the role of adrenergic beta receptors for skin pigmentation

THE ROLE OF ADRENERGIC BETA
RECEPTORS FOR SKIN
PIGMENTATION
FC17-10
Tomohiro Hakozaki, Tim Laughlin, Steven Zhao, Heather Matheny, Deb
Finlay, Jiazhen Wang, Sancai Xie
The Procter & Gamble Company, Ohio, USA

Introduction:
Changes in facial attributes by age
10 yrs
30 yrs 50 yrs
10 yrs
As we age facial skin attributes change
Hyperpigmented spots increase with age

Senile Lentigo – Typical Hyperpigmented Spot
Increasing by Age
Skin appearance
Normal skin
Senile Lentigo
Number of melanocytes
Rete Ridge on Senile Lentigo

Objectives
• Compare the gene expression patterns between age
spots (solar lentigines) vs. adjacent skin using gene chip
microarrays
• Investigate the role of adrenergic receptors for skin
pigmentation
• Screen ingredients for their ability to down-regulate the
adrenergic receptor beta pathway

Senile Lentigo Genomics Study Design
Normal skin
Senile Lentigo
• 9 females (39-76 yrs) who
has solar lentigo on
back/arm
• Take biopsy from senile
lentigo and adjacent
normal skin
• Genomics analysis with
Affymetrix Genechips
Affymetrix Genechips
Biopsy
Biopsy
RNA
RNA

46,000 gene expression data

Adrenergic Receptors
Up-regulated Genes in Solar Lentigo Skin
Acr Title Fold change spot vs. normal P-value
ADRB1 Adrenergic Receptor beta-1 14.5 0.044
• Adrenaline – related to stress deeply, a
hormone, increases heart rate, contracts
blood vessels, etc.
• Adrenergic receptor - 2 main groups, α
and β, with several subtypes
• Adrenergic Receptor beta-2 antagonist
accelerates skin barrier recovery and
reduces epidermal hyperplasia induced
by barrier disruption (JID 2003)
• Adrenergic Receptor beta-2 UVB
irradiation increases adrenaline release
by keratinocytes, which can strengthen
melanin synthesis (JID 2008)
Reference; wikipedia

Beta-adrenergic receptor agonists stimulated
melanin synthesis in B16 melanoma cells
• Beta adrenergic agonists (dobutamine, isoprenaline) stimulated
melanin synthesis in melanoma B16 cells, when cultured to
confluency and synthesizing sufficient melanin to detect with a
simple spectrophotometric assay.
Melanin Synthesis Activity with Beta Adrenergic Agonists in B16
Melanoma Cells (%control)
Melanin amount [% vs. control]
300
250
200
150
100
50
dobutamine (beta-1 agonist)
Isoprenaline (beta-1&2 agonist)
0
0.000001 0.00001 0.0001 0.001 0.01
w/v %

Dobutamine (ADR beta-1 agonist) up-regulated
pigmentation related gene transcription
in human ex vivo tissue
Dobutamine stimulated pigmentation related gene expression
Fold change vs. control
14
12
10
8
6
4
2
*
*
*
*
Control
Dobutamine 0.001%
Dobutamine 0.01%
*
*
0
* sig vs. control (p

Investigating the function of Adrenergic Receptor (ADR)
– applying siRNA technique to B16 melanoma cells
• Knocked down 9 ADR genes with siRNA and measure melanin synthesis
in B16 melanoma cell
Melanocyte
melanosome
Ø
Melanin Production
Genome
Nucleus
ADR
mRNA
Small interfering (si) RNA
for adrenergic receptor
genes interfere ADR gene
expression

ADR series knockout leads to reducing melanin
production in B16 melanoma cells
Effect of Specific Adrenergic Receptor Knockdown by siRNA in B16 Melanin Production
Reduce Melanin Synthesis
% Knockdown of melanin production vs. control .
100
80
60
40
20
0
-20
-40
-60
78
26 28 30
ADRA1A
ADRA1B
ADRA1D
ADRA2A
25
ADRA2B
Over-expressed
in solar
lentigo
69
63
53
ADRA2C
ADRB1
ADRB2
ADRB3
-47
90
Tyrosinase

Screening of Adrenergic Receptor Beta Antagonist
Using a Recombinant GPCR Cell Line
• Beta-arrestin platform utilizing a recombinant GPCR cell line (CHO cells)
• Takes advantage of the property that activated GPCRs recruit beta-arrestin a
shutdown mechanism. The GPCR is expressed as a fusion protein with a small
piece of beta-galactosidase on the cytoplasmic side.
• Beta-arrestin is also expressed as a fusion protein with the remainder of the beta
galactosidase enzyme. Upon recruitment of beta-arrestin, the beta-galactosidase
pieces are united to form an active enzyme, which is detectable with a substrate
mixture that produces a luminescent signal with beta-galactosidase action.

