Premature skin aging
induced by air pollution:
New in vitro insights
approaching realistic
conditions
Imke Meyer, Symrise AG
Anti-ageing Skin Care Conference
London, 07. June 2016
PM, the new UV?
The anti-air pollution proof
Conclusion
How do PM affect skin’s youth?
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3
2
2
1
Taking anti-pollution protection to a new level
PM, the new UV?
US 23%
Mexico 43%
Brazil 37%
Pakistan 37%
Spain 34%
UK 21%
Russia 56%
China 51%
India 40%
South Korea 39%
Japan 21%
Atmospheric pollution gets global awareness Perceived as a primary cause of skin and hair problems
2013+2014
Lack of sleep 1 Pollution & dirt,
chemicals in the air (car exhaust/ factories…)
2
Stress/ conflict 3
Sunrays 4
My age 5
Source: Cosmetic Ingredients Consumer Study, 11 countries, n = 2000-2500 per country; Q23_A. In your opinion what are the main causes of your skin, scalp and hair problems? 4
People understand Air Pollution does not stop at the surface
5 Source: Cosmetic Ingredients Consumer Study, 11 countries, n = 2000-2500 per country; Q23_A. In your opinion what are the main causes of your skin, scalp and hair problems?
(Applies in %).
More than 80% of consumers across the globe think
that the skin absorbs the pollution that is in the air.
Consumers in Brazil, Mexico, Pakistan and India are most
sensitive to dust and dirt, followed by Russia and China.
With greater urge to act in China, Pakistan, India, Mexico
and Brazil.
Atmospheric pollutants ? Emergence of new indexes
UV / Visible / IR O3 NO2 SO2
PM 2.5
& 10
6
SO2
UV / Visible / IR O3 NO2
Atmospheric pollutants ? Emergence of new indexes
PM 2.5
& 10
7
Among those,
PM have been pointed out
VEHICULAR EXHAUST /
TRAFFIC
COAL BURNING POWER
PLANTS & INDUSTRIAL
COMBUSTION
CIGARETTE
SMOKE
DOMESTIC HEATING
KITCHEN EMISSION
PM come from many different sources
8
Pigment spots
on forehead:
+16% by traffic
related particles
PM accelerate extrinsic skin aging major epidemiological study 2010 / Prof. Dr. Jean Krutmann
Vierkoetter et al., J Invest Dermatol (2010) 130, 2719–2726
Pigment spot
on forehead & cheeks:
+ 22% & +20% increased by soot,
respectively
Pigment spots
on cheeks:
+8% increased by PM10
(particulate matter)
9
PM surveillance Major population in cities is exposed to exceeding AQG levels
WHO’s Ambient Air Pollution database ‐ Update 2014 10
12% of the assessed population are exposed to PM10 or PM2.5 complying with AQG levels.
27% for the interim target 3 (IT‐3, 30 µg/m3 for PM10 and 15 µg/m3 for PM2.5),
49% for IT‐2 (50 µg/m3 for PM10 and 25 µg/m3 for PM2.5), and
62% for IT‐1 (70 µg/m3 for PM10 and 35 µg/m3 for PM2.5).
only
Consumers increasing demand
- to check the actual pollution levels,
- to check the forcast of air quality,
- and finally to protect skin by cosmetic products against these harmful
factors.
PM surveillance How do consumers adapt?
http://aqicn.org/forecast/beijing/ 11
How do PM affect skin’s youth ?
Classification of particles
according to diameter:
• PM10: < 10 µm
• PM2.5: < 2.5 µm
• Ultrafine particles: < 100 nm
Countries / cities are encouraged
by WHO to measure and monitor
PM10 and PM2.5
WHO Air quality guidelines for particulate matter,
ozone, nitrogen dioxide and sulfur dioxide –
Global update 2005
Their size is 25 times smaller than human hair
13
Keratinocyte 20µm
PM10 <10µm
PM2.5 <2.5µm
Human hair 50-70µm
However small, PM do not really penetrate the skin
14
But they «use»
the hair follicle
as a passage
to deeper layers.
E P I D E R M I S
D E R M I S
What is effected by PM?
15
In the skin inter alia reactive oxygen
species (ROS) are induced.
Also the biosynthesis of cytokines like
interleukin (IL-) 1α, 6 and 8 is enhanced.
