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CA 02603102 2008-08-07
ELASTIN PROTECTIVE POLYPHENOLICS AND METHODS OF USING THE SAME
Technical Field
In general, the present invention is directed to compositions and methods for
treating skin. More particularly, the present invention is directed to elastin
protective
polyphenolics and methods of using the same.
Background
Children with inherited diseases, characterized by impaired primary deposition
of elastic fibers (i.e. Costello Syndrome or Cutis Laxa) develop wrinkles and
deep dermal
creases. Similar, but steadily developing signs of premature skin aging can
also be observed
in individuals with Pseudoxanthoma Elasticum and in normal persons after
prolonged
exposure to sun. Histological analysis of wrinkled skin demonstrates
disappearance and
altered organization of elastic fibers due to premature proteolytic
degradation and impaired
remodeling (solar elastosis) of these components of dermal extracellular
matrix. This
observed loss of physiologically relevant elastic fibers is also affected by
the fact that fully
differentiated (adult) dermal fibroblasts lose their ability to synthesize
elastin and thus cannot
replace damaged elastic fibers. Since elastic fibers are solely responsible
for cutaneous
elasticity/resilience there is an obvious need for development of methods that
might protect
existing elastic fibers from premature degradation by elastolytic proteinases
and facilitate new
elastogenesis in skin.
A proteolytic digest of elastin to provide a mixture of small elastin-derived
peptides (ProK-60), manganese salts (MnC12, MnSO4 and MnPCA) and trivalent
iron (Ferric
Ammonium Citrate) have each been shown to individually stimulate the
production and
effective assembly of new tropoelastin into new elastic fibers in both primary
cultures of
human dermal fibroblasts and in organ cultures of human adult skin explants.
Yet even under
optimal conditions, the elastogenic process is not 100% efficient. A
significant fraction (30-
40%) of newly produced tropoelastin is not assembled into extracellular
elastic fibers.
Instead, these unassembled tropoelastin interact with the cell surface elastin
receptor and
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CA 02603102 2008-08-07
further stimulate new elastogenesis, pro-mitogenic signaling pathways and pro-
migratory
signaling pathways. Moreover, these unassembled tropoelastin molecules and the
soluble
products of proteolytic degradation of insoluble elastin can stimulate the
secretion of
elastolytic metalloproteinases. While stimulation of dermal fibroblast
proliferation and
migration can contribute to the overall anti-aging effect induced by factors
initially triggering
new elastogenesis, the simultaneous up-regulation of elastolytic enzymes may
cause rapid
degradation of newly produced elastin and existing elastic fibers. Hence there
is a need to
protect existing and new elastic fibers from premature enzymatic proteolysis.
It has now been shown that the treatment of cultured dermal fibroblasts with
ellagic acid or tannic acid significantly enhances their net deposition of
elastic fibers. This
effect is due to the fact that these reagents bind to the newly produced
elastin and protect it
from proteolytic degradation.
Ellagic acid and tannic acid are polyphenols found in a wide variety of fruits
and nuts such as raspberries, strawberries, walnuts, gapes, and black
currants. These
molecules possess potent ability to scavenge reactive oxygen species (ROS) and
reactive
nitrogen species (RNS). Both ROS and RNS, generated inside cells after
exposure to several
endogenous and exogenous agents, may cause direct or indirect damage of many
important
biomolecules, including elastin mRNA, by activation of local proteinases,
glycosidases or
RNAses. Moreover, tannic acid has been shown to bind to insoluble bovine and
porcine
elastin and inhibit their degradation by porcine pancreatic elastase and
recently, ellagic acid
was shown to decrease expression of pro-MMP-2 and pro-MMP-9, precursors of two
elastolytic enzymes.
The most extensively studied polyphenol, ellagic acid, exhibits minimal
solubility in water and moderate to better solubility in organic solvents such
as methanol and
DMSO, suggesting that ellagic acid may act as a good lipophilic antioxidant .
Experimental
data indicate that ellagic acid inhibits lipid peroxidation at much lower
concentrations than
vitamin E. This property, along with its ability to scavenge peroxyl radicals,
makes it a
probable chain-breaking antioxidant candidate.
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CA 02603102 2008-08-07
Epidemiological studies indicate that there is an inverse association between
the incidence of coronary heart diseases and fruit consumption, largely
attributed to the
antioxidant nature of phenolic compounds. Ellagic acid exhibits cardio-
protective properties
in the neoepinephrine myocarditis rat model, hepato-protective activity
against carbon
tetrachloride both in vitro and in vivo and reduced cytogenetic damage induced
by radiation,
hydrogen peroxide and mitomycin C.
Additional experimental studies have demonstrated that ellagic acid, tannic
acid and their derivatives, due to their planar structure, also bind to DNA by
intercalating into
the minor groove and exhibit anti-mutagenic, anti-cancer and anti-
proliferative activities. In
addition, ellagic acid induces G1 arrest and inhibits overall cell growth,
causing apoptosis in
several tumor cells. Ellagic acid has also been shown to inhibit chemically
induced cancer in
the lung, liver, skin and esophagus of rodents, including TPA-induced tumor
promotion in
mouse skin. Given the common etiopathogenic processes of mutagenesis,
carcinogenesis,
and teratogenesis induced by genotoxic chemicals, ellagic acid was also tested
for
embryoprotection and demonstrated that it can interrupt the critical
teratogenic events
induced by methylating agents.
Topical applications of ellagic acid have been used in therapeutic
preparations.
Gali, et. al. demonstrated that topical applications of tannic acid
practically inhibit tumor
promoter-induced omithine decarboxylase activity (ODA) in mouse epidermis in
vivo
suggesting that tannic acid and other polyphenols may be effective not only
against skin
tumor initiation and complete carcinogenesis, but also against the promotion
phase of skin
tumorigenesis. Moreover, tannic acid and its polyphenol derivatives have been
shown to
possess anti-inflammatory activities and to decrease infectivity of human
cells with papiloma
virus, human immunodeficiency virus, and Staphylococcus aureus.
Brief summary of the invention
The present invention provides evidence that the effectiveness of ellagic acid
or tannic acid to prevent premature proteolytic degradation of tropoelastin
and fully
polymerized elastin, thus facilitating more efficient elastogenesis. Thus,
embodiments of the
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CA 02603102 2011-04-07
present invention provide compositions and methods for treating aging or
damaged skin using
ellagic acid, tannic acid, or derivatives thereof.
In one aspect, there is provided a topical composition comprising an effective
amount of a polyphenolic compound and a peptide, wherein said polyphenolic
compound is
tannic acid, ellagic acid or a combination thereof.
In some embodiments, the effective amount of the polyphenolic compound
is from about 1 lig to about 10 fig.
In some embodiments, the peptide is ProK60, E91 or a combination thereof.
In another aspect, there is provided the use of an effective amount of a
polyphenolic compound in the manufacture of a medicament for the protection of
elastin
fibers from degradation, wherein said polyphenolic compound is tannic acid,
ellagic acid or a
combination thereof.
In some embodiments, the effective amount of the polyphenolic compound is
from about 1 pig to about 10 pig.
In some embodiments, the use further comprises a stimulator of elastogenesis
In some embodiments, the stimulator of elastogenesis is ProK60, E91 or a
combination thereof.
In some embodiments, the polyphenolic compound and the stimulator of
elastogenesis are formulated for administration simultaneously.
In some embodiments, the polyphenolic compound and the stimulator of
elastogenesis are formulated for administration sequentially.
In some embodiments, the use of the composition is for the protection of
elastin fibers from degradation.
In some embodiments, the peptide comprises the amino acid sequence X1-X2-
X3 -X4-X5-X6, wherein Xi is V or 1, X2 is G, X3 is A, L or V, X4 is M, S, or
A, X5 is P and X6
is G.
