U.S. patent application number 10/733150 was filed with the patent office on 2004-09-02 for methods and compositions using compounds from fetal cells and tissues to improve condition of skin.
Invention is credited to Soo, Chia.
Application Number | 20040170615 10/733150 |
Document ID | / |
Family ID | 32507946 |
Filed Date | 2004-09-02 |
United States Patent
Application |
20040170615 |
Kind Code |
A1 |
Soo, Chia |
September 2, 2004 |
Methods and compositions using compounds from fetal cells and
tissues to improve condition of skin
Abstract
Compositions comprising one or more compounds expressed by fetal
tissues for modulating skin conditions, methods of identifying the
compounds, and methods of making and using the compounds are
provided.
Inventors: |
Soo, Chia; (Beverly Hills,
CA) |
Correspondence
Address: |
Zhaoyang Li
Squire, Sanders & Dempsey L.L.P.
Suite 300
One Maritime Plaza
San Francisco
CA
94111
US
|
Family ID: |
32507946 |
Appl. No.: |
10/733150 |
Filed: |
December 10, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60432519 |
Dec 11, 2002 |
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Current U.S.
Class: |
424/93.21 |
Current CPC
Class: |
A61P 11/00 20180101;
A61K 8/735 20130101; A61K 35/48 20130101; A61P 17/00 20180101; A61P
17/16 20180101; A61Q 19/00 20130101; A61Q 19/08 20130101; A61L
15/40 20130101; A61K 8/73 20130101 |
Class at
Publication: |
424/093.21 |
International
Class: |
A61K 048/00 |
Claims
What is claimed is:
1. A method of promoting skin regeneration comprising: a)
identifying compounds expressed by fetal tissues that promote skin
regeneration, and b) applying to skin of a mammal a composition
comprising one or more compounds expressed by fetal tissues.
2. The method of claim 1 wherein the compounds expressed by fetal
tissue are selected from the group consisting of natural compounds
derived from fetal tissues, compounds produced by recombinantly
expressing the compounds expressed by fetal tissues in genetically
engineered cells, and combinations thereof.
3. A method of promoting skin regeneration comprising: a) comparing
compounds expressed by adult tissue and compounds expressed by
fetal tissues, b) identifying compounds with higher expression in
adult tissues, c) identifying compounds that block the expression
of the compounds with higher expression in adult tissues and
designating the compounds that block the expression of the
compounds with higher expression in adult tissues as blocking
compounds, and d) applying a composition comprising one or more of
the blocking compounds to skin of a mammal, wherein the blocking
compounds are selected from the group consisting of naturally
derived compounds, recombinantly produced compounds, and
combinations thereof.
4. A cosmetic, pharmaceutical, or dermatological skin care
composition comprising compounds expressed by fetal tissues and a
dermatologically or pharmaceutically acceptable carrier.
5. The skin care composition of claim 4 wherein the compounds
expressed by fetal tissues are selected from the group consisting
of natural compounds derived from fetal tissues, compounds produced
by recombinantly expressing the compounds expressed by fetal
tissues in genetically engineered prokaryotic or eukaryotic cells,
and combinations thereof.
6. The skin care composition of claim 5 wherein the compounds
expressed by fetal tissues are in a form selected from the group
consisting of cell lysates, extracts, media, and combinations
thereof.
7. The skin care composition of claim 5 wherein the compounds
expressed by fetal tissues are selected from the group consisting
of partially purified compounds, individually purified compounds,
and combinations thereof.
8. The skin care composition of claim 5 wherein the compounds
expressed by fetal tissues are first genetically modified prior to
recombinant expression in genetically engineered prokaryotic or
eukaryotic cells.
9. The skin care composition of claim 4 in a formulation selected
from the group consisting of a solution, a lotion, an ointment, a
cream, and combinations thereof.
10. The skin care composition of claim 4 wherein the
dermatologically or pharmaceutically acceptable carrier is selected
from the group consisting of a woven patch, a non-woven patch, a
liposomal delivery vehicle, and combinations thereof.
11. The skin care composition of claim 4 further comprising an
ingredient selected from the group consisting of hyaluronic acid,
lactose-1-phosphate, maltose-1-phosphate, mannose-6-phosphate, and
lactose-6-phosphate, and combinations thereof.
12. The skin care composition of claim 4 further comprising an
additional skin care active selected from the group consisting of
desquamatory actives, anti-acne actives, retinoids, peptides,
polypeptides, nucleic acids, growth factors, cytokines, hydroxy
acids, anti-oxidants, radical scavengers, chelators,
anti-inflammatory agents, topical anesthetics, tanning actives,
skin lightening agents, anti-cellulite agents, flavonoids,
antimicrobial actives, skin soothing agents, skin healing agents,
antifungal actives, sunscreen actives, conditioning agents,
structuring agents, thickening agents, and mixtures thereof.
13. A cosmetic, pharmaceutical, or dermatological skin care
composition comprising: a) from about 0.000000001% to about 10% by
weight of individually purified compounds expressed by fetal
tissues; b) about 1% to about 80% by weight of non-purified or
partially purified compounds expressed by fetal tissues in enriched
lysates, extracts, or media; b) from about 0.1% to about 10% by
weight of hyaluronic acid; c) from about 0.000001% to about 10% by
weight of at least one additional skin care active including
lactose-1-phosphate, maltose-1-phosphate, mannose-6-phosphate, and
lactose-6-phosphate; and d) a dermatologically or pharmaceutically
acceptable carrier.
14. The skin care composition of claim 13 further comprising
vesicular delivery systems.
15. A method of promoting skin regeneration comprising applying to
skin of a mammal a composition comprising an effective amount of a
small leucine rich proteoglycan compound selected from the group
consisting of fibromodulin (FM), lumican, decorin, biglycan, and
combinations thereof, wherein the skin of a mammal is in the
absence of a dermal wound.
16. The method of claim 15 wherein the skin of a mammal is
non-intact, epidermally injured skin.
17. A cosmetic, pharmaceutical, or dermatological skin care
composition that promotes the regeneration of skin of a mammal,
comprising an effective amount of a proteoglycan compound selected
from the group consisting of FM, lumican, decorin, biglycan, and
combinations thereof, wherein the skin of a mammal is in the
absence of a dermal wound.
18. The skin care composition of claim 17 further comprising a
cosmetically, dermatologically or pharmaceutically acceptable
carrier.
19. The skin care composition of claim 17 wherein the proteoglycan
compound is selected from the group consisting of a natural
proteoglycan compound, a recombinant proteoglycan compound, and
combinations thereof.
20. The skin care composition of claim 17 wherein the skin is
non-intact, epidermally injured skin.
21. The skin care composition of claim 18 wherein the skin is
non-intact, epidermally injured skin.
22. The skin care composition of claim 19 wherein the skin is
non-intact, epidermally injured skin.
23. The skin care composition of claim 17 comprising: a) from about
0.0001% to about 10% by weight of the proteoglycan compound which
is purified, and about 0.1% to about 80% by weight of a cell
lysate, extract, or media enriched with the proteoglycan compound;
b) from about 0.1% to about 10% by weight of hyaluronic acid; c)
from about 0.000001% to about 10% by weight of at least one
additional skin care active; and d) a carrier selected from the
group consisting of a dermatologically acceptable carrier, a
pharmaceutically acceptable carrier, a vesicular delivery system,
and combinations thereof.
24. The skin care composition of claim 19 comprising: a) from about
0.0001% to about 10% by weight of the proteoglycan compound which
is purified, and about 0.1% to about 80% by weight of a cell
lysate, extract, or media enriched with the proteoglycan compound;
b) from about 0.1% to about 10% by weight of hyaluronic acid; c)
from about 0.000001% to about 10% by weight of at least one
additional skin care active; and d) a carrier selected from the
group consisting of a dermatologically acceptable carrier, a
pharmaceutically acceptable carrier, a vesicular delivery system,
and combinations thereof.
25. The skin care composition of claim 20 comprising: a) from about
0.0001% to about 10% by weight of the proteoglycan compound which
is purified, and about 0.1% to about 80% by weight of a cell
lysate, extract, or media enriched with the proteoglycan compound;
b) from about 0.1% to about 10% by weight of hyaluronic acid; c)
from about 0.000001% to about 10% by weight of at least one
additional skin care active; and d) a carrier selected from the
group consisting of a dermatologically acceptable carrier, a
pharmaceutically acceptable carrier, a vesicular delivery system,
and combinations thereof.
26. The skin care composition of claim 21 comprising: a) from about
0.0001% to about 10% by weight of the proteoglycan compound which
is purified, and about 0.1% to about 80% by weight of a cell
lysate, extract, or media enriched with the proteoglycan compound;
b) from about 0.1% to about 10% by weight of hyaluronic acid; c)
from about 0.000001% to about 10% by weight of at least one
additional skin care active; and d) a carrier selected from the
group consisting of a dermatologically acceptable carrier, a
pharmaceutically acceptable carrier, a vesicular delivery system,
and combinations thereof.
27. The skin care composition of claim 22 comprising: a) from about
0.0001% to about 10% by weight of the proteoglycan compound which
is purified, and about 0.1% to about 80% by weight of a cell
lysate, extract, or media enriched with the proteoglycan compound;
b) from about 0.1% to about 10% by weight of hyaluronic acid; c)
from about 0.000001% to about 10% by weight of at least one
additional skin care active; and d) a carrier selected from the
group consisting of a dermatologically acceptable carrier, a
pharmaceutically acceptable carrier, a vesicular delivery system,
and combinations thereof.
28. A method of modulating skin conditions comprising a step
selected from the group consisting of: a) promoting collagen
organization, b) modulating skin inflammatory conditions, c)
modulating skin pigmentation, and d) combinations thereof.
29. The method of claim 28 comprising modulating the level of a
compound selected from the group consisting of small leucine rich
proteoglycans (SLRPs), glycosaminoglycans (GAGs), and combinations
thereof.
30. The method of claim 28 wherein the SLRPs are selected from the
group consisting of FM, lumican, decorin, biglycan, and
combinations thereof, and wherein the GAGs are selected from the
group consisting of dermatan sulfate, chondroitin sulfate, keratan
sulfate, and combinations thereof.
31. The method of claim 30 wherein the SLRPs are selected from the
group consisting of FM, lumican, decorin, biglycan, and
combinations thereof, the level of which is modulated by applying
to the skin a composition comprising an effective amount of one or
more of the SLRPs.
32. The method of claim 28 wherein the skin of mammal is intact
33. The method of claim 30 wherein the skin of mammal is
intact.
34. The method of claim 30 wherein the skin of mammal is
intact.
35. The method of claim 28 wherein the skin of mammal is
non-intact, epidermally injured skin.
36. The method of claim 30 wherein the skin of mammal is
non-intact, epidermally injured skin.
37. The method of claim 31 wherein the skin of mammal is
non-intact, epidermally injured skin.
38. The method of claim 30 wherein the SLRPs are selected from the
group consisting of FM, lumican, decorin, biglycan, and
combinations thereof and modulate collagen fibrillogenesis in
non-intact, dermally injured skin.
39. The method of claim 30 wherein the level of the dermatan
sulfate, chondroitin sulfate, keratan sulfate, and combinations
thereof is modulated by applying to the skin a composition
comprising one or more enzymes that modulate collagen
fibrillogenesis and interfibrillar spacing.
40. The method of claim 30 wherein the level of the dermatan
sulfate, chondroitin sulfate, keratan sulfate, and combinations
thereof is modulated by applying to the skin a composition
comprising one or more enzymes that modulate unorganized matrix
deposition by fibroblasts.
41. The method of claim 39 wherein the enzymes are selected from
the group consisting of chondroitinase AC, chondroitinase B,
endo-beta-galactosidases, keratanase, keratanase II, Bc keratanase
II, and combinations thereof.
42. The method of claim 40 wherein the enzymes are selected from
the group consisting of chondroitinase AC, chondroitinase B,
endo-beta-galactosidases, keratanase, keratanase II, Bc keratanase
II, and combinations thereof.
43. The method of claim 28, comprising modulating skin inflammatory
conditions, wherein the skin inflammatory conditions are selected
from the group consisting of non-allergic skin inflammatory
conditions, allergic skin inflammatory conditions, neurogenic skin
inflammatory conditions, UV radiation (UVR) induced skin
inflammatory conditions, miscellaneous skin inflammatory
conditions, and combinations thereof.
44. The method of claim 30, comprising modulating skin inflammatory
conditions or modulating skin pigmentation, wherein the modulation
of the level of FM, lumican, decorin, and/or biglycan modulates
TNF-alpha activity.
45. The method of claim 30, comprising modulating skin inflammatory
conditions or modulating skin pigmentation, wherein the modulation
of the level of dermatan sulfate modulates leukocytosis.
46. The method of claim 45 wherein the level of dermatan sulfate is
modulated by chondroitinase B.
47. The method of claim 30, comprising modulating skin
pigmentation, wherein the modulation of the level of dermatan
sulfate modulates melanocyte proliferation.
48. The method of claim 30 comprising modulating skin pigmentation,
wherein the level of the dermatan sulfate, chondroitin sulfate,
keratan sulfate, and combinations thereof is modulated by applying
to the skin a composition comprising one or more enzymes.
49. The method of claim 47 wherein the enzymes are selected from
the group consisting of chondroitinase AC, chondroitinase B,
endo-beta-galactosidases, keratanase, keratanase II, Bc keratanase
II, and combinations thereof.
50. The method of claim 30 by modulating skin pigmentation, wherein
the modulation of the level of a SLRP selected from the group
consisting of FM, lumican, decorin, biglycan and combinations
thereof modulates bFGF activity.
51. The method of claim 30 by modulating skin pigmentation, wherein
the modulation of the level of a SLRP selected from the group
consisting of FM, lumican, decorin, biglycan and combinations
thereof modulates melanocyte proliferation.
52. The method of claim 30 by modulating skin pigmentation, wherein
the modulation of the level of a SLRP selected from the group
consisting of FM, lumican, decorin, biglycan and combinations
thereof modulates melanocyte melanin production.
53. The method of claim 30 by modulating skin pigmentation, wherein
the modulation of the level of a SLRP selected from the group
consisting of FM, lumican, decorin, biglycan and combinations
thereof modulates stem cell factor.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The application claims priority to U.S. provisional
application Serial No. 60/432,519, filed on Dec. 11, 2002, the
teachings of which are incorporated herein.
FIELD OF THE INVENTION
[0002] The present invention relates to cosmetic skin care
compositions containing compounds expressed by fetal cells and
tissues that promote the condition of skin. The present invention
also relates to methods of identifying and producing compounds
expressed by fetal cells and tissues. The present invention also
relates to methods and compounds to promote the condition of skin
utilizing proteoglycans, which includes fibromodulin (FM), or any
functionally equivalent compound.
BACKGROUND OF THE INVENTION
[0003] Skin, among other things, is composed of epidermal and
dermal layers. The dermal layer provides the support and blood
supply for the epidermis. The dermal layer is also important in
maintaining the elasticity and appearance of the skin. The dermis
is largely comprised of cells and extracellular matrix ("ECM"). The
composition of the ECM is largely determined by fibroblasts that
elaborate various components such as collagens, elastins, and
proteoglycans. With increasing age, as well as exposure to the sun
and environmental contaminants, there is progressive thinning and
disruption of the supporting dermis. This leads directly to sagging
and consequent furrowing of the epidermis, i.e., the formation of
wrinkles. (See, for example, Oikarinen A. The aging of skin:
chronoaging versus photoaging. Photodermatol Photoimmunol Photomed
7: 3-4, 1990).
[0004] It is well established in the art that fetal skin is
fundamentally different from adult skin. For instance, after
injury, adult skin repairs through scar formation, a process
characterized by the replacement of injured tissues with a
disorganized deposition of collagen and various ECM components,
referred to collectively as a "scar." In contrast, fetal skin
repair occurs by cellular regeneration and restoration of normal
skin architecture through organized deposition of collagen and ECM
components to effect scarless repair. Studies have shown that the
capabilities for scarless skin repair is one quality of fetal skin,
and does not require the fetal immune system, fetal serum, or
amniotic fluid (Bleacher J C, et al., J Pediatr Surg 28: 1312-4,
1993); Ihara S, Motobayashi Y., Development 114: 573-82. 1992). For
example, isolated human fetal skin transplanted into athymic mice
heals without producing typical scar tissue (Adzick N S, Lorenz H
P., Ann Surg 220: 10-8. 1994).
[0005] Accordingly, it appears that specific molecules or
compositions in regenerating fetal skin that are minimally present
or not present at all in non-fetal skin (e.g., adult skin) are
important in regenerating and promoting the appearance of skin.
[0006] Numerous compounds and techniques have been described in the
art as being useful for promoting the condition of skin, especially
of "aged" or wrinkled skin. Topical compounds include retinoic acid
for stimulation of epidermal cell growth. Retinoids are well
recognized as anti-wrinkle actives which help to reduce the
subcutaneous effects of aging such as wrinkling, leatheriness,
looseness, roughness, dryness, and mottling (hyper pigmentation)
(see, U.S. Pat. Nos. 4,603,146 and 4,877,805). It has been
postulated that retinoids act by producing inflammation, which
causes thickening of the epidermis (acanthosis), and local
intercellular edema, leading to exfoliation and improved skin
texture and appearance. Use of L-ascorbic acid to stimulate
fibroblast cell growth and collagen production has also been
described (Hata R, Senoo H. J Cell Physiol 138:8-16. 1989).
Techniques for promoting the condition of skin include deliberate
methods of inducing skin irritation/injury through chemical (e.g.,
phenol peels), mechanical (e.g., dermabrasion), or thermal (e.g.,
lasers) means. Injury to the epidermis and/or dermis ultimately
results in new cell growth and ECM deposition that may improve the
overall appearance of skin.
[0007] Another skin conditions that often lead to skin damages is
inflammation. In principle, the inflammatory and immune responses
can be regulated through the use of drugs (In Goodman &
Gilman's The Pharmacological Basis of Therapeutics eds. Hardman et
al., Ninth Edition, McGraw-Hill publishing, 1996). Glucocorticoids
and aspirin-like drugs (non-steroidal anti-inflammatory agents,
NSAIDs) are the most widely used anti-inflammatory agents. NSAIDs
are typically used to treat symptoms of inflammation (e.g. pain and
fever). Corticosteroids are effective anti-inflammatory agents,
having effects on virtually all inflammatory cells, but manifest
significant adverse effects, such as inducing Cushingoid features,
skin thinning, increased susceptibility to infection, effects on
wound healing, and suppression of the
hypothalamic-pituitary-adrenal axis. Other anti-inflammatory drugs
presently available produce cytotoxic effects that reflect their
initial employment as cancer chemotherapeutics, typically
anti-neoplastic agents. Such drugs may kill cells indiscriminately,
particularly those that proliferate rapidly. Methotrexate, however,
is effective in treating rheumatoid arthritis at doses lower than
those used to treat cancer (cytoreductive dose). Immunosuppressive
agents, such as cyclosporin A and tacrolimus, are effective in
preventing allograft rejection, but their use in treating
autoimmune diseases has been limited by the development of severe
side effects, particularly nephrotoxicity.
[0008] With specific regard to skin, topical or oral
corticosteroids or antihistamines are the mainstays of therapy.
However, corticosteroids have many undesirable side effects as
listed above, while antihistamines may themselves elicit an
allergic reaction when applied topically or cause excessive
drowsiness when taken orally (Shai A, et al., Inflammation,
dermatitis and cosmetics. Handbook of Cosmetic Skin Care. London:
Martin Dunitz Ltd., pp. 135-146, 2001).
[0009] Hyperpigmentation is another common skin disease. Once
present, hyperpigmentation is very difficult to treat. Because
acquired hyperpigmentation can have a significant negative impact
on cosmetic and psychosocial issues, much attention has focused on
the treatment of hyperpigmentation. The current state of the art in
hyperpigmentation offers many modalities, but none are completely
satisfactory. The major limitation is that current modalities are
primarily skin "bleaching" compounds that are fairly ineffective at
treating established hyperpigmentation, especially dermal
hyperpigmentation (Reviewed in Briganti S, et al., Pigment Cell
Res. 16:101-110, 2003). A variety of other substances have been
proposed for the control or inhibition of skin pigmentation. Almost
all of these substances work by either bleaching existing pigment
or preventing new pigment synthesis by inhibiting the activity of
tyrosinase, the principle rate-limiting enzyme in the production of
melanin. For example, U.S. Pat. No. 6,123,959 describes the use of
aqueous compositions comprising liposomes and at least one
competitive inhibitor of an enzyme for the synthesis of melanin in
combination with at least one non-competitive inhibitor of an
enzyme for the synthesis of melanin. U.S. Pat. No. 6,132,740
describes the use of certain resorcinol derivatives as skin
lightening agents. WO 99/64025A1 describes compositions for skin
lightening which contain tyrosinase inhibiting extracts from
dicotyledonous plant species indigenous to Canada. U.S. Pat. No.
5,580,549 describes an external preparation for skin lightening
comprising 2-hydroxybenzoic acid derivatives and salts thereof as
inhibitors of tyrosinase. WO 99/09011A1 describes an agent for
inhibiting skin erythema and/or skin pigmentation, containing at
least one carbostyril derivative and salts thereof. U.S. Pat. Nos.
5,214,028 and 5,389,611 describes lactoferrin hydrolyzates for use
as a tyrosinase inhibitory agents. Additionally, a number of
compounds and plant extracts are reported to have activity against
hyperpigmentation, including ascorbic acid and derivatives thereof,
kojic acid and compounds related thereto, licorice (glycyrrhiza)
extract, and bearberry extract. While these chemical compounds and
extracts are active in the reversal and prevention of
hyperpigmentation, they can be irritating to the skin with
prolonged use.
[0010] Despite the proposal of all these substances, the main
products for treatment of hyperpigmentation contains hydroquinone,
a well known active substance for skin de-pigmentation (e.g., see
U.S. Pat. No. 6,139,854). However, hydroquinone can have serious
side effects if applied over a long period of time. For example,
the application of hydroquinone to skin may lead to permanent
de-pigmentation, and thus to increased photosensitivity of the skin
when exposed to ultraviolet light. Moreover, hydroquinone can be
metabolized to benzoquinones, which are potent haematotoxic,
genotoxic and carcinogenic compounds that can also induce the
formation of radical species, predisposing cells to oxidative
damage (Do Ceu Silva M, et al., Mutagenesis. 18:491-496, 2003). For
that reason, in some countries hydroquinone is only allowed to be
used for skin de-pigmentation in limited concentrations, and, in
other countries, the product is banned completely for this
application.
[0011] Therefore, there is a need for new and more effective
methods for modulating skin conditions such as treating skin aging,
inflammation and pigmentation which carry fewer significant and
undesirable side effects.
SUMMARY OF THE INVENTION
[0012] Described herein are novel cosmetic skin care compositions
containing compounds expressed by fetal tissues to promote the
condition of skin. The compounds may be delivered to skin by way
of, but not limited to, a solution, a lotion, an ointment, a cream,
a gel, or a skin peelable strip.
