NecroXNecroXMitochondria Protectant

<INCI name>Cyclopentylamino Carboxymethylthiazolylindole

LG Life Sciences2010, March

Non Confidential

Introduction

Mitochondria, energy factory

Mitochondria is the nature`s power generation factory where all the energies used in ourfactory where all the energies used in our daily life is created

Skin aging comes from the mitochondrial damage within skin cells

95% of oxygen that we take everyday is used in mitochondria to generate energyin mitochondria to generate energy

ROS, well known as harmful cell damaging g gfactor is mostly generated in mitochondria as a remain of oxygen consumption by mitochondria

Mitochondria protection is a key to prevent skin cell damage

Problem of traditional antioxidants

Problem of traditional antioxidants (Vit. C as example)

Benefit of NecroX

Radical+

Stable body+

Radical+

Stable body+Vitamin C +

UnstableoxidativeVitamin C

NecroX +Stable

oxidativeReductoneVitamin C

Toxicity(+)

Reductone

Toxicity( )Toxicity(+) Toxicity(-)

Functional anti-oxidant must be stable after reacting with radicals

Reacted product of NecroX is stable and safe

Problem of traditional antioxidants

Anti OxidantPotency

Water Solubility

SolutionStability

MitochondriaTargeting

Vitamin E`s

Vitamin C`s

Natural Extracts

NecroX

Technical Background

NoteNoteNecroX refers to INCI registered compoundNecroX derivative refers to compounds that have similar properties due to sharing its core chemical structureits core chemical structure

1. Mechanism of action

Dual mechanism of action

NecroX

Mitochondrial ROS scavenging effect Cell damage preventing effect

- Localizes in mitochondria

- Controlling ROS in skin cell mitochondria

- Shows dose dependant cell protection effect

on cells against various stresses

P i ll d h- Shows strong anti-oxidant effect - Preventing cell death process

NecroX derivative (μM)

2. Mitochondrial Antioxidant Effect

70

80

50

60

70

fect

(%

)

A

20

30

40

Inhi

bitio

n Ef

NecroX, mitochondria targeting effect0

10

20

0 1% 0 03% 0 003% 0 0003%

B C

A: We attached green fluorescence dye to NecroX derivative. It shows clear localization around nucleus.

B: Mitotracker is well known dye for mitochondria observation. Red area is where mitochondria exists

0.1%Vit. C

0.03%NecroX

0.003%NecroX

0.0003%NecroX

Strong antioxidant effect h l i

area is where mitochondria exists

C: We merged images A and B. They show apparent orange color, which came from the mixture of green and red color.

NecroX shows almost 20 times stronger anti oxidant effect compared to vitamin C in DPPH assay

NecroX is the most potent known mitochondria targeting anti oxidant

2. Mitochondrial Antioxidant Effect DHR123Imaging in H9C2

Positive Control(without tBHP )

No ROS

Negative Control (with tBHP)

High level of ROSNo ROS High level of ROS

tBHP + NecroX 10μM tBHP + NecroX 2.5μM tBHP + NecroX 0.6μM

What is tBHP?tert-Butyl hydroperoxide is an organic peroxide widely used in a variety of oxidation processes.

NecroX shows dose dependent ROS scavenging effect

3. Cell Damage Preventing Effect tBHP inducedstress in H9C2

Positive Control (no tBHP ) tBHP + NecroX 10μM

N ti C t l (tBHP)

Healthy Cells

Negative Control (tBHP)

Damaged Cells

NecroX shows protection effect against tBHP induced stress

3. Cell Damage Preventing Effect Retinol inducedStress in CCD986-sk

100

120

80

bili

ty (%

)

40

60Via

b

0

20

Retinol 50 IU Retinol 50 IU +Control Retinol 50 IU Retinol 50 IU + NecroX 100μM

Control

What is Retinol?Retinol is well known cosmetic ingredient mostly used in anti wrinkle product. Retinol helps skin cells create better, healthier skin cells and increase the amount of skin-support substances. The only problem with retinol is its unstable form, which shows toxicity on skin cells.

NecroX shows protection effect against retinoic acid induced stress

3. Cell Damage Preventing Effect Cold & WarmStress in H9C2

12030 μM

80

100

viva

l30 μM

10 μM

40

60

% s

urv 3 μM

1 μM

20

40

0

NecroX PositiveControl

NegativeControl

Cold & Warm stressNecroX was applied to H9C2 cells, and after treatment cells were exposed to 4˚C for 24 hours, and back into 37 ˚C for 24 hours again. We observed viability rate of cells.

NecroX shows protection effect against cold & warm temperature stress

3. Cell Damage Preventing Effect High fat stress (Stearic acid)

NecroX Derivative 1 NecroX Derivative 2

What is stearic acid?It is saturated fatty acid, high level of fatty acid leads to fibrosis of body organs which is typical type of cell damagetypical type of cell damage.

Where is stearic acid found?Found in many animal and vegetable food sources. Mainly in animal oils.

NecroX dose dependently protects cells from stearic acid insult.

Note: Experiment was done with NecroX derivative compounds from drug candidates, not from cosmetic ingredient candidate. NecroX derivatives shows similar properties due to sharing its core chemical structure

Safety

1. Skin Cell Toxicity

Cell90% ViabilityConcentrationConcentration

HaCaT 62.75 μM

Human keratinocyte 125.5 μM

B16F10 125.5 μM

CCD986-sk All concentration safe

TIB-196 31.375 μM

RBL-2H3(rat basophile leukemia)

62.5 μM( p

2. Phototoxiciy

Test system and method

Balb/c 3T3, Neutral Red assayTest article treatments for 60 min and irradiate at 365nm for 50 min (5J/cm2)

i iNo phototoxicity: PIF < 2 or MPE < 0.1

b bl h i iCriteria Probable phototoxicity: 2 < PIF < 5 or 0.1 < MPE < 0.15 Phototoxicity: PIF > 5 or MPE > 0.15

Test article NecroX

Test concentration (ug/ml)

-Irr, +Irr : 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500

Test article IC50 (-Irr) IC50(+Irr)

NecroX 70.7 71.3

PIF Mean MPE Mean Classif ication

0.9 0.01 negative

: +Irr response: -Irr responsemg/l = ug/ml

Summary

Lifestyle Stress, damaging cells

F d

Cell damaging factors from our daily life

Lifestyle stresses damage cells, make skin aging.Our innovative solution: NecroX reduces stress to

Food

Junk food

High fat Our innovative solution: NecroX reduces stress to

cells and protects against long term damaging

effect by direct mitochondrial anti oxidant effect Environmental

Tobacco smoke

and cell damage preventing effect.

NecroX

Car exhaust

Acid rain

Sun burn (UV)

Emotional

NecroX- Directly targeting mitochondria within cells

- ROS controlling effect (anti-oxidant)

Noise

Hard work

Fatigue

Excessive lifestyle

- Cell damage preventing effect

- Shields the cells from environmental influencesy

Thank You

Contact InformationEmail: NecroX.lgls@gmail.com