Journal of Cosmetics, Dermatological Sciences and Applications, 2013, 3, 234-241
http://dx.doi.org/10.4236/jcdsa.2013.33036 Published Online September 2013 (http://www.scirp.org/journal/jcdsa)
The Efficacy and Safety of Succinylated Atelocollagen and
Adenosi ne for the Treatment of Periorbital Wrinkles *
Dong Jin Ryu1, Jin Young Jung1, Kee Yang Chung1, Hwal Suh2, Sang Ho Oh1, Ju Hee Lee1#
1Department of Dermatology and Cutaneous Biology Research Institute, Yonsei University College of Medicine, Seoul, Korea; 2De-
partment of Medical Engineering, Yonsei University College of Medicine, Seoul, Korea.
Email: #juhee@yuhs.ac
Received July 24th, 2013; revised August 22nd, 2013; accepted August 30th, 2013
Copyright © 2013 Dong Jin Ryu et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The degradation of structural collagen contributes to the characteristic appearance of wrinkles. The anti-wrinkle effects
of a variety of substances have been studied, but the potential anti-wrinkle effects of topical applications of collagen for
periorbital wrinkles have not been investigated. To evaluate the effects of topical application of succinylated atelocol-
lagen on periorbital wrinkles and to compare the results of treatment with adenosine, a clinical study on Korean partici-
pants was carried out. Each participant’s right or left periorbital area was treated with either a solution containing suc-
cinylated atelocollagen and adenosine or a solution containing only succinylated atelocollagen for two months. A pla-
cebo solution was applied to the opposite periorbital area of each patient’s face for two months. Based on objective and
subjective measurements of clinical improvement, the assessment scores for treated sites were statistically significantly
higher than scores for placebo sites after two months of treatment. Analysis of silicone casts of periorbital wrinkles
demonstrated partial effects of succinylated atelocollagen on periorbital wrinkles. However, we did not observe any
effects of adenosine on periorbital wrinkles. Succinylated atelocollagen may be an effective treatment option for perior-
bital wrinkles, but further studies including a longer treatment period and larger subject group are needed to verify these
results.
Keywords: Adenosine; Succinylated Atelocollagen; Periorbital Wrinkles
1. Introduction
Skin aging manifests as changes in skin thickness, color,
elasticity, vascular dilatation and wrinkles. Epidermal
atrophy and the degeneration of dermal collagen and
elastic fibers lead to the formation of wrinkles and fine
lines. Due to mimetic muscle movement and the remod-
eling of bony tissues, deeper and more prominent wrin-
kles occur with aging [1]. Sunlight, smoking and pollut-
ants increase the production of collagenase, and an en-
zyme breaks down collagen through a signal transduction
cascade, resulting in the acceleration of wrinkle forma-
tion [2]. In the field of dermatology, there is increasing
demand for effective, convenient medications and cos-
metics that prevent or treat wrinkles and that have few
side effects.
Studies on a variety of agents that may be effective for
treating and preventing wrinkles are ongoing, with reti-
noids, vitamin C and adenosine considered especially ef-
fective [1]. Several anti-oxidants, including vitamin E,
ferulic acid, co-enzyme Q10, idebenone, green tea, sily-
marin and pycnogenol, are incorporated into skin care
products [1]. Asiaticoside, phosphatidylserine, ginseng
saponins isolated from red ginseng, and K6PC-5, a novel
sphingosine kinase activator, are under continued study,
having demonstrated antioxidative effects and wrinkle
improvement [3-5]. However, the potential anti-wrinkle
effects of topical applications of collagen, a component
of the dermis, have not been investigated.
Collagen is a fundamental component of the dermis,
and type I collagen makes up 80% of the collagen in the
skin [6]. As aging progresses, fragmented type I collagen
fibrils become prominent and disrupt the mechanical
properties of the skin, leading to wrinkle formation [7].
Therefore, supplying collagen, the major component of
the skin’s extracellular substrate, may offer an effective
fundamental treatment for wrinkles. However, collagen
*This research was supported by the grants from Dalim Corporation
(Seoul, Korea) 4-2008-0333 and 1-2011-0006. The authors have no
conflicts of interest to report.
#Corresponding author.
Copyright © 2013 SciRes. JCDSA
The Efficacy and Safety of Succinylated Atelocollagen and Adenosine for the Treatment of Periorbital Wrinkles 235
is insoluble in neutral solution and has a large molecular
weight (300 kDa), so it is not sure that collagen pene-
trates into the skin. In addition, when collagen is kept at
room temperature, it becomes denatured and loses its
triple helix structure, rendering it ineffective. Due to
these limitations, direct injections of small collagen par-
ticles dispersed in distilled water are often used by der-
matolo- gists rather than topical applications.