E P I D E R M I S
D E R M I S
PM
ROS
ROS
cyto-
kines
ROS
cyto-
kines
cyto-
kines
HM
Skin cells are receptive to PAHs Via the Aryl hydrocarbon Receptor (AhR)
16
AhR
AhR
AhR
The PAH-AhR toxic complex
can pass into the nucleus
PAHs bind to AhRs
Inside keratinocytes
Aryl Hydrocarbon (AhR) receptors PAH
The chain reaction alter the cell’s functions
17
OXIDATIVE STRESS
COLLAGEN ALTERATION
DARK SPOTS
INFLAMMATION
AhR
AhR
AhR
Inside the nucleus, it stimulates the expression
of CYP1A1, MMP-1, POMC and IL6 PAH
The Solution:
A molecule designed by Symrise
with the ideal architecture
Arylhydrocarbon Receptors (AhR)
preferably bind to :
Flat molecules made up of benzene rings
consisting mainly of hydrogen and carbon atoms
(aryl hydrocarbon)
The strategy :
Reversibly block the AhR from interacting with aryl
hydrocarbons
The idea to neutralize the AhR
18
by an AhR antagonist
BDDI, an AhR antagonist
Benzylidene Dimethoxydimethylindanone
19
BDDI temporarily
binds to the AhR
The AhR is no longer available
to connect to PAHs
PAHs are kept outside the nucleus
Adverse reactions are limited
PAH
BDDI
The anti-pollution proof
An original model, designed by Symrise Evaluate product efficacy against actual PM
21
Identify suitable surrogate samples for the study of authentic street particles. 1
National Institute of Standards and Technology, Gaithersburg, MD, USA
SRM 1650b
• Bulk diesel particulate material collected from the heat exchangers of a dilution tube facility, following 200 engine hours of particulate accumulation. Representative of heavy-duty diesel engine particulate emissions (DEP).
• Mean Particle Diameter: 0.18 μm
SRM 2975
• The material was collected from a filtering system designed specifically for diesel-powered forklifts.
• Mean Particle Diameter: 19.4 ± 0.3 µm
Both are associated with polycyclic aromatic hydrocarbons (PAHs) and nitro-substituted polycyclic aromatic hydrocarbons (nitro-PAHs).
2
Comparison of authentic street particulate matter collected at a traffic intensive road in Copenhagen, Denmark, on 5 weekdays in September 2000 to SRM2975 & SRM1650b 3
Danielsen et al, Particle and Fibre Toxicology 2008, 5:6
• NHEKs were stimulated with 1.5 µg/cm2 particulate matter for 6h or 24h.
• In the presence or absence of different actives. • Test-compounds have been applied 2h or 24h prior to
particulate matter.
• qRT-PCR is performed to determine gene expression (upregulation of gene expression)
Protocol:
In-vitro test protocol
22
Cells of different donors were used; inter alia • Keratinocytes from adult female Caucasian donor
(53 years) • Keratinocytes from adult female Asian donor
(67 years)
NHEKs were cultured in starved conditions for 24h prior to stimulation.
NHEKs: Normal Human Epidermal Keratinocytes
Gene expression
4 indicators Gene expression
23
PHASE I METABOLISM
Cyp1A1 (cytochrome P450, family 1,
subfamily A, polypeptide 1)
is upregulated by AhR
(aryl hydrocarbon receptor)
agonists like polycyclic
aromatic hydrocabons
(PAHs).
WRINKLE FORMATION
MMP-1 (matrix metallo proteinase-
1) is an enzyme involved
in degradation of collagen
UNEVEN SKIN TONE
POMC (proopiomelanocortin,
precursor of α-MSH and
ACTH) released by
keratinocytes, triggering
the melanin biosynthesis
in melanocytes
INFLAMMAGING
Cytocines like IL-6
(interleukin-6) are involved
in different premature
aging mechanisms of the
skin initiated by
inflammatory processes.
PROTECT FROM
THE APPEARANCE OF
WRINKLES
Characteristics:
• Synthetic material
• Recommended use level: 0.1 - 0.5%
• Light yellow to yellow powder
• Easy to formulate (soluble in oils at the recommended use level)
Characteristics of BDDI
24
INCI: Benzylidene Dimethoxydimethylindanone
Activity profile:
• AhR an tagon is t
• Protection against UVB induced premature aging via the
AhR antagonism
Regulatory: can be used worldwide except China DEFENDER
SKIN CELL
O
O
O
Both DEPs stimulate CYP1A1 gene expression significantly with SRM1650 as a stronger stimulant. BDDI prevents this increase significantly. Comparable results for Asian and Caucasian keratinocytes. All further experiments done with Caucasian keratinocytes.
Phase 1 Metabolism
25
*p<0.05 vs untreated
+ p<0.05 vs stimulated
NHEK: adult, female, Asian
SRM1650b SRM2975
* *
*
*
NHEK: adult, female, Caucasian
untreated 0% 0.00031% 0% 0.00031%
30
25
20
15
10
5
0
Cyp1A
1 g
ene
BDDI
untreated 0% 0.000077%
Prevention of melanin stimulation
26
+ *
SRM1650b stimulated
+
0.00031%
1.8
1.4
1
0.4
0.6
0.2
0
PO
MC
gene
1.6
1.2
0.8
BDDI significantly reduces the
Proopiomelanocortin (POMC)
gene expression induced by
DEP. POMC is released from
keratinocytes, triggers melanin
biosynthesis and is responsible
for the formation of dark spots.
