Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS FOR IMPROVING SKIN CONDITIONS COMPRISING HUMAN
GROWTH HORMONE AS AN ACTIVE INGREDIENT
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
The present invention relates to skin condition-improving compositions for
topical application to the skin, which comprises human growth hormone as an
active
ingredient and a method for improving skin conditions of a human.
DESCRIPTION OF THE RELATED ART
It is a generally known in the art that macromolecules such as human growth
hormone (molecular weight of about 22 kD), cannot pass through the skin
stratum
corneum. A molecular weight that can be delivered efficiently through the skin
epidermis is generally recognized to be no more than about 500 daltons or at
most 2
kD even with the help of skin penetration enhancers. Accordingly, applying
macromolecules such as human growth hormone topically to the intact skin and
expecting cosmetic or medical efficacies by the action of macromolecules have
been
considered unsuccessful.
Some attempts have been made to deliver proteins to the dermal layer of the
skin by topical application of liposomes encapsulating the proteins. It has
been
reported that the proteins encapsulated into lipsomes are delivered via the
dermis or
the hair follicles. For delivery through hair follicles, either a delivery
system in the
form of liposomes or a lipid composite comprising lipids such as fatty acids
has been
reported to be favorable. In addition, although efficiencies turned out to be
much
lower, an aqueous solution containing an organic solvent such as ethanol or an
aqueous solution containing a polymer such as polyethylene glycol, has also
been
tested as a facilitating medium for delivery through hair follicles. With
respect to
efficiencies of delivering proteins through the skin using liposomes carrying
proteins, a
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general principle has not been established yet, because cases of liposomal
protein
deliveries have been scarce and even in those rare cases, the efficiencies of
protein
deliveries varied widely depending on the empirical choices of the target
proteins and
the nature of the liposomes used. One thing to note is that, although the idea
of
delivering proteins through hair follicles is gaining more acceptances, even
when a
protein is delivered in a liposome-encapsulated form, liposomes may not pass
through
the infundibular portion of the hair follicles intact, rather they may undergo
different
morphological transformations or phase transitions owing to, first, the
characteristics
of the phospholipids conferring inside and outside pH's and charge valences of
the
liposomes, second, those of the proteins attached to or encapsulated by the
liposomes, and third, those of the constituting ingredients of the surrounding
tissues
making up the hair follicles. Thus as a whole, these three factors and their
complex
interactions seem to determine follicular delivery efficiencies of the
liposomes
containing the proteins in question through empirical formulations rather than
by a
general guiding principle at the moment. What is particularly noteworthy with
regard
to the present invention is the existence of a report stating human growth
hormone
receptors are located on the living cell layers of the epidermis and
throughout the
ancillary organs and tissues constituting and surrounding the hair follicles.
Human growth hormone is secreted from the anterior lobe of pituitary gland
and circulates with blood while it influences each organ of the human body. In
the
growth stage of a human, it is particularly involved in the growth of
skeleton, an
increase in muscles, the decomposition of fat, the growth of the internal
organs,
sexual growth and the like. In addition, it was suggested that when human
growth
hormone was administered by injection to adults at a physiological range of
blood
concentration, it would show various effects, such as the strengthening of the
heart
circulatory system, the enhancement of exercise ability, the strengthening of
muscles,
the reduction in abdominal fatness, the increase in libido, the improvement of
improvement of arteriosclerosis, and the improvement of geriatric depression.
It is
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known that the effects of human growth hormone on the human body are not
caused
by the human growth hormone itself, but rather are caused by the action of
insulin-
like growth factor-1 (IGF-1), the expression of which is stimulated by human
growth
hormone and which is produced mainly in the liver and secreted into blood.
This is
because the blood half-time of human growth hormone is about 15 minutes,
whereas
the blood half-time of IGF-1 is about 20 hours, indicating that IGF-1 can be
lasting
much longer than human growth hormone. Concretely speaking, human growth
hormone secreted from pituitary gland binds to human growth hormone-binding
protein present in blood, migrates with blood circulation, and meets a human
growth
hormone receptor present in each tissue of the human. At this time, the human
growth hormone is liberated from the human growth hormone-binding protein
while it
binds to the human growth hormone receptor, and the synthesis and secretion of
IGF-1 are stimulated as a result of signaling caused by the binding. The IGF-1
secreted into blood then binds to an IGF-1 binding protein, and circulates
with blood
flow while it binds to an IGF-1 receptor present in each tissue of the human
body,
thus exhibiting various physiological effects caused by the secretion of human
growth
hormone. Accordingly, if the effect of the injection agent human growth
hormone on
the skin will be actually shown, it will be an effect caused by the action of
IGF-1, and
thus will necessarily depend on the presence or absence of the IGF-1 receptor
on the
surface of dermal cells that can be brought into direct contact with blood.
Even if the
skin is considered to be influenced directly by human growth hormone, but not
by
IGF-1, the influence will necessarily be transferred by the human growth
hormone
receptor present at sites which are in contact with blood. Therefore, it is
considered
that expecting any effect on the skin by applying human growth hormone (having
molecular size that cannot pass through the skin) together with cosmetics to
the
normal skin surface that is not brought into direct contact with blood is not
common
sense. The present invention is a first report that, through a method of
applying
human growth hormone in the form of a cosmetic preparation to the normal skin
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surface that is not in direct contact with blood, but not a method of
transferring the
effect of human growth hormone through blood, the human growth hormone can
show cosmetic and medical effects on the skin, such as the improvement of
acne,
wrinkles, atopic skin, skin damage caused by UV light, dark spots, freckles,
dry skin
and oily skin, the reduction of hair follicles, and the stimulation of hair
growth.