Among the succinylated atelocollagen used in this re-
search, the telopeptide on both ends, which causes im-
mune reactions, was eliminated to minimize the immune
reactions in human body. Furthermore, the atelocollagen
was treated with succinic anhydride to electrically induce
an anionic status that improves solubility in water or
neutral solution. We performed a double blind, random-
ized, prospective, split-face clinical study on Korean par-
ticipants with periorbital wrinkles to evaluate the effects
of topical application of succinylated atelocollagen on
periorbital wrinkles. We compared the results of topical
atelocollagen treatment to the results of treatment with
adenosine, which is an approved anti-wrinkle treatment
ingredient.
2. Methods
2.1. Patients
The study protocol was approved by the Institutional
Review Board of Severance Hospital, Yonsei University
College of Medicine, Seoul, Korea and informed consent
was obtained from each participant. Thirty-two female
participants (mean age 49.1; age range 32 - 59 years)
with periorbital wrinkles were enrolled in a double-blind,
randomized, prospective, split-face clinical study. The
participants were stratified based on a baseline facial
grading scale, ranging from 0 to 5 (0, no evidence of
lines or wrinkles to 5, many deep lines below the eye
with several coarse wrinkles that extend into the cheek
area) [8]. Exclusion criteria included a history of keloid
scarring or other skin diseases, any laser procedures or
isotretinoin use within six months of study initiation,
pregnancy, or systemic diseases that are known to affect
metabolism. Patients with facial scale grades of 0 or 1
were also excluded.
2.2. Treatments
One side of each participant’s face (periorbital area) was
randomly assigned to be treated with either a solution
containing succinylated atelocollagen and adenosine (so-
dium hyaluronate, L-lysine, succinylated atelocollagen,
adenosine) (Group A), or a solution containing only suc-
cinylated atelocollagen (sodium hyaluronate, L-lysine,
succinylated atelocollagen) (Group B). A placebo (so-
dium hyaluronate, L-lysine) solution (Group P) was ap-
plied to the other side of each patient’s face. Composi-
tions of the test and placebo solutions are summarized in
Table 1. Subjects were instructed to apply the treatment
and placebo solutions twice a day for two months on
each side of the face in the periorbital area. Patients were
instructed to avoid the use of any bleaching or anti-
wrinkle agents during the course of treatment. Skin biop-
sies were obtained from eight subjects, four from each
group, for histological analyses of both periorbital areas
before and after the two-month-treatment period.
2.3. Assessment of Clinical Effects
Before treatment and after one and two months of treat-
ment, digital photographs were taken of each patient us-
ing identical camera settings and lighting conditions.
Two dermatologists, blinded to the patient’s treatment,
evaluated clinical improvements by studying these pho-
tographs, and the participants themselves subjectively
evaluated improvements at the same time points. Both
patients and clinicians followed a five-point grading
scale: 0 = no improvement, 1 = 1% 25%, 2 = 26%
50%, 3 = 51% 75%, 4 = 76% 100% improvement.
We used silicone casts (ReplicaTM, Cuderm Inc., Dal-
las, TX, USA) of the participants’ periorbital wrinkles to
assess skin texture before and after the two-month treat-
ment period. An adhesive ring locator was placed against
the periorbital area and filled with pre-mixed resin so that
the resin overflowed 2 - 3 mm onto the cardboard surface.
After the resin set and the cast was peeled from the skin
surface, the foam adhesive spacer layer was separated
from the cardboard frame and discarded. The casts were
labeled with the date, initials and group and stored in a
paper envelope until analysis. For analysis, a collimated
light source was directed at a 25˚ angle from the plane of
Table 1. Compositions of the test and placebo solutions.
Ingredients (gm/30 ml) Group A (collagen + adenosine) Group B (collagen) Group P (placebo)
Sodium hyaluronate 0.12 0.12 0.12
L-lysine 0.00006 0.00006 0.00006
Succinylated atelocollagen 0.12 0.12 -
Adenosine 0.012 - -
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The Efficacy and Safety of Succinylated Atelocollagen and Adenosine for the Treatment of Periorbital Wrinkles
236
the cast surface. Each cast was placed in a holder that
fixed the direction of the tab position of the cast.
The textures of shadows produced by oblique lighting
of the negative casts were analyzed via two assay meth-
ods. The first method involved measuring the luminance
along a set of 10 equal-length parallel lines (passes) run-
ning across the cast parallel to the lighting direction.