[0013] Although individual identification or purification of
compounds expressed by fetal tissues may be useful, the application
of this invention does not require the individual identification or
purification of the compounds. The present invention also relates
to methods of identifying compounds expressed by fetal tissues or
of identifying the conditions that promote expression of these
compounds. The present invention also relates to methods for
promoting expression of compounds expressed by fetal tissues
through modification of external cellular environments or through
recombinant expression.
[0014] The present invention also relates to methods of using
compositions containing compounds expressed by fetal tissues to
improve the condition of skin. The methods generally include the
step of topically applying the composition to the skin of a mammal
in need of treatment and where a safe, and effective amount of the
compositions is used.
[0015] Furthermore, the present invention describes novel cosmetic
skin care compositions containing a safe and effective amount of
FM, or functionally equivalent molecule in purified or enriched
extract form.
[0016] Other components which may be included with the compositions
containing compounds expressed by fetal tissues or FM, depending on
the formula, are safe and effective amounts of hyaluronic acid, an
additional skin care active, and a cosmetically acceptable,
dermatologically acceptable or pharmaceutically acceptable
carrier.
[0017] Representative embodiments of the compositions include, but
are not limited to hyaluronic acid, ECM peptides or polypeptides,
growth factors, and L-ascorbic acid.
[0018] These and other features, aspects, and advantages of the
present invention will become evident to those skilled in the art
from a reading of the present disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 shows the operative procedure for the fetal wound
model. A. A small part of the antimesenteric surface of the uterus
is incised and a purse-string suture is placed around the incision.
B and C. A full thickness wound is created on each embryo by
excising a 2 mm disc of tissue. Blue or green vital stain is
applied immediately after wounding for later wound
identification.
[0020] FIG. 2 shows the H&E staining of wounded E16 rat skin
with regenerative repair. A. 24 hours post-injury, 100.times..
There is minimal inflammatory infiltrate. B. 24 hours post-injury,
400.times.. The presence of blue vital dye in hair follicles near
the migrating epithelial edge suggests concurrent hair follicle
regeneration with wound re-epithelialization (black open arrow). C.
24 hours post-injury, 400.times.. Neutrophils (black solid arrows)
and lymphocytes (black open arrow) are the predominant cells of the
wound periphery and the center of the wound, respectively. D. 72
hours post-injury, 100.times.. The wound is entirely healed with
complete regeneration of the normal skin architecture. Normal
distribution of hair follicles (black open arrows) are observed in
the dermis. E. 72 hours post-injury, 400.times.. At higher
magnification, the previous wound site, as indicated by the
presence of blue vital stain in the dermis (black open arrows), is
indistinguishable from the non-wounded skin. F. Confocal
microscopic view of wounded E16 rat skin (Fa through Fc). Fa. 48
hours post-injury, 630.times.. Note the organized appearance of the
collagen fibers with a reticular lattice structure. Fb. 72 hours
post-injury, 630.times.. The wound site is completely
re-epithelialized with complete restoration of normal skin collagen
architecture and hair follicle regeneration. Fc. Non-wounded E19
skin [i.e., E16+72 hours], 630.times.. No difference is observed
between E16 skin, 72 hours post-wounding, and non-wounded E19 skin.
e: epidermis. h: hair follicle. d: dermis. Scale bars: A,D, 200
.mu.m; B,C,E, 50 .mu.m; Fa-Fc, 32 .mu.m. G. There is no significant
difference in total collagen density between E16 fetuses 72 hours
post-injury and non-wounded E19 (E16+72 hours) fetuses
(p>0.05).
[0021] FIG. 3 shows the H&E staining of wounded E 19 rat skin
with non-regenerative repair. A. 24 hours post-injury, 100.times..
There is moderate inflammatory infiltrate and increased red blood
cells. B. 24 hours post-injury, 400.times.. Re-epithelialization is
also noted at 24 hours after injury (black open arrow). C. 24 hours
post-injury, 400.times.. Monocytes (black open arrows) comprises
most of the inflammatory cells. D. 72 hours post-injury,
100.times.. The wound is completely re-epithelialized with
increased cellularity and neovascularity. Hair follicles (black
open arrows) are not observed in the repaired wound site (far left)
compared with unwounded site (far right). E. 72 hours post-injury,
400.times.. At higher magnification, blue vital dye (black open
arrows) within the repaired wound is visible. F. Confocal
microscopic view of E19 rat skin (Fa through Fc). Fa. 48 hours
post-injury, 630.times.. Large spaces among newly formed collagen
fibers within the dermis are noticeable. A thin layer of dense
collagen fibers is seen as basement membrane (white open arrows).
Fb. 72 hours post-injury, 630.times.. Disorganized collagen
deposition pattern with heterogeneously sized collagen fibers is
apparent in the healed dermal scar tissue. Not e the absence of
hair follicle regeneration. Fc. Non-wounded neonatal day 1 (N1)
skin [i.e., E19+72 hours, E21=term], 630.times.. Non-wounded N1
skin exhibited an organized collagen deposition pattern that is
significantly different from E19 skin, 72 hours post-wounding. e:
epidermis. h: hair follicle. d: dermis. Scale bars: A,D, 200 .mu.m;
B,C,E, 50 .mu.m; Fa-Fc, 32 .mu.m. G. There is significantly
increased total collagen density in wounded E19 fetuses 72 hours
post-injury relative to non-wounded N1 controls (p=0.00043).
[0022] FIG. 4 shows specific primer based reverse transcription
polymerase chain reaction (RT-PCR) screening of gene expression for
transforming growth factor (TGF)-.beta. ligands, receptors, and
modulators during fetal skin development. RT-PCR was performed on
RNA isolated from day 14, 16, 17, 18, 19, 20, and 21 fetal dorsal
skin (N=10-15 fetuses/time point). To determine relative changes in
mRNA levels during development, densitometry values for each blot
were corrected to GAPDH expression at each time point and
normalized by setting the highest value to one. The results are
depicted graphically as the mean (A,B,C). The transition period is
highlighted in gray. Unpaired two-tailed student's t test was
performed to detect statistically significant differences in gene
expression between E16 (beginning transition) and E18 (end
transition) fetal skin. A representative blot is shown for each
TGF-13 ligand (A'), receptor (B'), or modulator (C') with the
corresponding P value on the right and the transition period
highlighted in gray. Statistically significant differences in gene
expression between day 16 (beginning transition) and day 18 (end
transition) fetal skin are underlined (A',B',C'). Statistically
significant P values (<0.05) are marked with an "*". A
representative fetal GAPDH PCR reaction is shown (inset).
[0023] FIG. 5 shows graphically specific primer based RT-PCR
screening of gene expression for matrix metalloproteinases (MMP)
and their tissue-derived inhibitors (TIMPs) during repair. In
general, wounds that scar have a relative propensity towards
excessive ECM deposition rather than degradation (right), while
scarless wounds have a relative propensity towards less ECM
deposition.
[0024] FIG. 6 shows graphically specific primer based RT-PCR
screening of gene expression for FGFs during repair. In general,
wounds that scar appear to be associated with higher bFGF (FGF-2)
expression (right), while scarless wounds have relatively less bFGF
expression.
[0025] FIGS. 7A and B show graphically specific primer based RT-PCR
screening of gene expression for FM and decorin during repair. A.
FM transcripts increased markedly by 2.75-fold 24 hours after
injury in E16 fetuses (P<0.0001) that manifest regenerative
repair, but not in E18-wounded fetuses with non-regenerative
repair. B. In contrast, decorin transcripts are relatively higher
in E18 wounds when compared to E 16 wounds, although both E16 and
E18 wounds displayed decreased decorin levels relative to
non-wounded aged match controls.
[0026] FIG. 8. shows the results of applying and blocking compounds
identified through gene screening of fetal skin. In this case the
compound is FM. H&E staining shows that FM application to late
gestation wounds that normally heal without regeneration results in
complete scarless regenerative repair, while and anti-FM antibody
application to early gestation wounds that normally heal with
scarless regeneration results in non-regenerative repair. Wounds,
shown at 200.times. (left) and 400.times. (right) magnification,
were harvested at 72 hours along with controls. Red arrows indicate
permanent dye. Black arrows indicate hair follicles. A. Normally,
(E16) fetal wounds heal scarlessly and would be indistinguishable
from control (E19) skin (insets), if not for the presence of
permanent dye and disruption of the panniculus carnosus muscle
(blue arrows). Note the regeneration of hair follicles and absence
of inflammation. B. Treatment of E16 wounds with anti-FM antibody
induced scar formation. Hair follicles have failed to regenerate
and inflammation is present. C. Treatment of E16 fetal wounds with
an immunoglobulin G (IgG) control solution failed to induce
scarring. Note the hair follicles. D. Usually, E18 fetal wounds
heal with scar. In the scar, inflammatory cells (blue arrows) are
present, but hair follicles are absent. E. Treatment of E18 fetal
wounds with exogenous FM inhibited scar formation. These wounds can
only be identified by permanent dye and disruption of the
panniculus carnosus muscle (blue arrows), since they lack
significant inflammation and contain hair follicles. F. E18 wounds
treated with the collagen control solution still healed with scar
formation. Note the inflammatory cells (blue arrows) and absence of
hair follicles.
[0027] FIG. 9. shows confocal microscopy of FM and anti-FM antibody
treated fetal wounds. FM treated late gestation wounds demonstrate
an organized collagen architecture, while anti-FM antibody treated
wounds demonstrate a disorganized collagen architecture. Wounds and
controls were harvested 72 hours post-injury. Collagen fibers have
been stained with Sirius red and appear white. Red open arrows
indicate hair follicles. A. Unwounded skin from an E19 fetus
(200.times.) for comparison with the E16 wounds. B. Normally, E16
fetal wounds heal scarlessly (200.times.). The dermal collagen
fibers are thin, and their reticular arrangement is
indistinguishable from the organization of collagen in unwounded
skin. C. In contrast, the collagen fibers in anti-FM
antibody-treated E16 fetal wounds are thicker and more randomly
arranged (400.times.). D. Collagen fibers are thinner and have a
reticular arrangement in the E16 wounds treated with IgG control
solution (400.times.). E. Unwounded skin from an E21 fetus for
comparison with the E18 wounds--400.times. (left) and
1000.quadrature..times. (right). F. Usually, E18 fetal wounds heal
with scar. No hair follicles are seen in the scar (400.times.
(left)). Collagen fibers in the area of scar are thicker and are
arranged randomly with greater distances between fibers, which is
better seen at higher magnification (1000.times. (right)). G.
Addition of FM to E18 wounds, however, inhibits
scarring--400.times. (left) and 1000.times. (right). Here the
collagen fibers are thin, while the organization of collagen in the
wound bed is very similar to the architecture of unwounded E21
skin. H. Wounds treated with collagen control solution have
thicker, randomly arranged fibers with greater inter-fiber
distances--400.times. (left) and 1000.times. (right).
[0028] FIG. 10 shows relative mRNA expression of type I collagen in
FM-treated (E18) wounds compared with scarless (E16) wounds,
scarring (E18) wounds, and unwounded skin (E18). Consistent with
results FIG. 5, FM treated wounds with exhibited less ECM
deposition as exemplified by gene expression for type I collagen.
RNA was isolated from fetal tissue 12 hours post-wounding and
reduced-cycle, RT-PCR was performed. Relative mean mRNA expression
is depicted .+-.SEM. Student's t test was used to perform pair-wise
comparisons of means. P values<0.05 were considered significant.
Asterisks indicate significant differences between control
wound/skin and the FM-treated group.
[0029] FIG. 11 shows adult wounds treated with FM (0.4 mg/ml) or
PBS control solutions with significant improvement in collagen
architecture and increased dermal tissue regeneration. Wounds were
harvested 2 weeks post-injury, and tissue sections were stained
with either H&E or Sirius red (for confocal microscopy). Black
arrows mark the wound bed, and red arrows indicate permanent dye
and disruption of the panniculus carnosus muscle. A. H&E
staining of an adult wound treated with FM--200.times. (left) and
400.times. (right) magnification. At higher magnification, dermal
collagen fibers appear to have a parallel arrangement. B. Confocal
microscopy of the superficial dermis (near the epidermal-dermal
border) of a FM-treated adult wound--400.times. (left) and
1000.times. (right) magnification. The white collagen fibers have a
relatively uniform, linear appearance and lie parallel to the
overlying epidermis. C. Confocal microscopy of the deep dermis of a
FM-treated adult wound--400.times. (left) and 1000.times. (right)
magnification. Again, parallel organization of collagen fibers is
seen. D. H&E staining of an adult wound treated with control
PBS solution--200.times. (left) and 400.quadrature..times. (right)
magnification. The wound area is larger than in the FM-treated
wounds. E. Confocal microscopy of the superficial dermis of a
control adult wound illustrating the random deposition of collagen
fibers--400.times. (left) and 1000.times. (right) magnification. F.
Confocal microscopy of the deep dermis of a control adult
wound--400.times. (left) and 1000.times. (right) magnification.
Here the disorderly pattern of collagen deposition and great
variation in collagen fiber morphology is more clearly seen.
DETAILED DESCRIPTION
[0030] I. Definitions
[0031] The term "topical application," as used herein, means to
apply or spread the compositions of the present invention onto the
surface of the skin.
[0032] The term "dermatologically-acceptable," as used herein,
means that the compositions or components thereof so described are
suitable for use in contact with human skin without undue toxicity,
incompatibility, instability, allergic response, and the like.
[0033] The term "safe and effective amount" as used herein means an
amount of a compound or composition sufficient to significantly
induce a positive benefit, preferably a positive skin appearance or
feel benefit, including independently the benefits disclosed
herein, but low enough to avoid serious side effects, i.e., to
provide a reasonable benefit to risk ratio, within the scope of
sound judgment of the skilled artisan.
[0034] The term "FM" as used herein means not only FM, but also any
functionally equivalent molecule with or without genetic
modification.
[0035] The term "wild-type FM protein" as used herein means
non-genetically modified, naturally occurring FM present in
tissues.
[0036] The term "recombinant FM cDNA" as used herein means FM cDNA
(either genetically modified or not) that has been cloned into a
suitable expression vector (e.g., plasmid, adenovirus).
[0037] The term "genetically modified" refers to modification of
the DNA expressing a protein such as FM so as to increase a
property of the protein, e.g., transcription efficiency,
purification efficiency, biological activity by increasing binding
efficiency, resistance to proteolysis, etc.
[0038] The term "recombinantly expressed" as used herein means
fetal tissue derived cDNA (either genetically modified or not) that
has been cloned into a suitable expression vector (e.g., plasmid,
adenovirus) for purposes of obtaining protein expression from the
cDNA.
[0039] The term "lysates" as used herein means compositions
obtained through lysing cells using a suitable detergent.
[0040] The term "extracts" as used herein means compositions
obtained after further purification or concentration of cell lysate
material.
[0041] The term "media" as used herein means compositions isolated
from the external environment of unmodified or genetically modified
fetal cells or tissues.
[0042] The term "compounds" as used herein may be considered
equivalent to "molecules", although the term "molecules" is more
preferable when describing a single entity.
[0043] "Healthy skin" or "normal skin" refers to non-lesional skin,
i.e., with no visually obvious erythema, edema, hyper-, hypo-, or
uneven pigmentations, scale formation, xerosis, or blister
formation. Histologically, healthy or normal skin refers to skin
tissue with a morphological appearance comprising well-organized
basal, spinous, and granular layers, and a coherent multi-layered
stratum corneum. In addition, the normal or healthy epidermis
comprises a terminally differentiated, stratified squamous
epithelium with an undulating junction with the underlying dermal
tissue. Normal or healthy skin further contains no signs of fluid
retention, cellular infiltration, hyper- or hypoproliferation of
any cell types, mast cell degranulation, parakeratoses, etc., and
implies normal dendritic processes for Langerhans cells and dermal
dendrocytes. This appearance is documented in dermatological
textbooks, for example, Histopathology of the Skin, Lever and
Schaumburg-Lever (eds.), J.B. Lippincott Company (1991) and
Textbook of Dermatology, Champion et al., (eds.), 5th Ed. Blackwell
Scientific Publications (1992), especially Chapter 3 "Anatomy and
Organization of Human Skin"; Physiology, Biochemistry and Molecular
Biology of the Skin, Vols. I And II, Goldsmith (ed.), Oxford Press
(1991), the full disclosures of which are expressly and completely
incorporated herein by reference.
[0044] The term "promoting skin condition" includes
prophylactically promoting and/or therapeutically promoting skin
condition, including visible and/or tactile discontinuities in
skin. As used herein, prophylactically promoting skin condition
includes delaying, minimizing and/or preventing visible and/or
tactile discontinuities in skin. As used herein, therapeutically
promoting skin condition includes ameliorating (e.g., diminishing,
minimizing and/or effacing, discontinuities in skin). Promoting
skin condition involves improving skin appearance and/or feel.
[0045] II. Skin Conditions
[0046] A. Skin Inflammation
[0047] Skin inflammation as used herein generally includes
non-allergic skin inflammatory condition, allergic skin
inflammatory condition, neurogenic skin inflammatory condition,
TNF-alpha mediated conditions, UV radiation (UVR) induced skin
inflammatory conditions, and miscellaneous skin inflammatory
conditions.
[0048] "Non-allergic skin inflammatory condition" refers to an
inflammatory condition of the skin which is not solely mediated by
a specific antigen. Such conditions include, e.g., irritant contact
dermatitis, psoriasis, eczema, pruritus, seborrheic dermatitis,
nummular dermatitis, lichen planus, acne vulgaris, comedones,
polymorphs, nodulocystic acne, conglobata, senile acne, secondary
acne such as solar acne, medicinal acne or professional acne; other
types of keratinization disorders, for example, ichthyoses,
ichthyosiform conditions, Darier malady, palmoplantary
keratodermies, leucoplasies and leucoplasiform conditions and
lichen; other dermatologic disorders such as blistery dermatoses
and collagen maladies; and extrinsic aging of the skin, be it
photoinduced or not.
[0049] "Allergic skin inflammatory condition" refers to skin
inflammation caused by one or more allergen. "Allergen" refers
herein to a substance which induces symptoms of immediate
hypersensitivity by inducing IgE antibody responses and delayed
hypersensitivity reaction. Generally, such responses require a
sensitization of the immune system to the allergen. For instance,
mosquito bite-induced itch and inflammation is thought to result
from an IgE and IgG mediated allergic reaction to antigenic
materials in mosquito salivary glands. The primary target in
immediate allergic reactions are mast cells, which have high
affinity IgE receptors (Ohtsuka E, et al, Jpn J Pharmacol
86:97-105, 2001). Upon IgE-dependent stimulation, mast cells
release several pro-inflammatory mediators such as TNF-alpha, Kulka
M and Befus A D., Arch Immunol Ther Exp (Warsz) 51:111-110, 2003).
TNF-alpha is found pre-formed and stored in granules of mast cells
or newly synthesized following mast cell activation (luvone T, et
al., Br J Pharmacol. 128:700-704, 1999). TNF-alpha is a
multifunctional cytokine and a key mediator of immune and
inflammatory response and it has been found pre-formed and stored
in granules of mast cells or newly synthesized following mast cell
activation (Gordon J R, and Galli S J., Nature. 346:274-276,
1990)
[0050] "Neurogenic skin inflammatory condition" refers to an
inflammatory condition of skin related to proinflammatory
neuropeptide release (e.g., during times of emotional or
psychological stress) that may occur concomitant with or separate
from non-allergenic or allergenic inflammatory skin conditions. For
instance, stress induced acne has been proposed as an example of
neurogenic inflammation. The skin is innervated by primary afferent
sensory nerves, postganglionic cholinergic parasympathetic nerves,
and postganglionic adrenergic and cholinergic sympathetic nerves.
Sensory nerves are derived from the dorsal root ganglion and are
present in all parts of the skin representing the initial somatic
portion of the afferent sensory pathway. The cutaneous sensory
nervous system comprises a network of fine C fibers within the skin
that innervate multiple cell types and play an important role in
inflammation. The epidermis is also innervated by a
three-dimensional network of unmyelinated nerve fibers with free
branching endings that arise in the dermis. Sensory nerves not only
function as an afferent system to conduct stimuli from the skin to
the central nervous system, but they also act in an efferent
neurosecretory fashion to stimulate target tissues through their
terminals. Various stimuli, such as noxious stimuli, may directly
activate the peripheral endings of primary sensory neurons
generating impulses that are conveyed centrally as well as, through
antidromic axon-reflexes, peripherally. Upon release of
neuropeptides from sensory terminals, important visceromotor
inflammation and trophic effects occur in the peripheral tissues.
Normal human skin expresses a variety of neuropeptides that are
either directly derived from sensory neurons or from skin cells
such as keratinocytes, microvascular endothelial cells or
fibroblasts. In addition, immune cells that either constitutively
reside in the skin, such as mast cells, or that infiltrate the skin
during inflammation have been reported to produce neuropeptides.
Cutaneous nerve fibers can modulate inflammatory reactions through
the local release of neuropeptides, which are able to regulate both
acute and chronic aspects of cutaneous inflammatory processes, such
as vascular motility, cellular trafficking, activation and
trophism. Clinical evidence in support of a connection between
neuropeptides secretion and the development of inflammation is
found in various skin diseases, such as atopic dermatitis,
psoriasis and alopecia areata, and acne which are commonly
exacerbated during periods of emotional stress. Indeed, stress has
been shown to elicit the release of substance P, a potent
proinflammatory neuropeptide (Reviewed in Toyoda M, et al.,
Neuropeptides and sebaceous glands. Eur J Dermatol. 12:422-427,
2002). Several studies have demonstrated that mast cells are often
found in close contact with nerves and that there may be functional
interactions between MCs and the nervous system. In addition,
recent evidence suggests that substance P is an important mediator
in intimate nerve-mast cell cross talk. These findings suggest that
substance P endogenously released by dermal nerve fibers may be
important in the regulation of endothelial-leukocyte interaction
via mast cells. It has been demonstrated that the proinflammatory
effects induced by mast cell degranulation products is inhibited by
a blocking antiserum to TNF-alpha. Thus, a cascade of cellular
events involving mast cell degranulation and the release of
proinflammatory cytokines such as TNF-alpha will then induce
adhesion molecules such as E-selectin on the adjacent venular
endothelium that would then facilitate the local accumulation of
blood leukocytes and further augment the inflammatory response.
Thus, TNF-alpha may also modulate neurogenic inflammation.
[0051] "TNF-alpha mediated conditions" refers to local skin
disorders where TNF-alpha is a primary mediator leading to the
manifestation of the disorders. TNF-alpha, previously known as
cachectin, is produced by a large number of cells or tissues
including neutrophils, activated lymphocytes, macrophages, NK
cells, LAK cells, astrocytes, endothelial cells, smooth muscle
cells, mast cells, keratinocytes and other epithelial cell types.