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CA 02603102 2011-04-07
In some embodiments, the peptide is SEQ ID NO: 1 (Val-Gly-Ala-Met-Pro-
Gly), SEQ ID NO: 2 (Val-Gly-Leu-Met-Pro-Gly), SEQ ID NO: 3 (Val-Gly-Val-Met-
Pro-
Gly), SEQ ID NO: 4 (Val-Gly-Ala-Ser-Pro-Gly), SEQ ID NO: 5 (Val-Gly-Ala-Ala-
Pro-Gly),
SEQ ID NO: 6 (Val-Gly-Leu-Ser-Pro-Gly), SEQ ID NO: 7 (Val-Gly-Leu-Ala-Pro-
Gly), SEQ
ID NO: 8 (Val-Gly-Val-Ser-Pro-Gly), SEQ ID NO: 9 (Ile-Gly-Ala-Met-Pro-Gly),
SEQ ID
NO: 10 (Ile-Gly-Leu-Met-Pro-Gly), SEQ ID NO: 11 (Ile-Gly-Val-Met-Pro-Gly), SEQ
ID
NO: 12 (Ile-Gly-Ala-Ser-Pro-Gly), SEQ ID NO: 13 (Ile-Gly-Ala-Ala-Pro-Gly), SEQ
ID NO:
14 (Ile-Gly-Leu-Ser-Pro-Gly), SEQ ID NO: 15 (Ile-Gly-Leu-Ala-Pro-Gly), SEQ ID
NO: 16
(Ile-Gly-Val-Ser-Pro-Gly), SEQ ID NO: 17 (Ile-Gly-Val-Ala-Pro-Gly) or a
combination
thereof
In some embodiments, the stimulator of elastogenesis is a peptide comprising
the amino acid sequence X1-X2-X3-X4-X5-X 6, wherein X1 is V or I, X2 is G, X3
is A, L or V,
X4 is M, S, or A, X5 is P and X6 is G.
In some embodiments, the stimulator of elastogenesis is SEQ ID NO: 1 (Val-
Gly-Ala-Met-Pro-Gly), SEQ ID NO: 2 (Val-Gly-Leu-Met-Pro-Gly), SEQ ID NO: 3
(Val-
Gly-Val-Met-Pro-Gly), SEQ ID NO: 4 (Val-Gly-Ala-Ser-Pro-Gly), SEQ ID NO: 5
(Val-Gly-
Ala-Ala-Pro-Gly), SEQ ID NO: 6 (Val-Gly-Leu-Ser-Pro-Gly), SEQ ID NO: 7 (Val-
Gly-Leu-
Ala-Pro-Gly), SEQ ID NO: 8 (Val-Gly-Val-Ser-Pro-Gly), SEQ ID NO: 9 (Ile-Gly-
Ala-Met-
Pro-Gly), SEQ ID NO: 10 (Ile-Gly-Leu-Met-Pro-Gly), SEQ ID NO: 11 (Ile-Gly-Val-
Met-
Pro-Gly), SEQ ID NO: 12 (Ile-Gly-Ala-Ser-Pro-Gly), SEQ ID NO: 13 (Ile-Gly-Ala-
Ala-Pro-
Gly), SEQ ID NO: 14 (Ile-Gly-Leu-Ser-Pro-Gly), SEQ ID NO: 15 (Ile-Gly-Leu-Ala-
Pro-
Gly), SEQ ID NO: 16 (Ile-Gly-Val-Ser-Pro-Gly), SEQ ID NO: 17 (Ile-Gly-Val-Ala-
Pro-Gly)
or a combination thereof.
Brief Description of the Figures
The file of this patent contains at least one drawing executed in color.
Copies
of this patent with color drawing(s) will be provided by the Patent and
Trademark Office
upon request and payment of necessary fee.
Figure 1. Assessment of immunodetected and insoluble elastic fibers in
dermal fibroblast cultures. (a) Micrographs of immunodetected tropoelastin in
cultures
maintained in the presence and absence of EA and TA (1 ug/mL each). (b)
Results of
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CA 02603102 2011-04-07
morphometric evaluation of tropoelastin levels in fibroblast cultures. (c)
Evaluation of
metabolically labeled insoluble elastin in treated and control cultures. All
results were
obtained in 7 day-old cultures of dermal fibroblasts derived from a 36 year-
old caucasian
female. Data demonstrate that treatment with ellagic acid or tannic acid
significantly
increases a net deposition of extracellular elastic fibers as compared to
respective untreated
controls.
Figure 2. Pulse and chase experiment to evaluate pretreated tropoelastin/
elastin stability against non-specific enzymatic degradation. (a) Results of
morphometric
assessment of immuno-detectable elastin and (b) content of metabolically
labeled insoluble
elastin, detected at the respective ends of the indicated pulse and chase
periods, demonstrate
that cultures of dermal fibroblasts derived from a 26 year-old female, that
were incubated the
first seven days in the presence of EA and TA (1 g/mL each), sustain their
high net content
of insoluble elastin (metabolically pulsed with [31-1]-valine between day 4
and 7) even when
maintained for an additional seven days (chase period) in media containing
only 1% FBS and
no polyphenols. In contrast, 14 day-old control (untreated) cultures
demonstrate a significant
decrease in their net content of metabolically labeled insoluble elastin
(initially deposited at
the end of pulse period, at day 7). (c) The assessment of [31-1]-thymidine
incorporation and
assay of total DNA indicate that conditions of the chase period in which cells
were
maintained in 1% FBS caused inhibition of cellular proliferation.
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CA 02603102 2008-08-07
Figure 3. Evaluation of the protective effect of polyphenols against
elastolytic
degradation of insoluble elastin.
Results of in vitro assay demonstrate that samples of
insoluble [3H]-labeled elastin from bovine ligamentum nuchae, pretreated with
EA or TA (1
Rg/mL and 10 lig/mL each) demonstrate higher resistance to proteolytic
degradation by
indicated enzymes belonging to three different classes of proteinases
(elastases) capable of
elastin degradation.
Figure 4. Assessment of the effect of polyphenols on elastogenesis induced by
known elastogenic compounds. (a) Results of the quantitative assessment of
newly deposited
insoluble elastin (metabolically labeled with [31-1]-valine) detected in 7 day-
old cultures of
dermal fibroblasts derived from a healthy 50 year-old caucasian female.
Fibroblasts
maintained in the presence of stimulators of elastin synthesis, mixture of
small elastin-derived
peptides (ProK-60 25 p,g/mL) or Ferric Ammonium Citrate (FAC 20 1.1M),
significantly
increased their net deposition of insoluble elastin as compared with the
untreated control.
Additional treatment either with ellagic acid (1 pg/mL) or tannic acid (1
g/mL) cause further
proportional increase in net elastin content in all tested experimental
groups. (b)
Representative micrographs of 7 day-old cultures of dermal fibroblasts
(derived from 50 year-
old caucasian female) immuno-stained with anti tropoelastin antibody. Cultures
treated with
ProK-60 (25 Rg/mL) produced more elastic fibers than untreated control
cultures. Additional
treatment either with ellagic ecid (EA) or with tannic acid (TA) (both in
concentration
1 g/mL) caused a further increase in net deposition of immunodetectable
elastic fibers.
Figure 5. Assessment of binding of tannic acid to collagen type I. (a) Results
of triplicate (1 mg) aliquots of pure collage type I inclubated with 20 jig/m1
of tannic acid for
2 hour at 37C.
Initial concentration of tannic acid was confirmed by direct
spectrophotometric reading at 280 nm. This method demonstrated a dose-
dependent linear
increase in absorbance. At the end of incubation period the collagen type I
slurries were
separated by centrifugation and the concentration of TA in supernatants were
spectrophotometrically determined again at 280 nm. In each experimental group
means SD
were calculated and obtained values were statistically compared with beginning
concentrations of both polyphenols. (b) 1 mg of collagen type I (from rat
tail) sequestered
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CA 02603102 2008-08-07
75.5 0.001% (P<0.0001) of the TA (originally 20 it.g/mL) from solution,
suggesting that
tannic acid may also bind to collagen type I.
Detailed description of the invention
Before the present compositions and methods are described, it is to be
understood that this invention is not limited to the particular molecules,
compositions,
methodologies or protocols described, as these may vary. It is also to be
understood that the
terminology used in the description is for the purpose of describing the
particular versions or
embodiments only, and is not intended to limit the scope of the present
invention which will
be limited only by the appended claims.
It must also be noted that as used herein and in the appended claims, the
singular forms "a", "an", and "the" include plural reference unless the
context clearly dictates
otherwise. Thus, for example, reference to a "cell" is a reference to one or
more cells and
equivalents thereof known to those skilled in the art, and so forth. Unless
defined otherwise,
all technical and scientific terms used herein have the same meanings as
commonly
understood by one of ordinary skill in the art. Although any methods and
materials similar or
equivalent to those described herein can be used in the practice or testing of
embodiments of
the present invention, the preferred methods, devices, and materials are now
described.
Nothing herein is to be construed as an admission that the invention is not
entitled to antedate
such disclosure by virtue of prior invention.
