A CUTTING-EDGE MOLECULE FOR AA CUTTING-EDGE MOLECULE FOR A RADIANT SKIN TONE

v5

THE CULTURE OF A BRIGHTER SKIN (I)

Skin brighteners use and consumers’ demand is growing worldwideSkin brighteners use and consumers demand is growing worldwide.

• In Western countries, skin brighteners are applied for theprevention and treatment of irregular hyperpigmentation, resultingin an evener skin tone.

• In Asia, brighter skin is a symbol of beauty and femeninity. The useof skin brightening agents is widely extended by traditional beliefs.o The need of whitening the skin is so high that most of anti-aging

products contain also whitening ingredients and UV filters.

THE CULTURE OF A BRIGHTER SKIN (II)

Japan

Sales of Whitening d t 15 17% f

Chinaproducts are 15-17% of

Skin Care sales30% of people use Skin

Whitening products

Thailand

Even more population (60%) uses Skin

Whitening products

THE CULTURE OF A BRIGHTER SKIN (III)

Whitening and brightening d t t f 60% f

Anti-age spots

products account for 60% of new products among the top

ethic beauty launches (Mintel GNPD Beauty Innovation)

Even skin tone

GROWTH IN SKIN LIGHTENING BY MARKET

(US $ Milli )(US $ Million)

AC Nielsen, 2007

SKIN COLOUR

The colour of the skin and hair depends on the

• Melanins are pigmented biopolymers synthesised in

amount, distribution and type of melanin.

• Melanins are pigmented biopolymers synthesised inmelanocytes in the DEJ (Dermo-Epidermal Junction).

• Melanosomes with melanins migrate and they aretransferred to keratinocytes in skin.

melanins

MELANOCYTES

melanins

KERATINOCYTES

SKIN COLOUR

CHANGES IN SKIN PIGMENTATION

• Typical pigmentary changes appearduring intrinsic aging and photoaging:

HYPERPIGMENTATIONmelasma, freckles, age spots and senil lentigines

abnormal accumulation of melanin

acute or persistent UV exposure

THE SEARCH FOR A NEW SKIN BRIGHTENER

• Inhibition of melanin synthesis by inhibition of tyrosinase activity.

• Tyrosinase: key enzyme of melanogenesis.

melanins SKIN COLOUR

MELANOCYTES

Need for novel skin brightening agents with increased efficacy and improved safety profilesff y p f y p

THE IDEAL SKIN BRIGHTENER

According to Dooley1, new depigmentation products should include thefollowing desirable features:

PROPERTIES

following desirable features:

Inhibition of mammalian tyrosinase (human tyrosinase)

Lack of toxicologic or mutagenic potential (impeccable safety profile)

Clinical efficacy (proven in vivo)

Formulation stability (high stability)

Novelty and patent protection

PHOTOPROTECTIVE EFFECT prevention of UV-induced skin damage

1Dooley TP. Topical skin depigmentation agents: current products and discovery of novel inhibitors of melanogenesis. J Dermatolog Treat. 8:275-279, 1997.

CHROMABRIGHT® STABILITY

Tested in an O/W emulsionTested in an O/W emulsionqsp 100Water

%INGREDIENT

0.05CHROMABRIGHT®

3Polyacrylamide, C13-14 Isoparaffin, Laureth-7

10Mineral Oil (Paraffinum Liquidum)

100

120

0,05

0,06

60

80

ncen

trat

ion

(%)

0,03

0,04

cent

ratio

n (%

)0

20

40Con

0

0,01

0,02

Con

c0 months 1 month 2 months 3 months 4 months 6 months

Time

Room temp. 40 ºC

3,8 5,0 8,4

pH

room temp 60 ºC

Chromabright® proved to remain stable after 6 months and at different pH final formulations

CHROMABRIGHT® SAFETY

Completely safe profile:Completely safe profile:

o Ocular irritation (HET-CAM test).

o Phototoxicityo Phototoxicity.

o Cytotoxicity on human epidermal keratinocytes.

o Cytotoxicity on 3T3 fibroblasts.o Cytotoxicity on 3T3 fibroblasts.

o Bacterial reverse mutation test (Ames test).

o Cytotoxicity on human primary melanocytes.

o Skin sensitisation.

