U.S. patent application number 11/304886 was filed with the patent office on 2006-07-20 for material for promoting skin basement formation.
This patent application is currently assigned to Shiseido Company, Ltd.. Invention is credited to Satoshi Amano, Shinji Inomata, Yukiko Matsunaga.
Application Number | 20060159782 11/304886 |
Document ID | / |
Family ID | 18603568 |
Filed Date | 2006-07-20 |
United States Patent
Application |
20060159782 |
Kind Code |
A1 |
Amano; Satoshi ; et
al. |
July 20, 2006 |
Material for promoting skin basement formation
Abstract
A method for promoting skin basement membrane formation
comprises administering matrix metalloproteinase inhibitor or
matrix metalloproteinase inhibitor and matrix protein production
promoting agent. The matrix metalloproteinase inhibitor is
N-hydroxy-2(R)-[[(4-methoxyphenyl)sulfonyl](3-picolyl)amino]-3-methylbuta-
namide hydrochloride.
Inventors: |
Amano; Satoshi;
(Yokohama-shi, JP) ; Matsunaga; Yukiko;
(Yokohama-shi, JP) ; Inomata; Shinji;
(Yokohama-shi, JP) |
Correspondence
Address: |
SNIDER & ASSOCIATES
P. O. BOX 27613
WASHINGTON
DC
20038-7613
US
|
Assignee: |
Shiseido Company, Ltd.
Tokyo
JP
|
Family ID: |
18603568 |
Appl. No.: |
11/304886 |
Filed: |
December 16, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10648485 |
Aug 27, 2003 |
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11304886 |
Dec 16, 2005 |
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09979712 |
Nov 26, 2001 |
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PCT/JP01/02507 |
May 27, 2001 |
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10648485 |
Aug 27, 2003 |
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Current U.S.
Class: |
424/733 ;
424/745; 424/757; 424/771; 514/317 |
Current CPC
Class: |
A61K 36/65 20130101;
A61K 36/74 20130101; A61K 8/64 20130101; A61K 36/74 20130101; A61L
27/60 20130101; A61K 36/82 20130101; A61P 17/00 20180101; A61K
36/65 20130101; A61K 31/445 20130101; A61L 2300/414 20130101; A61L
27/22 20130101; A61K 31/445 20130101; A61L 2300/434 20130101; A61K
36/82 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2800/782 20130101; A61K 2300/00 20130101;
A61Q 19/00 20130101; A61L 27/54 20130101; A61K 31/00 20130101; A61K
45/06 20130101 |
Class at
Publication: |
424/733 ;
514/317; 424/745; 424/757; 424/771 |
International
Class: |
A61K 36/84 20060101
A61K036/84; A61K 36/53 20060101 A61K036/53; A61K 36/48 20060101
A61K036/48; A61K 36/52 20060101 A61K036/52; A61K 31/445 20060101
A61K031/445 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 27, 2000 |
JP |
2000-87574 |
Claims
1. A method for promoting a skin basement membrane formation in a
subject for which the skin basement membrane formation is
necessary, comprising administering a matrix metalloproteinase
inhibitor
N-hydroxy-2(R)-[[(4-methoxyphenyl)sulfonyl](3-picolyl)amino]-3-methylbuta-
namide hydrochloride to said subject.
2. A method for promoting a skin basement membrane formation in a
subject for which the skin basement membrane formation is
necessary, comprising administering a matrix metalloproteinase
inhibitor
N-hydroxy-2(R)-[[(4-methoxyphenyl)sulfonyl](3-picolyl)amino]-3-methylbuta-
namide hydrochloride and a matrix protein production promoting
agent to said subject.
3-6. (canceled)
7. The method for promoting a skin basement formation in accordance
with claim 1, further comprising mixing said matrix
metalloproteinase inhibitor with a matrix metalloproteinase
inhibitor selected from the group consisting of Thymus serpyllum
L., Valeriana fauriei Briquet or other similar plants
(Valerianaceae), Diospyros kaki Thunberg (Ebenaceae), and
Astragalus sinicus Linne (Leguminosae).
8. The method for promoting a skin basement formation in accordance
with claim 1, further comprising mixing said matrix
metalloproteinase inhibitor with a matrix metalloproteinase
inhibitor selected from the group consisting of Crataegus cuneata
Siebold et Zuccarini (Rosaceae), Paeonia suffruticosa Andrews
(Poeonia montan Sims) (Paconiaceae), Thea sinensis Linne var.
assamica Pierre (Thcaccae), and Eucalyptus globules Labillardiere
or its similar plants (Myrtaceae).
9. The method for promoting a skin basement formation in accordance
with claim 1, further comprising mixing said matrix
metalloproteinase inhibitor with a matrix metalloproteinase
inhibitor selected from the group consisting of Potentilla
tormentilla Schrk (Rosaceae), Tilia corda: a Mill., Tilia
platyphyllus Scop., and Tilia europaea Linne (Tiliaceae).
10. The method for promoting a skin basement formation in
accordance with claim 1, further comprising mixing said matrix
metalloproteinase inhibitor with a matrix metalloproteinase
inhibitor selected from the group consisting of Betula alba Linne
(Betulaceze), Origanum majorana L., Uncaria gambir Roxburgh
(Rubiaceae), and Juglans regia Linne var. sinensis De Candolie or
its similar plants (Juglandaceae).
11. The method for promoting a skin basement formation in
accordance with claim 1, further comprising mixing said matrix
metalloproteinase inhibitor with a matrix metalloproteinase
inhibitor selected from the group consisting of Sophora flavescens
Aiton (Leguminosae), Sanguisorba officinalis Linne (Rosaceae),
Hypericum perforatum Linne or Hypericum erectum Thunberg
(Guttiferae), and Thea sinensis Linne (Theaceae).
12. The method for promoting a skin basement formation in
accordance with claim 1, further comprising mixing said matrix
metalloproteinase inhibitor with a matrix metalloproteinase
inhibitor selected from the group consisting of Curcuma longa L
(Zingiberaceae), purified extracts of Curcuma longa L including
Symplocos racemosa and Cyperus rotundus, Cyperus scariosus,
Gaultheria fragrantissima, and Acacia formensia.
13. The method for promoting a skin basement formation in
accordance with claim 1, further comprising mixing said matrix
metalloproteinase inhibitor with a matrix metalloproteinase
inhibitor selected from the group consisting of Terminalia chebula,
Ficus bengalensis, Cassia fistula Linn, Lyonia ovalifolia,
Calophyllum inophyllum, and Ficus religiosa.
14. The method for promoting a skin basement formation in
accordance with claim 2, further comprising mixing said matrix
metalloproteinase inhibitor with a matrix metalloproteinase
inhibitor selected from the group consisting of Thymus serpyllum
L., Valeriana fauriei Briquet or other similar plants
(Valerianaceae), Diospyros kaki Thunberg (Ebenaceae), and
Astragalus sinicus Linne (Leguminosae).
15. The method for promoting a skin basement formation in
accordance with claim 2, further comprising mixing said matrix
metalloproteinase inhibitor with a matrix metalloproteinase
inhibitor selected from the group consisting of Crataegus cuneata
Siebold et Zuccarini (Rosaceae), Paeonia suffruticosa Andrews
(Poeonia montan Sims) (Paconiaceae), Thea sinensis Linne var.
assamica Pierre (Thcaccae), and Eucalyptus globules Labillardiere
or its similar plants (Myrtaceae).
16. The method for promoting a skin basement formation in
accordance with claim 2, further comprising mixing said matrix
metalloproteinase inhibitor with a matrix metalloproteinase
inhibitor selected from the group consisting of Potentilla
tormentilla Schrk (Rosaceae), Tilia corda: a Mill., Tilia
platyphyllus Scop., and Tilia europaea Linne (Tiliaceae).
17. The method for promoting a skin basement formation in
accordance with claim 2, further comprising mixing said matrix
metalloproteinase inhibitor with a matrix metalloproteinase
inhibitor selected from the group consisting of Betula alba Linne
(Betulaceze), Origanum majorana L., Uncaria gambir Roxburgh
(Rubiaceae), and Juglans regia Linne var. sinensis De Candolie or
its similar plants (Juglandaceae).
