U.S. patent application number 16/358862 was filed with the patent office on 2019-09-19 for method of selecting antioxidants for use in topically applied compositions.
The applicant listed for this patent is BAYER HEALTHCARE LLC. Invention is credited to Donathan G. Beasley, THOMAS A. MEYER.
Application Number | 20190285657 16/358862 |
Document ID | / |
Family ID | 40427148 |
Filed Date | 2019-09-19 |
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United States Patent
Application |
20190285657 |
Kind Code |
A1 |
MEYER; THOMAS A. ; et
al. |
September 19, 2019 |
METHOD OF SELECTING ANTIOXIDANTS FOR USE IN TOPICALLY APPLIED
COMPOSITIONS
Abstract
Antioxidant-containing compositions and methods for confirming
antioxidant activity of a composition formulated for topical
application to skin. Methods for testing a composition for ability
to inhibit both ultraviolet radiation-induced lipid peroxidation on
skin and ultraviolet radiation-induced reactive oxygen species
formation in the stratum corneum. Compositions and methods for
treating and preventing photodamage to skin.
Inventors: |
MEYER; THOMAS A.; (MEMPHIS,
TN) ; Beasley; Donathan G.; (MEMPHIS, TN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BAYER HEALTHCARE LLC |
Whippany |
NJ |
US |
|
|
Family ID: |
40427148 |
Appl. No.: |
16/358862 |
Filed: |
March 20, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14818212 |
Aug 4, 2015 |
10241121 |
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16358862 |
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13651894 |
Oct 15, 2012 |
9096882 |
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14818212 |
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12357006 |
Jan 21, 2009 |
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13651894 |
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61114758 |
Nov 14, 2008 |
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61023713 |
Jan 25, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 17/16 20180101;
A61K 2800/522 20130101; G01N 33/92 20130101; C12Q 1/025 20130101;
A61P 17/02 20180101; A61K 8/9789 20170801; A61K 49/00 20130101;
A61P 17/00 20180101; G01N 33/5088 20130101; A61K 8/37 20130101;
A61Q 17/04 20130101; A61K 8/9794 20170801; A61P 39/06 20180101;
G01N 33/502 20130101; A61K 8/678 20130101; G01N 33/5082
20130101 |
International
Class: |
G01N 33/92 20060101
G01N033/92; G01N 33/50 20060101 G01N033/50; A61Q 17/04 20060101
A61Q017/04; C12Q 1/02 20060101 C12Q001/02; A61K 8/97 20060101
A61K008/97; A61K 8/67 20060101 A61K008/67; A61K 8/37 20060101
A61K008/37; A61K 49/00 20060101 A61K049/00 |
Claims
1-2. (canceled)
3. A composition for topical application, wherein the composition
comprises at least one antioxidant compound that substantially
inhibits ultraviolet radiation-induced reactive oxygen species
formation in the upper layers of the epidermis and at least one
antioxidant compound that substantially inhibits ultraviolet
radiation-induced reactive oxygen species formation in the lower
layers of the epidermis.
4. The composition of claim 3, which comprises Vitamin E in an
amount of about 0.25% by weight.
5. The composition of claim 3, which comprises Vitamin E in an
amount of about 0.5% by weight.
6. The composition of claim 3, which comprises diethylhexyl
syringylidene malonate.
7. The composition of claim 4, which further comprises diethylhexyl
syringylidene malonate.
8. The composition of claim 5, which further comprises diethylhexyl
syringylidene malonate.
9. The composition of claim 3, which further comprises at least one
sunscreen active agent.
10. The composition of claim 9, wherein the sunscreen active agent
is chosen from the group consisting of para aminobenzoic acid,
avobenzone, cinoxate, dioxybenzone, homosalate, menthyl
anthranilate, octyl salicylate, oxybenzone, padimate O,
phenylbenzimidazole sulfonic acid, sulisobenzone, trolamine
salicylate, titanium dioxide, zinc oxide, diethanolamine
methoxycinnamate, digalloy trioleate, ethyl dihydroxypropyl PABA,
glyceryl aminobenzoate, lawsone with dihydroxyacetone, red
petrolatum, ethylhexyl triazone, dioctyl butamido triazone,
benzylidene malonate polysiloxane, terephthalylidene dicamphor
sulfonic acid, disodium phenyl dibenzimidazole tetrasulfonate,
diethylamino hydroxybenzoyl hexyl benzoate, bis diethylamino
hydroxybenzoyl benzoate, bis benzoxazoylphenyl ethylhexylimino
triazine, drometrizole trisiloxane, methylene bis-benzotriazolyl
tetramethylbutylphenol, bis-ethylhexyloxyphenol
methoxyphenyltriazine, 4-methylbenzylidenecamphor, isopentyl
4-methoxycinnamate, and mixtures thereof.
Description
FIELD OF THE INVENTION
[0001] This invention relates generally to compositions applied
topically to skin and hair for protection against ultraviolet
radiation. The invention also relates to methods of selecting
antioxidants for inclusion in such compositions.
[0002] This application is Divisional of U.S. patent application
Ser. No. 14/818,212, filed Aug. 4, 2015, which is a Continuation of
U.S. patent application Ser. No. 13/651,894, filed Oct. 15, 2012,
which is a Continuation of U.S. patent application Ser. No.
12/357,006, filed Jan. 21, 2009, which claims priority to U.S.
Provisional Patent Application No. 61/023,713, filed Jan. 25, 2008,
and U.S. Provisional Patent Application No. 61/114,758, filed Nov.
14, 2008, the disclosures of which are incorporated by reference in
their entireties.
BACKGROUND OF THE INVENTION
[0003] Exposure of skin to ultraviolet radiation (UVR) induces
formation of free radicals and oxidants (singlet oxygen, hydroxy
radical, hydrogen peroxide, peroxynitrite, superoxide anions, etc.)
collectively referred to as reactive oxygen species (ROS) (Hanson K
M, Clegg R M. Photochemistry and Photobiology, 2002, 76(1): 57-63;
Black H S. Photochem. Photobiol. 1987, 46, 213-221). Formation of
UV-induced ROS causes oxidative damage to lipids, proteins and DNA
(Vile G F and Tyrrell R M. Free Radic. Biol. Med, 1995, 18,
721-722; Chen Q, et al. Proc. Natl. Acad. Sci. USA, 1995, 92,
4337-4341).
[0004] Under normal circumstances, low levels of ROS are
neutralized by skin's constitutive zo antioxidant defenses.
However, research has shown that even sub-erythemal doses of UVR
generates such an abundance of ROS that skin's own antioxidant
defenses become overwhelmed, resulting in a build up of ROS that
are free to cause oxidation, which contributes to acute
(immunesuppression and photosensitivity disorders) and chronic
(photoaging and skin cancer) forms of skin damage (Thiele J J, et
al. J. Invest. Dermatol, 1998, 110(5), 756-761; Sander C. S, et al.
J. Invest. Dermatol, 2002, 118 (4), 618-625; Thiele J. J: Skin
Pharmacol. Appl. Skin Physiol, 2001, 14 (suppl. 1), 87-91; Sander C
S, et al. International Soc. Dermatol, 2004, 43, 326-335). EP
1591104 (STADA Pharmaceuticals AG) describes the use of
antioxidants in pharmaceutical formulations for protection against
infrared radiation.
[0005] Antioxidants (Aox) function to neutralize ROS. If the right
type and level are present within skin where ROS are being formed,
Aox should be able to neutralize ROS before they can attack and
oxidize other biomolecules. Accordingly, it would be useful to have
a method to determine which topical applied antioxidants can be
highly effective at neutralizing UVR-induced reactive oxygen
species (ROS) within skin. Further, it would be useful to have a
method to distinguish compounds which may only be effective in
solution to scavenge free radicals from compounds that may be
highly effective antioxidants on skin when exposed to UVR. Further,
it would be useful to have a method to determine the correct choice
and use-level of antioxidants in sunscreen products to provide
extra protection against skin damage caused by UVR-induced ROS. In
addition, it would be useful to have a composition that provides
protection from UVR-induced ROS both at the skin surface and deep
in the epidermis, for example as far as the basal layer. These and
other objectives are provided by the invention described
herein.
[0006] Accordingly the invention described herein provides, inter
alia, a method which comprises two unique ex vivo methods to assess
the ability of topically applied Aox to provide protection against
UVR-induced ROS formation within skin's outer layers. The first
method uses microscopy, e.g., fluorescence microscopy, to image and
quantify ROS formation in the inner layers of the epidermis, e.g.
through to the basal layers, by imaging sections of human skin. The
second method quantifies the extent to which the Aox containing
composition inhibits peroxidation of lipids in skin's outer layers.
This specification also demonstrates that a commonly used
laboratory test to measure efficacy of antioxidants in solution to
scavenge free radicals is not predictive of an Aox's ability to
function effectively on more complex biological substrates like
skin exposed to UVR. Thus, the present invention provides an
advantage over prior art methods to select Aox for use in sunscreen
products to ensure they provide a protective benefit.
[0007] All patent and non-patent references cited herein are hereby
incorporated in their entirety into this specification by reference
thereto. Identification or discussion of any reference in this
section or any part of this specification shall not be construed as
an admission that such reference is available as prior art to the
present application.
SUMMARY OF THE INVENTION
[0008] The present invention provides a method for confirming
antioxidant activity of a composition formulated for topical
application to skin, wherein the method comprises testing the
composition for ability to inhibit both ultraviolet
radiation-induced lipid peroxidation on skin and ultraviolet
radiation-induced reactive oxygen species formation throughout the
epidermis.
[0009] The invention also provides a method for screening compounds
for antioxidant behavior in a composition to be topically applied
to skin, wherein the screening method comprises determining the
compound's ability to inhibit both ultraviolet radiation-induced
skin lipid hydroperoxide formation and ultraviolet
radiation-induced reactive oxygen species formation throughout the
epidermis.
[0010] This invention also provides a composition for application
to skin or hair of a subject, wherein the composition comprises an
antioxidant compound or combination of antioxidant compounds,
wherein the antioxidant compound or combination of antioxidant
compounds substantially inhibit both ultraviolet radiation-induced
skin lipid peroxidation and ultraviolet radiation-induced reactive
oxygen species formation throughout the epidermis.
[0011] The invention further provides for a composition for topical
application, wherein the composition comprises at least one
antioxidant compound that substantially inhibits ultraviolet
radiation-induced reactive oxygen species formation in the upper
layers of the epidermis and at least one antioxidant compound that
substantially inhibits ultraviolet radiation-induced reactive
oxygen species formation in the lower layers of the epidermis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1--shows the extent to which antioxidants (Aox) inhibit
UVR-induced formation of lipid hydroperoxides (LOON) ex vivo using
tape strips to collect lipids from human skin in the presence of
different types and levels of Aox.