As used herein, the term "about" means plus or minus 10% of the numerical
value of the number with which it is being used. Therefore, about 50% means in
the range of
45%-55%. Generally speaking, the term "tissue" refers to any aggregation of
similarly
specialized cells which are united in the performance of a particular
function.
The term "cosmetic," as used herein, refers to a beautifying substance or
preparation which preserves, restores, bestows, simulates, or enhances the
appearance of
bodily beauty or appears to enhance the beauty or youthfulness, specifically
as it relates to the
appearance of tissue or skin.
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CA 02603102 2008-08-07
The term "modify" is used to convey that the present invention changes either
the appearance, form, characteristics and/or the physical attributes of the
tissue to which it is
being provided, applied or administered. The change in form may be
demonstrated by any of
the following alone or in combination: enhanced appearance of the skin;
increased softness of
the skin; increased turgor of the skin; increased texture of the skin;
increased elasticity of the
skin; decreased wrinkle formation and increased endogenous elastin production
in the skin,
increased firmness and resiliency of the skin.
As used herein, the terms "pharmaceutically acceptable", "physiologically
tolerable" and grammatical variations thereof, as they refer to compositions,
carriers, diluents
and reagents, are used interchangeably and represent that the materials are
capable of
administration upon a mammal without the production of undesirable
physiological effects
such as nausea, dizziness, rash, or gastric upset. In a preferred embodiment,
the therapeutic
composition is not immunogenic when administered to a human patient for
therapeutic
purposes.
"Providing" when used in conjunction with a therapeutic means to administer
a therapeutic directly into or onto a target tissue or to administer a
therapeutic to a patient
whereby the therapeutic positively impacts the tissue to which it is targeted.
Thus, as used
herein, the term "providing", when used in conjunction with a polyphenolic
compound, can
include, but is not limited to, providing a polyphenolic compound into or onto
the target
tissue; providing a polyphenolic compound systemically to a patient by, e.g.,
intravenous
injection whereby the therapeutic reaches the target tissue; and the like.
The term "skin" means that outer integument or covering of the body,
consisting of the dermis and the epidermis and resting upon subcutaneous
tissue.
As used herein, the term "therapeutic" means an agent utilized to treat,
combat, ameliorate, prevent or improve an unwanted condition or disease of a
patient. In
part, embodiments of the present invention are directed to improve the
functionality, the
appearance, the elasticity, and/or the elastin content of mammalian tissue. As
it applies to
skin, it is measured by turgor, tone, appearance, degree of wrinkles, and
youthfulness. As the
term applies to blood vessels it may be measured by the degree of elasticity
or proper
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CA 02603102 2008-08-07
vasomotor response (vasodilatation/vasoconstriction) of the vessel.
Accordingly, therapeutic
treatment of blood vessels may have implications in diseases associated with
visco-elasticity,
including hypertension, arteriosclerosis, angina, angiogenesis, myocardial
infarction, coronary
thrombosis, restenosis post angioplasty, and chronic obstructive pulmonary
disease.
The terms "therapeutically effective" or "effective", as used herein, may be
used interchangeably and refer to an amount of a therapeutic composition of
the present
invention--e.g., a polyphenolic compound. For example, a therapeutically
effective amount
of a composition comprising polyphenolic compound is a predetermined amount
calculated to
achieve the desired effect, i.e., to effectively promote elastin production,
cell proliferation, or
improved appearance, or improved tissue elasticity in an individual to whom
the composition
is administered.
As used herein, "tissue", unless otherwise indicated, refers to tissue which
includes Elastin as part of its necessary structure and/or function. For
example, connective
tissue which is made up of, among other things, collagen fibrils and elastin
fibrils satisfies the
definition of "tissue" as used herein. Additionally, elastin appears to be
involved in the
proper function of blood vessels, veins, and arteries in their inherent visco-
elasticity.
Skin is composed of a top layer, the epidermis, which is approximately 20 cell
layers or about 0.1 mm in thickness, and a lower layer, the dermis, which is
from about 1 to
about 4 mm in thickness and contains small blood vessels, collagen, elastin
and fibroblasts.
The dermis provides structural support and nutrients to the epidermis. Aging
has been shown
to increase cellular heterogeneity of the epidermal layer, however, it has
little effect on the
thickness of the epidermal layer. The supporting dermis, on the other hand, is
known to thin
with age and exposure to the sun and environmental contaminants (other
environmental
effects on the skin are discussed in U.S. Pat. No. 4,938,969 and U.S. Pat. No.
5,140,043). As
the dermal layer provides the support and blood supply for the epidermis, the
dermal layer is
important in maintaining the elasticity and appearance of the skin. Disruption
of the
supporting dermis leads directly to sagging and, consequently, furrowing of
the epidermis,
i.e., the formation of wrinkles.
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CA 02603102 2008-08-07
Deep wrinkles are also due to continual stretching and contraction of both the
dermis and epidermis. Currently, these deep wrinkles or furrows may only be
eliminated by
plastic surgery or by collagen injections directly beneath the depressed
areas. The fine
wrinkles that occur with age and prolonged exposure to the sun and other
environmental
contaminants are the direct result of deterioration of the supporting dermal
layer.
Elastin is secreted by the fibroblasts of the connective tissues and by the
vascular smooth muscle cells (i.e., arteries, veins and heart) and elastic
cartilage chondrocytes
(i.e., epiglottis and ear cartilage) into the extracellular matrix. In the
dermal connective
tissue, the elastin fibers are thin and sinuous. Elastin contained in the
dermis represents 5%
of its dry weight. Elastin is a large fibrous protein which is formed by
spiral filaments that
can be compared to springs. The spiral filaments consist of peptidic chains
that can stretch
out. The peptidic chains are connected to each other by very specific amino-
acids: desmosin
and isodesmosin, which builds between them, giving the molecule a reticular
aspect. After
stretching out, the molecules resume their original shape due to this cross
linking, which is
essential to molecular elasticity.
The biosynthesis of elastin begins with the embryonic period and continues
through adulthood, at which time our body stops producing elastin. Thus,
elastin is no longer
renewed. With aging, the elastic fibers progressively degenerate and separate
into fragments.
The skin progressively loses its elasticity, resulting in fine lines and
wrinkles. This damage to
our elastic tissue cannot be avoided and is part of the natural
(physiological) aging process.
This process begins relatively early, but accelerates considerably after age
40.
Elastin owes its properties to its thin structure which resembles that of
rubber.
Elastin is the protein responsible for our skin's essential elasticity and
tonicity. Its decrease
means the skin starts sagging, allowing fine lines, folds and wrinkles to
appear and grow.
One embodiment of the present invention provides compositions comprising at
least one polyphenolic compound, or derivatives thereof, preferably ellagic
acid or tannic acid
or a combination thereof. The polyphenolic compound may be present in an
effective
amount, for example, to stimulate elastogenesis or protect elastin fibers from
degradation. In
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CA 02603102 2008-08-07
one embodiment, an effective amount is from about 0.011.1g to about 100 kg,
preferably from
about 1 kg to about 10 g.
Compositions of the present invention may further include a stimulator of
elastogenesis. Such stimulators of elastogenensis include, but are not limited
to, elastin
derived peptides, plant derived peptides, bovine derived peptides, manganese,
iron, copper
and combinations thereof.
Embodiments of the present invention may further comprise an agent selected
from anti-inflammatory agents, sunscreens, sunblocks, stimulators of protein
synthesis, cell
membrane stabilizing agents, moisturizing agents, coloring agents, opacifying
agents and
combinations thereof.
A further embodiment of the present invention provides compositions
comprising at least one polyphenolic compound, or derivatives thereof,
preferably ellagic acid
or tannic acid and optionally one stimulator of elastogenesis. A stimulator of
elastogenesis
may be, for example, small elastin-derived peptides including, but not limited
to, ProK-60 or
other small elastin-derived peptides as set forth in co-pending U.S.
Application No.
10/778,253 entitled "Elastin Digest Compositions and Methods Utilizing the
Same" filed
February 13, 2004. Such compositions may be useful to significantly increase
the net
deposition of insoluble elastic fibers, thereby enhancing the skin's
elasticity and decreasing
the appearance of fine lines and/or wrinkles. Thus, further embodiments of the
present
invention provide compositions and methods to compensate for the loss of
elastic
components in the dermis.
The result of aging on skin, whether or not it has been accelerated by
environmental damage (such as radiation, pollution) is a deterioration of the
dermal layer--
fewer fibroblasts, less collagen, less elastin and less circulatory support.