18. The method for promoting a skin basement formation in
accordance with claim 2, further comprising mixing said matrix
metalloproteinase inhibitor with a matrix metalloproteinase
inhibitor selected from the group consisting of Sophora flavescens
Aiton (Leguminosae), Sanguisorba officinalis Linne (Rosaceae),
Hypericum perforatum Linne or Hypericum erectum Thunberg
(Guttiferae), and Thea sinensis Linne (Theaceae).
19. The method for promoting a skin basement formation in
accordance with claim 2, further comprising mixing said matrix
metalloproteinase inhibitor with a matrix metalloproteinase
inhibitor selected from the group consisting of Curcuma longa L
(Zingiberaceae), purified extracts of Curcuma longa L including
Symplocos racemosa and Cyperus rotundus, Cyperus scariosus,
Gaultheria fragrantissima, and Acacia formensia.
20. The method for promoting a skin basement formation in
accordance with claim 2, further comprising mixing said matrix
metalloproteinase inhibitor with a matrix metalloproteinase
inhibitor selected from the group consisting of Terminalia chebula,
Ficus bengalensis, Cassia fistula Linn, Lyonia ovalifolia,
Calophyllum inophyllum and Ficus religiosa.
21. The method for promoting a skin basement formation in
accordance with claim 1, further comprising mixing said matrix
metalloproteinase inhibitor with a matrix metalloproteinase
inhibitor selected from the group consisting of Symplocos racemosa,
a purified extract of Curcuma longa L.
22. The method for promoting a skin basement formation in
accordance with claim 2, further comprising mixing said matrix
metalloproteinase inhibitor with a matrix metalloproteinase
inhibitor selected from the group consisting of Symplocos racemosa,
a purified extract of Curcuma longa L.
23. The method for promoting skin basement formation in accordance
with claim 2, wherein said matrix protein production promoting
agent is selected from the group consisting of one or more of
soybean lysolecithin transforming growth factor .alpha. (TGFa), and
transforming growth factor .beta.1 (TGFb1).
Description
[0001] This application claims the priority of Japanese Patent
Application No. 2000-87574, filed on Mar. 27, 2000, which is
incorporated herein by reference. This application is a
continuation of U.S. patent application Ser. No. 10/648,485, filed
on Aug. 27, 2003, which is a continuation of U.S. patent
application Ser. No. 09/979,712, filed on Nov. 26, 2001 and later
abandoned (International Patent Application Number PCT/JP200102507
filed Mar. 27, 2001).
FIELD OF THE INVENTION
[0002] The present invention relates to a skin basement membrane
stabilizing agent. In addition, the present invention relates to
artificial skin formation promoting agent and a production method
for artificial skin.
BACKGROUND ART
[0003] In the field of cosmetics and dermatology, a wide variety of
measures have been proposed and attempted to alleviate or treat
injuries caused by the effects of the external environment and
aging including the exposure of the skin to sunlight. For example,
examples of skin changes accompanying aging primarily include the
formation of wrinkles, and hardening or decreased resiliency.
[0004] Primary interest is being directed at decreased function of
collagen fibers and elastic fibers composed of collagen, elastin
and glucosaminoglycans in the skin dermis as the cause of such
changes. In the past, the use of hydroxycarboxylic acids (e.g.,
Japanese Patent No. 2533339), and the use of lysophospholipids
(e.g., Japanese Unexamined Patent Publication No. 8-67621) or that
described in the Journal of the Japan Society of Fats and Oils,
Vol. 46, No. 9 (1997), pp. 13-19 have been proposed as means for
preventing or repairing such changes.
[0005] In the former publication, keratin and wrinkles have been
suggested as being able to be eradicated by preventing decreases in
collagen fibers. On the other hand, in the latter publication,
lysophospholipids are suggested to demonstrate whitening effects by
accelerating the production ability of glycosaminoglycans (and more
specifically, hyaluronic acid) in human fibroblasts.
[0006] The most powerful effect of the above external environment
on skin aging is produced by ultraviolet rays present in sunlight,
and these ultraviolet rays have clearly been established to be a
factor that promotes aging. Ultraviolet rays are known to induce
skin changes referred to as photoaging that is characterized by
deep wrinkles (Scharffetter-Kochanek, Advances in Pharmacology,
1997, 38, 639-655). Ultraviolet rays have a diverse range of
effects on the skin, including damage to genetic DNA, induced
production of active oxygen, and more recently, induced production
of matrix-metalloproteinases (Fisher, et al., Nature, 1996, 379,
335-339).
[0007] Due to the multifunctional nature of ultraviolet rays, the
mechanism by which this photoaging induced by ultraviolet rays
occurs has not been adequately elucidated. Deep wrinkles
corresponding to human photoaged skin have been clearly shown to
form on the skin on the backs of hairless mice following continuous
irradiation of ultraviolet rays at an energy level not enough to
cause erythema. Substances having an effect on wrinkles have also
been evaluated using this mouse model (Moloney, et al., Photochem.
Photobiol. 1992, 56, 495-504). However, the wrinkle formation
mechanism has still not been adequately elucidated and its
elucidation is awaited.
[0008] On the other hand, Koivukangas, et al. reported in 1994 that
levels of gelatinase, an enzyme that decomposes the basement
membrane, becomes elevated in skin irradiated with ultraviolet rays
(Acta Derm. Venereol. 1994, 74, 279-282). In addition, it has also
been reported that the basement membrane exhibits structural
changes at sites of the skin exposed to sunlight, and that
reduplication is observed particularly frequently (Lavker, J.
Invest. Dermal. 1979, 73, 59-66). This suggests the possibility
that ultraviolet rays contained in sunlight have an effect on
basement membrane structure by increasing the amount of basement
membrane decomposing enzymes produced in the skin.
[0009] However, there is no specific means known for promoting the
formation of skin basement membrane structure.
[0010] Artificial skin is important as an alternative to natural
skin that has been damaged by some cause, or as an experimental
material for testing the action and pharmacological effects of
pharmaceuticals and cosmetics on the skin. In either of these
applications, artificial skin is desired that has a structure that
imitates the structure of natural skin as closely as possible.
[0011] Natural skin is roughly composed of the epidermis, dermis
and basement membrane present between them. One example of a known
production method of artificial skin that imitates natural skin
involves the formation of an epidermal layer by culturing normal
human epidermal keratinocytes on contracted type I collagen gel
having a structure that resembles the dermis and contains human
fibroblasts. In this method, however, there was the problem of
inadequate formation of a basement membrane between the collagen
gel imitating the dermis and the epidermal layer imitating the
epidermis.
DISCLOSURE OF THE INVENTION
[0012] Thus, the object of the present invention is to provide a
novel means for stabilizing skin basement membrane, and to provide
a novel means for achieving adequate formation of basement membrane
in the production of artificial skin.
[0013] As a result of various studies to solve the above problems,
the inventors of the present invention found that the formation of
a skin basement membrane structure is promoted by administering
matrix metalloproteinase inhibitor, or both matrix
metalloproteinase inhibitor and matrix protein production promoting
agent, thereby leading to completion of the present invention.
[0014] Thus, the present invention provides a skin basement
membrane formation promoting agent that contains matrix
metalloproteinase inhibitor.
[0015] In addition, the present invention provides a skin basement
membrane formation promoting agent containing matrix
metalloproteinase inhibitor and matrix protein production promoting
agent.
[0016] As a result of various studies to solve the above problems,
the inventors of the present invention found that, in culturing to
produce artificial skin, formation of a basement membrane can be
promoted by adding matrix metalloproteinase inhibitor, or both
matrix metalloproteinase inhibitor and matrix protein production
promoting agent to the medium, thereby leading to completion of the
present invention.
[0017] Thus, the present invention provides an artificial skin
formation promoting agent containing matrix metalloproteinase
inhibitor.
[0018] In addition, the present invention provides an artificial
skin formation promoting agent containing matrix metalloproteinase
inhibitor and matrix protein production promoting agent.
[0019] In addition, the present invention provides an artificial
skin production method comprising the addition of matrix
metalloproteinase inhibitor to an artificial skin formation
medium.
[0020] Moreover, the present invention provides an artificial skin
production method comprising the addition of matrix
metalloproteinase inhibitor and matrix protein production promoting
agent to an artificial skin formation medium.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a photograph representing a biological form of the
cross-section of artificial skin comparing the case of addition of
matrix metalloproteinase CGS27023A (10 .mu.M) or MMP inhibitor (300
.mu.M) with a control to which neither is added in culturing for
the formation of artificial skin.
