Modulation of Aquaporins to Deliver Consumer Benefits: Applications for Skin Care
E. A. Jewell-Motz, Ph.D., J. R. Kaczvinsky, Ph.D., K. M . Lammers, M.S., S. Xie, Ph.D., R. M. Osborne, Ph.D
P&G Beauty , Cincinnati, Ohio USA
Caffeine Stimulates AQP3 Protein
Expression in vitro
Aquaporin channel
(Science 17 October 2003:
Vol. 302. no. 5644, pp. 383 –
384)
AQP3 is localized to
human skin
1.1
1
0.9
Age (years)
In vivo Expression of AQP 3
• 20 Caucasian female subjects, 18-55 years old
• RNA isolated from hip biopsies & 1 leg biopsy
• AQP RNA expression quantitated via
GeneChip™ assay
Objective
● Determine whether identified materials
promote a skin moisturizing benefit in humans
when formulated into product.
Caffeine Stimulates AQP3 Activity
in vitro
*
0.4
0.3
0.2
0.1
0
x
70
po
st
-t
60
2d
50
W
EE
K
40
0.5
B
30
ELISA for In Vitro AQP Expression
• Cultured human neonatal keratinocytes were cultured in
96 well plates and treated at 37°C for 48-96 hrs
• Fixed cells were incubated with anti-AQP3 antibody.
• A peroxidase coupled secondary antibody was added,
and AQP3 protein content was determined after
treatment with Horseradish Peroxidase Chromogen
TMB.
130
% Increase in
Glycerol Transport
0
20
*
el
in
e
2500
0.6
as
100uM
In vivo Dry Skin Grade Improvements
(vs. moisturizer alone)
Control
aquaporin-3 expression and activity in skin
cells.
Stratum corneum
1.2
0.8
● Identify compounds that stimulate
Stratum basale
1.3
3
5000
1.4
W
EE
K
7500
Product Formulated with Caffeine
Delivers Skin Moisturization Benefit
in Human Clinical Studies
1.5
2
r = -0.446
p = 0.063
1.6
W
EE
K
10000
AQP3 Signal
Aquaporins (AQP) are an important class of
proteins that regulate the transport of water
and other small solutes across plasma
membranes. First discovered in the early
1990’s (1), there are now 13 distinct AQP
family members that have been identified in
a variety of mammalian tissues, many of
which are relevant to the consumer products
industry. In particular, two aquaporins
(AQP3 and AQP9) are expressed in human
skin cells (2, 3). AQP3 has been implicated
as playing a key role in the transport and
distribution of epidermal water and glycerol
(3,4) and the regulation of keratinocyte
differentiation (5), both critical processes for
maintaining skin hydration, barrier function
and overall skin health.
Fold Change in AQP Protein
AQP3 levels decrease with age
Conclusions
1
Background
120
110
100
90
80
Control
100uM
Glycerol Transport Assay
● MatTek’s Epiderm cultures were treated with test
compound for 48 hours before adding glycerol to the
medium in the lower chamber.
● Glycerol levels within the upper chamber medium were
determined 24 hours later (Free Glycerol Reagent, Sigma,
St. Louis, MO).
● Measurement in the glycerol assay was converted into a
percentage increase over the control (where the control is
equal to 100%). Each mean and SEM were derived from at
least three separate experiments, performed in duplicate.
Clinical Design
●40 female subjects
●4 test sites per leg (8 total per subject), 5x4 cm2 area,
2 ul/cm2, total 40 ul treatment twice/day except
weekends- 1x per day
●Assessments performed at baseline, 1, 2, and 3
weeks then 2, 7, and 11 day regression assessments
●Technical measures via expert visual grading for
dryness and redness,
●Panelists were asked not to consume caffeine or
herbal drinks
• AQP3 is expressed in human skin
• An age-associated decrease in AQP3
correlates with an age-associated
increase in dry skin.
• Caffeine increases both the protein
expression and glycerol transport
activity of AQP3 as determined in
vitro.
• Moisturizer formulated with caffeine
delivers a moisturization benefit to dry
skin in clinical testing.
• Modulation of aquaporins may be
beneficial for a variety of consumer
products applications including, but
not limited to, anti-aging, dry mouth
relief, urinary incontinence, or relief for
diarrhea.
References
1. Preston G. M., Carroll T.P., Guggino W. P. and
Agre P., Science 256, 385-387 (1992).
2. Sougrat R., Morand M., Gondran C., Barre P.,
Gobin R., Bonte F., Dumas M., and Verbavatz
J., J. Invest. Derm., 118(4), 678-685 (2002).
3. Sugiyama Y., Ota Y., Hara M., and Inoue S.,
Biochim. Biophys. Acta 1522, 82-88 (2001).
4. Hara-Chikuma M. and Verkman A. S., Biol Cell,
97(7), 479-486 (2005).
5. Zheng X. and Bollinger Bollag W., J. Invest.
Derm., 121(6), 1487-1495 (2003).