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Abstract There is a concern that peptides in cosmetic creams marketed as anti-aging/anti-wrinkle may penetrate into the deep layers of the skin and potentially stimulate biological activity. Claims for one cosmetic peptide, acetyl hexapeptide-8 (Ac-EEMQRR-amide), suggest interference with neuromuscular signaling as its anti-wrinkle mechanism of action. Therefore, the skin penetration of commercially available Ac-EEMQRR-amide from a cosmetic formulation (oil-in-water (O/W) emulsion) was determined in hairless guinea pig (HGP) and human cadaver skin assembled into in vitro diffusion cells. An O/W emulsion containing 10% Ac-EEMQRR-amide was applied to skin at a dose of 2 mg/cm(2). After a 24-h exposure, the skin surface was washed to remove unabsorbed peptide. Skin disks were tape stripped to determine the amount of peptide in the stratum corneum. Removal of the stratum corneum layers was verified by confocal microscopy. The epidermis was heat separated from the dermis and each skin fraction was homogenized. Skin penetration of Ac-EEMQRR-amide was measured in skin layers by hydrophilic interaction liquid chromatography with tandem mass spectrometry using electrospray ionization (ESI) in the positive mode. Stable isotopically labeled hexapeptides were used as internal standards for the quantitation of native hexapeptides to correct for matrix effects associated with ESI. The results (percent of applied dose) showed that the majority of the Ac-EEMQRR-amide was washed from the surface of both HGP and human skin. Ac-EEMQRR-amide that penetrated skin remained mostly in the stratum corneum of HGP (0.54%) and human (0.22%) with the peptide levels decreasing as each layer was removed by tape stripping. Total Ac-EEMQRR-amide found in the epidermis of HGP and human skin was similar at 0.01%. No peptide was detected in the dermis or buffer collected underneath the skin for both human and HGP. There was no hexapeptide metabolite (H2N-EEMQRR-amide) detected in any layers of HGP skin, human skin or buffer collected underneath the skin. This skin penetration data will be useful for evaluating the safety of cosmetic products containing small peptide cosmetic ingredients.
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Cutaneous and Ocular Toxicology
ISSN: 1556-9527 (Print) 1556-9535 (Online) Journal homepage: http://www.tandfonline.com/loi/icot20
In vitro skin penetration of acetyl hexapeptide-8
from a cosmetic formulation
Margaret E. K. Kraeling, Wanlong Zhou, Perry Wang & Oluwatosin A.
Ogunsola
To cite this article: Margaret E. K. Kraeling, Wanlong Zhou, Perry Wang & Oluwatosin A.
Ogunsola (2015) In vitro skin penetration of acetyl hexapeptide-8 from a cosmetic formulation,
Cutaneous and Ocular Toxicology, 34:1, 46-52, DOI: 10.3109/15569527.2014.894521
To link to this article: http://dx.doi.org/10.3109/15569527.2014.894521
Published online: 22 Apr 2014.
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ISSN: 1556-9527 (print), 1556-9535 (electronic)
Cutan Ocul Toxicol, 2015; 34(1): 46–52
!2014 Informa Healthcare USA, Inc. DOI: 10.3109/15569527.2014.894521
RESEARCH ARTICLE
In vitro skin penetration of acetyl hexapeptide-8 from a cosmetic
formulation
Margaret E. K. Kraeling
1
, Wanlong Zhou
2
, Perry Wang
2
, and Oluwatosin A. Ogunsola
1
1
US Food and Drug Administration, CFSAN/Office of Applied Research and Safety Assessment, Division of Toxicology, Laurel, MD, USA and
2
US Food and Drug Administration, CFSAN/Office of Regulatory Science, College Park, MD, USA
Abstract
There is a concern that peptides in cosmetic creams marketed as anti-aging/anti-wrinkle
may penetrate into the deep layers of the skin and potentially stimulate biological activity.
Claims for one cosmetic peptide, acetyl hexapeptide-8 (Ac-EEMQRR-amide), suggest interfer-
ence with neuromuscular signaling as its anti-wrinkle mechanism of action. Therefore, the
skin penetration of commercially available Ac-EEMQRR-amide from a cosmetic formulation
(oil-in-water (O/W) emulsion) was determined in hairless guinea pig (HGP) and human cadaver
skin assembled into in vitro diffusion cells. An O/W emulsion containing 10% Ac-EEMQRR-amide
was applied to skin at a dose of 2 mg/cm
2
. After a 24-h exposure, the skin surface was washed
to remove unabsorbed peptide. Skin disks were tape stripped to determine the amount of
peptide in the stratum corneum. Removal of the stratum corneum layers was verified
by confocal microscopy. The epidermis was heat separated from the dermis and each skin
fraction was homogenized. Skin penetration of Ac-EEMQRR-amide was measured in skin layers
by hydrophilic interaction liquid chromatography with tandem mass spectrometry using
electrospray ionization (ESI) in the positive mode. Stable isotopically labeled hexapeptides
were used as internal standards for the quantitation of native hexapeptides to correct for
matrix effects associated with ESI. The results (percent of applied dose) showed that the
majority of the Ac-EEMQRR-amide was washed from the surface of both HGP and human skin.