[0022] FIG. 2 is a photograph representing a biological form of the
cross-section of artificial skin that shows the results of
performing hematoxylin and eosin staining (H & E) and
immunostaining (detection of laminin (LN5)) on formed artificial
skin in the case of adding matrix metalloproteinase inhibitor
CGS27023A (shown as CGS) only, the case of adding CGS and
transforming growth factor .alpha., a matrix protein production
promoting agent (CGS+TGFa), the case of adding CGS and transforming
growth factor .beta.1, a matrix protein production promoting agent
(CGS+TGFb1), and the case of adding both TGFa and TGFb1 to CGS
(CGS+TGFa+TGFb1) to the medium during culturing for formation of
artificial skin.
[0023] FIG. 3 is a photograph used in place of a drawing
representing a biological form of a fragment of artificial skin
showing the results of performing immunostaining (detection of type
IV collagen (IVC) and detection of type VII collagen (VIIC)) in the
same experiment as FIG. 2.
[0024] FIG. 4 is a photograph representing a biological form of
cross-sections of artificial skin formed in the case of adding
matrix metalloproteinase inhibitor CGS27023A and soy bean lecithin,
a matrix protein production promoting agent (CGS+soy bean
lecithin), and the case of not adding the above substances
(control) to the medium during culturing for the formation of
artificial skin.
EMBODIMENTS FOR CARRYING OUT THE INVENTION
[0025] Matrix Metalloproteinase Inhibitor
[0026] There are no particular restrictions on the matrix
metalloproteinase inhibitor used in the present invention, and any
substance may be used provided it has such inhibitory activity.
Examples of matrix metalloproteinase include gelatinase,
collagenase, stromelysin and matrilysin. Thus, a substance
(inhibiting) such as gelatinase, collagenase, stromelysin or
matrilysin can be selected for the matrix metalloproteinase
inhibitor.
[0027] Specific examples of matrix metalloproteinase inhibitors
include substance CGS27023A
N-hydroxy-2(R)-[[(4-methoxyphenyl)sulfonyl](3-picolyl)amino]-3-methylbuta-
namide hydrochloride (J. Med. Chem. 1997, Vol. 40, p. 2525-2532),
and MMP-inhibitor (p-NH.sub.2-Bz-Gly-Pro-D-Leu-Ala-NHOH) (FN-437)
(BBRC, 1994, Vol. 199, p. 1442-1446).
[0028] Moreover, specific examples of metalloproteinase inhibitor
of the present invention that can be used include various plant
extracts and purified products obtained therefrom. Examples of such
plants include Thymus serpyllum L., Valeriana faureie Briquet or
other similar plants (Valerianaceae), Diospyros kaki Thunberg
(Ebenaceae), Astragalus sinicus Linne (Leguminosae) Crataegus
cuneata Siebold et Zuccarini (Rosaceae), Paeonia suffruticosa
Andrews (Poeonia montan Sims) (Paconiaceae), Thea sinensis Linne
var. assamica Pierre (Thcaccae), Eucalyptus globulus Labillardiere
or its similar plants (Myrtaceae), Potentilla tormentilla Schrk
(Rosaceae), Tilia corda: a Mill., Tilia platyphyllus Scop., Tilia
europaea Linne (Tiliaceae), Betula alba Linne (Betulaceze),
Origanummajorana L., Uncaria gambir Roxburgh (Rubiaceae), Juglans
regia Linne var. sinensis De Candolie or its similar plants
(Juglandaceae), Sophora flavescens Aiton (Leguminosae), Sanguisorba
officinalis Linne (Rosaceae), Hypericum perforatum Linne or
Hypericum erectum Thunberg (Guttiferae), Thea sinensis Linne
(Theaceae), Curcuma longa L (Zingiberaceae), purified extracts of
Curcuma longa L including Symplocos racemosa and Cyperus rotundus,
Cyperus scariosus, Gaultheria fragrantissima, Acacia fornensia,
Terminalia chebula, Ficus bengalensis, Cassia fistula Linn, Lyonia
ovalifolia, Calophyllum inophyllum and Ficus religiosa.
[0029] Extracts of these plants are obtained from the root, leaves,
stem or flower in the case of herbaceous plants, or from the root,
buds, bark, fruits, leaves or flowers in the case of woody
plants.
[0030] Extracts from these plants are obtained by drying the plant
material as necessary and cutting or crushing as necessary,
followed by extracting into an aqueous extracting agent or organic
solvent. Examples of aqueous extracting agents that can be used
include cold water, warm water and hot water at the boiling point
or lower temperature, while examples of organic solvents that can
be used include methanol, ethanol, 1,3-butanediol and ether at
normal temperature or heated.
[0031] Examples of matrix proteins in the present invention include
laminin, type IV collagen, type VII collagen, parlecan and nidogen,
which are constituent components of the basement membrane.
[0032] Matrix Protein Production Promoting Agent
[0033] Examples of the matrix protein production promoting agent
used in the present invention include, in the case of promoting the
production of these proteins, soy bean lysolecithin, transforming
growth factor .alpha. (TGF.alpha.), transforming growth factor
.beta.1 (TGFb1), transforming growth factor .beta.2 (TGFb2),
transforming growth factor .beta.3 (TGFb3) and epithelium growth
factor (EGF).
[0034] In the case the artificial skin formation promoting agent or
skin basement membrane stabilizing agent of the present invention
contains matrix metalloproteinase inhibitor and matrix protein
production promoting agent, the ratio of matrix metalloproteinase
inhibitor and matrix protein production promoting agent, although
varying according to their types and activity, is roughly
1:10.sup.6 to 10.sup.6:1.
[0035] Artificial Skin
[0036] Any arbitrary medium that is conventionally used for the
production of artificial skin can be used for the basal medium used
to produce artificial skin in the present invention, and examples
of such media include Dulbecco's Modified Eagle's Medium (DMEM)
containing 10% fetal calf serum, DMEM-Ham's F12 (3:1) medium
containing 10% fetal calf serum, 5 .mu.g/ml of transferrin, 5
.mu.g/ml of insulin, 2 nM tri-iodotyrosine, 0.1 nM cholera toxin
and 0.4 .mu.g/ml of hydrocortisone, and keratinocyte growth medium
(KGM) and DMEM containing 10% fetal calf serum mixed at a ratio of
1:1. Although varying according to the particular type, the amount
of matrix metalloproteinase inhibitor added to these basal media is
about 1 nmol/L to 10.sup.-2 mole/L. In addition, the amount of
matrix protein production promoting agent added to the basal medium
is about 1 ng/L to 1 g/L.
[0037] In the production of artificial skin of the present
invention, contracted type I collagen gel containing human
fibroblasts is first allowed to stand undisturbed on a metal mesh.
The contracted type I collagen containing human fibroblasts can be
prepared in, for example, the manner described below. After
preparing a collagen solution in which fibroblasts are suspended
over ice, the collagen is gelled in a Petri dish. Next, the gel is
peeled from the walls of the Petri dish and the collagen gel is
contracted in a CO.sub.2 incubator.
[0038] Next, epidermal cells, such as normal human epidermal
keratinocytes, are cultured on the above collagen gel to form
epidermal. Formation of an epidermal layer by culturing epidermal
cells can be carried out in the following manner. Contracted
collagen gel is placed on a metal mesh, and a glass ring is placed
on this gel. A suspension of epidermal keratinocytes originating in
human foreskin is placed in the glass ring while preventing liquid
leaking. The keratinocytes are adhered in a CO.sub.2 incubator,
followed by removal of the ring. The above medium is filled to the
boundary of the epidermal layer and cornified layer is formed by
continuing culturing while exposing the epidermal layer to the
air.
[0039] According to this method, artificial skin is obtained that
approximates natural skin in which an adequate basement membrane is
formed between a dermal layer and epidermal layer composed of
contracted type I collagen gel containing fibroblasts.
[0040] Skin Basement Membrane Formation Promoting Agent
[0041] According to the result of a study on basement membrane
structural changes of facial skin for which cosmetics are primarily
used that focused on a report of structural changes characterized
by reduplication of the basement membrane accompanying aging as one
of the changes that accompany aging (Lavker, J., Invest. Dermal.
1979, 73, 59-66), structural changes in the basement membrane begin
to occur in the late twenties and have been found to accumulate
with age. These structure changes of basement membrane are skin
changes that precede skin changes accompanying aging, such as the
formation of wrinkles, hardening or decreased resiliency.