Skin tissue consists of the epidermis, the dermis and the hypodermis. The
epidermis determines the properties of the skin, and is frequently susceptible
to
damage directly from the external environment, and thus the repair and
regeneration
of the epidermis are highly important. The epidermis consists of a layer of
epidermal
cells. The skin epidermal cells are also called "keratinocytes", because the
skin
epidermal cells synthesize intermediate filament protein keratin that
strengthens the
epidermis, during their differentiation. These cells are layered with
differentiation
while migrating toward the epidermis, and become flat while organs inside the
cells
gradually disappear and they become dead cells. A cell layer located at the
innermost
portion of the epidermis is contiguous to the basal lamina and called the
"stratum
basale", and the cells forming the layer are called "basal cells", among which
epidermal stem cells are present. The cells of the stratum basale
differentiate into
the epidermis while they sequentially form the stratum spinosum, the stratum
granulosum, the stratum lucidum and the stratum corneum, the stratums being
divided into a living cell layer at the lower position with respect to the
stratum
granulosum, and a dead cell layer at the upper position. Flattened scale-like
tissues
outside the stratum corneum are also called "squames" where keratins are
densely
filled. Cells located from the outside of the stratum granulosum to the
stratum
corneum are reinforced with a layer of cross-linked protein whose plasma
membrane
is thin and tough. While the epidermal cells are proliferated from the
epidermal stem
cells and differentiated into the stratum corneum, they are internally
reinforced by the
cross-linking of keratins and are also linked by keratins with desmosomes
firmly linked
with other cells in the same layer so as to maintain the entire layer
structure thereof.
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The epidermal cells are differentiated into the outer epidermis while they
produce and
secrete lipid so as to form double-layered tissue on a plasma membrane
cornified with
protein, thus preventing the skin surface from the external environment, like
saran
wrap. Since the human epidermis is replaced at two-week interval, the
proliferation
5 ability of skin stem cells forming the epidermis can be considered to be
huge.
It was recently reported that the multipotent stem cells of the skin are
located
at the bulge region, which lies just below the sebaceous gland of hair
follicles. These
bulge stem cells serve as the basis for making epidermal stem cells, hair
matrix stem
cells, and sebaceous glands stem cells. The bulge stem cells are located only
in the
bulge region and express a special combination of protein while maintaining
their
property as stem cells. The epidermal stem cells maintain the epidermis while
they
proliferate and differentiate. When a hair falls out, the hair matrix stem
cells will
proliferate and differentiate to make a new hair. However, the epidermal stem
cells,
the hair matrix stem cells or the sebaceous glands stem cells have limitations
in their
proliferation ability or the ability to maintain the ability of stem cells,
and thus, for
example, most of the epidermal stem cells will differentiate after they
proliferate 3-6
times. On the other hand, although the bulge stem cells proliferate slower
than the
other three stem cells, it seems that the bulge stem cells can indefinitely
proliferate
during the life of human beings while making the epidermal stem cells, the
hair matrix
stem cells and the sebaceous glands stem cells and, at the same time,
maintaining
their stem cell character. The fact that the expression and action of human
growth
hormone occurs at the location of the bulge stem cells has been disclosed for
the first
time through the present invention, and this novel finding is quite
significant,
considering that human growth hormone exhibits the effects of improving
various skin
conditions by the present invention.
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DETAILED DESCRIPTION OF THIS INVENTION
The present inventors have made intensive researches to develop a substance
capable of improving skin conditions, and as a result, found that the topical
application of human growth hormone to the skin can greatly improve skin
conditions,
thereby completing the present invention.
Accordingly, it is an object of the present invention to provide a skin
condition-improving composition for topical application to the skin.
It is another object of the present invention to provide a method for
improving skin conditions.
Other objects and advantages of the present invention will become apparent
from the detailed description to follow taken in conjugation with the appended
claims.
In one aspect of this invention, there is provided a composition formulated
for
topical administration to skin for improving skin conditions, which comprises
human
growth hormone and a cosmetically or pharmaceutically acceptable carrier,
wherein
the skin condition is acne, wrinkles, dark spots, poor skin elasticity, poor
hair growth,
skin aging or poor skin moisture.
The present inventors have conducted studies and efforts to find a novel use
of human growth hormone and, as a result, found that the topical application
of
human growth hormone to the skin can greatly improve skin conditions. Current
therapy with human growth hormone is performed mainly to treat dwarfism and
human growth hormone deficiency using an injection method. In the prior art,
because of the high molecular weight of human growth hormone and a prejudice
for
the action pathway of human growth hormone, it was not noticed that the
topical
application of human growth hormone to the normal skin makes it possible to
expect
the effect thereof. The present invention greatly deviates from this
conventional
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common sense or knowledge in the art and is characterized in that the effect
of the
topical application of human growth hormone to the normal skin has been found
for
the first time. The present invention is the first invention of applying human
growth
hormone to the skin through two routes, i.e., hair follicles and the
epidermis.
The present invention is the first report showing that, by a method of
applying human growth hormone to the normal skin surface opposite to a region
that
is in contact with blood, but not by a method of transferring the effect of
human
growth hormone through blood, human growth hormone can show cosmetic and
medical effects on the skin, such as the improvement of acne, wrinkles, atopic
skin,
skin damage caused by UV light, dark spots, freckles, dry skin and oily skin,
the
reduction of hair follicles, and the stimulation of hair growth.
Human growth hormone (hGH) which is used as an active ingredient in the
present invention may be any polypeptide showing human growth hormone
activity.
For example, any one selected from the group consisting of mature hGH, Met-
hGH,
hGH variants, modified-hGH, hGH fragments and hGH analogues may be used.
Preferred is mature hGH or Met-hGH. The mature hGH refers to a human growth
hormone having the amino acid sequence of the major human growth hormone
present in human blood, the Met-hGH refers to a human growth hormone having
methionine added to the N-terminus of mature hGH, the hGH variants refer to
human
growth hormones having the amino acid sequences of human growth hormones other
than the major human growth hormone present in the human body, the modified
hGH
refers to a human growth hormone modified by adhesion of an additive such as
pegylation or glycation to at least one amino acid residue of human growth
hormone,
the hGH fragments indicate human growth hormones obtained by deleting a
portion
of the amino acid sequences of human growth hormones by a genetic engineering
method or biochemical method, and the hGH analogue refers to a human growth
hormone obtained by modifying the amino acid sequence of human growth hormone
into another amino acid sequence having properties similar thereto by a
genetic
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engineering method. As used herein, the phrase "having human growth hormone
activity" can be specified according to one of the following two methods. In
one
method, it can be specified according to whether human growth hormone causes
signaling by binding to a human growth hormone-binding protein or a human
growth
hormone receptor, and in another method, it can be specified according to
whether
biological effects caused by the action of human growth hormone are shown.