Ac-EEMQRR-amide that penetrated skin remained mostly in the stratum corneum of HGP
(0.54%) and human (0.22%) with the peptide levels decreasing as each layer was removed by
tape stripping. Total Ac-EEMQRR-amide found in the epidermis of HGP and human skin was
similar at 0.01%. No peptide was detected in the dermis or buffer collected underneath the skin
for both human and HGP. There was no hexapeptide metabolite (H
2
N-EEMQRR-amide)
detected in any layers of HGP skin, human skin or buffer collected underneath the skin. This
skin penetration data will be useful for evaluating the safety of cosmetic products containing
small peptide cosmetic ingredients.
Keywords
Acetyl hexapeptide-8, peptide, skin
penetration
History
Received 10 January 2014
Accepted 11 February 2014
Published online 22 April 2014
Introduction
The cosmetic industry has been marketing anti-wrinkle
and anti-aging skin care cosmetics containing various small
peptides. The anti-aging peptides have been referred to as
repairing peptides, relaxing peptides and firming peptides
1
or
signal peptides, enzyme inhibitor peptides, neurotransmitter
inhibitor peptides and carrier peptides
2
. In general, these
peptides are claimed to improve the appearance of fine lines
and wrinkles and improve the tone and texture of skin. These
peptides are also claimed to ‘‘act’’ on different functions in
skin. Acetyl hexapeptide-8 (Ac-EEMQRR-amide), one of the
most commonly used synthetic anti-aging peptides, has a
chain of six amino acids acetylated at the N-terminal residue
(N-acetyl-L-alpha-glutamyl-L-alpha-glutamyl-L-methionyl-L-
glutaminyl-L-arginyl-L-argininamide) and a molecular
weight of 875 Da. Formerly known as acetyl hexapeptide-3,
acetyl hexapeptide-8 is marketed by the trade name of
ArgirelineÕ
2,3
.
Acetyl hexapeptide-8 is claimed to be a neurotransmitter
inhibitor peptide and is used in anti-wrinkle formulations
applied to the area around the eyes. Acetyl hexapeptide-8 is a
synthetic peptide patterned from the N-terminal end of the
synaptosomal-associated protein of the 25 kDa (SNAP25)
receptor. Acetyl hexapeptide-8 competitively inhibits the
SNAP25 component of the N-ethyl-maleimide-sensitive
factor attachment protein receptor (SNARE) complex
2–4
.
Other synthetic peptides that mimic the protein SNAP25
have been shown to be specific inhibitors of neurosecretion
5,6
and Ac-EEMQRR-amide appears to inhibit Ca
2+
-dependent
exocytosis mimicking the activity of botulinum neurotoxin
(BoNT) type A
4,7
. When the SNARE complex is destabilized,
the release of neurotransmitters is disrupted, calming the
Address for correspondence: Margaret E. K. Kraeling, Division of
Toxicology, US Food and Drug Administration, CFSAN/Office of
Applied Research and Safety Assessment, 8301 Muirkirk Road, Laurel,
MD 20708, USA. E-mail: margaret.kraeling@fda.hhs.gov
Downloaded by [FDA Library] at 12:55 11 February 2016
muscle contraction and reducing the formation of lines and
wrinkles. BoNT type A, which is also a treatment for reducing
the appearance of specific facial lines and wrinkles
8
,
irreversibly destroys the SNAP25 protein in the SNARE
complex and the effects last longer
9
. Marketed as an
alternative or concomitant treatment to injections of BoNT
type A, Ac-EEMQRR-amide can be applied topically with
potential lower toxicity
2,4
.
Determination of the extent of penetration of peptides from
topical applications is challenging. Peptides have many amide
bonds and high molecular weights (above 500 Da) which limit
their ability to permeate skin. They are often charged at
physiological pH and are inherently hydrophilic
2,10
. Also, the
stratum corneum of the skin is lipophilic and serves as a
barrier to hydrophilic compounds. Small lipophilic peptides
will diffuse more readily into the stratum corneum, epidermal
and dermal layers
11
. Penetration enhancement techniques
have been developed to facilitate the penetration of peptides
through the skin
10
. These are mostly applications for
enhancing drug delivery and treatment of disease. For
cosmetic applications or uses, the peptides should not be
delivered into the deep layers of the skin.
