CORUM Whitening agents

New Generation Vitamin C Derivative CORUM 9515 3-O-Ethyl Ascorbic Acid

AS POTENT AS VITAMIN C WITH BETTER STABILITY

CORUM 9515

INCI: 3- O – Ethyl ascorbic acid Also known as:

3- O – ethyl ascorbic ether 3- O – ethyl ascorbic acid

Physical Properties  Purity > 99.0%  White crystalline powder  Water soluble  Metabolized by the human body in the same manner as L- ascorbic acid (Vitamin C)  Excellent stability

CAS: 86404-04-8 MW: 204.18

The Functions of CORUM 9515 Skin lightening & Balance skin tone Reduce dark spot & age spot Anti-photoaging Collagen synthesis Reversing auto-oxidation Radical scavenging DNA protection Easy to penetrate the epidermis

CORUM 9515 Efficacy Studies 

In-vitro tyrosinase inhibition



Anti-photoaging (Anti-inflammation test)



Reducing activity



Stimulation of collagen synthesis



In-vitro whitening activity



Radical scavenging effect



Ex-vivo melanin assay

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DNA protection

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In-vivo whitening efficacy

 Skin penetration profile

CORUM 9515 Whitening Mechanism

TRP-2

Three melanin-generating enzymes:

1. 2. 3.

Tyrosinase TRP-2 (DOPAchrome tautomerase) TRP-1 (DHICAoxidase)

 Inhibit the reaction center Cu+2 ion of tyrosinase  Inhibit the activity of TRP-2

 Reduce oxidized melanin pigments

Ref: Briganti, S et al. (2003) Chemical and Instrumental Approaches to Treat Hyperpigmentation Pigment Cell Res 16:101–110.

TRP-1

Efficacy Test: In-vitro tyrosinase (I) inhibition Reaction:

Enzyme level

Tyrosine Assay 100

Tyrosine Inhibition Activity IC50

99.8

93.8

100

Inhibition (%)

Inhibition (%)

80 60 37.6

40 20

2.1

9.7

75 5 mg/ml 72.6%

50

7.5 mg/ml 81.4%

10 mg/ml 90.3%

2 mg/ml 51.4%

25 1 mg/ml 37.6%

0 Vit.C

Corum 9515

AA2G

MAP

α-Arbutin

0 0

Sample: 1 mg/ml (0.1%)

4

Concentration (mg/ml)

0.1 % CORUM 9515 can inhibit tyrosinase activity up to 37.6 %. 2 mg/mL of CORUM 9515 can reach 50% inhibition. Ref: Corum Internal Report. Feb 2009.

8

12

Efficacy Test: In-vitro tyrosinase (II) inhibition Reaction:

Enzyme level

DOPA Assay 100

Tyrosine Inhibition Activity IC50

99.1

100

Inhibition (%)

Inhibition (%)

80 54.8

60

31.9

40 22.3 20

2 mg/ml 67.1% 50

8 mg/ml 92.9%

1 mg/ml 47.1% 0.5 mg/ml 40.7%

25

-19.9

0 Vit.C -20

75

4 mg/ml 83.3%

Corum 9515

AA2G

MAP

Sample: 1 mg/ml (0.1%)

α-Arbutin

0 0

5 Concentration (mg/ml)

0.1 % Corum 9515 can inhibit tyrosinase activity up to 54.8%. 1 mg/mL of Corum 9515 can reach 50% inhibition. Ref: Corum Internal Report. Feb 2009.

10

Efficacy Test: In-vitro Tyrosinase, Trp-2 inhibition Protein level

Tyrosinase inhibition

Tyrosinase Level (%)

100

47.5% Inhibition rate

80 60 40

Test: 20

 Western blot – Tyrosinase  In-vitro melanogenesis inhibition

0 Control 1.

Control 2.

without α-MSH

1 % Corum 9515

1 % AA2G

(with α-MSH)

with α-MSH

Result : 1 % Corum 9515 can inhibit tyrosinase activity up to 47.5 % in protein level.

Ref: Corum Internal Report. Aug 2012.