Variations in luminance were treated as indicators of
surface roughness Rz, the average maximum difference
in the luminance value for five equal-length segments in
each of the ten lines traversing the sample; and Ra, the
average deviation of the luminance curve about the mean
luminance for the same ten lines, with “R” parameters
reported in units of brightness (gray levels) ranging from
0 to 255 and increase with increasing roughness; FNum,
number markers indicative of fine and coarse lines per
mm; and IDL, the integrated developed length of the
luminance traces of the ten scan lines. The IDL is the
total length of the luminance lines as a proportion of the
straight-line distance and it increases with the roughness
of the surface. For the second assay method, the cast im-
age area was divided into ten equal width bands or sub-
areas. Shadow-like features were distinguished in each of
these bands when their luminance values were less than
the detection threshold. Four parameters were determined
from the detected features: spacing, or the mean distance
in millimeters between adjacent detected features, which
increases with disappearance of wrinkles; breadth, or the
average breadth of the detected features in millimeters,
which is proportional to the depth of the wrinkles pro-
ducing shadows and decreases as wrinkles become shal-
lower; shadows, or the percent of the sampled replica
area with luminance values less than the detection
threshold, which the relative area of shadow cast by the
wrinkles in the cast (shadows decrease with smoothing of
the skin); and NumWr, or the total number of shadowy
features detected in the ten bands or sub-areas, which
decreases with smoothing of the skin.
2.4. Assessment of Histological Changes
Skin biopsies were taken from eight volunteers before
treatment and after the two-month-treatment period using
a 3 mm biopsy punch. At the first visit, specimens were
obtained from crow’s feet at a 1.5 cm distance from the
lateral margin of both eyes. The biopsies performed after
the two-month-treatment period were taken directly next
to the previous biopsy sites. The specimens were fixed in
formalin and embedded in paraffin. Skin samples were
stained with hematoxylin and eosin to measure the epi-
dermal thickness, Masson trichrome to evaluate changes
in dermal collagen, monoclonal anti-human procollagen
type I antibody (Abcam Inc., Cambridge, MA, USA) to
demonstrate changes in collagen formation and mono-
clonal anti-human Ki-67 antibody (Abcam Inc.) to eva-
luate changes in cellular proliferation in the skin. Images
of each section were taken at a magnification of X200
with a 12.5 megapixel digital camera (DP70, Olympus
Optical Co., Tokyo, Japan) connected to a light micro-
scope (BX40, Olympus Optical Co.). The images were
analyzed by MetaMorph (Molecular Devices, Sunnyvale,
CA, USA).
2.5. Statistical Analysis
Statistical analysis was performed using the Wilcoxon
signed-rank and rank-sum tests for evaluation of objec-
tive and subjective improvement scores and histological
changes. One sample t-test and an independent group
t-test were used to evaluate changes in the casts. P-values
less than 0.05 were considered statistically significant
and P-values less than 0.10 were considered directionally
significant.
3. Results
3.1. Degree of Clinical Improvement
Twenty-eight of 32 participants completed the two-
month study. Four patients dropped out for personal rea-
sons without experiencing any side effects. The numbers
of patients in each group were 15, 13 and 28 in groups A,
B and P respectively. The mean clinical improvement
scores, evaluated by two blinded dermatologists, at the
one-month treatment assessment were 1.53 ± 0.64, 1.08 ±
0.86 and 0.57 ± 0.50 in groups A, B and P respectively.
The mean clinical improvement scores at the two-month
treatment assessment were 2.27 ± 0.96, 2.31 ± 1.25 and
0.96 ± 0.74 in groups A, B and P respectively (Figure 1).
A significant difference in clinical improvement was
observed between groups A and P (P = 0.0005), but we
did not observe a significant difference between groups
B and P at the one-month treatment assessment (P >
Figure 1. Mean clinical improvement scores assessed by
physicians (grading scale: 0 = no improvement, 1 = 1% -
25%, 2 = 26% - 50%, 3 = 51% - 75%, 4 = 76% - 100% im-
provement).
Copyright © 2013 SciRes. JCDSA
The Efficacy and Safety of Succinylated Atelocollagen and Adenosine for the Treatment of Periorbital Wrinkles 237
0.05). Significant differences were observed between
groups A and P, and between groups B and P at the two-
month treatment assessment (P = 0.0005, P = 0.0039).
There were no significant differences between groups A
and B at the one-month or two-month treatment assess-
ments (P > 0.05).
