, the quantitative determination of the skin damage<br />

in vivo is difficult.<br />

Skin models should be used.<br />

Significant parameters should be found.<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

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CONTENT<br />

Parameters for skin damage/irritation<br />

Free Radical Status of the skin<br />

Different skin models<br />

Correlation between different parameters<br />

Antioxidative Power of the skin<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

4


Introduction – skin irritants and skin protection<br />

Free Radical Status and skin irritation<br />

Radical formation in<br />

the control skin<br />

(treated with<br />

protector)<br />

Radical-boosting<br />

due to irritants<br />

Skin Protector and<br />

irritant<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

5


Generation and propagation of free radicals inside the skin:<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

6


Radicals are formed where the chemical or physical noxa are present<br />

Source of oxidative stress<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

7


Example 1: Dihydroxyacetone<br />

Induces the Maillard reaction<br />

Radical intermediates are formed inside the epidermis/dermis<br />

(AGE-products)<br />

UV-radiation induces more free radicals in the DHA-treated skin<br />

Maillard reaction<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

8


Example 2 : Detergents<br />

Induce membrane disorder and dehydration<br />

Radical intermediates are formed inside the sc /epidermis<br />

UV-radiation induces more free radicals in the SDS-treated skin<br />

detergents<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

9


Example 3: UV radiation<br />

Induces oxygen free radicals and lipid peroxides inside the skin.<br />

UV radiation<br />

spectrum<br />

wave length λ<br />

energy ε<br />

UV C<br />

UV B<br />

UV A<br />

C<br />

B<br />

A<br />

100 nm – 280 nm<br />

280 nm – 320 nm<br />

320 nm – 400 nm<br />

12.3 – 9.8 eV<br />

9.8 – 9.2 eV<br />

9.2 – 8.1 eV<br />

IR<br />

760 nm – 1 mm<br />

1.6 – 10 -3 eV<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

10


4. RSF - principle<br />

UV<br />

O 2<br />

.- .<br />

OH L . LOO . R .<br />

Direct<br />

measurement (ESR)<br />

inflammation<br />

pigmentation<br />

DNA-damage<br />

Collagen disorder<br />

Protein disorder<br />

Apoptosis ...<br />

Measurements of<br />

biological endpoints (MED,<br />

PPD,...)<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

11


The Radical Status - principle<br />

Skin biopsy<br />

(pig ear, cow udder,<br />

Human skin, 3D skin models,…)<br />

Application of protective<br />

formulations<br />

- Sunscreens<br />

- anti-ageing products<br />

- antioxidants<br />

Application of noxa<br />

- coolants, acids, detergents<br />

- electromagnetic radiation<br />

- temperature<br />

<strong>Lab</strong>elling with a radical indicator<br />

Induction of free radicals via UV-radiation<br />

Quantification of these FR by ESR spectrometry directly in the skin<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

12


Electron Spin Resonance spectroscopy<br />

Quantitative and qualitative analysis of FR / ROS.<br />

Quantitative and qualitative detection of antioxidative reactions.<br />

Inpedendence from physical – chemical properties of the sample.<br />

Rapid and covenient analysis.<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

13


Choice of the skin model<br />

Pig skin<br />

BUS model<br />

No aerobic metabolism<br />

Active aerobic metabolism<br />

Biochemical parameters (MTT, PGE 2 )<br />

Repetitive application<br />

Similar to human skin<br />

(mammalian)<br />

Long application period(5 h)<br />

Repetetive application<br />

similar to human skin (mammalian)<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

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<strong>SEPAWA</strong> <strong>2011</strong><br />

15


Dose-effect relationship<br />

PIG SKIN<br />

Triton-X 100<br />

260<br />

240<br />

220<br />

200<br />

FR, %<br />

180<br />

160<br />

140<br />

120<br />

100<br />

80<br />

0,36788 1 2,71828 7,38906<br />

ED 50<br />

= 1,19%<br />

Triton X, %<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

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PIG SKIN<br />

Nr.<br />

Code<br />

FREE<br />

RADICALS, %<br />

Effective<br />

concentration<br />

of Triton X<br />

1<br />

Untreated skin<br />

100 ± 41<br />

0<br />

2<br />

Treated with 10 % Triton X<br />

205 ± 45<br />

10 %<br />

3<br />

Placebo (O/W)<br />

100 ± 31<br />

0<br />

4<br />

10% Triton X and Placebo<br />

165 ± 36<br />

1,19 %<br />

5<br />

10% Triton X and Product A<br />

181 ± 54<br />

1,24 %<br />

6<br />

10% Triton X and Product B<br />

89 ± 6 *<br />

0<br />

Tab. 2<br />

* Different from the treated skin (10% Triton, negative control) at a significance (p