[0013] FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, and 2I--shows that the
addition of antioxidants to sunscreens significantly reduces
UV-induced formation of ROS within the stratum corneum. Relative to
the control, the SPF 30 sunscreen formula reduced ROS by 39%
whereas the SPF 30 formula with 0.5% vitamin E and 0.1% Emblica as
antioxidants reduced ROS by 73%. The extent of ROS formation is
color coded, with blue indicating low and orange or red indicating
high ROS levels.
[0014] FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, and 3I--shows two
photon fluorescence microscopy on skin layers to demonstrate
ability of Vitamin E and Emblica antioxidants to reduce UV-induced
formation of ROS within the epidermis. A composition containing
0.5% Vitamin E and 0.1% Emblica provide little protection against
free radical formation in the lower (basal) layer of the
epidermis.
[0015] FIGS. 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, 4I, and 4J--shows two
photon fluorescence microscopy on skin layers to demonstrate effect
of various known antioxidants to reduce UV-induced formation of ROS
within the epidermis. Tested antioxidant extracts actually
increased rather than decreased free radicals when exposed to
UVR.
[0016] FIGS. 5A, 5B, 5C, and 5D--shows two photon fluorescence
microscopy on skin layers to demonstrate effect of various known
antioxidants to reduce UV-induced formation of ROS within the
epidermis after 1 MED of UVR. A composition containing 0.5% Vitamin
E and 0.9% Oxynex ST are shown to inhibit formation of ROS within
lower epidermal layers.
[0017] FIGS. 6A, 6B, 6C, and 6D--shows two photon fluorescence
microscopy on skin layers to demonstrate effect of various known
antioxidants to reduce UV-induced formation of ROS within the
epidermis after 4 MED of UVR. A composition containing 0.5% Vitamin
E and 0.9% Oxynex ST are shown to inhibit formation of ROS within
lower 30 epidermal layers.
DETAILED DESCRIPTION
[0018] The present invention provides a method for confirming
antioxidant activity of a composition formulated for topical
application to skin, wherein the method comprises testing the
composition for ability to inhibit both ultraviolet
radiation-induced lipid peroxidation on skin and ultraviolet
radiation-induced reactive oxygen species formation throughout the
epidermis.
[0019] The invention also provides a method for screening compounds
for antioxidant behavior in a composition to be topically applied
to skin, wherein the screening method comprises determining the
compound's ability to inhibit both ultraviolet radiation-induced
skin lipid hydroperoxide formation and ultraviolet
radiation-induced reactive oxygen species formation throughout the
epidermis.
[0020] In certain embodiments of the methods of the invention
determining inhibition of ultraviolet radiation-induced reactive
oxygen species formation in the skin comprises imaging skin
throughout the epidermis down to the basal layer using two-photon
fluorescence intensity imaging.
[0021] In certain embodiments of the method of the invention
determining inhibition of UVR-induced skin lipid hydroperoxide
formation comprises determining percent lipid hydroperoxide
inhibition of the compound in comparison to placebo.
[0022] In certain embodiments the methods of the invention comprise
the steps of applying to distinct areas of skin of a subject an
antioxidant containing composition and a placebo composition to
produce an antioxidant skin site containing antioxidant and skin
lipids and a placebo skin site containing placebo and skin lipids;
applying a strip to the antioxidant skin site and the placebo skin
site to produce an antioxidant strip sample containing antioxidant
and skin lipids and a placebo strip sample containing placebo and
skin lipids; removing said strip samples from the skin and exposing
said strip samples to UVR to form a UVR-induced antioxidant/lipid
reaction product on the antioxidant strip sample and a UVR-induced
placebo/lipid reaction product on the placebo strip sample;
separately contacting the antioxidant strip sample and the placebo
strip sample with solvent to prepare a first extract containing
UVR-induced antioxidant/lipid reaction product and a second extract
containing UVR-induced placebo/lipid reaction product; assaying
said first and second extracts for lipid hydroperoxide content for
each extract; and comparing the lipid hydroperoxide content of the
first extract to the lipid hydroperoxide content of the second
extract.
[0023] In certain embodiments the methods of the invention comprise
the further steps of applying placebo to two distinct sites on the
skin of the subject; producing strip samples from each site;
subjecting strip samples from only one of the two placebo sites to
UVR to produce a subset of irradiated placebo strip samples and a
subset of nonirradiated placebo strip samples; separately
contacting nonirradiated placebo strip samples with solvent to
prepare a third extract containing placebo and skin lipids; and
assaying said third extract for lipid hydroperoxide content to
determine background lipid hydroperoxide formation.
[0024] In certain embodiments the methods of the invention comprise
comparing the lipid hydroperoxide content of the first extract to
the lipid hydroperoxide content of the second extract comprises
calculating percent lipid hydroperoxide formation by the following
formula:
% LF = ( LOOH 1 - LOOH 3 ) ( LOOH 2 - LOOH 3 ) .times. 100
##EQU00001##
wherein %LF is the percent lipid hydroperoxide formation, LOOH1 is
the lipid hydroperoxide content of the first extract, LOOH2 is the
lipid hydroperoxide content of the second extract, and LOOH3 is the
lipid hydroperoxide content of the third extract.
[0025] The art recognizes numerous compounds as having antioxidant
properties. As used herein, the term "antioxidant" refers to
compounds or combinations of compounds determined by the methods of
the invention to have a %LF that is less than 100%. As used herein
the term "prooxidant" refers to compounds or combinations of
compounds that have a %LF that is greater than 100%. As
demonstrated herein, certain compounds referred to in the art as
antioxidants actually have a prooxidant behavior when tested
according to the methods of the invention, making them unsuitable
as ingredients in compositions for topical application,
particularly in sunscreens, unless present in the combinations as
described herein.
[0026] The compositions of the invention containing the appropriate
Aox can comprise any form readily known by those of ordinary skill
in the art of preparing cosmetic compositions. Examples of such
include, but are not limited to, nonionic vesicle dispersions,
emulsions, creams, milks, gels, cream gels, ointments, suspensions,
dispersions, powders, solids, sticks, foams or sprays. In certainly
preferred embodiments, the composition can comprise an anhydrous or
aqueous solid or paste, emulsion, suspension, or dispersion.
Preferable forms of the compositions include an oil-in-water
emulsion, a water-in-oil emulsion, an alcohol solution, or an
aerosol formulation.
[0027] Thus, the subject invention also provides a cosmetic
composition for topical application to human skin and/or hair,
comprising an appropriate Aox and amount of Aox determined by the
methods described herein. Non-limiting examples of such cosmetic
compositions may include such products as moisturizers, cleansers,
conditioners, shampoo, body wash, styling gel/lotion, eye cream and
eye liner, blush, mascara, foundation, nail polish, polish remover,
eye shadow, lipstick, lip gloss, lip liners, lip balms, makeup
remover, nail treatment, foot care compositions, acne treatment,
redness/rosacea treatment, varicose/spider vein treatment,
anti-aging compositions, sunless tanning compositions, after-sun
compositions, concealers, hair color and bleaching compositions,
skin fading/lighteners, body firming lotion, shaving cream, after
shave, relaxer, antiperspirants and deodorants, exfoliants, scrubs,
liquid hand soap, bubble bath, pain and wound treatment
compositions, insect repellant, anti-itch and rash cream, styling
mousse and foams, perfume, lubricants, body oil, body spray, baby
lotion, diaper cream, baby soap, baby shampoo, baby oil, baby
wipes, hair-loss treatment, hair spray, depilatory, hair growth
inhibitors, hair removal waxes, personal cleansing, cologne, oil
controller, and hand sanitizer.
[0028] Examples of antioxidants useful in the compositions of the
invention include, but are not limited to, Diethylhexyl
syringylidene malonate, Vitamin E, diisopropyl vanillidene malonate
(also referred to as DIPVM) and related compounds (described in
U.S. Pat. Nos. 6,602,515; 6,831,191; 6,936,735; 7,150,876; and
7,166,273), Tetrahydrocurcumenoids, Soybean zymbiozome fermentum,
Red clover extract, Vitis vinifera (grape) seed extract/Brand B,
Green tea extract, Pikea robusta extract, Tocopherol (and) vitis
vinifera (grape) seed extract, Vitis vinifera (grape) seed
extract/Brand A, Phylanthus emblica fruit extract and combinations
thereof. Amounts of antioxidants to be added to the compositions of
the invention are generally between about 0.01% by weight to about
10.0% by weight, preferably between about 0.1% by weight to about
5.0% by weight. Exact amounts can be determined by one of ordinary
skill in the art according to testing methods described herein.
[0029] In certain embodiments the composition of the invention can
comprise Vitamin E alone as an antioxidant in an amount greater
than about 0.05% by weight, in an amount of about 0.1% by weight or
greater, in an amount of about 0.25% by weight or greater, and in
an amount of about 0.5% by weight or greater. In certain
embodiments the composition of the invention can comprise Vitamin E
as an antioxidant and at least one additional antioxidant
compound.
[0030] In certain embodiments the composition of the invention may
comprise Vitamin E in combination with a pro-oxidant compound as
determined by the methods of the invention, where the presence of
Vitamin E in these embodiments will counteract the pro-oxidant
effects of these compounds to form an antioxidant combination. In
certain embodiments of this composition Vitamin E is present in an
amount greater than about 0.05% by weight, in an amount of about
0.1% by weight or greater, in an amount of about 0.25% by weight or
greater, and in an amount of about 0.5% by weight or greater.
Examples of such pro-oxidant compounds that will be useful in the
compositions of the invention that comprise Vitamin E include, but
are not limited to, Rosemary officinalis oleoresin, Rosa Gallica
extract, Bioactive Photosynthetic complex from green plants,
Thermus Thermophillus ferment, ergothiotaine and combinations
thereof. Amounts of pro-oxidants to be added to the compositions of
the invention are generally between about 0.01% by weight to about
10.0% by weight, preferably between about 0.1% by weight to about
5.0% by weight. Exact amounts can be determined by one of ordinary
skill in the art according to testing methods described herein.