[0042] Thus, it is essential for expression of normal skin function
that epidermal basal cells be securely bound to the basement
membrane, and promotion of basement membrane formation is
considered to be important for this. As a result of studying
wrinkle formation inhibitors using an ultraviolet radiation aged
mouse model based on the presumption of routine ultraviolet rays,
activity that inhibits wrinkle formation was found in drugs that
are able to inhibit matrix metalloproteinase. Thus, agents that
promote regeneration and repair of the basement membrane in the
skin are effective as inhibitors of skin aging.
[0043] Thus, according to the present invention, a skin activating
composition or basement membrane formation promoting artificial
skin culturing liquid is newly provided that contains one type or a
mixture of two or more types of the above matrix metalloproteinase
inhibiting compound, or one type or a mixture of two or more types
of this matrix metalloproteinase inhibiting compound and one type
or a mixture of two or more types of a compound that demonstrates
activity which increases the production of matrix protein, at a
concentration sufficient for exhibiting effects that promote
regeneration, repair and formation of skin basement membrane. This
skin activation refers to, for example, the prevention or
improvement of decreased skin function accompanying structural
changes in basement membrane caused by aging and so forth, and more
specifically, skin wrinkling and hardening.
[0044] The above concentration sufficient for exhibiting effects
that promote regeneration, repair and formation of skin basement
membrane refers may vary according to the type of compounds used,
other components or vehicles used to prepare said composition, and
the duration of use.
[0045] The skin basement membrane formation promoting agent of the
present invention can contain active ingredient in the form of
matrix metalloproteinase inhibitor, or matrix metalloproteinase
inhibitor and matrix protein production promoting agent, at
0.000001 to 60 wt %, and preferably 0.00001 to 60 wt %, relative to
the composition.
[0046] The skin basement membrane stabilizing agent of the present
invention can be in the form of an aqueous solution, oil, other
solution, milky liquid, cream, gel, suspension, microcapsules,
powder, granules, capsules or solid, and after being prepared in
any of these forms according to methods which are themselves known,
can be coated, adhered, sprayed, injected, consumed or inserted
into the body in the form of a lotion, milky liquids, cream,
ointment, paste, poultice, aerosol, injection, medication
(including tablets, powders, granules, pills, syrups and troaches)
or suppository. Among these preparation forms, skin topical
preparations such as lotions, milky liquids, creams, ointments,
pastes, poultices and aerosols are considered to be preparation
forms that are suitable for the composition of the present
invention. The skin topical preparations described here include
prescription drugs, non-prescription drugs and cosmetics, and are
to be used with the same meaning hereinafter.
[0047] Vehicles and fragrances routinely used when preparing such
compositions, as well as oils, surfactants, antiseptics,
sequestering agents, water-soluble polymers, thickeners, powder
ingredients, ultraviolet defensive agents, moisturizing agents,
pharmacologically effective ingredients, antioxidants, pH
adjusters, cleaning agents, drying agents, emulsifiers and so forth
can be suitably blended into the composition of the present
invention. In the case of blending each of these ingredients into
the skin activating composition of the present invention, it is
necessary to blend them within a range that does not impair the
desired effect of the present invention.
[0048] Examples of the above oil include liquid oils, solid oils,
waxes, hydrocarbon oils, higher fatty acids, higher alcohols,
synthetic ester oils and silicones.
[0049] More specifically, examples of liquid oils include avocado
oil, tsubaki oil, primrose oil, turtle oil, macadamia nut oil, corn
oil, mink oil, olive oil, rape seed oil, egg yolk oil, sesame oil,
persic oil, wheat germ oil, sasanqua oil, castor oil, linseed oil,
safflower oil, cottonseed oil, perilla oil, soy bean oil, peanut
oil, theine oil, kaya oil, rice bran oil, Chinese wood oil,
Japanese wood oil, hohoba oil, germ oil, triglycerin, trioctanoic
glycerin and triisopalmitic glycerin; examples of solid oils
include cacao butter, coconut oil, horse tallow, hardened coconut
oil, palm oil, beef tallow, goat tallow, hardened beef tallow, palm
heart oil, pork tallow, beef bone tallow, haze heart oil, hardened
oil, beef leg tallow, haze wax and hardened castor oil; examples of
waxes include beeswax, candelilla wax, cotton wax, carnauba wax,
bayberry wax, tree wax, spermaceti, montan wax, bran wax, lanolin,
kapok wax, lanolin acetate, liquid lanolin, sugar cane wax,
isopropyl lanolin fatty acid, hexyl laurate, reduced lanolin,
jojoba wax, hard lanolin, shellac wax, POE lanolin alcohol ether,
POE lanolin alcohol acetate, POE cholesterol ether, lanolin fatty
acid polyethylene glycol and POE hydrogenated lanolin alcohol
ether; and examples of hydrocarbon oils include liquid paraffin,
ozokerite, squalene, pristan, paraffin, ceresin, squalene,
Vaseline.TM. (petroleum jelly) and microcrystalline wax.
[0050] Examples of higher fatty acids include lauric acid, myristic
acid, palmitic acid, stearic acid, behenic (beheninic) acid, oleic
acid, 12-hydroxystearic acid, undecylenic acid, tolic acid,
isostearic acid, linoleic acid, linolenic acid, eicosapentanoic
acid (EPA) and docosahexanoic acid (DHA).
[0051] Examples of higher alcohols include lauryl alcohol, cetyl
alcohol, stearyl alcohol, behenyl alcohol, myristyl alcohol, oleyl
alcohol, cetostearyl alcohol and other straight chain alcohols, as
well as monostearyl glycerin ether (vatyl alcohol),
2-decyltetradecinol, lanolin alcohol, cholesterol, phytosterol,
hexyldodecanol, isostearyl alcohol, octyldecanol and other branched
chain alcohols.
[0052] Examples of synthetic ester oils include isopropyl
myristate, cetyl octanoate, octyldodecyl myristate, isopropyl
palmitate, butyl stearate, hexyl laurate, myristyl myristate, decyl
oleate, hexyldecyl dimethyloctanoate, cetyl lactate, myristyl
lactate, lanolin acetate, isocetyl stearate, isocetyl isostearate,
cholesteryl 12-hydroxystearate, ethylene glycol di-2-ethylhexylate,
dipentaerythritol fatty acid ester, n-alkylglyol monoisostearate,
glycerin di-2-heptylundecanoate, trimethylolpropane
tri-2-ethylhexylate, trimethylolpropane triisostearate,
pentaneerythritol tetra-2-ethylhexylate, glycerin
tri-2-ethylhexylate, trimethylolpropane triisostearate,
cetyl-2-ethyl hexanoate, 2-ethylhexyl palmitate, glycerin
trimyristate, tri-2-heptylundecanoic glyceride, castor oil fatty
acid methyl ester, oleic oil, cetostearyl alcohol, acetoglyceride,
2-heptylundecyl palmitate, diisobutyl adipate,
N-lauroyl-L-glutamate-2-octyldodecyl ester, di-2-heptylundecyl
adipate, ethyl laurate, di-2-ethylhexyl sebacate, 2-hexyldecyl
myristate, 2-hexyldecyl palmitate, 2-hexyldecyl adipate,
diisopropyl sebacate, 2-ethylhexyl succinate, ethyl acetate, butyl
acetate, amyl acetate and triethyl citrate.
[0053] Examples of silicones include dimethyl polysiloxane,
methylphenyl polysiloxane, methylhydrogen polysiloxane and other
linear polysiloxanes, decamethyl polysiloxane, dodecamethyl
polysiloxane, tetramethyltetrahydrogen polysiloxane and other
cyclic polysiloxanes, and silicon resin and silicone rubber that
form a three-dimensional mesh structure.
[0054] The skin basement membrane stabilizing agent of the present
invention may contain anionic surfactant, cationic surfactant,
amphoteric surfactant or nonionic surfactant and so forth either
alone or in combination.