PIG SKIN<br />

Excess in FR<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

250<br />

18<br />

200<br />

150<br />

100<br />

50<br />

0<br />

10% Triton X<br />

no Placebo Product A Product B<br />

treatment<br />

free radicals, %<br />

Inhibition<br />

of FR


700<br />

PIG SKIN<br />

600<br />

0,5 % NaOH<br />

500<br />

free radicals, %<br />

400<br />

300<br />

200<br />

100<br />

0<br />

without P 1 P 2 P 3 P 4 P 5 P 6<br />

protectors<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

19


Correlation between inducible FR and cell vitality:<br />

BUS model<br />

Cytotoxic and<br />

inflammatory effects<br />

measured by MTT / PGE 2<br />

-<br />

test<br />

Free radicals<br />

measured by ESR<br />

spectroscopy<br />

MTT score<br />

PGE 2 score<br />

Radical Status of the skin<br />

Protection factor<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

20


BUS model<br />

Noxa: coolant<br />

Protector: HERWESAN AiO,<br />

2mg/cm 2 , 20 min prior to the application of the coolant<br />

260<br />

240<br />

220<br />

Graph<br />

correlation:<br />

R = 0,997<br />

2,5<br />

2,0<br />

Free Radicals (%)<br />

200<br />

180<br />

160<br />

140<br />

120<br />

100<br />

80<br />

1,5<br />

1,0<br />

0,5<br />

0,0<br />

-0,5<br />

MTT score<br />

untreated<br />

Protector<br />

Coolant<br />

Protector + Coolant<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

21


nOctan 5h<br />

Protector + nOctan 5h<br />

SDS 10% 5h<br />

Protector + SDS 5h<br />

Triton X 10% 5h<br />

Protector+ Triton X 5h<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

22<br />

Protector + Toluol 5h<br />

Toluol 5 h<br />

Protector 5 h<br />

control 5h<br />

350<br />

300<br />

250<br />

200<br />

150<br />

100<br />

50<br />

0<br />

Toluol<br />

controls<br />

noxe treatments<br />

protection + noxe BUS model<br />

n Octan<br />

SDS<br />

Triton X<br />

free radicals, %


Protection factor: FR (unprotected skin)<br />

FR (protected skin)<br />

BUS model<br />

Irritant<br />

Toluol<br />

n-Octan<br />

SDS 10%<br />

Triton-X 10%<br />

Protection Factor of the Skin<br />

Protection Product<br />

After 1 h<br />

5,7<br />

1,4<br />

2,8<br />

1,7<br />

After 5 h<br />

1,5<br />

5,1<br />

1,1<br />

1,5<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

23


4<br />

3<br />

MTT score<br />

24<br />

P + Triton X<br />

Triton-X<br />

P + Toluol<br />

Toluol<br />

protector<br />

control<br />

BUS model<br />

220<br />

200<br />

180<br />

160<br />

140<br />

120<br />

Free radicals, %<br />

100<br />

80<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

2<br />

1<br />

0


5<br />

4<br />

5h<br />

BUS model<br />

total score MTT+PGE 2<br />

3<br />

2<br />

1<br />

0<br />

R = 0,98<br />

80 100 120 140 160 180 200 220<br />

FR<br />

Good correlation between the inducible FR and the cell vitality<br />

and irritation parameters.<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

25


time, t<br />

26<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

Skin Antioxidative Retension (SAR)<br />

Penetration time<br />

produkt<br />

ESR measurement<br />

PIG SKIN<br />

Penetration time<br />

marker<br />

before UV<br />

0,3 MED UV<br />

0,5 MED UV<br />

0,7 MED UV<br />

1 MED UV<br />

1 MED UV + sunscreen<br />

1 0 0<br />

8 0<br />

6 0<br />

4 0<br />

2 0<br />

0<br />

rel. SAP, %


BUS model<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

120<br />

100<br />

27<br />

80<br />

60<br />

40<br />

20<br />

0<br />

**<br />

**<br />

control Triton X Toluol<br />

treatment<br />

Antioxidative Power of the skin, SAP (%)


Conclusion<br />

• The Radical Staus Factor is a valuable tool for the<br />

determination of the skin‘s defence system.<br />

• The inducibility of free radicals is directly related to<br />

the cell vitality, the inflammation process and the<br />

antioxidative systems of the skin.<br />

• The Radical Status is able to detect both<br />

toxic/negative and the protective/positive effect of<br />

topically applied substances, raw materials, or final<br />

product.<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

28


Thank you for your kind attention!<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

29


Pig ear Application of sunscreen 20 min time<br />

ESR tissue cell<br />

Biopsy 4 mm<br />

UV irradiation<br />

ESR measurement<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

30


<strong>SEPAWA</strong> <strong>2011</strong><br />

31


Collaborations<br />

• BASF SE<br />

• Botanica GmbH, Schweiz<br />

• Crodarom, France<br />

• Forschungsinstitut für molekulare Pharmakologie FMP, Berlin, Deutschland<br />

• GABA International, Schweiz<br />

• GfN-Selco GmbH, Deutschland<br />

• Hallstar, USA<br />

• Henkel KGaA, Deutschland<br />

• Kuhs GmbH, Deutschland<br />

• LEO Pharma, Denmark<br />

• Lipotec S.A., Spain<br />

• Mann&Schröder GmbH, Germany<br />

• Marico s.l., India<br />

• MAXIM Markenprodukte GmbH, Deutschland<br />

• Merck KGaA, Deutschland<br />

• Mibelle Cosmetics AG, Schweiz<br />

• Qenax AG, Schweiz<br />

• ROVI Cosmetics International GmbH, Deutschland<br />

• Schering-Plough, USA<br />

• Semper Idem GmbH, Deutschland<br />

• Spirig Pharma AG, Schweiz<br />

• SLI Chemicals GmbH<br />

• The Dial Corp., USA<br />

• Unilever Thai Trading Lim., Thailand<br />

• Weleda AG<br />

• Universitätsklinik Charité, Dermatologie, Berlin, Deutschland<br />

• Technische Hochschule Berlin, Deutschland<br />

<strong>SEPAWA</strong> <strong>2011</strong><br />

32

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