Note: Descriptions are shown in the official language in which they were submitted.
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TOPICAL APPLICATION OF
1-HYDROXYL 3,5-BIS(4'HYDROXYL STYRYL)BENZENE
FIELD OF THE INVENTION
The invention relates to compositions and methods for the topical application
of 1-
hydroxyl 3,5-bis(4'hydroxyl styryl)benzene.
BACKGROUND OF THE INVENTION
It is known to provide active agents to the skin for purposes of treating the
signs of
skin aging, providing anti-inflammatory benefits to the skin, or lightening
the skin.
A particular class of anti-inflammatory agents is those that inhibit the cell
transcription factor nuclear kappa-B (NFKB). For example, it is known that
certain
substituted resorcinols such as 4-hexyl resorcinol and tetrahydrocurcuminoids
are NFKB
inhibitors. Such compounds provide anti-aging benefits when applied to the
skin.
However, only a relatively small group of compounds have been identified as
both
effective and cosmetically acceptable.
It is known to apply anti-inflammatory compounds to the skin to provide anti-
pigmentation benefits. A particular class of anti-inflammatory agents used for
this
purpose is agents that reduce the amount of melanin in melanocytes by means of
inhibiting inflammatory mediator molecules. However, only a relatively small
group of
compounds have been identified as suitable for topical use to regulate melanin
formation
in skin.
SUMMARY OF THE INVENTION
The inventors have now surprisingly found that 1-hydroxyl 3,5-bis(4'hydroxyl
styryl)benzene is a potent NFKB inhibitor and is suitable for topical
application to skin,
and that 1-hydroxyl 3,5-bis(4'hydroxyl styryl)benzene and cosmetically
acceptable salts
thereof may be used to treat signs of skin aging, are inhibitors of melanin
formation in
melanocytes, and may be applied to skin in need of skin lightening treatment.
In one aspect, the invention provides a composition comprising 1-hydroxyl 3,5-
bis(4'hydroxyl styryl)benzene or a cosmetically acceptable salt thereof; and a
cosmetically
acceptable topical carrier comprising an ingredient selected from the group
consisting of
wetting agents, emulsifiers, emollients, humectants, and fragrances.
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The invention further provides a method of treating human skin, comprising
topically applying to said human skin a composition comprising 1-hydroxyl 3,5-
bis(4'hydroxyl styryl)benzene or a cosmetically acceptable salt thereof
The invention also provides a method of treating a sign of skin aging,
comprising
topically applying to skin in need of such treatment a composition comprising
1-hydroxyl
3,5-bis(4'hydroxyl styryl)benzene or a cosmetically acceptable salt thereof
The invention provides a method of lightening skin, comprising the step of
topically applying to skin in need of skin lightening treatment a composition
comprising
1-hydroxyl 3,5-bis(4'hydroxyl styryl)benzene or a cosmetically acceptable salt
thereof
The invention further provides a method of inhibiting the growth of a
microorganism, comprising applying to said microorganism 1-hydroxyl 3,5-
bis(4'hydroxyl styryl)benzene or a cosmetically acceptable salt thereof
Other features and advantages of the present invention will be apparent from
the
detailed description of the invention and from the claims.
DETAILED DESCRIPTION OF THE INVENTION
It is believed that one skilled in the art can, based upon the description
herein,
utilize the present invention to its fullest extent. The following specific
embodiments are
to be construed as merely illustrative, and not limitative of the remainder of
the disclosure
in any way whatsoever.
Unless defined otherwise, all technical and scientific terms used herein have
the
same meaning as commonly understood by one of ordinary skill in the art to
which the
invention belongs. Also, all publications, patent applications, patents, and
other references
mentioned herein are incorporated by reference. Unless otherwise indicated, a
percentage
or concentration refers to a percentage or concentration by weight (i.e., %
(W/W). Unless
stated otherwise, all ranges are inclusive of the endpoints, e.g., "from 4 to
9" includes the
endpoints 4 and 9.
Products described herein may optionally be in finished packaged form. In one
embodiment, the package is a container such as a plastic, metal or glass tube
or jar
containing the composition. The product may further contain additional
packaging such
as a plastic or cardboard box for storing such container. In one embodiment,
the product
comprises a composition of the invention and contains instructions directing
the user to
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apply the composition to the skin to treat the signs of skin aging as
discussed infra. Such
instructions may be printed on the container, label insert, or on any
additional packaging.
As used herein, "topically applying" means directly laying on or spreading on
outer skin, the scalp, or hair, e.g., by use of the hands or an applicator
such as a wipe,
roller, or spray.
As used herein, "cosmetically acceptable" means that the ingredients the term
describes are suitable for use in contact with tissues (e.g., the skin or
hair) without undue
toxicity, incompatibility, instability, irritation, allergic response, or the
like.
As used herein, "cosmetic" 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.
As used herein, "skin in need of treatment for the signs of aging" means a
skin that
is, but not limited to, sagging, loose, lax, rough, wrinkly, thinned, or
uneven. Improving
the signs of aging means improving the firmness of the skin, improving the
texture of the
skin, improving the appearance of wrinkles in skin, improving the skin tone,
or the
treating external aggressions in skin.
As used herein, "improving the firmness of skin" means the enhancing of the
firmness or elasticity of the skin, preventing the loss of firmness or
elasticity of skin, or
preventing or treating sagging, lax and loose skin. The firmness or elasticity
of the skin
can be measured by use of a cutometer. See Handbook of Non-Invasive Methods
and the
Skin, eds. J. Serup, G. Jemec & G. Grove, Chapter 66.1 (2006). The loss of
skin elasticity
or firmness may be a result of a number of factors, including but not limited
to aging,
environmental damage, or the result of an application of a cosmetic to the
skin.
As used herein, "improving the texture of skin" means the smoothing of the
surface of the skin to remove either bumps or crevasses on the skin surface.
As used herein, "improving the appearance of wrinkles in skin" means
preventing,
retarding, arresting, or reversing the process of wrinkle formation in skin.
As used
herein, "wrinkle" includes fine lines, fine wrinkles, or coarse wrinkles.
Examples of
wrinkles include, but are not limited to, fine lines around the eyes (e.g.,
"crow's feet"),
forehead and cheek wrinkles, frown-lines, and laugh-lines around the mouth.
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As used herein, "uneven skin" means a condition of the skin associated with
diffuse or mottled pigmentation, which may be classified as hypemigmentation,
such as
post-inflammatory hypemigmentation.
As used herein, "blotchiness" means a condition of the skin associated with
redness or erythema.
As used herein, "treatment of external aggressions in skin" means the
reduction or
prevention of the damage from external aggressions in skin. Examples of
external
aggressions include, but are not limited to, damage to the skin from the use
of cleansers
(e.g., topical cleansers containing surfactants), make-up, shaving as well as
environmental
damage such as from UV light (e.g., sundamage from sunlight or damage from non-
natural sources such as UV lamps and solar simulators), ozone, exhaust,
pollution,
chlorine and chlorine containing compounds, and cigarette smoke. Effects of
external
aggressions on the skin include, but are not limited to, oxidative and/or
nitrosative damage
to and modifications on lipids, carbohydrates, peptides, proteins, nucleic
acids, and
vitamins. Effects of external aggressions on the skin also include, but are
not limited to,
loss of cell viability, loss or alteration of cell functions, and changes in
gene and/or protein
expression.
As used herein, "improving the skin tone" means the lightening of the
appearance
of the skin (e.g., lightening pigmented marks or lesions, reducing skin
sallowness, and/or
evening the color of the skin).