This particular cytokine governs a wide variety of biological
activities including: cytotoxic effects against tumors, activation
of neutrophils, normal proliferation of cells, inflammatory,
immunological, and antiviral responses. A membrane-bound form of
TNF-alpha has been located in lymphocytes or monocytes where it is
involved in intracellular signaling and activation. The specific
overproduction of TNF-alpha is known to be an important determinant
for a number of diseases, infections, and inflammatory conditions
including rheumatoid arthritis, cachexia, endotoxin shock,
inflammatory bowel disease, Crohn's disease, psoriasis, contact
dermatitis, adult respiratory distress syndrome, infections,
transplantation, ischemic/reperfusion damage, diseases involving
eosinophils (e.g. asthma, allergy, etc.), graft-versus-host
reactions, bone resorption, inflammatory bowel disease, multiple
sclerosis (MS), diabetes, AIDS and Alzheimer's disease and/or the
weight loss associated with Alzheimer patients (Reviewed in Beutler
B., Tumor Necrosis Factors, The Molecules And Their Emerging Role
In Medicine Raven Press, 1992, and European Cytokine Network, 5(2)
(1994).
[0052] "UVR mediated skin inflammatory condition" refers to an
inflammatory condition of skin from excessive UVR exposure.
[0053] "Miscellaneous skin inflammatory condition" refers to an
inflammatory condition of skin not otherwise specified above
affecting intact or non-intact skin. Skin infections are examples
of inflammatory skin conditions occurring in intact skin or
non-intact skin. Wounds are examples of inflammatory skin
conditions occurring in non-intact skin.
[0054] As noted above, the largest organ in the body, the skin,
also makes TNF-alpha. Since skin represents the border to a hostile
environment, it needs an arsenal of biological weapons to combat
such insults as chemical irritants, bacteria, insect bites,
sunlight and physical trauma. Pro-inflammatory cytokines stand as
ready messengers to inform and direct the immune system upon
challenge.
[0055] B. Skin Pigmentation
[0056] The difference in skin color between different individuals
and races are determined by the amount and distribution of melanin
produced by melanocytes. In fact, differences in skin shade and
color are determined not by the number and density of the
melanocytes, which are basically identical in all humans of any
race, but by their degree of melanogenic activity, the number and
size of the melanosomes, the type of melanin deposited onto
melanosomes, and the donation of mature melanosomes to surrounding
keratinocytes. (Reviewed in Abdel-Malek Z., The Pigmentary System.
Physiology and Pathophysiology. Eds. Nordlun J J, et al., pp.
115-122, 1998).
[0057] Regulation of Skin Pigmentation
[0058] Melanin production and cell proliferation by melanocytes is
regulated by several factors including ultraviolet radiation (UVR),
steroid hormones, inflammatory mediators, growth factors, peptide
hormones, and melanotropins (Reviewed in Abdel-Malek Z, 1998).
Exposure to UVR stimulates the synthesis of a variety of hormones,
cytokines, and growth factors by epidermal cells or keratinocytes.
Keratinocytes exposed to UVR produce interleukin (IL)-1 and tumor
necrosis factor (TNF)-alpha, two major inflammatory cytokines.
Moreover, TNF-alpha has been shown to play an important role in the
formation of sunburn (i.e., apoptotic) keratinocytes. The synthesis
of basic fibroblast growth factor (bFGF) by keratinocytes was also
enhanced by UVR treatment. Basic FGF is mitogenic for human
melanocytes. In addition, UVR may also increase keratinocyte
endothelin (ET)-1, -melanocyte stimulating hormone ({tilde over
(.quadrature.)}MSH, and adrenocorticotropic hormone (ACTH)
synthesis. ET-1 is a potent mitogen and melanogen for melanocytes
that is regulated by IL-1, TNF-alpha, or UVR, while MSH and ACTH
which are mitogenic and melanogenic may function as transducers for
the melanogenic effects of UVR. ET-1 may also act synergistically
with bFGF and {tilde over (.quadrature.)}MSH to stimulate
melanocyte proliferation.
[0059] The effects of sex steroid hormones (androgens and
estrogens) on cutaneous pigmentation have been recognized for a
along time. The increased pigmentation of the areola and genitalia
has been attributed mostly to these hormones. Changes in the levels
of the female sex hormones during pregnancy have been implicated in
the skin darkening, seen in melasma (Abdel-Malek Z, 1998).
[0060] The clinical observation of post-inflammatory
hyperpigmentation has also implicated immune inflammatory mediators
in this phenomenon. Inflammatory cytokines such as IL-1 and
TNF-alpha increase production and secretion of endothelins by
keratinocytes (Manaka L, et al., Br J Dermatol 145:895-903, 2001).
Other inflammatory mediators of the cyclooxygenase pathway such as
prostaglandin E have been found to increase in skin following UV
exposure and to increase melanogenesis (Abdel-Malek Z, 1998).
[0061] Although many molecules are involved in regulation of
melanogenesis, it is clear from the preceding section that the
inflammatory cytokine TNF-alpha plays a major role as an initial
mediator in the regulation of skin pigmentation upon exposure to
UVR and upon other conditions that may induce or increase skin
inflammation such as injury, acne, insect bites, etc.
TNF-upregulation may then induce the expression of other molecules
that may further increase the inflammatory and/or melanogenetic
stimuli. For instance, TNF-alpha stimulates inflammatory mediator
prostaglandin E2 production by human synovial cells and dermal
fibroblasts (Dayer, J M, et al., J Exp Med. 162:2163-2168, 1985) as
well as melanogenetic mediator endothelin 1. Thus, the inhibition
of TNF-alpha activity may be useful in preventing an entire cascade
of inflammatory and melanogenetic stimuli that can result in
undesirable effects on skin pigmentation.
[0062] In addition, the regulation of bFGF may also have important
implications for skin color. Basic FGF which is induced by UVR and
is mitogenic for melanocytes also enhances stem cell factor (SCF)
production (Sugimoto Y, et al., J Cell Physiol. 181:285-294, 1999).
SCF induces melanocytic hyperplasia with increased melanocyte
number and increased melanin (Grichnik J M, et al., J Am Acad
Dermatol. 33:577-583, 1995). Overexpression of SCF in the skin and
serum of systemic sclerosis patients is associated with
hyperpigmentation (Kihira C, et al., J Dermatol Sci. 20:72-78, 1998
and Yamamoto T, et al., Br J Dermatol. 144:199-200, 2001). Thus,
modulation of bFGF may not only affect bFGF, but SCF effects on
melanocyte proliferation and melanin production as well.
[0063] C. Dermal Collagen--Organization
[0064] Dermal Components
[0065] The dermal layer provides the support and blood supply for
the epidermis. The dermal layer is also important in maintaining
the elasticity, thickness, and appearance of the skin. The dermis
is largely comprised of fibroblast cells and ECM. Immune cells such
as mast cells, polymorphonuclear leukocytes, lymphocytes, and
macrophages are also present in the dermis. The composition of the
ECM is largely determined by fibroblasts that elaborate various
components such as collagens, elastins, and other matrix proteins.
The ECM acts as a scaffold for cell adhesion, proliferation,
migration, and differentiation and gives mechanical strength and
elasticity to tissue (Kuwaba K, et al., J Dermatol Sci. 29:185-194,
2002). The major component of ECM is collagen whose functions will
be further detailed in the next section. Closely associated with
dermal collagen are elastin fibers which are found at the periphery
of collagen bundles and endow the skin with recoil properties
(i.e., the skin's ability to "spring back" after being stretched).
It is believed that damage to the elastin fibers leads to the
decreased skin elasticity seen in aged skin. Other matrix proteins
include glycoproteins such as fibronectin and tenascin which
influence cell migration, adhesion, and orientation,
glycosaminoglycans (GAGs) such as hyaluronic acid, dermatan
sulfate, and heparin sulfate which may be important for cell
growth, membrane receptor function, and adhesion, and proteoglycans
such as decorin (Baumann L. Basic science of the dermis. Cosmetic
Dermatology: Principles and Practice. Hong Kong: The McGraw Hill
Companies, Inc., pp. 9-12, 2002). GAGs and proteoglycans have been
shown to be key regulators of a variety of cellular behaviors and
will be discussed further below.
[0066] Dermal Collagens--General Characteristics
[0067] Collagens comprise the most abundant proteins in the ECM.
Collagens are the major structural element of all connective
tissues where they contribute to the stability and structural
integrity of tissues. Over 21 different collagens have been
described. Based on their structure and supramolecular
organization, they have been divided into fibril-forming collagens
(types I, II, III, V, and XI), basement membrane collagen (type
IV), microfibrillar collagen (type VI), anchor fibrils (type VII),
hexagonal network-forming collagens (types VIII and X),
fibril-associated collagens (types IX, XII, XIV, XIX, XX, and XXI),
transmembrane collagens (types XIII and XVII), and multiplexins
(types XV, XVI, and XVIII). Despite their high structural
diversity, all members of the collagen family have a characteristic
right-handed triple helix composed of three .quadrature.-chains.
About 90% of total collagens are fibril-forming collagens.
(Reviewed in Gelse K, Poschl E, and Aigner T. Collagens-structure,
function, and biosynthesis. Adv Drug Del Rev 55, 1531-1546, 2003).
Type I collagen comprises 80-85% of the dermal matrix and is
responsible for the tensile strength of the dermis. Type I collagen
is decreased in photoaged skin and increased in skin after dermal
injury (e.g., trauma, dermabrasion). Type III collagen is the
second most abundant dermal collagen, comprising 10-15% of the
dermal matrix and is important for skin compliance (Baumann L.
Basic science of the dermis. Cosmetic Dermatology: Principles and
Practice. Hong Kong: The McGraw Hill Companies, Inc., pp. 9-12,
2002).
[0068] Dermal Collagen--Organization
[0069] Because the major component of ECM is collagen, the
mechanical, physiological, and biological properties of ECM are
affected by the supra-molecular structure of collagen such as the
organization of collagen molecules into fibrils, of fibrils into
bundles, and of bundles into a tissue-specific matrix, the
structure or organization of collagen can profoundly impact the
function or appearance of various tissues (Kuwaba et al., 2002).
Innate mutations in the collagen molecule or molecules involved in
collagen fibrillogenesis can lead to collagen disorganization and
disease entities such as Ehlers Danlos (Ameye L, and Young M F.
Mice deficient in small leucine-rich proteoglycans: novel in vivo
models for osteoporosis, osteoarthritis, Ehlers-Danlos syndrome,
muscular dystrophy, and corneal diseases. Glycobiology.
12:107R-116R, 2002). Acquired conditions such as prolonged exposure
to UVR can also lead to destruction of normal tissue architecture
and replacement by disorganized collagen with thinning and
wrinkling of the skin. Lastly, acquired injury or disease to the
normal collagen architecture of various tissues can lead to the
production and deposition of disorganized collagen during the
repair process. Examples of this include hepatic cirrhosis,
pulmonary fibrosis, and dermal scar formation. Thus, many disparate
processes, with or without over skin injury, can lead to collagen
disorganization and the promotion of collagen organization can
potentially be used to treat many different clinical
conditions.
[0070] Glycosaminoglycans
[0071] GAGs constitute a considerable fraction of the
glycoconjugates found on cellular membranes and in the ECM of
virtually all mammalian tissues. Their ability to bind and alter
protein-protein interactions or enzymatic activity makes them
important determinants of cellular responsiveness in development,
homeostasis, and disease. Although heparin sulfate, heparin, and
hyaluronic acid have been more commonly studied, dermatan sulfate
is the predominant GAG expressed in skin accounting for as much as
0.3% dry weight of skin. In addition, dermatan sulfate, also known
as chondroitin sulfate B, has been shown to promote bFGF and FGF-7
activity. Dermatan sulfate and dermatan sulfate associated
proteoglycans such as decorin are markedly upregulated after
injury. Dermatan sulfate derived from wounds activates endothelial
leukocyte adhesion through stimulation of ICAM-1. Indirectly, the
production of dermatan sulfate proteoglycans such as decorin and
biglycan have been associated with increased scarring (Reviewed in
Trowbridge J M, and Gallo R L. Dermatan sulfate: new functions from
an old GAG. Glycobiology. 12:117R-125R, 2002; Trowbridge J M, et
al., J Biol Chem. 277:42815-42820, 2002). Thus, dermatan sulfate is
associated with increased leukocytosis which can contribute to
inflammation as well as increased bFGF which can contribute to
melanocyte proliferation. In addition, treatment with
chondroitinase B, a lyase that degrades dermatan sulfate as its
sole substrate, inhibited bFGF mediated fibroblast proliferation
(Denholm E M, et al., Eur J Pharmacol. 400:145-153, 2000; Pojasek
K, et al., J Biol Chem. 277:31179-31186, 2002). Therefore,
modulation of GAG levels, either directly through degradation with
enzymes or indirectly through modulation of their associated
proteoglycans can potentially minimize inflammation and
hyperpigmentation. Though not wishing to be bound by a particular
theory, the chondroitinase B mediated decrease in dermatan sulfate
levels may decrease bFGF mediated effects on fibroblast and
melanocyte proliferation with resultant promotion of skin
regeneration, collagen organization, decreased hyperpigmentation,
as well as decrease ICAM-1 mediated effects with resultant
inhibition of leukocytosis and inflammation.
[0072] In addition, there is evidence suggesting that the size of
GAGs such as dermatan sulfate change during repair processes and
that the size of a particular GAG can have potential implications
for collagen organization. Specifically, while small leucine rich
proteoglycans (SLRPs) are known to impact formation of collagen
fibrils, the size of particular GAGs on SLRPs can potentially
impact the spacing of the collagen fibrils (i.e., interfibrillar
distance) as will as the diameter of collagen fibrils. For example,
elongated GAGs were associated with enlarged interfibrillar spaces
with thin collagen fibrils, while normal sized GAGs were associated
with tightly packed, thick collagen bundles during repair in adult
mice (Kuwaba K, et al., J Dermatol Sci. 29:185-194, 2002). Thus
modulation of GAG length through various enzymes specific for a
particular GAG can be used to further promote collagen
organization. For example, keratan sulfates (another type of GAG)
can be modulated through use of various keratan sulfate degrading
enzymes (Reviewed in Yamagishi K, et al., J Biol Chem.
278:25766-25772, 2003).
[0073] Small Leucine-Rich Proteoglycans
[0074] Another important class of matrix proteins are SLRPs that
have been shown to bind to transforming growth factor-beta
(TGF-beta) and to regulate collagen fibrillogenesis. Decorin and
biglycan are two members of the SLRP family that have already been
discussed above. The SLRP family is rapidly growing and includes at
least 13 members (Reviewed in Ameye L and Young M F. Mice deficient
in small leucine-rich proteoglycans: novel in vivo models for
osteoporosis, osteoarthritis, Ehlers-Danlos syndrome, muscular
dystrophy, and corneal diseases. Glycobiology 12:107R-116R, 2002).
Most SLRPs can be grouped into three classes. Decorin and biglycan
are representative of class I SLRPs in that they contain a unique
N-terminal cysteine sequence and carry one and two chondroitin or
dermatan sulfate chains, respectively. Unlike class I, class II
SLRPs contain a different N-terminal cysteine sequence and
generally carry polylactosamine or keratan sulfate chains. FM,
lumican, keratocan, and osteoadherin are examples of class II
SLRPs. Class III SLRPs exhibit a characteristic N-terminal cysteine
sequence and contain sulfated tyrosine residues in the N-terminal
end.
[0075] In general, class I SLRPs tends to be more ubiquitous than
class II members with the distribution of class II SLRPs being the
most tissue specific. Several SLRPs bind to collagens type I, II,
V, VI, XII, and XIV to modulate collagen fibrillogenesis. In
addition, at least three SLRPs (decorin, biglycan, and FM) bind to
TGF-beta, a multifunctional cytokine involved in inflammation
apoptosis, cell proliferation, differentiation, and scar formation.
Based on these findings, several patents have been filed concerning
the ability to reduce scar tissue or wound contraction by
neutralizing TGF-.quadrature.eta activity through the application
of potential TGF-betamodulators within the decorin proteoglycan
family, including decorin, biglycan and FM (U.S. Pat. No.
6,509,314; U.S. Pat. No. 5,583,103; U.S. Pat. No. 5,958,411; U.S.
Pat. No. 5,654,270; and U.S. Pat. No. 5,824,655).
[0076] In addition, because decorin production by fibroblasts
appears to diminish with age and photodamage, and because lack of
decorin in skin is associated with decreased tensile strength and
skin fragility, several patents exist that specifically mention
decorin, but not other SLRPs, in the context of preventing or
treating skin aging. For instance boosting decorin synthesis in
skin by topical application of conjugated linoleic acid,
petroselinic acid, and other compounds (U.S. Pat. No. 6,551,602;
U.S. Pat. No. 6,455,057; U.S. Pat. No. 6,440,434; U.S. Pat. No.
6,423,325; U.S. Pat. No. 6,287,553; U.S. Pat. No. 6,042,841) or
actual use of decorin in cosmetic or dermatologic compositions
(U.S. publication No. 20030124152).
[0077] Existing knockout mice models demonstrate that although
SLRPs belong loosely to the same proteoglycan family, they have
distinctly different effects that are not interchangeable. For
instance, decorin (class I SLRP) deficient mice demonstrate skin
fragility, while FM (class II SLRP) deficient mice demonstrate no
known skin defects (Ameye L and Young M, 2002, Glycobiology, 12(9):
107R-116R). Furthermore, targeted disruption of biglycan (class I
SLRP) results in diminished bone mass and no described skin
abnormalities, while lumican (class II SLRP) knockout mice
demonstrate corneal opacity. Thus, although decorin, biglycan, FM,
and lumican belong to the same SLRP family with decorin/biglycan
and FM/lumican belonging to the same class of proteoglycans, each
member has distinct biological functions with different effects on
collagen fibrillogenesis, structure, and organization that are not
interchangeable.
[0078] In terms of tissue distribution, decorin and biglycan are
ubiquitous, although they show a quite divergent localization
within tissues, with decorin found more in the ECM of tissues where
it is bound to type I collagen (Vogel K G, et al., Biochem J
223:587-597, 1984) and biglycan localized more closely around cells
(Bianco P, et al., J Histochem Cytochem. 38:1549-1563, 1990).
Decorin is the dominant dermatan sulfate proteoglycan distributed
on the surface of collagen fibrils in skin (Kuwaba K, et al., J
Dermatol Sci. 29:185-194, 2002). FM has a somewhat more restricted
distribution with high concentrations in cartilage, tendon and
sclera, while low in skin and mineralized bone (Heinegard D, et
al., J Biol Chem. 261:13866-13872, 1986). Lumican is found mainly
in the cornea (Reviewed in Ameye and Young, 2002).
[0079] D. Aging
[0080] With increasing age, there is decreased ability of
fibroblasts to proliferate and to synthesis collagen and other ECM
proteins such as proteoglycans (Takeda K, et al., J Cell Physiol.
153:450-459, 1992). For instance, type I collagen and decorin
production is decreased in aged skin (Hunzelmann N, et al., Biochim
Biophys Acta. 1360:64-70, 1997 and Carrino D A, et al., J Biol
Chem. 278:17566-17572, 2003). In addition, older fibroblasts also
exhibit higher basal and induced steady-state mRNA levels of
interstitial collagenase (Burke E M, et al., Exp Gerontol.
29:37-53, 1994). Thus, the relative balance between ongoing ECM
deposition (e.g., type I collagen, decorin) and degradation (e.g.,
collagenase) is tilted towards overall ECM degradation with age.
This lead to progressive thinning and disruption of the supporting
dermis that then results in sagging and consequent furrowing of the
epidermis, i.e., the formation of wrinkles. On a microscopic level,
the collagen in aged skin is characterized by thickened fibrils
organized in rope-like bundles, which are in disarray as compared
to the organized pattern seen in younger skin (See, for example,
Oikarinen A., Photodermatol Photoimmunol Photomed 7: 3-4,
1990).
[0081] Besides chronoaging, accelerated skin aging as a result of
sun and/or environmental contaminants exposure can also occur. For
example, photoaging occurs as a result of UVR exposure. UVR
exposure initiates an inflammatory reaction in skin that is
mediated in large part by TNF-alpha as well as other factors
discussed herein. TNF-alpha has been shown to inhibit collagen and
fibronectin synthesis in dermal fibroblasts (Mauviel A, et al., J
Invest Dermatol. 96:243-249, 1991; Mauviel A, et al., FEBS Lett.
236:47-52, 1988) as well as promote collagen degradation (Dayer J
M, et al., J Exp Med. 162:2163-2168, 1985; Siwik D A, et al., Circ
Res. 86:1259-1265, 2000)--both of which contribute to skin aging.
UVR exposure dramatically up-regulates the production of several
types of collagen degrading enzymes known as matrix
metalloproteinases (MMPs) (interstitial collagenase also belongs
within this category of enzymes). Because MMPs degrades collagen,
long-term elevations in MMPs as a result of UVR exposure likely
results in the disorganized and clumped collagen seen in photoaged
skin. Thus MMPs may represent a mechanism by which collagen type I
levels are reduced following UV exposure (Baumann L. Photoaging.
Cosmetic Dermatology: Principles and Practice. Hong Kong: The
McGraw Hill Companies, Inc., pp. 13-20, 2002). Significant levels
of UVR also lead to degradation of the dermis. Furthermore, UVR
also induces bFGF, which among other effects, is known to stimulate
plasminogen activator and collagenase activity that facilitate ECM
breakdown (Reviewed in Abraham J A and Klagsbrun M. Modulation of
wound repair of members of the fibroblast growth factor family. Ed.
Clark R A F. The Molecular And Cellular Biology Of Wound Repair.
Vol. xxiii. New York: Plenum Press, pp. 195-248, 1996) modulation
of bFGF activity by FM may also prevent or minimize the effects of
photoaging including collagen disorganization.
[0082] Thus, both normal and accelerated aging is associated with
an overall decrease in ECM production (e.g., collagen, decorin,
fibronectin) and an increase in ECM degradation (e.g., MMPs and
plasminogen activator) that leads to progressive ECM thinning and
collagen disorganization and clumping. Therefore methods to prevent
decreased ECM production or increased ECM degradation (e.g., by
inhibition of TNF-alpha and/or bFGF) or methods to promote collagen
organization can be useful in promoting the condition of skin,
especially of aged skin.
[0083] III. Methods and Compositions for Promoting Skin
Regeneration
[0084] In one aspect of the present invention, described herein is
a method for modulating skin conditions such as promoting skin
regeneration. The method comprising a step which can be promoting
collagen organization, modulating skin inflammatory conditions,
modulating skin pigmentation, and combinations thereof.
[0085] In one embodiment, the method of modulating skin conditions
can be achieved by, for example, modulating the level of a compound
which can be SLRPs, GAGs, MMPs or combinations thereof in skin of a
mammal. The SLRPs can be, for example, FM, lumican, decorin,
biglycan, and combinations thereof, the GAGs can be, for example,
dermatan sulfate, chondroitin sulfate, keratan sulfate, and
combinations thereof, and the MMPs can be, for example, MMP-1 or
MMP-9, or combinations thereof. The level of SLRPs can be modulated
by applying to the skin a composition comprising an effective
amount of one or more of the SLRPs. The skin can be intact or
non-intact with or without a dermal injury or non-intact skin with
epidermal injury. The level of the dermatan sulfate, chondroitin
sulfate, keratan sulfate, and combinations can be modulated, for
example, by applying to the skin a composition comprising one or
more enzymes that modulate collagen fibrillogenesis and
interfibrillar spacing and/or enzymes that modulate unorganized
matrix deposition by fibroblasts. Exemplary enzymes that modulate
collagen fibrillogenesis, interfibrillar spacing, and/or
unorganized matrix deposition by fibroblasts include, but are not
limited to, chondroitinase AC, chondroitinase B,
endo-beta-galactosidases, keratanase, keratanase II, Bc keratanase
II, and combinations thereof. The level of MMPs can be modulated by
applying to the skin a composition comprising an effective amount
of one or more of the MMPs to modulate collagen degradation.