In the present invention, human growth hormone can be applied to the skin
as an aqueous solution or with a carrier. One of the surprising
characteristics of the
present invention is that, as illustrated in Example XII and FIG. 10b, even
when an
aqueous solution of human growth hormone itself is applied directly to the
skin, the
desired effect of improving skin conditions can be somewhat achieved. Even
when an
aqueous solution of human growth hormone itself is topically applied to the
skin, the
human growth hormone will reach the location of bulge stem cells in hair
follicles and
can provide the effect of improving skin conditions.
According to a preferred embodiment of the present invention, a composition
according to the present invention has a phospholipid or liposome composition,
and
preferably a liposome composition. It is preferable that human growth hormone
as
an active ingredient be encapsulated in liposome and applied to the skin.
According
to a more preferred embodiment of the present invention, the present
composition
has a nanoliposome composition. As used herein, the term "nanoliposome" refers
to
a liposome having the form of conventional liposome and a mean particle
diameter of
20-1000 nm. According to a preferred embodiment of the present invention, the
mean particle diameter of the nanoliposome is 50-500 nm, more preferably 50-
350
nm, and most preferably 50-250 nm.
Liposome is defined as a spherical phospholipid vesicle of colloidal particles
which are associated with themselves, and liposomes composed of amphiphilic
molecules each having a water soluble head (hydrophilic group) and a water
insoluble
tail (hydrophobic group) show a structure aligned by spontaneous binding
caused by
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the interaction therebetween, and are classified, according to the size and
lamellarity
thereof, into SUV (small unilamellar vesicle), LUV (large unilamellar vesicle)
and MLV
(multi lamellar vesicle). The liposomes showing various lamellarities as
described
above have a double membrane structure similar to the cell membrane.
The (nano)liposome in the present invention can be prepared using
phospholipid, polyol, a surfactant, fatty acid, salt and/or water.
Phospholipid which is a component used in the preparation of the present
(nano)liposome is used as biphilic lipid, and examples thereof include natural
phospholipids (e.g., egg yolk lecithin, soybean lecithin, and sphingomyelin)
and
synthetic phospholipids (e.g., dipalmitoylphosphatidylcholine or hydrogenated
lecithin),
the lecithin being preferred. More preferably, the lecithin is a naturally
derived
unsaturated or saturated lecithin extracted from soybean or egg yolk.
Polyols which can be used in the preparation of the present (nano)liposome
are not specifically limited and preferably include propylene glycol,
dipropylene glycol,
1,3-butylene glycol, glycerin, methylpropanediol, isoprene glycol, pentylene
glycol,
erythritol, xylitol and sorbitol.
The surfactant which can be used in the preparation of the present
(nano)liposome may be any surfactant known in the art, and examples thereof
include anionic surfactants (e.g., alkyl acyl glutamate, alkyl phosphate,
alkyl lactate,
dialkyl phosphate and trialkyl phosphate), cationic surfactants, amphoteric
surfactants
and nonionic surfactants (e.g., alkoxylated alkylether, alkoxylated
alkylester,
alkylpolyglycoside, polyglycerylester and sugar ester).
The fatty acids which can be used in the preparation of the present
(nano)liposome are higher fatty acids, and preferably saturated or unsaturated
fatty
acid having a C12_22 alkyl chain, and examples thereof include lauric acid,
myristic acid,
palmitic acid, stearic acid, oleic acid and linoleic acid.
The salt which is used in the preparation of the present (nano)liposome may
be any salt known in the art, and examples thereof include phosphate salt,
sulfate salt,
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nitrate salt, chloride salt, hydroxide salt, sodium salt, potassium salt,
calcium salt,
ammonium salt, amino acid salt, and amino acid.
Water which is used in the preparation of the present (nano)liposome is
generally deionized distilled water.
5 According to a preferred embodiment of the present invention, the present
(nano)liposome is prepared only with phospholipid, salt and water, as
described in
detail in Examples below.
According to a preferred embodiment of the present invention, the present
hGH-containing nanoliposome is prepared through a process comprising the steps
10 of: (a) dissolving a phospholipid capable of forming liposome (preferably,
yellow yolk
lecithin or soybean lecithin) in a buffered aqueous solution of salt
containing human
growth hormone; and (b) passing the aqueous solution containing human growth
hormone and phospholipid through a high-pressure homogenizer while gradually
increasing the content of the phospholipid and the pressure of the high-
pressure
homogenizer as the number of the passages increases, thus preparing a human
growth hormone-containing nanoliposome.
The aqueous solution containing human growth hormone is preferably a
buffer solution having a pH of 6-8, and more preferably about 7, for example,
sodium
phosphate buffer solution. If the sodium phosphate buffer solution is used,
the
concentration thereof will preferably be 5-100 mM, more preferably 5-60 mM,
even
more preferably 10-30 mM, and most preferably about 20 mM.
The most prominent feature of the present process is that the mixture of the
phospholipid and the hGH-containing aqueous solution is passed through the
high-
pressure homogenizer several times, in which the amount of the phospholipid
and the
pressure of the homogenizer are increased in a stepwise manner as the number
of
the passages increases. According to a preferred embodiment of the present
invention,
the pressure of the homogenizer is stepwisely increased from 0 to 1000 bar,
and
preferably from 0 to 800 bar. The pressure may be increased by 50 bar or 100
bar,
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preferably 100 bar. According to a preferred embodiment of the present
invention,
the amount of the phospholipid is stepwisely increased from 5 to 40 w/v(%),
and
more preferably from 5 to 30 w/v(%).
Through the high-pressure homogenization process including these stepwise
increases in phospholipid content and pressure, an hGH-containing nanoliposome
is
prepared and a liquid hGH-containing nanoliposome is preferably prepared.