[0055] Examples of anionic surfactants include fatty acid soaps
such as soap base, sodium laurate and sodium palmitate; higher
alkyl sulfate esters such as sodium lauryl sulfate and potassium
lauryl sulfate; alkyl ether sulfate ester salts such as POE
triethanol amine lauryl sulfate and POE sodium lauryl sulfate;
N-acylsarcosinic acids such as sodium lauroylsarcosine; higher
fatty acid amide sulfonates such as sodium N-myristoyl-N-taurine,
sodium coconut oil fatty acid methyltauride and sodium
laurylmethyltauride; phosphate esters such as POE sodium oleyl
ether phosphate and POE stearyl ether phosphate; sulfosuccinates
such as sodium di-2-ethylhexylsulfosuccinate, sodium
monolauroylmonoethanolamide polyoxyethylene sulfosuccinate and
sodium laurylpolypropylene glycol sulfosuccinate; alkylbenzene
sulfonates such as linear sodium dodecylbenzene sulfonate, linear
dodecylbenzene sulfonate triethanolamine and linear dodecylbenzene
sulfonate; N-acylglutamates such as monosodium N-lauroylglutamate,
disodium N-stearoylglutamate and monosodium
N-myristoyl-L-glutamate; higher fatty acid ester sulfate esters
such as sodium hardened coconut oil fatty acid glycerin sulfate;
sulfated oils such as Turkey red oil oil; as well as POE alkyl
ether carboxylic acid, POE alkylaryl ether carboxylic acid,
.alpha.-olefin sulfonate, higher fatty acid ester sulfonate,
secondary alcohol sulfate ester, higher fatty acid alkylolamide
sulfate ester, sodium lauroylmonoethanolamide succinate,
N-palmitoylaspartate ditriethanolamine and sodium casein.
[0056] Examples of cationic surfactants include alkyl trimethyl
ammonium salts such as stearyl trimethyl ammonium chloride and
lauryl trimethyl ammonium chloride, dialkyl dimethyl ammonium salts
such as distearyl dimethyl ammonium chloride, alkyl pyridinium
salts such as (N,N'-dimethyl-3,5-methylene piperidinium) chloride
and cetyl pyridinium chloride, alkyl quaternary ammonium salts,
alkyl dimethylbenzyl ammonium salts, alkyl isoquinolinium salts,
dialkyl morpholinium salts, POE alkyl amines, alkyl amine salts,
polyamine fatty acid derivatives, amyl alcohol fatty acid
derivatives, benzalkonium chloride and benzetonium chloride.
[0057] Examples of amphoteric surfactants include imidazoline-based
amphoteric surfactants such as
2-undecyl-N,N,N-(hydroxyethylcarboxymethyl)-2-imidazoline sodium
and 2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy-2-sodium
salt, and betaine-based amphoteric surfactants such as
2-heptadecyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine,
lauryldimethylaminoacetate betaine, alkyl betaine, amidobetaine and
sulfobetaine.
[0058] Examples of lipophilic nonionic surfactants include sorbitan
fatty acid esters such as sorbitan monooleate, sorbitan
monoisostearate, sorbitan monolaurate, sorbitan monopalmitate,
sorbitan monostearate, sorbitan sesquioleate, sorbitan trioleate,
penta-2-ethylhexylic diglycerol sorbitan and tetra-2-ethylhexylic
diglycerol sorbitan, glycerin polyglycerin fatty acids such as
glycerin mono-cottonseed oil fatty acid, glycerin monoerucate,
glycerin sesquiolate, glycerin monostearate, glycerin
.alpha.,.alpha.'-oleic pyroglutamate, glycerin monostearate and
malic acid, propylene glycol fatty acid esters such as propylene
glycol monostearate, hardened castor oil derivatives, glycerin
alkyl ether and polyoxyethylene-methylpolysiloxane copolymers.
[0059] Examples of hydrophilic nonionic surfactants include POE
sorbitan fatty acid esters such as POE sorbitan monooleate, POE
sorbitan monostearate, POE sorbitan monooleate and POE sorbitan
tetraoleate, POE sorbitol fatty acid esters such as POE sorbitol
monolaurate, POE sorbitol monooleate, POE sorbitol pentaoleate and
POE sorbitol monostearate, POE glycerin fatty acid esters such as
POE glycerin monostearate, POE glycerin monoisostearate and POE
glycerin triisostearate, POE fatty acid esters such as POE
monooleate, POE distearate, POE monodioleate and ethylene glycol
distearate, POE alkyl ethers such as POE lauryl ether, POE oleyl
ether, POL stearyl ether, POE behenyl ether, POE 2-octyldodecyl
ether and POE cholestanol ether, POE alkylphenyl ethers such as POE
octylphenyl ether, POE nonylphenyl ether and POE dinonylphenyl
ether, plurallonics such as pluronic, POE-POP alkyl ethers such as
POE-POP cetyl ether, POE-POP 2-decyltetradecyl ether, POE-POP
monobutyl ether, POE-POP hydrogenated lanolin and POE-POP glycerin
ether, tetra POE-tetra POP ethylene diamine condensation products
such as tetronic, POE castor oil/hardened castor oil derivatives
such as POE castor oil, POE hardened castor oil, POE hardened
castor oil monoisostearate, POE hardened castor oil triisostearate,
POE hardened castor oil monopyroglutamate monoisostearate diester
and POE hardened castor oil maleate, POE beeswax-lanolin
derivatives such as POE sorbitol beeswax, alkanol amides such as
coconut oil fatty acid diethanol amide, lauric monoethanol amide
and fatty acid isopropanol amide, POE propylene glycol fatty acid
esters, POE alkyl amines, POE fatty acid amides, sucrose fatty acid
esters, POE nonylphenyl formaldehyde compounds, alkylethoxydimethyl
amine oxides and trioleyl phosphates.
[0060] Examples of the above preservatives include methyl
parabenzene, ethyl parabenzene and butyl parabenzene.
[0061] Examples of the above sequestering agents include sodium
edetate and EDTA.
[0062] Examples of the above water-soluble polymers include natural
polymers, semi-synthetic polymers, synthetic polymers and inorganic
polymers.
[0063] Examples of natural water-soluble polymers include plant
polymers such as gum arabic, tragacanth gum, galactan, guar gum,
carob gum, karaya gum, carageenan, tamarind gum, xanthane gum,
pectin, agar, queenseed marmelo, algea colloid (brown algae
extract), starch (rice, corn, potato, wheat) and glycyrrhizic acid,
microbial polymers such as xanthane gum, dextran succinoglucan and
pluran, and animal polymers such as collagen, casein, albumin and
gelatin.
[0064] Examples of semi-synthetic water-soluble polymers include
starch-based polymers such as dextrin, carboxymethyl starch and
methylhydroxypropyl starch, cellulose-based polymers such as methyl
cellulose, nitrocellulose, ethyl cellulose, methylhydroxypropyl
cellulose, hydroxyethyl cellulose, cellulose dimethyl dialkyl
(12-20) ammonium sulfate, hydroxypropyl cellulose, sodium
carboxymethyl cellulose (CMC), crystalline cellulose and powdered
cellulose, alginate-based polymers such as sodium alginate and
propylene glycol alginate ester.
[0065] Examples of synthetic water-soluble polymers include
vinyl-based polymers such as polyvinyl alcohol, polyvinyl methyl
ether, polyvinyl pyrrolidone, carboxyvinyl polymer and alkyl
denatured carboxyvinyl polymer, polyoxyethylene-based polymers such
as polyethylene glycol 2000, 4000 and 6000, polyoxyethylene
polyoxypropylene copolymer-based polymers, acrylic polymers such as
sodium polyacrylate, polyethylene acrylate and polyacrylamide,
polyethylene imines and cationic polymers.
[0066] Examples of inorganic water-soluble polymers include
bentonite, magnesium aluminum silicate, raponite, hectrite and
silicic anhydride.
[0067] Examples of the above powder component include inorganic
powders such as talc, kaolin, mica, sericite, muscovite,
phlogopite, synthetic mica, lepidolite, biotite, lithia mica,
vermiculite, magnesium carbonate, calcium carbonate, aluminum
silicate, barium silicate, calcium silicate, magnesium silicate,
strontium silicate, metal tungstate, magnesium, silica, zeolite,
barium sulfate, baked calcium sulfate (baked gypsum), calcium
phosphate, fluoroapatite, hydroxyapatite, ceramic powder, metal
soap (zinc myristate, calcium palmitate, aluminum stearate) and
boron nitride, organic powders such as polyamide resin powder
(Nylon powder), polyethylene powder, polymethyl methacrylate
powder, polystyrene powder, styrene and acrylic acid copolymer
resin powder, benzoguanamine resin powder, polytetrafluoroethylene
powder and cellulose powder, inorganic white pigments such as
titanium dioxide and zinc oxide, inorganic red pigments such as
iron oxide (rouge) and iron titanate, inorganic brown pigments such
as .gamma.-iron oxide, inorganic yellow pigments such as yellow
iron oxide and ocher, inorganic black pigments such as black iron
oxide, carbon black and low order titanium oxide, inorganic violet
pigments such as mango violet and cobalt violet, inorganic green
pigments such as chromium oxide, chromium hydroxide and cobalt
titanate, inorganic blue pigments such as ultramarine and Prussian
blue, pearl pigments such as titanium oxide coated mica, titanium
oxide coated bismuth oxychloride, titanium oxide coated talc,
colored titanium oxide coated mica, bismuth oxychloride and fish
scale foil, metal powder pigments such as aluminum powder and
copper powder, zirconium, barium or aluminum lake organic pigments
such as red no. 201, red no. 202, red no. 204, red no. 205, red no.