A major concern associated with adding anti-wrinkle
peptides into cosmetics is whether peptides penetrate into
the deep layers of human skin and potentially stimulate
biological activity. In a penetration study carried out on
human epidermal membranes placed in static cells, it was
reported that 30% of the applied dose of an aqueous solution
of Ac-EEMQRR-amide appeared in the receptor chamber
after 2 h
4
. Although the starting concentration of Ac-
EEMQRR-amide was not stated, the peptide appeared to
have permeated the human epidermal membranes. The
activity of small peptides patterned after SNAP25 has been
mostly tested in in vitro cell cultures including chromaffin
cells
4–7
. The activity and efficacy of Ac-EEMQRR-amide has
been tested in a few clinical studies. In the most documented
study, a 10% O/W emulsion of Ac-EEMQRR-amide versus
placebo was applied twice daily to 10 healthy volunteers
4
.
The Ac-EEMQRR-amide treated group showed nearly a 30%
versus 10% for control improvement in periorbital rhytids
after 30 d as measured by silicone replica analysis
4
. In another
clinical study in 14 volunteers, a cream containing 5%
ArgirelineÕsolution (0.05%) was applied twice daily around
the eyes for 28 d. ArgirelineÕshowed a mean wrinkle
reduction of 16.26%
2
.
If substantial quantities of these peptides do penetrate
into the deep layers of the skin, then these anti-aging claims
might be considered as drug claims and not cosmetic claims.
If the peptides do not penetrate the skin, the cosmetic industry
may need to consider modifying their marketing claims.
In order to assess the exposure of consumers to potentially
bioactive cosmetic peptides, we examined the skin penetra-
tion of commercially available Ac-EEMQRR-amide from a
cosmetic O/W emulsion formulation in viable hairless guinea
pig (HGP) and excised human cadaver skin assembled into
in vitro diffusion cells. Ac-EEMQRR-amide was mixed
into the emulsion at a concentration of 10%. After a 24-h
exposure, the surface of the skin was washed to remove
unabsorbed peptide and skin was separated into various layers
for analysis. In support of the skin penetration study, a rapid
liquid chromatography with tandem mass spectrometry
(LC–MS/MS) method was developed for the determination
of Ac-EEMQRR-amide in skin matrices, cream emulsion
and washing fluids containing surfactants
12,13
. This analytical
method uses hydrophilic interaction liquid chromatography–
solid phase extraction (HILIC-SPE) for sample preparation
and cleanup. The peptides were then chromatographically
separated using an analytical HILIC column followed by
MS/MS determination in the electrospray ionization (ESI)
positive mode. Stable isotopically labeled hexapeptides
were used as internal standards for the quantitation of
native hexapeptides to correct for matrix effects associated
with ESI. This method can also simultaneously quantitate a
corresponding hexapeptide metabolite, H
2
N-EEMQRR-
amide, that may be formed during the skin penetration
process
14
. The quantification of peptide amounts in these
skin penetration studies will be useful for evaluating the
safety of cosmetic products containing short-chain peptides.
Materials and methods
Materials
Ac-EEMQRR-amide, Ac-EEMQRR^-amide, H
2
N-EEMQRR-
amide and H
2
N-EEMQRR^-amide were custom-synthesized
by New England Peptide LLC (Gardner, MA). In all cases,
R^ stands for
15
N
4
and
13
C
6
, which are universally labeled
components. LC–MS grade acetonitrile (ACN), methanol
(MeOH) and water were obtained from Fisher Scientific
(Fair Lawn, NJ). Formic acid (FA, 97%, ACS) was obtained
from Acros Organic (Morris Plains, NJ). Trifluoroacetic acid
(TFA, reagent Plus, 99%) was obtained from Sigma-Aldrich
(St. Louis, MO). Ammonium hydroxide (NH
4
OH) was
obtained from Mallinckrodt Baker (Phillipsburg, NJ).
Nitrogen gas was provided by an LC/MS Gas Generator
Source SF 5000 (Parker Balston, Haverhill, MA).
Composition of the O/W emulsion (g/100 g emulsion)
The O/W emulsion vehicle used in the skin absorption studies
is comprised of the following ingredients and concentrations:
polyglyceryl-3 distearate (BASF Corp., Parsippany, NJ) 3%,
mineral oil light (Penreco, Karns City, PA) 10%, cetearyl
alcohol (Henkel Corp., Hoboken, NJ) 3%, propylene glycol
(Aldrich Chemical Co., Milwaukee, WI) 5%, potassium
phosphate–sodium hydroxide buffer (pH 7) 78%, methyl
paraben (Pfaltz & Bauer, Inc., Stamford, CT) 0.5% and propyl
paraben (Pfaltz & Bauer, Inc.) 0.5%
15
.
Composition of the receptor fluid HHBSS
The receptor fluid to be used in this study for perfusion
through the diffusion cell system is HEPES-buffered Hank’s
balanced salt solution (HHBSS). The receptor fluid was used
at a pH of 7.4 and was sterilized by vacuum filtration through
a 0.2 mm filter. The composition of the receptor fluid was as
follows: CaCl
2
(anhydrous): 140 mg/mL, dextrose: 1000 mg/
mL, HEPES: 5960 mg/L, MgS0
4
7H
2
O: 200 mg/mL, KCl:
400 mg/mL, KH
2
PO
4
(monobasic): 60 mg/mL, NaHCO
3
:
350 mg/mL, NaCl: 7000 mg/mL, Na
2
HPO
4
(dibasic): 50 mg/
mL and gentamicin sulfate: 50 mg/mL
16
.
