The present invention relates to a composition containing
a chemokine, particularly, an interferon-inducible T-cell
alpha chemoattractant (ITACT) which can decrease the gene
expression of a factor related to melanin pigment production
in melanocytes, thereby exhibiting a skin whitening effect.
【BACKGROUND ART】
Melanocytes are present in the basal epidermis of skin,
and play a role in producing a melanin pigment by external
factors such as UV, transferring the produced melanin pigment
to peripheral keratinocytes, thereby inhibiting DNA damage of
the keratinocytes. However, the activity of these melanocytes
is abnormally regulated by genetic factors, hormones and
various disease factors, resulting in excessive pigmentation
and hyperplasia beyond the normal level of pigment production,
thereby leading to hyperpigmentation disorders such as
freckles or lentigines or contributing as a factor for
hypopigmentation disorders such as vitiligo.
The activation of excessive melanocytes is a state in
which the homeostasis is lost in melanin pigment production
3
due to chronic exposure of external stimuli such as
ultraviolet rays. The skin is an elaborately regulatedimmune
system, and secretes a variety of cytokines, chemokines
and other inflammatory mediators. The ultraviolet rays
stimulate immune cells constituting the skin such as keratinforming
cells, macrophages, T cells and the like to induce the
secretion of immune substances. Such immune substances not
only create a chronic inflammatory state by accumulating
immune cells to the microenvironment surrounding the periphery
of the melanocytes, but also have an effect on the melanocytes
themselves such as proliferation and migration of the
melanocytes, and excessive production of melanin pigment.
Generally, it is well known that a cytokine, which is
known as an immunological mediator, plays an important role in
skin physiology such as atopic dermatitis and contact
dermatitis, while a cytokine, which is an immunoreactive
mediator with a small size of about 8 to 14 kD, shows a
selective chemotaxis to surrounding cells, and has a function
of inducing migration of cell and accumulation in a target
organ, is not well known for its role in skin physiology.
Similarly, studies on various immune substances have been
conducted for an immuno-modulating therapy to regulate
hyperpigmentation disorders caused by hyperactivation of
melanocytes, but the direct influence of ITAC, which is a skin
immune cell-derived factor and a chemokine, on melanocytes is
4
not well known.
Immune responses caused by infections or internal or
external injuries are well known for very precise control
mechanisms. However, if a problem arises in the ability to
precisely control the immune system, a chronic inflammation
will occur, and such changes in the microenvironment will
affect the activity of the surrounding cells.
Recently, it has been reported that the change in the
cytokine mediating the immune response modifies the epigenetic
pattern, thereby modifying the genes. And research on the
treatment of chronic inflammation and further on the treatment
of cancer caused by the the inflammation is actively carried
out through epigenetic control substances. However,
symptomatic genetic approaches to the melanocytes themselves
or cells surrounding microenvironment regulation by the
chemokine are a nonexistent state.
[Prior Art Document]
(Patent Document 1) Korean Patent Publication No. 10-
2013-0056955 (Publication date: May 31, 2013)
【DETAILED DESCRIPTION OF THE INVENTION】
【Technical Problem】
The present inventors have made extensive effort to find
out a method for regulating the activity of melanocytes or
cells surrounding microenvironment through the regulation of
5
the immune system, and as a result, they have discovered that
the activation of melanocytes can be regulated by treating a
chemokine, particularly ITAC, thereby completing the present
invention.
Accordingly, one object of the present invention is to
provide a composition which exhibits a skin whitening effect
by regulating the activation of melanocytes by a chemokine.
【Technical Solution】
To achieve the above object, the present invention
provides a skin whitening composition containing an
interferon-inducible T-cell alpha chemoattractant.
【ADVANTAGEOUS EFFECT】
The composition of the present invention can provide a
skin whitening effect by regulating the activation of
melanocytes, and further is effective in alleviating and
treating skin hyperpigmentation disorders such as freckles and
lentigines.
【BRIEF DESCRIPTION OF THE DRAWINGS】
FIG. 1 shows graphs illustrating the result of a
decrease in gene expression of factors related to melanin
pigment production in human melanocytes treated with ITAC.
FIG. 2 is a graph showing the result of an increase in
6
gene expression, protein content and enzyme activity of HDAC5
in human melanocytes treated with ITAC.