As used herein, "skin in need of reducing skin inflammation" means a skin
exhibiting redness or erythema, edema, or being reactive or sensitive to
external elements.
External elements include, but are not limited to, sun rays (UV, visible, IR),
microorganisms, atmospheric pollutants such as ozone, exhaust pollutants,
chlorine and
chlorine generating compounds, cigarette smoke, cold temperature, heat.
Inflammatory
disorders and related conditions which may be treated or prevented by use of
the
compositions of this invention include, but are not limited to the following:
arthritis,
bronchitis, contact dermatitis, atophic dermatitis, psoriasis, seborrheic
dermatitis, eczema,
allergic dermatitis, polymorphous light eruptions, inflammatory dermatoses,
folliculitis,
alopecia, poison ivy, insect bites, acne inflammation, irritation induced by
extrinsic factors
including, but not limited to, chemicals, trauma, pollutants (such as
cigarette smoke) and
sun exposure, secondary conditions resulting from inflammation including but
not limited
to xerosis, hyperkeratosis, pruritus, postinflammatory hyperpigmentation,
scarring and the
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like. Preferably, the inflammatory disorders and related conditions which may
be treated
or prevented using the methods of the invention are arthritis, inflammatory
dermatoses,
contact dermatitis, allergic dermatitis, atopic dermatitis, polymorphous light
eruptions,
irritation, including erythema induced by extrinsic factors, acne
inflammation, psoriasis,
seborrheic dermatitis, eczema, poison ivy, insect bites, folliculitus,
alopecia, and
secondary conditions and the like.
As used herein, the term "lightening the skin" refers generally to lightening,
brightening, whitening, and/or evening of the skin tone, skin color, and/or
shade of skin,
and/or to the reduction in sallowness, and/or to the lightening and/or fading
of
hyperpigmented marks and/or lesions including, but not limited to, pigmented
spots,
melanin spots, age spots, sun spots, senile lentigos, freckles, lentigos
simplex, pigmented
solar keratosis, seborrhoeic keratosis, melasma, acne marks, post-inflammatory
hyperpigmentation, lentigines, ephelides, combinations of two or more thereof
and the
like. In certain embodiments, "lightening the skin" also refers to increased
skin radiance,
glow, translucency and/or luminescence and/or obtaining a more radiant,
glowing,
translucent or luminous skin tone appearance or a less yellow or sallow skin
tone. In
certain preferred embodiments, "lightening the skin" refers to lightening and
evening the
skin tone, increasing skin radiance and/or lightening age spots.
As used herein, the term "skin in need of skin lightening treatment" refers
generally to skin that exhibits one or more property selected from the group
consisting of:
skin having a measured Individual Typology Angle (ITA) value below 41 as
determined
per the COLIPA GUIDELINE: GUIDELINE FOR THE COLORIMETRIC
DETERMINATION OF SKIN COLOUR TYPING AND PREDICTION OF THE
MINIMAL ERYTHEMAL DOSE (MED) WITHOUT UV EXPOSURE published in
2007, which is incorporated herein by reference and further described below,
darkened
and/or sallow skin, including skin darkened by UV, skin with uneven skin tone,
or skin
with one or more hyperpigmented marks and/or lesions including, but not
limited to,
pigmented spots, melanin spots, age spots, sun spots, senile lentigos,
freckles, lentigos
simplex, pigmented solar keratosis, seborrhoeic keratosis, melasma, acne
marks, post-
inflammatory hyperpigmentation, lentigines, ephelides, combinations of two or
more
thereof and the like. In the COLIPA guidelines, skin color is defined function
of the ITA
value as: very light skin >55; Light skin 41-55, Intermediate 28-41, and Tan
skin <28. In
certain preferred embodiments, "skin in need of skin lightening" refers to
individuals with
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a skin having an ITA value of less than 41, such as about 40 or less, about 35
or less,
about 30 or less, or more preferably about 28 or less. In certain other
preferred
embodiments, the present invention is directed to compositions and methods for
use on
skin in need of skin lightening treatment selected from sallow and/or darkened
skin. In
certain other preferred embodiments, the present invention is directed to
compositions and
methods for use on skin in need of skin lightening treatment selected from the
group
consisting of age spots, freckles, marks left after acne, and combinations of
two or more
thereof.
As used herein, unless otherwise specified, all percentages of ingredients in
compositions are weight percent of active/solids ingredient based on the total
weight of
composition.
As used herein, "substantially free" of an ingredient means containing less
than
about 1% by weight, such as less than about 0.5% by weight, such as less than
about
0.25% by weight, such as less than about 0.1% by weight of such ingredient. In
one
embodiment, "substantially free" means completely free of such ingredient.
Compositions of the present invention are suitable for treating human skin,
e.g.,
skin on the face or body, for signs of skin aging, or for inflammation. In a
particularly
preferred embodiment, a composition according to the invention is used to
treat the
presence of lines and wrinkles and/or loss of elasticity. Compositions of the
present
invention are suitable for treating human skin, e.g., skin on the face or
body, to lighten the
skin.
1-Hydroxyl 3,5-bis(4'hydroxyl styryl)benzene
Compositions of the present invention comprise 1-hydroxyl 3,5-bis(4'hydroxyl
styryl)benzene or a cosmetically acceptable salt thereof
1-Hydroxyl 3,5-bis(4'hydroxyl styryl)benzene is a curcumin analog having the
structure below:
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OH
0 0 /
*
HO OH
As described in US Patent No. 7,745,670, 1-hydroxyl 3,5-bis(4'hydroxyl
styryl)benzene can be made by reacting 1-(bromomethyl)-4-methoxybenzene with
triethyl phosphate using an Arbuzov reaction to produce diethyl [(4-
methoxyphenyl)methyl]phosphonate. This is coupled with 5-methoxybenzene-1,3-
dicarbaldehyde-using sodium hydride as base in THF, followed by reaction with
boron
trichloride and dichloromethane to replace methoxy groups with hydroxyls.
Salts of 1-hydroxyl 3,5-bis(4'hydroxyl styryl)benzene can be made by, for
example, reacting the 1-hydroxyl 3,5-bis(4'hydroxyl styryl)benzene with a base
such as
piperazine, or another base, to produce at least some phenoxide salt of 1-
hydroxyl 3,5-
bis(4'hydroxyl styryl)benzene.
Topical Compositions
Generally, the 1-hydroxyl 3,5-bis(4'hydroxyl styryl)benzene or salt thereof is
present in the composition in a cosmetically effective amount, such as from
about 0.01%
to about 10%, preferably from about 0.1% to about 5%, more preferably from
about 0.2%
to about 2%, even more preferably from about 0.5% to about 1.5%, by weight of
the
composition.
The compositions of the present invention are applied topically to human skin
and/or hair.
The compositions may be spreadable. They may be topically applied by
spreading, for example spreading over the skin or hair, in particular over
skin of the face
or hands.
In one embodiment, a composition of the invention is topically applied without
a
voltage.
In addition to 1-hydroxyl 3,5-bis(4'hydroxyl styryl)benzene, the composition
may
further include a cosmetically acceptable topical carrier that may be from
about 50% to
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about 99.99%, by weight, of the composition (e.g., from about 80% to about
99%, by
weight, of the composition). In a preferred embodiment of the invention, the
cosmetically
acceptable topical carrier includes water.
The cosmetically acceptable topical carrier may be unsuitable for ingestion.
The cosmetically acceptable topical carrier may include an ingredient selected
from one or more of the following five classes: wetting agents, emulsifiers,
emollients,
humectants, and fragrances. In certain embodiments, the cosmetically
acceptable topical
carrier includes ingredients from two or more of the above-mentioned classes,
such as
ingredients from at least three or more of such classes.
