Scancos 2010

Evaluating the activity of a new plant-based soothing active

• Why design a new soothing active?• Product Brief

· Goals and ideas• In Vitro

· Initial screening

· Primary testing – Inflammation in-vitro

· Cellular resilience

· Photosensitivity

· Hyperosmotic shock

· Barrier function ECIS• In Vivo

· Histamine

· TEWL

· Erythema• Summary

Why design a new soothing active?

CLR gap fillingMarket demandsOpportunityTrend forecastingNew testing hardwareNiche areas of efficacy

Product Brief

GoalsSafe, significant measurable effects, natural, unique/protectable

Project CandidatesPhragmites kharka (Reed)Poria Cocus ( Mushroom)

Variables Raw material

Source, growing conditions, part of plant,sub-speciesManufacture

Isolation, separation, physical form, carrierSynergism

Targeted combinations

In vitro Test ResultsEffect on UV-induced inflammation

SyriCalm™ CLR

SyriCalm™ CLR

Reduction of UV-induced proinflammatory mediator production

70

80

90

100

Control 1.0% SyriCalm™ CLR 1.5% SyriCalm™ CLR

Keratinocytes were irradiated with UV light (2 J/cm² UVA; 0.2 J/cm² UVB) after pretreatment with

different dosages of SyriCalm™ CLR.

The release of IL-8 and TNFα by irradiated cells without pretreatment with SyriCalm™ CLR is set at

100%.

Method: Luminescence ELISA

IL-8 (%)

0

10

20

30

40

50

60

70

80

90

100

Control 1.0% SyriCalm™ CLR 1.5% SyriCalm™ CLR

TNFα (%)

In vitro Test ResultsCellular resilience against

UV irradiation

SyriCalm™ CLR

SyriCalm™ CLR

Protection against UV-inducedATP depletion

ATP (RLU)

Keratinocytes were

irradiated with different

dosages of UV light after

pretreatment with

different dosages of

SyriCalm™ CLR.

Control cells were

irradiated without

pretreatment with

SyriCalm™ CLR.

Method:

Luciferase/Luceferin

assay 150

160

170

180

190

200

210

220

230

240

250

Control 0.25% SyriCalm™ CLR1.0% SyriCalm™ CLR 1.5% SyriCalm™ CLR

1.0 J/cm2 UVA

0.1 J/cm2 UVB

2.0 J/cm2 UVA

0.2 J/cm2 UVB

In vitro Test ResultsEffect on Photosensitivity

SyriCalm™ CLR

SyriCalm™ CLR

Photosensitivity

Reactive oxygen species

• Decrease in cell viability• Inflammation

UV

Hypericin

SyriCalm™ CLR

Hypericin phototoxicity: Protection against decrease in cell viability

70

75

80

85

90

95

100

105

110

Control A Control B 0.25% SyriCalm™ CLR 0.50% SyriCalm™ CLRCell viability (%)

Keratinocytes were

treated with hypericin

(0.5 µM) and UV

irradiation (0.25 J/cm²

UVA; 0.025 J/cm² UVB)

with and without

pretreatment with

SyriCalm™ CLR.

Related to control,

without presence of

hypericin and

SyriCalm™ CLR (100%).

Method: MTT assay

(absorption: 570 nm)UV + + + +Hypericin – + + +

SyriCalm™ CLR

Hypericin phototoxicity: Reduction of IL-8 expression

IL-8 (RLU)

Keratinocytes were treated

with and without SyriCalm™

CLR, then irradiated with

0.25 J/cm² UVA; 0.025

J/cm² UVB.

RLU (relative luminescence

units)

Method: Luminescence

ELISA1000

2000

3000

4000

5000

6000

7000

8000

Control 0.5% SyriCalm™ CLR1.0% SyriCalm™ CLR 1.5% SyriCalm™ CLR

without hypericin with 0.5µM hypericin

SyriCalm™ CLR

Epidermal integrity

The barrier function of the skin is not only dependent on the quality of the Stratum Corneum.

External stress leads to alterations in cell shape and volume and intercellular anchoring in the epidermis• loss in barrier function• inflammation

In vitro Test ResultsEffect on cellular Taurine efflux during hyperosmotic stress

SyriCalm™ CLR

SyriCalm™ CLR

Hyperosmotic stress - consequences

Keratinocytes in the Stratum Granulosum are subjected to constantly fluctuating osmotic conditions • degradation of intra- and intercellular protein structures• impaired protein synthesis• impaired DNA repair mechanisms• induction of apoptotic mechanisms• further loss of barrier function, etc.

Healthy keratinacytes in the SG are vital to skin function.