Consequently, the
normal stretching and contraction of the skin leads to damage of the dermis
that is not readily
corrected, resulting in wrinkling. Further embodiments of the present
invention provide
methods and compositions for increasing the deposition of insoluble elastin
fibers, therein
reducing the effects of radiation, including, but not limited to, ultraviolet
radiation, or other
environmental damage.
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CA 02603102 2008-08-07
Dermatologists and cosmetologists have directed their efforts to improving the
appearance of skin using agents known to stimulate the growth and
proliferation of epidermal
cells. Newly proliferated cells provide more structure and hold more moisture,
giving the
skin a younger appearance. One method of causing new skin cell proliferation
is
accomplished by use of an irritant or chemical peel in which the uppermost
layers of the
epidermis are caused to slough off, leading to proliferation and replacement
with new
epidermal cells. While such treatment is recognized to provide some cosmetic
improvement,
it does not address the major causative factor--the compromised supporting
dermal layer.
Thus, embodiments of the present invention also provide methods and
compositions for the
enhanced deposition of insoluble elastin fibers, therein providing the dermal
support and
elasticity necessary for smooth, supple skin.
One embodiment of the invention is a stable, effective topical composition
comprising at least one polyphenolic compound, or derivative thereof
Preferably, the
polyphenolic compound is selected from tannic acid, ellagic acid, derivatives
thereof, salts
thereof and combinations thereof
Another embodiment of the present invention is a method of treating damage
to skin, such as often arises due ultraviolet light exposure and/or aging. The
method includes
applying the present topical composition to a damaged portion of the skin, for
example, but
not limited to, topically applying compositions of the present invention to
the locus of
wrinkles. These topical polyphenolic based compositions are particularly
effective for
reducing epidermal wrinkling resulting from intrinsic aging, photo damage, or
other
environmental damage.
Such compositions may also be used prophylactically to reduce photo-induced
damage which can result from exposure of skin to sunlight and other harmful
irradiation.
Another embodiment of the invention is a method of prophylactically applying
the compositions of the invention for the protection of the skin against
damage which may
occur due to radiation or other environmental insults/exposure.
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CA 02603102 2011-04-07
A further embodiment provides a method for treating and/or reducing wrinkles
and/or fine lines by contacting skin with a composition of the present
invention.
Another embodiment is a method for stabilizing insoluble elastin fibers
present in the skin by contacting the skin with a topical composition of the
invention. An
object of the present invention is to provide a composition useful in
minimizing early and
acute ultraviolet radiation damage, as well as late and chronic radiation
induced photo
damage which together may enhance or cause photoaging of the skin.
It is yet a further aspect to provide in the form of a topical carrier, at
least one
polyphenolic compound, or derivative thereof, including, but not limited to,
tannic acid
and/or ellagic acid, which is effective in increasing the amount of insoluble
elastin
deposition, therein restoring that elastin which is degraded upon exposure to
free radicals.
It is another aspect to provide factors, including but not limited to, a
source of
small elastin-derived peptides (for example, ProK-60), a manganese component
(for example,
Mn-PCA, manganese sulfate, manganese gluconate), an iron component (for
example, ferric
ammonium citrate), a copper component (for example, copper-PCA, copper
sulfate), bovine-
derived peptides or plant-derived peptides to promote elastogenesis for skin
repair and wound
healing that occurs due to photoaging processes in the skin, such as those
that can occur from
acute sunburn and/or chronic exposure to ultraviolet radiation. Suitable
manganese and iron
components are described in U.S. Patent No. 7,666,829; suitable plant-derived
peptides are
described in U.S. Patent No. 7,723,308; Pub. U.S. 2006/014503; U.S. 7,566,693;
Pub. U.S.
2010/0016417; Pub. U.S. 2006/012027 and WO 2007/030145; and suitable bovine-
derived
peptides are described in U.S. Patent No. 7,566,693; Pub. U.S. 2010/0016417;
Pub. U.S.
2006/012027 and WO 2007/030145.
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CA 02603102 2008-08-07
A further embodiment of the present invention provides a composition
comprising an effective amount of a polyphenolic compound and an elastin
derived peptide is
provided. The polyphenolic compound may be selected from tannic acid, ellagic
acid and
combinations thereof. An effective amount of the polyphenolic compound is
preferably from
about li_tg to about 10 [lg. In a preferred embodiment, the elastin derived
peptide is selected
from ProK60, E91 and a combination thereof.
In a further embodiment of the present invention a composition comprising an
effective amount of a polyphenolic compound and an elastogenic plant-derived
peptide is
provided. The polyphenolic compound may be selected from tannic acid, ellagic
acid and a
combination thereof An effective amount of the polyphenolic compound is
preferably from
about ll_tg to about 10 lig.
Another embodiment is a method for protecting elastin fibers from degradation
comprising administering an effective amount of a polyphenolic compound to a
subject in
need thereof The polyphenolic compound is selected from tannic acid, ellagic
acid and a
combination thereof An effective amount of the polyphenolic compound is
preferably from
about lp.g to about 10 [lg.
In another embodiment, the method may further comprise administering a
stimulator of elastogenesis selected from elastin-derived peptides, plant-
derived peptides,
bovine-derived peptides, manganese, iron, copper and combinations thereof
The
polyphenolic compound and the stimulator of elastogenesis may be administered
simultaneously or sequentially.
The preparation of a pharmacological composition that contains active
ingredients dispersed therein is well understood in the art. Typically such
compositions if
desired, may be prepared as sterile compositions either as liquid solutions or
suspensions,
aqueous or non-aqueous, however, suspensions in liquid prior to use can also
be prepared.
The active ingredient of the present invention may be mixed with excipients
which are pharmaceutically acceptable and compatible with the active
ingredient and in
amounts suitable for use in the therapeutic methods described herein. Various
excipients may
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CA 02603102 2008-08-07
be used as carriers for the peptide compositions of the present invention as
would be known
to those skilled in the art. For example, compounds may be dissolved
excipients such as
water comprising solutions, alcohol comprising mixtures, intravenous and
saline comprising
mixture, dextrose, glycerol, ethanol or the like and combinations thereof. In
addition, if
desired, the composition can contain minor amounts of auxiliary substances
such as wetting
or emulsifying agents, pH buffering agents and the like which enhance the
effectiveness of
the active ingredient.
Formulations comprising polyphenolics, for example ellagic acid or tannic
acid, may be prepared by mixing such excipients with the polyphenolic. The
polyphenolic
compounds in the formulation may comprise from about 0.0002 to about 90% by
weight of
the formulation. These formulations may be employed directly as a constituent
of therapeutic
or cosmetic treatments, such as emulsions, lotions, sprays, ointments, creams
and foam
masks. Final products may contain up to 10% by weight but preferably 0.001 to
5% of such a
solution though of course more concentrated or more dilute solutions may also
be used in
greater or lesser amounts. For example, an eye cream may comprise about 0.1%
(w/w) and a
facial cream may comprise about 0.01% (w/w) of a polyphenolic compound in an
excipient.
A therapeutic composition of the present invention can include
pharmaceutically acceptable salts of the components therein. Pharmaceutically
acceptable
salts include the acid addition salts that are formed with inorganic acids
such as, for example,
hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric,
mandelic and the
like. Salts formed with the free carboxyl groups can also be derived from
inorganic bases
such as, for example, sodium, potassium, ammonium, calcium or ferric
hydroxides, and such
organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol,
histidine, procaine
and the like.
Physiologically tolerable carriers and excipients are well known in the art.
Other equivalent terms include physiologically acceptable or tissue
compatible. Exemplary
of liquid carriers are sterile aqueous solutions that contain no materials in
addition to the
active ingredients and water, or contain a buffer such as sodium phosphate at
physiological
pH value, physiological saline or both, such as phosphate-buffered saline.
Still further,
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CA 02603102 2008-08-07
aqueous carriers can contain more than one buffer salt, as well as salts such
as sodium and
potassium chlorides, dextrose, propylene glycol, polyethylene glycol and other
solutes.
Thus, the dosage ranges for the administration of polyphenolic are those large
enough to produce the desired effect in which the condition to be treated is
ameliorated. The
dosage should not be so large as to cause adverse side effects. Generally, the
dosage will vary
with the age, condition, and sex of the patient, and the extent of the disease
in the patient, and
can be determined by one of skill in the art. The dosage can be adjusted in
the event of any
complication.