220, red no. 226, red no. 228, red no. 405, orange no. 203, orange
no. 204, yellow no. 205, yellow no. 401, yellow no. 404, red no. 3,
red no. 104, red no. 106, red no. 227, red no. 230, red no. 401,
red no. 505, orange no. 205, yellow no. 4, yellow no. 5, yellow no.
202, yellow no. 203, green no. 3 and blue no. 1, natural pigments
such as chlorophyll and .beta.-carotene, and colorants such as
titanium yellow, carsamine and saffron red.
[0068] Examples of the above ultraviolet defensive agents include
both "ultraviolet absorbers", which are substances that chemically
absorb ultraviolet rays, and "ultraviolet blockers", which are
substances that scatter and reflect ultraviolet rays by physical
action.
[0069] Namely, examples of long-wavelength ultraviolet (UVA)
absorbers include anthranilic acid-based ultraviolet absorbers such
as methyl anthranilate and homomenthyl-N-acetylanthranilate,
benzophenone-based ultraviolet absorbers such as
2,4-dihydroxybenzophenone, 2,2-dihydroxy-4-methoxybenzophenone,
2,2'-dihydroxy-4,4'-dimethoxybenzophenone,
2,2',4,4'-tetrahydroxybenzophenone,
2-hydroxy-4-methoxybenzophenone,
2-hydroxy-4-methoxy-4'-methylbenzophenone,
2-hydroxy-4-methoxybenzophenone-5-sulfonic acid,
4-phenylbenzophenone,
2-ethylhexyl-4'-phenyl-benzophenone-2-carboxylate,
2-hydroxy-4-n-octoxybenzophenone and
4-hydroxy-3-carboxybenzophenone, benzotriazole-based ultraviolet
absorbers such as 2,2'-hydroxy-5-methylphenylbenzotriazole,
2-(2'-hydroxy-5'-t-octylphenyl)benzotriazole and
2-(2'-hydroxy-5'-methylphenyl)benzotriazole, dianisoylmethane and
4-methoxy-4'-t-butyldibenzoylmethane.
[0070] Among these long-wavelength ultraviolet absorbers,
4-methoxy-4'-tert-butyldibenzoylmethane,
2-hydroxy-4-methoxybenzophenone and 2-hydroxy-4-methoxybenzophenone
derivatives such as 2-hydroxy-4-methoxybenzophenone-5-sulfonate are
preferable because they are long-wavelength ultraviolet absorbers
having superior safety and effectiveness.
[0071] In addition, examples of medium-wavelength ultraviolet (UVB)
absorbers include benzoic acid-based ultraviolet absorbers such as
paraminobenzoic acid (to be abbreviated as PABA), PABA monoglycerin
ester, N,N-dipropoxy PABA ethyl ester, N,N-diethoxy PABA ethyl
ester, N,N-dimethyl PABA ethyl ester, N,N-dimethyl PABA butyl ester
and N,N-dimethyl PABA amyl ester, salicylic acid-based ultraviolet
absorbers such as dipropylene glycol salicylate, ethylene glycol
salicylate, myristyl salicylate, methyl salicylate, amyl
salicylate, menthyl salicylate, homomenthyl salicylate, octyl
salicylate, phenyl salicylate, benzyl salicylate and p-isopropanol
phenyl salicylate, cinnamic acid-based ultraviolet absorbers such
as octyl cinnamate, ethyl-4-isopropyl cinnamate,
methyl-2,5-diisopropyl cinnamate, ethyl-2,4-diisopropyl cinnamate,
methyl-2,4-diisopropyl cinnamate, propyl-p-methoxycinnamate,
isopropyl-p-methoxycinnamate, isoamyl-p-methoxycinnamate,
octyl-p-methoxycinnamate (2-ethylhexyl-p-methoxycinnamate),
2-ethoxymethyl-p-methoxycinnamate, cyclohexyl-p-methoxycinnamate,
ethyl-.alpha.-cyano-.beta.-phenylcinnamate,
2-ethylhexyl-.alpha.-cyano-.beta.-phenylcinnamate,
glycerylmono-2-ethylhexanoyl-diparamethoxycinnamate,
methoxycinnamate octyl,
3,4,5-trimethoxycinnamate-3-methyl-4-[methylbis(trimethylsiloxy)si-
lyl]butyl and p-dimethoxycinnamate monoethyl ester, camphor
derivatives such as 3-(4'-methylbenzylidene)-d,1-camphor,
3-benzylidene-d,1-camphor and
5-(3,3-dimethyl-2-norvolnilidene)-3-penten-2-one, urocanic acid,
urocanic ethyl ester, 2-phenyl-5-methylbenzooxazole and
dibenzaladine.
[0072] Moreover, examples of ultraviolet blockers include titanium
oxide (TiO.sub.2), talc (MgSiO.sub.2), carmine (FeO.sub.2),
bentonite, kaolin and zinc oxide (ZnO).
[0073] Examples of the above moisturizing agents include
polyethylene glycol, propylene glycol, glycerin, 1,3-butylene
glycol, hexylene glycol, xylitol, sorbitol, maltitol, chondroitin
sulfuric acid, hyaluronic acid, mucoitin sulfuric acid, charoninic
acid, atherocollagen, cholesteryl-12-hydroxystearate, sodium
lactate, bile acid salt, d1-pyrrolidone carboxylate, short chain
soluble collagen, diglycerin (EO) PO addition product, Chestnut
rose Fruit extract, milfoil extract and melilote extract.
[0074] Examples of the above pharmacologically effective
ingredients include whiteners such as albutin, vitamin C and its
derivatives, kojic acid placental extract, glutathione and
saxifrage extract, antiphlogistics such as glycyrrhizic acid
derivatives, glycyrrhetinic acid derivatives, salicylic acid
derivatives, hinokitiol, zinc oxide and allantoin, activators such
as royal jelly, actinolite, cholesterol derivatives and calf blood
extract, circulation promoters such as nonylic urenylamide,
nicotinic benzyl ester, nicotinic .beta.-butoxyethyl ester,
capsisin, zingerone, cantharidin, ichthammol, caffeine, tannic
acid, .alpha.-borneol, nicotinic tocopherol, inositol
hexanicotinate, cyclandelate, cinnarizine, tolazoline,
acetylcholine, verapamil, cepharanthin and .gamma.-olizanol,
antiseborrheics such as sulfur and thianthol, and for various
purposes, Phellodendron Bark extract component, goldthread extract
component, Lithospermum Root extract component, peony extract
component, gentian extract component, birch extract component, sage
extract component, loquat extract component, carrot extract
component, aloe extract component, mallow extract component, iris
extract component, grape extract component, Coix Seed extract
component, sponge cucumber extract component, lily extract
component, saffron extract component, Cnidium Officinale Root
extract component, Ginger Root extract component, Saint John's-wort
extract component, Restharrow Root extract component, rosemary
extract component, garlic extract component, red pepper extract
component, dried orange peel, Japanese angelica root and so forth,
vitamin A substances such as retinol and retinol acetate, vitamin
B.sub.2 substances such as riboflavin butyrate and riboflavin
adenine nucleotide, vitamin B2 substances such as pyridoxine
hydrochloride and pyridoxine dioctanoate, vitamin C substances such
as L-ascorbic acid, L-ascorbate dipalmitate ester,
L-ascorbate-2-sodium sulfate, L-ascorbate phosphate ester and
dipotassium DL-.alpha.-tocopherol-L-ascorbate phosphate diester,
pantothenic acids such as calcium pantothenate, D-pantothenyl
alcohol, pantothenyl ethyl ether and acetylpentothenyl ethyl ether,
vitamin D substances such as ergocalciferol and cholecalciferol,
nicotinic acids such as nicotinic acid, nicotinic amide and benzyl
nicotinate, vitamin E substances such as .alpha.-tocopherol and
DL-.alpha.-tocopherol succinate, and other vitamins such as vitamin
P and biotin.