Screening of ADRB Antagonist
Undecylenoyl Phenylalanine works as an antagonist of ADRB1 & 2
N-undecyl-10-L-phenylalanine
Effect of Undecylenoyl Phenylalanine
on Adrenergic Receptor B1
Effect of Undecylenoyl Phenylalanine
on Adrenergic Receptor B2
100
90
120
% Inhibition vs. control
80
70
60
50
40
30
20
10
0
1.44E-05 0.00036 0.009 0.045
Undecylenoyl Phenylalanine concentration [%]
% inhibition vs. control
100
80
60
40
20
0
0.0003 0.0009 0.0028 0.0084
Undecylenoyl Phenylalanine concentration [%]

Topical application of 1% Undecylenoyl Phenylalanine
containing moisturizer reduced melanin amount in
ex vivo human tissue model
1% Undecylenoyl Phenylalanine-containing Moisturizer
Reduces Melanin Amount in Human Skin Explant Model
Melanin amount measured by image analysis of
stained cross-sectional images (day 6)
800
700
600
500
400
300
200
100
0
1% UP in Vehicle Vehicle moisturizer
• Tissue from abdominal, Skin type III, Tissue was cultured with treatment for 6 days, N=6 samples for each

ADRB Signaling Pathway in Melanocyte
• Adrenergic receptors beta-1 and -2 intersect a known pigmentation pathway at cAMP production caused
by activated Gs Protein. The cascade proceeds downstream to MITF upregulation which turns on
pigmentation related gene expressions including tyrosinase.
Keratinocyte
Adrenaline
Cytokines
Keratinocyte
alpha-MSH
Endothelin-1
SCF
Melanosome
transfer
Undecyleno
yl
Phenylalan
ine
X
ADRB1/2
MC1R
cAMP
ETR
PKC
PKA
c-kit
MAP kinase
Melanin synthesis↑
Melanocyte

Evaluation of Undecylenoyl Phenylalanine Effect on Facial
Hyper-pigmented Spots in Human Clinical Study
Product
Design
Subject
Measure
• Vehicle moisturizer
• 1% Undecylenoyl phenylalanine containing formula in Vehicle
• 5% Niacinamide containing formula in Vehicle (control)
• Split face, round robin design
• Double blinded, left-right randomized, vehicle controlled
• 8 weeks product treatment period (0, 4, 8 week visits)
• Females having moderate hyperpigmented spots symmetrically on
both sides of the face
• 25-60 years
• 110 observations per treatment
• Capture facial images followed by computer image analysis for spot
area and skin color

Computer image analysis to detect hyperpigmented spots
Captured facial image and masking area
(shown in green)
Detected hyperpigmented spot area
(shown in white)

Topical application of 1% Undecylenoyl Phenylalanine containing
moisturizer reduced facial hyperpigmented spot appearance among
Chinese females vs. its vehicle moisturizer
• Both undecylenoyl phenylalanine (UP)-containing moisturizer and Niacinamidecontaining
moisturizer significantly reduced appearance of facial spot compared to
Vehicle (p

Conclusions
• A series of in vitro and ex vivo studies demonstrated that
Adrenergic receptors beta 1 and 2 play a key role in melanogenesis
and potentially for age spot formation
• Undecylenoyl phenylalanine works effectively to suppress
melanogenesis via down-regulating the adrenergic receptor beta
pathway in vitro
• An Undecylenoyl phenylalanine moisturizer reduced the
appearance of facial hyperpigmented spots in a clinical study
Acknowledgements
• University of Cincinnati for solar lentigo samples
• This work was funded by P&G Beauty & Grooming

This work was funded by P&G Beauty & Grooming