FIG. 3 shows graphs illustrating the result of a
decrease in melanin pigment production in human melanocytes
into which an HDAC5 over-expression system is introduced.
【DETAILED DESCRIPTION OF THE EMBODIMENTS】
The present invention relates to a composition capable
of providing a skin whitening effect by regulating the gene
expression of the factors related to melanin pigment
production by controlling the activation of melanocytes. The
composition of the present invention contains, as an active
ingredient, a chemokine, particularly an interferon-inducible
T -cell alpha chemoattractant (ITAC; SEQ ID NO: 1).
The ITAC used in the present invention can be obtained
from a number of suitable origins (e.g., human recombinant
ITAC, Product No. 672-IT of R & D system). This can be
produced by recombinant DNA methodology, for example by a
method in which a gene encoding human ITAC is cloned and
expressed in a host system, while permitting the production of
large quantities of pure human ITAC. Biologically active
building blocks or fragments of ITAC may also be used in the
present invention.
The composition of the present invention contains ITAC
as an active ingredient in an amount of 0.0001 to 0.0005% by
7
weight based on the total weight of the composition. If ITAC
is contained in an amount of less than 0.0001% by weight, it
is not sufficient to inhibit the activity of tyrosinase for
whitening of the skin, whereas if it is contained in an amount
exceeding 0.0005% by weight, it is not suitable for containing
and formulating other ingredients.
The composition according to the present invention
provides an excellent skin whitening effect, and is also
effective in alleviating and treating skin hyperpigmentation
disorders such as symptoms of freckles or lentigines, and
post-inflammatory pigmentation such as acne.
The composition according to the present invention is a
composition for external skin application, and may be
formulated into a conventional external preparation, a
cosmetic formulation or a pharmaceutical formulation.
The composition according to the present invention may
be formulated by containing a cosmetically or dermatologically
acceptable medium or base. It may be provided in any form
suitable for topical application, for example, in the form of
solutions, gels, solids, paste anhydrous products, emulsions
obtained by dispersing oil phase in aqueous phase,
suspensions, microemulsions, microcapsules, microgranules or
ionic (liposomes) and non-ionic vesicle dispersants, or in the
form of creams, skins, lotions, powders, ointments, sprays or
conceal stick. It may also be used in the form of a foam or
8
an aerosol composition further containing a compressed
propellant. These compositions may be prepared according to a
conventional method in the art.
The cosmetic composition according to the present
invention may contain adjuvants commonly used in cosmetic or
dermatological science such as fatty substances, organic
solvents, solubilizing agents, thickening agents, gelling
agents, softening agents, antioxidants, suspending agents,
stabilizing agents, foaming agents, flavoring agents,
surfactants, water, ionic or non-ionic emulsifiers, fillers,
sequestering agents, chelating agents, preservatives,
vitamins, blockers, wetting agents, essential oils, dyes,
pigments, hydrophilic or lipophilic active agents, lipid
vesicles or any other ingredient commonly used in cosmetics.
Such adjuvants are introduced in the amounts commonly used in
the cosmetic or dermatological fields.
Further, the composition according to the present
invention may be formulated as a pharmaceutical composition by
further containing a suitable pharmaceutically acceptable
carrier, excipient and diluent.
The pharmaceutical dosage form of the present invention
is not particularly limited, but may be used alone or in
combination with other pharmaceutically active compounds.
The pharmaceutical composition according to the present
invention may be formulated into any form suitable for
9
pharmaceutical preparations, in addition to transdermal dosage
forms such as lotions, ointments, gels, creams, patches and
sprays according to conventional methods.
The preferred dosage of the pharmaceutical composition
of the present invention varies depending on the age, gender,
weight, symptom, severity of disease, drug form, route and
duration of administration of a subject, but can be
appropriately selected by those skilled in the art. However,
for a desired effect, the pharmaceutical composition of the
present invention may be administered in the range of 1 mg/kg
per day to 5000 mg/kg per day, but is not limited thereto.
The administration may be performed once or multiple times a
day. In addition, the dose may be increased or decreased
according to the age, gender, weight, severity of disease,
route of administration and the like. Accordingly, the dose
does not, in any way, limit the scope of the invention.
Further, the composition of the present invention may
contain a skin absorption promoting-substance to increase the
skin whitening effect.