The improvement scores assessed by the physicians
and by participants were similar (Figure 2). However, at
the one-month treatment assessment, no significant dif-
ferences were observed among the three groups (P > 0.05)
by patients. At the two-month treatment assessment, par-
ticipant evaluations were significantly different between
groups A (Figure 3) and P (Figure 4) (P = 0.01), and
between groups B (Figure 5) and P (P = 0.001). There
were no significant differences between groups A and B
at the two-month treatment assessment (P > 0.05). The
Figure 2. Mean clinical improvement scores assessed by
participants (grading scale: 0 = no improvement, 1 = 1% -
25%, 2 = 26% - 50%, 3 = 51% - 75%, 4 = 76% - 100% im-
provement).
Figure 3. Comparison of clinical improvement of periorbi-
tal wrinkles and cast images of representative subject in
group A before treatment (a) and two months after the
treatment (b). Cast images before treatment (c) and two
months after the treatment (d).
Figure 4. Comparison of clinical improvement of periorbi-
tal wrinkles and cast images of representative subject in
group P before treatment (a) and two months after the
treatment (b). Cast images before treatment (c) and two
months after the treatment (d).
Figure 5. Comparison of clinical improvement of periorbi-
tal wrinkles and cast images of representative subject in
group B before treatment (a) and two months after the
treatment (b). Cast images before treatment (c) and two
months after the treatment (d).
treatments were well tolerated and there were no re-
markable complications for any of the participants.
3.2. Cast Image Analysis
A total of 112 casts were evaluated. The casts repre-
sented treated sites for groups A and B, and placebo sites
for group P at two visit times: baseline (BL) and after the
two-month treatment period (V2). The results are sum-
marized in Table 2. Changes from baseline were calcu-
lated by subtracting each subject’s BL values from the
appropriate V2 values. Table 3 summarizes the mean
changes from baseline for groups A, B and P. Changes
from baseline were significant for three measured pa-
Copyright © 2013 SciRes. JCDSA
The Efficacy and Safety of Succinylated Atelocollagen and Adenosine for the Treatment of Periorbital Wrinkles
Copyright © 2013 SciRes. JCDSA
238
Table 2. The results of cast image analysis at baseline (BL) and after two months of treatment (V2) (Group A, succinylated
atelocollagen + adenosine; group B, succinylated atelocollagen; group P, placebo).
Group A Group B Group P
BL V2 BL V2 BL V2
Rz 128.7 ± 38.0 109.6 ± 23.9 139.5 ± 32.6 113.0 ± 28.5 144.7 ± 24.2 126.8 ± 32.7
Ra 24.5 ± 8.2 19.7 ± 4.9 26.9 ± 8.3 20.5 ± 6.5 27.9 ± 6.1 24.3 ± 7.1
FNum 0.476 ± 0.093 0.439 ± 0.113 0.399 ± 0.098 0.386 ± 0.109 0.469 ± 0.112 0.469 ± 0.111
IDL 6.95 ± 2.64 5.48 ± 1.55 6.84 ± 2.00 5.28 ± 1.67 6.93 ± 1.49 5.88 ± 1.66
Spacing 1.948 ± 1.381 1.946 ± 1.053 1.840 ± 1.259 2.239 ± 1.177 1.389 ± 0.325 2.005 ± 1.288
Breadth 0.188 ± 0.030 0.198 ± 0.042 0.247 ± 0.058 0.206 ± 0.042 0.222 ± 0.043 0.216 ± 0.039
Shadows 4.2 ± 3.6 3.1 ± 2.0 5.4 ± 3.7 2.7 ± 2.7 5.8 ± 3.8 4.4 ± 3.2
NumWr 71.5 ± 52.4 57.3 ± 35.6 73.7 ± 40.3 46.4 ± 43.9 83.3 ± 36.1 72.1 ± 47.7
Table 3. Mean changes from baseline in groups A, B and P, and treatment effects calculated by subtracting value of group P
from value of group A or B. (Group A; succinylated atelocollagen + adenosine, group B; succinylated atelocollagen, group P;
placebo.).
Mean changes from baseline Treatment effects
Group A Group B Group P Group A Group B
Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD
Rz 19.1 ± 40.5** 26.5 ± 37.1* 17.8 ± 27.7* 9.2 ± 51.4 20.8 ± 41.3**
Ra 4.8 ± 8.4* 6.4 ± 8.2* 3.6 ± 6.5* 1.1 ± 10.5 5.3 ± 9.0**
FNum 0.038 ± 0.096 0.014 ± 0.117 0.001 ± 0.089 0.035 ± 0.129 0.018 ± 0.134
IDL 1.47 ± 2.80** 1.56 ± 2.82** 1.04 ± 1.59* 0.33 ± 3.11 1.39 ± 2.85
Spacing 0.002 ± 1.665 0.372 ± 1.768 0.623 ± 1.193* 0.615 ± 2.188 0.334 ± 1.978
Breadth 0.01 ± 0.056 0.041 ± 0.059* 0.006 ± 0.045 0.008 ± 0.063 0.026 ± 0.069
Shadows 1.1 ± 4.0 2.7 ± 3.3* 1.4 ± 3.2* 0.3 ± 4.3 1.2 ± 2.8
NumWr 14.1 ± 57.2 27.3 ± 48.2** 11.2 ± 42.0 4.1 ± 66.3 14.8 ± 49.4
*P < 0.05, **P < 0.10.
