TranskinTM A novel skin penetration enhancing peptide

for the transdermal delivery of protein growth factors

Sanghwa Lee Skin Science Research Center LG Household & Healthcare

1. Introduction

2. Development of TranskinTM

3. Development of TranskinTM-Conjugated

Growth Factors

4. Safety, stability and efficacy

5. Summary

- Contents -

Company Introduction Founded in 1947, LG Household & Health Care, as the first company of LG Group, is the Korea’s leading company in Fast Moving Consumer Goods industry.

. Founded in 1947

. Annual sales of 4.3 billion US$ (2013)

. 6,500+ employees (with overseas subsidiaries)

Oral Care

Hair Care

Skin Care

Laundry Products

Paper Products

Cleaning & Hygiene

WHOO

OHUI

su:m

Soo Rye Han

ISA KNOX

LacVert

Cathy Cat

VONIN

CAREZONE

Beyond

The Face shop

VOV

Health & Nutrition Supplement

Vitamins, Health Drinks & Other Supplements

Coca-Cola

Fanta

Georgia Coffee

Sprite

Vitamin Water

Nestea

Powerade

Minute Maid

Soonsoo

Etc.

LG-Unicharm Sanitary Napkins & Diapers

LG-HAI (USA)

LG-Shanghai (China)

LG Beijing (China)

LG Hangjou (China)

LG-VINA (Vietnam)

LG-Taiwan (Taiwan)

Raw Materials : Stearic Acid, Glycerin, etc.

HAITAI Beverage

Bong Bong

SunKist

Sunny10

DK

Blackbean Tea

Home care

Personal Care Nutri-health Beverage Subsidiaries Cosmetics

LG H&H Structure

Company Introduction

Premium

(Prestige)

Mid to High-end

(Masstige)

Mass Affordable

VONIN

LacVert SOORYEHAN ISA KNOX

OHUI WHOO

LG H&H’s prestige brands are mainly sold through department stores and door-to-door sales

LG H&H operates retail outlets that sells its masstige brands. As of 3Q 2010, there were 1,065 outlets nationwide

In 2010, LG H&H acquired a leading mass affordable cosmetic company (THE FACE SHOP), strengthening the company’s offerings in the mass affordable segment

SU:M

CAREZONE

THEFACESHOP

BELIF

Specialized skin-care products made from high-quality Oriental herbal materials, such as ginseng

Functional cosmetics competing with foreign brands, targeted for younger generation

Offers mid to entry cosmetic products

838 domestic stores and 320 overseas stores as of 3Q 2010

Launched China operations in 2006. Currently have over 100 stores retail presences in 10 provinces

Specialized skin-care products marketed as “Cosmedical” products

Offers lab-based skin-care products

Targeted 20’s~30’s

Mid-range version of Whoo, with traditional flavor

Targeted mid 20’s~mid 30’s

Offers specialized skin-care products for men

Source: Company filings and website Core brands Non-core brands

LG H&H offers a Wide Range of Skin-Care and Cosmetics Products from Mass Affordable to Prestige

Beautiful

Launched in 2007 with the concept of fermentation

Rapidly gaining market traction (67% YtoY growth in 2010 3Q)

Launched in August 2010

Offers natural herb-based cosmetic products

Focused on moisturizing products

Targeted early 20’s ~ early 30’s

Cosmetics

BEYOND

Launched in 2005 Eco-Friendly Brand with wide range of products including skincare, hair, body and color cosmetics

Cosmetic Market leader, offering the best skin science technology

Company Introduction O HUI brand

The first regenerating line, applying stem cell technology in skin care products.

• Collaboration with CHA stem cell institute, which is one of the top stem cell

research centers in the world

• The First contains “Stem Cell Formula” as one of the active ingredients

• Stem cell formula is consisted of selected growth factors and cytokines

secreted by stem cells proved to be helpful to skin regeneration

Company Introduction O HUI The First

Introduction

• EGF: Epidermal Growth Factor (M.W. 6k Da)

• hGH: Human Growth Hormone (M.W. 22k Da)

• TGFβ: Transforming Growth Factor (M.W. 25k Da)

• FGF: Fibroblast Growth Factor (M.W. 27k Da)

• PDGF: Platelet Derived Growth Factor (M.W. 30k Da)

• Cell proliferation

• ECM(Extra Cellular Matrix) production

• Wound healing

Growth factors are used in cosmetic formulations

Commonly used Growth factors

• High molecular weight Protein

• Poor skin penetration, especially across the stratum corneum

• Could not achieve in vivo efficacy as expected by in vitro data

• Use of liposome

▷ Low entrapment efficiency

• Introduction of PTD1)