[0075] Furthermore, these pharmacological components can be widely
blended within a range over which the desired pharmacological
effect of the present invention is not impaired by that
blending.
[0076] The composition of the present invention prepared in this
manner prevents decreased function accompanying structural changes
of basement membrane, and promotes activation of the skin.
EXAMPLES
[0077] The following provides a more detailed explanation of the
present invention through its examples.
Example 1
Production of Artificial Skin Using Matrix Metalloproteinase
Inhibitor
[0078] Collagen gel was obtained by preparing 10 ml of a collagen
solution of suspended fibroblasts (0.3 to 1.times.10.sup.5
cells/ml) originating in human dermis (product I-AC of Koken Co.,
Ltd. was used for the collagen) on ice followed by gelling the
collagen at 37.degree. C. in a 60 mm Petri dish. Next, the gel was
peeled from the walls of the Petri dish and the collagen gel was
contracted in a CO.sub.2 incubator.
[0079] This contracted collagen gel equivalent to dermis was placed
on a metal mesh and a glass ring (inner diameter: 12 mm) was placed
over this gel. 0.4 ml of a suspension of epidermal keratinocytes
(1.times.10.sup.6/ml) (mixed medium of KGM-DMEM containing 5% fetal
calf serum (1:1)) were placed inside the glass ring to prevent
leakage of liquid. After adhering keratinocytes in a CO.sub.2
incubator overnight, the ring was removed on the following day. The
above medium was filled to the boundary of the epidermal layer and
culturing was continued while exposing the epidermal layer to air
to produce a skin model having a stratified epidermis exhibiting
corneal layer formation.
[0080] After inoculating the epidermic cells, the medium was
changed to that containing either (1) 10 .mu.M CGS27023A (matrix
metalloproteinase inhibitor) or (2) 300 .mu.M MMP-inhibitor
(p-NH.sub.2-Bz-Gly-Pro-D-Leu-D-Ala-NHOH (FN-437) (BBRC, 1994, 199,
p. 1442-1446) (acquired from Cabbiochem. Novabiochem Corporation)
(matrix metalloproteinase inhibitor)) starting in the first week,
and then replaced with medium containing the same type and same
concentration of matrix metalloproteinase inhibitor every 2-3 days
after that and cultured for 2 weeks. In addition, (3) similar
culturing was carried out without adding matrix metalloproteinase
inhibitor for use as a control.
[0081] Those results are shown in FIG. 1. As is clear from the
figure, although a basement membrane-like structure was not
observed directly beneath the basal cells of the epidermis in
control (3), in the case of adding matrix metalloproteinase
inhibitors (1) CGS27023A compound or (2) MMP-inhibitor, promotion
of basement membrane formation was clearly observed.
[0082] A similar experiment was carried out for extracts of the
plants indicated below instead of the above matrix
metalloproteinase inhibitor compounds to confirm promotion of
basement membrane formation.
[0083] Similar effects were confirmed for Thymus serpyllum L.,
Valeriana fauriei Briquet or other similar plants (Valerianaceae),
Diospyros kaki Thunberg (Ebenaceae), Astragalus sinicus Linne
(Leguminosae), Crataegus cuneata Siebold et Zuccarini (Rosaceae),
Paeonia suffruticosa Andrews (Poeonia montan Sims) (Paconiaceae),
Thea sinensis Linne var. assamica Pierre (Thcaccae), Eucalyptus
globulus Labillardiere or its similar plants (Myrtaceae),
Potentilla tormentilla Schrk (Rosaceae), Tilia corda: a Mill.,
Tilia platyphyllus Scop., Tilia europaea Linne (Tiliaceae), Betula
alba Linne (Betulaceze), Origanum majorana L., Uncaria gambir
Roxburgh (Rubiaceae), Juglans regia Linne var. sinensis De Candolie
or its similar plants (Juglandaceae), Sophora flavescens Aiton
(Leguminosae), Sanguisorba officinalis Linne (Rosaceae), Hypericum
perforatum Linne or Hypericum erectum Thunberg (Guttiferae), Thea
sinensis Linne (Theaceae), Curcuma longa L (Zingiberaceae),
purified extracts of Curcuma longa L including Symplocos racemosa
and Cyperus rotundus, Cyperpus scariosus, Gaultheria
fragrantissima, Acacia formensia, Terminalia chebula, Ficus
bengalensis, Cassia fistula Linn, Lyonia ovalifolia, Calophyllum
inophyllum and Ficus religiosa.
Example 2
Production of Artificial Skin Using Both Matrix Metalloproteinase
Inhibitor and Matrix Protein Production Promoting
Agent
[0084] Although the same method as Example 1 was repeated, the
following substances were used as test substances. [0085] (1) 10
.mu.M matrix metalloproteinase inhibitor CGS27023A only; [0086] (2)
10 .mu.M matrix metalloproteinase inhibitor CGS27023A and 10 ng/ml
of matrix metalloproteinase protein production promoting agent,
Transforming Growth Factor .alpha. (TGFa); [0087] (3) 10 .mu.M
matrix metalloproteinase inhibitor CGS27023A and 10 ng/ml of matrix
protein production promoting agent, Transforming Growth Factor
.beta.1 (TGFb1); [0088] (4) 10 .mu.M matrix metalloproteinase
inhibitor CGS27023A, 10 ng/ml of matrix protein production
promoting agent, Transforming Growth Factor .alpha. (TGFa) and 10
ng/ml of Transforming Growth Factor .beta.1 (TGFb1); and [0089] (5)
culturing of a control to which none of the above were added.
[0090] The formed artificial skin was stained by hematoxylin-eosin
(H & E) and immunostaining (using anti-laminin 5 (LN5)
antibody, anti-type IV collagen (IVC) antibody, and anti-type VII
collagen (VIIC) antibody). Those results are shown in FIG. 2, FIG.
3 and Table 1. Furthermore, the results for H & E and LN5 are
shown in FIG. 2, while the results for IVC and VIIC are shown in
FIG. 3.
[0091] In the control, adhesion of epidermis/dermis joined portions
was weak, the epidermis easily peeled from the dermis and there
were voids observed in the tissue. In contrast, in the group
treated with matrix metalloproteinase inhibitor CGS27023A, adhesion
between the epidermis and dermis was clear. In addition, in the
case of adding matrix protein production promoting agent,
Transforming Growth Factor .alpha. (TGFa), Transforming Growth
Factor .beta.1 (TGFb1) or both (TGFa+TGFb1) in addition to matrix
metalloproteinase inhibitor CGS27023A, staining of type VII
collagen in particular was promoted significantly, thereby
confirming that adhesion between the epidermis and dermis was
further promoted. TABLE-US-00001 TABLE 1 Effects of Various Drugs
on Basement Membrane Formation in a Skin Model Deposition of
basement membrane component Basement Treatment Type IV Type VII
membrane conditions Laminin 5 collagen collagen formation
Non-treatment + + - - control group CGS27023A ++ ++ + + CGS27023A +
++ ++ ++ ++ soybean lecithin CGS27023A + TGF.alpha. ++ ++ ++
CGS27023A + TGF.beta.1 ++ ++ ++ CGS27023A + ++ ++ ++ TGF.alpha. +
TGF.beta.1
[0092] Culturing was carried out in the same manner as above using
the following plant extracts instead of the above matrix
metalloproteinase inhibitor CGS27023A.