The composition of the present invention is useful in the improvement in
various skin conditions. Preferably, the present composition is effective in
the
improvement in skin conditions including acne, wrinkle, dark spots, skin
elasticity, hair
growth, skin aging, skin moisture and proliferation of dermal stem cell. More
specifically, the improvements in skin conditions refer to the treatment of
acne,
improvement of wrinkle, removal of dark spots, improvement of skin elasticity,
promotion of hair growth, prevention of skin aging, improvement of moisture-
retaining property of skin or promotion of dermal stem cell proliferation.
More
preferably, the skin condition improved by the present invention is acne,
wrinkle or
hair growth.
The present composition may be provided as a cosmetic or pharmaceutical
composition.
The cosmetic compositions of this invention for improving skin conditions
may be formulated in a wide variety of forms, for example, including a
solution, a
suspension, an emulsion, a paste, an ointment, a gel, a cream, a lotion, a
powder, a
soap, a surfactant-containing cleanser, an oil, a powder foundation, an
emulsion
foundation, a wax foundation and a spray.
The cosmetically acceptable carrier contained in the present cosmetic
composition, may be varied depending on the type of the formulation. For
example,
the formulation of ointment, pastes, creams or gels may comprise animal and
vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives,
polyethylene glycols, silicones, bentonites, silica, talc, zinc oxide or
mixtures of these
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substances. In the formulation of powder or spray, it may comprise lactose,
talc, silica,
aluminum hydroxide, calcium silicate, polyamide powder and mixtures of these
substances. Spray may additionally comprise the customary propellants, for
example,
chlorofluorohydrocarbons, propane/butane or dimethyl ether.
The formulation of solution and emulsion may comprise solvent, solubilizer
and emulsifier, for example water, ethanol, isopropanol, ethyl carbonate,
ethyl
acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol,
oils, in
particular cottonseed oil, groundnut oil, maize germ oil, olive oil, castor
oil and
sesame seed oil, glycerol fatty esters, polyethylene glycol and fatty acid
esters of
sorbitan or mixtures of these substances. The formulation of suspension may
comprise liquid diluents, for example water, ethanol or propylene glycol,
suspending
agents, for example ethoxylated isosteary alcohols, polyoxyethylene sorbitol
esters
and poly oxyethylene sorbitan esters, micocrystalline cellulose, aluminum
metahydroxide, bentonite, agar and tragacanth or mixtures of these substances.
The formulation of soap may comprise alkali metal salts of fatty acids, salts
of fatty acid hemiesters, fatty acid protein hydrolyzates, isethionates,
lanolin, fatty
alcohol, vegetable oil, glycerol, sugars or mixtures of these substances.
Furthermore, the cosmetic compositions of this invention may contain
auxiliaries as well as carrier. The non-limiting examples of auxiliaries
include
preservatives, antioxidants, stabilizers, solubilizers, vitamins, colorants,
odor
improvers or mixtures of these substances.
Where the present composition is formulated to provide a pharmaceutical
composition, it may comprise a pharmaceutically acceptable carrier including
carbohydrates (e.g., lactose, amylose, dextrose, sucrose, sorbitol, mannitol,
starch,
cellulose), gum acacia, calcium phosphate, alginate, gelatin, calcium
silicate,
microcrystalline cellulose, polyvinylpyrrolidone, water, salt solutions,
alcohols, gum
arabic, syrup, vegetable oils (e.g., corn oil, cotton-seed oil, peanut oil,
olive oil,
coconut oil), polyethylene glycols, methyl cellulose, methylhydroxy benzoate,
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propylhydroxy benzoate, talc, magnesium stearate and mineral oil, but not
limited to.
The pharmaceutical compositions of this invention, further may contain wetting
agent,
sweetening agent, emulsifier, buffer, suspending agent, preservatives,
flavors,
perfumes, lubricant, stabilizer, or mixtures of these substances. Details of
suitable
pharmaceutically acceptable carriers and formulations can be found in
Remington's
Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by
reference.
The pharmaceutical composition of this invention is formulated for topical
application onto skin.
The correct dosage of the pharmaceutical compositions of this invention will
be varied according to the particular formulation, the mode of application,
age, body
weight and sex of the patient, diet, time of administration, condition of the
patient,
drug combinations, reaction sensitivities and severity of the disease. It is
understood
that the ordinary skilled physician will readily be able to determine and
prescribe a
correct dosage of this pharmaceutical compositions. According to a preferred
embodiment of this invention, the suitable dosage unit is to administer once a
day
with 0.001-100 ng/cm2(unit surface area of skin), most preferably, 0.1-2
ng/cm2.
According to the conventional techniques known to those skilled in the art,
the pharmaceutical compositions of this invention can be formulated with
pharmaceutical acceptable carrier and/or vehicle as described above, finally
providing
several forms including a unit dosage form. Most preferably, the
pharmaceutical
composition is a solution comprising nanoliposomes.
The present composition acts on epidermal stem cell to increase the number
of hair follicles so as to stimulate of hair growth, proliferates the
keratinocytes of the
epidermal layer to greatly inhibit skin aging, improves the skin damaged by UV
light
and wrinkles formed by UV light, remodels the connective tissue of the dermal
layer
to improve skin firmness and improve wrinkles, and shows the effects of
treating acne
and removing dark spots. Taken together, the composition of this invention can
greatly improve skin conditions. In addition, the present composition is very
safe to
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the human body, and has excellent stability when it is prepared in the form of
a
nanoliposome.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an electron microscope photograph of a human growth hormone
(hGH)-containing liposome cream formulation (formulation A) prepared in
Example I.
FIG. 2 is a gel permeation chromatogram of an hGH-containing liposome
(Lipo-hGH) of formulation B prepared in Example I.
FIG. 3 shows the results of SDS-PAGE for hGH encapsulated with Lipo-hGH
of formulation B prepared in Example I.
FIG. 4 is a reverse-phase HPLC chromatogram of hGH encapsulated in Lipo-
hGH of formulation B prepared in Example I.
FIG. 5 is a reverse-phase HPLC chromatogram of phospholipid in Lipo-hGH of
formulation B prepared in Example I.
FIG. 6 is a graphic diagram showing safety test results for Lipo-hGH of
formulation B prepared in Example I.
FIG. 7 is a graphic diagram showing safety test results for an hGH-containing
liposome according to the present invention.