DOI: 10.3109/15569527.2014.894521 Skin penetration of a cosmetic peptide 47
Downloaded by [FDA Library] at 12:55 11 February 2016
Tritiated water for skin barrier test
Tritiated water (specific activity 55.5 mCi/mmol; 97% purity)
was obtained from New England Nuclear (Boston, MA) and
applied to skin in diffusion cells at concentrations of
0.17–0.26 mCuries in a volume of 200 mL. Radioactivity was
measured on a Beckman LS 6500 liquid scintillation counter
(Beckman Instruments, Inc., Fullerton, CA).
Percutaneous absorption experiments
In vitro percutaneous absorption studies were conducted based
on methods described in detail for HGP and human skin in
flow-through diffusion cells
15–19
. Peptide skin absorption
studies were conducted using HGPs, obtained from Charles
River Labs (Wilmington, MA) and human cadaver abdominal
skin obtained from the National Disease Research Interchange
(NDRI) (Philadelphia, PA). Full-thickness frozen skin from
NDRI was stored at 80±2C until use. The skin was thawed
to room temperature, subcutaneous fat was removed and the
skin surface was gently washed with a 1% (v/v) liquid detergent
(PalmoliveÕ‘‘Original’’ formula) solution. The skin was cut
with a Padgett dermatome (Integra Life Sciences, Plainsboro,
NJ) to a thickness of approximately 250–400 mm
17,18
. Skin
disks were prepared with a punch and placed epidermal-side up
in Teflon flow-through diffusion cells. The skin in the
diffusion cells was perfused with HHBSS, pH 7.4, receptor
fluid at a f low rate of approximately 1.5 mL/h for the duration
of the study with the skin surface temperature of the stratum
corneum maintained at 32 ± 2 C. The surface area of skin in
the diffusion cell was 0.64 cm
2
and the receptor well volume
was 260 mL.
Prior to application of the test article, the integrity of
the barrier of human skin in diffusion cells was verified by a
20-min exposure to [
3
H]-water and subsequent determination
of the percent absorption of the radiolabel
19
. Three to four
diffusion cells were tested to verify the barrier integrity of the
human skin. If the percent of the applied dose of [
3
H]-water
absorbed through skin in the majority of the diffusion cells
was greater than the historical limit of 0.35%, the skin was
not used
19
.
Peptide skin absorption was determined from an O/W
emulsion. Ac-EEMQRR-amide was formulated in an O/W
emulsion at a concentration of 10% and applied to disks
of viable HGP skin and human cadaver skin mounted in
diffusion cells. The emulsion was applied to the skin at a dose
rate of approximately 2 mg/cm
2
. A minimum of four diffusion
cells per experiment was run for the peptide in emulsion
formulation and two cells for the blank (control) emulsion
formulation. The peptide in emulsion formulation was tested
on skin from four HGP and three human cadaver donors
(in replicates of four diffusion cells). The blank (control)
emulsion formulation was also tested on skin from four HGP
and three human donors. Receptor fluid fractions were
collected at 6-h intervals for a total of 24 h after dosing
using a fraction collector. After 24 h, the surface of the skin
was washed three times with 0.1 mL 1% soap solution and
rinsed two times with 0.2 mL distilled water. The skin was
gently rubbed with cotton tipped applicators to remove
unabsorbed material from the surface of the skin. The skin
was then removed from the diffusion cell and the stratum
corneum was removed in layers by successive tape stripping
using Scotch Magic
TM
tape. The viable epidermal layer of
skin was then separated from the dermal layer by heat
separation. The skin was wrapped in Saran
TM
Wrap plastic
wrap and submerged in a 60 ± 1 C water bath for 90 s. The
skin was unwrapped and the epidermis was slowly peeled
from the dermis
15
. Each epidermis and dermis was homo-
genized on ice using a Polytron tissue homogenizer
(Kinematica Inc., Bohemia, NY) at full speed for three 10-s
bursts. Following completion of the skin penetration studies,
all samples were collected and analyzed for peptide content
by HILIC–MS/MS.
Hydrophilic interaction liquid chromatography–
tandem mass spectroscopy
The LC/MS–MS system and preparation of standard solutions
has been described previously
12,13
. For the dosing emulsion,
the frozen sample was thawed and equilibrated at room
temperature. A 1.00–2.00 mg test portion was weighed into a
20-mL disposable scintillation vial using a Sartorius SC-2
microbalance (Goettingen, Germany). Ten milliliters of
extraction solution (0.1:0.1:85:15 TFA:FA:ACN:H
2
O) was
added to the vial. The vial was vortexed for 3 min. After
sonicating for 10 min, the vial was vortexed for another 3min
followed by filtration of the supernatant through a 0.2 mm
PTFE filter (Pall Life Sciences, Port Washington, NY). The
filtered solution was diluted 100 times with the extraction
solution. One hundred microliters of internal standards
(1000 ng/mL Ac-EEMQRR^-amide and 2500 ng/mL H2N-
EEMQRR^-amide) and 900 mL of the diluted solution were
transferred into an autosampler vial and mixed well for
LC/MS–MS injection. Each sample was prepared in triplicate
and each preparation was injected three times consecutively.