Efficacy Test: In-vitro Tyrosinase, Trp-2 inhibition Protein level

Trp-2 inhibition 100

Trp-2 Level (%)

80

72.6% Inhibition rate

60

40

Tests:

20

 Western blot – Trp-2  In-vitro melanogenesis

0 Control 1. without α-MSH

Control 2.

1 % Corum 9515 with α-MSH

1 % AA2G

inhibition (with α-MSH)

Result : 1 % Corum 9515 can inhibit Trp-2 activity up to 72.6 % in protein level.

Ref: Corum Internal Report. Aug 2012.



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

Reducing activity




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


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

DNA protection





Efficacy Test: Ferric Reducing Ability

TRP-2

TRP-1

Ref: Briganti, S et al. (2003) Chemical and Instrumental Approaches to Treat Hyperpigmentation Pigment Cell Res 16:101–110.

Efficacy Test: Ferric Reducing Ability  pKa value greater - give electron easier - better reducing ability  2’ carbon without ethyl group bonded has better reducing ability

2 1

3

Ascorbic acid (reduced form of Vitamin C)

2

3

2

3

2

3

Ethyl Ascorbic Acid

AA2G

MAP

MW: 204.18

MW: 338.26

MW: 759.22

Efficacy Test: Ferric Reducing Ability e-

Fe 3 + +

100

100

Fe 2+

100

Inhibition (%)

80

60

52.6

40

31.9

20 1 0

Vit.C

Corum 9515

AA2G

Sample: 10 mg/ml (1%) Ref: Corum Internal Report. Feb 2009.

MAP

α-Arbutin








Reducing activity















DNA protection





Efficacy Test: In-vitro whitening activity study - melanin assessment by IDEA Method: 12 hours contact with the product and stimulation during 48 hours with theophylline at 0.5 mM. Result: 49.75% whitening effect, 20mg/ml Corum 9515 60

Inhibition (%)

49.75

38.21

40 32.8

20

0 5 mM Kojic acid

15 mg/ml Corum 9515

Ref: “Study of the effect of a test item on depigmentation of cells in culture: melanin assessment”, IDEA, Aug, 2008.

20 mg/ml Corum 9515








Reducing activity

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










DNA protection





Efficacy Test: Ex-vivo Melanin Assay MelanoDerm™

by BioInnovation Laboratories, Inc (USA) Purpose  using an in-vitro tissue model of the human epidermis prepared from cultured human keratinocytes and melanocytes.  both water-soluble and water-insoluble materials  skin darkening agents or skin lightening agents

Efficacy Test: Ex-vivo Melanin Assay Day 0

Day 9

Untreated Tissue

Melanin (μg/tissue)

30

3 % Corum 9515

Melanin Content (μg/tissue) 23.4 16.7

20

10

0

Untreated

3 % Corum 9515



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

Reducing activity

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


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




DNA protection





Efficacy Test: In-vivo whitening efficacy by SPINCONTROL

Method : Chromametric Analysis

Skin type I

Skin type II

Skin type III

Skin type IV

Ref: Chardon, A., Cretois, I., Hourseau, C., Skin colour typology and sun tanning pathways Int. J. Cosm. Sci, (1991), 13, 191-208

Efficacy Test: In-vivo whitening efficacy by SPINCONTROL

Method:  Chromametry (CR-300)  2% Corum 9515 cream

35

Mean Individual Typological Angel

Volunteers:  20 healthy Asian  Female  25-40 years old  Skin type III

+4.0% 34

33

32

T0

Result: Significant increase on mean ITA

Ref: “In vivo evaluation of the efficacy of one whitening lotion in healthy Asian subjects by chromatography”, Spincontrol Asia, November, 2007.

T +28 Days

Efficacy Test: In-vivo whitening efficacy


Efficacy Test: In-vivo whitening efficacy









Reducing activity

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










DNA protection





Mechanism of Photoaging

Efficacy Test:

Anti-inflammation IL-6 test Principle

Stimulate by External Material (e.g. 40 mJ/cm² UVB)

Cytokines Release

Regulate Inflammatory reaction IL6

Corum 9515 100

IL-6 Production (%)

100

100% Inhibition rate

50

18 5

1 0 Control 1

Control 2

1% Corum 9515

0.5% Corum 9515

UVB irradiated Ref: Corum Internal Report.Sep 2012.