FIG. 8 shows test results for the wrinkle-reducing effect of the present hGH-
encapsulated liposome on nude mice having UV-induced wrinkles.
FIG. 9 shows analysis results for the activity of human growth hormone
encapsulated in an hGH-encapsulating liposome according to the present
invention.
FIG. 10a is a photograph showing the localization of human growth hormone,
which occurs when the present hGH-encapsulating nanoliposome is delivered to
the
skin through hair follicles in Sprague Dawley rats.
FIG. 10b is a photograph showing the effect of the present hGH-
encapsulated nanoliposome on the dermal layer and hair follicles of the skin
of
Sprague Dawley rats.
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FIG. 11a is a photograph showing the effect of the present hGH-
encapsulated nanoliposome on the epidermis and dermis of the skin of ICR mice.
FIG. 11b is a photograph showing that the present hGH-encapsulated
nanoliposome induces the remodeling of connective tissue in the dermal layer
of ICR
5 mice.
FIG. 12 is a photograph showing the effect of the present hGH-encapsulated
nanoliposome on artificial human skin.
FIG. 13 shows analysis results for the particle size distribution of an hGH-
encapsulated nanoliposome according to the present invention.
10 FIG. 14 is a graphic diagram showing the wrinkle-reducing effect of the
present hGH-encapsulated nanoliposome.
The following specific examples are intended to be illustrative of the
invention
and should not be construed as limiting the scope of the invention as defined
by
15 appended claims.
EXAMPLES
Example I: Preparation of various human growth hormone-containing
liposome (Lipo-hGH) formulations
Formulation A (cream formulation): human growth hormone-containing
cream formulation
Phospholipid used in formulation A was lipoid S100 (Lipoid GmbH, Germany)
or lipoid S75 (Lipoid GmbH, Germany).
The heat exchanger of a high-pressure homogenizer (max. output 5 L/hr,
highest pressure 1200 bar, Model HS-1002; manufactured by Hwasung Machinery
Co.,
Ltd., South Korea) was placed in ice water such that the temperature of the
outlet of
the homogenizer did not exceed 30 C, and the inside of the homogenizer was
then
washed with distilled water so as to be ready to operate. Then, to 100 ml of a
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solution of human growth hormone (LG Life Sciences, Ltd) dissolved in a buffer
solution (20 mM NaH2PO4 pH 6.5-7.5, 1 mM EDTA) at a concentration of 1 mg/ml,
phospholipid was added at a ratio of 5 w/v% and sufficiently hydrated and
stirred.
The stirred solution was passed through the homogenizer three times or more at
room temperature and a low pressure of 0 bar. To the solution passed through
the
homogenizer, phospholipid was added to a ratio of 6 w/v% and sufficiently
hydrated
and stirred. The stirred solution was passed through the homogenizer three
times or
more at 100 bar. Then, to the solution passed through the homogenizer in the
condition of 100 bar, phospholipid was added to a ratio of 7 w/v%,
sufficiently
hydrated and stirred, and passed through the homogenizer three times or more
at
200 bar. Then, to the solution passed through the homogenizer in the condition
of
200 bar, phospholipid was added to a ratio of 8 w/v%, sufficiently hydrated
and
stirred, and passed through the homogenizer three times or more at 300 bar. To
the
solution passed through the homogenizer in the condition of 300 bar,
phospholipid
was added to a ratio of 9 w/v%, sufficiently hydrated and stirred, and passed
through
the homogenizer three times or more at 400 bar. Then, to the solution passed
through the homogenizer in the condition of 400 bar, phospholipid was added to
a
ratio of 10 w/v%, sufficiently hydrated and stirred, and passed through the
homogenizer three times or more at 500 bar. Then, to the solution passed
through
the homogenizer in the condition of 500 bar, phospholipid was added to a ratio
of 11
w/v%, sufficiently hydrated and stirred, and passed through the homogenizer
thre
times or more at 600 bar. Then, to the solution passed through the homogenizer
in
the condition of 600 bar, phospholipid was added to a ratio of 12 w/v%,
sufficiently
hydrated and stirred, and passed through the homogenizer three times or more
at
800 bar, thus preparing a human growth hormone-containing liposome (Lipo-hGH)
cream formulation.
FIG. 1 shows an electron microscope photograph of the human growth
hormone-containing liposome cream formulation prepared in this Example. The
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liposome cream formulation prepared in this Example was coated with gold and
observed with a scanning electron microscope (HITACHI S 2500). In the
observation
result, the shape of the bent and connected background was presumed as gel,
and
small spherical grains were estimated as nanosize (0.02-0.3 m) loposomes.
Formulation B (liposome formulation): Human growth hormone (hGH)-
containing liposome formulation
Phospholipid used in the preparation of formulation B was soybean lecithin
(ShinDongBang Corp., South Korea), Metarin P (Degussa Texturant Systems
Deutschland GmbH & Co. KG), Nutripur S (Degussa Texturant Systems Deutschland
GmbH & Co. KG) or Emultop (Degussa Texturant Systems Deutschland GmbH & Co.
KG).
The heat exchanger of a high-pressure homogenizer (max. output 5 L/hr,
highest pressure 1200 bar, Model HS-1002; manufactured by Hwasung Machinery
Co.,
Ltd., South Korea) was placed in ice water such that the temperature of the
outlet of
the homogenizer did not exceed 30 C, and the inside of the homogenizer was
then
washed with distilled water so as to be ready to operate. Then, to 100 ml of a
solution of human growth hormone (LG Life Sciences, Ltd.) dissolved in a
buffer
solution (20 mM NaH2PO4 pH 6.5-7.5, 1 mM EDTA) at a concentration of 1 mg/ml,
phospholipid was added at a ratio of 10 w/v% and sufficiently hydrated and
stirred.
The stirred solution was passed through the homogenizer three times or more at
room temperature and a low pressure of 0 bar. Then, to the solution passed
through
the homogenizer, phospholipid was added to a ratio of 14 w/v%, sufficiently
hydrated
and stirred, and passed through the homogenizer three times or more at 100
bar.