[0031] In one embodiment the subject invention, the composition can
be in the form of an aerosol, wherein the composition is combined
with at least one propellant, which may be any suitable gas that
can be compressed or liquefied within a spray dispensing canister
and which expands or volatilizes to vapor or gas form upon exposure
to ambient temperature and pressure conditions to deliver the
composition in an aerosol form. Suitable propellants include
hydrocarbons having 1 to 5 carbon atoms, including but not limited
to methane, ethane, propane, isopropane, butane, isobutane, butene,
pentane, isopentane, neopentane, pentene, hydrofluorocarbons
(HFCs), chlorofluorocarbons(CFCs), nitrogen, ethers including
dimethyl ether, and any mixtures thereof. Those of ordinary skill
in the art recognize that in a closed container such as an aluminum
can or glass bottle, propellants such as dimethyl ether condense to
the liquid state at ambient temperature. Thus, the composition in
the aerosol container is liquid formulation which can contain
dissolved propellant, undissolved liquid propellant and gaseous
propellant. All of this is under pressure due to the vapor pressure
of the propellant. In the practice of the subject invention, the
propellant can be present in an amount up to about 90 weight
percent, preferably from about 2 weight percent to about 50 weight
percent, and more preferably about 5 weight percent to about 40
weight percent, most preferably 30 weight percent, based on the
total weight of the aerosol composition.
[0032] The compositions of the invention can also comprise aerosol
foams or so-called mousse compositions. For example, U.S. Pat. No.
6,627,585 describes a mousse-forming cleansing shampoo composition
comprising a foamable concentrate comprising at least one
surfactant, dispersed particles of a water-insoluble conditioning
agent, an aqueous carrier; and an aerosol propellant. U.S. Pat. No.
6,264,964 describes a cosmetic composition including a crosslinked
non-emulsifying polysiloxane elastomer and a carboxyvinyl polymer
which is in the form of an aerosol foam in a pressurized system.
The propellant may be introduced into the mousse composition at the
time of filling by using a standard aerosol dispenser, e.g. a spray
can arrangement.
[0033] The subject invention contemplates the incorporation of Aox
with sunscreen actives in sunscreen and sunblock products and any
other topically applied composition where the addition of sunscreen
active agents and/or Aox would not detract from the efficacy of the
product nor affect the sunscreening ability of the sunscreen active
agents.
[0034] The compositions of the present invention may contain a wide
range of additional, optional components which are referred to
herein as "cosmetic components", but which can also include
components generally known as pharmaceutically active agents. The
CTFA Cosmetic Ingredient Handbook, Seventh Edition, 1997 and the
Eighth Edition, 2000, which is incorporated by reference herein in
its entirety, describes a wide variety of cosmetic and
pharmaceutical ingredients commonly used in skin care compositions,
which are suitable for use in the compositions of the present
invention. Examples of these functional classes disclosed in this
reference include: absorbents, abrasives, anticaking agents,
antifoaming agents, antioxidants, binders, biological additives,
buffering agents, bulking agents, chelating agents, chemical
additives, colorants, cosmetic astringents, cosmetic biocides,
denaturants, drug astringents, external analgesics, film formers,
fragrance components, humectants, opacifying agents, pH adjusters,
plasticizers, reducing agents, skin bleaching agents,
skin-conditioning agents (emollient, humectants, miscellaneous, and
occlusive), skin protectants, solvents, foam boosters, hydrotropes,
solubilizing agents, suspending agents (nonsurfactant), sunscreen
agents, ultraviolet light absorbers, SPF boosters, waterproofing
agents, and viscosity increasing agents (aqueous and
nonaqueous).
[0035] In the practice of the invention, the composition may
contain one or more sunscreen active agents. For purposes of the
present invention, a "sunscreen active agent" or "sunscreen active"
shall include all of those materials, singly or in combination,
that are regarded as acceptable for use as active sunscreening
ingredients based on their ability to absorb UV radiation. Such
compounds are generally described as being UV-A, UV-B, or UV-A/UV-B
active agents. Approval by a regulatory agency is generally
required for inclusion of active agents in formulations intended
for human use. Those active agents which have been or are currently
approved for sunscreen use in the United States include organic and
inorganic substances including, without limitation, para
aminobenzoic acid, avobenzone, cinoxate, dioxybenzone, homosalate,
menthyl anthranilate, octyl salicylate, oxybenzone, padimate O,
phenylbenzimidazole sulfonic acid, sulisobenzone, trolamine
salicylate, titanium dioxide, zinc oxide, diethanolamine
methoxycinnamate, digalloy trioleate, ethyl dihydroxypropyl PABA,
glyceryl aminobenzoate, lawsone with dihydroxyacetone, red
petrolatum. Examples of additional sunscreen actives that have not
yet been approved in the US but are allowed in formulations sold
outside of the US include ethylhexyl triazone, dioctyl butamido
triazone, benzylidene malonate polysiloxane, terephthalylidene
dicamphor sulfonic acid, disodium phenyl dibenzimidazole
tetrasulfonate, diethylamino hydroxybenzoyl hexyl benzoate, bis
diethylamino hydroxybenzoyl benzoate, bis benzoxazoylphenyl
ethylhexylimino triazine, drometrizole trisiloxane, methylene
bis-benzotriazolyl tetramethylbutylphenol, and
bis-ethylhexyloxyphenol methoxyphenyltriazine,
4-methylbenzylidenecamphor, and isopentyl 4-methoxycinnamate.
However, as the list of approved sunscreens is currently expanding,
those of ordinary skill will recognize that the invention is not
limited to sunscreen active agents currently approved for human use
but is readily applicable to those that may be allowed in the
future.
[0036] In one embodiment of the invention the additional sunscreen
active agent comprises a photoprotecting effective amount of
particulates of at least one inorganic pigment or nanopigment,
non-limiting examples of which include titanium dioxide, zinc
oxide, iron oxide, zirconium oxide, cerium oxide, or mixture
thereof.
[0037] The compositions of the invention may also include materials
that also increase the SPF of the final composition by such
mechanisms as UV radiation scattering and dispersion. Such
materials are referred to herein as "UV-radiation scattering
agents" and comprise materials that exhibit UV absorbing activity
or exhibit no UV absorbing activity. An example of such
UV-radiation scattering agents include polymeric materials, such as
the product known as SunSpheres.TM. (Rohm and Haas; Philadelphia,
Pa.) which are described by their manufacturer as hollow
styrene/acrylates copolymer spheres manufactured by emulsion
polymerization. The polymer spheres are said to raise SPF values
across the UVA and UVB region by dispersing and/or scattering the
incident UV radiation throughout the film of sunscreen present on a
surface, such as human skin. It is understood that the spheres
cause less UV radiation to penetrate into the skin by redirecting
the radiation towards the UV-absorbing sunscreen actives in the
sunscreen formulation, where the radiation reacts with the
sunscreen active molecules and the energy is dissipated as heat. As
used herein, the terms "spheres" or "scattering agents" are not
limited by chemical makeup or shape, but comprise any agent that
produces the effect of lengthening the path of incident UV
radiation, increasing the statistical likelihood that the radiation
will contact a sunscreen active molecule, i.e., a UV absorbing
active agent. These materials may also include UV absorbing
materials that also exhibit scattering properties such as ZnO
(examples include Z-Cote.TM. products available from BASF),
TiO.sub.2 (examples include the SolaveilTM products available from
Uniqema (New Castle, Del., USA)), compounds such as methylene
bis-benzotriazolyl tetramethylbutylphenol, ("Tinasorb.TM. M"
available from Ciba Specialty Chemicals, Inc. (Basel, Switzerland).
UV radiation scattering agents are typically present in the
formulation in amounts up to about 10% by weight, preferably in
ranges of about 0.5% to about 7.0% by weight, in particularly
preferred ranges of 3% to about 5% by weight.
[0038] As used herein, the terms "sunless-tanning agent" or
"self-tanning compositions" refer to compositions which, when
applied to human skin, impart thereto an appearance similar to that
achieved by exposing the skin to natural or artificial sunlight.
Examples of sunless tanning active agents are described in U.S.
Pat. Nos. 6,482,397, 6,261,541, and 6,231,837. Such sunless tanning
compositions typically comprise, in addition to an artificial
tanning effective amount of a self tanning agent, effective amounts
of a composition coloring agent and a cosmetically acceptable
carrier adapted for topical application to human skin. The self
tanning agents can also include those compositions generally
accepted in the art for application to human skin, and which, when
so applied, react therein with amino acids so as to form pigmented
products. Such reactions give the skin a brown appearance similar
to the color obtained upon exposing it to sunlight for periods of
time sufficient to tan the skin. Suitable self tanning agents
include, without limitation, alpha-hydroxy aldehydes and ketones,
glyceraldehyde and related alcohol aldehydes, various indoles,
imidazoles and derivatives thereof, and various approved
pigmentation agents. Presently preferred herein as self tanning
agents are the alpha-hydroxy aldehydes and ketones. Most
preferably, the self tanning agent is dihydroxyacetone ("DHA").
Other suitable self tanning agents include, without limitation,
methyl glyoxal, glycerol aldehyde, erythrulose, alloxan,
2,3-dihydroxysuccindialdehyde, 2,3-dimethoxysuccindialdehyde,
2-amino-3-hydroxy-succindialdehyde and
2-benzylamino-3-hydroxysuccindialdehyde.
[0039] Suitable emulsifiers or surfactants include pharmaceutically
acceptable, non-toxic, non-ionic, anionic and cationic surfactants.
Examples of suitable non-ionic surfactants include glycerol fatty
acid esters such as glycerol monostearate, glycol fatty acid esters
such as propylene glycol monostearate, polyhydric alcohol fatty
acid esters such as polyethylene glycol (400) monooleate,
polyoxyethylene fatty acid esters such as polyoxyethylene (40)
stearate, polyoxyethylene fatty alcohol ethers such as
polyoxyethylene (20) stearyl ether, polyoxyethylene sorbitan fatty
acid esters such as polyoxyethylene sorbitan monostearate, sorbitan
esters such as sorbitan monostearate, alkyl glycosides such as
cetearyl glucoside, fatty acid ethanolamides and their derivatives
such as the diethanolamide of stearic acid, and the like. Examples
of suitable anionic surfactants are soaps including alkali soaps,
such as sodium, potassium and ammonium salts of aliphatic
carboxylic acids, usually fatty acids, such as sodium stearate.
Organic amine soaps include organic amine salts of aliphatic
carboxylic acids, usually fatty acids, such as triethanolamine
stearate. Metallic soaps include salts of polyvalent metals and
aliphatic carboxylic acids, usually fatty acids, such as aluminium
stearate. Other classes of suitable anionic surfactants include
sulfated fatty acid alcohols such as sodium lauryl sulfate,
sulfated oils such as the sulfuric ester of ricinoleic acid
disodium salt, and sulfonated compounds such as alkyl sultonates
including sodium cetane sulfonate, amide sulfonates such as sodium
N-methyl-N-oleyl laurate, sulfonated dibasic acid esters such as
sodium dioctyl sulfosuccinate, alkyl aryl sulfonates such as sodium
dodecylbenzene sulfonate, alkyl naphthalene sulfonates such a
sodium isopropyl naphthalene sulfonate, petroleum sulfonate such as
aryl napthalene with alkyl substitutes. Examples of suitable
cationic surfactants include amine salts such as octadecyl ammonium
chloride, quartemary ammonium compounds such as benzalkonium
chloride.