▷ Works on cell membrane, but not on stratum corneum

Introduction

Growth factors in Cosmetics - Limitations

Attempts to enhance skin penetration

An innovative method needed

1) Protein Transduction Domain

Bacteriophage M 13 Library1)

• M.W. of bacteriophage M13 : 1.6 x 107 Da

• Random peptide sequence fused to N-terminal of

gene III coat protein

• Selection of Bacteriophage penetrated real pig skin

• Peptide sequence facilitate the penetration of

bacteriophage could also enhance the

penetration of growth factors Random peptide sequence

Development of TranskinTM

Phage display : Principles and Assumptions

1) http://www.mikeblaber.org/oldwine/bch5425/lect33/lect33.htm

• 3 rounds of selection using Franz cell and Porcine skin

• DNA sequence analysis of selected bacteriophage

• Amino acid sequence determination form DNA sequence

• 10 candidate sequences, for example, ‘NGSLNTHLAPIL’

• Named “TranskinTM”

Epidermis

Dermis

Stratum corneum

0.7

mm

Phage display : Selection Procedures

Development of TranskinTM

Characterization of TranskinTM Peptides

G: Neutral B: Hydrophobic R: Hydrophilic Amino acids

Transkin Peptide

NGSLNTHLAPIL

PTD1) Sequences

GRKKRRQRRRPQ

RRRRRRRRR

1) Protein Transduction Domain

Vs.

Development of TranskinTM

NGSLNTHLAPIL

ACTGSTQHQCG

TranskinTM (S3-2)

Control peptide

GGGVGVAPGK-FITC Penetrated peptide

+ Rel

ativ

e Fl

uore

scen

ce

0

2

4

6

8

10

12

14

16

0 1X 5X 10X

Control S3-2

Enhancer Peptide

*

*

*

Skin Penetration of TranskinTM Peptide

Development of TranskinTM

Development of TranskinTM-Growth Factors

Mechanism of TranskinTM

Buffer Transkin

SC SC

Intact SC layer

Disrupting SC Layers?

☞ No Disrupting SC layers

• Disruption of Lipids or Proteins in the SC1)

▷ Integrity of SC structure

• Modulation of their organization

▷ Order of SC structure

Lipid

Aqueous Keratin

Organization of SC lipids Normal Disorders

• X-ray scattering • FTIR • DSC Analysis

Transcelluar Intracelluar

1. FTIR2) Spectrum

2940 2900 2860 2820

Abso

rban

ce

wavenumber

182% --- Transkin --- SPACE --- Buffer 178%

Modulation of SC organization?

2. DSC3) also shows a different pattern ☞ Increase of the Fluidity of SC

1) Stratum Corneum 2) Fourier Transform Infrared Spectroscopy 3) Differential Scanning Calorimeter Analysis

Development of TranskinTM-Growth Factors

Effect of TranskinTM Treatment on Skin Permeation

0 ㎛ 10 ㎛ 20 ㎛ 30 ㎛ XYZ

Cumulative XZ

FITC4) labelled Peptide (2.2 kDa)

Buff

er

Tran

skin

SC

ED

DM

SC

ED

DM

TranskinTM could enhance the partitioning of drugs into the SC layer

• Expression vector construction of TranskinTM peptide fused to N-terminal of EGF/hGH

• Expression of fusion protein in E. coli

• Purification of TranskinTM-EGF and TranskinTM-hGH

• Validation of Biological activity and Skin penetration

Scheme of production of TranskinTM-growth factors

Vector construction Host strain setup Purified growth factors

Development of TranskinTM-Growth Factors

Production of TranskinTM-EGF, TranskinTM-hGH

• TranskinTM-EGF, TranskinTM-hGH were validated by N-terminal sequencing

• Same biological activities as their mother forms

EGF TranskinTM-EGF

Development of TranskinTM-Growth Factors

Biological Equivalence of Transkin-Growth Factors

T-EGF1) 0

Act

ivity

(x10

6 IU

/mg)

EGF

2.5-

2.0-

1.5-

1.0-

0.5-

1) T-EGF: Transkin conjugated EGF

• Expression of Extracellular matrix related genes assessed by Real-time PCR

Development of TranskinTM-Growth Factors

Biological Equivalence of Transkin-Growth Factors

Hyaluronan synthase 3 Laminin Elastin

EGF 10ng/mL

0

2-

Rel

ativ

e Act

ivity

4-

Control

6-

EGF 1ng/mL

T-EGF 10ng/mL

T-EGF 1ng/mL

hGH 102ng/mL

0

2-

Rel

ativ

e Act

ivity

2-

Control

4-

hGH 10ng/mL

T-hGH 102ng/mL

T-hGH 10ng/mL

EGF 10ng/mL

0

2-

Rel

ativ

e Act

ivity

4-

Control

6-

EGF 1ng/mL

T-EGF 10ng/mL

T-EGF 1ng/mL

• 5-fold enhancement of skin penetration

Development of TranskinTM-Growth Factors

Enhanced Penetration of TranskinTM-Growth Factors

(A)