rameters in Group A (Figure 3) and six parameters in
Group B (Figure 5). The changes for both groups repre-
sented improvements in texture (increased smoothness),
with decreases from baseline for Rz, Ra and IDL (both
groups A and B), Breadth, Shadows and NumWr (Group
B only). In group P, changes from baseline were signifi-
cant for five measured parameters (Figure 4). The
changes represented improvements in texture: decreases
from baseline for Rz, Ra, IDL and Shadows parameters,
with an increase in the Spacing parameter.
Similarly, the differences between treated and placebo
site pairs (treatment effects) were calculated by subtract-
ing the appropriate baseline corrected values for each
subject. These results are shown in Table 3. Treatment
effects were directionally significant for two parameters
for group B only. Significant treatment effects for Rz and
Ra were in the direction of active smoother compared to
group P.
Groups A and B were also compared. A direct com-
parison of group A and B treatment effects confirmed
that the differences in Ra parameters between groups
were directionally significant, with group B smoother
than group A (P = 0.0923).
3.3. Histological Changes
Changes in histological features between baseline and
after two months of treatment are shown in Table 4.
Changes from baseline were calculated by subtracting
each subject’s BL values from the V2 values.
Epidermal thickness increased after two months of
treatment compared with vales before treatment in all u
The Efficacy and Safety of Succinylated Atelocollagen and Adenosine for the Treatment of Periorbital Wrinkles 239
Table 4. Quantitative changes in histological features between baseline and after two months of treatment. (Group A; suc-
cinylated atelocollagen + adenosine, group B; succinylated atelocollagen, group P; place bo. ).
Change Group A Group B Group P
Epidermal thickness (μm) 10.23 ± 7.60 13.15 ± 5.65 6.96 ± 10.15
Collagen (OD) 707.07 ± 1354.46 873.87 ± 331.01 756.72 ± 476.35
Ki-67 (OD) 224.28 ± 102.03* 181.49 ± 283.44 102.47 ± 266.31
Procollagen type I (OD) 346.29 ± 570.80 406.87 ± 665.45 529.29 ± 618.73
OD, optical density. *P < 0.05.
groups. The periorbital areas treated with succinylated
atelocollagen and adenosine containing solution or only
succinylated atelocollagen containing solution showed
greater increases in epidermal thickness. However, there
were no significant differences among the three groups
(P > 0.05).
Quantitative image analyses of pre- and post-treatment
biopsies revealed that after the two-month treatment pe-
riod, the quantities of collagen fibers of each group had
increased compared with baseline (Table 4). However,
there were no significant differences among the three
groups (P > 0.05).
Quantitative image analyses of Ki-67 expression on
pre- and post-treatment biopsies showed an increase after
the two-month treatment period in groups A and B.
However, there was a decrease in the optical density of
Ki-67 expression in group P (Table 4). There was a sig-
nificant difference between groups A and P (P = 0.0455),
but there were no significant differences between groups
A and B or between groups B and P (P > 0.05) (Figure
6).
Immunohistochemical staining of procollagen type I
expression revealed that procollagen expression was in-
creased in all groups. Quantitative analyses of procolla-
gen type I expression showed that the changes from
baseline were 346.3 ± 570.8, 406.9 ± 665.5 and 529.3 ±
618.7 in groups A, B, and P respectively. However, there
were no significant differences among the three groups
(P > 0.05).
4. Discussion
Wrinkle formation represents both photoaging and chro-
nological aging. Periorbital wrinkles are generated early
in the formation of wrinkles. At the molecular level, ul-
traviolet (UV) irradiation activates cytokine receptors
and other growth factors on the surface of fibroblasts and
keratinocytes. Activated receptors and growth factors
stimulate transcription factor AP-1 through a signal
transduction cascade, and AP-1 stimulates the transcrip-
tion of matrix metalloproteinase (MMP) genes. MMPs
break down collagen, the main component of the dermal
extracellular matrix, and other structural proteins [2]. The
Figure 6. Immunohistochemical staining of Ki-67 expres-
sion (original magnification X200). (a) Before treatment
and (b) after two months of treatment using succinylated
atelocollagen and adenosine-containing solution; (c) before
treatment and (d) after two months of treatment using suc-
cinylated atelocollagen-containing solution; (e) before treat-
ment and (f) after two months of treatment using placebo
solution.