For the cotton tip applicator samples used for washing
the skin, the weighing step was omitted. For the blank
emulsion, the dilution step was omitted but the remainder
of the extraction procedure was the same as that for the
Ac-EEMQRR-amide containing emulsion. For stratum cor-
neum samples on the tape strips contained in a 20-mL vial,
the samples were taken out of the freezer and equilibrated at
room temperature. Five hundred microliters of internal
standards (1000 ng/mL Ac-EEMQRR^-amide and 2500 ng/
mL H
2
N-EEMQRR^-amide) and 4.50 mL of extraction
solution (0.1:0.1:85:15 TFA:FA:ACN:H
2
O) were added to
the vial. The vial was vortexed for 3 min, sonicated for 10 min
and vortexed for another 3min followed by filtration of the
supernatant through a 0.2 -mm PTFE filter into a vial for
LC/MS–MS injection.
Frozen epidermis and dermis samples in HHBSS were
thawed and equilibrated at room temperature. Six hundred
microliters of internal standards (1000 ng/mL Ac-EEMQRR^-
amide and 2500 ng/mL H2N-EEMQRR^-amide) was added to
the tubes. The tube was vortexed for 3 min, sonicated for
10 min and vortexed for another 3 min. The sample was then
transferred to a 15-mL centrifuge tube followed by rinsing the
test tube three times with HHBSS buffer and collecting all
the solutions into the centrifuge tube. The final volume of the
sample was adjusted to 6 mL with HHBSS buffer. The tube
was vortexed for 3 min, sonicated for 10 min and vortexed for
48 M. E. K. Kraeling et al. Cutan Ocul Toxicol, 2015; 34(1): 46–52
Downloaded by [FDA Library] at 12:55 11 February 2016
another 3 min. A one milliliter test portion was pipetted into a
50-mL centrifuge tube. In order to match HILIC-SPE mode,
9 mL of 0.05% ammonium hydroxide in 90:10 ACN–H
2
O
was added to the tube followed by vortexing the tube
for 3 min, sonicating for 10 min and vortexing for 3 min.
The sample was then subjected to clean up by using an
HILIC cartridge and injected into the LC–MS/MS as
previously described
12,13
.
For receptor fluid samples containing HHBSS, the samples
were thawed and equilibrated at room temperature. Nine
hundred microliters of receptor f luid plus 100 mL of internal
standards (1000 ng/mL Ac-EEMQRR^-amide and 2500 ng/
mL H
2
N-EEMQRR^-amide) were added to a 50-mL centri-
fuge tube followed by vortexing for 1min. In order to match
HILIC-SPE mode, 9.0 mL of 0.05% ammonium hydroxide in
90:10 ACN–H
2
O was added to the tube followed by vortexing
for 3 min, sonicating for 10 min and vortexing for 3 min. The
sample was subjected to clean up using an HILIC cartridge
and injected into the LC–MS/MS
12,13
.
Confocal microscopy
Confocal microscopy was used to confirm the amount
of stratum corneum removed by tape stripping. At the end
of 24-h skin absorption studies, some skin disks were
removed from the diffusion cells, the dosed area of the skin
disk was tape stripped, cut and snap frozen in liquid nitrogen.
The sections were further embedded in Tissue-Tek optimal
cutting temperature media (Sakura Finetek) for cryosection-
ing. Slices (10–20 mm) of the frozen skin were cut in a
cryostat (Leica CM 1900, Leica Microsystems, Inc., Buffalo
Grove, IL). Samples were then analyzed on a laser scanning
confocal microscope (Leica SP5 II confocal microscope,
Leica Microsystems, Inc., Buffalo Grove, IL, 543nm laser
line, 1% power attached to an Inverted Microscope, 40
objective) to determine layers of stratum corneum removed.
Results
In order to assess the safety of Ac-EEMQRR-amide, skin
penetration of Ac-EEMQRR-amide was conducted under
conditions that simulate consumer exposure. In a study
looking at the preparation and stability of cosmetic formula-
tions with Ac-EEMQRR-amide, the release of Ac-EEMQRR-
amide from an O/W cream formulation was significantly
higher (50%) than the gel formulation (20%) after 5 h
3
.