In one embodiment, the cosmetically acceptable topical carrier includes water,
an
emulsifier, and an emollient.
The compositions may be made into a wide variety of product types that include
but are not limited to lotions, creams, gels, sticks, sprays, ointments,
cleansing liquid
washes and solid bars, shampoos and hair conditioners, hair fixers, pastes,
foams,
powders, mousses, shaving creams, wipes, patches, hydrogels, film-forming
products,
facial masks and skin masks, films and make-up such as foundations, and
mascaras.
These product types may contain several types of cosmetically acceptable
topical carriers
including, but not limited to solutions, suspensions, emulsions such as
microemulsions
and nanoemulsions, gels, solids and liposomes.
The compositions useful in the present invention can be formulated as
solutions.
Solutions typically include an aqueous or organic solvent (e.g., from about
50% to about
99.99% or from about 90% to about 99% of a cosmetically acceptable aqueous or
organic
solvent). Examples of suitable organic solvents include humectants (e.g.,
water-retaining
or hygroscopic materials) such as propylene glycol, pentylene glycol,
polyethylene
glycol, polypropylene glycol, glycerol, 1,2,4-butanetriol, sorbitol esters,
1,2,6-hexanetriol;
as well as ethanol, and mixtures thereof Solutions can optionally include a
wetting agent,
such as to provide foam, e.g, an anionic, non-ionic, or cationic wetting
agent.
Compositions useful in the subject invention may be formulated as a solution
comprising an emollient. Such compositions preferably contain from about 2% to
about
50% of an emollient(s). As used herein, "emollients" refer to materials used
for the
prevention or relief of dryness, such as by preventing the transepidermal loss
of water
from the skin. Examples of emollients include hydrophobic compounds such as
vegetable
oils, mineral oils (e.g., petrolatum), fatty esters (e.g., isopropyl
palmitate, c12-c15 alkyl
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benzoate) including those fatty esters of glycerol, silicone oils (e.g.,
dimethicone) and the
like.
A lotion can be made from such a solution. Lotions typically contain from
about
1% to about 20% (e.g., from about 5% to about 10%) of an emollient(s) and from
about
50% to about 90% (e.g., from about 60% to about 80%) of water.
Another type of product that may be formulated from a solution is a cream. A
cream typically contains from about 5% to about 50% (e.g., from about 10% to
about
20%) of an emollient(s) and from about 45% to about 85% (e.g., from about 50%
to about
75%) of water.
Although it is preferred that the composition of the present invention
includes
water, the composition may alternatively be anhydrous or an ointment that
includes no
water but organic and/or silicone solvents, oils, lipids and waxes. An
ointment may
contain a simple base of animal or vegetable oils or semi-solid hydrocarbons.
An
ointment may contain from about 2% to about 10% of an emollient(s) plus from
about
0.1% to about 2% of a thickening (gelling) agent(s).
The composition may be formulated as an emulsion. If the topical carrier is an
emulsion, from about 1% to about 10% (e.g., from about 2% to about 5%) of the
topical
carrier contains an emulsifier(s). Emulsifiers may be nonionic, anionic or
cationic.
Examples of suitable emulsifiers include those typically identified as such in
the art of
personal care and cosmetic formulations, e.g., cationic emulsifiers such as
disteryldimonium chloride, non-ionic emulsifiers such as stereth-2, stereth-
21; anionic
emulsifiers such as potassium cetyl phosphate; polymeric emulsifiers such as
acryloyldimethyltaurateNP copolymers, and the like.
Lotions and creams can be formulated as emulsions. Typically such lotions
contain from 0.5% to about 5% of an emulsifier(s). Such creams typically
contain from
about 1% to about 20% (e.g., from about 5% to about 10%) of an emollient(s);
from about
20% to about 80% (e.g., from 30% to about 70%) of water; and from about 1% to
about
10% (e.g., from about 2% to about 5%) of an emulsifier(s).
Single emulsion skin care preparations, such as lotions and creams, of the oil-
in-
water type and water-in-oil type are well-known in the cosmetic art and are
useful in the
subject invention. Multiphase emulsion compositions, such as the water-in-oil-
in-water
type or the oil-in-water-in-oil type, are also useful in the subject
invention. In general,
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such single or multiphase emulsions contain water, emollients, and emulsifiers
as essential
ingredients.
The compositions of this invention can also be formulated as a gel (e.g., an
aqueous, alcohol, alcohol/water, or oil gel using a suitable gelling
agent(s)). Suitable
gelling agents for aqueous and/or alcoholic gels include, but are not limited
to, natural
gums, (cross-linked) acrylic acid and acrylate polymers and copolymers, and
cellulose
derivatives (e.g., hydroxymethyl cellulose and hydroxypropyl cellulose).
Suitable gelling
agents for oils (such as mineral oil) include, but are not limited to,
hydrogenated
butylene/ethylene/styrene copolymer and hydrogenated
ethylene/propylene/styrene
copolymer. Such gels typically contains between about 0.1% and 5%, by weight,
of such
gelling agents.
The compositions of the present invention can also be formulated into a solid
formulation (e.g., a wax-based stick, soap bar composition, powder, or a wipe
containing
powder).
The compositions useful in the subject invention may contain, in addition to
the
aforementioned components, a wide variety of additional oil-soluble materials
and/or
water-soluble materials conventionally used in compositions for use on skin
and hair, at
their art-established levels.
Additional Cosmetically Active Agents
In one embodiment, the composition includes an additional NFKB-inhibitor such
as a substituted resorcinol, (E)-3-(4-methylphenylsulfony1)-2-propenenitrile
(such as "Bay
11-7082," commercially available from Sigma-Aldrich of St. Louis, Missouri), a
tetrahydrocurcuminoid (such as Tetrahydrocurcuminoid CG, available from
Sabinsa
Corporation of Piscataway, NJ), paulownin, extracts of Paulownia wood (for
example the
wood of Paulownia tomentosa, P aulownia fortunei, Paulownia elongate,
Paulownia
taiwaniana, andl orP aulownia kawakamii,), and combinations thereof
In one embodiment, the composition further contains another cosmetically
active
agent. As used herein, a "cosmetically active agent" is a compound (e.g., a
synthetic
compound or a compound isolated from a natural source or a natural extract)
that has a
cosmetic or therapeutic effect on the skin including, but not limiting to anti-
aging actives,
anti-inflammatory agents, tropoelastin promoters, anti-acne agents, anti-
microbial agents,
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anti-inflammatory agents, anti-mycotic agents, external analgesics,
sunscreens,
antioxidants, keratolytic agents, vitamins, skin lightening agents and skin
firming agents.
In one embodiment, the composition includes a skin-lightening agent such as a
tyrosinase inhibitor, melanin-degradation agent, melanosome transfer
inhibiting agent
including PAR-2 antagonists, retinoids, antioxidants, tranexamic acid,
tranexamic acid
cetyl ester hydrochloride, skin bleaching agent, linoleic acid, adenosine
monophosphate
disodium salt, Chamomilla extract, allantoin, opacifier, talc or silica, zinc
salt, or the like,
or other agent as described in Solano et al. Pigment Cell Res. 19 (550-571)
and Ando et al.
Int J Mol Sci 11(2566-2575).
