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 !