Then, to the solution passed through the homogenizer, phospholipid was added
to a
ratio of 18 w/v%, sufficiently hydrated and stirred, and passed through the
homogenizer three times or more at 200 bar. Then, to the solution passed
through
the homogneizer, phospholipid was added to a ratio of 20 w/v%, sufficiently
hydrated
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and stirred, and passed through the homogenizer three times or more at 300
bar.
Then, to the solution passed through the homogenizer, phospholipid was added
to a
ratio of 22 w/v%, and sufficiently hydrated and stirred, and passed through
the
homogenizer three times or more at 400 bar. Then, to the solution passed
through
the homogenizer, phospholipid was added to a ratio of 24 w/v%, sufficiently
hydrated
and stirred, and passed through the homogenizer three times or more at 500
bar.
Then, to the solution passed through the homogenizer, phospholipid was added
to a
ratio of 26 w/v%, sufficiently hydrated and stirred, and passed through the
homogenizer three times or more at 600 bar. Then, to the solution passed
through
the homogenizer, phospholipid was added to a ratio of 28 w/v%, sufficiently
hydrated
and stirred, and passed through the homogenizer three times or more at 700
bar.
Then, the solution passed through the homogenizer at 700 bar was passed
through
the homogenizer three times or more at 800 bar and discharged from the
homogenizer. The discharged solution was subjected to high-speed
centrifugation at
15,000 x g for 30 minutes, and the supernatant was separated. At this time,
human
growth hormone which has not been encapsulated in liposome was removed by gel
permeation chromatography (GE Healthcare, USA), thus obtaining liquid phase
liposome (see FIG. 2).
Formulation B prepared using a solution of distilled water and buffer solution
(20 mM NaH2PO4, 1 mM EDTA, pH 6.0-7.5) did not show a difference in the
physical
properties and stability of liposome. Also, the obtained formulation was
stored in
more than 10 w/v% of soybean lecithin at 15-30 C for a long period of time
and, as
a result, the phase separation into a lipid layer (lower) and an aqueous
solution
(upper) occurred. However, in less than 10 w/v% of soybean lecithin, it had
excellent
stability without phase separation.
Example II: FPLC separation and SDS-PAGE analysis
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For the analysis of the human growth hormone-containing liposome of
formulation B prepared in Example I, FPLC (Acta explorer, Amersham Bioscience)
was
equipped with a superdex 200 HR/30 column at room temperature, and the column
was equilibrated with two times the column volume of a buffer solution (20 mM
NaH2PO4, 1 mM EDTA and 150 mM NaCl). Then, the human growth hormone-
containing liposome was separated into fractions which were then collected and
analyzed by SDS-PAGE. As shown in FIG. 3, the band of human growth hormone
could be observed at about 22 kDa.
Example III: Quantification of human growth hormone in liposome
HPLC (Shimazu) was equipped with a C18 Delta pack column (Waters, USA),
and reverse phase-HPLC was performed by concentration gradient (B 60-10% : 0-
25
min, B 60%: 25.01-30 min) at a flow rate of 1 ml/min using 0.1% TFA
acetonitrile as
solvent A and 0.1% TFA H2O as solvent B. A standard sample (international
standard
human growth hormone NIBSC code 98/574) was quantified using a fluorescence
detector (excitation: 295 nm, range: 270-300 nm; emission: 350 nm, range: 300-
400
nm) in conditions of oven temperature of 55 C and run time of 30 min. Then, a
sample was pretreated by disrupting the human growth hormone-containing
liposome
solution with a sonicator and adding a buffer solution (50 mM Tris-CI pH 8.0,
1 mM
EDTA, 8 M urea, 2% Tween 20) thereto in the same volume as the sample and then
pipetting the mixture, and was quantified by HPLC using the fluorescence
detector
(see FIG. 4).
From the quantification results, it can be seen that the Lipo-hGH of
formulation B prepared in Example I contained about 3.69 pg/ml of human growth
hormone.
Example IV: Analysis of phospholipid content
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HPLC (Shimazu) was equipped with a Spherisorb S5 NH2 column (Waters),
and HPLC was performed by isocratic gradient at a flow rate of 1 ml/min using
a
mixed solvent of 60% acetonitrile, 30% methanol and 5% H20. Phospholipid was
completely dissolved in a mixed solvent of methanol: chloroform (90%:10%) and
5 quantified using a UV light detector (215 nm) in conditions of oven
temperature of 35
C and run time of 20 min. In the same manner, the present human growth
hormone-containing liposome solution was completely dissolved in a mixed
solvent of
methanol: chloroform (90%:10%) and then quantified by HPLC (see FIG. 5).
From the quantification results, it can be seen that the Lipo-hGH of
10 formulation B prepared in Example I contained about 3.26 mg/ml of
phospholipid.
Example V: Stability test
A stability test for the human growth hormone-containing liposome of
formulation B prepared in Example I was performed in the following manner. The
15 present Lipo-hGH containing 0.1% methyl paraben was analyzed for stability
by
placing it in brown color bottles, standing the bottles at 4 C and 15-30 C,
respectively, and quantifying the content of hGH by HPLC at one-week
intervals. As
can be seen in FIG. 6, the present Lipo-hGH after 10 months of storing had
initial hGH
contents of 87.5% at 4 C and 75% at room temperature. This suggests that the
20 present Lipo-hGH has excellent stability.
Example VI: Safety test
To test the safety of the present human growth hormone-containing
liposome (formulation B prepared in Example I), cytotoxicities for human
keratinocyte
cell line HaCaT (DKFZ, Germany) and human embryonic fibroblast HEF (gift from
Prof.
Lee, Jaeyong, Department of Biochemistry, School of Medicine, Hallym
University)
were examined.
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HaCaT and HEF were suspended in 10% FBS/DMEM (FD media at
concentrations of 1 x 105 cells/ml and 5 x 104 cells/ml, respectively. 1 ml of
each of
the suspensions was added to a 24-well plate and then cultured in a 5% CO2
incubator at 37 C for one day. After one day of the culture, the upper-layer
medium
was carefully removed, and a suitable amount of 10% FD medium and various
concentrations of samples were added to the wells of the plate and allowed to
react in
a 5% CO2 incubator at 37 Cfor one day. The samples used were a buffer solution
(containing 20 mM Na-Pi, pH 7.0, 1 mM EDTA and 0.1% methyl paraben), liposome,
human growth hormone and the Lipo-hGH of formulation B prepared in Example I.