Examples of suitable tyrosinase inhibitors include but, are not limited to,
vitamin
C and its derivatives, vitamin E and its derivatives, kojic acid, arbutin,
resorcinols,
hydroquinone, flavones e.g., licorice flavanoids, licorice root extract,
mulberry root
extract, dioscorea coposita root extract, saxifraga extract and the like,
ellagic acid,
salicylates and derivatives, glucosamine and derivatives, fullerene,
hinokitiol, dioic acid,
acetyl glucosamine, 5,5'-dipropyl-bipheny1-2,2'-diol (magnolignan), 4-(4-
hydroxypheny1)-2-butanol (4-HPB), combinations of two or more thereof, and the
like.
Examples of vitamin C derivatives include, but are not limited to, ascorbic
acid
and salts, ascorbic acid-2-glucoside, sodium ascorbyl phosphate, magnesium
ascorbyl
phosphate, and natural extract enriched in vitamin C.
Examples of vitamin E derivatives include, but are not limited to, alpha-
tocopherol, beta, tocopherol, gamma-tocopherol, delta-tocopherol, alpha-
tocotrienol, beta-
tocotrienol, gamma-tocotrienol, delta-tocotrienol and mixtures thereof,
tocopherol acetate,
tocopherol phosphate and natural extracts enriched in vitamin E derivatives.
Examples of resorcinol derivatives include, but are not limited to,
resorcinol, 4-
substituted resorcinols like 4-alkylresorcinols such as 4-butyresorcinol
(rucinol), 4-
hexylresorcinol (SYNOVEA HR, SYNTHEON), phenylethyl resorcinol (SYMWHITE,
SYMRISE), 1-(2,4-dihydroxypheny1)-3-(2,4-dimethoxy-3-methylpheny1)-propane
(nivitol, UNIGEN) and the like and natural extracts enriched in resorcinols.
Examples of salicylates include, but are not limited to, 4-methoxy potassium
salicylate, salicylic acid, acetylsalicylic acid, 4-methoxysalicylic acid and
their salts.
In certain preferred embodiments, the tyrosinase inhibitors include a 4-
substituted
resorcinol, a vitamin C derivative, or a vitamin E derivative. In more
preferred
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embodiments, the tyrosinase inhibitor comprises phenylethyl resorcinol, 4-
hexyl
resorcinol, or ascorbyl-2-glucoside.
Examples of suitable melanin-degradation agents include, but are not limited
to,
peroxides and enzymes such as peroxidases and ligninases. In certain preferred
embodiments, the melanin-inhibiting agents include a peroxide or a ligninase.
Examples of suitable melanosome transfer inhibiting agents include PAR-2
antagonists such as soy trypsin inhibitor or Bowman-Birk Inhibitor, vitamin B3
and
derivatives such as niacinamide, essential soy, whole soy, soy extract. In
certain preferred
embodiments, the melanosome transfer inhibiting agents includes a soy extract
or
niacinamide.
Examples of retinoids include, but are not limited to, retinol (vitamin A
alcohol),
retinal (vitamin A aldehyde), retinyl acetate, retinyl propionate, retinyl
linoleate, retinoic
acid, retinyl palmitate, isotretinoin, tazarotene, bexarotene, adapalene,
combinations of
two or more thereof and the like. In certain preferred embodiments, the
retinoid is
selected from the group consisting of retinol, retinal, retinyl acetate,
retinyl propionate,
retinyl linoleate, and combinations of two or more thereof In certain more
preferred
embodiments, the retinoid is retinol.
Other skin lightening agents include vitamin B5, vitamin B12, glycolic acid
and
extracts of Paulownia wood (for example the wood of Paulownia tomentosa,
Paulownia
fortunei, Paulownia elongate, Paulownia taiwaniana, andl orPaulownia
kawakamii).
Examples of antioxidants include, but are not limited to, water-soluble
antioxidants such as sulfhydryl compounds and their derivatives (e.g., sodium
metabisulfite and N-acetyl-cysteine, glutathione), lipoic acid and
dihydrolipoic acid,
stilbenoids such as resveratrol and derivatives, lactoferfin, iron and copper
chelators and
ascorbic acid and ascorbic acid derivatives (e.g., ascoby1-2-glucoside,
ascorbyl palmitate
and ascorbyl polypeptide). Oil-soluble antioxidants suitable for use in the
compositions of
this invention include, but are not limited to, butylated hydroxytoluene,
retinoids (e.g.,
retinol and retinyl palmitate), tocopherols (e.g., tocopherol acetate),
tocotrienols, and
ubiquinones. Natural extracts containing antioxidants suitable for use in the
compositions
of this invention, include, but not limited to, extracts containing flavonoids
and
isoflavonoids and their derivatives (e.g., genistein and diadzein), extracts
containing
resveratrol and the like. Examples of such natural extracts include grape
seed, green tea,
black tea, white tea, pine bark, feverfew, parthenolide-free feverfew, oat
extracts,
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blackberry extract, cotinus extract, soy extract, pomelo extract, wheat germ
extract,
hesperedin, grape extract, portulaca extract, licochalcone, chalcone, 2,2'-
dihydroxy
chalcone, primula extract, propolis, and the like.
Other Materials
Various other materials may also be present in the composition, as known in
the
art. These include humectants, pH adjusters, chelating agents (e.g., EDTA),
fragrances,
dyes and preservatives (e.g., BHT, benzyl alcohol).
In one embodiment, the composition is substantially free, preferably
completely
free, of preservatives. This is because, advantageously, 1-hydroxyl 3,5-
bis(4'hydroxyl
styryl)benzene and salts thereof are active for inhibiting and/or killing
microorganisms.
Accordingly, compositions according to the invention can be formulated with
little or no
conventional preservatives. Conventional preservatives for example are
selected from the
group consisting of parabens, benzoic acid and salts thereof, phenoxyethanol,
isothaizolones, imidazolidinyl urea, iodo propynyl butyl carbamate, DMDM
hydantoin,
caprylyl glycol, dehydroacetic acid, sorbic acids and salts thereof,
chlorophensin, glyceryl
caprylate, phenylpropanol, sodium levulinate, anisic acid, ethylhexylglycerin,
benzyl
alcohol and combinations thereof
The composition and formulations and products containing such compositions of
the present invention may be prepared using methodology that is well known by
an artisan
of ordinary skill.
Methods of Use
Compositions of the present invention may be topically applied to human skin,
e.g., skin that is in need of treatment for one or more signs of skin aging as
described
above. In one embodiment, the compositions are applied to skin in need of
treatment for
lines and wrinkles and/or loss of elasticity. The compositions may be applied
to the skin
in need of such treatment according to a suitable treatment regimen, e.g.,
every month,
every week, every other day, every day, twice a day, or the like.
1-hydroxyl 3,5-bis(4'hydroxyl styryl)benzene, cosmetically acceptable salts
thereof and compositions containing the same may be used to provide
antimicrobial
effects (e.g., antibacterial, antifungal, antiviral, or anti-parasitic
effects).
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In one embodiment, the invention provides a method of inhibiting the growth of
a
microorganism, comprising applying to said microorganism 1-hydroxyl 3,5-
bis(4'hydroxyl styryl)benzene, a cosmetically acceptable salt thereof, or a
composition
containing the same.
Microorganisms that may be inhibited or killed by such application include
gram
positive bacteria, gram negative bacteria and fungi including Staphylococcus
aureus,
Staphylococcus epidermic/is, Propionibacterium acnes, Escherichia colt,
Pseudomonas
aeruginosa, Candida albicans, and Aspergillus niger.
It is believed that one skilled in the art can, based upon the description
herein,
utilize the present invention to its fullest extent. The following specific
embodiments are
to be construed as merely illustrative, and not limitative of the remainder of
the disclosure
in any way whatsoever. The following non-limiting examples further illustrate
the
invention.