Our percutaneous penetration studies of Ac-EEMQRR-amide
were conducted using a 10% concentration of Ac-EEMQRR-
amide in a simple O/W emulsion formulation. More complex
formulations containing different surfactants may have dif-
ferent Ac-EEMQRR-amide penetration results. Formulation
differences affected the percutaneous absorption of alpha
hydroxy acids used in cosmetics
15
.
The results of the skin penetration study conducted
using HGP skin are given in Table 1. Almost all of the
Ac-EEMQRR-amide was washed off from the surface
of the HGP skin (99.4%) after 24 h. From Ac-EEMQRR-
amide that did penetrate HGP skin, about 0.54% of
the applied peptide remained in the stratum corneum
(1.2 mg), while 0.01% (0.02 mg) was absorbed into the viable
epidermis.
The results of the skin absorption study of Ac-EEMQRR-
amide using human skin are given in Table 2. These results
also provide evidence that the majority of the Ac-EEMQRR-
amide did not penetrate and was removed from the human
cadaver skin after washing (99.7% recovered). From the Ac-
EEMQRR-amide that did penetrate human skin, 0.22% of the
applied peptide remained in the stratum corneum (0.48 mg).
Although there was less than half the concentration of
Ac-EEMQRR-amide in the stratum corneum of human
skin compared to HGP, there is no significant difference in
Ac-EEMQRR-amide concentrations (t-test, SigmaStat, Systat
Software, San Jose, CA). The amount of Ac-EEMQRR-amide
Table 2. Amount and percent of applied dose of peptide recovered and penetrated in human skin 24h after dosing.
Amount of acetyl
hexapeptide-8
measured (mg)
Percent of applied
dose acetyl hexa-peptide-8
recovered
Percent of applied
dose acetyl hexa-peptide-8
penetrated
Cotton-tipped applicators (washes) 192.4 ± 4.91 99.7 ± 0.04
Stratum corneum 0.48 ± 0.51 0.25 ± 0.03 0.22 ± 0.03
Viable epidermis 0.015 ± 0.009 0.01 ± 0.01 0.01 ± 0.01
Dermis None detected None detected None detected
Receptor fluid None detected None detected None detected
Dose ¼222.4 ± 5.4 mg acetyl hexapeptide-8. Values are four replicates from three human cadaver donors (n¼3).
Table 1. Amount and percent of applied dose of peptide recovered and penetrated in HGP Skin 24h after dosing.
Amount of acetyl
hexa-peptide-8
measured (mg)
Percent of applied
dose acetyl
hexa-peptide-8 recovered
Percent of applied
dose acetyl
hexa-peptide-8 penetrated
Amount of amine
hexa-peptide
measured (mg)
Cotton-tipped applicators (washes) 215.0 ± 2.7 99.43 ± 0.20 None detected
Stratum corneum 1.2 ± 0.43 0.56 ± 0.20 0.54 ± 0.23 None detected
Viable epidermis 0.02 ± 0.01 0.01 ± 0.003 0.01 ± 0.004 None detected
Dermis None detected None detected None detected None detected
Receptor fluid None detected None detected None detected None detected
Dose ¼235.5 ± 20.8 mg acetyl hexapeptide-8. Values are four replicates from four animals (n¼4).
DOI: 10.3109/15569527.2014.894521 Skin penetration of a cosmetic peptide 49
Downloaded by [FDA Library] at 12:55 11 February 2016
detected in the human epidermis (0.015 mg) was similar to
that in HGP skin at 0.02 mg.
The stratum corneum of HGP and human skin was
removed layer by layer using successive tape stripping
15
.
Figure 1 shows the amount of Ac-EEMQRR-amide in each
tape stripped layer of stratum corneum removed from
HGP and human skin. For both skin types, the amounts of
Ac-EEMQRR-amide recovered in the stratum corneum
decreases as the layers approach the viable epidermal layer.
There is significantly more Ac-EEMQRR-amide in the first
three layers of stratum corneum in HGP than in human
skin. By the fourth tape strip, Ac-EEMQRR-amide levels are
not as significantly different and become more similar
as more layers of stratum corneum are removed. Again,
the overall total amounts of Ac-EEMQRR-amide in HGP and
human stratum corneum are not significantly different.
Confocal microscopy images show that eight tape strips
removed all stratum corneum for HGP skin (Figure 2) and
that 13 tape strips removed most, but not all, of the stratum
corneum for excised human cadaver skin (Figure 3).
Ac-EEMQRR-amide was not detected in the dermal layers
of HGP or human skin or in any of the receptor fluid fractions
collected over 24 h. There was also no H
2
N-EEMQRR-amide
metabolite detected in any of the skin layers or receptor
fluid fractions of HGP skin.
Discussion
Data on the skin penetration of Ac-EEMQRR-amide are
limited. In one penetration study using epidermal membranes
placed in static diffusion cells, Ac-EEMQRR-amide was
Figure 2. Tape stripping HGP skin eight
times completely removes all of the stratum
corneum (40magnification). SC, stratum
corneum; E, epidermis; D, dermis.