[0086] In another embodiment, the method of modulating skin
conditions can be achieved by modulating skin inflammatory
conditions or modulating skin pigmentation. The skin inflammatory
conditions can be, for example, non-allergic skin inflammatory
conditions, allergic skin inflammatory conditions, neurogenic skin
inflammatory conditions, UVR induced skin inflammatory conditions,
miscellaneous skin inflammatory conditions, and combinations
thereof. The skin inflammatory conditions and/or skin pigmentation
can be achieved by, for example, the modulation of the level of FM,
lumican, decorin, and/or biglycan, which may modulate TNF-alpha
activity, or modulation of the level of dermatan sulfate, which may
modulate leukocytosis in the skin. The level of dermatan sulfate
can be modulated by one or more enzyme, for example, chondroitinase
B. The skin pigmentation may also be achieved via modulation of the
level of dermatan sulfate, which modulates bFGF activity. Basic FGF
activity can then in turn directly modulate melanocyte
proliferation or indirectly by enhancing production of SCF. SCF can
directly modulate melanocyte proliferation and melanin production.
Alternatively, skin pigmentation can be modulated by modulating the
level of dermatan sulfate, chondroitin sulfate, keratan sulfate,
and combinations thereof by applying to the skin a composition
comprising one or more enzymes such as chondroitinase AC,
chondroitinase B, endo-beta-galactosidases, keratanase, keratanase
II, Bc keratanase II, and combinations thereof.
[0087] In another aspect of the present invention, it is disclosed
herein compositions comprising one or more compounds expressed by
fetal tissues which are effective for promoting skin regeneration
and methods of using the compositions. In particular, the present
invention provides a composition that improves, minimizes,
prevents, and or treats visible and/or tactile discontinuities in
skin. Such discontinuities may be induced or caused by internal
and/or external factors, and include the signs of skin aging
described herein. Promoting skin condition is understood to
include, but not be limited to: 1) treatments that improves,
minimizes, prevents, and or treats skin aging as manifested by
wrinkling, sagging, uneven pigmentation, loss of elasticity or
resiliency; 2) treatments that result in smoother, softer skin
texture or appearance; 3) treatments that improves, minimizes,
prevents, and or treats skin inflammation; 4) treatments that
improves, minimizes, prevents, and or treats hyperpigmentation or
uneven skin pigmentation from causes not related to skin aging
(e.g., acne, insect bites, etc); 5) treatments that promote ECM
organization; and 6) treatments that promote skin regeneration.
[0088] In still another aspect of the present invention, methods
are provided for promoting the condition of skin utilizing SLRPs,
which among others includes FM, lumican, decorin, and biglycan as
well as utilizing enzymes to modulate GAGs associated with
proteoglycans. In one embodiment, the present invention includes
skin regeneration and dermal collagen organization as well as
methods to prevent or minimize skin inflammation and
hyperpigmentation.
[0089] In a further aspect of the present invention, it is provided
a method for identifying compounds expressed by fetal tissues for
promoting skin regeneration, compositions comprising one or more of
the compounds thus identified, and methods of using the
compositions for promoting skin regeneration. The method may
further include isolating the compound expressed by fetal tissue,
identifying the compound, recombinantly expressing the compound,
and then applying the compound to the skin of a mammal.
[0090] In still a further embodiment of the present invention, a
method of promoting skin regeneration includes comparing compounds
expressed by adult tissue and compounds expressed by fetal tissue.
Compounds expressed only in adult tissue are selected and other
compounds are selected to block expression of the compounds
expressed only in adult tissue. The blocking compounds are then
applied to skin of a mammal.
[0091] Cosmetic skin care compositions are also provided that may
include a compound expressed by fetal tissues. Other skin care
compositions are provided that may include a proteoglycan such as
FM, an enzyme such as chondroitinase B, and an enzyme such as
MMP-1.
[0092] All percentages and ratios used herein are by weight of the
total composition and all measurements made are at 25.degree.
Celsius, unless otherwise designated. In certain instance
compositions containing enzymes may be expressed by units of
specific activity (IU) for a given weight (e.g., IU/mg) or a given
volume (e.g., IU/ml)
[0093] The compositions of the present invention are useful for
topical application and for promoting skin condition. The compounds
expressed by fetal tissues can be used as individually purified or
partially purified or used directly without purification in the
form of cell lysates, extracts, and culture media. In one
embodiment of the present invention, compounds expressed by fetal
cells or tissues may be isolated directly through tissue culture
media or cell lysates and further concentrated or purified.
Although individual identification or purification of compounds
expressed by fetal tissues may be useful, the application of this
invention does not require the individual identification or
purification of the compounds. The tissue culture media or cell
lysate, which may or may not be further concentrated or purified,
may then be formulated into cosmetic compositions to improve the
condition of skin.
[0094] In a representative embodiment, the composition may
comprise: a) from about 0.0001% to about 10% by weight of the
proteoglycan compound which is purified, and about 0.1% to about
80% by weight of a cell lysate, extract, or media enriched with the
proteoglycan compound; b) from about 0.1% to about 10% by weight of
hyaluronic acid; c) from about 0.000001% to about 10% by weight of
at least one additional skin care active; and d) a carrier which
can be a cosmetically acceptable carrier, a dermatologically
acceptable carrier, a pharmaceutically acceptable carrier, a
vesicular delivery system, and combinations thereof.
[0095] A. Compounds Expressed By Fetal Tissues
[0096] Early gestation fetal skin has an innate ability to heal
through a process of true tissue regeneration rather than scar
formation. Non-coincidentally, the process of tissue regeneration
is also characterized by a paucity of inflammation. Thus, the use
of a fetal skin model can be used to identify molecules that are
important to the inherent ability of early gestation fetal skin to
heal through regeneration rather than scar.
[0097] It is well documented in the art that fetal skin is
fundamentally different from adult skin. For instance, after
injury, adult skin repairs through marked inflammation and scar
formation, a process characterized by the replacement of injured
tissues with a disorganized deposition of collagen and various ECM
components, referred to collectively as a "scar." In contrast,
fetal skin repair occurs by cellular regeneration and restoration
of normal skin architecture through organized deposition of
collagen and ECM components to effect scarless repair with minimal
inflammation (Mackool, R. J., Gittes, G. K., and Longaker, M. T.
Scarless healing. The fetal wound. Clin Plast Surg 25:357-365,
1998). Studies have shown that the capabilities for scarless skin
repair is one quality of fetal skin, and does not require the fetal
immune system, fetal serum, or amniotic fluid (Bleacher J C, Adolph
V R, Dillon P W, Krummel T M. Isolated fetal mouse limbs:
gestational effects on tissue repair in an unperfused system. J
Pediatr Surg 28: 1312-4; discussion 1314-5. 1993; Ihara S,
Motobayashi Y. Wound closure in fetal rat skin. Development 114:
573-82. 1992). For example, isolated human fetal skin transplanted
into athymic mice heals without producing typical scar tissue
(Adzick N S, Lorenz H P, Ann Surg 220: 10-8. 1994).
[0098] Accordingly, specific molecules or compositions in
regenerating fetal skin that are minimally present or not present
at all in non-fetal skin (e.g., adult skin) are important in
regenerating and promoting the condition of skin. Specifically,
given the lack of significant inflammation in fetal skin, some of
these molecules or compositions may also exert anti-inflammatory
effects by preventing or minimizing inflammation. Also, given the
lack of unorganized fibrous tissue deposition and organization in
fetal skin, some of these molecules or compositions may also
prevent excessive ECM production and/or promote ECM organization
with restoration of normal collagen architecture (Whitby D J and
Ferguson M W, Development 112:651-668, 1991).
[0099] Although a method for identifying genes important for skin
ageing and/or skin stress (WO 02/053773 A3) and genes important for
skin homeostasis (WO 02/053774 A3) has been disclosed in the prior
art, the methods involved are completely different from the present
invention. For instance, it has been demonstrated that the capacity
for tissue regeneration and scarless repair is confined to specific
time points during the fetal period (Ihara S, et al., Development
110: 671-680, 1990). Only early gestation mammals have the capacity
to heal without scar. Late gestation fetuses and neonatal animals
have already lost the capacity for tissue regeneration and exhibit
and "adult-type" wound healing response characterized by scar (Soo
C, et al., Am J of Pathol. 157:423-433. 2000). Thus from a
molecular gene screening perspective, the comparison of "old and
young skin" as specified in WO 02/053773 A3, would not identify the
genes necessary for tissue regeneration as that capacity is already
lost in late gestation fetuses and certainly lost in "young" skin.
This is supported by the observation that cleft lip repair in
infants with "young" skin is followed significant scar formation,
and that the only instance of scarless cleft lip repair has been in
early gestation fetal animal models (Longaker M T, et al., Plast
Reconstr Surg 90:750-756, 1992). The prior art does not describe
using fetal tissues and wound models for identification of
compositions, especially of cosmetic compositions, to improve the
condition of skin. In addition, the prior art as stated in WO
02/053773 A3) does not describe a skin regenerative formulation of
compositions comprising compounds expressed by fetal tissues.
[0100] a) Methods for Preparation of Fetal Tissue in a Fetal
Model
[0101] Female Sprague Dawley (SD) rats (.about.300 gm) were mated.
Detection of a vaginal plug as evidence of pregnancy was considered
day 0.5 of gestation (term=21.5 days). For creation of the fetal
wounds, pregnant rats were anesthetized on days 16 and 18.5 or 19
of gestation. Fetal rat skin transitions from scarless fetal-type
repair to adult-type repair with scar between day 16 (E16) and day
18 (E18) of gestation (term=21.5 days). E19 fetal rats were chosen
to avoid potential overlaps with the E 16 to E18 transition period.
Anesthesia consisted of 1% Ketamine at a dose of 10-20 mg/kg and
0.1% Xylazine at a dose of 0.3 mg/kg. The pregnant animals were
shaved and a midline laparotomy performed. Each uterine segment was
externalized and a 7-0 nylon purse-string suture was placed through
all layers of the uterine wall on the non-placental surface. The
myometrium and amniotic sac was then incised within the
purse-string utilizing microsurgical scissors. Subsequently, a 2-mm
excisional wound was made on the dorsum of the fetuses by grasping
the skin with microsurgical forceps and excising the skin with
scissors. Blue or green vital stain was applied to the excisional
sites for later wound identification. Warm sterile normal saline
was then applied through the hysterotomy and the purse-string
closed (FIG. 1). The maternal fascia and skin was then closed in
two layers using 2-0 synthetic absorbable suture.
[0102] For histology, E16 and E19 fetal wounds were harvested at
12, 24, 36, 48, and 72 hours post-operatively. Non-wounded skin
from each of the wound harvest time points were used as controls
(e.g., E17 control skin for E16+24 hours wounds). A total of four
animals from two separate pregnancies were utilized for each time
point. All tissue specimens were fixed in 4% paraformaldehyde,
dehydrated through graded ethanol, embedded in paraffin, and cut
into 5 .mu.m sections for Hematoxylin and Eosin (H & E)
staining and immunohistochemistry, or into 7 .mu.m sections for
confocal laser scanning (CLSM) microscopy.
[0103] For RNA analysis, E16 and E19 fetal wounds were harvested at
24 and 72 hours after injury. Non-wounded skin from each of the
wound harvest time points were used as controls (e.g., E19 control
skin for E16+72 hour wounds). A total of 20 wounds were utilized
for each time point. The isolated tissue was immediately frozen in
liquid nitrogen and stored at -70.degree. C. until RNA
extraction.
[0104] b) Methods for Confirmation of Tissue Regeneration in a
Fetal Model
[0105] Both H & E staining to evaluate overall wound appearance
and CLSM to analyze collagen architecture and fibril arrangement
were used (FIGS. 2 and 3). CLSM techniques were performed as
previously described (Beanes S R, et al., Plast Reconstr Surg.
109:160-170., 2002).
[0106] Total collagen density per healed wound site was calculated
using Image Pro.RTM. Plus by dividing total collagen surface area
by total wound surface area (Media Cybernetics, Silver Spring, Md.)
for both E16 (n=10) and E19 (n=8) wounds at 72 hours after injury.
For comparison, total collagen density in non-injured skin from
age-mated controls [e.g., E19 (E16+72 hours) and neonatal day1
(E19+72 hours) animals] was also determined. Means and standard
deviations were calculated and unpaired two-tailed Student's t test
was performed to detect statistically significant differences in
total collagen density. A p value of <0.05 was considered
significant.
[0107] As can be seen from FIGS. 2A-E, E16 wounds demonstrated
regenerative scarless repair on H&E with minimal dermal
inflammation and dermal cellularity and organized collagen
architecture on CLSM that was comparable to normal, unwounded skin
(FIG. 2F). Digital imaging analysis verified that 72 hour
post-injury E16 wounds and non-injured E19 (E16+72 hours) skin did
not display significantly different collagen densities (p>0.05)
(FIG. 2G). In contrast, E19 wounds demonstrated non-regenerative
repair with scar and absent hair follicle regeneration as well as
increased and prolonged inflammation and increased dermal
cellularity (FIGS. 3A-E). CLSM revealed a disorganized collection
of dense, heterogeneous collagen fibrils in the completely healed
wound (FIG. 3F). Digital imaging analysis verified that there was
significantly increased collagen density in E19 wounds 72 hours
after injury relative to non-wounded neonatal day 1 skin (E19+72
hours) (p=0.00043) (FIG. 3G). These studies indicate that early
gestation E16 wounds exhibit a capacity for scarless regenerative
repair that is lost in late gestation E19 wounds. Human fetal skin
from 15 to 22 weeks (2 .sup.nd trimester) also exhibited a capacity
for scarless regenerative repair. (Reviewed in Dang C, et al., Clin
Plast Surg. 30:13-23, 2003).
[0108] c) Method for Direct Compound Derivation from Human Fetal
Tissue Culture Systems
[0109] As discussed above, human fetal skin between 15 to 22 weeks
possesses the capability for scarless skin repair and tissue
regeneration. Thus, non-genetically modified compounds expressed by
fetal tissues may be obtained from fetal skin organ cultures, two-
or three-dimensional fetal cell cultures, and media from cultured
fetal cells/tissues. These compounds may be in the form of lysates,
extracts, or media. Methods of cell and tissue culturing, as well
as methods of obtaining cellular lysates or extracts, are well
known in the art. (Refer to Pollard J W, Walker J M (1997) Basic
cell culture protocols, 2nd ed. Humana Press, Totowa, N.J for more
specific details on cell culture). Human fetal cell culture media
may be isolated and the resultant supernatant processed. Cell
culture supernatant processing is well known to those of ordinary
skill in the art and can include, but is not limited to,
concentration of the supernatant, specific compound purification
from the supernatant, and sterilization of the supernatant. The
method of cell culture supernatant processing should ensure optimal
preservation of biologic activity of the compounds expressed by
fetal tissues. Aseptic processing and other efforts to promote
sterilization are also desirable and needed. The following are
examples intended to clarify, but not limit, the scope of the
invention.
[0110] Human Fetal Cell Culture
[0111] Fetal skin fibroblasts may be isolated from fetal skin
specimens by placing small strips of fetal skin (dermal side down)
into a cell culture plate. Fetal skin fibroblasts migrate from the
pieces of skin and attach to the culture plate. Following
attachment of the fibroblasts to the culture plate, the pieces of
skin are then discarded. The fetal skin fibroblasts are allowed to
grow to the desired confluency and are isolated according to
standard techniques.
[0112] Human Fetal Cell Media Preparation
[0113] Fetal cell media (or supernatant) can be obtained by pouring
or aspirating the fluid from the fetal tissue or cell cultures.
Following removal, the resulting supernatant can be further
processed. Examples of such processing may include, but are not
limited to, concentration by a water flux filtration device or
defiltration (see section below "Example of Protein Purification
from Fetal Cellular Media or Lysate" for more information about
further processing).
[0114] Human Fetal Cell Lysate Preparation
[0115] After allowing fetal skin fibroblasts to grow to 70-80%
confluency in a cell culture plate, cell lysate preparation is
carried out as follows: (1) culture plates containing the fetal
fibroblasts are thoroughly washed in phosphate buffered solution
("PBS") in order to remove serum; (2) the fetal fibroblast cells
are then incubated approximately 3 minutes with a dissociating
enzyme such as trypsin to facilitate detachment from the culture
plates; (3) the detached cells are then pelleted by centrifugation
and then lysed using a detergent such as sodium dodecyl sulfate
("SDS"). The supernatant is then dialyzed to remove the traces of
SDS.
[0116] Intracellular products are also isolated by chemical (e.g.,
organic solvents), enzymatical (e.g., lysozyme and EDTA),
mechanical, or physical cell disruption methods (e.g.,
homogenization, ultrasonication, high pressure homogenization,
agitation with abrasion). Combinations of mechanical and
non-mechanical methods are also contemplated.
[0117] Protein Purification from Human Fetal Cellular Media or
Lysate
[0118] Purification of the extracellular (cellular media) or
intracellular (lysate) products can be performed using a variety of
methods to facilitate product isolation or to remove undesired
contaminants. One method is solid-liquid phase separation (e.g.,
centrifugation/sedimentati- on, extraction, filtration). Another
method is concentration (e.g., evaporation, ultrafiltration,
adsorption, precipitation). Yet another method is chromatography
(e.g., size exclusion, ion-exchange chromatography,
chromatofocusing, hydrophobic interaction, affinity chromatography,
immobilized metal-ion affinity chromatography, covalent
chromatography.) These techniques are all readily carried out by
one of ordinary skill in the art. Sterilization techniques such as
filtration or heat or irradiation can also be applied if necessary.
(Refer to Ratledge C, Kristiansen B (2001) Basic biotechnology, 2nd
ed. Cambridge University Press, Cambridge, U.K. for more
information on protein purification).
[0119] d) Method for Indirect Compound Derivation from Fetal Wound
Models through Gene Recombinant Technology
[0120] In another embodiment of the present invention, compounds
expressed by fetal tissues or conditions that promote expression of
these compounds are identified in mammalian skin (human or
non-human). Fetal tissues and cells display distinctly different
patterns of gene expression from adult cells. At the protein level,
this can result in differential production of distinct ECM
components, growth factors, cytokines, and enzymes (Sullivan K M,
Lorenz H P, Meuli M, Lin R Y, Adzick N S. A model of scarless human
fetal wound repair is deficient in transforming growth factor beta.
J Pediatr Surg 30:198-202; discussion 202-3, 1995). For instance,
fetal skin fibroblasts produce higher ratios of type III relative
to type I collagen and different profiles of proteoglycans, as well
as more hyaluronic acid (Mast B A, Diegelmann R F, Krummel T M,
Cohen I K. Scarless wound healing in the mammalian fetus. Surg
Gynecol Obstet 174: 441-51. 1992). Proteoglycans are core proteins
carrying one or more GAG chains with key roles in ECM assembly,
cellular interactions, and growth factor storage (Ruoslahti E.
Proteoglycans in cell regulation. J Biol Chem 264:13369-72,
1989).
[0121] Gene expression differences between fetal and adult tissues
may be identified through standard molecular biology techniques.
For example, Northern blot, subtractive hybridization, differential
display PCR, microarray, and real time PCR may be utilized to
identify gene expression differences. (The following are sample
references for some of the various techniques: DD-PCR--Liang L,
Arthur B P. Differential display of eukaryotic messenger RNA by
means of the polymerase chain reaction. Science 257:967-971, 1992;
Microarrray--Zhang X, et al., Craniosynostosis in transgenic mice
overexpressing Nell-1. J Clin Invest 110: 861-70. 2002; Subtractive
hybridization--Diatchenko L, et al., Suppression subtractive
hybridization: a method for generating differentially regulated or
tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA
93: 6025-30. 1996). Using the various differential gene expression
identification techniques, a molecular "blue print" of the specific
events in fetal skin regeneration can be elucidated and specific
molecules identified as being up-regulated or down-regulated during
fetal regenerative repair. Please refer to FIGS. 4-7 for examples
of specific primer PCR based techniques of candidate gene
screening.
[0122] Once identified, up-regulated molecules in early fetal, but
not late fetal or adult tissues or cells may then be isolated and
identified for subsequent product development. These up-regulated
molecules may be "added back" to confer more "fetal-like"
properties to adult skin. Conversely, molecules up-regulated in
late fetal or adult tissues, but not early fetal tissues, may also
be identified for subsequent product development. One may "block or
inhibit" the identified up-regulated molecules in adult tissues to
confer more "fetal-like" properties to adult skin.
[0123] The following are intended to clarify, but not limit the
scope of the present invention. The molecules to be "added back"
may be directly derived from fetal tissue or fetal cell culture
media, lysates, or extracts (see the above discussion, supra).
Alternatively, the molecules to "add back" may be "recombinantly
expressed" (with or without preceding genetic modification) in
genetically engineered cells for the purpose of increasing its
expression. Up-regulated molecules in fetal cells or tissues may
then be utilized in skin compositions individually or in
combination. Conversely, up-regulated molecules identified in
adult, but not fetal tissues (i.e., the molecules to "block or
inhibit") may be targeted using antisense RNA molecules or known
inhibitors or modulators (e.g., chondroitinase B for modulation of
dermatan sulfate). The antisense RNA molecules or known inhibitors
may then be subsequently used in the skin care composition.
[0124] Screening Normal Rat Fetal Tissue as a Function of
Gestation
[0125] Gene expression screening of uninjured fetal skin as a
function gestation may allow for the identification of molecule
important for fetal skin homeostasis in the absence of injury. FIG.
4 shows the downregulation of FM and upregulation of decorin with
increasing gestational age and loss of regenerative healing, which
are unexpected to one with ordinary skill in the art, showing
decorin upregulation temporally associates with non-regenerative
repair in late gestation fetuses. The other molecules screened by
this methodology [i.e., TGF-beta ligands and receptors as well as
latent TGF-beta binding protein-1 (LTBP-1)] are included as
examples of screening using specific primer based PCR.
[0126] Screening Wounded Rat Fetal Tissue
[0127] Gene expression screening of injured E16 relative to E19
fetal skin allows for potential identification of molecules that
are critical to skin regeneration. FIG. 5 shows the results of PCR
based screening for MMPs and TIMPs. Scarless wounds are
characterized by relatively higher MMP-1 and MMP-9 expression and
less TIMP-1. This demonstrates that relatively increased ECM
degradation relative to excessive ECM deposition is important to
scarless repair. Since excessive accumulation of ECM constitutes on
essential component of a scar, compounds that can prevent excessive
ECM deposition and promote ECM degradation such as MMPs may be
useful to skin regeneration after injury characterized by scar
formation. FIG. 6 shows the results of PCR based screening for
FGFs. Scarless wounds are characterized by minimal FGF induction in
the initial 48 hours after injury relative to wounds that scar. In
particular, FGF-2 (which is bFGF) is relatively decreased in
scarless fetal wounds. This suggests that modulation of bFGF may be
useful to skin regeneration. FIG. 7 shows the results of PCR based
screening for decorin and FM. It shows that FM, but not decorin, is
upregulated in E16 regenerative wounds, indicating that modulation
of FM, rather than decorin, can be used to promote skin
regeneration.