The compositions are administered in a manner compatible with the dosage
formulation, and in a therapeutically effective amount. A therapeutic amount
of a
polyphenolic-based composition is an amount sufficient to produce the desired
result, and can
vary widely depending upon the disease condition and the potency of the
therapeutic
compound. In the present invention the desired result is an improvement in
elasticity of the
tissue as determined by an improvement in the elastin content of the tissue,
improved capacity
and function of the tissue, or improved appearance, suppleness, and/or tone of
the tissue being
treated. The quantity to be administered depends on the subject to be treated,
the capacity of
the subject's system to utilize the active ingredient, and the degree of
therapeutic effect
desired. Precise amounts of active ingredient required to be administered
depend on the
judgment of the practitioner and are peculiar to each individual. However,
suitable dosage
ranges for systemic application are disclosed herein and depend on the
conditions of
administration. Suitable regimes for administration are also variable, but are
typified by an
initial administration followed by repeated doses at one or more time
intervals by a
subsequent administration. Where a single composition is not available for a
treatment, or
where such a composition is not desirable, administration of composition may
also comprise
the application of several different compositions sequentially to achieve a
desired therapeutic
effect.
Topical carriers are employed which should be both non-irritating to the skin
and which are suitable for delivering the active components to the skin.
Further, suitable
topical carriers should be those which do not inhibit the antioxidant activity
of the active
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CA 02603102 2008-08-07
ingredients thus reducing the efficiency of the composition for protecting the
skin from the
effects of acute and chronic ultraviolet radiation. Further, such carriers
must be of
sufficiently high purity and sufficiently low toxicity to render them suitable
for chronic
topical administration to the skin and be free of bacterial contaminants.
The active ingredients described herein can be incorporated in any suitable
pharmacologically acceptable carrier which is suitable for topical
administration to the human
skin. As such, the pharmacologically acceptable carrier must be of sufficient
purity and have
sufficiently low toxicity to render it suitable for administration to a human
noting that,
generally, the carrier can represent up to 99.99% and typically from at least
approximately
80% of the total composition. Thus, the phrase "pharmaceutically acceptable"
refers to
molecular entities and compositions that do not produce an allergic or similar
untoward
reaction when administered to a human. The pharmaceutically acceptable
carriers and
additives employed in the present compositions are compatible with at the
tannic acid and/or
ellagic acid compounds and compositions described herein containing such
compounds.
Typical compositions for use herein include a wide variety of physical forms.
These include, but are not limited to, solutions, lotions, creams, oils, gels,
sticks, sprays,
ointments, balms, patches and pastes. Generally, such carrier systems can be
described as
being solutions, creams, emulsions, gels, solids and aerosols.
Solvents are generally employed in the preparation of suitable topical
compositions. Such solvents can either be aqueous or organic based and, in
either case, the
solvent must be capable of having dispersed or dissolved therein the above-
described active
components while not being irritating to the user. Water is a typical aqueous
solvent while
suitable organic solvents include propylene glycol, battalion glycol,
polyethylene glycol,
polypropylene glycol, glycerol, 1,2,4-butanetriol, sorbitol esters, 1,2,6-
hexanetriol, ethanol,
isopropanol, butanediol and mixtures thereof. Solvents can be included in the
overall
composition in amounts ranging from 0.1% to 99% and preferably from 2.0% to
75%. It is
noted that compositions of the present invention can be produced in the form
of an emollient.
A wide variety of suitable emollients are known and may be used herein. In
this regard,
reference is made to U.S. Pat. No. 5,296,500.
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CA 02603102 2008-08-07
Alternatively, the polyphenolic compound can be formulated as a lotion
containing from about 0.01% to 10% of the above described active ingredients.
Further, it
may be formulated from a solution carrier system as a cream. A cream of the
present
invention would preferably comprise from about 0.1% to 15% and preferably from
1% to 5%
of the above described active ingredients. Lotions and creams can be
formulated as
emulsions as well as solutions.
It is contemplated that the polyphenolic compounds described above may be
used as a lotion or cream emulsion of the oil-in-water type or as a water-in-
oil type, both of
which being extremely well known in the cosmetic field. Multi-phase emulsions
such as the
water-in-oil type is disclosed in U.S. Pat. No. 4,254,105 may also be
employed.
It is further contemplated that the polyphenolic compounds be formulated
from a solution carrier system as an ointment. An ointment may comprise a
simple base of
animal or vegetable oils or semi-solid hydrocarbons (oleaginous). Ointments
may also
comprise absorption ointment bases which absorb water to form emulsions.
Ointment
carriers may also be water soluble. An ointment may comprise from 1% to 99% of
an
emollient plus to about 0.1% to 99% of a thickening agent. Reference is again
made to U.S.
Pat. No. 5,296,500 and the citations contained therein for a more complete
disclosure of the
various ointment, cream and lotion formulations for use herein.
The compositions can include one or more of a variety of optional ingredients,
such as, but not limited to, anti-inflammatory agents, sunscreens/sunblocks,
stimulators of
protein synthesis, cell membrane stabilizing agents (i.e., carnitine),
moisturizing agents,
coloring agents, opacifying agents and the like, so long as they do not
interfere with the
elastin stabilizing properties of the polyphenolic compounds, or derivatives
thereof. The
formulation can also include, other active ingredients, such as antibiotics,
analgesics, anti-
allergenics and the like. The formulation is commonly applied to the skin as a
lotion or
cream to be rubbed on body tissue over the desired area. For optimum efficacy
treatment in
accordance with the presented method should be initiated as early as possible
following
exposure to sunlight or other radiation source. The formulation is generally
applied to the
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CA 02603102 2011-04-07
skin once or twice daily. As noted elsewhere herein, the present composition
may also be
used to inhibit and/or minimize the effects of aging and/or photo damage on
the skin.
In the compositions provided herein the polyphenolic compounds, or
derivatives thereof, such as tannic acid and/or ellagic acid, is present in an
amount from about
0.01 to 80 weight percent, further from about 0.1 to 20 weight percent, and
further from about
0.5 to 10 weight percent.
The optional source of small elastin-derived peptides (such as, but not
limited
to ProK-60 or E91) component stimulates new elastogenesis, supplementing
elastic tissue,
and consequently, reduction of wrinkles and other skin conditions related to
loss of elasticity.
The source of small elastin-derived peptides component, when used in the
composition is
generally present in an amount from about .001 to about 10 weight percent,
preferably from
about 0.005 to about 0.1 weight percent of the composition.
The optional manganese component may be any magnesium compound, or a
pharmaceutically acceptable salt thereof, but preferably is MnCli, MnSO4
and/or MnPCA,
wherein the manganese component is typically present in an amount from about
0.001 to 10
weight percent, preferably from about 0.0012 to 0.012 weight percent of the
composition.
A trivalent iron component, such as, but not limited to, Ferric Ammonium
Citrate (FAC) may also be included in the composition. The trivalent iron
component
stimulates new elastogenesis and assists in treatment of elastic tissue
defects. The trivalent
iron, when included in the composition, is generally present in an amount from
about 0.001 to
about 10 weight percent of the composition.
Copper may also be included in the composition. In preferred embodiments,
copper may be present in about 0.001 to about 10 weight percent, more
preferably from about
0.005 to about 0.1 weight percent of the composition.
Elastogenic plant-derived peptides may also be present in the composition.
Such peptides are more fully described in U.S. Patent No. 7,723,308; Pub. U.S.
2006/014503;
U.S. 7,566,693; Pub. U.S. 2010/0016417; Pub. U.S. 2006/012027 and WO
2007/030145,
U.S. Patent No. 7,566,693; Pub. U.S. 2010/0016417; Pub. U.S. 2006/012027 and
WO
2007/030145
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CA 02603102 2011-04-07
and U.S. Patent No. 7,723,308; Pub. U.S. 2006/014503; WO 2006/113681; U.S.
7,566,693;
Pub. U.S. 2010/0016417; Pub. U.S. 2006/012027 and WO 2007/030145. Such
peptides may
be sextapeptide comprising the sequence Xi-X2-X3 -X4-X5-X 6, wherein X1 is V
or I, X2 is G,
X3 is A, L or V, X4 is M, S, or A, X5 is P and X6 is G. Such a plant-derived
peptide or
synthetic plant-derived peptide may be present in the composition. In
preferred
embodiments, the peptide may be present in about 0.0001 to about 0.01 weight
percent, more
preferably from about 0.0004 to about 0.002 weight percent of the composition.
Upon formulation, compositions of the present invention may be administered
in a manner compatible with the dosage formulation and in such amount as is
therapeutically
effective. The formulations are easily administered in a variety of dosage
forms such as
direct topical application, application via a transdermal patch and the like.