[0040] An emollient is an oleaginous or oily substance which helps
to smooth and soften the skin, and may also reduce its roughness,
cracking or irritation. Typical suitable emollients include mineral
oil having a viscosity in the range of 50 to 500 centipoise (cps),
lanolin oil, coconut oil, cocoa butter, olive oil, almond oil,
macadamia nut oil, aloe extracts such as aloe vera lipoquinone,
synthetic jojoba oils, natural sonora jojoba oils, safflower oil,
corn oil, liquid lanolin, cottonseed oil and peanut oil.
Preferably, the emollient is a cocoglyceride, which is a mixture of
mono, di- and triglycerides of cocoa oil, sold under the trade name
of Myritol 331 from Henkel KGaA, or Dicaprylyl Ether available
under the trade name Cetiol OE from Henkel KGaA or a
C.sub.12-C.sub.15 Alkyl Benzoate sold under the trade name Finsolv
TN from Finetex. One or more emollients may be present ranging in
amounts from about 1 percent to about 10 percent by weight,
preferably about 5 percent by weight. Another suitable emollient is
DC 200 Fluid 350, a silicone fluid, available Dow Corning Corp.
[0041] Other suitable emollients include squalane, castor oil,
polybutene, sweet almond oil, avocado oil, calophyllum oil, ricin
oil, vitamin E acetate, olive oil, silicone oils such as
dimethylopolysiloxane and cyclomethicone, linolenic alcohol, oleyl
alcohol, the oil of cereal germs such as the oil of wheat germ,
isopropyl palmitate, octyl palmitate, isopropyl myristate,
hexadecyl stearate, butyl stearate, decyl oleate, acetyl
glycerides, the octanoates and benzoates of (C.sub.12-C.sub.15)
alcohols, the octanoates and decanoates of alcohols and
polyalcohols such as those of glycol and glyceryl, ricinoleates
esters such as isopropyl adipate, hexyl laurate and octyl
dodecanoate, dicaprylyl maleate, hydrogenated vegetable oil,
phenyltrimethicone, jojoba oil and aloe vera extract.
[0042] Other suitable emollients which are solids or semi-solids at
ambient temperatures may be used. Such solid or semi-solid cosmetic
emollients include glyceryl dilaurate, hydrogenated lanolin,
hydroxylated lanolin, acetylated lanolin, petrolatum, isopropyl
lanolate, butyl myristate, cetyl myristate, myristyl myristate,
myristyl lactate, cetyl alcohol, isostearyl alcohol and isocetyl
lanolate. One or more emollients can optionally be included in the
formulation.
[0043] A humectant is a moistening agent that promotes retention of
water due to its hygroscopic properties. Suitable humectants
include glycerin, polymeric glycols such as polyethylene glycol and
polypropylene glycol, mannitol and sorbitol. Preferably, the
humectant is Sorbitol, 70% USP or polyethylene glycol 400, NF. One
or more humectants can optionally be included in the formulation in
amounts from about 1 percent to about 10 percent by weight,
preferably about 5 percent by weight.
[0044] A dry-feel modifier is an agent which when added to an
emulsion, imparts a "dry feel" to the skin when the emulsion dries.
Dry feel modifiers can include talc, kaolin, chalk, zinc oxide,
silicone fluids, inorganic salts such as barium sulfate, surface
treated silica, precipitated silica, fumed silica such as an
Aerosil available from Degussa Inc. of New York, N.Y. U.S.A.
Another dry feel modifier is an epichlorohydrin cross-linked
glyceryl starch of the type that is disclosed in U.S. Pat. No.
6,488,916.
[0045] It may be advantageous to incorporate additional thickening
agents, such as, for instance, various Carbopols available from
Noveon Co. Particularly preferred are those agents which would not
disrupt the lamellar structure in the formulation of the final
product, such as non-ionic thickening agents. The selection of
additional thickening agents is well within the skill of one in the
art.
[0046] Additional natural or synthetic substances can also added to
the compositions of the invention to protect from or delay its
deterioration due to the action of oxygen in the air (oxidation).
They may also reduce oxidation reactions in skin tissue. Such
substances prevent oxidative deterioration which may lead to the
generation of rancidity and nonenyzymatic browning reaction
products. Typical suitable substances include propyl, octyl and
dodecyl esters of gallic acid, butylated hydroxyanisole (BHA,
usually purchased as a mixture of ortho and meta isomers),
butylated hydroxytoluene (BHT), green tea extract, uric acid,
cysteine, pyruvate, nordihydroguaiaretic acid, Vitamin A, Vitamin E
and Vitamin C and their derivatives. One or more such substances
can optionally be included in the composition in an amount ranging
from about 0.001 to about 5 weight percent, preferably about 0.01
to about 0.5 percent.
[0047] Chelating agents are substances used to chelate or bind
metallic ions, such as with a heterocylic ring structure so that
the ion is held by chemical bonds from each of the participating
rings. Suitable chelating agents include ethylene
diaminetetraacetic acid (EDTA), EDTA disodium, calcium disodium
edetate, EDTA trisodium, albumin, transferrin, desferoxamine,
desferal, desferoxamine mesylate, EDTA tetrasodium and EDTA
dipotassium, or combinations of any of these.
[0048] Fragrances are aromatic substances which can impart an
aesthetically pleasing aroma to the sunscreen composition. Typical
fragrances include aromatic materials extracted from botanical
sources (i.e., rose petals, gardenia blossoms, jasmine flowers,
etc.) which can be used alone or in any combination to create
essential oils. Alternatively, alcoholic extracts may be prepared
for compounding fragrances. However, due to the relatively high
costs of obtaining fragrances from natural substances, the modern
trend is to use synthetically prepared fragrances, particularly in
high-volume products. One or more fragrances can optionally be
included in the sunscreen composition in an amount ranging from
about 0.001 to about 5 weight percent, preferably about 0.01 to
about 0.5 percent by weight. Additional preservatives may also be
used if desired and include well known preservative compositions
such as benzyl alcohol, phenyl ethyl alcohol and benzoic acid,
diazolydinyl, urea, chlorphenesin, iodopropynyl and butyl
carbamate, among others.
[0049] The compositions of the invention can further comprise skin
protectant active agents. Suitable examples include (with preferred
weight percent ranges), Allantoin (0.5 to 2 percent); Aluminum
hydroxide gel (0.15 to 5 percent); Calamine (1 to 25 percent);
Cocoa butter (greater than 50); Cod liver oil (5 to 14 percent);
Colloidal oatmeal; Dimethicone (1 to 30 percent); Glycerin (20 to
45 percent); Hard fat (greater than 50); Kaolin (4 to 20 percent);
Lanolin (12.5 to 50 percent); Mineral oil (greater than 50
percent); Petrolatum (greater than 30 percent); Sodium bicarbonate;
Topical starch (10 to 98 percent); White petrolatum (greater than
30 percent); Zinc acetate (0.1 to 2 percent); Zinc carbonate (0.2
to 2 percent); and Zinc oxide (1 to 25 percent).
[0050] The compositions of the invention may further include insect
repelling components. The most widely used insect repelling active
agent for personal care products is N,N-Diethyl-m-toluamide,
frequently called "DEET" and available in the form of a concentrate
containing at least about 95 percent DEET. Other synthetic chemical
repellents include ethyl butylacetylaminoproprionate (also known as
IR 3535), dimethyl phthalate, ethyl hexanediol, indalone,
di-n-propylisocinchoronate, bicycloheptene, dicarboximide and
tetrahydrofuraldehyde. Certain plant-derived materials also have
insect repellent activity, including citronella oil and other
sources of citronella (including lemon grass oil), limonene,
rosemary oil and eucalyptus oil. Choice of an insect repellent for
incorporation into the sunscreen emulsion will frequently be
influenced by the odor of the repellent. The amount of repellent
agent used will depend upon the choice of agent; DEET is useful at
high concentrations, such as up to about 15 percent or more, while
some of the plant-derived substances are typically used in much
lower amounts, such as 0.1 percent or less.
[0051] Topical application of the compositions of the invention
described herein to the hair or skin of a human will provide
enhanced protection against deleterious effects of ultraviolet
radiation (UVR). Thus, the subject invention further provides a
method for protecting human skin and/or hair against the
deleterious effects of solar radiation, more particularly UVR,
which method comprises topically applying thereto an effective
amount of the compositions as described herein containing
sunscreens and one or more antioxidants. An esthetically beneficial
result of exposure of skin to UVR (i.e., light radition wavelengths
of from 280 nm to 400 nm) is the promotion of tanning of the human
epidermis. Another benefit of sun exposure comes from production of
vitamin D within the skin. UVR is typically divided into UV-A
(light wavelengths from 320 to 400 nm) and UV-B (wavelengths
ranging from 280 to 320 nm) regions. Overexposure to UV-B
irradiation is generally understood to lead to skin burns and
erythema. In addition, overexposure to UV-A radiation may cause a
loss of elasticity of the skin and the appearance of wrinkles,
promoting premature skin aging. Such irradiation promotes
triggering of the erythemal reaction or amplifies this reaction in
certain individuals and may even be the source of phototoxic or
photoallergic reactions. It is increasingly believed that
overexposure to UV-A may also lead to melanoma. Thus, the
application of the compositions of the invention to the skin and/or
hair of an individual will provide enhanced UVR photoprotection
(UV-A and/or UV-B) of the skin and/or hair of the individual.
[0052] The invention further provides a method of treating and/or
reversing photodamage of skin by applying the compositions of the
invention to skin that will be or has been exposed to UVR. The term
"treating and/or reversing photodamage" is intended to mean
obtaining an improvement in one or more attributes of skin
condition such as dryness, texture, elasticity/firmness/resiliency,
lines/wrinkles, skin tone/clarity, uniformity of pigmentation,
and/or erythema which condition is exacerbated by exposure to
UVR.
[0053] The sunscreen containing compositions of the invention are
intended to provide a sun protection factor (SPF) rating of at
least 2, with additional preferable embodiments having a sun
protection factor of at least 5, at least 10, at least 15, at least
20, at least 25, at least 30, at least 35, at least 40, at least
45, at least 50, at least 55, at least 60, at least 65, at least
70, at least 75, at least 80, at least 85, at least 90, at least
95, and at least 100. The sunscreen containing compositions of the
invention are also intented to provide U.S. FDA UV-B "star ratings"
of at least one star, at least two stars, at least three stars and
up to four stars.