* vs untreated p<0.001
+ vs stimulated p<0.001
BDDI
untreated 0% 0.000077%
Skin preservation against inflammaging
27
+
*
SRM1650b stimulated
+
0.00031%
1.8
1.4
1
0.4
0.6
0.2
0
IL-6
ge
ne
1.6
1.2
0.8
BDDI significantly reduces the
DEP-induced expression of
inflammation gene markers
such as Interleukin-6 (IL-6),
linked to an inflammatory
response of the skin
(inflammaging).
* vs untreated p<0.001
+ vs stimulated p<0.001
BDDI
28
PM 2.5
& 10
The AhR chain reaction
is triggered by
But also by
UV O3
BDDI, broad protection vs. atmospheric pollution
P R O T E C T O R STEM CELL
&
VERSATILE A N T I -
I N F L A M M A G I N G
Characteristics:
• Natural material, standardized
• Recommended use level: up to 0.2%
• Yellow liquid
• Easy to formulate (oil-soluble)
Characteristics of BIO3040
29
Ginger root extract obtained by eco-friendly supercritical CO2 extraction (no solvent) with additional steps of enrichment INCI: Zingiber Officinale (Ginger) Root Extract
Activity profile:
• Stem cell protector
• Poten t an t i -ox idant p roper t ies
• Poten t an t i - in f lammatory p roper t ies
• Skin tone evening efficacy (clinically proven)
• no AhR antagonism*
Regulatory: can be used worldwide
* Yoshida et al. (2014) J Agric Food Chem 62(24): 5492-5499.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
untreated 0% 0.000004% 0.00002% 0.0001%
PO
MC
gene
BIO3040
SRM1650b stimulated
Prevention of melanin stimulation
30
BIO3040 significantly reduces
the Proopiomelanocortin
(POMC) gene expression
induced by DEP. POMC is
released from keratinocytes,
triggers melanin biosynthesis
and is responsible for the
formation of dark spots.
* vs untreated p<0.05
+ vs stimulated p<0.05
+
* +
+
Gene expression
Skin preservation against inflammaging
31
BIO3040 significantly reduces
the DEP-induced expression of
inflammation gene markers
such as Interleukin-6 (IL-6),
linked to an inflammatory
response of the skin
(inflammaging).
* vs untreated p<0.05
+ vs stimulated p<0.05 0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
untreated 0% 0.000004% 0.00002% 0.0001%
IL-6
gene
BIO3040
+
*
+
+
SRM1650b stimulated
Gene expression
• HaCaT keratinocytes were stimulated with 3,75 µg/cm2 particulate matter for 6h.
• In the presence or absence of different actives. • Test-compounds have been applied 2h prior to
particulate matter.
• ELISA technique is performed to determine protein expression.
Protocol:
In-vitro test protocol
32
Human HaCaT keratinocytes were cultured in 96well plates 24h prior to stimulation with particulate matter.
HaCaT cells:
Protein biosynthesis
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
untreated 0% 0.0000025% 0.000025% 0.00025%IL
-1 a
lpha p
rote
in [
pg/
well]
BIO3040
Skin preservation against inflammaging
33
BIO3040 significantly reduces
the DEP-induced biosynthesis
of interleukin-1α (IL-1α), a key
mediator of inflammatory
processes in the skin
(inflammaging).
The results on gene level are
confirmed on protein level.
* vs untreated p<0.05
+ vs stimulated p<0.05
* +
+
SRM1650b stimulated
Protein biosynthesis
Summary & Outlook
— SRM1650b and SRM2975 are appropriate surrogates for authentic street particulate
matter.
— Both DEPs were able to stimulate AhR pathway in epidermal keratinocytes measured as
upregulated Cyp1A1 expression.
— Induction of premature aging was measured by upregulation of POMC, IL-6 and MMP-1
gene expression after incubation with SRM1650b.
Anti-Air Pollution
35
Conclusion
— The presented methodology is applicable for the development of potent anti-air
pollution actives by covering different pathways induced by an appropriate
surrogate.
Next Steps
— Confirmation of results ex vivo on human skin explants.
— In use clinical study in a polluted city.
Anti-Air Pollution
36
Summary & Outlook
Summary
Cyp1A1 POMC IL-6 MMP-1
BDDI + + + +
BIO3040 ++ ++
Symrise, always inspiring more…
in collaboration with
Leibniz-Institut für Umweltmedizinische Forschung (IUF) an der
Heinrich-Heine Universitaet GmbH
Prof. Dr. Jean Krutmann
Dr. Susanne Grether-Beck