Example 1: NFKB-Inhibition
NFKB-INHIBITION TESTS were performed on various concentrations of 1-
hydroxyl 3,5-bis(4'hydroxyl styryl)benzene and a vehicle control (DMSO). The 1-
hydroxyl 3,5-bis(4'hydroxyl styryl)benzene was prepared as described in US
Patent No.
7,745,670.
The NF-M3 INHIBITION TEST was conducted as follows. Rat cardiac myoblasts
H9c2 cells were purchased from ATCC (Manassas, VA.). Cultures were maintained
in
Dulbecco's modified Eagle's medium (DMEM, Invitrogen Life Technologies,
Carlsbad,
CA.) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 50
ug/ml
streptomycin (Invitrogen life technologies, Carlsbad, CA.). 1x104 cells grown
in 96-well
plates were transiently transfected with 0.45ug total DNA per well using
Lipofectamine
2000 (Invitrogen life technologies, Carlsbad, Calif). In all transfections, a
construct with
the thymidine kinase promoter and the Renilla luciferase reporter gene (pRL-
TK,
Promega, Madison Wis.) was included as an internal control in addition to the
NF-kB
luciferase promoter. One day after transfection, cells were treated with the
indicated
samples (in DMSO as vehicle) at indicated concentrations and stimulated with
100 ng/mL
of Tumor Necrosis Factor-a (TNFa , Sigma-Aldrich, St Louis, MO) for
approximately 24
hours before they were lysed for luciferase assays, using Dual-Luciferase
Reporter System
from Promega (Madison, Wis.), following manufacturer's protocol. Briefly, the
firefly
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luciferase activity was measured first (representing NF-kB promoter activity),
followed by
the renilla luciferase (internal control), using luminometer LMAX, from
Molecular
Devices (Sunnyvale, Calif.). The ratio of these two luciferase activities
(RLU) was used to
evaluate the activity of each promoter:
NF-KB Inhibition = [1¨( RLUsample /RLUcontrol)] * 100
where RLUsample and RLUcontrol are the normalized luciferase activity ratios
of the
sample and control, respectively.
The results are shown in Table 1, in which NF-kB Gene Reporter Activation
(Luminescence, L) is reported. Percent NF-kB Inhibition is also reported.
TABLE 1
NF-1(13 Gene Reporter Percent NF-1(13
Activation
Inhibition
(Luminescence, L)
Untreated 131.5
TNFa (10Ong/m1) Stimulated, "Lcontrol" 452.4
TNFa + Vehicle (0.1% DMSO) 588.3 0%
TNFa + 1-hydroxyl 3,5-bis(4'hydroxyl 585.5 0.5%
styryl)benzene (0.2 ug/ml)
TNFa + 1-hydroxyl 3,5-bis(4'hydroxyl 458.6 22.1%
styryl)benzene (0.5 ug/ml)
TNFa + 1-hydroxyl 3,5-bis(4'hydroxyl 283.8 51.7%
styryl)benzene (1 ug/ml)
TNFa + 1-hydroxyl 3,5-bis(4'hydroxyl 170.5 71.0%
styryl)benzene (2 ug/ml)
TNFa + 1-hydroxyl 3,5-bis(4'hydroxyl 22.6 96.1%
styryl)benzene (4 ug/ml)
1-Hydroxyl 3,5-bis(4'hydroxyl styryl)benzene showed a strong reduction in NF-
kB mediated inflammatory response in human skin cells.
Example 2: Anti-Inflammatory Activity
The topical anti-inflammatory activities of 1-hydroxyl 3,5-bis(4'hydroxyl
styryl)benzene and its piperazine salt were evaluated as follows.
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Epidermal equivalents (EPI 200 HCF), multilayer and differentiated epidermis
consisting of normal human epidermal keratinocytes, were purchased from MatTek
(Ashland, MA). Upon receipt, epidermal equivalents were incubated for 24 hours
at 37 C
in maintenance medium without hydrocortisone. Equivalents were topically
treated
(2mg/cm2) with 1-hydroxyl 3,5-bis(4'hydroxyl styryl)benzene thereof in a 70:30
Ethanol-
Propylene glycol vehicle two hours before exposure to solar ultraviolet light
(1000W-
Oriel solar simulator equipped with a 1-mm Schott WG 320 filter; UV dose
applied: 70
kJ/m2 as measured at 360nm). The equivalents were incubated for 24 hours at 37
C with
maintenance medium then supernatants were analyzed for IL-8 cytokine release
using
commercially available kits (Millipore Corp., Billerica, MA).
The results are shown in Table 2.
TABLE 2
Sample Change over vehicle % inhibition over UV
alone (Normalized to treatment
100)
Vehicle (70/30: Ethanol + 100 28
Propylene Glycol)
Vehicle + UV 134 42
UV + 1-hydroxyl 3,5- 82 21 39%
bis(4'hydroxyl
styryl)benzene - Free Phenol
(0.5%)
UV + 1-hydroxyl 3,5- 80 26 40%
bis(4'hydroxyl
styryl)benzene - Free Phenol
(1%)
UV + 1-hydroxyl 3,5- 85 14 37%
bis(4'hydroxyl
styryl)benzene - Piperazine
salt (0.5%)
UV + 1-hydroxyl 3,5- 81 31 40%
bis(4'hydroxyl
styryl)benzene - Piperazine
salt (1%)
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1-Hydroxyl 3,5-bis(4'hydroxyl styryl)benzene and its piperazine salt showed
strong reduction in UV-induced inflammation in human skin cells.
Example 3: Induction of Elastin and CollagenlA gene expression
Primary human dermal fibroblasts (Lifeline Cell Technologies, Frederick, MD)
were grown until confluence in DMEM media (Invitrogen/Life Technologies,
Carlsbad,
CA) with 10% FBS and 1% Penicillin-Streptomycin on 24 well tissue culture
plates,
followed by treatment with 1-hydroxyl 3,5-bis(4'hydroxyl styryl)benzene
(0.1ug/mL)
with and without TNF-a for 48 hr. Post-treatment, cells were lysed using RLT
buffer and
total RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA).
Quantitative
PCR was performed using the Gene Amp PCR System 9700 and 7500 Real Time PCR
System cycler (Applied Biosystems)/Life Technologies, Carlsbad, CA). Elastin
(Qiagen)
and Collagenlal primers (custom sequence -sense: 5'-TCC-CCA-GCT-GTC-TTA-TGG-
CT-3' and anti-sense: 5'-CAG-GCA-CGG-AAA-TTC-CTC-C-3') were used. The
housekeeping gene GAPDH was used for normalization (custom primer sequence: F
5'-
ATC-TCT-GCC-CCC-TCT-GCT-G-3' and R 5'-ATG-GTT-CAC-ACC-CAT-GAC-GA-
3'; Invitrogen/Life Technologies, Carlsbad, CA). Fold-changes in PCR
(normalized to
GAPDH) were calculated from untreated.
The results are shown in Tables 3A and 3B.
TABLE 3A
Collagen lA Elastin
Normalized Fold-change in
Normalized Fold-change in
PCR PCR
(Mean) (Mean)
Untreated 1.0 1.0
1-hydroxyl 3,5- 2.96 6.23
bis(4'hydroxyl
styryl)benzene (0.1ug/mL)
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TABLE 3B
Collagen lA Elastin
Normalized Fold-change in
Normalized Fold-change in
PCR PCR
(Mean) (Mean)
Untreated 1.0 1.0
TNF-a (lOng/mL) 0.52 0.41
TNF-a (lOng/mL) + 1- 1.36 1.12
hydroxyl 3,5-
bis(4'hydroxyl
styryl)benzene (0.1ug/mL)
1-Hydroxyl 3,5-bis(4'hydroxyl styryl)benzene increased the expression of genes
associated with collagen and elastin production.