[0054] The invention will be further described by means of the
following examples, which are not intended to limit the invention,
as defined by the appended claims, in any manner.
Experimental
[0055] DPPH (.alpha.,.alpha.-diphenyl-.beta.-picrylhydrazyl) Free
Radical Test.
[0056] DPPH is a stable free radical that when dissolved in
solution forms an intense purple color. When reduced by an
antioxidant, the purple color fades until it finally disappears as
DPPH is completely reduced. The extent to which the color fades can
be easily measured and used to rank the relative effectiveness of
different materials purported to have antioxidant properties.
Measurements were recorded for antioxidant raw materials in simple
methanol solutions. Samples were prepared by dissolving
antioxidants at various concentrations in methanol. After sample
preparation, 125 .mu.l of the sample or pure methanol as a control
were pipetted into sample test tubes followed by 2 ml of methanol
and vortexed. Then 2 ml of DPPH stock solution (0.25 mM in
methanol) was added to each tube (giving a total volume of 4.125 ml
for each sample) and vortexed. Immediately after addition of DPPH,
test tubes were covered and placed into a 30.degree. C. water bath
for 20 minutes. After the 20 minute incubation, the absorbance of
each sample was recorded at 517 nm using a Perkin Elmer Lambda 40
spectrophotometer. All samples were prepared in triplicate and
their mean absorbance values were used to express the efficacy of
antioxidants at various concentrations in terms of antioxidant
reducing units (ARU) by using the following equation:
ARU=(Absorbance Methanol Control-Absorbance sample).times.10
ARU values range in magnitude from 0 for "no" efficacy to about 15
for raw materials that have high antioxidant efficacy.
[0057] Antioxidant effectiveness for a variety of oil and
water-soluble raw materials purported to have antioxidant
properties appears in Table 1. The raw materials include well-known
antioxidants such as vitamin E, in addition to popular plant
extracts such as green tea, rose, grape and mushrooms, among
others. Effectiveness is expressed as antioxidant reducing units
(ARU), which span values of 0 for no efficacy to 12 for
antioxidants with high efficacy to reduce the DPPH radical.
Examples of both oil and water soluble antioxidants spanned this
range.
TABLE-US-00001 TABLE 1 Antioxidant Level (%) ARU Solubility
Rosemary officinalis oleoresin [ROO] * 0.1 0.0 O Arjunolic acid
(1%) 1.0 0.4 O Diethylhexyl syringylidene malonate 1.0 5.7 O
Vitamin E [Vit. E] * 0.5 6.5 O Tetrahydrocurcumenoids [THC]* 0.5
9.0 O Bioactive photosynthetic complex from 0.1 0.0 W green plants
[BPSC]* Thermus thermophillus ferment 1.0 0.0 W [Thermus]*
Ergothiotaine [ET]* 0.1 0.0 W Rosa gallica extract [Rosa G]* 1.0
0.0 W Foeniculum vulgare (fennel) seed Extract 5.0 0.0 W Soybean
zymbiozome fermentum 1.0 0.0 W Shitake mushroom extract 5.0 0.0 W
Helianthus annuus (sunflowler) extract 1.0 0.0 W Red clover extract
1.0 0.6 W Vitis vinifera (grape) seed extract/Brand B 0.1 1.9 W
Green tea extract [GT]* 0.1 2.2 W Pikea robusta extract 1.0 2.2 W
Tocopherol (and) vitis vinifera (grape) 1.0 7.6 W seed extract
n-Acetyl cysteine [NAC] * 0.1 9.9 W Vitis vinifera (grape) seed
extract/Brand A 0.1 11.5 W Phylanthus emblica fruit extract 0.1
11.5 W [Emblica]*
[0058] Compared with vitamin E, some materials clearly possess
lower (ARU<6.5) while others higher (ARU>6.5) antioxidant
effectiveness. Based upon ARU values alone, it would be expected
that antioxidants with ARU>6.5 would be superior to vitamin E in
their ability to neutralize UVR-induced formation of ROS within the
skin. However, recognizing that ROS covers a wide range of reactive
compounds, including free radicals but also other oxidants like
hydrogen peroxide, singlet oxygen or peroxynitrite, results from
the DPPH free radical test may not adequately predict antioxidant
effectiveness on skin that is exposed to UVR. We, therefore,
selected several antioxidants (denoted with *) from Table 1
covering a range of ARU values to test in model systems that more
closely mimic intact human skin exposed to UVR to understand if Aox
effectiveness in solution as measured by the DPPH free radical test
translated to similar levels of effectiveness on skin.
Ex Vivo Tape Strip Method to Assess Antioxidants Ability to Inhibit
Lipid Peroxidation.
[0059] To determine if antioxidants (Aox) maintain their
effectiveness on skin in the presence of UVR, we devised a novel
and more relevant model that utilizes human skin lipids as
substrates for UVR-induced peroxidation. Lipids removed from skin
on broad pieces of tape serve as the substrates for subsequent
exposure to UVR. By applying a standard lotion with or without Aox
to skin prior to tape-stripping, skin lipids can be collected on
tape strips in the presence or absence of Aox that essentially
maintains the same proximity that lipids and Aox had on skin.
Following UVR exposure, the extent to which the presence of Aox
protect lipids against peroxidation from ROS can be measured
relative to lipids in the absence of antioxidants.
[0060] Human volunteers were recruited for the test and asked not
to apply any products to their arms for at least two days before
the test Prior to any product treatments, inner aspects of
subjects' left and right forearms were wiped with a Kimwipe.TM.
moistened with isopropanol to remove any residues that might be on
the surface of the skin. Arms were wiped only once applying gentle
pressure and then allowed to dry at least 10 minutes before
proceeding. A template (90 mm.times.50 mm) was positioned on each
inner forearm such that two areas could be clearly delineated.
Using a superfine tip Sharpie.TM. pen, a mark was placed at each
corner of the template to outline each application site, with two
sites delineated per forearm. Using a fingercot, either placebo or
antioxidant lotion was applied (100 mg) to a delineated site on a
forearm. Care was taken to insure that products were applied evenly
within the entire application area. After application, sites were
allowed to air-dry for 30 minutes during which subjects were
instructed not to allow any clothing to come into contact with test
areas.
[0061] After lotions dried for 30 minutes, each site was
tape-stripped using a 4.0 inch piece of Scotch.RTM. Brand No. 800
Prescription Label tape (1.5 inches wide). One end of the tape was
folded over to provide an edge that did not adhere to skin for easy
removal. The piece of tape was positioned over a site and then
using a finger the tape was gently pressed onto the skin to make
good contact. Then the tape was quickly removed from the subject's
arm. After removal, all tapes were stored in a dark location such
as a drawer until they were either irradiated with UV or extracted
with isopropanol (i.e., nonirradiated control). Select tape strips
of skin of each subject were irradiated with a dose of 10
joules/cm.sup.2 using a 1000 W Xe arc solar simulator (WG320
filtered). An Optronics OL-754 spectroradiometer was used to adjust
the output of the solar simulator to deliver a constant dose of
UVR. After irradiation, tapes were trimmed to a length of three
inches and placed in 20 ml glass scintillation vials. Then four ml
of isopropanol was added to each vial, after which they were
capped. Vials were then shaken vigorously again and placed in a
-20.degree. C. freezer to extract overnight. The next day samples
were shaken before aliquots were removed for lipid hydroperoxide
(LOOH) analysis.
[0062] Each tape extract was assayed for total LOOH content using a
Lipid Hydroperoxide Assay Kit (Kamiya Biomedical Company,Thousand
Oaks, CA) following manufacturer's directions. Lipid hydroperoxides
were then quantitated by measuring methylene blue formation at 675
nm using a spectrophotometer. Standard curves were prepared using
cumene hydroperoxide and were linear over the range of LOOH
detected in these experiments. Each extract was assayed in
triplicate and the results presented here represent the mean of
those analyses. The standard deviations were typically less than
10%.
[0063] The extent to which antioxidant (Aox) or placebo lotions
inhibited UVR-induced lipid hydroperoxide (LOOH) formation was
calculated by inserting the values of LOOH determined from the four
application sites on each volunteer into the following
equation:
% LOOH Formation = ( LOOH Aox irradiated - LOOH unirradiated
placebo ) ( LOOH irradiated placebo - LOOH unirradiated placebo )
.times. 100 ##EQU00002##
[0064] Calculation of "% LOOH formation" enables each subject to be
his own internal control and normalizes the data with respect to
the area of the tape (3 inches) used to strip skin. In this way,
values for % LOOH formation can be compared between sites on
different people.
[0065] Data were analyzed using paired t-tests to determine if
antioxidant or placebo treatments yielded significantly different
results. An alpha level of 0.05 and a power of 80% was used for all
statistical tests.
[0066] Results for select antioxidants (* in Table 1) to protect
lipids from UVR-induced ROS formation appear in FIG. 1, which
reveals several striking features. Both water and oil soluble
materials can protect lipids against UVR-induced peroxidation;
however, the raw material must be able to partition into the lipid
bilayers to be protective. Vitamin E protects skin lipids in a dose
dependant manner (yellow bars). Tetrahydrocurcurminoids (THC) are
as efficacious as Vitamin E. Vitamin E in combination with Emblica,
GT or THC (orange bars) protect about as well as Vit. E alone
(yellow bars). Surprisingly, some materials (red bars) increase
rather than decrease lipid hydroperoxide (LOOH) levels, acting as
pro-oxidants as opposed to antioxidants. Addition of Vitamin E can
reduce pro-oxidant properties (blue bars) of antioxidants but not
to the same degree as observed with Vitamin E alone.
[0067] These results demonstrate that the DPPH free radical test by
itself is not predictive of an Aox's ability to function
effectively on skin to protect lipids from peroxidation from
UV-induced ROS formation.
Two-Photon Fluorescence Microscopy Imaging of Stratum Corneum.