SyriCalm™ CLR

Hyperosmotic stress in the epidermis

SC SG SS SB

Water gradient in normal skin

Water gradient in dry and diseased skin

The higher the gradient in the SG, the more

hyperosmotic stress

SyriCalm™ CLR

Hyperosmotic stress - Strategies

Isotonic Hypertonic

Taurine

fast

slow

fast

Electrolytes

Electrolytes

Taurine

SyriCalm™ CLR

Hyperosmotic stress - Challenges

Isotonic Hypertonic

Electrolytes

Electrolytes

Taurine

Taurine

SyriCalm™ CLR

Experiment – Protection against Taurine

efflux

TT

T T T T T T

T

T T T T T T

T T T

T T T

T

T T T T T T T

Taurine influx

0 - 48hrWashing of medium at t = 48hr

Taurine efflux

48 – 48+48hr

SyriCalm™ CLR

Experiment – Protection against Taurine

efflux

Method: evaluation of counts per minute with TopCount NXT™ Microplate Scintillation

counter (Perkin elmer) in MicroScint 40 Scintilation cocktail (Perkin Elmer)

0h 24h 48h 48+2h 48+24h 48+48h

Hyperosmotic stress

Taurine influx

Taurine efflux

1st application

2nd

3rd

SyriCalm™ CLR

Protection against Taurine efflux

Taurine efflux (%)

3D epidermal skin models

were established in a

CELLnTEC PCT medium,

after which they were

exposed to a hyperosmotic

medium (400 mOsm) and

treated with SyriCalm™ CLR

and analyzed according

mentioned scheme.

The osmolarity of the

medium was kept at 400

mOsm during the whole

experiment.

Taurine efflux is expressed

as percentage radioactivity of

the cell medium related to

total radioactivity (cell lysate

+ consecutive samples of the

medium)

0

10

20

30

40

50

60

70

80

90

100

48+24h 48+48h

non-treated treated (1st+2nd) treated (3rd) treated (1st+2nd+3rd)

SyriCalm™ CLR

Protection against IL-8 expression during

hypo-osmotic stress

IL-8 (%)

Keratinocytes were

incubated in a hypo-

osmolary medium (250

mOsm) for 30 min and

12 hours in the

presence of different

concentrations of

SyriCalm™ CLR.

Method: Luminescence

ELISA

0

200

400

600

800

1000

1200

1400

untreated Control 0.25% SyriCalm™ CLR 1.5% SyriCalm™ CLR

12 hours30 min

In vitro Test ResultsEffect on epidermal integrity

SyriCalm™ CLR

SyriCalm™ CLR

Determining epidermal integrity with ECIS

External stress leads to a disturbance of epidermal integrity

• loss in cellular volume

• degradation of intra- and inter cellular structural proteins,

potentially leading to cell-detachment

External stresses

SyriCalm™ CLR

Determining epidermal integrity with ECIS

ECIS: Electric Cell-substrate Impedance Sensing

An automated non-invasive method to monitor cell behaviour and attachment (barrier

function)

Principle:A confluent layer of keratinocytes will have

a certain resistance (impedance) against

conducting an electrical current.

Change in cell volume and shape and loss

of intercellular attachment lead to a loss of

impedance. Regaining the impedance is a

measure for regaining barrier function.

SyriCalm™ CLR

Protection against loss of epidermal integrity

after an electrical pulse

Change in Impedance

(%):

A confluent layer of

Human Keratinocytes

was applied on an

Electrode Array Chip

and incubated with

SyriCalm™ CLR for 24

hours without FCS.

The chip was

connected to an ECIS

instrument.

Values of resistance

were collected before

and after an Elevated

Field Pulse was

applied (t=0, 15 kHz, 6

V, 5 sec). The

impedance before the

pulse is set at 100%.

70

75

80

85

90

95

100

105

110

-0,02 0 0,02 0,04 0,06 0,08 0,1 0,12 0,14

1.5% SyriCalm™ CLR Control

70

75

80

85

90

95

100

105

110

-0,02 0 0,02 0,04 0,06 0,08 0,1 0,12 0,14

1.5% SyriCalm™ CLR Control

70

75

80

85

90

95

100

105

110

-0,02 0 0,02 0,04 0,06 0,08 0,1 0,12 0,14

1.5% SyriCalm™ CLR Control

t (hour)

-0.02 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14

SyriCalm™ CLR

85

90

95

100

105

-0,5 0 0,5 1 1,5 2 2,5

1.5% SyriCalm™ CLR Control

85

90

95

100

105

-0,5 0 0,5 1 1,5 2 2,5

1.5% SyriCalm™ CLR Control

85

90

95

100

105

-0,5 0 0,5 1 1,5 2 2,5

1.5% SyriCalm™ CLR Control

Protection against loss of epidermal integrity

after UV irradiation

t (hour)

Impedance (%)

A confluent layer of

Human Keratinocytes

was applied on an

Electrode Array Chip

and incubated with

SyriCalm™ CLR for 24

hours without FCS.