For topical administration in an aqueous solution, for example, the
compositions may be used directly on the skin without any toxic effects to the
patient.
Alternatively, the compositions of the invention may be dissolved or
resuspended in a
suitable buffer prior to mixing, if necessary.
In general, routine experimentation will determine specific ranges for optimal
therapeutic effect for each composition and each administrative protocol, and
administration
to specific individuals will be adjusted to within effective and safe ranges
depending on the
condition and responsiveness of the individual to initial administrations.
Some variation in dosage will necessarily occur depending on the condition of
the subject being treated. The person responsible for administration will, in
any event,
determine the appropriate dose for the individual subject.
Moreover, for human
administration, preparations should meet sterility, pyrogenicity, general
safety and purity
standards as required by the FDA.
While the making and using of various embodiments of the present invention
are discussed in detail below, it should be appreciated that the present
invention provides
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CA 02603102 2008-08-07
many applicable inventive concepts which can be embodied in a wide variety of
specific
contexts. The specific embodiments discussed herein are merely illustrative of
specific ways
to make and use the invention and do not delimit the scope of the invention.
Various
modifications and combinations of the illustrative embodiments, as well as
other
embodiments of the invention, will be apparent to persons skilled in the art
upon reference to
the description.
EXAMPLE 1
Materials. All chemical-grade reagents were obtained from Sigma (St. Louis,
MO). aMEM medium, fetal bovine serum (FBS), 0.2% trypsine-0.02% EDTA and other
cell
culture products were obtained from GIBCO Life Technologies (Burlington,
Canada).
Polyclonal antibody to tropoelastin and BA4 monoclonal antibody to VGVAPG were
purchased from Elastin Products Company, Inc. (Owensville, MO). Monospecific
polyclonal
anti-AKAAAKAAAKA antibody was a gift of Dr. Barry Starcher from the University
of
Texas. Secondary antibody fluorescein-conjugated goat anti-rabbit (GAR-FITC)
was
purchased from Sigma (St. Louis, MO). DNeasy Tissue system for DNA assay and
RNeasy
Mini Kit for isolation of total RNA were purchased from Qiagen (Mississauga,
Canada).
Expression probe for elastin was purchased from Applied Biosystems (Foster
City, CA). The
radiolabeled reagents, [31-1]-valine, and [31-1]-thymidine were purchased from
Amersham
Canada Ltd. (Oakville, Canada).
Cell Cultures. Biological effects of ellagic acid and tannic acid were tested
in
cultures of dermal fibroblasts derived from punch biopsies of healthy skin
from Caucasian
females of different ages ranging from 4-52 years old. All fibroblasts were
originally isolated
by allowing them to migrate out of skin explants and then passaged by
trypsinization and
maintained in alpha-minimum essential medium supplemented with 20 mM Hepes, 1%
antibiotics and antimycotics, 1% L-Glutamate and 2% fetal bovine serum (FBS).
In all
experiments, consecutive passages 3-5 were tested. Cells were densely plated
(50 x105 cells
/dish) to reach confluency and then cultured for 7 days in the presence and
absence of ellagic
acid (dissolved in DMSO) and tannic acid (dissolved in water), both in
concentration of 1
p,g/mL. This optimal concentration was chosen after a series of pilot
experiments indicated
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CA 02603102 2008-08-07
that 1 Ag/m1 of EA and TA induced optimal effect on net deposition of elastin
and did not
trigger any change in cellular proliferation rate nor affect basic metabolic
performance.
In a parallel series of experiments, cultures of dermal fibroblasts were
treated
with well established stimulators of tropoelastin synthesis, ProK-60 (25
pg/m1) and Ferric
Ammonium Citrate (20 [iM), and simultaneously incubated in the presence and
absence of
ellagic acid or tannic acid (both in concentration 1 g/m1).
Assessment of Cellular Proliferation. Cellular proliferation rates of control,
ellagic acid and tannic acid treated fibroblasts were assessed at the end
point by counting of
trypsinized cells, by total DNA assay using the DNeasy Tissue System from
Qiagen and by
assessment of [31-1]-thymidine incorporation, which was added to all cultures
(2 piCi/well) for
the last 24 hours.
Assessment of Elastin mRNA levels. Fibroblasts were cultured to confluency
in medium with 2% FBS and then in serum-free medium for 24 hours. The medium
was
changed again and cells were incubated for the next 24 hours in the presence
and absence of
ellagic acid or tannic acid (both in concentration 1 1.tg/mL). At the end of
the incubation
period total RNA was extracted using TRI-reagent. Steady-state levels of
elastin mRNA were
analyzed by semi quantitative PCR and by Northern Blot using a human elastin
cDNA
recombinant H-11 probe. In all experiments, performed in triplicate, the
loading control was
routinely performed.
Assessment of elastic fibers content by immunohistochemistry. Seven-day-old
and 14-day-old confluent cultures of fibroblasts, which produce abundant ECM,
were
assessed. All cultures were fixed in cold 100% methanol at -20 C for 30 min,
then incubated
for 1 hour with 2 jig/m1 of polyclonal antibody to tropoelastin. Cultures were
then incubated
for an additional hour with appropriate fluorescein-conjugated secondary
antibody (GAR-
FITC). Nuclei were counterstained with propidium iodide. Morphometric analysis
of five
separate cultures in each experimental group, immunostained with antibodies
recognizing
extracellular matrix components was performed using a computerized video
analysis system
(Image-Pro Plus software 3.0, Media Cybernetics, Silver Spring, MD).
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CA 02603102 2008-08-07
Radioactive metabolic labeling and quantification of newly deposited
insoluble elastin. Quintuplicate, 4 day-old cultures of dermal fibroblasts
maintained in the
presence or absence of 1 1.1g/mL of ellagic acid or tannic acid were
additionally exposed for
the 3 following days to 20 tiCi [311]-valine. At the end of incubation period
the contents of
radioactive NaOH-insoluble elastin was assessed separately in each culture by
scintillation
county. Final results reflecting amounts of metabolically labeled, insoluble
elastin were
expressed as CPM/ Rg DNA. DNA was determined with the DNeasy Tissue System
from
Qiagen Proteolytic.
Pulse and chase experiments aimed at the assessment of durability of newly
deposited metabolically-labeled elastin. In this series of experiments, dermal
fibroblasts,
plated as described above in media with 10% FBS, were maintained in the
presence and
absence of 1 pg/mL of EA or TA for the first 7 days and pulsed with 20 Ci
[3H]-valine
between day 4 and 7. While quadruplicate 7-day-old cultures from each
experimental group
were directly processed for the assessment of radioactive NaOH-insoluble
elastin, parallel
quadruplicate cultures from each experimental group were transferred to media
containing
only 1% FBS, which did not stimulate proliferation and new elastogenesis, and
maintained
for the next seven days (chase period) in the absence of ellagic acid or
tannic acid. At day 14
these cultures were terminated and the net content of the radioactive NaOH-
insoluble elastin
was assessed as described above.
Organ cultures of explants derived from surgical biopsies of human skin In
order to further test whether ellagic acid and tannic acid would penetrate
into skin tissue and
enhance elastogenesis, fragments of normal skin (from 30 and 34 year old
females) obtained
during plastic surgery procedures were tested in organ culture system. Skin
fragments were
cut into multiple 1mm2 pieces and placed on top of metal grids immersed in
culture medium
containing 5% FBS and maintained for 10 days in the presence and absence of 1
1..tg/mL of
ellagic acid or tannic acid alone or combined with 25 jig/ml of ProK-60. The
media were
changed every second day. All organ cultures were fixed in 1% buffered
formalin and their
transversal serial histological sections were stained with Movat's
pentachrome.
Morphometric analysis was performed as described above. In each analyzed group
(three
explants from each patient) low-power fields (1 mm2) of 20 serial sections
stained with
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CA 02603102 2008-08-07
Movat's pentachrome were analyzed and all structures stained black (elastic
fibers) were
counted.
Assessment of tropoelastin integrity by western blots. To determine the
influence of tannic acid and ellagic acid on the integrity of soluble
tropoelastin, dermal
fibroblasts obtained from three different donors (initially plated at 50,000
cells/dish) were
cultured to confluency in medium with 5% FBS and then triplicate cultures were
incubated
for the next 24 hours in the presence and absence of ellagic acid or tannic
acid (both in
concentration 1 i_tg/mL). At the end of the incubation period conditioned
media were
collected and then the soluble proteins present in the intracellular
compartments were
extracted with 0.5 M acetic acid in the presence of proteinase inhibitors in
the following final
concentrations: 2 mM benzamidine, 2 mM EACA, 2 mM PMSF, 1 mM EDTA and 1 mg/ml
Trasylol. Extraction was carried out for six hours at 4 C and the insoluble
material was
pelleted by centrifugation. The supernatant was dialyzed exhaustively (4000
kDa cutoff
membrane) at 4 C against water containing proteinase inhibitors, then
lyophilized.