Chromabright® showed no signs of toxicity in any of the tests above

CHROMABRIGHT® EFFICACY

IN VITROo Inhibition of mushroom tyrosinase activity.

It is the most used assay to assess potential depigmenting agents.

IN VITRO

o Inhibition of endogenous human tyrosinase activity.

This assay should be considered much better than mushroom tyrosinase.

o Depigmenting effect on human melanocytes.

o Melanogenesis inhibition on human epidermal melanocytes.

o Photoprotective effect on human epidermal keratinocytes.

o Skin brightening effect on human volunteers.

IN VIVO

IN VITRO EFFICACY (I)

1. Tyrosinase inhibition1. Tyrosinase inhibition • L-Dopa (tyrosinase substrate) was digested in thepresence of 1mM Chromabright® and the enzyme.

-20120

dop

on

• Absorbance variations were measured at 475 nm.

• Kojic acid 0.1mM was used as positive control.Mushroom tyrosinase

55.5% 37.0%

0

20

40

60

8040

60

80

100

% inhibition oachrom

e prod

rom

e pr

oduc

tio

37% inhibition of mushroom tyrosinase activity80

100

1200

20

Control Kojic acid CHROMABRIGHT®

of uction

% do

pach

r

(0.1mM) (1mM)

tyrosinase activity

-20120dopa

tion

Endogenous human tyrosinase

69.0%

43.0%

0

20

40

60

8040

60

80

100

% inhibition oachrom

e produrom

e pr

oduc

t

43% inhibition of endogenous human tyrosinase activity

80

100

1200

20

Control Kojic acid CHROMABRIGHT®

of uction

% do

pach

(0.1mM) (1mM)

IN VITRO EFFICACY (II)

2. Depigmenting effect on human melanocytes cultures2. Depigmenting effect on human melanocytes cultures

• Incubation of plated human melanocytes for 5 days.

• Daily addition of fresh medium containing 0.1mMChromabright® or Kojic Acid45 9%

% Lightening efficacy

Chromabright or Kojic Acid.

• Kojic acid 0.1mM was used as positive control.

• The lightening efficacy was assigned by counting thecells showing melanin staining and the total number of

ll

40%

50%

30.2%

45.9%

cells.

10%

20%

30%

Chromabright® exhibits a better depigmenting effect than Kojic Acid

on human melanocytes(0.1mM)

0%Kojic acid CHROMABRIGHT®

(0.1mM)

IN VITRO EFFICACY (III)

3. 3. MelanogenesisMelanogenesis inhibition on human inhibition on human melanocytesmelanocytes cultures (I)cultures (I)

• Primary human melanocytes (HEMn-DP) cells were seeded andallowed to grow for 2 weeks.

• Melanocytes were treated on days 1, 3, 6, 8, 10, 13, 15 and 17 with:120

o Chromabright® (5µM, 10µM, 100µM, 150µM and 200µM)

o Hydroquinone (10µM)

o MAP (Magnesium Ascorbyl Phosphate) (10µM)60

80

100

anin

(pg

/cel

l)

o Kojic Acid (10µM)

o Arbutin (10µM)

• Control: medium without treatment.0

20

40

% o

f m

ela

• After 20 days of culture, melanin concentration was determined bymeasurement of absorbance at 450nm and values were normalisedrespect to the number of cells per well.