0.25% Corum 9515



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

Reducing activity

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







DNA protection





Collagen and its function What is collagen

 The main protein of connective tissue  Make up 25% - 35% of the whole-body protein content  Different types of collagen

Functions

 Impart strength, support and skin elasticity

Why important

 As skin ages, it produces less collagen and loses its elasticity

Wrinkle formation + Aged skin

Less collagen

Abundant collagen Epidermis

Dermis

The role of ascorbic acid in collagen synthesis Proline Monooxygenase

Proline

Hydroxyproline

makes up collagen

Fe3+

Fe2+

Corum 9515

Inhibition of oxidation (%)

Semidehydroascorbyl radical

Ascorbate

Ferric Reducing Ability 100

100

100

 Proline residues convert into hydroxyproline during the synthesis of collagen

80 52.6

60

 Hydroxylation requires Vitamin C

31.9

40 20

1

0

Vit C

AA2G

Arbutin

MAP

Corum 9515

Efficacy Test:

Stimulation of natural collagen synthesis by IDEA, France After 48hrs contacts  TGF β1 is a very strong collagen synthesis stimulator  It is tested with type I collagen 40

30

Stimulation (%)

30

27

20

10

0 10 ng/ml TGF β1

500 ppm Corum 9515

Result: Corum 9515 has a similar effect on collagen synthesis as TGF β1 Ref: “Activity study of a test item on collagen synthesis from human fibroblast culture”, IDEA, June, 2008.



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

Reducing activity















DNA protection

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Corum 9515 Free-Radical Scavenging Mechanism What are free radicals

Cause

Why important

Normal oxygen atom

Electron lose Create free radical

 Atoms, molecules, or ions with unpaired electrons  Due to its unpaired electrons, they are often highly reactive  Often causes chain reactions

 UV rays, stress & environmental pollution etc.  It can participate in unwanted side reactions resulting in cell damage  Many form of cancer are thought to be the result of reactions between free radicals and DNA

Free radical set-off chain reaction

Erosion of cell membrane

Cell membrane

Ref . Stephaniepizzo, (2009). Anti-Up? A Quick View into Antioxidants, Free Radicals, and Your Body

Corum 9515 can neutralize free-radical

Efficacy Test: Radical scavenging test ROS assay after UVB irradiated

Principle

100

100

ROS Production (%)

79.6 64.6 55.1 50

25.6

0

Control 485 nm

Control

5000 ppm 500 ppm 50 ppm Corum 9515 Corum 9515 Corum 9515 UVB irradiated

CORUM 9515 shows great ability on ROS inhibition. 528 nm

Ref: Corum Internal Report. Feb 2013.



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

Reducing activity

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





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

DNA protection

a. In-vitro, Comet Assay




b. Ex-vivo


Efficacy Test: DNA Protection Comet Assay

 Method: Single Cell Gel Electrophoresis Assay – Comet Assay  Theory: Strong alkali A

T

A

A

C

C

T

T

A

T

T

G

G

A

DNA damaged by H2O2

Single cell electrophoresis

-

+ No damage

Serious damage

A A

T

A

T

A

T

T

C G

C

T

G

A

DNA disintegrates

Efficacy Test: DNA Protection Comet Assay

Untreated

Control (H2O2)

Protection rate 25 AA2G

21.9

Corum 9515

AA2G 10,000 ppm

20

C-9515 5,000 ppm

AA2G 5,000 ppm

Protection Rate (%)

C-9515 10,000 ppm

15

10

5

8.9

9.4

4.4

0 5000 ppm

10000 ppm

Result: CORUM 9515 shows high efficiency on DNA protection activity.

What are Sun Burn Cells? Sunburn cells (SBC) are keratinocytes undergoing apoptosis as a protective mechanism against the carcinogenic effects of UVB irradiation which irreversibly damages their DNA or other chromophores.