[0128] Individual Gene Cloning
[0129] Gene cloning techniques are well described in the art
(Please refer to Wu W. Methods in Gene Biotechnology. Boca Raton:
CRC Press, 1997 for more information). Briefly, for genes in which
the coding sequence is known, specific primer PCR based techniques
may be used for gene cloning. From these sequences, PCR primers can
be designed that flank the coding sequence. After amplification,
PCR products can then be ligated into various expression plasmids
for bacterial, yeast, or mammalian cells. For genes in which the
coding sequence is unknown, which is often the case for genes
sequences identified using differential display, isolation of the
full length cDNA clones can be accomplished by 5' Rapid
Amplification of cDNA Ends (RACE) as previously described (Soo C,
et al., J Cell Biochem. 74:1-10, 1999). For instance the complete
cDNA sequence for human FM was obtained through GenBank by the
following accession numbers: NM.sub.--002023, BC035281, or X75546.
The full-length coding cDNA for the human FM was amplified by
reverse transcription-polymerase chain reaction (RT-PCR) from the
total RNA of human fibroblast. The specific primers used for the
PCR were designed on the basis of published human FM cDNA sequence
on GenBank Accession No NM.sub.--002023 and the 1.1 kb PCR fragment
was confirmed by full-length sequencing.
[0130] Mammalian Expression Plasmid Construction and Expression for
FM
[0131] The p3xFLAG-CMV-14 (Sigma) expression vector for the
mammalian system was chosen by virtue of its convenience of
subsequent protein purification. The 3xFLAG sequence was attached
at the C-terminal end of the human FM cDNA insect in frame to
render the production of C-terminal flag-tagged recombinant
protein. The final construct of HFM expression plasmid,
p3FLAG-CMV-HFM, was further confirmed by selective restriction
digestion and DNA sequencing.
[0132] The CHO-K1 cell line (ATCC, Manassas, Va.) was used for the
production of recombinant human FM (rhFM). The stable transfected
cell line was established following extensive selection with G418
in the medium and screening by the immunofluorescent cytochemisty
with anti-Flag antibody for the expression of the recombinant
protein. The rhFM was purified through affinity chromatography of
anti-flag agarose column with competitive elution by 3xFLAG peptide
to maintain the rhFM in natural form. The purified protein contains
native KS side chains which was confirmed by Western blot in terms
of the molecular size. The ability of rhFM to bind TGF-.beta.1 and
bFGF was verified by ELISA binding assay. The hydrophilic flag
peptide of the rhFM can be readily removed by digestion with
enterokinase if necessary.
[0133] Bacterial Expression Plasmid Construction and Expression for
FM
[0134] The pFLAG-MAC (Sigma) expression vector for the E. coli
system was used for its easy purification of the recombinant
protein from the bacterial cytoplasm. Within this amino-terminal
flag-tagged vector, the human FM cDNA insert was driven by the tac
promoter and the flag tag in the fusion protein can be removed with
the enterokinase if necessary. As same as in the mammalian system,
the full-length coding cDNA fragment of the human FM for the
construct was amplified by RT-PCR from the total RNA of human
fibroblast. The final construct of HFM expression plasmid,
pFLAG-MAC-HFM, was further confirmed by selective restriction
digestion and DNA sequencing.
[0135] The E. coli strain BL21 was used for the production of rhFM.
The transformation and selection of the positive colony was carried
out under the standard protocols. The large scale bacterial culture
with an OD600 of about 2.0 was harvested by centrifuge at
5,000.times.g for 10 minutes. The bacterial cells were lysed with
CelLytic B lysis buffer (Sigma) supplemented with DNase I at 5
ug/ml. The rhFM was purified from the crude extract of bacteria
through affinity chromatogrophy of anti-flag agarose column with
competitive elution by 3XFLAG peptide. The molecular weight and
purity of the rhFM was confirmed by Western blot. The ability of
rhFM to bind TGF-.beta.1 and bFGF was verified by ELISA binding
assay. The hydrophilic flag peptide of the rhFM can be readily
removed by digestion with enterokinase if necessary.
[0136] Recombinant Expression of Individual Genes in Prokaryotic
Cells
[0137] A vector containing the gene of interest is constructed for
later insertion into prokaryotic cells. A strong promoter (such as
T7) or a Lac promoter can be used to induce high transcription
efficiency. A potential affinity binding sequence such as a
histidine affinity tag will be inserted into the N-terminal having
a protease cleavage site. Additional sequences such as maltose
binding protein, may be inserted to increase solubility.
(Hildebrand A, Romaris M, Rasmussen L M, Heinegard D, Twardzik D R,
Border W A, Ruoslahti E. Interaction of the small interstitial
proteoglycans biglycan, decorin and FM with transforming growth
factor beta. Biochem J 302 (Pt 2): 527-34, 1994).
[0138] Recombinant Expression of Individual Genes in Eukaryotic
Cells
[0139] Eukaryotic systems such as yeast, baculovirus, and mammalian
systems allow post-translational modification of gene products. A
strong promoter such as AOX1 in yeast, or cytomegalovirus ("CMV")
in mammalian cells can be used to induce high transcription
efficiency. An affinity tag can be inserted at either the N or C
terminal of the translational product. Secretory sequences can also
be inserted at the N-terminal to increase the secretion of desired
recombinant molecules into the cell culture medium.
[0140] Large scale expression of recombinant proteins can also be
performed through established commercial companies using
bioengineered plant systems (e.g., www.VentriaBio.com).
[0141] Binding Assay of Flag-Tagged Human FM (HFM) and Decorin
(HDC)
[0142] Flat bottom multi-well plates were coated with human
recombinant TGF-beta1 (Sigma) or human recombinant bFGF (Sigma) at
50 .mu.l/well (0.5 .mu.g/ml) diluted in coating buffer (0.5 mM
sodium carbonate buffer, pH9.3) overnight at 4 C. The coated wells
were then emptied and 200 .mu.l of binding buffer (50 mM Tris/HCl,
pH7.4, 150 mM NaCl, 2% BSA and 0.05% Tween20) was added to each
coated well to block non-specific binding sites by incubation for 2
hours at 37C. The wells were emptied and washed three times by
entirely filling each well with wash buffer (PBS, 0.1% Tween20) and
then flicking out the contents and slapping the plate upside down
three times on a paper towel. The affinity purified flag-tagged
human FM and decorin from plasmid transfected CHO-K1 cells were
added in a 100 .mu.l/well of binding buffer and incubated for 1
hour at 37C followed by overnight at 4C. The wells were emptied and
washed as before and incubated with 100 .mu.l/well of anti-flag
biotinylated M2 mAb (Sigma) diluted at 1 .mu.g/ml in TBS-Ca buffer
(50 mM Tris/HCl, pH7.4, 150 mM NaCl, and 1 mM CaCl2) for 1.5 hours
at room temperature. The wells were emptied and washed again as
before and 100 .mu.l/well of detecting Streptavidin-HRP (Dako
Corp.) was incubated for another 1.5 hours at room temperature. The
wells were washed four times and emptied completely before adding
100 .mu.l/well of developing buffer (100 .mu.g/ml
Tetramethylbenzidine, 0.003% H202 in sodium acetate buffer, pH6.0).
The reaction was stopped by adding 100 .mu.l of 1N hydrochloric
acid to each well after 15-30 minutes for development. Binding
capacity of purified human FM and decorin to the
surface-immobilized hrTGF-beta1 or hrbFGF was determined by the
absorbance value at 450 nm using an ELISA plate reader (Fisher).
All the experiments were performed in triplicate. Both flag-tagged
bacterial alkaline phosphatase (BAP-Flag)(Sigma) and purified
lysate from parental CHO-K1 cells (ConL) were used as negative
controls.
[0143] Readings at 450 nm for TGF-beta1 Binding (Triplicate)
[0144] Blank 0.121, 0.111, 0.113
[0145] ConL 0.151, 0.163, 0.164
[0146] BAP 0.120, 0.137, 0.130
[0147] HDC 0.205, 0.207, 0.206
[0148] HFM 0.787, 0.722, 0.741
[0149] Readings at 450 nm for bFGF Binding (Triplicate)
[0150] Blank 0.123, 0.107, 0.123
[0151] ConL 0.207, 0.242, 0.198
[0152] BAP 0.138, 0.120, 0.122
[0153] HDC 0.233, 0.282, 0.244
[0154] HFM 0.796, 0.729, 0.763
[0155] The binding of HSM to TGF-beta1 was well described in the
art. It is not known in the art that HFM is equally capable of
binding to bFGF. This ability of HSM to bind bFGF is particularly
remarkable as the interaction of bFGF with keratan sulfate SLRPs
such as FM has not been described. To date, the only known
interaction between SLRPs and bFGF has been with dermatan sulfate
moieties present on decorin and not the decorin core protein itself
(Zamfir A, et al., Glycobiology. 13:733-742, 2003). Since bFGF is
also a potent mitogen for fibroblasts, this ability of FM to bind
bFGF can be particularly useful in methods to treat conditions of
excessive fibroblast proliferation as in scars or to treat
conditions of excessive melanocyte proliferation or activity as in
hyperpigmentation. Furthermore, since UVR results in bFGF induction
and bFGF, among other effects, is known to stimulate plasminogen
activator and collagenase activity that facilitate ECM breakdown
(Reviewed in Abraham J A and Klagsbrun M. Modulation of wound
repair of members of the fibroblast growth factor family. Ed. Clark
R A F. The Molecular And Cellular Biology Of Wound Repair. Vol.
xxiii. New York: Plenum Press, pp. 195-248, 1996), modulation of
bFGF activity by FM may also prevent or minimize the effects of
photoaging including collagen disorganization.
[0156] Genetic Modification Prior to Recombinant Expression
[0157] Mutations or genetic modifications can be created in both
the non-coding or non-essential regions of a defined gene sequence
(e.g., promoter region, untranslated 3' regions) as well as the
coding or essential regions of a defined gene sequence. By
"essential" it is meant the portion(s) of the gene that is/are
critical to the gene carrying out its intended function. Genetic
modifications in the non-coding regions are generally made to
enhance the overall transcriptional or translational efficiency of
a gene and to increase the ease of purification of the final
protein product--these changes generally do not affect the
functional characteristics of the gene. In contrast, genetic
modifications of the coding region are generally made for purposes
of modifying the translated product to increase or decrease desired
functions of the gene (e.g., modify affinity for target molecule,
modify skin penetration characteristics, modify post-translational
processing, modify half-life of molecule). Both techniques for
non-coding or coding modification are all well described in the
art.
[0158] B. Small Leucine Rich Proteoglycans
[0159] SLRPs are a class of compounds with different functions that
can be used to promote skin regeneration. Although FM was mentioned
for reduction of dermal scarring and wound contraction, by
definition these are conditions that occur with dermal injury.
There are many instances were application of FM or equivalent may
be desirable to improve the condition of skin that do not involve
the requirement for reduction of dermal scarring or wound
contraction.
[0160] a) Treatment of Intact, Aged Skin to Promote Skin
Regeneration
[0161] It is known in the prior art that decorin may improve the
appearance of aged skin. But as stated above, decorin and FM are
different classes of SLRPs with different functions. In addition FM
null mice did not have obvious skin deficits and there is minimal
FM expression in non fetal skin. Therefore it is completely
unexpected and novel that FM would have such a central role in
collagen organization and skin regeneration.
[0162] Although boosting decorin synthesis in skin by topical
application of conjugated linoleic acid, petroselinic acid, and
other compounds (U.S. Pat. No. 6,551,602; U.S. Pat. No. 6,455,057;
U.S. Pat. No. 6,440,434; U.S. Pat. No. 6,423,325; U.S. Pat. No.
6,287,553; U.S. Pat. No. 6,042,841) or actual use of decorin in
cosmetic or dermatologic compositions (20030124152) has been
described in the prior art, there is no mention of the use of FM.
The existing prior art has focused on decorin because decorin
production by fibroblasts appears to diminish with age and
photodamage, and because lack of decorin in skin is associated with
decreased tensile strength and skin fragility (Takeda K, et al., J
Cell Physiol. 153:450-459, 1992; Carrino D A, et al., Arch Biochem
Biophys. 373:91-101, 2000). Thus, several patents exist that
specifically mention decorin, but not other SLRPs such as FM, in
the context of preventing or treating skin aging.
[0163] The use of FM to improve the condition of skin is not
obvious from the existing prior art. For instance, as expected from
the close relationship between decorin and skin, decorin knockout
mice demonstrate distinct skin fragility. Meanwhile, FM knockout
mice as expected did not demonstrate any discernable skin
abnormalities and minimal FM has been detected in skin adult skin.
Moreover, in injured adult skin decorin, but not FM, is
upregulated--indicating a close relationship between decorin
expression and the adult non-regenerative repair response. In
addition, FM and decorin belong to two different classes of SLRPs
that bind to different regions of the collagen molecule.
[0164] Therefore, the upregulation of FM, but not decorin, during
regenerative repair in early gestation fetal skin as evidenced by
organized collagen deposition, while non-regenerative repair with
disorganized collagen arrangement was not accompanied by FM
upregulation in late gestation fetal skin (see FIGS. 4 and 7).
Moreover it was even more surprising that elimination of FM alone
by using anti-FM antibodies was enough to prevent organized
collagen deposition in early gestation fetal animals that normally
exhibit scarless repair and that addition of FM alone was enough to
promote organized collagen deposition in late gestation fetal
animals that normally exhibit repair with scar (FIGS. 8 and 9).
Consistent with the previous data on the lack of excessive matrix
accumulation in scarless fetal wounds, type I collagen mRNA
expression was decreased in late gestation wounds following FM
treatment (FIG. 10). Application of FM to adult wounds also
significantly improved overall dermal structure and collagen
organization (FIG. 11). This confirms that our method to use
compounds from fetal tissues, or identified through fetal tissues,
to improve skin condition is applicable to adult skin.
[0165] b) Treatment of Non-Intact Skin with Epidermal Injury
[0166] The use of decorin, or functionally equivalent molecules
such as FM, to prevent dermal scarring or wound contraction as been
well described in the prior art. A scar is a fibrous or connective
tissue deposition that by definition only occurs with dermal
injury. Dermal injury initiates a cascade of wound healing
responses that involves hemostasis, inflammation, proliferation,
and remodeling. Normal dermal injury repair is characterized by
connective tissue deposition by fibroblasts and wound contraction
by myofibroblasts that ultimately result in scar (Mast B A. The
skin. In Wound Healing: Biochemical and Clinical Aspects. Eds.
Cohen K I, Diegelmann R F, Lindblad W J. Philadelphia: W B Saunders
Company, p. 344-355, 1992). For instance, U.S. Pat. No. 6,509,314
teaches that "dermal scarring is a process following a variety of
dermal injuries that results in the excessive accumulation of
fibrous tissue comprising collagen, fibronectin, and proteoglycans.
The induction of fibrous matrix accumulation is a result of growth
factor release at the wound site by platelets and inflammatory
cells. The principle growth factor believed to induce the
deposition of fibrous scar tissue is . . . TGF-beta. Decorin binds
and neutralizes a variety of biological functions of TGF-beta,
including the induction of ECM."
[0167] In contrast, epidermal injuries alone do not scar or cause
wound contraction. Partial thickness wounds such as abrasions or
superficial burns do not penetrate the dermis and therefore,
neither fibrous or excessive connective tissue deposition (i.e.
scar) nor wound contraction plays a role in epithelial healing
(Mast B A. The skin. In Wound Healing: Biochemical and Clinical
Aspects. Eds. Cohen K I, Diegelmann R F, Lindblad W J.
Philadelphia: W B Saunders Company, p. 344-355, 1992). Thus, the
normal wound cascade of hemostasis, inflammation, proliferation,
and remodeling does apply because first of all, there are no blood
vessels in the epidermis to injure. However epidermal injury or
irritation can initiate an inflammatory response that can affect
melanocytes and Langerhans cells that are contained within the
epithelial layer. Stimulation and/or injury to melanocytes can
stimulate or disrupt the process of pigment production by
melanocytes. Overstimulation of melanocyte pigment production can
lead to epidermal or dermal hyperpigmentation, while injury to
melanocytes can lead to hypopigmentation. The processes of
hyperpigmentation and hypopigmentation directly relate to the
process of pigment production by melanocytes and are completely
different from the processes of scar formation and wound
contraction which are mediated by fibroblasts producing collagen
and other ECM components and myofibroblasts, respectively. Thus,
because of the completely different mechanisms involved in scar
formation or wound contraction relative to pigmentary problems, it
follows that use of FM for treatment of potential complications
associated with epidermal inflammation or injury is not obvious
from the prior art.
[0168] c) Treatment to Promote Collagen Organization
[0169] With specific regard to intact skin, promotion of collagen
organization may potentially be used to treat conditions of
disorganized collagen formation such as chronological aging or
photoaging. With specific regard to non-intact skin, promotion of
collagen organization may potentially be used to treat conditions
of disorganized collagen formation such as dermal scarring.
[0170] As mentioned previously, the use of decorin, or functionally
equivalent molecules such as FM, to prevent dermal scarring (U.S.
Pat. No. 5,654,270; U.S. Pat. No. 6,509,314) or wound contraction
as been well described in the prior art (U.S. Pat. No. 5,510,328;
U.S. Pat. No. 5,851,994).
[0171] Thus, the prior art teaches that excessive fibrous matrix
accumulation is a central component of scar and that this is
mediated primarily by TGF-beta. Furthermore, the prior art teaches
that improvement of scar by decorin or related molecules is
primarily related to the ability to regulate TGF-beta activity.
Thus the prior art teaches that decorin or related molecules can
modulate the quantity of ECM accumulation. This reduction in the
quantity of ECM accumulation will aid the treatment of pathological
entities characterized by excessive fibrous tissue deposition such
as glomerulonephritis and dermal scar. However, reduction in ECM
quantity alone or even inhibition of TGF-beta activity alone is not
enough to completely eliminate scar. This is because although
TGF-beta is known to have a direct effect on the production of ECM,
it has no known effects on the organization of the ECM.
[0172] TGF-betas are multifunctional cytokines with widespread
effects on cell growth and differentiation, embryogenesis, immune
regulation, inflammation, and wound healing (Border W A, et al.,
Kidney International. Supplement. 49:S59-61, 1995). In terms of
cutaneous repair, TGF-beta1 and TGF-beta2 are known to promote
scar, while TGF-beta3 may reduce scar (Lin R Y, et al., Ann Surg.
222:146-154, 1995; Shah M, et al., J Cell Sci. 108 (Pt 3):985-1002,
1995). TGF-beta has been implicated in the ontogenetic transition
from scarless fetal-type repair with minimal inflammation to
adult-type, non-regenerative repair with significantly increased
inflammation. Adult-type repair with scar is characterized by
excessive quantity of matrix deposition and decreased quality of
matrix deposition. A number of strategies designed to neutralize
TGF-beta1, including antibodies against TGF-beta1 and TGF-beta2,
antisense TGF-beta1 oligodeoxynucleotides, and viral gene therapy,
have been shown to reduce, but not completely eliminate, scarring
in adult animals (Choi B M, et al., Immunol Cell Biol. 74:144-150,
1996; Shah M, et al., Lancet. 339:213-214, 1992; Shah M, et al., J
Cell Sci. 107 (Pt 5):1137-1157, 1994; Isaka Y, et al., Nat Med.
2:418-423., 1996; Elepfandt P, et al., Neurosci Lett. 322:107-110,
2002). This indicates that inhibition of TGF-beta activity with its
associated reduction in ECM quantity alone is not enough to
completely eliminate scar.
[0173] It has been shown that deficient TGF-beta1 expression as a
sole mechanism for scarless fetal repair is overly simplistic. It
has also been shown that even scarless E16 wounds exhibit initial
TGF-beta1 and -.beta.2 upregulation after injury (Soo C, et al., Am
J Pathol. in press). Moreover, although TGF-beta is widely
recognized as a pro-fibrotic peptide that can increase the quantity
of ECM, there are no data indicating that TGF-beta can directly
impact the quality or organization of the ECM. This indicates that
other factors directly involved in ECM structure and organization
may also be important to regenerative fetal repair.
[0174] However, there are other conditions, not necessarily
associated with skin injury, that result also in fibrous connective
tissue deposition. One example is systemic sclerosis, a connective
tissue disease characterized by fibrosis of the skin, subcutaneous
tissue, and various internal organs due primarily to excessive
accumulation of type I and III collagen (Kuroda K, and Shinkai H.,
Arch Dermatol Res. 289:481-485, 1997.
[0175] Thus, innate or acquired abnormal collagen structure or
organization can lead to the dysfunction of various tissues. For
instance, mutations in the collagen molecule itself leading to
abnormal collagen structure can give rise to various congenital
diseases syndromes such as chondrodysplasias, osteogenesis
imperfecta, Ehler's Danlos Syndrome, or epidermolysis bullosa
(Reviewed in Gelse K, et al., Adv Drug Deliv Rev. 55:1531-1546,
2003), while mutations the SLRPs that regulate collagen
fibrillogenesis can give rise to various abnormalities such as
osteoarthritis, an Ehler's Danlos-like phenotype, muscular
dystrophy, and corneal diseases. (Ameye L, and Young M F.,
Glycobiology. 12:107R-116R, 2002). Meanwhile, injury or disease can
lead to acquired disorganization of collagen architecture that then
generates further diseases. In these instances, the disorganized
collagen can be collectively termed a "scar". Scar formation is
central to the pathogenesis of many human diseases, including liver
cirrhosis, pulmonary fibrosis, and ischemic heart disease.
[0176] There are two different processes that result in scar. One
is matrix accumulation, without matrix accumulation there is
essentially no substance to form a scar. Scar formation can form
with just excessive matrix deposition even without an inciting
injury event. For instance, one TGF-beta model of
glomerulonephritis in the kidney is based on excessive ECM
accumulation that overwhelms the normal balance of matrix
deposition and degradation. Another example is systemic sclerosis,
a connective tissue disease characterized by fibrosis of the skin,
subcutaneous tissue, and various internal organs due primarily to
excessive accumulation of type I and III collagen. The other
process that results in scar is lack of matrix organization.
[0177] Accordingly, there are two strategies to treat scar. One is
decreasing matrix accumulation. The other is promoting matrix
organization. However, reduction of scarring comprises more than
the reduction of matrix accumulation. Arguably, the organization
pattern of the accumulated matrix is more important than the amount
of matrix present per se. This is supported by experimental
evidence that neutralization of TGF-beta alone is wounds by
anti-TGF-beta antibodies is not enough to eliminate scar. Thus,
other mechanisms not related to TGF-beta mediated matrix
accumulation are required for scarless repair.