Am

ount

of

pro

tein

(ng/m

l)

EGF vs. TranskinTM-EGF

0

50

100

150

200

250

300

350

EGF S3-2 EGF

*

Am

ount

of

pro

tein

(ng/m

l)

0

100

200

300

400

500

600

hGH S3-2 hGH

*

(B) hGH vs. TranskinTM-hGH

• 100-fold increase in skin absorption

Development of TranskinTM-Growth Factors

Enhanced Penetration of TranskinTM-Growth Factors

200 ppm treatment

Concentration in skin

Absorption rate (%)

Relative absorption rate (fold)

hGH 0.055 0.028 1

TranskinTM-hGH 6.2 3.1 107

D.M

E.

D S.C

a b

100

㎛

100

㎛

Development of TranskinTM-Growth Factors

Safety of TranskinTM-Growth Factors

Sample IC50 Category

TranskinTM-hGH IC50≥ 1% Extremely Low

TranskinTM-EGF IC50≥ 1% Extremely Low

Cytotoxicity (MTT Assay)

Sample Relative Fluorescence Intensity Category

TranskinTM-hGH CD54 RFI ≤ 200, CD86 RFI ≤ 150 Not a sensitizer

TranskinTM-EGF CD54 RFI ≤ 200, CD86 RFI ≤ 150 Not a sensitizer

Allergy Test (h-CLAT Assay)

Sample Irritation

index Category

Control (Emulsion Type) 0.05 Not an irritant TranskinTM-EGF 100 ppm, Transkin-hGH 100 ppm 0.03 Not an irritant

Patch Test

Development of TransomeTM

Idea for Stabilization of TranskinTM-Growth Factors

• Stabilization Tech.

1. Liposome 2. Immobilization 3. Microemulsion

Microemulsion

Transkin-GF1) located on the surface

w/o o/w

Transkin-GF Lecithin

Water

Surfactant

Hydrophilic drug

Oil

Hydrogenated Lecithin

1) Transkin-GF: Transkin conjugated Growth Factor

Development of TransomeTM

Preparation of Microemulsion with TranskinTM-GF

Ternary System

S+CoS : Surfactant+ Co-surfactant

50% water 50% Mix A

C

B A

O/W μE2)

S+CoS

Water Oil

60% Oil 40% S-CoS

20% water 80% Mix A

20 nm

mean size 25.2 ㎚

Particle analyze

• Transparent • Very Stable • Low viscosity

1) TranskinTM-GF: Transkin conjugated Growth Factor 2) μE: Microemulsion

Development of TransomeTM

Enhanced the Stability and Permeation of TranskinTM-GF El

astin

Syn.

Rat

e (%

) 4-

2-

0-

Activity

Elas

tin

Syn.

Rat

e (%

) 80-

40-

0- 25°C 45 °C

Stability (8 weeks)

8ⅹ

T-EGF T-EGF-μE2)

Permeation

Rela

tive

Pe

netrat

ion

150-

100-

0-

127ⅹ

EGF T-EGF

T-EGF-μE

T-EGF T-EGF-μE

• 8-fold increase the stability of TranskinTM-Growth Factors

Efficacy of TranskinTM-GF containing cosmetic preparation

Wrinkle Improvement

Dermal Density

10-

5-

0- control sample

10-

5-

0-

1.7ⅹ

0W

4W

Impro

vem

ent (%

)

2.4ⅹ

0W

4W

control sample

Impro

vem

ent (%

)

0.25

0.27

0.29

0.31

0.33

0.35

0 w 4 w

Skin Elasticity

*

6

8

10

12

14

16

18

0 w 4 w

*

Ra

R7 * p<0.05

* p<0.05

• Peptide sequence ‘N-G-S-L-N-T-H-L-A-P-I-L’ facilitating skin penetration of protein was developed and named “TranskinTM” peptide using phage display method

• Transkin peptide was conjugated to EGF and hGH

- 5-fold increase in skin penetration, 100-fold increase in skin absorption

- maintain same biological activities

- show low cytotoxicity, no allergic potential or skin irritation

• To enhance the stability of TranskinTM- GF, they were incorporated into microemulsion system, TransomeTM

• Cosmetic preparation containing TranskinTM- GF improved dermal density, skin elasticity and facial wrinkles

• TranskinTM- GFs were introduced to OHUI “The First” as one of the active ingredients

Summary

Thank You !