Concentrated preparations of the conditioned media and cell extracts from all
analyzed
cultures were analyzed for their protein content, and then samples containing
equal amounts
of protein (20 ug/sample) were suspended in 2 x SDS sample buffer with DTT,
resolved by
SDS PAGE, routinely transferred to nitrocellulose and immunoblotted with
specific anti-
tropoelastin antibody.
Immuno-precipitation of radioactive tropoelastin¨like peptides.
This
experiment was aimed at elucidating whether binding of ellagic acid or tannic
acid to
tropoelastin molecules would block two characteristic elastin domains
responsible for orderly
self-aggregation and cross-linking respectively. Instead of very unstable
tropoelastin,
triplicate samples of a [31-I]-yaline-labeled recombinant polypeptide
containing linear amino
acid sequences encoded by human tropoelastin gene exons; 20421-23-24)2 were
used. 100 pd
samples of this radioactive recombinant polypeptide modeled after human
elastin dissolved in
PBS (specific radioactivity 1000 CPM/ sample) were incubated in the presence
and absence
of 1 p,g/mL of ellagic acid or tannic acid, at room temperature for 6 hours.
Aliquots of all
control and experimental samples were then immuno-precipitated with (BA4)
monoclonal
antibody (recognizing VGVAPG and other similar domains, encoded by exons 20
and 24,
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CA 02603102 2011-04-07
responsible for self-aggregation of tropoelastin) and with monospecific
polyclonal anti-
AKAAAKAAAKA antibody recognizing the exon 21- and 23-encoded cross-linking
sequences. It was anticipated that in case that binding of ellagic acid or
tannic acid would
block one or both of these crucial domains present in the tested radioactive
recombinant
peptide, it could not be immunoprecipitated with anti- VGVAPG or anti-
AKAAAKAAAKA
antibodies.
Proteolytic degradation protection assay of insoluble elastin. To determine
whether ellagic acid or tannic acid may directly protect fully cross-linked
"insoluble elastin"
against elastolytic activity of several elastases, an in vitro assay measuring
degradation of an
insoluble [3H]-elastin substrate was used. Briefly, insoluble elastin was
purified from bovine
ligamentum nuchae using a modification of the hot alkali technique and was
shown by amino
acid analysis to be free of microfibrillar protein and other contaminants.
Sequencing of
insoluble, digested elastin was performed using an Applied Biosystems model
473A protein
sequencer equipped with a model 610A data analysis program. The stock of this
pure
insoluble elastin preparation was washed twice with water and twice with
acetonitrile and
then labeled with sodium [3H]-borohydride and stored at -20 C. Before each
experiment, the
[31-1]-elastin substrate suspended in PBS was boiled for 5 min and extensively
washed to
remove all unbound radioactivity. Then, its 100 jig aliquots (specific
activity 300 CPM/lug)
were suspended in serum free culture medium and pre-incubated for I hour in
the presence
and absence of 1 1.1g/mL or 10 iag/mL of ellagic acid or tannic acid. All
samples of
radioactive elastin were then submitted to three 5 min washes in serum free
culture medium
prior to their 18 hour incubation at 37 C with aliquots (50 ng) of human
leukocyte elastase
(HLE), porcine pancreatic elastase (PPE), MMP-2, or Papaine dissolved in the
assay buffer
TM
(50 mM Tris-HCL, pH 7.5 containing 150 mM NaCl, 10 mM CaC12, 0.02% Brij and
0.02%
sodium azide). Each treatment was tested in quadruplicate samples. At the end
of the
incubation, all samples were microcentrifuged (8000 x g for 5 min) and 100
1.1.1 aliquots of
supernatant containing the solubilized degradation products were mixed with 4
ml
scintillation fluid and counted in triplicates in liquid scintillation
counter. The radioactivity
(cpm/sample) released into the supernatant reflecting the degradation of [3H]-
elastin substrate
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CA 02603102 2008-08-07
was assessed and the mean and standard deviations were calculated from
sixtiplicate
assessments from 3 different experiments.
Assessement of TA and EA binding to insoluble elastin. In order to directly
show that both polyphenols bind to elastin, triplicate (1 mg) aliquots of our
above mentioned
preparation of pure insoluble elastin were incubated with 20 1.1g/m1 of
ellagic acid or tannic
acid for 2 hr at 37 C. The initial concentration of both polyphenols were
confirmed by a
direct spectrophotometric reading at 280 nm. This method demonstrated a dose-
dependent
linear increase in absorbancy. At the end of incubation period the insoluble
elastin slurries
were separated by centrifugation and the concentrations of polyphenols in
supernatants were
determined again. The detected differences between the initial and final
concentration of
polyphenols in supernatants from particular samples directly indicated that
both ellagic acid
and tannic acid bound to elastin slurries during the incubation period. In
each experimental
group means SD were calculated and obtained values were statistically
compared with
beginning concentrations of both polyphenols.
Statistical analysis. In all above mentioned quantitative assays, means and
standard deviations (expressed as Mean + SD) were calculated and statistical
analyses were
carried out by ANOVA to establish whether detected differences were
statistically significant.
Results.
Ellagic acid and tannic acid enhance deposition of elastin by dermal
fibroblasts. Results of immunohistochemical analysis (Figure la and b) and
quantitative
assessment of metabolically labeled insoluble elastin (Figure 1 c) indicated
that 7 day old
monolayer cultures of dermal fibroblasts maintained with ellagic acid or
tannic acid contain
thicker elastic fibers and a higher net content of NaOH-insoluble elastin than
untreated
control cultures. Moreover, results of morphometric analysis demonstrated that
both ellagic
acid and tannic acid caused a significant (p<0.005) increase (67 +6 % and 96
12%
respectively) in net elastogenesis observed in organ cultures of human skin
explants
maintained for 10 days with 5% FBS. Explants maintained for 10 days in culture
media
containing tannic acid contain thicker and longer elastic fibers than those
present in explants
maintained only in control medium or medium with ProK-60 (data not shown).
Interestingly,
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CA 02603102 2008-08-07
the presence of tannic acid seems to particularly enhance elastogenesis in
cells protruding
from the stratum basale, toward the papillary dermis, and in cells surrounding
small
capillaries. Results of semi-quantitative PCR and Northern blotting indicated,
however, that
treatment of cultured dermal fibroblasts with ellagic acid or tannic acid did
not induce any
increase in the transcription of their elastin gene (data not shown) nor
change their
proliferation rate, as assessed by incorporation of radioactive thymidine and
total DNA
content (data not shown). Despite this finding, results of western blotting,
with anti-
tropoelastin antibody, showed that both cell extracts and conditioned media,
of dermal
fibroblasts incubated with ellagic acid or tannic acid, contained more intact
70 kDa
tropoelastin and less immuno-detectable degradation products of lower
molecular weight than
untreated counterparts (data not shown). This finding gave evidence that both
polyphenols
protected newly produced tropoelastin from premature intracellular and
pericellular
degradation by endogenous proteinases.
Ellagic acid and tannic acid protect elastin from proteolytic degradation.
Results of a pulse and chase experiment (Figure 2a and b) demonstrated that
cultures of
dermal fibroblasts, exposed for seven days to ellagic acid and tannic acid,
sustain their high
net content of insoluble elastin (metabolically pulsed with [31-1]-valine
between day 4 and 7)
when maintained for an additional seven days (chase period) in media
containing only 1%
FBS (no ellagic acid or tannic acid), which did not stimulate proliferation
(Figure 2c) and new
elastogenesis. In contrast, 14 day-old control (untreated) cultures
demonstrated a significant
decrease in their net content of metabolically labeled insoluble elastin
(detected at the end of
pulse period, at day 7). Moreover, results of an in vitro elastolytic assay
demonstrated that
samples of purified insoluble elastin (purity confirmed by amino acid
analysis), pretreated
with ellagic acid or tannic acid, were more resistant to proteolytic
degradation by all tested
elastolytic enzymes belonging to the serine proteinase (human leukocyte
elastase and porcine
pancreatic elastase), metallo-proteinase (MMP-2) and cysteine proteinase
(papaine) families
(Figure 3). These data suggested that ellagic acid and tannic acid protected
newly deposited
and purified insoluble elastin against degradation by both endogenous and
exogenous
elastolytic enzymes, respectively, through association with elastin.