[0093] Thymus serpyllum L., Valeriana fauriei Briquet or other
similar plants (Valerianaceae), Diospyros kaki Thunberg
(Ebenaceae), Astragalus sinicus Linne (Leguminosae), Crataegus
cuneata Siebold et Zuccarini (Rosaceae), Paeonia suffruticosa
Andrews (Poeonia montan Sims) (Paconiaceae), Thea sinensis Linne
var. assamica Pierre (Thcaccae), Eucalyptus globulus Labillardiere
or its similar plants (Myrtaceae), Potentilla tormentilla Schrk
(Rosaceae), Tilia corda: a Mill., Tilia platyphyllus Scop., Tilia
europaea Linne (Tiliaceae), Betula alba Linne (Betulaceze),
Origanum majorana L., Uncaria gambir Roxburgh (Rubiaceae), Juglans
regia Linne var. sinensis De Candolie or its similar plants
(Juglandaceae), Sophora flavescens Aiton (Leguminosae), Sanguisorba
officinalis Linne (Rosaceae), Hypericum perforatum Linne or
Hypericum erectum Thunberg (Guttiferae), Thea sinensis Linne
(Theaceae), Curcuma longa L (Zingiberaceae), purified extracts of
Curcuma longa L including Symplocos racemosa and Cyperus rotundus,
Cyperus scariosus, Gaultheria fragrantissima, Acacia formensia,
Terminalia chebula, Ficus bengalensis, Cassia fistula Linn, Lyonia
ovalifolia, Calophyllum inophyllum and Ficus religiosa.
[0094] As a result, results were obtained that were similar to the
case of using CGS27023A as matrix metalloproteinase inhibitor. In
addition, similar results were also obtained for Symplocos
racemosa, a purified extract of Curcuma longa L.
[0095] Moreover, a structure resembling a basement membrane was
continuously observed directly beneath the basal cells in the case
of using 30 .mu.g/ml of soy bean lecithin instead of Transforming
Growth Factor for the matrix protein production promoting agent.
Those results are shown in FIG. 4.
Example 3
Preparation of Artificial Skin Culture Medium
[0096] Matrix metalloproteinase inhibitor CGS27023A or
MMP-inhibitor were added to a concentration of 10 .mu.M to basal
medium comprising a 1:1 mixture of KGM (keratinocyte growth
medium), typically used to culture epidermic cells, and Dulbecco's
Modified Eagle's Medium containing 10% fetal calf serum, typically
used to culture fibroblasts. Furthermore, KGM was prepared by
adding HEPES (6.7 g/L), NaHCO.sub.3 (1.2 g/L), insulin (5 mg/L),
transferrin (10 mg/L), hydrocortisone (0.5 mg/L) and
phosphorylethanolamine (14.1 mg/L) to MCDB153 medium (10.93 g/L)
and adjusting the pH to 7.4 followed by the addition of 20 g of
bovine pituitary extract (Far East Pharmaceutical).
Example 4
Preparation of Artificial Skin Culture Medium
[0097] At least one type of Transforming Growth Factor .alpha.
(TGFa) or Transforming Growth Factor .beta.1 (TGFb1) were added as
matrix protein production promoting agent to the artificial skin
culture medium in Example 3 to a concentration of 10 ng/ml.
[0098] In addition, soy bean lecithin was added at 30 .mu.g/ml in
place of the above TGFa or TGFb1.
Preparation Example 1
Cream
[0099] TABLE-US-00002 Polyoxyethylene (addition of 20 moles) cetyl
alcohol ether 1.0 Methylphenyl polysiloxane (20 cs) 2.0 Liquid
paraffin 3.0 2-hydroxy-4-methoxybenzophenone 5.0 Active ingredient
0.2 Propylene glycol 5.0 Glycerin 2.0 Ethyl alcohol 15.0
Carboxyvinyl polymer 0.3 Hydroxypropyl cellulose 0.1
2-aminomethylpropanol 0.1 Antiseptic As suitable Fragrance As
suitable Ion exchanged water As suitable
[0100] (Production Method)
[0101] Propylene glycol, glycerin, ethyl alcohol, carboxyvinyl
polymer, hydroxypropyl cellulose and 2-aminomethylpropanol were
added to ion exchanged water followed by heating to 70.degree. C.
(aqueous phase).
[0102] Methylphenyl polysiloxane, liquid paraffin, polyoxyethylene
cetyl alcohol ether, antiseptic, 2-hydroxy-4-methoxybenzophenone,
active ingredient (such as lipidure and/or CGS27023A) and fragrance
were mixed and prepared to 70.degree. C. (oily phase).
[0103] After gradually adding the aqueous phase to the oily phase,
pre-emulsifying and obtaining homogeneous emulsified particles
using a homomixer, the emulsion was deaerated and cooled to obtain
a cream.
Preparation Example 2
Cream
[0104] TABLE-US-00003 Polyoxyethylene (addition of 20 moles) cetyl
alcohol ether 1.0 Methylphenyl polysiloxane (20 cs) 2.0 Liquid
paraffin 3.0 2-hydroxy-4-methoxybenzophenone 5.0 Active ingredient
0.2 Propylene glycol 5.0 Glycerin 2.0 Ethyl alcohol 15.0
Carboxyvinyl polymer 0.3 Hydroxypropyl cellulose 0.1
2-aminomethylpropanol 0.1 Antiseptic As suitable Fragrance As
suitable Ion exchanged water As suitable
[0105] (Production Method)
[0106] Propylene glycol, glycerin, ethyl alcohol, carboxyvinyl
polymer, hydroxypropyl cellulose and 2-aminomethylpropanol were
added to ion exchanged water followed by heating to 70.degree. C.
(aqueous phase).
[0107] Methylphenyl polysiloxane, liquid paraffin, polyoxyethylene
cetyl alcohol ether, antiseptic, 2-hydroxy-4-methoxybenzophenone,
active ingredient (such as lipidure and/or CGS27023A) and fragrance
were mixed and prepared to 70.degree. C. (oily phase).
Preparation Example 3
Milky Liquid
[0108] TABLE-US-00004 Wt % Cetyl alcohol 1.0 Beeswax 0.5 Vaseline
(Petroleum Jelly) 2.0 Squalene 6.0 Dimethyl polysiloxane 2.0 Ethyl
alcohol 5.0 Glycerin 4.0 1,3-butylene glycol 4.0 Active ingredient
0.1 Tranexamic acid 1.0 Polyoxyethylene (10) monooleic ester 1.0
Glycerol monostearic ester 1.0 Queenseed extract (5% aqueous
solution) 20.0 Antiseptic As suitable Fragrance As suitable Ion
exchanged water As suitable
[0109] (Production Method)
[0110] Glycerin and 1,3-butylene glycol were added to ion exchanged
water, mixed and heated to 70.degree. C. (aqueous phase). Cetyl
alcohol, beeswax, Vaseline (petroleum jelly), squalene, dimethyl
polysiloxane, active ingredient (such as lipidure or CGS27023A),
tranexamic acid, polyoxyethylene (10) monooleic ester, glycerol
monostearic ester and antiseptic were prepared in the form of a
mixture and heated to 70.degree. C. (oily phase). After adding the
aqueous phase to the oily phase and performing preliminary
emulsification, adding and stirring queenseed extract and ethyl
alcohol and obtaining homogeneous emulsified particles using a
homomixer, the emulsion was deaerated, filtered and cooled to
obtain a milky liquid.
Preparation Example 4
Milky Liquid
[0111] TABLE-US-00005 Wt % Cetyl alcohol 1.0 Beeswax 0.5 Vaseline
(Petroleum Jelly) 2.0 Squalene 6.0 Dimethyl polysiloxane 2.0 Ethyl
alcohol 5.0 Glycerin 4.0 1,3-butylene glycol 4.0 Active ingredient
0.1 Tranexamic acid 1.0 Polyoxyethylene (10) monooleic ester 1.0
Glycerol monostearic ester 1.0 Queenseed extract (5% aqueous
solution) 20.0 Antiseptic As suitable Fragrance As suitable Ion
exchanged water As suitable
[0112] (Production Method)
[0113] Glycerin and 1,3-butylene glycol were added to ion exchanged
water, mixed and heated to 70.degree. C. (aqueous phase). Cetyl
alcohol, beeswax, Vaseline (petroleum jelly), squalene, dimethyl
polysiloxane, active ingredient (such as lipidure or CGS27023A),
tranexamic acid, polyoxyethylene (10) monooleic ester, glycerol
monostearic ester and antiseptic were prepared in the form of a
mixture and heated to 70.degree. C. (oily phase). After adding the
aqueous phase to the oily phase and performing preliminary
emulsification, adding and stirring queenseed extract and ethyl
alcohol and obtaining homogeneous emulsified particles using a
homomixer, the emulsion was deaerated, filtered and cooled to
obtain a milky liquid.
[0114] After gradually adding the aqueous phase to the oily phase,
pre-emulsifying and obtaining homogeneous emulsified particles
using a homomixer, the emulsion was deaerated and cooled to obtain
a cream.
* * * * *