【BEST MODE】
Hereinbelow, the present invention will be described in
detail by way of Examples and Test Examples. However, these
Examples are given for illustrative purposes only, and the
scope of the invention is not intended to be limited by these
10
Examples. Further, modifications, substitutions and insertions,
etc. conventionally known in the art can be made to the
invention, without deviating from the scope of the present
invention.
[Reference Example 1] Preparation of melanocytes
Human melanocytes purchased from Cascade (Normal Human
Epidermal Melanocyte-Moderately pigmented, Product No. C-102-
5C) were seeded into a 100 cm2 plate in a number of 6 x 105
cells, 254 medium (Product No. M-254-500, Gibco) containing
HMGS (Human Melanocyte Growth Supplement; Product No. S-002-5,
Gibco) was used as a basal medium, and the cells were
incubated at 37°C in a 5% CO2 incubator. When the density
reached about 70 to 80%, the cells were subcultured, and the
subcultured melanocytes were provided to the experiments
carried out thereafter.
[Test Example 1] Whether gene expression of factors
related to melanin pigment production in human melanocytes is
reduced by ITAC
The human melanocytes were treated with ITAC to determine
whether the pigment production of the melanocytes was reduced
by ITAC. The Product No. 672-IT manufactured by R & D system
was used for ITAC.
The human melanocytes cultured in Reference Example 1
11
above were treated with ITAC at a concentration of 200 ng/ml,
and non-treated melanocytes were cultured at 37°C and 5% CO2
for 48 hours as a control group. The total mRNA of the
cultured human melanocytes was extracted to synthesize cDNA,
and Real-time PCR (Applied Biosystems, 7500 Fast) was
performed using the synthesized cDNA.
The real-time PCR was carried out by repeating the cycle
at 95°C for 15 seconds and at 60°C for 60 seconds for a total
of 40 cycles. A relative value was measured as to how much
the expression of the corresponding gene was increased
compared to the gene expression of the control group, and the
difference in relative gene expression between the samples was
measured using tyrosinase (TYR), MITF, TYRP1, DCT, MART1 and
gp100 primers, which are the typical melanin pigment
production-stimulating factors. The primers were TaqMan
primers manufactured by Applied Biosystems as follows:
tyrosinase (TYR, Product No.: Hs01099965_m1), MITF (Product
No.: Hs01117294_m1), TYRP1 (Product No.: Hs00167051_m1), DCT
(Product No.: Hs01095856_m1), MART1 (Product No.:
Hs00194133_m1), gp100 (PMEL, Product No.: Hs00173854_m1). The
measurement results are shown in FIG. 1.
Based on the results shown in FIG. 1, it was confirm
that the expression levels of the pigment productionstimulating
factors were reduced by the treatment of ITAC and
that the expression level of tyrosinase (TYR), TYRP1, DCT,
12
MART1 and gp100 were significantly reduced. Based on this, it
can be confirmed that ITAC has the ability to inhibit melanin
pigment production by the mechanism.
[Test Example 2] Increase in expression level and enzyme
activity of HDAC5, a histone deacetylase, by ITAC
The human melanocytes cultured in Reference Example 1
were treated with ITAC at a concentration of 50 ng/ml (Product
No. 672-IT, R & D system) for 48 hours. cDNA was synthesized
from the RNA isolated therefrom, and the change in mRNA level
was measured by Q-PCR (Applied biosystems, 7500 Fast) using a
primer of HDAC5 (histone deacetylase 5). The Q-PCR was
carried out by repeating the cycle at 95°C for 15 seconds and
at 60°C for 60 seconds for a total of 40 cycles, and a
relative value was measured as to how much the expression of
the corresponding gene was increased compared to the gene
expression of the control group. The HDAC5 primer used in the
experiment above was TaqMan primer (Product No. Hs00608366_m1)
manufactured by Applied Biosystems.
Based on the measurement results shown in FIG. 2A, it
can be confirmed that when 50 ng/ml of ITAC was treated, the
expression level of HDAC5 mRNA was remarkably increased
compared to the non-treated control group.
Further, the human melanocytes cultured in Reference
Example 1 were treated with 50 ng/ml of ITAC (Product No. 672-
13
IT, R & D system) for 48 hours. 30 μg of the cell lysate was
loaded, and the level of protein expression of HDAC5 was
confirmed by western blotting.