[0068] Two-photon fluorescence intensity imaging was performed ex
vivo on pieces of human breast skin (.about.0.5 cm.times.0.5 cm) to
detect and quantify levels of UV-induced reactive oxygen species
(ROS) in the stratum corneum using procedures described previously
(Hanson K M, Clegg R M. Photochemistry and Photobiology, 2002,
76(1): 57-63). Test formulas were applied to the surface of skin
samples at 2 mg/cm.sup.2 using the tip of a glass rod. Prior to
irradiation, skin samples were incubated in 100 pmolar
dihydrorhodamine (DHR) in phosphate-buffered saline-ethanol. DHR
partitions into the tissue where it reacts with UV-induced ROS to
produce highly flourescent rhodamine-123, which is subsequently
imaged and quantified as a measure of UV-induced ROS formation. At
least two unique areas are imaged from each skin sample and at each
depth. A base formula without sunscreen actives or antioxidants was
used as a control
[0069] The images shown in FIG. 2 demonstrate the extent of
UV-induced ROS formation that resulted within the full thickness of
stratum corneum after each formula was applied to intact pieces of
human skin and irradiated. The images demonstrate clearly that
exposure to 4 MEDs of full spectrum UV radiation generates abundant
ROS formation. Application of an SPF 30 broad spectrum sunscreen
formula prior to irradiation reduced ROS formation by 39% relative
to the control formula by virtue of its ability to absorb UV before
it can interact with skin to generate ROS. However, application of
an SPF 30 formula plus antioxidants (0.5% vitamin E, 0.1% Emblica)
reduced ROS formation by a total of 73% relative to the control
formula, which represents an additional reduction in ROS of 34%
compared with the formula that only contains the sunscreen
actives.
[0070] Thus, these results demonstrate convincingly that addition
of antioxidants of the right type and level can complement
sunscreens as an additional strategy to protect skin from the
harmful effects of UV-induced ROS formation. Exposure of skin to
UVR can generate an abundance of ROS even through a protective
layer of broad spectrum SPF 30 sunscreen. With the power to
neutralize ROS, antioxidants (Aox) can provide measurable and
meaningful levels of protection against the damaging effects of ROS
and in this way significantly augment the protective power of
sunscreens provided, however, that Aox for use in sunscreens are
selected appropriately.
Protection through Basal Layer with Antioxidant Combination
[0071] A. Lipid Peroxidation Inhibition
[0072] Vitamin E is highly effective at neutralizing UV-induced ROS
in the outer layer of epidermis, the stratum corneum. Placebo and
experimental formulations were prepared to compare the
formulations' ability to inhibit lipid peroxidation. The
formulations used are shown in Table 2.
TABLE-US-00002 TABLE 2 Placebo and Antioxidant Lotions Used in Tape
Strip Studies Placebo Placebo Antioxidant Antioxidant Lotion Lotion
Lotion Lotion Ingredient %, w/w %, w/w %, w/w %, w/w Part A USP
purified 86.75 86.75 86.15 85.35 water Simulgel NS 2.00 -- 2.00
Sepigel 305 -- 2.00 2.00 -- Sodium cetearyl 0.250 0.25 0.25 0.25
sulfate Emblica 0.1 -- Part B Octyl palmitate 10.0 -- 10.0
Isopropyl laurate -- 10.0 10.0 -- Vitamin E -- 0.50 0.50 Oxynex ST
-- -- 0.90 Part C Germaben II 1.00 1.00 1.00 1.00
The formulations were prepared by adding sodium cetearyl sulfate to
the water of part A and mixing, followed by addition of either
Simulgel NS or Sepigel 305 and mixing thoroughly. Then the Part B
ingredients were added with mixing, followed by Germaben II in Part
C. After all ingredients are added, the emulsion was mixed
thoroughly.
[0073] Using the ex vivo tape strip method as described above, a
composition containing vitamin E, by itself or in combination with
Emblica, was found to be highly effective at protecting skin's
lipids on its outer surface from UV-induced oxidation mediated by
ROS (Table 3).
TABLE-US-00003 TABLE 3 % Inhibition of Antioxidant LOOH 0.05% vit E
-3.40 0.10% vit E 28.4 0.25% vit E 61.0 0.1% Emblica 19.6 0.3%
Emblica 10.9 0.05% vit E + 0.1% 28.1 Emblica 0.10% vit E + 0.1%
47.8 Emblica 0.25% vit E + 0.1% 65.0 Emblica 0.50% vit E + 0.1%
74.4 Emblica
[0074] B. Two Photon Fluorescence Microscopy
[0075] Two photon fluorescence microscopy was then conducted on a
formulation containing Vitamin E and Emblica to determine ability
to inhibit formation of ROS. The methods used were similar to those
as described above, using confocal microscopy to visualize cells at
different depths within intact pieces of skin and then fluorescence
to quantify the extent of ROS formation. In the present experiment,
however, instead of using human breast skin, the skin used was the
EpiDerm.TM. Skin Model (MatTek Corporation, Mass. USA), which
consists of normal, human-derived epidermal keratinocytes which
have been cultured to form a multilayered, highly differentiated
model of the human epidermis. Ultrastructurally, the EpiDerm.TM.
Skin Model closely parallels human skin, thus providing a useful in
vitro model to study the ability of antioxidants to neutralize ROS
formed during exposure to UVR down to the basal layer.
[0076] Prior to UVR exposure, pieces of EpiDerm.TM. skin were
treated with dihydrorhodamine, which partitions throughout the
aqueous and lipid regions of the tissue. Upon exposure to UVR,
dihydrorhodamine in the tissue reacts chemically with ROS wherever
it forms to generate a highly fluorescent molecule. The
fluorescence is subsequently detected and quantified to provide an
indication of the level of UV-induced ROS formed in deeper layers
of the skin. By applying antioxidants topically before irradiation
ROS formation can be measured and compared to the ROS formed after
a placebo lotion without antioxidants was applied to the skin. In
this way, the efficacy of antioxidants to neutralize ROS within
deeper layers of the skin can be measured and their relative
efficiencies established.
[0077] Using this method, the experimental formulation containing
vitamin E and emblica was tested to determine its ability to
neutralize ROS within the basal layer or bottom layer of the
epidermis after exposure to 4 MED. The experimental formulation and
placebo lotion used in this experiment were prepared as
follows.
TABLE-US-00004 TABLE 4 Placebo Lotion Part A USP purified water
60.04% Acrylates/c10-30 alkyl acrylate crosspolymer 0.30% Part B
Propylene glycol 5.00% Disodium EDTA 0.01% Triethanolamine, 99%
0.35% Part C Octyl palmitate 29.00% PVP/Eicosene copolymer 2.00%
Stearic acid 0.50% Polyglyceryl-3 distearate 0.29% Methylparaben
0.30% Sorbitan isostearate 0.71% Propylparaben 0.10% Dimethicone,
50 cst 0.40% Part D Benzyl alcohol 1.00%
TABLE-US-00005 TABLE 5 Antioxidant Lotion Part A Usp purified water
58.54% Acrylates/c10-30 alkyl acrylate crosspolymer 0.30% Part B
Propylene glycol 5.00% Disodium EDTA 0.01% Triethanolamine, 99%
0.35% Part C Octyl palmitate 29.00% Pvp/eicosene copolymer 2.00%
Stearic acid 0.50% Polyglyceryl-3 distearate 0.29% Methylparaben
0.30% Sorbitan isostearate 0.71% Vitamin E, dl alpha tocopherol
0.50% Emblica 0.10% Propylparaben 0.10% Dimethicone, 50 cst 0.40%
Part D Benzyl alcohol 1.00%
A water phase was created by adding Acrylates/C10-30 Alkyl Acrylate
Crosspolymer to water of Part A while stirring and mixed until
clear and lump-free. While mixing, the propylene glycol and
disodium EDTA were added to the water phase mixture of Part A and
mixed well for 10 minutes. Triethanolamine of Part B was then added
to the water phase mixture and continued mixing well. Separately an
oil phase was created by mixing the ingredients of Part C together
and heat to 140-145.degree. F. while mixing well. The oil phase was
then added to the water phase and continued mixing to form an
emulsion. The emulsion was cooled to room temperature and then
benzyl alcohol of part D was added to the cool emulsion and mixed
thoroughly. Additional water was added QS to weight. The difference
between the placebo and experimental formulation was the addition
of antioxidants into the oil phase.
[0078] As shown in FIG. 3, while highly effective in the outer
layers of the epidermis, the combination of 0.5% vitamin E and 0.1%
emblica only reduced ROS formation in the basal layer of epidermis
by about 5%.
[0079] We next evaluated a wide range of ingredients used within
the cosmetic industry for their claimed antioxidant ability, and
were surprised to observe that many of them behaved as pro-oxidants
at the basal layer on skin exposed to UVR. As demonstrated in FIG.
4, rather than decreasing levels of ROS, these ingredients
increased levels of ROS by up to 250% within the basal layer of
epidermis relative to a placebo lotion without any antioxidant.
Many of these ingredients that behaved as pro-oxidants represented
natural plant extracts from fennel seeds, rose and white grapes.
Together these results demonstrated that antioxidants used in
suncare products where intentional sun exposure occurs need to be
selected judiciously. Moreover the tests demonstrate that the
methods of the invention reveal that not all ingredients identified
as "antioxidants" provide actual antioxidant properties in real
world use with UVR exposures.
[0080] We next tested an experimental formulation containing a
combination of antioxidant vitamin E and diethylhexyl syringylidene
malonate (Oxynex.RTM. ST, Merck KGaA, Germany) to determine whether
the combination would be effective protection against UV-induced
ROS formation throughout the epidermis. Placebo and experimental
formulations were prepared similar to the methods described
above:
TABLE-US-00006 TABLE 6 Placebo Lotion Part A USP purified water
60.04% Acrylates/c10-30 alkyl acrylate crosspolymer 0.30% Part B
Propylene glycol 5.00% Disodium EDTA 0.01% Triethanolamine, 99%
0.35% Part C Octyl palmitate 29.00% PVP/Eicosene copolymer 2.00%
Stearic acid 0.50% Polyglyceryl-3 distearate 0.29% Methylparaben
0.30% Sorbitan isostearate 0.71% Propylparaben 0.10% Dimethicone,
50 cst 0.40% Part D Benzyl alcohol 1.00%
TABLE-US-00007 TABLE 7 Antioxidant Lotion Part A Usp purified water
58.54% Acrylates/c10-30 alkyl acrylate crosspolymer 0.30% Part B
Propylene glycol 5.00% Disodium EDTA 0.01% Triethanolamine, 99%
0.35% Part C Octyl palmitate 29.00% Pvp/eicosene copolymer 2.00%
Stearic acid 0.50% Polyglyceryl-3 distearate 0.29% Methylparaben
0.30% Sorbitan isostearate 0.71% Vitamin E, dl alpha tocopherol
0.50% Diethylhexyl syringylidene malonate 0.90% Propylparaben 0.10%
Dimethicone, 50 cst 0.40% Part D Benzyl alcohol 1.00%
[0081] A water phase was created by adding Acrylates/C10-30 Alkyl
Acrylate Crosspolymer to water of Part A while stirring and mixed
until clear and lump-free. While mixing, the propylene glycol and
disodium EDTA were added to the water phase mixture of Part A and
mixed well for 10 minutes. Triethanolamine of Part B was then added
to the water phase mixture and continued mixing well. Separately an
oil phase was created by mixing the ingredients of Part C together
and heat to 140-145.degree. F. while mixing well. The oil phase was
then added to the water phase and continued mixing to form an
emulsion. The emulsion was cooled to room temperature and then
benzyl alcohol of part D was added to the cool emulsion and mixed
thoroughly. Additional water was added QS to weight. The difference
between the placebo and experimental formulation was the addition
of antioxidants into the oil phase.