After the reaction, the viability of the cells was measured using 3-(4,5-
dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT: Sigma, USA) (Shearman et al.,
Proc. Natl.
Acad. Sci. 91(4):1470-4(1994), Shearman et al., J. Neurochem. 65(1):218-
27(1995)
and Kaneko et al., J. Neurochem. 65(6):2585-93(1995)). The MTT reaction
products
were measured for absorbance at 570 nm using an ELISA reader (Molecular
Devices,
USA). The cell viability by each of the samples was expressed as a value
relative to
the absorbance of a well not containing the samples, taken as 100 % (FIG. 7).
As can be seen in FIG. 7, the present hGH-containing nanoliposome had no
effect the cell viability of HaCaT and HEF, indicating that it is a
formulation very safe
to a living body.
Example VII: Analysis for proliferation of nanoliposome formulation Lipo-
hGH in Nb2 cells
To the well of a 96-well plate containing 50 l of the Nb2 noble rat lymphoma
cell line (NIBSC ECACC #97041101 ) at a concentration of 1 x 105 cells/ml, S-
hGH
(standard human growth hormone, NIBSC code 98/574), a sample comprising a
1000-fold dilution of a pretreated solution (obtained by disrupting a liposome
solution
containing no human growth hormone with a sonicator and adding a solution
(containing 50 mM Tris-CI pH 8.0, 1 mM EDTA, 8 M urea, 2% Tween 20) thereto in
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the same volume as the sample and then pipetting the mixture) added S-hGH, or
a
sample comprising a 1000-fold dilution of the Lipo-hGH (N-hGH; formulation B
prepared in Example I) subjected to a sample pretreatment process (comprising
disrupting a liposome solution containing no human growth hormone with a
sonicator
and adding a solution (containing 50 mM Tris-Cl pH 8.0, 1 mM EDTA, 8 M urea,
2%
Tween 20) thereto in the same volume as the sample and then pipetting the
mixture),
was added. Each of the samples was cultured in a 5% CO2 incubator at 37 Cfor 5
days, and the amount of the proliferated cells was measured using MTT. The
mean
absorbance of the group containing hGH was calculated as a value relative to
the
mean absorbance of the control group containing no hGH, taken as 100%.
As shown in FIG. 9, the human growth hormone encapsulated in the present
Lipo-hGH maintained its original activity.
Example VIII: Analysis of particle size distribution
The Lipo-hGH of formulation B separated by gel permeation chromatography
in the above Example was analyzed for particle size distribution at a
refractive index
of 1.52 using a particle size analyzer (Mastersizer 2000/ Malvern Instruments
Ltd.)
(see FIG. 13). As represented in FIG. 13, the present Lipo-hGH showed the
largest
distribution at a particle size of 0.193 m, indicating that the Lipo-hGH of
formulation
B is present in the nanometer size.
Example IX: Analysis of wrinkle-improving effect
4-week-old nude mice (purchased from Korea Research Institute of Chemical
Technology) were tested using the Lipo-hGH (N-hGH). An animal breeding chamber
was kept at a temperature of 22 2 Cand a humidity of 55-60% in a 12-hr
light/12-
hr dark cycle, and the animals were permitted free access to solid feed
(Central Lab.
Animal Inc., Seoul, Korea) and water sterilized by irradiation and were
acclimated for
about 2 weeks. In order to induce wrinkles on the backside of these nude mice,
20
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mJ of UVB was irradiated to the mice three times a week for 8 weeks. Then, to
the
UVB-irradiated back, each of a sample solution and a control solution were
applied
using a cosmetic brush for 8 weeks. Then, a wrinkle-improving effect was
evaluated
according to the Donald method (Hyun-Seok Kim et. al, Mech. Ageing Dev. (2005.
8.16 In press)).
The test results are shown in FIGS. 8 and 14. In FIG. 8, the control group
(n=3) was not treated with anything, the UVB-control group (n=3) was treated
with
20 mJ of UVB to induce only wrinkles, the liposome (n=3) was treated with 20
mJ of
UVB to induce wrinkle and was treated with liposome, and the Lipo-hGH group
(n=3)
was treated with 20 ml of UVB to induce wrinkles and treated with the present
Lipo-
hGH. As shown in FIGS. 8 and 14, the present Lipo-hGH had the effect of
effectively
removing the UV-induced wrinkles, which was clearly shown starting from 2
weeks
after the topical application of the present Lipo-hGH.
Example X: Analysis of acne treatment effect
The acne treatment effect of the present human growth hormone-containing
liposome was examined in the following manner.
Sixty 15-40-year-old women were randomly divided into three groups, and
then allowed to use each of the hGH-containing liposome formulation B of
Example I
(formulation 1), a comparative solution containing only liposome (formulation
2) and
a comparative buffer solution (formulation 3) first after face washing two
times
(morning and evening) a day for 3 weeks. In addition, there was no particular
limitation on usually used cosmetics. Then, the improvement of acne was
evaluated
based on the user's opinion according to the following criteria. The rest
results are
shown in Table 1 below. Evaluation criteria: +++ (had a very good improvement
effect); ++ (had a significant improvement effect); + (had a slight
improvement
effect); (had no improvement effect, but not became worse); and - (became
worse).
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TABLE 1
Period Formulation 1 Formulation 2 Formulation 3
151 week + t t
2nd week ++ + f
3rd week +++ + f
As can be seen in Table 1, the present formulation had a very good effect on
the improvement of acne, which started to be clearly shown 2 weeks after the
application of the formulation. Furthermore, the present composition did not
substantially cause irritation to the skin, for example, erythema or itching.
Example XI: Analysis of dark spot removal effect
The dark spot removal effect of the present human growth hormone-
containing liposome was tested in the following manner.