Figure 3. Tape stripping human skin 13 times
does not remove all of the stratum corneum
(40magnification). SC, stratum corneum;
E, epidermis; D, dermis.
Figure 1. Average amount of Ac-EEMQRR-amide in the descending
layers of stratum corneum in HGP and human skin. HGP skin tape
stripped 8 times; human skin tape stripped 13 times.
50 M. E. K. Kraeling et al. Cutan Ocul Toxicol, 2015; 34(1): 46–52
Downloaded by [FDA Library] at 12:55 11 February 2016
able to cross the membrane
4
. Thirty percent of the applied
dose of an aqueous solution of Ac-EEMQRR-amide appeared
in a receptor chamber after 2 h
4
. The concentration of
Ac-EEMQRR-amide used in the penetration study was not
clear, but the study showed that diffusion across epidermal
membranes was possible.
Most biological activity and efficacy attributed to
Ac-EEMQRR-amide has been described in the results of
clinical studies. In one study, skin topography analyses
were performed on silicone imprints from around the eyes
of 10 human volunteers treated twice daily for 30d with
an O/W emulsion containing 10% Ac-EEMQRR-amide
4
.
Confocal laser scanning microscopy was used to analyze
the imprints to assess the skin surface before and after
Ac-EEMQRR-amide treatment. Three dimensional recon-
structions of optical sections were performed. Quantitative
analysis and normalization of the silicon replicas (skin
topography images) showed that the depth of the skin
wrinkles decreased by 30% while the O/W emulsion treatment
alone reduced the depth of skin wrinkles by 10%
4
. In another
anti-wrinkle test (silicon imprints of the treated areas and
measurement by confocal microscopy) on healthy volunteers,
a cream containing 5% ArgirelineÕsolution (0.05%) applied
twice daily for 28d showed a 16.3% mean wrinkle reduction
2
.
Acetyl hexapeptide-8 was used recently in a clinical
study for the treatment of blepharospasm (BSP)
20
.
Blepharospasm is a focal dystonia, for which injectable
BoNT is the only effective treatment. In a double blind,
placebo controlled trial, a topical cream application of 0.005%
Ac-EEMQRR-amide was applied daily to patients with BSP.
Patients receiving BoNT therapy at regular 3-month intervals
were included for comparison. The results indicated that
topical Ac-EEMQRR-amide application extended the dur-
ation of action of concomitant BoNT therapy versus placebo,
as measured by return to baseline values of the Jankovic
Blepharospasm Rating Scale scores. Although the results
were not significantly different between treatment (BoNT
with peptide) and control groups (BoNT only), one-third of
the patients in the treatment group had a significantly longer
interval between the need for follow-up BoNT injections than
the usual 3-month interval (range: 3.3–7.1 months)
20
.
In our in vitro skin penetration studies, we were able to
measure Ac-EEMQRR-amide penetration by HILIC–MS/MS
and quantitate the amounts of Ac-EEMQRR-amide or
metabolite in skin layers and complex matrices. In the skin
penetration assessments with both HGP and human skin, it
was apparent that most of the Ac-EEMQRR-amide was
washed off the surface of the skin with over 99% recovered
in the cotton-tipped applicators. The small amount of
Ac-EEMQRR-amide penetration that did occur remained
mostly in the stratum corneum for both HGP and human
skin. In HGP skin, total Ac-EEMQRR-amide penetration in
stratum corneum exceeded that in human skin by more
than twofold (1.2 and 0.48 mg, respectively), although the
difference was not significant (t-test). Rodent skin is often
found to be more permeable than human skin
17,21
. The
stratum corneum on the HGP is not as thick and compact as
human skin as demonstrated in this study. Only eight tape
strips were needed to completely remove the HGP stratum
corneum while 13 tape strips was sometimes not enough to
completely remove the stratum corneum from human skin.
Separation of the stratum corneum from the epidermis of the
human cadaver skin also limited the number of tape strips
that could be performed. Although the barrier properties of
the human cadaver stratum corneum were acceptable in our
skin penetration studies based on the tritiated water results,
at the end of the experiments the stratum corneum became
separated from the epidermis more readily as more tape
strips were applied. In past penetration studies, removing
10 tape strips of the surface layers of animal and human
skin typically represented test chemical that remained in the
stratum corneum
15,17
. In these studies, the amount of peptide
remaining in the lower keratinocyte layers of the human
stratum corneum was included as peptide remaining in the
epidermis.
