Transcript Slide 1

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