activation of MMP-1 and MMP-9 leads to the partial
degradation of collagen, which regulates procollagen
type I synthesis and down-regulates procollagen type I
formation [9]. AP-1 stimulated by UV irradiation also
interferes with collagen synthesis through down-regu-
lation of procollagen type I gene expression [10]. Colla-
gen provides skin with its tensile strength, so the degra-
dation of structural collagen and reduced collagen syn-
thesis are likely to be major contributors to the charac-
teristic appearance of wrinkles [11]. In chronological
aging, molecular features similar to those in photoaging
have been identified [2].
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The Efficacy and Safety of Succinylated Atelocollagen and Adenosine for the Treatment of Periorbital Wrinkles
240
As mentioned above, some substances have been
proven to repair and prevent wrinkles. Many controlled
studies have demonstrated that topical applications of
retinoids reduce the severity of wrinkles [12]. All-trans-
retinoic acid, a first-generation retinoid, induces produc-
tion of procollagen types I and III through induction of
TGF-β, a key player in fibroblast collagen synthesis [11].
Increases in levels of collagen types I and III provide
resiliency and strength to skin. Another constituent,
adenosine, has also been shown to be effective in treating
wrinkles [13]. Adenosine acts as a regulator of cellular
and organ function, and interacts with a family of 4G
protein-coupled receptors, A1, A2A, A2B and A3. After
binding to A2A receptors, collagen production is stimu-
lated by fibroblasts via a MEK-1/MAPK-mediated path-
way and collagen degradation is reduced by down-regu-
lation of MMP expression [14].
Increased collagen synthesis and decreased collagen
degradation are the keystones of strategies for preventing
or improving the appearance of wrinkles. However, there
are no reported clinical studies that evaluate the effect of
topical collagen on periorbital wrinkles. Collagen en-
hances the attachment and proliferation of fibroblasts
[15]. Fibroblasts bind to intact collagen fibrils via cell
surface integrin receptors and this binding creates dy-
namic mechanical tension within fibroblasts and main-
tains their shape. Mechanical tension and cell shape are
critical for proper cellular function and the synthesis of
procollagen fibrils [7]. Recently, there has been an in-
crease in the use of collagen for the treatment of chronic
wounds [16].
In this study, we investigated the efficacy of topical
application of succinylated atelocollagen-containing so-
lution and compared these effects with the effects of
adenosine on periorbital wrinkles. The succinylated ate-
locollagen-containing solution that we tested was com-
posed of succinylated atelocollagen, L-lysine, sodium
hyaluronate and purified water. The size of the suc-
cinylated atelocollagen molecule is 300 nm × 2.4 nm.
The telopeptide consists of 12 - 27 amino acids in a col-
lagen molecule, and the sequence of the telopeptide is
different between individuals. Complete removal of the
telopeptide helps prevent immune reactions and reduces
the size of the molecule [17]. Succinylated atelocollagen
was treated with succinic anhydride to create an electri-
cally anionic surface that improves solubility in water or
neutral solution. L-lysine has been widely used as a cell
adhesion agent and mediator for drug delivery. Because
L-lysine has polycationic sites, it adheres to anionic sites
on the cell surface and the succinylated atelocollagen.
Therefore, combining the succinylated atelocollagen with
L-lysine increases skin penetration. Brown et al. [18]
showed that hyaluronic acid, which has hydrophilic
properties and a large molecular weight (360 - 400 kDa),
can be absorbed from the surface of intact skin by pas-
sive diffusion and active transport and be observed in the
deeper layers of epidermis and dermis. Succinylated ate-
locollagen has better solubility and similar molecular
weight so it may be absorbed through intact human skin.
Hyaluronic acid is also a component of the extracellular
matrix and has the ability to induce fibroblasts to prolif-
erate and to produce more collagen [19]. In addition,
succinylated atelocollagen and hyaluronic acid can hold
water within their molecules. Interspersed water has a
bulking effect and may improve periorbital wrinkles.
Based on the objective measurements of clinical im-
provement, groups A and B showed more significant im-
provement than group P after a two-month treatment
period. However, we did not observe any significant ef-
fects after two months of adenosine treatment. The sub-
jective assessments of improvement by the participants
were similar to the objective assessments of clinical im-
provement. In the analysis of the casts, the treated sites
for both groups A and B exhibited consistent smoothing
of the periorbital wrinkles. In paired comparisons with
group P, only group B showed directionally significant
improvement for two parameters of roughness. Although
the differences were not statistically significant, other
parameters were in the direction of active smoother
compared to group P. It was confirmed that only one
parameter of roughness (Ra) in group B was lower than
in Group A (P = 0.0923). Overall, the results suggest that
the treatment effect in group B was somewhat superior to
the effects in groups A or P.