Skin absorption is defined as the amount of test com-
pound in skin that would be considered systemically avail-
able. In order to have a biological effect, Ac-EEMQRR-amide
would have to be absorbed at least into the epidermis where
it can be taken up into the bloodstream. In our results, 0.01%
of the applied dose of Ac-EEMQRR-amide was absorbed
into the epidermis, corresponding to 0.02 mg (31 ng/cm
2
)
and 0.015 mg (23 ng/cm
2
) of Ac-EEMQRR-amide for HGP
and human skin, respectively. It is not known whether
these amounts of Ac-EEMQRR-amide found in the epider-
mis of each skin disk are sufficient to elicit a physiological
effect. Synthetic peptides patterned after the C-terminus
and N-terminus of SNAP25 have been shown to be specific
inhibitors of neurosecretion at micromolar concentrations
when used in vitro
4–7
.A2mM concentration of Ac-
EEMQRR-amide appeared to inhibit formation of the
SNARE complex more than the one micromolar concentra-
tion. In chromaffin cells, 100 mM concentrations of Ac-
EEMQRR-amide inhibited 30% of the total catecholamine
exocytosis with 110 mM reported as the IC50
4
.
There was no detection of the amine hexapeptide metab-
olite, H
2
N-EEMQRR-amide, in any HGP and human skin
layers or receptor fluids. The HGP skin was used as the viable
skin model to determine whether conversion of the parent
Ac-EEMQRR-amide during absorption would occur in the
epidermis. Even though the epidermal thickness of HGP
(29 mm) is less than that of human skin (100 mm)
22
, HGP skin
can remain viable and metabolically active for 24 h in the
diffusion cells
16
. Human cadaver skin is not metabolically
active, so no metabolite would be expected to be formed using
the non-viable human cadaver skin.
Conclusions
In order to assess the safety of a commonly used cos-
metic peptide, the in vitro percutaneous penetration of
Ac-EEMQRR-amide was conducted using HGP and human
skin in diffusion cells. HILIC with tandem mass spectroscopy
was successfully used to analyze and quantify Ac-EEMQRR-
amide and metabolite H
2
N-EEMQRR-amide. This analytical
method was particularly successful in quantifying peptides in
various complex matrices, including emulsion ingredients,
surfactants and different layers of the skin. Most of the
Ac-EEMQRR-amide was washed off from the surface of the
skin for both HGP and human skin, after a 24-h exposure.
DOI: 10.3109/15569527.2014.894521 Skin penetration of a cosmetic peptide 51
Downloaded by [FDA Library] at 12:55 11 February 2016
Almost all of the Ac-EEMQRR-amide that did penetrate skin
was located on and in the stratum corneum. There was less
Ac-EEMQRR-amide in human stratum corneum probably due
to the better barrier properties of human skin. Skin absorption
of Ac-EEMQRR-amide was similar in HGP and human skin,
with 0.01% Ac-EEMQRR-amide reaching the epidermal
layer. It is not known whether these small amounts of
Ac-EEMQRR-amide found in the epidermis of HGP or
human skin are sufficient to elicit a physiological effect.
With the exception of color additives cosmetic products
and ingredients are not subject to Food and Drug
Administration premarket approval authority. Understanding
the skin absorption of potentially biologically active peptides
contained in cosmetics is important for the assessment of
safety and cosmetic claims. These skin penetration data will
be useful for evaluating the safety of cosmetic products
containing small peptide cosmetic ingredients.
Acknowledgements
Conduct of these studies was performed under the guidance of
the Institutional Animal Care and Use Committee and the
Research Involving Human Subjects Committee (RIHSC)
Protocol # 12-004F and with support from the Center for
Food Safety and Applied Nutrition, Office of Cosmetics and
Colors. Authors wish to thank Jeffrey J. Yourick, Robert L.
Sprando, Alexander J. Krynitsky and Jeanne I. Rader (U.S.
Food and Drug Administration, Center for Food Safety and
Applied Nutrition) in support of this research.
Declaration of interest
The opinions and conclusions expressed in this paper are
solely the views of the authors and do not necessarily reflect
those of the US Food and Drug Administration.
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Synopsis Alpha hydroxy acids (AHAs) are used in many cosmetic products as exfoliants, moisturizers, and emollients. The activity of AHAs on skin is likely influenced by their ability to be absorbed into the different layers of skin. The absorption of a homologous series of AHAs was measured through hunnan skin by using in vitro diffusion cell techniques. The (4C) radiolabeled compounds were applied to the skin in an oil-in-water emulsion vehicle. The absorption of the AHAs was measured at pH 3.0, to simulate the pH of the most acidic cosmetic formulations, and at pH 7.0, to observe the effect of complete ionization of AHAs on skin penetration. Much greater absorption of the AHAs was seen at pH 3.0. We also observed substantial absorption into the various skin layers (stratum corneum, viable epidermis and dermis) as well as the receptor fluid. Total absorption of glycolic acid and lactic acid was similar (27-30%). Absorption of the longer-chain AHAs decreased to 21.0% and 19.3%, for 2-hydroxyoctanoic and 2-hydroxydecanoic acids, respectively. At the end of the 24-h studies, these longer-chain AHAs did not form a depot in the skin. The stratum corneum was shown to have a pH gradient with an average pH near 7 at the viable epidermal layer. Therefore, the AHAs ionize to polar molecules as they enter and diffuse through the stratum corneum.
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