Sixty 40-60-year-old women were randomly divided into three groups, and
then allowed to use each of the hGH-containing liposome formulation B of
Example I
(formulation 1), a comparative solution containing only liposome (formulation
2) and
a comparative buffer solution (formulation 3) first after face washing two
times
(morning and evening) a day for 8 weeks. In addition, there was no particular
limitation on usually used cosmetics. The improvement of dark spots was
evaluated
based on the user's opinion according to the following criteria. The test
results are
shown in Table 2 below. Evaluation criteria: +++ (had a very good improvement
effect); ++ (had a significant improvement effect); + (had a slight
improvement
effect); (had no improvement effect, but not became worse); and - (became
worse).
TABLE 2
Period Formulation 1 Formulation 2 Formulation 3
lst week t t f
2nd week t f f
3`d week + t t
4`h week + t t
5th week ++ + t
6th week ++ + t
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7th week ++ + t
8th week ++ + t
As indicated in Table 2, the present formulation had a significantly excellent
effect on the improvement of dark spots, which started to be clearly shown
from
about 3-5 weeks after the application of the formulation. Furthermore, the
present
composition did not substantially cause irritation to the skin, for example,
erythema or
5 itching.
Example XII: Analysis of localization of nanoliposome formulation Lipo-hGH
and effect thereof on skin
The abdominal region of a Sprague Dawley rat was divided into six zones
10 (circles each having a radius of 1 cm) and treated with the following
samples: 0.1%
methyl-paraben buffer solution, 0.1% liposome, 0.001 U hGH, 0.0001 U hGH,
0.001 U
Lipo-hGH, and 0.0001 U Lipo-hGH.
The animal was treated with each of the samples in an amount of 50 l two
times at 24-hour intervals seven times in total. At 24 hours after treatment
with the
15 last sample, tissue was extracted from the rat. The extracted tissue was
sectioned to
a thickness of 40 m and treated with a polyclonal rabbit anti-human growth
hormone primary antibody (DAKO, U.S.A.) and then with a biotin-conjugated anti-
rabbit secondary antibody (VECTOR. VECTASTAIN ABC kit (RABBIT IgG), U.S.A.) at
room temperature for 30 minutes. Next, the sectioned tissue was treated with a
20 VECTASTAIN ABC reagent (VECTOR, U.S.A.) at room temperature for 30 minutes
and
subjected to a color development reaction with a DAB substrate
(Diaminobenzidine,
Sigma, USA). The sectioned tissue was dehydrated with 78% ethanol, 85% ethanol
and 95% ethanol in order and then treated with xylene for 5 minutes. The
tissue was
fixed on a slide glass, and then the location of human growth hormone
contained in
25 Lipo-hGH was observed.
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As shown in FIG. 10a, the human growth hormone encapsulated in the
present Lipo-hGH or the rat growth hormone originally contained in the rat was
found
at locations considered as the bulge stem cells of hair follicles.
Also, as shown in FIG. 10b, the dermal layer of the rat skin applied with the
present Lipo-hGH (containing 0.0001 U hGH) widened, and the number of hair
follicles on the dermal layer increased. Furthermore, it can be found in FIG.
12 that,
even the hGH aqueous solution was applied to the skin, the hGH reached the
location
of bulge stem cells in hair follicles, and this finding was very surprising
considering
the technical level and common sense in the art. This result a possibility of
achieving
the improvement of skin conditions, even when not only hGH encapsulated in
liposome, but also an hGH aqueous solution itself, are applied to the skin.
Example XIII: Analysis of effect of nanoliposome formulation Lipo-hGH on
mouse skin
The effect of the present nanoliposome formulation Lipo-hGH (prepared in
Example I) on the skin of ICR mice was analyzed by H&E (Hematoxylin & Eosin)
staining. For this purpose, after removing the hairs of the back of ICR mice,
the back
regions divided with respect to the vertebra were treated with a control group
and the
present Lipo-hGH at 4-hr intervals for 2 weeks: group 1 (n=3); untreated group
2
(n=3); a group (n=3) treated with liposome/0.1 U of the present Lipo-hGH;
group 3
(n=3) treated with liposome/0.01 U of the present Lipo-hGH; group 4 (n=3)
treated
with liposome/0.001 U of the present Lipo-hGH. After 2 weeks of the treatment,
tissues were extracted from the mice. The extracted tissues were made into
paraffin
blocks and sectioned to a thickness of 4 m, and the sectioned tissues were
placed on
a slide glass. Then, the sections were deparaffined and treated with a
hematoxylene
solution at room temperature for 10 minutes and then with an eosin solution at
room
temperature for 1 minute. Next, the sections were dehydrated with 78% ethanol,
85% ethanol, 95% ethanol and 100% ethanol in order and then treated with
xylene
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for 5 minutes. The tissues were immobilized, and then the stained tissues were
observed under a microscope.
As represented in FIG. 11a, the proliferation of cells in the epidermal layer
of
the skin treated with the present nanoliposome formulation Lipo-hGH was
greatly
increased, and the remodeling of connective tissues in the dermal layer
occurred to
form more compact connective tissues. FIG. 11b is a photograph taken at 400x
magnification and clearly shows that the remodeling of connective tissues in
the
dermal layer occurred.
Example XIV: Analysis of effect of nanoliposome formulation Lipo-hGH on
artificial skin
Neoderm-EDTM (Tego Science, South Korea) was used to analyze the effect
of the present nanoliposome formulation Lipo-hGH on artificial skin. Neoderm-
ED TM is
a human skin model for in vitro tests and consists of an epidermal and dermal
matrix.
Test groups were as follows: group 1 untreated; group 2 treated only with
buffer
solution; groups 3 and 4 treated with liposome; and groups 5 and 6 treated
with
0.001 unit and 0.01 unit, respectively, of the present Lipo-hGH. Paraffin
embedding
and H&E staining were performed in the same manner as in the above Example.
Finally, the stained tissues were observed under a microscope.
As shown in FIG. 12, the proliferation of cells in the keratinocyte layer of
Neoderm-ED TM treated with the present nanoliposome formulation Lipo-hGH was
actively made.
Having described specific examples of the present invention, it is to be
understood that such examples are only preferred embodiments and should not be
construed as limiting the scope of the invention. Therefore, the substantive
scope of
the invention may be determined by appended claims and their equivalents.