[0178] Other known functions for the decorin family of
proteoglycans include in vitro effect on collagen fibril formation.
However, even in vitro, there are apparently different effects. For
instance, decorin and FM interact with different sites on the
collagen molecule. Meanwhile in vivo, SLRP knockout animals
demonstrate different morphology indicating different functions and
tissue distributions of the SLRPs in vivo. For instance, FM
knockout mice have no discernable skin abnormalities and minimal FM
has been detected in skin. FM, in contrast to decorin is not
elevated during non-regenerative type repair.
[0179] Therefore the surprising and novel aspect of this is the
surprising upregulation of FM during regenerative repair in the
fetus and absence of FM during non-regenerative repair with scar.
Even more surprising was the upregulation of decorin with
non-regenerative repair in adult and late gestation animals.
Moreover it was even more surprising that elimination of FM alone
was enough to prevent organized collagen deposition and addition of
FM alone was enough to promote organized collagen deposition in
fetal animals.
[0180] Treatment of Intact or Non-Intact Skin to Decrease
Inflammation
[0181] A novel aspect of this invention is that a heretofore
unrecognized sequence of events in which modulation of decorin and
biglycan can modulate TNF-.alpha., a major inflammatory cytokine
involved in multiple inflammatory skin conditions and modulation of
corresponding dermatan sulfate moieties on decorin or biglycan can
modulate leukocytosis, a major component of the inflammatory
response. Thus, modulation of decorin, biglycan, and/or
corresponding dermatan sulfate moieties can impact a myriad of
inflammatory conditions associated with intact or non-intact skin.
For instance, mild exposure to UV radiation (i.e., sunburn) and
skin rubbing or scratching are examples of actions that can induce
skin inflammation without a break in epidermal integrity
(definition of intact skin). Other examples of inflammatory skin
conditions that may be accompanied by intact or non-intact skin
include, but are not limited to non-allergenic skin inflammatory
conditions, allergic skin inflammatory conditions, neurogenic skin
inflammatory conditions, UVR induced skin inflammatory conditions,
or miscellaneous skin inflammatory conditions. Thus, a composition
comprising one or more of SLRPs and/or enzymes to modulate GAGs
such as dermatan sulfate can be used to treat inflammatory
conditions of the skin. Preferred embodiments include decorin
and/or chondroitinase B.
[0182] Treatment of Intact or Non-Intact Skin to Decrease
Hyperpigmentation
[0183] A novel aspect of this invention is that a heretofore
unrecognized sequence of events in which modulation of decorin and
FM can modulate bFGF, a potent melanocyte mitogen and modulate
TNF-alpha, a major inflammatory cytokine. Another novel aspect of
this invention is that a heretofore unrecognized sequence of events
in which modulation of corresponding dermatan sulfate moieties on
decorin or biglycan can modulate leukocytosis, a major component of
the inflammatory response as well as modulate bFGF activity.
[0184] While not wishing to be bound by any particular theory,
modulation of bFGF activity by modulating decorin and/or FM levels
or by modulating dermatan sulfate levels through use of enzymes
such as chondroitinase B, can directly impact bFGF mediated
melanocyte proliferation. In addition, modulation of bFGF activity
will also modulate SCF activity, a potent inducer of melanocyte
proliferation and melanin production. Moreover, epidermal injuries
or irritation can initiate an inflammatory response that may also
stimulate melanocytes with resultant hyperpigmentation or injure
melanocytes with resultant melanocyte cell death and
hypopigmentation. The ability to decrease inflammation by
modulating TNF-alpha, may potentially diminish melanocyte
stimulation or injury with correspondingly decreased potential for
hyperpigmentation or hypopigmentation.
[0185] Although the ability of certain SLRPs, e.g., decorin,
biglycan, and FM, to bind TGF-beta has been described in the prior
art in the context of decreasing ECM accumulation in the kidney,
TGF-beta has not been implicated in problems of excessive
pigmentation. In fact, TGF-beta1 strongly inhibits normal
melanocyte proliferation and DNA synthesis in vitro (Krasagakis K,
et al., Anticancer Res. 14:2565-2571, 1994). In addition,
hyperpigmentation is a process completely different from scar
formation. Scar formation involves excessive accumulation of
fibrous tissue manufactured by fibroblasts and occurs with dermal
injury. Hyperpigmentation, in contrast, involves excessive
production and deposition of the pigment melanin by melanocytes and
can occur with or without actual skin injury. In cases associated
with injury, there is usually an accompanying inflammatory
component. In cases without actual disruption of skin integrity, an
inflammatory component may or may not be present depending on the
degree of UVR exposure. Thus, hyperpigmentation can occur with or
without associated skin inflammation and with or without associated
skin injury, while scar formation is associated prerequisitely with
dermal injury
[0186] One embodiment of the invention would be toward application
to acne lesions and insect bites which can induce significant
hyperpigmentation in certain ethnic groups such as Asians, Latinos,
can blacks. Another embodiment of the invention would be
application to inflammatory skin conditions with the potential for
hyperpigmentation such as, but not limited to, non-allergenic skin
inflammatory conditions, allergic skin inflammatory conditions,
neurogenic skin inflammatory conditions, UVR induced skin
inflammatory conditions, or miscellaneous skin inflammatory
conditions.
[0187] C. Fibromodulin
[0188] In one aspect of the present invention, a composition
comprising FM can be used to promote skin regeneration. FM, one of
several components expressed by fetal tissue and a SLRP,
dramatically improves the organization of dermal collagen in skin
without evidence of skin irritation. Purified FM or FM enriched
cellular extracts, when applied topically, improved the condition
of skin without irritation. In fact, application of FM resulted in
a significant reduction of skin inflammation and inflammatory
cytokine expression. Although the use of FM has been described in
the art for reducing dermal scarring associated with acute
cutaneous injury (see, U.S. Pat. Nos. 5,654,270 and 5,510,328), the
current state of the art fails to describe the novel, cosmetic skin
care use of FM or functionally equivalent molecules for promoting
the condition of non-scarred skin. In addition, the current state
of the art also fails to describe the novel, cosmetic or
pharmacological use of FM, or functionally equivalent molecules for
decreasing skin inflammation and hyperpigmentation.
[0189] In a representative embodiment of the present invention,
specific compounds expressed by fetal tissues such as FM can be
isolated from native tissues (wild-type form) or from suitable
expression vehicles such as bacteria or yeast (recombinant
form--with or without modification of the coding region) and then
formulated into cosmetic or non-cosmetic compositions to improve
the condition of skin. Examples of specific compositions are
described herein. The following are intended to clarify, but not
limit the scope of the invention.
[0190] The FM useful for promoting skin regeneration can be
wild-type or recombinant FM. Purified wild-type FM protein can be
obtained from commercially available sources (Sigma-Aldrich Corp.,
St Louis, Mo.). Recombinant FM is obtained by cloning the FM cDNA,
preferably human, into a suitable expression vector (e.g., plasmid,
adenovirus). The cDNA for human FM is known in the art. (GenBank
accession number X75546).
[0191] In one embodiment, the coding sequence of the recombinant FM
cDNA may also be genetically modified prior to recombinant
expression to enhance specific characteristics using techniques
well known in the art. For example, site-specific mutagenesis can
be used to increase the binding affinity of the protein to its
receptors by using oligonucleotide primers. In addition,
hydrophilic and secretory sequence such as Ig kappa-chain or
histidine and GST tag sequences can be added to increase
purification efficiency. The non-coding sequence of the recombinant
FM cDNA may also be genetically modified to enhance specific
characteristics using techniques well known in the art. For
example, native mammalian promoters may not be efficient enough to
produce large amount of proteins. In such a case, CMV and SV40
promoters can be inserted into mammalian systems to increase
transcription efficiency. In addition, a SV40 or .beta.-globin poly
A sequence can be added to the 3' end to increase stability and
protein production efficiency.
[0192] In another embodiment, FM may be isolated from
non-genetically modified cells or genetically modified cells.
Methods of cell and tissue culturing, as well as methods of
obtaining cellular lysates or extracts, are well described in the
art and may be performed by one of ordinary skill in the art (Refer
to Pollard J W, Walker J M. Basic cell culture protocols, 2nd ed.
Humana Press, Totowa, N.J, 1997 for more information). Cell culture
media enriched with FM may be isolated and the resultant
supernatant processed. Such processing are apparent to one of
ordinary skill in the art and can include, but are not limited to,
concentration of the supernatant, specific compound purification
from the supernatant, sterilization of the supernatant. The methods
should ensure optimal preservation of biologic activity of the
compounds expressed by fetal tissues. Aseptic processing and other
efforts to promote sterilization are also desirable.
[0193] Representative embodiments of the present invention may also
contain, but are not limited to hyaluronic acid, ECM peptides or
polypeptides, growth factors, L-ascorbic acid, or carbohydrate
moieties such as lactose-1-phosphate, maltose-1-phosphate,
mannose-6-phosphate, and lactose-6-phosphate. It is understood that
the term hyaluronic acid includes its derivatives and broadly
refers to naturally occurring, microbial and synthetic derivatives
of acidic polysaccharides of various molecular weights constituted
by residues of glucuronic acid and N-acetyl-D-glucosamine.
Hyaluronic acid has been described as a skin conditioning agent for
use in skin care compositions (see U.S. Pat. No. 6,444,647). It is
also believed to play an important role in fetal tissue
regeneration. (Burd D A, Greco R M, Regauer S, Longaker M T,
Siebert J W, Garg H G. Hyaluronan and wound healing: a new
perspective. Br J Plast Surg 44:579-84, 1991). Monosaccharide
carbohydrate moieties such as lactose-1-phosphate,
maltose-1-phosphate, mannose-6-phosphate, and lactose-6-phosphate
have been described as being useful in preventing or minimizing
inflammation (DiCorleto P E, and de la Motte C A., J Immunol.
143:3666-3672, 1989; Crestani B, et al., Am J Physiol.
264:L391-400, 1993; Bartlett M R, et al., Immunol Cell Biol.
72:367-374, 1994; Davis R H, et al., J Am Podiatr Med Assoc.
84:77-81, 1994).
[0194] In one representative embodiment of the present invention,
hyaluronic acid is used with compounds expressed by fetal tissues
for skin conditioning purposes and to potentiate the effects of
cosmetic skin care compositions containing compounds expressed by
fetal tissues. A representative skin care composition may comprise,
for example, from about 0.1% to about 10% by weight of hyaluronic
acid.
[0195] D. Dosages
[0196] The amount of the compounds expressed by fetal tissues
included in the composition described herein varies with the skin
conditions of a mammal. Generally, the compositions and ranges by
weight depend on several factors including: the molecular weight of
the compound(s), the purity of the compound(s), the bioactivity of
the compound(s), and the degradation profile of the compound(s).
For instance, enzymes, growth factors and cytokines, can be
relatively small molecules. Hence they will exhibit relatively
higher bioactivity for a given weight; however, growth factors and
cytokines are also easily degraded in the absence of any protective
delivery vehicles and thus, must be provided in higher dosages by
weight for biological efficacy in circumstances lacking a delivery
vehicle. In contrast other molecules such as collagens or
proteoglycans that have potentially ECM structural functions are
generally larger with potentially less bioactivity for a given
molecular weight, and are more resistant to degradation. Thus,
other molecules such as collagens or proteoglycans may be provided
in higher or lower dosages by weight depending on the factors
outlined above. In one embodiment, the composition may comprise
about 0.0001% to about 10% by weight of the proteoglycan compound
which is purified, and about 0.1% to about 80% by weight of a cell
lysate, extract, or media enriched with the proteoglycan compound.
In another embodiment, the composition may comprise about 0.0001%
to about 10% by weight of the enzyme or growth factor compound
which is purified, or of 0.000000001% to about 0.0001% by weight of
the enzyme or growth factor compound which is purified, and about
0.1% to about 80% by weight of a cell lysate, extract, or media
enriched with the enzyme or growth factor compound. In another
embodiment, the composition may comprise about 0.001 IU/ml to about
1 IU/ml of the enzyme compound which is purified, or about IU/ml to
about 1000 IU/ml of the enzyme compound which is purified.
[0197] E. Additional Skin Care Actives
[0198] The compositions of the present invention may contain a safe
and effective amount of one or more additional skin care actives
selected from, but not limited to, the group consisting of
desquamatory actives, anti-acne actives, retinoids, hydroxy acids,
peptides, polypeptides, growth factors, cytokines, anti-oxidants,
radical scavengers, chelators, anti-inflammatory agents, topical
anesthetics, tanning actives, skin lightening agents,
anti-cellulite agents, flavonoids, antimicrobial actives, skin
soothing agents, skin healing agents, antifungal actives, sunscreen
actives, conditioning agents, structuring agents, thickening
agents, and mixtures thereof. The amount of the additional skin
care actives may vary with the specific skin conditions to be
modulated. In one embodiment, the composition may contain from
about 0.000001% to about 10% by weight of at least one additional
skin care active.
[0199] In a representative embodiment, where the composition is to
be in contact with human keratinous tissue, the additional
components should be suitable for application to keratinous tissue,
that is, when incorporated into the composition they are suitable
for use in contact with human keratinous tissue without undue
toxicity, incompatibility, instability, allergic response, and the
like within the scope of sound medical judgment. The CTFA Cosmetic
Ingredient Handbook, Second Edition (1992) describes a wide variety
of nonlimiting cosmetic and pharmaceutical ingredients commonly
used in the skin care industry, which are suitable for use in the
compositions of the present invention. Examples of these ingredient
classes include: abrasives, absorbents, aesthetic components such
as fragrances, pigments, colorings/colorants, essential oils, skin
sensates, astringents, etc. (e.g., clove oil, menthol, camphor,
eucalyptus oil, eugenol, menthyl lactate, witch hazel distillate),
anti-acne agents, anti-caking agents, antifoaming agents,
antimicrobial agents (e.g., iodopropyl butylcarbamate),
antioxidants, binders, biological additives, buffering agents,
bulking agents, chelating agents, chemical additives, colorants,
cosmetic astringents, cosmetic biocides, denaturants, drug
astringents, external analgesics, film formers or materials (e.g.,
polymers), for aiding the film-forming properties and substantivity
of the composition (e.g., copolymer of eicosene and vinyl
pyrrolidone), opacifying agents, pH adjusters, propellants,
reducing agents, sequestrants, skin bleaching and lightening agents
(e.g., hydroquinone, kojic acid, ascorbic acid, magnesium ascorbyl
phosphate, ascorbyl glucosamine), skin-conditioning agents (e.g.,
humectants, including miscellaneous and occlusive), skin soothing
and/or healing agents (e.g., panthenol and derivatives (e.g., ethyl
panthenol), aloe vera, pantothenic acid and its derivatives,
allantoin, bisabolol, and dipotassium glycyrrhizinate), skin
treating agents, thickeners, and vitamins and derivatives
thereof.
[0200] In any embodiment of the present invention, however, the
actives useful herein can be categorized by the benefit they
provide or by their postulated mode of action. However, it is to be
understood that the actives useful herein can in some instances
provide more than one benefit or operate via more than one mode of
action. Therefore, classifications herein are made for the sake of
convenience and are not intended to limit the active to that
particular application or applications listed.
[0201] Desquamation Actives
[0202] A safe and effective amount of a desquamation active may be
added to the compositions of the present invention, more preferably
from about 0.1% to about 10%, even more preferably from about 0.2%
to about 5%, also preferably from about 0.5% to about 4%, by weight
of the composition.
[0203] Desquamation actives enhance the skin appearance benefits of
the present invention. For example, the desquamation actives tend
to improve the texture of the skin (e.g., smoothness). One
desquamation system that is suitable for use herein contains
sulfhydryl compounds and zwitterionic surfactants and is described
in U.S. Pat. No. 5,681,852, to Bissett, incorporated herein by
reference. Another desquamation system that is suitable for use
herein contains salicylic acid and zwitterionic surfactants and is
described in U.S. Pat. No. 5,652,228 to Bissett, incorporated
herein by reference. Zwitterionic surfactants such as described in
these applications are also useful as desquamatory agents herein,
with cetyl betaine being particularly representative.
[0204] Anti-Acne Actives
[0205] The compositions of the present invention may contain a safe
and effective amount of one or more anti-acne actives. Examples of
useful anti-acne actives include resorcinol, sulfur, salicylic
acid, benzoyl peroxide, erythromycin, zinc, etc. Further examples
of suitable anti-acne actives are described in further detail in
U.S. Pat. No. 5,607,980 to McAtee et. al.
[0206] Anti-Wrinkle Actives/Anti-Atrophy Actives
[0207] The compositions of the present invention may further
contain a safe and effective amount of one or more anti-wrinkle
actives or anti-atrophy actives. Exemplary
anti-wrinkle/anti-atrophy actives suitable for use in the
compositions of the present invention include sulfur-containing D
and L amino acids and their derivatives and salts, particularly the
N-acetyl derivatives, a representative example of which is
N-acetyl-L-cysteine; thiols (e.g. ethane thiol); hydroxy acids
(e.g., alpha-hydroxy acids such as lactic acid and glycolic acid or
beta-hydroxy acids such as salicylic acid and salicylic acid
derivatives such as the octanoyl derivative), phytic acid, lipoic
acid; lysophosphatidic acid, skin peel agents (e.g., phenol and the
like), and retinoids which enhance the keratinous tissue appearance
benefits of the present invention, especially in regulating
keratinous tissue condition (e.g., skin condition).
[0208] As used herein, "retinoid" includes all natural and/or
synthetic analogs of Vitamin A or retinol-like compounds which
possess the biological activity of Vitamin A in the skin as well as
the geometric isomers and stereoisomers of these compounds. The
retinoid is preferably retinol, retinol esters (e.g.,
C.sub.2-C.sub.22 alkyl esters of retinol, including retinyl
palmitate, retinyl acetate, retinyl propionate), retinal, and/or
retinoic acid (including all-trans-retinoic acid and/or
13-cis-retinoic acid), more preferably retinoids other than
retinoic acid. These compounds are well known in the art and are
commercially available from a number of sources (e.g., Sigma
Chemical Company (St. Louis, Mo.), and Boerhinger Mannheim
(Indianapolis, Ind.)). Other retinoids which are useful herein are
described in U.S. Pat. No. 4,677,120 to Parish et al.; U.S. Pat.
No. 4,885,311 to Parish et al.; U.S. Pat. No. 5,049,584 to Purcell
et al; U.S. Pat. No. 5,124,356 to Purcell et al.; and U.S. Pat. No.
Reissue 34,075 to Purcell et al. Other suitable retinoids are
tocopheryl-retinoate [tocopherol ester of retinoic acid (trans- or
cis-), adapalene {6-[3-(1-adamantyl)-4-methoxyphenyl]-2-n- aphthoic
acid}, and tazarotene (ethyl 6-[2-(4,4-dimethylthiochroman-6-yl)--
ethynyl]nicotinate). Representative retinoids are retinol, retinyl
palmitate, retinyl acetate, retinyl propionate, retinal and
combinations thereof.
[0209] The compositions preferably contain from or about 0.005% to
or about 2%, more preferably 0.01% to or about 2%, retinoid.
Retinol is preferably used in an amount of from or about 0.01% to
or about 0.15%; retinol esters are preferably used in an amount of
from or about 0.01% to or about 2% (e.g., about 1%); retinoic acids
are preferably used in an amount of from or about 0.01% to or about
0.25%; tocopheryl-retinoate, adapalene, and tazarotene are
preferably used in an amount of from or about 0.01% to or about
2%.
[0210] Peptides/Polypeptides
[0211] As used herein, any naturally occurring, enzyme digested, or
synthesized amino acid sequences of more than 3, but equal to or
less than 34 amino acids is referred to as a "peptides", while
"polypeptides" refers any naturally occurring, enzyme digested, or
synthesized amino acid sequences of more than 34 amino acids.
[0212] Peptides, including but not limited to, di-, tri-, tetra-,
and pentapeptides and derivatives thereof, as well as enzymatically
cleaved fragments of ECM components such as collagen, elastins, may
be included in the compositions of the present invention in amounts
that are safe and effective. Also useful herein are naturally
occurring and commercially available compositions that contain
peptides.
[0213] When included in the present compositions, peptides are
preferably included in amounts from about 1.times.10.sup.-6% to
about 10%, more preferably from about 1.times.10.sup.-6% to about
0.1%, even more preferably from about 1.times.10.sup.-5% to about
0.01%, by weight of the composition. In certain compositions where
the peptide is CARNOSINE.RTM., the compositions preferably contain
from about 0.1% to about 5%, by weight of the composition, of such
peptides.
[0214] Growth Factors/Cytokines
[0215] Although compounds expressed by fetal tissues may include
growth factors and cytokines, representative embodiments of the
present composition may also include a safe and effective amount of
additional growth factors or cytokines not necessarily expressed by
fetal tissues. Cell growth stimulating compounds or factors are
herein described as natural or exogenous compounds which have a
stimulating effect on the elaboration and growth of specific cell
lines. These include anabolic growth hormones, such as human growth
hormone and thyroid stimulating hormone, or on specific cell lines
such as granulocytes, platelets or erythrocytes. Specifically, with
regard to promoting epidermal growth, such as in skin tissue repair
or wound healing, various factors have been identified as growth
factors, including but not limited to: epidermal growth factor
(EGF), fibroblast growth factor (FGF), transforming growth factor
(TGF), vascular endothelial cell growth factor (VEGF), and
insulin-like growth factor (IGF).
[0216] Anti-Oxidants/Radical Scavengers
[0217] The compositions of the present invention may include a safe
and effective amount of an anti-oxidant/radical scavenger. The
anti-oxidant/radical scavenger is especially useful for providing
protection against UV radiation which can cause increased scaling
or texture changes in the stratum corneum and against other
environmental agents which can cause skin damage.
[0218] A safe and effective amount of an anti-oxidant/radical
scavenger may be added to the compositions of the subject
invention, preferably from about 0.1% to about 10%, more preferably
from about 1% to about 5%, of the composition.
[0219] Anti-oxidants/radical scavengers such as ascorbic acid
(vitamin C) and its salts, ascorbyl esters of fatty acids, ascorbic
acid derivatives (e.g., magnesium ascorbyl phosphate, sodium
ascorbyl phosphate, ascorbyl sorbate), tocopherol (vitamin E),
tocopherol sorbate, tocopherol acetate, other esters of tocopherol,
butylated hydroxy benzoic acids and their salts,
6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
(commercially available under the tradename TROLOX.RTM.), gallic
acid and its alkyl esters, especially propyl gallate, uric acid and
its salts and alkyl esters, sorbic acid and its salts, lipoic acid,
amines (e.g., N,N-diethylhydroxylamine, amino-guanidine),
sulfhydryl compounds (e.g., glutathione), dihydroxy fumaric acid
and its salts, lycine pidolate, arginine pilolate,
nordihydroguaiaretic acid, bioflavonoids, curcumin, lysine,
methionine, proline, superoxide dismutase, silymarin, tea extracts,
grape skin/seed extracts, melanin, and rosemary extracts may be
used. Representative anti-oxidants/radical scavengers are selected
from tocopherol sorbate and other esters of tocopherol, more
preferably tocopherol sorbate. For example, the use of tocopherol
sorbate in topical compositions and applicable to the present
invention is described in U.S. Pat. No. 4,847,071 to Donald L.