[0082] As shown in the Table 8 and FIGS. 5 and 6, in vitro ROS
results demonstrate that a lotion containing both Vitamin E and
Oxynex.RTM. ST together provided substantially higher protection
from UV-induced ROS formation than can be achieved by Vitamin E and
Emblica.
TABLE-US-00008 TABLE 8 % ROS Neutralized Antioxidant in the Basal
Layer After 1 MED Exposure 0.5% vitamin E + 0.1% Emblica ND 0.5%
vitamin E + 0.9% Oxynex 47 ST After 4 MED Exposure 0.5% vitamin E +
0.1% Emblica 5 0.5% vitamin E + 0.9% Oxynex 33 ST ND = not
determined.
The addition of Oxynex.RTM. ST to a lotion containing Vitamin E
neutralized 33% ROS after exposure to 4 MED of UVR and neutralized
47% ROS after 1 MED UVR within the basal layer. These results are
significantly better than 5% ROS neutralization for a lotion
containing only Vitamin E and Emblica.
[0083] The compositions containing the combination of 0.5% Vitamin
E and 0.9% Oxynex.RTM. ST were also tested in the lipid
peroxidation tests described above and shown to prevented
UV-induced lipid peroxidation by 75%. This is comparable to results
obtained for 0.5% Vitamin E plus 0.1% Emblica. However, although
the compositions were similar in ability to inhibit lipid
peroxidation, by combining Vitamin E with Oxynex.RTM. ST we have
observed an unexpected increase in protection from ROS formation
across the full thickness of epidermis.
[0084] These results also confirm that Oxynex.RTM. ST maintains its
antioxidant capability within skin when exposed to UVR as opposed
to becoming a pro-oxidant. Taken together, these results
demonstrate the unexpected benefits of combining Vitamin E with
Oxynex.RTM. ST for protection against UV-induced ROS formation
within the full thickness of epidermis.
Cosmetic Clinical Efficacy Evaluation of High SPF Antioxidant
Formulation
[0085] Two topical antioxidant sunscreen formulation were generated
according to the methods of the invention containing the sunscreen
and antioxidant loads:
TABLE-US-00009 Ingredient SPF 70 SPF 30 Sunscreen Homosalate 15%
15% Octocrylene 10% 2% Avobenzone 3% 2% Oxybenzone 6% 5% Octisalate
5% 5% Antioxidant Diethylhexyl sringylidene 0.9% -- malonate
Vitamin E 0.5% 0.5% Emblica -- 0.01% All percentages are w/w.
[0086] In vivo SPF testing conducted according to the U.S. F.D.A.
approved testing protocols determined that the first formulation
rated as at least SPF 70 and the second was at least SPF 30. For
convenience they will be referred to herein as SPF 70 and SPF 30.
Methods of in vitro and in vivo measurement of SPF are describe,
e.g., in U.S. Patent Application Publication Nos. 20070160549 and
20080081024.
[0087] A clinical efficacy evaluation was conducted to determine
the effect of the high SPF antioxidant formulations produced as
described above on various indications of skin health including,
skin dryness (moisture), skin texture (roughness, smoothness),
elasticity (i.e., firmness or resiliency), skin tone and clarity,
uniformity of pigmentation, fine lines and wrinkles, erythema,
photodamage, and hidden damage (subclinical pigmentation). The
study is a single-blind, parallel, randomized, controlled, twelve
(12) week use test with an additional baseline equilibration period
of seven (7) days. One-hundred-nine (109) subjects were enrolled
and one-hundred-five (105) continued on the study. Four (4)
subjects were discontinued due to inability to make all regularly
scheduled visits. No adverse experiences have been reported. The
results below demonstrate significant improvement over baseline
condition of the skin as a result of 12 weeks of use of the tested
products.
[0088] Qualified subjects were divided into two (2) product groups
and an untreated control group. An expert clinical evaluator graded
the face of each subject at each visit to assess individual
parameters that contribute to the visual and tactile properties of
premature aging, as well as to provide an overall global assessment
of degree of visible photodamage. The clinical grading scores at
baseline were used to confirm that the subject presents with mild
to moderate photo-damage and therefore were qualified for
participation.
[0089] Specific attributes were quantified using
bioinstrumentation; silastic resin replicas with image analysis to
also measure fine lines and wrinkles, a Nova Meter for determining
moisture content, and a Dermalab suction device to measure skin
elasticity. A trained photographer photographed each subject at
baseline, 2, 4, 8 and 12 weeks using a fixed angle standard and
cross-polarized light Canfield clinical camera apparatus to
document appearance of a specific site on the side of the face
(including crow's feet area). The photographer took full facial UV
reflected photographs, at baseline and at the 12-week visit or
until the product had washed out and no longer fluoresces
(whichever was later). The expert clinical evaluator graded the UV
photographs taken at baseline and 12-weeks (.+-.days to washout) to
assess degree of subclinical "hidden" damage present and then
globally assessed the amount of change compared to baseline.
[0090] Test compositions were overwrapped to hide the identity of
the manufacturer and labeled with the appropriate test article
codes and use directions. Approximately one-half the study product
was delivered prior to the start of the study and the second half
of the product was delivered prior to the mid-point of the
study.
[0091] Seven to ten days prior to initiation of the treatment
period, subjects underwent a baseline equilibration period, during
which they discontinued the use of all facial sunscreens, skin
treatment products, their current facial cleansing bar or cream,
and any moisturizing facial cosmetic products; use Camay soap
daily, each morning for any facial cleansing and as needed
throughout the day; and refrain from use of tanning beds for the
duration of the study.
[0092] Following the equilibration period, subjects were qualified
by presenting with sufficient signs of dryness and extrinsic skin
aging. Following qualification, subjects were randomly assigned to
one of two test groups or to the untreated control group. For the
duration of the study subjects assigned to both the treatment and
non-treatment groups wash their faces only with the Camay soap
provided. Subjects in both of the treatment groups applied the
assigned test article to their face (and neck if desired) once
daily (each morning), then reapply as needed.
[0093] Subjects recorded application times each day on a diary
provided by the study site at each visit. Subjects in the
non-treatment group recorded the number of times that they cleanse
and apply their usual moisturizer, sunscreen and color cosmetic
products during the study. Diaries were collected and redistributed
in the same manner as outlined for the treatment groups.
[0094] All subjects had clinical skin evaluations, Nova Meter,
Dermalab, Silastic resin replicas, and standard light photography
after 2, 4, 8 and 12 weeks. Subjects have UV photos taken only at
baseline and 12 weeks (.+-.days for washout) by an expert clinical
evaluator who was not aware of product assignment, nor which
subjects were in the treatment groups and which subjects were in
the non-treated control group.
[0095] Evaluations were made at each visit for several indicators
of photodamage listed below. Grading scales are outlined in each of
the categories
A. Overall Assessment of Degree of Photodamage.
[0096] Subjects were graded on a scale of 0-10 with 0 representing
no photodamage and 10 representing severe photodamage. The results
were as follows:
TABLE-US-00010 TABLE 9 MEAN OVERALL PHOTODAMAGE SCORE .+-. S.D. (%
IMPROVEMENT) SPF 30 SPF 70 CONTROL BASELINE 4.80 .+-. 0.97 4.72
.+-. 0.84 4.71 .+-. 0.79 (n = 39) (n = 39) (n = 27) WEEK 2 4.41
.+-. 0.73* (8) 4.45 .+-. 0.51* (5) 4.44 .+-. 0.60* (6) (n = 39) (n
= 39) (n = 27) WEEK 4 4.04 .+-. 0.62* (16) 4.15 .+-. 0.68* (12)
4.14 .+-. 0.60* (12) (n = 39) (n = 39) (n = 27) WEEK 8 4.40 .+-.
0.70* (8) 4.42 .+-. 0.51* (6) 4.48 .+-. 0.64.sup.T (5) (n = 39) (n
= 37) (n = 26) WEEK 12 3.97 .+-. 0.49* (17) 3.86 .+-. 0.38* (18)
3.95 .+-. 0.38* (16) (n = 39) (n = 39) (n = 27) *Significantly
different than baseline value, p .ltoreq. 0.050. .sup.TTrendwise
significantly different than baseline value, p-0.150-0.051.
B. Facial Dryness.
[0097] Subjects were given grades of 0- 4 as follows: [0098] 0
normal skin; no signs of dryness [0099] 1 mild dryness; slight but
definite dryness, fine scaling present may have a powdery or ashy
appearance [0100] 2 moderate dryness; somewhat coarser scaling,
some cracking evident as uplifted scales.
[0101] 3 marked dryness; marked coarse scaling, cracking evident as
uplifted scales. [0102] 4 severe dryness;very marked; very coarse
scaling; cracking progressing to fissuring; ertyhema may be
present. The results were as follows:
TABLE-US-00011 [0102] TABLE 10 MEAN DRYNESS SCORE .+-. S.D. (%
IMPROVEMENT) SPF 30 SPF 70 CONTROL BASE- 0.97 .+-. 0.16 1.05 .+-.
0.22 1.07 .+-. 0.26 LINE (n = 39) (n = 39) (n = 27) WEEK 2 0.20
.+-. 0.52* (79) 0.23 .+-. 0.48* (78) 0.11 .+-. 0.32* (90) (n = 39)
(n = 39) (n = 27) WEEK 4 0.12 .+-. 0.40* (88) 0.15 .+-. 0.36* (86)
0.18 .+-. 0.48* (83) (n = 39) (n = 39) (n = 27) WEEK 8 0.33 .+-.
0.57* (66) 0.10 .+-. 0.31* (91) 0.26 .+-. 0.53* (76) (n = 39) (n =
37) (n = 26) WEEK 12 0.00 .+-. 0.00* (100) 0.00 .+-. 0.00* (100)
0.11 .+-. 0.32* (90) (n = 39) (n = 39) (n = 27)
[0103] C. Texture (Roughness/Smoothness)
[0104] Subjects were graded from a score of 0, indicating smooth,
even surface, to 10 indicating a rough, coarse, uneven surface. The
results were as follows:
TABLE-US-00012 TABLE 11 MEAN TEXTURE SCORE .+-. S.D. (%
IMPROVEMENT) SPF 30 SPF 70 CONTROL BASELINE 3.68 .+-. 0.69 3.86
.+-. 0.61 3.64 .+-. 0.63 (n = 39) (n = 39) (n = 27) WEEK 2 2.24
.+-. 1.00* (39) 2.56 .+-. 0.94* (34) 2.35 .+-. 1.02* (35) (n = 39)
(n = 39) (n = 27) WEEK 4 2.58 .+-. 0.97* (30) 2.25 .+-. 1.17* (42)
2.30 .+-. 1.02* (37) (n = 39) (n = 39) (n = 27) WEEK 8 2.62 .+-.