Analysis of histological features revealed increased
epidermal thickness, collagen and procollagen type I
density with treatment in all groups. There were no sig-
nificant differences among the three groups. The density
of Ki-67 in group A was significantly higher than that of
group P. Ki-67 protein is a cellular marker for prolifera-
tion and can be exclusively detected within the cell nu-
cleus during interphase [20]. It is thought that adenosine,
which was included in group A’s treatment, may affect
the proliferation of keratinocytes. However, we did not
observe proliferation of dermal fibroblasts with Ki-67
staining. This may be due to the short treatment time or
the low penetration rate of the treatment solution. We
were not able to distinguish the effects of succinylated
atelocollagen or adenosine on histological features. The
lack of significant differences among the three groups
may be due to the small number of subjects (each group,
n = 4) and relatively short duration of treatment.
Although degree of clinical improvement showed anti-
wrinkle efficacy of succinylated atelocollagen, the objec-
tive measures such as cast image analysis and histology-
cal changes did not support it sufficiently. Analysis of
cast images showed significant improvement on some
parameters between the areas treated with succinylated
Copyright © 2013 SciRes. JCDSA
The Efficacy and Safety of Succinylated Atelocollagen and Adenosine for the Treatment of Periorbital Wrinkles
Copyright © 2013 SciRes. JCDSA
241
atelocollagen-treated and the areas treated with placebo.
However, we did not observe any effects of adenosine on
periorbital wrinkles. This may be due to the small num-
ber of subjects or to the short treatment period.
5. Conclusion
The results of our study show that succinylated atelo-
collagen may be an effective treatment option for perior-
bital wrinkles, but further studies with a longer treatment
period and larger subject group are needed to verify these
results.
REFERENCES
[1] L. Baumann, “Skin Aging and Its Treatment,” The Jour-
nal of Pathology, Vol. 211, No. 2, 2007, pp. 241-251.
doi:10.1002/path.2098
[2] G. J. Fisher, S. Kang, J. Varani, Z. Bata-Csorgo, Y. Wan,
S. Datta and J. J. Voorhees, “Mechanisms of Photoaging
and Chronological Skin Aging,” Archives of Dermatology,
Vol. 138, No. 11, 2002, pp. 1462-1470.
doi:10.1001/archderm.138.11.1462
[3] A. Chiu and A. B. Kimball, “Topical Vitamins, Minerals,
and Botanical Ingredients as Modulators of Environ-
mental and Chronological Skin Damage,” British Journal
of Dermatology, Vol. 149, No. 4, 2003, pp. 681-691.
doi:10.1046/j.1365-2133.2003.05540.x
[4] H. Y. Park, J. K. Youm, M. J. Kwon, B. D. Park, S. H.
Lee and E. H. Choi, “K6PC-5, a Novel Sphingosine
Kinase Activator, Improves Long-Term Ultraviolet Light-
Exposed Aged Murine Skin,” Experimental Dermatology,
Vol. 17, No. 10, 2008, pp. 829-836.
doi:10.1111/j.1600-0625.2008.00708.x
[5] Y. G. Kim, M. Sumiyoshi, M. Sakanaka and Y. Kimura,
“Effects of Ginseng Saponins Isolated from Red Ginseng
on Ultraviolet B-Induced Skin Aging in Hairless Mice,”
European Journal of Pharmacology, Vol. 602, No. 1,
2009, pp. 148-156. doi:10.1016/j.ejphar.2008.11.021
[6] J. Varani, M. K. Dame, L. Rittie, S. E. G. Fligiel, S. Kang,
G. J. Fisher and J. J. Voorhees, “Decreased Collagen Pro-
duction in Chronologically Aged Skin: Roles of Age-
Dependent Alteration in Fibroblast Function and Defec-
tive Mechanical Stimulation,” The American Journal of
Pathology, Vol. 168, No. 6, 2006, pp. 1861-1868.
doi:10.2353/ajpath.2006.051302
[7] G. J. Fisher, T. Quan, T. Purohit, Y. Shao, M. K. Cho, T.