Based on the results shown in FIG. 2B, it was confirmed
that although no change was observed in the expression of
GAPDH which is a control group, the level of protein
expression of HDAC5 was significantly increased when ITAC was
treated.
Furthermore, in order to examine the effect of
increasing the levels of mRNA and protein of HDAC5 by ITAC on
enzyme activity, 50 ng/ml or 200 ng/ml of ITAC (Product No.
672-IT, R & D system) was treated to the human melanocytes
cultured in Reference Example 1 above for 48 hours to prepare
a cell lysate. The cell lysate was maintained at a low
temperature until just before the experiment to maintain the
enzyme activity. 10 μl of assistant reagent buffer(HDAC assay
buffer) for HDAC analysis or 10 μl of assistant reagent buffer
containing 4 μM of TSA (trichostatin A), which is a HDAC
inhibitor and a control group, was aliquoted into a 96-well
plate. After adding 20 μl of the cell lysate, the resultant
was stored in an incubator at 37°C for 10 minutes so that the
maximum enzyme activity could be achieved.
Then, 10 μl of 4 mM substrate was added and reacted for
1 hour. 20 μl of an enzymatic activity amplification buffer
(activator solution) was added and reacted at room temperature
14
for 20 minutes, and the absorbance was measured at 405 nm
(BioTek, Synergy 2). Here, the enzyme activity was
quantitatively calculated as the activity (%) relative to the
control group. On the other hand, in this experiment, it was
concluded that when the enzyme activity of HDAC5 was inhibited
by 70 to 100% in the experimental group containing TSA, which
is a broad-spectrum HDAC inhibitor, this experiment was
considered to work effectively, and the enzyme activity of
HDAC5 was measured using the HDAC assay kit (17-374)
manufactured by Upstate.
Based on the measurement results shown in FIG. 2C, it
can be confirmed that ITAC increases the enzyme activity of
HDAC5 in a concentration-dependent manner in the range of 50
to 200 ng/ml used in the present invention.
[Test Example 3] Inhibition of melanin pigment production
by HDAC5
The melanocytes, which excessively produce HDAC5 protein
compared to the cells in control group using an HDAC5
overexpression vector, were centrifuged (Eppendorf, centrifuge
5415R, Germany), and the pellet was isolated therefrom and
observed for color.
As a result of comparing it with the non-treated group
used as a control group, as shown in FIG. 3 A, the
overexpression of HDAC5 reduced the amount of melanin pigment
15
production in a concentration-dependent manner.
The content of the pellet was dissolved with sodium
hydroxide, and the absorbance was measured at OD490 which is
specific for melanin pigment (BioTek, Synergy2) and corrected
by the total protein content.
Based on the results shown in FIG. 3, it can be
confirmed that the overexpression of HDAC5 induces a reduction
in the amount of melanin in a concentration-dependent manner.

【CLAIMS】
【Claim 1】
A skin whitening composition containing an interferoninducible
T-cell alpha chemoattractant as an active
ingredient.
【Claim 2】
The skin whitening composition of claim 1, characterized
in that the interferon-inducible T-cell alpha chemoattractant
is contained in an amount of 0.0001 to 0.0005% by weight based
on total weight of the composition.
【Claim 3】
The skin whitening composition of claim 1, characterized
in that the composition inhibits the gene expression of a
factor related to melanin pigment production in a melaninforming
cell.
【Claim 4】
The skin whitening composition of claim 1, characterized
in that the composition increases the gene expression of
histone deacetylase.
【Claim 5】
The skin whitening composition of claim 1, characterized
in that the composition improves a skin hyperpigmentation
disorder.
【Claim 6】
The composition of claim 5, characterized in that the
We Claim:
17
skin hyperpigmentation disorder is freckles or lentigines.
【claim 7】
The composition of claim 5, characterized in that the
skin hyperpigmentation disorder is caused by post-inflammatory
pigmentation.
【claim 8】
The skin whitening composition of any one of claims 1 to
7, characterized in that the composition is a composition for
external skin application.
【Claim 9】
The skin whitening composition of any one of claims 1 to
7, characterized in that the composition is a cosmetic
composition.
【Claim 10】
A composition for preventing and treating
hyperchromatism containing an interferon-inducible T-cell
alpha chemoattractant as an active ingredient.
【Claim 11】
A use of a composition, which contains an interferoninducible
T-cell alpha chemoattractant as an active
ingredient, for skin whitening.