Chromabright® inhibited melanogenesis at all tested concentrations in a dose-dependent mannerconcentrations, in a dose-dependent manner

IN VITRO EFFICACY (IV)

3. 3. MelanogenesisMelanogenesis inhibition on human inhibition on human melanocytesmelanocytes cultures (II)cultures (II)

By optical microscopy

Control 200µMControl 200µM

Hydroquinone 10µM10µM Chromabright® melanogenesis

Similar activity at the same concentration, but Chromabright®

did t t t t i it

Chromabright melanogenesis inhibition efficacy was higher than

Arbutin, Kojic Acid and MAP

at the same concentration (10µM)

while Hydroquinone cytotoxic effect was clearly observed, at the dosages tested.

did not present cytotoxicity at the same concentration (10µM).

IN VITRO EFFICACY (V)

4. Cellular 4. Cellular photoprotectionphotoprotection • Test based on the determination of the protective effect ofa chemical when tested in the presence of a cytotoxic doseof simulated solar light.

• Irradiation to cells implies a decreased uptake of the vitaldye Neutral Red (NR).

NRU photoprotection test in Human Epidermal Keratinocytes

190% increase in cell viability

Chromabright® helps to prevent the skin-damaging effects of UV radiation.

IN VIVO EFFICACY (I)

1. Brightening effect1. Brightening effect • Measurements were taken before application, after 30 and 60 daysof treatment.

• Parameters used to evaluate the effects:

• L* (Luminance): represents the relative brightness fromtotal darkness (L*=0) to absolute white (L*=100)

ITAº (I di id l T l l A l ) t i ki l

o 20 Asian female volunteers, aged 18 to 46.

o A cream containing 0.1% Chromabright® wasapplied on one side of the face twice daily and

• ITAº (Individual Typologycal Angle): categorises skin colour,obtained combining L* and b* (yellow-blue colour axis)

a placebo cream on the other side for 60 days.

o Evaluation: means of a Chromameter CR-300.

A cream containing 0.1% Chromabright®

induced a significant brightening effect induced a significant brightening effect after 30 and 60 days

IN VIVO EFFICACY (II)

2. 2. DepigmentingDepigmenting effecteffect• % clinical improvement:

o Score 0: 0%

o 10 volunteers, aged 18 to 70, with melasma and/or actiniclentigines applied a cream containing 0.5% Chromabright® ontheir face and/or hands twice a day for 60 days.

Cli i l d i hi t l f d t th

o Score 1: 1-25%

o Score 2: 26-50%

o Score 3: 51-75%

S 4 76 100%o Clinical and iconographic controls were performed at thebeginning of the study, and after 30 and 60 days of application.

o Score 4: 76-100%

3 50 3.40

2.00

2.50

3.00

3.50

2.20 2.11

3.11

ore T30

0 days 30 days

0.50

1.00

1.50Sco

T600 days 30 days

0.00MELASMA LENTIGINES

0 days 60 days80% of the volunteers with melasma and y y

77.8% with lentigines experienced a significant improvement after 60 days

COSMETIC BENEFITS

Safety & efficacytogether

High efficacy in short time

Photoaging prevention

SafetyPure moleculep

High stability and keeps

purity of 100%Cellular

photoprotection demonstrated

Completely safe toxicological

profileo Significant brightening effect in only 2 months

demonstratedo High depigmenting power

after 30 days

TECHNICAL INFORMATION

DESCRIPTIONA new patented ingredient designed for skin brightening applications that A new patented ingredient designed for skin brightening applications that shows neither cytotoxic effects, nor any irritation or sensitisation reaction.

APPEARANCEPowder.

INCIDimethylmethoxy Chromanyl PalmitateDimethylmethoxy Chromanyl Palmitate.

PROPERTIESIt induces a significant lightening effect on the skin, at the same time that It induces a significant lightening effect on the skin, at the same time that fights against photoaging.

APPLICATIONSChromabright® can be incorporated in cosmetic formulations containing oil or silicon phases where a brightening effect on the skin is desired.

DOSAGEDOSAGE0.1-0.5%

A CUTTING-EDGE MOLECULE FOR A RADIANT SKIN TONE

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