Nuclear shrinkage

Nuclear fragmentation

Nuclear fading

Sun burn cells are indicated by arrow.

Efficacy Test: DNA Protection

after UVB irradiation on human living skin explants by BIO-EC, France Explants preparation:  From a 45-year-old Caucasian woman  12 skin explants of an average diameter of 11 mm (±1mm) Number of sun burn cell in the epidermis

Spongiosis is slight in the basal layers

Some cells with picnotic nuclei.

*Sunburn cell are indicated by circles

Number of Sunburn Cells/cm

75

53.3 50 35.4 25

0 Control

4% Corum 9515

with UV irradiation

Result: Corum 9515 shows high efficiency on DNA protection activity after UVB irradiation.



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

Reducing activity

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

DNA protection





Skin Penetration Profile Human Epidermis Model EPISKIN® ：In Vitro Skin Penetration Test in vitro reconstructed human epidermis which is similar to the in vivo human epidermis. ※ Episkin is a skin model as alternatives to animal testing.

Applications:  Percutaneous absorption

 In vitro skin irritation

 In vitro skin corrosion

 Pharmacology /Toxicology

Test materials:  2 % solution

 2 % Whitening cream

Skin Penetration Profile Human Epidermis Model EPISKIN® ：In Vitro Skin Penetration Test Cumulated permeated amount Test materials: 2 % solution Cumulated permeated amount (μg‧cm-2)

40 35 30 Corum 9515

25 20

AA2G

15

10 5 0 0

1

2

3

4 5 Time (hours)

6

7

8

Result: CORUM 9515 shows better skin penetration ability compared with AA2G.

Skin Penetration Profile Human Epidermis Model EPISKIN® ：In Vitro Skin Penetration Test Penetration rate ‧Ratio (Corum 9515 vs. AA2G) Test materials: 2 % solution

6

Corum 9515

5

AA2G

better in penetration

3.00 Ratio of penertration rate (CORUM 9515 vs AA2G)

Penetration rate (%)

Corum 9515 is 2.4 times

4 3 2 1

2.50 2.00 1.50

1.00 0.50 0.00

0 1

2

3

4

5

6

Time(hours)

7

8

1

2

3

4

5

6

7

Time(hours)


8

Skin Penetration Profile

Human Epidermis Model EPISKIN® ：In Vitro Skin Penetration Test Cumulated permeated amount Test materials: 2 % Cream Cumulated permeated amount (μg‧cm-2)

14 12 10 8

Corum 9515

6

AA2G

4 2 0

0

1

2

3

4 5 Time (h)

6

7

8


Skin Penetration Profile Human Epidermis Model EPISKIN® ：In Vitro Skin Penetration Test Penetration rate ‧Ratio (Corum 9515 vs. AA2G)

6

Corum 9515

5

AA2G

Corum 9515 is 5 times

6.00

better in penetration

5.00

Ratio of penertration rate (Corum 9515 vs AA2G)

Penetration rate (%)

Test materials: 2 % Cream

4.00

4

3.00

3

2.00

2

1.00

1 0

0.00 1

2

3

4 5 6 Time(hours)

7

8

1

2

3

4 5 6 Time(hours)


7

8

Stability Tests  Heat-stability (45 ̊C , 1 month) - color (Transmittance in 440nm ) - purity (HPLC)

 Photo-stability (sunlight, 1 month)

 Buffer system and pH effect (45 ̊C, 56 days and RT, 90 days)

Stability: Heat-Stability 0 month 2% 45C without buffer 1 Month 45 ̊C Stability (Transmittance at 440nm ) C-9515

α-Arbutin

AA2G

Vit-C

1 month 2% 45C without buffer

C-9515

α-Arbutin

AA2G

Vit-C

Transmittance %T

100

96.8 90.9

85.1

80 60 41.6 40 20 0 Corum 9515

α-Arbutin

AA2G

Sample: 2%

Ref: Corum Internal Report, November, 2007.