Example 4: Inhibition of TNF-a - Induced MMP-9 levels
The ability of 1-hydroxyl 3,5-bis(4'hydroxyl styryl)benzene to inhibit TNF-a
induced MMP-9 levels was tested at different concentrations as follows. The
formation of
TNF-a induced MMP-9 is involved in the undesirable breakdown of extracellular
matrix
in human skin.
Epidermal equivalents (EPI 200 HCF) were purchased from MatTek (Ashland,
MA). Upon receipt, the epidermal equivalents were incubated for 24 hours at 37
C in
maintenance medium without hydrocortisone. The equivalents were topically
treated
(2mg/cm2 ) with 1-hydroxyl 3,5-bis(4'hydroxyl styryl)benzene extracts in a 70%
ethanol/30% propylene glycol vehicle 2 hours before treatment with TNF-a
(10Ong/mL).
the equivalents were incubated for 48 hours at 37 C with maintenance medium
then
supernatants were analyzed for MMP -9 using commercially available kits (R&D
Systems, Minneapolis, MN).
The results are shown in Table 4.
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TABLE 4
Treatment (Dose, as % w/v) Mean of MMP-9 Percent Inhibition of
Release (ng/ml) MMP-9 Production
(over TNF-a alone)
Untreated 1422 611
TNF-a alone 3557 1181
TNF-a + 1-hydroxyl 3,5- 1392 627 60.9
bis(4'hydroxyl
styryl)benzene (0.25%)
TNF-a + 1-hydroxyl 3,5- 1012 249 71.6
bis(4'hydroxyl
styryl)benzene (0.5%)
1-Hydroxyl 3,5-bis(4'hydroxyl styryl)benzene inhibited the formation of TNF-a
induced MMP-9.
Example 5: Melanogensis Inhibition Test
Ultraviolet (UV) radiation from the sun is the most important external
stimulus for
melanin formation leading to skin darkening. Reducing melanin formation in the
skin
may be achieved by inhibiting multiple steps of the melanin biogenesis
process.
Tyrosinase is the key regulatory enzyme of melanogenesis. Agents that inhibit
tyrosinase
will reduce melanin synthesis.
1-Hydroxyl 3,5-bis(4'hydroxyl styryl)benzene (0.0001, 0.001, and 0.01% w/v)
was tested for tyrosinase inhibition as follows.
Mushroom tyrosinase samples (Sigma, T7755, 10 U/reaction) were treated with
the samples of 1-hydroxyl 3,5-bis(4'hydroxyl styryl)benzene and control
samples.
Reaction was initiated by addition of 10 mM L-dopa (Research Organics, #2111D)
at 37
C. Tyrosinase activity was measured by recording light absorption (optical
density) at
492 nm after 30 min for both control samples (no exposure to test compound)
and test
samples that were exposed to the test compound.
Percent Inhibition of Tyrosinase was related to the reduction in absorption at
492
nm (relative to the control) by the following formula:
Percent Inhibition of Tyrosinase = [ Teoniroi - Tsample Teontrol)] *100
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where Tcontroi is the tyrosinase activity of the control and Tsampie is the
tyrosinase activity in
the presence of test compound. Samples were tested in duplicate and Percent
Inhibition of
Tyrosinase was averaged and standard deviation was reported. The control had
an
average optical density of 0.622.
The results are provided in Table 5.
TABLE 5
Percent Inhibition of Tyrosinase
activity (mean standard
deviation)
1-hydroxyl 3,5-bis(4'hydroxyl 3.3 1.95
styryl)benzene - Free Phenol (0.0001%)
1-hydroxyl 3,5-bis(4'hydroxyl 8.21 1.27
styryl)benzene - Free Phenol (0.001%)
1-hydroxyl 3,5-bis(4'hydroxyl 11.84 3.22
styryl)benzene - Free Phenol (0.01%)
1-hydroxyl 3,5-bis(4'hydroxyl 2.03 3.94
styryl)benzene - Piperazine salt (0.0001%)
1-hydroxyl 3,5-bis(4'hydroxyl 11.52 3.16
styryl)benzene - Piperazine salt (0.001%)
1-hydroxyl 3,5-bis(4'hydroxyl 16.64 2.76
styryl)benzene - Piperazine salt (0.01%)
1-hydroxyl 3,5-bis(4'hydroxyl styryl)benzene showed a substantial and dose
dependent inhibition of tyrosinase enzyme activity.
Example 6: Inhibition of UV-induced Melanogenesis
One or more samples of B16(F10) cells were prepared and each pre-treated with
a
test sample followed by UVB exposure as described below. Upon treatment, UVB
stimulated melanogenesis in the cells and test compounds were evaluated based
on their
ability to inhibit or slow down the rate of melanogenesis. The cells were
lysed for protein
measurement at 595nm and melanin content at 470nm. The potency of the test
compounds
were determined by comparing the % inhibition achieved by the test compounds
against
the treated control.
On a first day, murine melanoma B16(F10) cells were seeded in 60mm plates with
a density of ¨1 million cells per plate and incubated for 48hrs at 37 C, 5%
CO2. On day
2, the cells with a confluency rate of 90-100% were treated with test compound
at a
predetermined concentration (e.g. 25 [tg/mL) for two hours (for test compound
samples
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only) followed by exposure to UVB 200mJ/cm2 (for test samples and treated
control). The
cells were harvested on day 3 (24 h post UVB irradiation for test samples and
treated
control) and lysed in protein lysis buffer (50mM Tris, pH 8, 2mM EDTA, 150mM
NaC1,
and 1%TRITON X 100 - a nonionic surfactant purchased from BioRad Cat.#: 161-
0407),
and centrifuged.
The resulting supernatant was mixed well with a protein dye assay (Bio-rad
protein assay reagent) and a spectrophotometer (Molecular Devices VERSAmax)
was
used to determine the optical density (OD) of the sample at 595nm, which was
recorded as
the "protein assay OD." The cell pellet remaining after removal of the
supernatant was
dissolved in alkaline DMSO buffer, and the resulting solution was similarly
tested using a
spectrophotometer for melanin absorbance assay at 470 nm. The absorbance was
recorded as the "melanin assay OD."
Control samples of B16(F10) murine melanoma cells were prepared and harvested
as indicated above, but without addition of any test sample and without
exposure to UVB
(untreated control). Other samples were prepared and harvested as indicated
above, but
without addition of test sample and exposed to UVB as described below (treated
control).
Three samples each of the untreated control, treated control, and each test
sample
were made and the Melanin OD and Protein OD measured for each. The normalized
melanin for each untreated control (3 samples), treated control (3 samples)
and test sample
(3 samples for each test compound) was calculated via the following equation:
Normalized Melanin = melanin assay OD/protein assay OD.
The averages of the Normalized Melanin of the untreated controls and treated
controls
were calculated (sum of the three calculated values/3).
The Induction value of the Control (which can be thought of as the UV-induced
increase in melanin, without the benefit of any test sample to reduce
melanogensis) was
determined by subtracting the average Normalized Melanin of untreated control
from the
average Normalized Melanin of treated control. Similarly, the Induction value
of each test
sample (which can be thought of as the amount of additional melanin over and
above the
baseline of the sample not treated with UV) wass then calculated by
subtracting average
Normalized Melanin of untreated control from the Normalized Melanin of the
test sample.