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CA 02603102 2008-08-07
Ellagic acid and tannic acid bind to elastin and tropoelastin. Results of a
spectrophotometric assay (displaying a linear concentration curve for both
ellagic acid and
tannic acid at an absorbance of 280 nm), comparing concentrations of both
polyphenols
before and after incubation with insoluble elastin, demonstrated that 1 mg of
pure insoluble
elastin, isolated from ligamentum nuchae, absorbed 87 3% of the tannic acid
and 81 2% of
the ellagic acid in solutions having had initial concentrations of 20m/mL of
each polyphenol.
This finding implied that both polyphenols bind to insoluble elastin.
Additional results
showed that preincubation of a [3F1]-valine-labeled recombinant peptide,
containing the most
characteristic hydrophobic and cross-link generating domains of tropoelastin,
with ellagic
acid and tannic acid did not preclude its effective (practically identical)
immunoprecipitation
with respective anti-VGVAPG and anti-AKAAAKAAAKA antibodies (data not shown).
This indicated that these polyphenols associate with and protect tropoelastin
in a way which
does not obscure hydrophobic domains (eg. VGVAPG), necessary for self-
aggregation, nor
KAAAK sequences participating in crosslinking.
Ellagic acid and tannic acid enhance elastogenic effect of selected
stimulators
of elastogenesis. Results of immunostaining and metabolic labeling established
that addition
of ellagic acid or tannic acid to fibroblast cultures simultaneously treated
with known
stimulators of elastin gene expression, ProK-60 or Ferric Ammonium Chloride
(FAC),
significantly enhanced their net deposition of insoluble elastin as estimated
in 7 day old
monolayer cultures (Figure 4). Morphometric analysis additionally demonstrated
that both
polyphenols significantly (p<0.02) enhanced elastogenic effect of ProK-60
observed in 10
day-old organ cultures of human skin explants (Ellagic Acid ¨ 22 4% and
Tannic Acid =
35 6%). Representative micrographs depicting synergistic effect of ProK-60 and
tannic acid
are presented in Figure 4d.
Discussion. Results demonstrate that the two polyphenols, ellagic acid and
tannic acid, used in concentration of 1 g/ml, did not modulate cellular
proliferation of
normal human dermal fibroblasts, despite the fact that anti-proliferative
properties of both of
these compounds were reported in cultures of various normal and malignant cell
lines when
used in higher doses. Dermal fibroblasts treated with both acids did not
demonstrate any
increase in levels of elastin mRNA yet facilitated a significant increase in
net elastic fiber
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CA 02603102 2008-08-07
content detected by immunochemistry and metabolic labeling of insoluble
elastin. It was
speculated that both ellagic acid and tannic acid might bind to intracellular
tropoelastin and to
newly assembled crosslinked elastin and protect them from proteolytic
degradation by
fibroblast-secreted proteolytic enzymes engaged in early remodeling of
extacellular matrix.
The hypothesis regarding potential preferential binding of both polyphenols to
intracellular
tropoelastin and extracellular elastin polymer was based on a previously
described
observation that addition of 0.25% tannic acid to glutaraldehyde fixative
dramatically
enhanced contrast of intracellular secretory vesicles containing tropoelastin
and contrast of
extracellular elastic fibers in tissues observed under the electron
microscope. In fact, in the
pre-immunostaining era, treatment with tannic acid became a widely accepted
method for
ultrastructural identification of elastic fibers that were previously
described as "electron
lucent and amorphous" under electron microscopy. The hypothesis was further
supported by
results of our pulse-and-chase experiment where ellagic acid and tannic acid
pre-treated
tropoelastin and insoluble elastin, deposited by dermal fibroblasts, remained
resistant against
non-specific endogenous degradation in the absence of ellagic acid or tannic
acid in culture
medium. Results also show that pre-incubation of [41]-labeled pure insoluble
elastin, with
either ellagic acid or tannic acid, significantly reduced its rate of
degradation (in the absence
of either polyphenol in the digest buffer) by several exogenous elastolytic
enzymes including
porcine pancreatic elastase, human leukocyte elastase, papaine and the UVB-
inducible MMP-
2. Results of this protection assay are consistent with recent observations
that addition of
tannic acid to the glutaraldehyde fixation process increased the stability of
porcine aortic
explants exposed to pancreatic elastase digestion. Moreover, the
spectrophotometric study
demonstrated that pure insoluble elastin, isolated from ligamentum nuchae,
binds both ellagic
acid and tannic acid, sequestering them from their solvent solutions. Results
of this study are
in further agreement the observation of similar binding of tannic acid by pure
aortic elastin
over time.
It has been previously shown that plant derived polyphenols, tannins and their
synthetic derivatives, can inhibit human leukocyte elastase and MMP-2/-9
activity in several
tumor cells. Other studies directly demonstrated that tannic acid specifically
inhibits the
chymotrypsin-like activity of purified 20S proteasomes and the activity of
tissue-type
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CA 02603102 2008-08-07
plasminogen activator, urokinase-type plasminogen activator and plasmin
activity. It was
speculated that a certain fraction of the described protective effect may be
due to a direct
inhibition of proteolytic enzymes by both polyphenols absorbed by or released
from the
elastin substrate.
The practical biological significance of the observations is that ellagic acid
or
tannic acid added to cultures of living cells facilitate normal secretion of
tropoelastin and its
assembly into elastic fibers by protecting intra- and extracellular
tropoelastin from
degradation by unspecific proteinases. Since ellagic acid and tannic acid did
not block
domains responsible for self-aggregation and subsequent cross-linking of this
protein, it is
speculated that ellagic acid and tannic acid may act in concert with the 67-
kDa elastin binding
protein (EBP) that acts as a protective molecular chaperone for intracellular
tropoelastin.
Moreover, the fact that ellagic acid and tannic acid significantly decreased
degradation of
newly produced elastin, in dermal fibroblast cultures, and fully cross-linked
elastin, from
ligamentum nuchae, indicate that both polyphenols may enhance longevity of
elastic fibers.
Given the presented beneficial effects of both tested polyphenols, both may be
used in topical preparations aimed at prevention of elastin degradation
characteristic of
normal aging and after chronic exposure to sunlight (photoaging). Since
ellagic acid and
tannic acid did not negatively interfere with the action of two known
stimulators of new
elastogenesis, but rather enhanced their net effect, their use in combination
with compounds
aimed at restoring cutaneous elastic fibers in aged skin or skin of patients
afflicted by diseases
caused by elastin gene insufficiency (i.e. Williams-Beuren Syndrome (WBS) and
Cutis Laxa)
may also appear beneficial. Results of our recent experiments (data not shown)
indicated that
tannic acid and ellagic acid prevented rapid, MMP-dependent degradation of
tropoelastin
produced by dermal fibroblasts derived from three WBS patients, yielding a
significant (21-
26%) net increase in deposition of insoluble elastin by these cells. Thus,
results of the in vitro
studies, presented herein, and lack of clinical side effects of polyphenolic
compounds fully
encourage their use in in vivo protection of existing elastic fibers and more
efficient
elastogenesis in skin.
In summary, results of in vitro studies demonstrate the use of tannic acid and
ellagic acid and other polyphenolic compounds in skin care products aimed at
initiation of
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CA 02603102 2008-08-07
new elastogenesis and protection of existing elastic fibers from normal aging-
related and UV-
induced proteolytic degradation.
EXAMPLE 2
In order to directly prove that tannic acid also binds to collagen type 1,
triplicate (1 mg) aliquots of pure collagen type I were incubated with 20
pig/m1 of TA for 2 h
at 37 C. The initial concentration of tannic acid was confirmed by direct
spectrophotometric
reading at 280 nm. This method adopted from Gori et al. demonstrated a dose-
dependent
linear increase in absorbance. At the end of incubation period the collagen
type I slurries
were separated by centrifugation and the concentration of tannic acid in
supernatants were
spectrophotometrically determined again at 280 nm. In each experimental group
means +SD
were calculated and obtained values were statistically compared with beginning
concentrations of both polyphenols.
Results. As shown in Figure 5, the binding studies demonstrate that 1 mg of
collagen type I (from rat tail) sequestered 75.5 +0.001% (P<0.0001) of the
tannic acid
(originally 20 lig/mL) from solution, suggesting that tannic acid may also
bind to collagen
type 1.
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