The chip was

connected to an ECIS

instrument.

Values of resistance

were collected before

and after UV irradiation

(t=0, 2 J/cm² UVA+0.2

J/cm² UVB). The

impedance before the

irradiation was set at

100%.

-0.5 0 0.5 1 1.5 2 2.5

In vitro Test ResultsEffect on UV induced degradation of

E-Cadherin

SyriCalm™ CLR

SyriCalm™ CLR

E-Cadherin in the epidermis

Actin

α-Catenin

β-Catenin

p120ctn

E-Cadherin

Trans-membrane protein,

essential in the formation and

maintenance of cell junctions

in the epidermis

• plays a crucial role in the

formation of tight

junctions and

desmosomes

• part of adherens

junctions

SyriCalm™ CLRSyriCalm™ CLR

Reduction of UV-induced E-Cadherin degradation

Soluble E-Cadherin

(pg/ml)

Keratinocytes were

irradiated with UV light (1

J/cm² UVA; 0.1 J/cm²

UVB) after pretreatment

with 0.5% of SyriCalm™

CLR.

Method: ELISA

2000

2200

2400

2600

2800

3000

3200

Control SyriCalm™ CLR 0.5%

1 J/cm² UVA

0.1 J/cm² UVB

Ground state

In vivo Test ResultsEffect on histamine-induced itching

SyriCalm™ CLR

SyriCalm™ CLR

Effect on histamine-induced itching

0

0,5

1

1,5

2

2,5

15 min 30 min 60 min

untreated

Posit ive control (Fenist il®, 0.1% Dimethindene Maleate)

3% SyriCalm™ CLR2.5

2

1.5

1

0.5

0

Itching (Scoring system)

Histamine was applied

intracutaneously on a

designated area on the inner

forearm of 10 volunteers

(33–79 years old) via a lancet

stitch. The application of the

test product took place

immediately after the insult

with histamine. The itching

was assessed by a scoring

system

15, 30 and 60 min after

application of the test

product.

In vivo Test ResultsEffect on UV-induced increase

in TEWL

SyriCalm™ CLR

SyriCalm™ CLR

Effect on UV-induced increase in TEWL

TEWL (g/hm2)

On designated skin areas

on the inner side of the

thigh of 10 volunteers (33

- 79 years old) the TEWL

was measured. Then

these areas were exposed

to

2.0 MED of UVB

irradiation, inducing loss

of barrier function. 24

hours after that the TEWL

was measured again.

Directly after that the test

products were applied for

4 days, twice daily. Every

day the TEWL was

measured (method:

Tewameter TM 210,

Courage & Khazaka,

Germany)

Application of products

SyriCalm™ CLR

Effect on UV-induced increase in TEWL -

Trends

TEWL (%)

Trends in reduction of

TEWL as a function of

time.

Comparison between 3%

SyriCalm™ CLR and

Positive control.

The TEWL measured at

maximum Erythema was

set at 100%.

50

55

60

65

70

75

80

85

90

95

100

Posit ive control (Bepanthen®, 5% Panthenol and Lanolin)

3% SyriCalm™ CLR

Day 3Day 2Day 1 Day 4TEWL

max

erythema

In vivo Test ResultsEffect on UV-induced erythema

SyriCalm™ CLR

SyriCalm™ CLR

Effect on UV-induced erythema

Skin redness (%)

On designated skin areas on

the inner side of the thigh of

10 volunteers (33 - 79 years

old) the skin redness was

measured. Then these areas

were exposed to 2.0 MED of

UVB irradiation, inducing

erythema. 24 hours after that

the redness of the skin was

measured again. Directly after

that the test products were

applied for 4 days, twice daily.

Every day the skin redness

was measured. The maximum

erythema is set at 100%.

(Method: Chromameter CR

200, Minolta, Japan)

40

50

60

70

80

90

100

Day 1 Day 2 Day 3 Day 4

Positive control (Bepanthen®, 5% Panthenol and Lanolin)

3% SyriCalm™ CLR

SyriCalm™ CLR

SummaryIn vitro:• Protection against external stresses

o Increase in cell viability o Increase of cell energy levelo Improvement of cellular structureo Supports barrier functiono Fights against the effects of

hyperosmotic stress• Reduction of photosensitivity• Anti-inflammatory actionIn vivo:• Protection against histamine & UV-induced stress

o Accelerated recovery of skin barrier function (TEWL) o Accelerated reduction of erythemao Accelerated reduction in itching

INCI: Water, Phragmites Kharka Extract,

Poria Cocos Extract

Dosage: 3%

Recommended pH: 3.0 – 8.0

Preserved with sodium benzoate