Vit.C

Stability: Crystalline Powder Heat-Stability

99.9

60 Days 45 ̊C Purity

 UV detection at 214nm  Corum 9515 crystalline powder

99.9

99.9

99.9

99.9

80 Area (%)

 HPLC system

100

60 40 20 0 Day 0 Day 1 Day 15 Day 30 Day 60

Result: CORUM 9515 crystalline powder remains stable under 45 ̊C for 60 days





Stability: CORUM 9515 Photo-Stability 0 month 2% under sunlight without buffer

C-9515

Vit-C

α -Arbutin

AA2G

1 month 2% under sunlight without buffer

C-9515

Ref: Corum Internal Report, November, 2007.

Vit-C

α -Arbutin

AA2G




Stability under different pH & buffer Purity at pH 4 120

Preparation: 2% buffer solution

Area (%)

PURITY assay by HPLC

100 80

A

60

B

40

C

20 0 Day 0 Day 1 Day 7 Day 14 Day 28 Day 56

Day: 56 days at 45 ̊C

Purity at pH 6 120 100 Area (%)

Buffer system: A - Sodium Citrate – Citric Acid B - Na2HPO4- Citric Acid C - NaHCO3 – Citric Acid

80

A

60

B

40

C

20 0 Day 0 Day 1 Day 7 Day 14 Day 28 Day 56


pH Stability under different buffer Stability of pH value in different buffer system (pH 4)

Stability

Preparation: 2% buffer solution

8

pH Value

pH

10

A 6

B

4

C

2

D

0

Day: 56 days at 45 ̊C

Day 1

Day 7 Day 14 Day 28 Day 56

Stability of pH value in different buffer system (pH 6) 10 8

pH Value

Buffer system: A - Sodium Citrate - Citric Acid B - Na2HPO4 - Citric Acid C - NaHCO3 - Citric Acid D -NaOH - Citric acid

Day 0

A

6

B 4

C D

2

0 Day 0 Ref: Corum Internal Report. Feb 2009.

Day 1

Day 7

Day 14

Day 28

Day 56

Anti-oxidant lessen color change Corum 9515 Ethyl Ascorbic Acid is a powerful anti-oxidant Thus a stronger Anti-oxidant is recommended to be used in formulation  prevent losing Corum 9515’s effect before it reaches its target area

Anti-oxidant Corum 9515 (Ethyl Ascorbic Acid) Other ingredients

Anti-oxidant lessen color change 45 ̊C for 6 months

C-9515 Sodium bisulfite

Control 0%

2%

2%

2%

0%

0%

0.15%

0.2%

 Sodium bisulfite can lessen color change.

 But ! Too much sodium bisulfite will accelerate color change.

Minimize Color Change - Chelating Agent Metal Ions promotes oxidation. Chelating agents work to trap and inactivate these metal ions with the formation of chelating complexes in the solution

105 100

Transmittance

95

EDTMP was found to minimize oxidation remarkably in solutions containing Corum 9515 according to our experiments.

90 85

Blank, pH 4.78

80 75

Control, pH 4.78

70

0.1% EDTMP, pH 4.72

65

0.05%EDTMP, pH 4.72

60

0

30

Day

60

90

Clarity of Solution After 90 Days

CORUM 9515 Toxicological Information  Skin Irritancy Test by IDEA, France 2% CORUM 9515 on the external face of the arm maintained over 48 hours with the help of a semi-occlusive patch. CORUM 9515 is found to be non-irritant after 48 hours semi-occlusive patch test.

 Cytotoxicity Test by Evic, France Cytotoxicity test on CORUM 9515 diluted with 10% distilled water. CORUM 9515 diluted at 10 % with distilled water was judged negligible.

 AMES Test by Vivotecnia, Spain CORUM 9515 were found to be non mutagenic and non pro-mutagenic.

CORUM 9515 Summary  Effective and stable skin lightening agent  Balance the skin tone  Reduce dark spot  Prevent photoaging  Increase collagen synthesis

 Excellent anti-oxidation properties  Scavenge radical  DNA protection

Thank you for your attention!

CORUM Whitening agents

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