The Percent Inhibition for each test sample was then calculated via the
equation:
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Percent Inhibition = 100 x [(Induction value of Control ¨ Induction value with
Test
Sample)/Induction value of Control].
The average Percent Inhibition was calculated as the sum of the three
resulting
Inhibition % values for each test sample divided by three.
The calculation sequence for Percent Inhibition are demonstrated using the
example of 1-hydroxyl 3,5-bis(4'hydroxyl styryl)benzene - Free Phenol
(0.0001%), in
Table 6A, below.
Table 6A: Example Calculations of Percent Inhibition of UV-induced
Melanogenesis
Average Normalized Melanin Untreated 0.122
control
Average Normalized Melanin UVB 0.234
treated control
Induction value of control 0.234-0.122=0.112
Normalized Melanin value 0.19 (Si)
(duplicate test sample Si & S2)
0.187(S2)
Induction value with Test sample 0.19-0.122 = 0.068
0.187 ¨0.122 = 0.065
Percent Inhibition for Test sample [(0.112 ¨ 0.068)/0.112] x 100 = 39.05%
(Si)
[(0.112 ¨ 0.065)/0.112] x 100 = 41.93%
(S2)
Ave = (39.05 + 41.93)/2 = 40.49%
1-Hydroxyl 3,5-bis(4'hydroxyl styryl)benzene and its piperazine salt were
evaluated according to the Inhibition of UV-induced Melanogenesis Test
described above.
The results are reported in Table 6B.
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Table 6B: Percent inhibition of UV-induced Melanogenesis
Percent inhibition of UV-
induced melanogenesis (mean
SD)
1-hydroxyl 3,5-bis(4'hydroxyl 18.93 5.92
styryl)benzene - Free Phenol (0.00001%)
1-hydroxyl 3,5-bis(4'hydroxyl 33.27 4.39
styryl)benzene - Free Phenol (0.00005%)
1-hydroxyl 3,5-bis(4'hydroxyl 40.49 2.04
styryl)benzene - Free Phenol (0.0001%)
1-hydroxyl 3,5-bis(4'hydroxyl 13.93 11.33
styryl)benzene - Piperazine salt
(0.00001%)
1-hydroxyl 3,5-bis(4'hydroxyl 33.52 1.54
styryl)benzene - Piperazine salt
(0.00005%)
1-hydroxyl 3,5-bis(4'hydroxyl 55.70 4.44
styryl)benzene - Piperazine salt (0.0001%)
1-Hydroxyl 3,5-bis(4'hydroxyl styryl)benzene and its piperazine salt showed a
strong inhibition in melanogenesis that was UV-induced.
Example 7: Inhibition of Pigmentation
Skin epidermal equivalent tissues obtained from MatTek's MelanoDermTM System
were used as follows. MatTek's MelanoDermTM System consists of normal, human-
derived epidermal keratinocytes (NHEK) and melanocytes (NHM) which have been
cultured to form a multilayered, highly differentiated model of the human
epidermis.
Specifically, MEL-300-B tissues, each 9mm in diameter were used in the
following tests.
1-Hydroxyl 3,5-bis(4'hydroxyl styryl)benzene and its piperazine salt were
prepared in an appropriate vehicle and applied topically to the skin model
daily. The
experiment lasted for eight days. Measurement was taken on day 9.
The Degree of Lightness for each skin model tissue (L- Value) was measured
using a spectrophotometer (Konica Minolta CM-2600d). The AL (degree of
lightness as
compared to vehicle-treated control) for each test sample was calculated using
following
formula:
AL = L-value of treated sample ¨ L-value of control sample
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The results are shown in Table 7.
Table 7
Degree of Lightness (AL)
1-hydroxyl 3,5-bis(4'hydroxyl 1.46 0.71
styryl)benzene - Piperazine salt (0.1%)
1-hydroxyl 3,5-bis(4'hydroxyl 2.83 0.41
styryl)benzene - Piperazine salt (0.25%)
1-hydroxyl 3,5-bis(4'hydroxyl 4.60 0.61
styryl)benzene - Piperazine salt (0.5%)
1-hydroxyl 3,5-bis(4'hydroxyl 4.23 0.64
styryl)benzene - Free Phenol (0.5%)
1-Hydroxyl 3,5-bis(4'hydroxyl styryl)benzene and its piperazine salt showed
inhibition in melanogenesis in epidermal equivalent tissues.
Example 8
The activity of 1-hydroxyl 3,5-bis(4'hydroxyl styryl)benzene was evaluated
against representative gram positive bacteria, gram negative and fungal
species as follows.
A 4% (40mg/mL) stock solution was prepared by weighing 200mg of 1-hydroxyl
3,5-bis(4'hydroxyl styryl)benzene powder (test article) in an amber-vial and
dissolving it
in 5mL DMSO by vortexing at room temperature.
Antimicrobial susceptibility testing to determine the Minimum Inhibitory
Concentration (MIC) of the test article was performed by broth assay based on
the
standard National Committee for Clinical Laboratory Standards (NCCLS) protocol
(NCCLS, 2000). The MIC in this assay was defined as the lowest concentration
of the test
article that inhibited the visible growth of the microorganism under study.
Seven different microorganisms were tested: three (3) gram positive bacteria
(Staphylococcus aureus (MO-012-015), Staphylococcus epidermic/is (MO-012-045),
and
Propionibacterium acnes (ATCC# 6919)), two (2) gram negative bacteria
(Escherichia
coli (ATCC# 8739) and Pseudomonas aeruginosa (ATCC# 9027)), and two (2) fungal
species (Candida albicans (ATCC# 10231) and Aspergillus niger (ATCC# 16404)).
All
species, minus the Staphylococcus spp., were obtained from the American Type
Culture
Collection (ATCC). The two (2) Staphylococcus spp. were obtained as clinical
isolates.
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The MIC for each of the seven strains was determined for the test article
using
dilutions made from the original 4% w/v stock of the test article to yield
preparations of a
1.00% w/v starting concentration and a 0.25% w/v starting concentration for
the assay.
The antimicrobial susceptibility test was performed in a 96-well microtitre
plate.
In this assay, two-fold dilutions of the test article starting concentrations
were performed
sequentially along the first 10 rows of the microtiter plate (rows 11 and 12
served as
positive and negative controls for test organism).
A suspension of inoculum was created for each test organism and adjusted to
the
turbidity of an optical density at 600 nm (0D600). This inoculum was diluted
1:100 and
100u1 was added to each well of the microtiter plate, to yield a final
inoculum of ¨1x106
CFU/well.
Plates were covered and incubated at 35 C for bacteria (Propionibacterium
acnes
incubated under anaerobic conditions), and 25 C for fungi. Microplates were
examined
48 hours later and determined for MIC (Propionibacterium acnes plates were
incubated
for ¨72 hours, and fungal spp. plates were incubated for ¨120 hours). The MIC
was the
lowest concentration of antimicrobial agent that yielded no growth by visual
reading after
incubation.
The Minimal Biocide Concentration (MBC) assay is designed to determine the
lowest concentration at which the test article will demonstrate bactericidal
or fungicidal
properties against a particular microorganism. After the MIC plates were
analyzed, a 96
Solid Pin Multi-Blot Replicator was flame sterilized, cooled, and aligned so
each pin was
dipped into one well of the MIC microtiter plate. The replicator was then used
to stamp a
fresh agar plate, so that a small aliquot of the volume from the well of the
MIC microtiter
plate was impregnated in the agar matrix of the MBC plate. Microplates were
examined
48 hours later and determined for MBC (Propionibacterium acnes plates were
incubated
for ¨72 hours, and fungal spp. plates were incubated for ¨120 hours). The
minimum
biocide concentration (MBC) was defined as the minimum concentration of
biocide in
which there is no bacterial or fungal growth.