Bissett, Rodney D. Bush and Ranjit Chatterjee.
[0220] Chelators
[0221] The compositions of the present invention may also contain a
safe and effective amount of a chelator or chelating agent. As used
herein, "chelator" or "chelating agent" means an active agent
capable of removing a metal ion from a system by forming a complex
so that the metal ion cannot readily participate in or catalyze
chemical reactions. The inclusion of a chelating agent is
especially useful for providing protection against UV radiation
which can contribute to excessive scaling or skin texture changes
and against other environmental agents which can cause skin
damage.
[0222] A safe and effective amount of a chelating agent may be
added to the compositions of the subject invention, preferably from
about 0.1% to about 10%, more preferably from about 1% to about 5%,
of the composition. Exemplary chelators that are useful herein are
disclosed in U.S. Pat. No. 5,487,884 to Bissett et al.; PCT
Publication No. WO91/16035, Bush et al.; and PCT Publication No.
WO91/16034, Bush et al. Representative chelators useful in
compositions of the subject invention are furildioxime,
furilmonoxime, and derivatives thereof.
[0223] Flavonoids
[0224] The compositions of the present invention may optionally
contain a flavonoid compound. Flavonoids are broadly disclosed in
U.S. Pat. Nos. 5,686,082 and 5,686,367, both of which are herein
incorporated by reference. Flavonoid compounds useful herein are
commercially available from a number of sources, e.g., Indofine
Chemical Company, Inc. (Somerville, N.J.), Steraloids, Inc.
(Wilton, N.H.), and Aldrich Chemical Company, Inc. (Milwaukee,
Wis.). Mixtures of the the flavonoid compounds may also be
used.
[0225] The herein described flavonoid compounds are preferably
present in the instant invention at concentrations of from about
0.01% to about 20%, more preferably from about 0.1% to about 10%,
and still more preferably from about 0.5% to about 5%.
[0226] Anti-Inflammatory Agents
[0227] A safe and effective amount of an anti-inflammatory agent
may be added to the compositions of the present invention,
preferably from about 0.1% to about 10%, more preferably from about
0.5% to about 5%, of the composition. The anti-inflammatory agent
enhances the skin appearance benefits of the present invention,
e.g., such agents contribute to a more uniform and acceptable skin
tone or color. The exact amount of anti-inflammatory agent to be
used in the compositions will depend on the particular
anti-inflammatory agent utilized since such agents vary widely in
potency.
[0228] Exemplary of anti-inflammatory agents are, but not limited
to, steroidal anti-inflammatory and non-steroidal agents. The
variety of compounds encompassed by these groups are well-known to
those skilled in the art. For example, one may refer to standard
texts for anti-inflammatory agents, including Rainsford K D (1985)
Anti-inflammatory and anti-rheumatic drugs. CRC Press, Boca Raton,
Fla. and Scherrer R A, Whitehouse M W (1974) Antiinflammatory
agents; chemistry and pharmacology. Academic Press, New York.
[0229] In addition, natural or synthetic modulators of transforming
growth factor beta, or other major inflammatory growth factors may
also minimize inflammation when applied as a cosmetic product.
(Logan A, Frautschy S A, Gonzalez A M, Sporn M B, Baird A. Enhanced
expression of transforming growth factor beta 1 in the rat brain
after a localized cerebral injury. Brain Res 587:216-25, 1992;
Border W A, Noble N A, Yamamoto T, Harper J R, Yamaguchi Y,
Pierschbacher M D, Ruoslahti E. Natural inhibitor of transforming
growth factor-beta protects against scarring in experimental kidney
disease. Nature 360:361-4, 1992).
[0230] In one embodiment, the so-called "natural" anti-inflammatory
agents are useful in methods of the present invention. Such agents
may suitably be obtained as an extract by suitable physical and/or
chemical isolation from natural sources (e.g., plants, fungi,
by-products of microorganisms) or can be synthetically prepared.
For example, candelilla wax, bisabolol (e.g., alpha bisabolol),
aloe vera, plant sterols (e.g., phytosterol), Manjistha (extracted
from plants in the genus Rubia, particularly Rubia Cordifolia), and
Guggal (extracted from plants in the genus Commiphora, particularly
Commiphora Mukul), kola extract, chamomile, red clover extract, and
sea whip extract, may be used.
[0231] Anti-Cellulite Agents
[0232] The compositions of the present invention may also contain a
safe and effective amount of an anti-cellulite agent. Suitable
agents may include, but are not limited to, xanthine compounds
(e.g., caffeine, theophylline, theobromine, and aminophylline).
[0233] Topical Anesthetics
[0234] The compositions of the present invention may also contain a
safe and effective amount of a topical anesthetic. Examples of
topical anesthetic drugs include benzocaine, lidocaine,
bupivacaine, chlorprocaine, dibucaine, etidocaine, mepivacaine,
tetracaine, dyclonine, hexylcaine, procaine, cocaine, ketamine,
pramoxine, phenol, and pharmaceutically acceptable salts
thereof.
[0235] Tanning Actives
[0236] The compositions of the present invention may contain a
tanning active. When present, it is preferable that the
compositions contain from about 0.1% to about 20%, more preferably
from about 2% to about 7%, and still more preferably from about 3%
to about 6%, by weight of the composition, of dihydroxyacetone as
an artificial tanning active.
[0237] Dihydroxyacetone, which is also known as DHA or
1,3-dihydroxy-2-propanone, is a white to off-white, crystalline
powder.
[0238] The compound can exist as a mixture of monomers and dimers,
with the dimers predominating in the solid crystalline state. Upon
heating or melting, the dimers break down to yield the monomers.
This conversion of the dimeric form to the monomeric form also
occurs in aqueous solution. Dihydroxyacetone is also known to be
more stable at acidic pH values. (See Windholz M, Merck & Co
(1983) The Merck index: an encyclopedia of chemicals, drugs, and
biologicals, 10th ed. Merck, Rahway, N.J, entry 3167, p. 463 and
"Dihydroxyacetone for Cosmetics", E. Merck Technical Bulletin,
03-304 110, 319 897, 180 588.)
[0239] Skin Lightening Agents
[0240] The compositions of the present invention may contain a skin
lightening agent. When used, the compositions preferably contain
from about 0.1% to about 10%, more preferably from about 0.2% to
about 5%, also preferably from about 0.5% to about 2%, by weight of
the composition, of a skin lightening agent. Suitable skin
lightening agents include those known in the art, including kojic
acid, arbutin, ascorbic acid and derivatives thereof (e.g.,
magnesium ascorbyl phosphate or sodium ascorbyl phosphate), and
extracts (e.g., mulberry extract, placental extract). Skin
lightening agents suitable for use herein also include those
described in PCT Publication No. WO95/34280, in the name of
Hillebrand, corresponding to PCT Application No. U.S. WO95/07432,
filed Jun. 12, 1995; and U.S. Pat. No. 6,068,834 filed in the names
of Kvalnes, Mitchell A. DeLong, Barton J. Bradbury, Curtis B.
Motley, and John D. Carter, corresponding to PCT Publication No.
WO95/23780.
[0241] Skin Soothing and Skin Healing Actives
[0242] The compositions of the present invention may comprise a
skin soothing or skin healing active. Skin soothing or skin healing
actives suitable for use herein include panthenoic acid derivatives
(including panthenol, dexpanthenol, ethyl panthenol), aloe vera,
allantoin, bisabolol, and dipotassium glycyrrhizinate. A safe and
effective amount of a skin soothing or skin healing active may be
added to the present composition, preferably, from about 0.1% to
about 30%, more preferably from about 0.5% to about 20%, still more
preferably from about 0.5% to about 10%, by weight of the
composition formed.
[0243] Antimicrobial and Antifungal Actives
[0244] The compositions of the present invention may contain an
antimicrobial or antifungal active. Such actives are capable of
destroying microbes, preventing the development of microbes or
preventing the pathogenic action of microbes. A safe and effective
amount of an antimicrobial or antifungal active may be added to the
present compositions, preferably, from about 0.001% to about 10%,
more preferably from about 0.01% to about 5%, and still more
preferably from about 0.05% to about 2%.
[0245] Examples of antimicrobial and antifungal actives include
B-lactam drugs, quinolone drugs, ciprofloxacin, norfloxacin,
tetracycline, erythromycin, amikacin, 2,4,4'-trichloro-2'-hydroxy
diphenyl ether, 3,4,4'-trichlorobanilide, phenoxyethanol, phenoxy
propanol, phenoxyisopropanol, doxycycline, capreomycin,
chlorhexidine, chlortetracycline, oxytetracycline, clindamycin,
ethambutol, hexamidine isethionate, metronidazole, pentamidine,
gentamicin, kanamycin, lineomycin, methacycline, methenamine,
minocycline, neomycin, netilmicin, paromomycin, streptomycin,
tobramycin, miconazole, tetracycline hydrochloride, erythromycin,
zinc erythromycin, erythromycin estolate, erythromycin stearate,
amikacin sulfate, doxycycline hydrochloride, capreomycin sulfate,
chlorhexidine gluconate, chlorhexidine hydrochloride,
chlortetracycline hydrochloride, oxytetracycline hydrochloride,
clindamycin hydrochloride, ethambutol hydrochloride, metronidazole
hydrochloride, pentamidine hydrochloride, gentamicin sulfate,
kanamycin sulfate, lineomycin hydrochloride, methacycline
hydrochloride, methenamine hippurate, methenamine mandelate,
minocycline hydrochloride, neomycin sulfate, netilmicin sulfate,
paromomycin sulfate, streptomycin sulfate, tobramycin sulfate,
miconazole hydrochloride, ketaconazole, amanfadine hydrochloride,
amanfadine sulfate, octopirox, parachlorometa xylenol, nystatin,
tolnaftate, zinc pyrithione and clotrimazole.
[0246] Representative examples of actives useful herein include
those selected from salicylic acid, benzoyl peroxide, 3-hydroxy
benzoic acid, glycolic acid, lactic acid, 4-hydroxy benzoic acid,
acetyl salicylic acid, 2-hydroxybutanoic acid, 2-hydroxypentanoic
acid, 2-hydroxyhexanoic acid, cis-retinoic acid, trans-retinoic
acid, retinol, phytic acid, N-acetyl-L-cysteine, lipoic acid,
azelaic acid, arachidonic acid, benzoylperoxide, tetracycline,
ibuprofen, naproxen, hydrocortisone, acetominophen, resorcinol,
phenoxyethanol, phenoxypropanol, phenoxyisopropanol,
2,4,4'-trichloro-2'-hydroxy diphenyl ether,
3,4,4'-trichlorocarbanilide, octopirox, lidocaine hydrochloride,
clotrimazole, miconazole, ketoconazole, neocycin sulfate, and
mixtures thereof.
[0247] Sunscreen Actives
[0248] Exposure to ultraviolet light can result in excessive
scaling and texture changes of the stratum corneum. Therefore, the
compositions of the subject invention may optionally contain a
sunscreen active. As used herein, "sunscreen active" includes both
sunscreen agents and physical sunblocks. Suitable sunscreen actives
may be organic or inorganic.
[0249] Inorganic sunscreens useful herein include the following
metallic oxides; titanium dioxide having an average primary
particle size of from about 15 nm to about 100 nm, zinc oxide
having an average primary particle size of from about 15 nm to
about 150 nm, zirconium oxide having an average primary particle
size of from about 15 nm to about 150 nm, iron oxide having an
average primary particle size of from about 15 nm to about 500 nm,
and mixtures thereof. When used herein, the inorganic sunscreens
are present in the amount of from about 0.1% to about 20%,
preferably from about 0.5% to about 10%, more preferably from about
1% to about 5%, by weight of the composition.
[0250] A wide variety of conventional organic sunscreen actives are
suitable for use herein. Balsam M S, Sagarin E (1972) Cosmetics,
Science and Technology, 2d edn. Wiley-Interscience, New York,
discloses numerous suitable actives.
[0251] More representative organic sunscreen actives useful in the
compositions useful in the subject invention are
2-ethylhexyl-p-methoxyci- nnamate, butylmethoxydibenzoyl-methane,
2-hydroxy-4-methoxybenzo-phenone, 2-phenylbenzimidazole-5-sulfonic
acid, octyldimethyl-p-aminobenzoic acid, octocrylene and mixtures
thereof.
[0252] Also particularly useful in the compositions are sunscreen
actives such as those disclosed in U.S. Pat. No. 4,937,370 to
Sabatelli and U.S. Pat. No. 4,999,186 to Sabatelli & Spirnak.
The sunscreening agents disclosed therein have, in a single
molecule, two distinct chromophore moieties which exhibit different
ultra-violet radiation absorption spectra. One of the chromophore
moieties absorbs predominantly in the UVB radiation range and the
other absorbs strongly in the UVA radiation range.
[0253] Representative members of this class of sunscreening agents
are 4-N,N-(2-ethylhexyl)methyl-aminobenzoic acid ester of
2,4-dihydroxybenzophenone; N,N-di-(2-ethylhexyl)-4-aminobenzoic
acid ester with 4-hydroxydibenzoylmethane;
4-N,N-(2-ethylhexyl)methyl-aminoben- zoic acid ester with
4-hydroxydibenzoylmethane; 4-N,N-(2-ethylhexyl)methyl-
-aminobenzoic acid ester of
2-hydroxy-4-(2-hydroxyethoxy)benzophenone;
4-N,N-(2-ethylhexyl)-methylaminobenzoic acid ester of
4-(2-hydroxyethoxy)dibenzoylmethane;
N,N-di-(2-ethylhexyl)-4-aminobenzoic acid ester of
2-hydroxy-4-(2-hydroxyethoxy)benzophenone; and
N,N-di-(2-ethylhexyl)-4-aminobenzoic acid ester of
4-(2-hydroxyethoxy)dibenzoylmethane and mixtures thereof.
[0254] Especially representative sunscreen actives include
4,4'-t-butylmethoxydibenzoylmethane,
2-ethylhexyl-p-methoxycinnamate, phenyl benzimidazole sulfonic
acid, and octocrylene.
[0255] A safe and effective amount of the organic sunscreen active
is used, typically from about 1% to about 20%, more typically from
about 2% to about 10% by weight of the composition. Exact amounts
will vary depending upon the sunscreen or sunscreens chosen and the
desired Sun Protection Factor ("SPF").
[0256] Particulate Material
[0257] The compositions of the present invention may contain a
particulate material, preferably a metallic oxide. These
particulates can be coated or uncoated, charged or uncharged.
Charged particulate materials are disclosed in U.S. Pat. No.
5,997,887 to Ha, et al., which is incorporated herein by reference.
Particulate materials useful herein include; bismuth oxychloride,
iron oxide, mica, mica treated with barium sulfate and TiO.sub.2,
silica, nylon, polyethylene, talc, styrene, polypropylene,
ethylene/acrylic acid copolymer, sericite, titanium dioxide,
bismuth oxychloride, iron oxide, aluminum oxide, silicone resin,
barium sulfate, calcium carbonate, cellulose acetate, polymethyl
methacrylate, and mixtures thereof.
[0258] Inorganic particulate materials (e.g., TiO.sub.2, ZnO, or
ZrO.sub.2) are commercially available from a number of sources. One
example of a suitable particulate material contains the material
available from U.S. Cosmetics (TRONOX TiO.sub.2 series, SAT-T
CR837, a rutile TiO.sub.2). Preferably, particulate materials are
present in the composition in levels of from about 0.01% to about
2%, more preferably from about 0.05% to about 1.5%, still more
preferably from about 0.1% to about 1%, by weight of the
composition.
[0259] Conditioning Agents
[0260] The compositions of the present invention may contain a
conditioning agent selected from humectants, moisturizers, or skin
conditioners. A variety of these materials can be employed and each
can be present at a level of from about 0.01% to about 20%, more
preferably from about 0.1% to about 10%, and still more preferably
from about 0.5% to about 7% by weight of the composition. These
materials include, but are not limited to, guanidine; urea;
glycolic acid and glycolate salts (e.g. ammonium and quaternary
alkyl ammonium); salicylic acid; lactic acid and lactate salts
(e.g., ammonium and quaternary alkyl ammonium); aloe vera in any of
its variety of forms (e.g., aloe vera gel); polyhydroxy alcohols
such as sorbitol, mannitol, xylitol, erythritol, glycerol,
hexanetriol, butanetriol, propylene glycol, butylene glycol,
hexylene glycol and the like; polyethylene glycols; sugars (e.g.,
melibiose) and starches; sugar and starch derivatives (e.g.,
alkoxylated glucose, fucose, glucosamine); hyaluronic acid;
lactamide monoethanolamine; acetamide monoethanolamine; panthenol;
allantoin; and mixtures thereof. Also useful herein are the
propoxylated glycerols described in U.S. Pat. No. 4,976,953 to Orr
et al.
[0261] Also useful are various C.sub.1-C.sub.30 monoesters and
polyesters of sugars and related materials. These esters are
derived from a sugar or polyol moiety and one or more carboxylic
acid moieties. Such ester materials are further described in, U.S.
Pat. No. 2,831,854, U.S. Pat. No. 4,005,196, to Jandacek; U.S. Pat.
No. 4,005,195 to Jandacek; U.S. Pat. No. 5,306,516 to Letton et
al.; U.S. Pat. No. 5,306,515 to Letton et al.; U.S. Pat. No.
5,305,514 to Letton et al.; U.S. Pat. No. 4,797,300 to Jandacek et
al.; U.S. Pat. No. 3,963,699 to Rizzi et al.; U.S. Pat. No.
4,518,772 to Volpenhein; and U.S. Pat. No. 4,517,360 to
Volpenhein.
[0262] Structuring Agents
[0263] In one embodiment, the compositions hereof, and especially
the emulsions hereof, may contain a structuring agent. Structuring
agents are particularly representative in the oil-in-water
emulsions of the present invention. Without being limited by
theory, it is believed that the structuring agent assists in
providing rheological characteristics to the composition which
contribute to the stability of the composition. For example, the
structuring agent tends to assist in the formation of the liquid
crystalline gel network structures. The structuring agent may also
function as an emulsifier or surfactant. Representative
compositions of this invention contain from about 0.1% to about
20%, more preferably from about 0.1% to about 10%, still more
preferably from about 0.5% to about 9%, of one or more structuring
agents.
[0264] Representative structuring agents are those having an HLB of
from about 1 to about 8 and having a melting point of at least
about 45.degree. C. Suitable structuring agents are those selected
from saturated C.sub.14-C.sub.30 fatty alcohols, saturated
C.sub.16-C.sub.30 fatty alcohols containing from about 1 to about 5
moles of ethylene oxide, saturated C.sub.16-C.sub.30 diols,
saturated C.sub.16-C.sub.30 monoglycerol ethers, saturated
C.sub.16-C.sub.30 hydroxy fatty acids, C.sub.14-C.sub.30
hydroxylated and nonhydroxylated saturated fatty acids,
C.sub.14-C.sub.30 saturated ethoxylated fatty acids, amines and
alcohols containing from about 1 to about 5 moles of ethylene oxide
diols, C.sub.14-C.sub.30 saturated glyceryl mono esters with a
monoglyceride content of at least 40%, C.sub.14-C.sub.30 saturated
polyglycerol esters having from about 1 to about 3 alkyl group and
from about 2 to about 3 saturated glycerol units, C.sub.14-C.sub.30
glyceryl mono ethers, C.sub.14-C.sub.30 sorbitan mono/diesters,
C.sub.14-C.sub.30 saturated ethoxylated sorbitan mono/diesters with
about 1 to about 5 moles of ethylene oxide, C.sub.14-C.sub.30
saturated methyl glucoside esters, C.sub.14-C.sub.30 saturated
sucrose mono/diesters, C.sub.14-C.sub.30 saturated ethoxylated
methyl glucoside esters with about 1 to about 5 moles of ethylene
oxide, C.sub.14-C.sub.30 saturated polyglucosides having an average
of between 1 to 2 glucose units and mixtures thereof, having a
melting point of at least about 45.degree. C.
[0265] Some representative structuring agents of the present
invention are selected from stearic acid, palmitic acid, stearyl
alcohol, cetyl alcohol, behenyl alcohol, stearic acid, palmitic
acid, the polyethylene glycol ether of stearyl alcohol having an
average of about 1 to about 5 ethylene oxide units, the
polyethylene glycol ether of cetyl alcohol having an average of
about 1 to about 5 ethylene oxide units, and mixtures thereof. More
representative structuring agents of the present invention are
selected from stearyl alcohol, cetyl alcohol, behenyl alcohol, the
polyethylene glycol ether of stearyl alcohol having an average of
about 2 ethylene oxide units (steareth-2), the polyethylene glycol
ether of cetyl alcohol having an average of about 2 ethylene oxide
units, and mixtures thereof. Even more representative structuring
agents are selected from stearic acid, palmitic acid, stearyl
alcohol, cetyl alcohol, behenyl alcohol, steareth-2, and mixtures
thereof.
[0266] Thickening Agent (Including Thickeners and Gelling
Agents)
[0267] In one embodiment, the compositions of the present invention
can contain one or more thickening agents, preferably from about
0.1% to about 5%, more preferably from about 0.1% to about 4%, and
still more preferably from about 0.25% to about 3%, by weight of
the composition.
[0268] Nonlimiting classes of thickening agents include those
selected from the following: carboxylic acid polymers, crosslinked
polyacrylate polymers, polyacrylamide polymers, polysaccharides,
and gelling agent gums.
[0269] Carboxylic acid polymers are crosslinked compounds
containing one or more monomers derived from acrylic acid,
substituted acrylic acids, and salts and esters of these acrylic
acids and the substituted acrylic acids, wherein the crosslinking
agent contains two or more carbon-carbon double bonds and is
derived from a polyhydric alcohol. Polymers useful in the present
invention are more fully described in U.S. Pat. No. 5,087,445, to
Haffey et al.; U.S. Pat. No. 4,509,949 to Huang et al; U.S. Pat.
No. 2,798,053 to Brown; and in Wenninger J A, McEwen G N, Cosmetic
Toiletry and Fragrance Association (1993) International cosmetic
ingredient dictionary, 5th ed. Cosmetic Toiletry and Fragrance
Association, Washington, D.C.
[0270] Crosslinked polyacrylate polymers are useful as thickeners
or gelling agents including both cationic and nonionic polymers,
with the cationics being generally representative. Examples of
useful crosslinked nonionic polyacrylate polymers and crosslinked
cationic polyacrylate polymers are those described in U.S. Pat. No.
5,100,660 to Hawe et al.; U.S. Pat. No. 4,849,484 to Heard; U.S.
Pat. No. 4,835,206 to Farrar et al.; U.S. Pat. No. 4,628,078 to
Glover et al.; U.S. Pat. No. 4,599,379 to Flesher et al.; and EP
228,868 to Farrar et al.
[0271] Polyacrylamide polymers, especially nonionic polyacrylamide
polymers including substituted branched or unbranched polymers.