He, J. Varani, S. Kang and J. J. Voorhees, “Collagen
Fragmentation Promotes Oxidative Stress and Elevates
Matrix Metalloproteinase-1 in Fibroblasts in Aged Hu-
man Skin,” The American Journal of Pathology, Vol. 174,
No. 1, 2009, pp. 101-114.
doi:10.2353/ajpath.2009.080599
[8] D. L. Bissett, J. E. Oblong and C. A. Berge, “Niacina-
mide: A B Vitamin that Improves Aging Facial Skin Ap-
pearance,” Dermatologic Surgery, Vol. 31, No. s1, 2005,
pp. 860-865.
[9] J. Varani, D. Spearman, P. Perone, S. E. Fligiel, S. C.
Datta, Z. Q. Wang, Y. Shao, S. Kang, G. J. Fisher and J. J.
Voorhees, “Inhibition of Type I Procollagen Synthesis by
Damaged Collagen in Photoaged Skin and by Colla-
genase-Degraded Collagen in Vitro,” The American Jour-
nal of Pathology, Vol. 158, No. 3, 2001, pp. 931-942.
doi:10.1016/S0002-9440(10)64040-0
[10] G. J. Fisher, S. Datta, Z. Wang, X. Y. Li, T. Quan, J. H.
Chung, S. Kang and J. J. Voorhees, “c-Jun-Dependent
Inhibition of Cutaneous Procollagen Transcription Fol-
lowing Ultraviolet Irradiation is Reversed by All-Trans
Retinoic Acid,” The Journal of Clinical Investigation,
Vol. 106, No. 5, 2000, pp. 663-670.
doi:10.1172/JCI9362
[11] M. Singh and C. E. Griffiths, “The Use of Retinoids in
the Treatment of Photoaging,” Dermatologic Therapy,
Vol. 19, No. 5, 2006, pp. 297-305.
doi:10.1111/j.1529-8019.2006.00087.x
[12] M. Samuel, R. C. Brooke, S. Hollis and C. E. M. Griffiths,
“Interventions for Photodamaged Skin,” Cochrane Data-
base System Review, Vol. 25, No. 1, 2005, CD001782.
[13] M. L. Abella,Evaluation of Anti-Wrinkle Efficacy of
Adenosine-Containing Products Using the FOITS Tech-
nique,” International Journal of Cosmetic Science, Vol.
28, No. 6, 2006, pp. 447-451.
doi:10.1111/j.1467-2494.2006.00349.x
[14] E. S. Chan, P. Fernandez, A. A. Merchant, et al., “Adeno-
sine A2A Receptors in Diffuse Dermal Fibrosis: Patho-
genic Role in Human Dermal Fibroblasts and in a Murine
Model of Scleroderma,” Arthritis & Rheumatism, Vol. 54,
No. 8, 2006, pp. 2632-2642. doi:10.1002/art.21974
[15] S. Srivastava, S. D. Gorham and J. M. Courtney, “The
Attachment and Growth of an Established Cell Line on
Collagen, Chemically Modified Collagen, and Collagen
Composite Surfaces,” Biomaterials, Vol. 11, No. 3, 1990,
pp. 162-168. doi:10.1016/0142-9612(90)90149-K
[16] J. U. Shin, Y. J. Choi, M. R. Roh, K. Y. Chung and H.
Suh, “Diabetic Ulcers Treated with Bi-Layered Collagen
Membrane,” Korean Journal of Dermatology, Vol. 47,
No. 7, 2009, pp. 831-834.
[17] J. U. Shin, M. R. Roh, D. K. Rah, N. K. Ae, H, suh and K.
Y. Chung, “The Effect of Succinylated Atelocollagen and
Ablative Fractional Resurfacing Laser on Striae Disten-
sae,” Journal of Dermatological Treatment, Vol. 22, No.
2, 2011, pp. 113-121. doi:10.3109/09546630903476902
[18] T. J. Brown, D. Alcorn and J. R. Fraser, “Absorption of
hyaluronan applied to the surface of intact skin,” Journal
of Investigative Dermatology, Vol. 113, No. 5, 1999, pp.
740-746. doi:10.1046/j.1523-1747.1999.00745.x
[19] M. A. Mariggiò, A. Cassano, A. Vinella, et al., “En-
hancement of Fibroblast Proliferation, Collagen Biosyn-
thesis and Production of Growth Factors as a Result of
Combining Sodium Hyaluronate and Aminoacids,” In-
ternational Journal of Immunopathology and Pharma-
cology, Vol. 22, No. 2, 2009, pp. 485-492.
[20] T. Scholzen and J. Gerdes, “The Ki-67 Protein: From the
Known and the Unknown,” Journal of Cellular Physiol-
ogy, Vol. 182, No. 3, 2000, pp. 311-322.
doi:10.1002/(SICI)1097-4652(200003)182:3<311::AID-J
CP1>3.0.CO;2-9