MIC and MBC values were calculated for the test article against each test
organism. The results are shown in Table 8.
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Table 8
Test S. aureus S. P. acnes E. coil P. C. A.
epidermidis aeruginosa albicans niger
MIC 0.0004% 0.0008% 0.0008% 0.031% 0.25% <0.002% 0.50%
MBC 0.0008% 0.0008% 0.0008% >1.0% >1.0% <0.002% >1.0%
Data are presented as MIC or MBC (minimum inhibitory concentration or
minimum biocide concentration) in values of (%, w/v), and demonstrates 1-
hydroxyl 3,5-
bis(4'hydroxyl styryl)benzene was found to inhibit the growth of gram positive
bacteria,
gram negative bacteria, and fungal species. The compound also demonstrated
biocidal
activity against gram positive bacteria and yeast species. These data further
suggest that
1-hydroxyl 3,5-bis(4'hydroxyl styryl)benzene has enhanced activity against
gram positive
bacteria and yeast compared to gram negative bacteria and mold.
Example 9
A composition according to the invention is prepared by blending the
ingredients
in Table 9.
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TABLE 9
Trade Name INCI Name %wt
Deionized Water Water 70.64
Sodium Chloride Sodium Chloride 0.01
1-hydroxyl 3,5-bis(4'-hydroxyl
1.00
styryl)benzene
Snow White Petrolatum Petrolatum 4.00
ISOFOL 28 Dodecylhexadecanol 2.50
DOW CORNING Q7-
9120 (20 CS) Dimethicone 1.25
KESSCO IPP Isopropyl PaImitate .00
VARISOFT TA-100 Distearyldimonium Chloride 5.00
Glycerin Glycerin 12.00
Benzyl Alcohol Benzyl Alcohol 0.60
Water is added to a process vessel. Mixing is begun and salt is added and
mixed
until dissolved. Heat is applied and mixing continued until to 85 C is
reached. 1-
hydroxyl 3,5-bis(4'-hydroxyl styryl)benzene is solublized in glycerin, then
added while
mixing is continued and the temperature is maintained at 85 C.
Distearyldimonium
chloride is added, along with petrolatum and dodecylhexadecanol, dimethicone,
and
isopropyl palmitate. The composition is mixed at 85 C for another 10-15
minutes. The
composition is then removed from heat, mixed and cooled. At 40 C, benzyl
alcohol is
added, q.s. with water, mixed and cooled to 30-35 C. The composition is then
filled into
packaging.
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A composition according to the invention is prepared by blending the
ingredients
in Table 10.
TABLE 10
Trade Name INCI Name % Wt
Deionized Water Water 70.55
Snow White Petrolatum Petrolatum 4.00
ISOFOL 28 Dodecylhexadecanol 2.50
Dow Corning Q7-9120 Dimethicone 1.25
(20 CS)
BHT BHT 0.10
Kessco IPP Isopropyl Palmitate 3.00
Varisoft TA-100 Distearyldimonium Chloride 5.00
1-hydroxyl 3,5-bis(4'hydroxyl 1.0
styryl)benzene
Glycerin Glycerin 12.00
Retinol 10S Gylcine Sofa (Soybean) Oil and Retinol 1.00
Benzyl Alcohol Benzyl Alcohol 0.60
Water is added to a process vessel and the temperature is set to 85 C. Mixing
is
begun and glycerin is added and mixed until dissolved. VARISOFT TA-100 is
added,
along with petrolatum and ISOFOL 28, DOW CORNING Q7-9120 20 CS, and isopropyl
palmitate. The composition is mixed at 85 C for another 10-15 minutes. The
composition is then removed from heat, mixed and cooled.
A composition according to the invention is prepared by blending the
ingredients
in Table 11.
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TABLE 11
Trade Name INCI Name % Wt
Deionized Water Water 73
HYDROLITE 5 Pentylene glycol 5
1-hydroxyl 3,5-bis(4'-hydroxyl 5
styryl)benzene
NATRULON OSF Carthamus Tinctorius Oleosome 10
FINSOLV TN C12-15 Alkyl Benzoate 4
ARISTOFLEX AVC Ammonium 2
AcryloyldimethyltaurateNP
Copolymer
Tanacetum parthenium Chrysanthemum Parthenium 1
(Feverfew) Leaf/Flower/Stem
extract
Juice
1-hydroxyl 3,5-bis(4'-hydroxyl styryl)benzene is weighed and dissolved in
HYDROLITE 5 and deionized water is added to form Phase A. Oleosomes and
FINSOLV TN are mixed to form Phase B. Phase B is added to Phase A very slowly
under continuous mixing. Mixing is continued for 15 minutes until a uniform
emulsion is
formed. ARISTOFLEX AVC is added to the emulsion under continuous mixing at
high
speed to obtain a thick, smooth and homogenous formulation.
A composition according to the invention is prepared by blending the
ingredients
in Table 12.
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TABLE 12
Trade Name INCI Name % Wt
Deionized Water Water 67.70
Carbomer Cross-linked polyacrylic acid 0.60
VERSENE NA Disodium EDTA 0.20
Brij 72 Steareth-2 0.75
Brij 721 Steareth-21 1.50
FINSOLV TN C12-15 Alkyl Benzoate 2.00
Dimethicone NF Dow corning Q7-9120 Silicone Fluid 5.00
(20 cst)
PHENONIP XB Phenoxyethanol, Methylparaben, 1.00
Ethylparaben, Propylparaben
LYS'LASTINE Peucedanum graveolens (10% active) 10.00
SYMMATRIX Maltodextrin, Rubus Fruticosus 10.00
(Blackberry) Leaf Extract (10%
active)
1-hydroxyl 3,5-bis(4'- 0.25
hydroxyl styryl)benzene
Glycerin Glycerin 1.0
An oil phase is prepared by adding FINSOLV TN to a clean glass beaker.
Agitation is begun and the vessel is heated to 55-60 C. When the oil phase
reaches 55 C
or higher, Brij 72 and Brij 721 are added. When the oil phase reaches 55-60
C, it is held
at that temperature and mixed for 15 min (or until uniform). The temperature
is then held
at 55-60 C with mixing until addition to water phase.
A water phase is prepared by adding water to a clean glass beaker. Agitation
is
begun and the vessel is heated to 55-60 C. Disodium EDTA is added. At 55-60
C, the
ingredients are mixed for 15 min or until homogeneous. The temperature is then
held at
55-60 C with mixing for phasing.
The oil phase is added to the water phase with increased agitation and then
mixed
at high speed for 10-20 min. At 50 C or lower, dimethicone is added. At 40 C
or lower,
PHENONIP XB is added. The phases are then mixed for 10 min or until uniform.
Sodium hydroxide is added (target pH is 5.4). The composition is then mixed
for 10 min
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or until uniform. LYS'LASTINE and SYMMATRIX are then added. 1-hydroxyl 3,5-
bis(4'-hydroxyl styryl)benzene is weighed and dissolved in glycerin and added
to the
mixture, which is mixed until uniform. Water is then added to QS and the
composition is
mixed for 10 additional minutes.
It is understood that while the invention has been described in conjunction
with the
detailed description thereof, that the foregoing description is intended to
illustrate and not
limit the scope the invention, which is defined by the scope of the appended
claims. Other
aspects, advantages, and modifications are within the claims.
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