More representative among these polyacrylamide polymers is the
nonionic polymer given the CTFA designation polyacrylamide and
isoparaffin and laureth-7, available under the Tradename Sepigel
305 from Seppic Corporation (Fairfield, N.J.). Other polyacrylamide
polymers useful herein include multi-block copolymers of
acrylamides and substituted acrylamides with acrylic acids and
substituted acrylic acids. Commercially available examples of these
multi-block copolymers include Hypan SR150H, SS500V, SS500W, and
SSSA100H, from Lipo Chemicals, Inc., (Patterson, N.J.).
[0272] "Polysaccharides" refer to gelling agents which contain a
backbone of repeating sugar (i.e., carbohydrate) units. Nonlimiting
examples of polysaccharide gelling agents include those selected
from cellulose, carboxymethyl hydroxyethylcellulose, cellulose
acetate propionate carboxylate, hydroxyethylcellulose, hydroxyethyl
ethylcellulose, hydroxypropylcellulose, hydroxypropyl
methylcellulose, methyl hydroxyethylcellulose, microcrystalline
cellulose, sodium cellulose sulfate, and mixtures thereof. Also
useful herein are the alkyl substituted celluloses. In these
polymers, the hydroxy groups of the cellulose polymer is
hydroxyalkylated (preferably hydroxyethylated or hydroxypropylated)
to form a hydroxyalkylated cellulose which is then further modified
with a C.sub.10-C.sub.30 straight chain or branched chain alkyl
group through an ether linkage. Typically these polymers are ethers
of C.sub.10-C.sub.30 straight or branched chain alcohols with
hydroxyalkylcelluloses. Examples of alkyl groups useful herein
include those selected from stearyl, isostearyl, lauryl, myristyl,
cetyl, isocetyl, cocoyl (i.e. alkyl groups derived from the
alcohols of coconut oil), palmityl, oleyl, linoleyl, linolenyl,
ricinoleyl, behenyl, and mixtures thereof. Representative among the
alkyl hydroxyalkyl cellulose ethers is the material given the CTFA
designation cetyl hydroxyethylcellulose, which is the ether of
cetyl alcohol and hydroxyethylcellulose. This material is sold
under the tradename NATROSOL.RTM. CS Plus from Aqualon Corporation
(Wilmington, Del.). Other useful polysaccharides include
scleroglucans which are a linear chain of (1-3) linked glucose
units with a (1-6) linked glucose every three units, a commercially
available example of which is CLEAROGEL.RTM. CS11 from Michel
Mercier Products Inc. (Mountainside, N.J.).
[0273] Other thickening and gelling agents useful herein include
materials which are primarily derived from natural sources.
Nonlimiting examples of these gelling agent gums include acacia,
agar, algin, alginic acid, ammonium alginate, amylopectin, calcium
alginate, calcium carrageenan, camitine, carrageenan, dextrin,
gelatin, gellan gum, guar gum, guar hydroxypropyltrimonium
chloride, hectorite, hyaluroinic acid, hydrated silica,
hydroxypropyl chitosan, hydroxypropyl guar, karaya gum, kelp,
locust bean gum, natto gum, potassium alginate, potassium
carrageenan, propylene glycol alginate, sclerotium gum, sodium
carboyxmethyl dextran, sodium carrageenan, tragacanth gum, xanthan
gum, and mixtures thereof.
[0274] Additional Actives
[0275] The compositions of the present invention can contain
additional skin care actives not comprising a specific category
include, but are not limited to a safe and effective amount of
farnesol and phytantrriol.
[0276] Farnesol is a naturally occurring substance which is
believed to act as a precursor and/or intermediate in the
biosynthesis of squalene and sterols, especially cholesterol.
Farnesol is also involved in protein modification and regulation
(e.g., farnesylation of proteins), and there is a cell nuclear
receptor which is responsive to farnesol.
[0277] Chemically, farnesol is
3,7,11-trimethyl-2,6,10-dodecatrien-1-ol and as used herein
"farnesol" includes isomers and tautomers of such. Farnesol is
commercially available, e.g., under the names farnesol (a mixture
of isomers from Dragoco, 10 Gordon Drive, Totowa, N.J.) and
trans-trans-famesol (Sigma Chemical Company, P.O. Box 14508, St.
Louis, Mo.).
[0278] When present in the compositions of the present invention,
the composition preferably contains from about 0.001% to about 50%,
by weight of the composition, more preferably from about 0.01% to
about 20%, even more preferably from about 0.1% to about 15%, even
more preferably from about 0.1% to about 10%, still more preferably
from about 0.5% to about 5%, and still more preferably from about
1% to about 5% of farnesol.
[0279] Phytantriol is the common name for the chemical known as
3,7,11,15,tetramethylhexadecane-1,2,3,-triol. Phytantriol is
commercially available from BASF (1609 Biddle Avenue, Whyandotte,
Mich.). For example, phytantriol is useful as a spider vessel/red
blotchiness repair agent, a dark circle/puffy eye repair agent,
sallowness repair agent, a sagging repair agent, an anti-itch
agent, a skin thickening agent, a pore reduction agent, oil/shine
reduction agent, a post-inflammatory hyperpigmentation repair
agent, wound treating agent, an anti-cellulite agent, and
regulating skin texture, including wrinkles and fine lines.
[0280] In the compositions of the present invention, the
phytantriol preferably is included in an amount from about 0.001%
to about 50% by weight of the composition, more preferably from
about 0.01% to about 20%, even more preferably from about 0.1% to
about 15%, even more preferably from about 0.2% to about 10%, still
more preferably from about 0.5% to about 10%, and still more
preferably from about 1% to about 5%.
[0281] F. Cosmetically, Dermatologically or Pharmaceutically
Acceptable Carriers
[0282] The composition provided herein may optionally include a
cosmetically acceptable, dermatologically acceptable, or
pharmaceutically acceptable carriers. Cosmetically acceptable,
dermatologically acceptable, or pharmaceutically acceptable
carriers are well known in the art (Shai A, et al. Principles of
preparation of medical and cosmetic products. Handbook of Cosmetic
Skin Care. London: Martin Dunitz Ltd., pp. 19-31, 2001).
[0283] In one embodiment, the topical compositions of the present
invention also have a dermatologically acceptable carrier. A safe
and effective amount of carrier is from about 50% to about 99.99%,
preferably from about 80% to about 99.9%, more preferably from
about 90% to about 98%, and even more preferably from about 90% to
about 95% of the composition.
[0284] The carrier can be in a wide variety of forms. For example,
emulsion carriers, including, but not limited to, oil-in-water,
water-in-oil, water-in-oil-in-water, and oil-in-water-in-silicone
emulsions, are useful herein.
[0285] Emulsions according to the present invention generally
contain a solution as described above and a lipid or oil. Lipids
and oils may be derived from animals, plants, or petroleum and may
be natural or synthetic (i.e., man-made). Representative emulsions
also contain a humectant, such as glycerin. Emulsions will
preferably further contain from about 0.01% to about 10%, more
preferably from about 0.1% to about 5%, of an emulsifier, based on
the weight of the carrier. Emulsifiers may be nonionic, anionic or
cationic. Suitable emulsifiers are disclosed in, for example, U.S.
Pat. No. 3,755,560 to Dickert et al.; U.S. Pat. No. 4,421,769 to
Dixon et al.; and McCutcheon's Detergents and Emulsifiers, North
American Edition, pages 317-324 (1986).
[0286] The emulsion may also contain an anti-foaming agent to
minimize foaming upon application to the keratinous tissue.
Anti-foaming agents include high molecular weight silicones and
other materials well known in the art for such use.
[0287] Suitable emulsions may have a wide range of viscosities,
depending on the desired product form. Exemplary low viscosity
emulsions, which are representative, have a viscosity of about 50
centistokes or less, more preferably about 10 centistokes or less,
still more preferably about 5 centistokes or less.
[0288] The compositions useful for the methods of the present
invention are generally prepared by conventional methods such as
are known in the art of making topical compositions. Such methods
typically involve mixing of the ingredients in one or more steps to
a relatively uniform state, with or without heating, cooling,
application of vacuum, and the like.
[0289] G. Vesicular Delivery Systems
[0290] The major obstacle for topical drug delivery is the low
diffusion rate of drugs across the stratum corneum. The natural
function of the skin is to protect the body for unwanted influences
from the environment. The main barrier of the skin is located in
the outermost layer of the skin, the stratum corneum. Since the
lipids regions in the stratum corneum form the only continuous
structure, substances applied onto the skin always have to pass
these regions. In order to increase transport across the skin,
various vesicular delivery systems such as gel-state, liquid-state,
and elastic vesicles have been described (Reviewed in Verma D D, et
al., Eur J Pharm Biopharm. 55:271-277, 2003; Verma D D, et al., Int
J Pharm. 258:141-151, 2003; Miyazaki S, et al., J Pharm Pharm Sci.
6:238-245, 2003; Takahashi A, et al., Int J Pharm. 246:179-186,
2002; Barry B W., Adv Drug Deliv Rev. 54 Suppl 1:S31-40, 2002;
Barry B W., Eur J Pharm Sci. 14:101-114, 2001; Jain S, et al., Drug
Dev Ind Pharm. 29:1013-1026, 2003).
[0291] IV. Methods of Using Compositions for Regulating Skin
Condition
[0292] The compositions of the present invention are useful for
promoting mammalian skin condition. Such regulation of keratinous
tissue conditions can include prophylactic and therapeutic
regulation. For example, such regulating methods are directed to
thickening keratinous tissue (i.e., building the epidermis and/or
dermis layers of the skin and where applicable the keratinous
layers of the nail and hair shaft) and preventing and/or retarding
atrophy of mammalian skin, preventing and/or retarding the
appearance of spider vessels and/or red blotchiness on mammalian
skin, preventing and/or retarding the appearance of dark circles
under the eye of a mammal, preventing and/or retarding sallowness
of mammalian skin, preventing and/or retarding sagging of mammalian
skin, softening and/or smoothing lips, hair and nails of a mammal,
preventing and/or relieving itch of mammalian skin, regulating skin
texture (e.g. wrinkles and fine lines), and improving skin color
(e.g. redness, freckles).
[0293] Regulating keratinous tissue condition involves topically
applying to the keratinous tissue a safe and effective amount of a
composition of the present invention. The amount of the composition
which is applied, the frequency of application and the period of
use will vary widely depending upon the fetal compounds and skin
care active and/or other components of a given composition and the
level of regulation desired (e.g., in light of the level of
keratinous tissue damage present or expected to occur).
[0294] In a representative embodiment, the composition is
chronically applied to the skin. By "chronic topical application",
this means continued topical application of the composition over an
extended period during the subject's lifetime, preferably for a
period of at least about one week, more preferably for a period of
at least about one month, even more preferably for at least about
three months, even more preferably for at least about six months,
and more preferably still for at least about one year. While
benefits are obtainable after various maximum periods of use (e.g.,
five, ten or twenty years), it is representative that chronic
application continue throughout the subject's lifetime. Typically
applications would be on the order of about once per day over such
extended periods, however application rates can vary from about
once per week up to about three times per day or more.
[0295] A wide range of quantities of the compositions of the
present invention can be employed to provide a skin appearance
and/or feel benefit. Quantities of the present compositions which
are typically applied per application are, in mg
composition/cm.sup.2 skin, from about 0.1 mg/cm.sup.2 to about 10
mg/cm.sup.2. A particularly useful application amount is about 1
mg/cm.sup.2 to about 2 mg/cm.sup.2.
[0296] Regulating keratinous tissue condition is preferably
practiced by applying a composition in the form of a skin lotion,
cream, gel, foam, ointment, paste, emulsion, spray, conditioner,
tonic, cosmetic, lipstick, foundation, nail polish, after-shave, or
the like which is preferably intended to be left on the skin or
other keratin structure for some esthetic, prophylactic,
therapeutic or other benefit (i.e., a "leave-on" composition).
After applying the composition to the skin, it is preferably left
on the skin for a period of at least about 15 minutes, more
preferably at least about 30 minutes, even more preferably at least
about 1 hour, still more preferably for at least several hours,
e.g., up to about 12 hours. Any part of the external portion of the
face, hair, and/or nails can be treated, e.g., face, lips,
under-eye area, eyelids, scalp, neck, torso, arms, hands, legs,
feet, fingernails, toenails, scalp hair, eyelashes, eyebrows, etc.
The composition can be applied with the fingers or with an
implement or device (e.g., pad, cotton ball, applicator pen, spray
applicator, and the like).
[0297] Another approach to ensure a continuous exposure of the skin
to at least a minimum level of fetal compounds and skin care
actives is to apply the compound by use of a patch applied. Such an
approach is particularly useful for problem skin areas needing more
intensive treatment (e.g., facial crows feet area, frown lines,
under eye area, and the like). The patch can be occlusive,
semi-occlusive or non-occlusive and can be adhesive or
non-adhesive. The composition can be contained within the patch or
be applied to the skin prior to application of the patch. The patch
can also include additional actives such as chemical initiators for
exothermic reactions such as those described in U.S. Pat. Nos.
5,821,250, 5,981,547, and 5,972,957 to Wu, et al. The patch is
preferably left on the skin for a period of at least about 5
minutes, more preferably at least about 15 minutes, more preferably
still at least about 30 minutes, even more preferably at least
about 1 hour, still more preferably at night as a form of night
therapy.
EXAMPLES
[0298] The following examples further describe and demonstrate
embodiments within the scope of the present invention. The examples
are given solely for the purpose of illustration and are not to be
construed as limitations of the present invention, as many
variations thereof are possible without departing from the spirit
and scope of the invention.
[0299] In one embodiment, compounds expressed by fetal cells or
tissues are isolated directly through tissue culture media or cell
lysates and further concentrated or purified. Although individual
identification or purification of compounds expressed by fetal
tissues is useful, the application of this invention does not
necessarily require the individual identification or purification
of the compounds. The tissue culture media or cell lysate, with or
without further concentration or purification, is then formulated
into cosmetic compositions to improve the condition of skin
according to the examples below. In another embodiment of the
present invention, compounds expressed by fetal tissues or
conditions that promote expression of these compounds are
identified. Once identified, the fetal compounds are isolated from
native tissues (wild-type form) or from suitable expression
vehicles such as bacteria or yeast (recombinant form--with or
without modification of the coding region) and formulated into
cosmetic compositions to improve the condition of skin according to
the examples below.
[0300] The ingredient "Fetal Tissue Compounds" listed in the table
below refers to, but is not necessarily limited to, fetal tissue
culture media, lysates, and extracts that may or may not have
undergone prior identification of each individual component. "Fetal
Tissue Compounds" also refer to compounds directly derived from
fetal tissues or compounds obtained through recombinant means with
or without prior genetic modification.
1TABLE 1 Examples of Skin Care Compositions Using Fetal Tissue
Compounds Ingredient Ex. 1 Ex. 2 Ex. 3 Ex. 4 Ex. 5 Ex. 6 PHASE A:
Water U.S.P. qs to 100 qs to 100 qs to 100 qs to 100 Qs to 100 qs
to 100 Disodium EDTA 0.15 0.15 0.15 0.15 0.15 0.15 Methyl Paraben
0.25 0.25 0.25 0.25 0.25 0.25 Allantoin 0.20 0.20 0.20 0.20 0.20
0.20 Glycerin 5.0 5.0 5.0 5.0 5.0 5.0 PHASE B: Cetyl Alcohol 0.30
0.30 0.30 0.30 0.30 0.30 Stearyl Alcohol 0.50 0.50 0.50 0.50 0.50
0.50 Behenyl Alcohol 0.40 0.40 0.40 0.40 0.40 0.40 Propyl Paraben
0.10 0.10 0.10 0.10 0.10 0.10 Famesol 5.0 5.0 5.0 5.0 5.0 5.0
Phytantriol 5.0 5.0 5.0 5.0 5.0 5.0 PHASE C Sepigel 305 2.0 2.0 2.0
2.0 2.0 2.0 PHASE D Titanium 0.5 Dioxide PHASE E Benzyl Alcohol
0.50 0.50 0.50 0.50 0.50 0.50 Dimethicone/Di 0.50 0.50 0.50 0.50
0.50 0.50 methiconol PHASE F Sodium 0.15 0.30 0.30 0.60 0.60 0.60
Hyaluronate Ascorbic Acid 5.0 5.0 5.0 5.0 5.0 5.0 PHASE G Fetal
Tissue 5.0 10.0 20.0 30.0 45.0 60.0 Compounds 80
[0301] To obtain a suitable skin composition, the Phase A
components listed in the table above are blended with a suitable
mixer (e.g., Tekmar model RW20DZM). The components are heated,
while stirring to a temperature of 70-80.degree. C. Separately, the
B phase components are blended with a suitable mixer and heated to
70-75.degree. C. and maintained while mixing. Phase B components
are added to Phase A components while mixing well to emulsify. When
the emulsion is at approximately 60.degree. C., Phase C component
is added while continuing to mix emulsion. The emulsion is allowed
to cool to approximately 40.degree. C. while stirring. At
approximately 50.degree. C., Phase D and E components are added to
the emulsion and mixing continued. At approximately 40.degree. C.
Phase F components are added while continuing to mix emulsion. The
emulsion is allowed to cool to approximately 30.degree. C. while
stirring, and Phase G component is added. The emulsion is then
milled using a suitable mill (Tekmar T-25) for approximately 5
minutes resulting in a uniform product.
[0302] In yet another embodiment of the present invention, specific
compounds expressed by fetal tissues such as FM are isolated from
native tissues (wild-type form) or from suitable expression
vehicles such as bacteria, yeast, or mammalian cells (recombinant
form--with or without modification of the coding region) and then
formulated into cosmetic compositions to improve the condition of
skin according to the table and steps below. For example, wild-type
or recombinant FM can be in either purified or partially purified
or non-purified forms. Purified is understood to mean the presence
of primarily FM protein. Partially or non-purified forms of FM may
also contain other fetal compounds in the form of, but not limited
to media, lysates, or extracts that improve the condition of
skin.
[0303] A composition using purified FM and the following
ingredients is prepared in making a reparative creme.
2TABLE 2 Examples of Skin Care Compositions Using Purified FM
Ingredient Ex. 1 Ex. 2 Ex. 3 Ex. 4 Ex. 5 Ex. 6 PHASE A: Water
U.S.P. qs to 100 qs to 100 qs to 100 qs to 100 qs to 100 Qs to 100
Disodium EDTA 0.15 0.15 0.15 0.15 0.15 0.15 Methyl Paraben 0.25
0.25 0.25 0.25 0.25 0.25 Allantoin 0.20 0.20 0.20 0.20 0.20 0.20
Glycerin 5.0 5.0 5.0 5.0 5.0 5.0 PHASE B: Cetyl Alcohol 0.30 0.30
0.30 0.30 0.30 0.30 Stearyl Alcohol 0.50 0.50 0.50 0.50 0.50 0.50
Behenyl Alcohol 0.40 0.40 0.40 0.40 0.40 0.40 Propyl Paraben 0.10
0.10 0.10 0.10 0.10 0.10 Famesol 5.0 5.0 5.0 5.0 5.0 5.0
Phytantriol 5.0 5.0 5.0 5.0 5.0 5.0 PHASE C Sepigel 305 2.0 2.0 2.0
2.0 2.0 2.0 PHASE D Titanium Dioxide 0.5 PHASE E Benzyl Alcohol
0.50 0.50 0.50 0.50 0.50 0.50 Dimethicone/Dimethiconol 0.50 0.50
0.50 0.50 0.50 0.50 PHASE F Sodium Hyaluronate 0.15 0.30 0.30 0.60
0.60 0.60 Ascorbic Acid 5.0 5.0 5.0 5.0 5.0 5.0 PHASE G Purified FM
0.001 0.01 0.1 1 5 10
[0304] The A phase components are blended with a suitable mixer
(e.g., Tekmar model RW20DZM). Phase A components are heated while
stirring to a temperature of 70-80.degree. C. Separately, the B
phase components are blended with a suitable mixer, heated to
70-75.degree. C. and maintained while mixing. Phase B components
are added to Phase A components while mixing well to emulsify. When
emulsion is at approximately 60.degree. C., Phase C components is
added while continuing to mix emulsion. The emulsion is allowed to
cool to approximately 40.degree. C. while stirring. At
approximately 50.degree. C., Phase D and E components are added to
the emulsion and mixing continued. At approximately 40.degree. C.
Phase F components is added while continuing to mix emulsion. The
emulsion is allowed to cool to approximately 30.degree. C. while
stirring, and Phase G component is then added. The emulsion is then
milled using a suitable mill (Tekmar T-25) for approx. 5 minutes
resulting in an uniform product.
[0305] Alternatively, composition using partially or non-purified
FM enriched lysates, extracts, or media, and the following
ingredients are prepared in making a reparative creme using the
ingredients below and the same steps described previously.
3TABLE 3 Examples of Skin Care Compositions Using Partially or
Non-Purified FM Ingredient Ex. 1 Ex. 2 Ex. 3 Ex. 4 Ex. 5 Ex. 6
PHASE A: Water U.S.P. qs to qs to qs to qs to qs to Qs to 100 100
100 100 100 100 Disodium EDTA 0.15 0.15 0.15 0.15 0.15 0.15 Methyl
Paraben 0.25 0.25 0.25 0.25 0.25 0.25 Allantoin 0.20 0.20 0.20 0.20
0.20 0.20 Glycerin 5.0 5.0 5.0 5.0 5.0 2.5 PHASE B: Cetyl alcohol
0.30 0.30 0.30 0.30 0.30 0.30 Stearyl alcohol 0.50 0.50 0.50 0.50
0.50 0.50 Behenyl alcohol 0.40 0.40 0.40 0.40 0.40 0.40 Propyl
Paraben 0.10 0.10 0.10 0.10 0.10 0.10 Famesol 5.0 5.0 5.0 5.0 5.0
2.5 Phytantriol 5.0 5.0 5.0 5.0 5.0 2.5 PHASE C: 2.0 2.0 2.0 2.0
2.0 2.0 PHASE D: Titanium Dioxide 0.5 PHASE E: Benzyl alcohol 0.50
0.50 0.50 0.50 0.50 0.50 Dimethicone/Dimethiconol 0.50 0.50 0.50
0.50 0.50 0.50 PHASE F: Sodium hyaluronate 0.15 0.30 0.30 0.60 0.60
0.60 Ascorbic acid 5.0 5.0 5.0 5.0 5.0 -- PHASE G: FM Enriched
Lysates, 0.1 1 10 20 40 80 Extracts, or Media
[0306] Many alterations and modifications may be made by those
having ordinary skill in the art without departing from the spirit
and scope of the invention. Therefore, it must be understood that
the illustrated embodiment has been set forth only for the purposes
of example and that it should not be taken as limiting the
invention as defined by the following claims. The following claims
are, therefore, to be read to include not only the combination of
elements which are literally set forth, but all equivalent elements
for performing substantially the same function in substantially the
same way to obtain substantially the same result. The claims are
thus to be understood to include what is specifically illustrated
and described above, what is conceptually equivalent, and also what
essentially incorporates the essence of the invention.
* * * * *
References