0.87* (29) 2.88 .+-. 0.95* (25) 2.70 .+-. 0.87* (26) (n = 39) (n =
37) (n = 26) WEEK 12 2.52 .+-. 0.74* (32) 2.35 .+-. 0.73* (39) 2.64
.+-. 0.64* (28) (n = 39) (n = 39) (n = 27) *Significantly different
than baseline value, p .ltoreq. 0.050.
D. Elasticity/Firmness/Resiliency
[0105] Subjects were graded from a score of 0, indicating firm,
resilient, taut skin, to 10 indicating skin that was loose,
flaccid, no turgor. The results were as follows:
TABLE-US-00013 TABLE 12 MEAN ELASTICITY/FIRMNESS/RESILIENCY SCORE
.+-. S.D. (% IMPROVEMENT) SPF 30 SPF 70 CONTROL BASELINE 5.16 .+-.
1.13 5.18 .+-. 0.94 5.21 .+-. 1.03 (n = 39) (n = 39) (n = 27) WEEK
2 4.24 .+-. 1.13* (18) 4.19 .+-. 1.06* (19) 4.21 .+-. 1.20* (19) (n
= 39) (n = 39) (n = 27) WEEK 4 4.15 .+-. 0.97* (20) 4.00 .+-. 0.80*
(23) 4.30 .+-. 1.08* (18) (n = 39) (n = 39) (n = 27) WEEK 8 3.77
.+-. 0.94* (27) 3.81 .+-. 0.92* (26) 4.05 .+-. 1.00* (22) (n = 39)
(n = 37) (n = 26) WEEK 12 3.33 .+-. 0.92* (36) 3.29 .+-. 1.03* (37)
3.67 .+-. 0.87* (30) (n = 39) (n = 39) (n = 27) *Significantly
different than baseline value, p .ltoreq. 0.050.
E. Lines and Wrinkles
[0106] Subjects were graded from a score of 0, indicating no lines
or wrinkles, to 10 indicating coarse skin containing numerous
wrinkles. The results were as follows:
TABLE-US-00014 TABLE 13 MEAN FINE LINES/WRINKLES SCORE .+-. S.D. (%
IMPROVEMENT) SPF 30 SPF 70 CONTROL BASELINE 4.12 .+-. 1.42 4.78
.+-. 1.32 4.44 .+-. 1.58 (n = 39) (n = 39) (n = 27) WEEK 2 3.92
.+-. 1.18 (5) 4.28 .+-. 0.72* (11) 4.37 .+-. 1.21 (2) (n = 39) (n =
39) (n = 27) WEEK 4 4.12 .+-. 0.95 (0) 4.15 .+-. 0.80* (13) 4.07
.+-. 1.29.sup.T (8) (n = 39) (n = 39) (n = 27) WEEK 8 3.55 .+-.
0.90* (14) 3.75 .+-. 0.74* (22) 3.81 .+-. 0.97* (14) (n = 39) (n =
37) (n = 26) WEEK 12 3.28 .+-. 0.89* (20) 3.58 .+-. 0.69* (25) 3.49
.+-. 1.06* (21) (n = 39) (n = 39) (n = 27) *Significantly different
than baseline value, p .ltoreq. 0.050. .sup.TTrendwise
significantly different than baseline value, p-0.150-0.051.
F. Skin Tone/Clarity
[0107] Subjects were graded from a score of 0, indicating clear,
radiant, translucent skin, to 10 indicating skin that was sallow,
dull and/or had uneven skin tone. The results were as follows:
TABLE-US-00015 TABLE 14 MEAN SKIN TONE SCORE .+-. S.D. (%
IMPROVEMENT) SPF 30 SPF 70 CONTROL BASELINE 5.57 .+-. 0.81 5.40
.+-. 0.65 5.44 .+-. 0.85 (n = 39) (n = 39) (n = 27) WEEK 2 5.07
.+-. 0.69* (9) 5.11 .+-. 0.60* (5) 5.33 .+-. 0.56 (2) (n = 39) (n =
39) (n = 27) WEEK 4 4.90 .+-. 0.70* (12) 4.78 .+-. 0.77* (12) 4.84
.+-. 0.76* (11) (n = 39) (n = 39) (n = 27) WEEK 8 4.62 .+-. 0.68*
(17) 4.50 .+-. 0.69* (17) 4.53 .+-. 0.79* (17) (n = 39) (n = 37) (n
= 26) WEEK 12 4.14 .+-. 0.75* (26) 3.92 .+-. 0.80* (27) 3.98 .+-.
0.62* (27) (n = 39) (n = 39) (n = 26) *Significantly different than
baseline value, p .ltoreq. 0.050.
G. Uniformity of Pigmentation
[0108] Subjects were graded from a score of 0, indicating uniform,
even pigmentation, to 10 indicating skin that was uneven, blotchy
or mottled. The results were as follows:
TABLE-US-00016 TABLE 15 MEAN UNIFORMITY OF PIGMENTATION SCORE .+-.
S.D. (`% IMPROVEMENT) SPF 30 SPF 70 CONTROL BASELINE 5.07 .+-. 0.94
4.90 .+-. 0.99 4.95 .+-. 1.09 (n = 39) (n = 39) (n = 27) WEEK 2
4.80 .+-. 0.74.sup.T (5) 4.67 .+-. 0.77.sup.T (5) 4.76 .+-. 0.73
(4) (n = 39) (n = 39) (n = 27) WEEK 4 4.99 .+-. 0.89 (2) 4.81 .+-.
0.90 (2) 4.84 .+-. 0.84 (2) (n = 39) (n = 39) (n = 27) WEEK 8 4.42
.+-. 0.66* (13) 4.38 .+-. 0.59* (11) 4.37 .+-. 0.78* (12) (n = 39)
(n = 37) (n = 26) WEEK 12 4.14 .+-. 0.73* (18) 4.04 .+-. 0.78* (18)
3.97 .+-. 0.69* (20) (n = 39) (n = 39) (n = 27) *Significantly
different than baseline value, p .ltoreq. 0.050. .sup.TTrendwise
significantly different than baseline value, p-0.150-0.051.
H. Erythema
[0109] Subjects were graded from a score of 0, indicating no
erythema or normal tone, to 10 indicating skin that was marked,
very red. The results were as follows:
TABLE-US-00017 TABLE 16 MEAN NOVA METER VALUE .+-. S.D. (%
IMPROVEMENT) SPF 30 SPF 70 CONTROL BASELINE 202.55 .+-. 87.93
203.12 .+-. 88.65 218.92 .+-. 114.92 (n = 39) (n = 39) (n = 27)
WEEK 2 179.17 .+-. 64.82.sup.T (-12) 180.15 .+-. 74.05.sup.T (-11)
196.22 .+-. 86.17 (-10) (n = 39) (n = 39) (n = 27) WEEK 4 160.17
.+-. 42.24* (-21) 177.0 .+-. 72.68* (-13) 191.74 .+-. 77.53 (-12)
(n = 39) (n = 39) (n = 27) WEEK 8 158.97 .+-. 39.01* (-22) 150.10
.+-. 38.63* (-26) 166.73 .+-. 57.76* (-24) (n = 39) (n = 37) (n =
26) WEEK 12 169.89 .+-. 45.02* (-16) 182.35 .+-. 60.00.sup.T (-10)
179.59 .+-. 62.21* (-18) (n = 39) (n = 39) (n = 27) *Significantly
different than baseline value, p .ltoreq. 0.050. .sup.TTrendwise
significantly different than baseline value, p = 0.150-0.051.
[0110] Facial skin condition was measured on all subjects using
Dermalab, Novameter and replica image analysis at baseline and
weeks 2, 4, 8 and 12 as follows.
[0111] Elasticity was measured on one side of the face (same
location at each visit) on all subjects using the Dermalab (Cortex
Technology, Denmark), which applies a negative pressure to the skin
surface and calculates the height to which the skin can be drawn up
and the rate at which it returns to equilibrium thus providing a
measurement of elasticity. Dermalab measurements took place on the
opposite side of the face as image analysis replicas.
[0112] Moisturization was measured on one side of the face (same
location at each visit) to document hydration levels of the skin
surface. The relative degree of skin hydration is assessed using
the Dermal Phase Meter 9003 (NOVA meter). Measurements are made by
applying an alternating voltage to the skin with a closely spaced
pair of electrodes and measuring the impedance. Changes in water
content change the impedance of the capacitive circuit. The first
two consecutive readings within 10% were recorded. The same side of
the face is measured at each visit. The test room temperature and
humidity will be recorded during each set of readings.
[0113] Contour (surface textural) analysis provides a method for
quantifying skin augmentation, the cosmetic action of reducing
lines and wrinkles. For this procedure, skin replicas made of the
crow's feet area were analyzed for contour and surface texture
using image analysis. Skin replicas of the crow's feet area were
prepared using silastic resin impression materials (Cuderm).
Silastic resin is a rapidly curing liquid applied using 1 cm
diameter replica rings which stay intact after application and
removal.
[0114] Facial skin condition was documented for all subjects using
standard and cross polarized light photography at baseline and
weeks 2, 4, 8 and 12. Hidden damage accumulated below the skin
surface was evaluated by expert graders, based on UV photos taken
at baseline and 12-weeks.
[0115] Digital photographs using both visible and cross-polarized
light are taken of all subjects at all visits. Subjects' faces are
positioned in the Canfield stereotactic repositioning apparatus and
photographs are taken using the Canfield Clinical Systems camera
and flash system. The camera used was a Nikon D80 SLR 35 mm model
with a 60 mm macro Nikkor lens and a modified SB-23 flash head. The
camera is set in Aperture priority automatic at f I6. For each
subject at each time interval, a slate was photographed at 1:6
magnification identifying the subject and time interval. A frontal
photo was taken at 1:6 magnification and two lateral 45.degree.
angle photos of each side of the face is taken at 1:4 and 1:3
magnification using standard lighting and repeated using
cross-polarized light.
[0116] Subjects placed their heads in the Canfield stereotactic
repositioning device and have photographs taken using the Canfield
Clinical Systems camera system. The camera used was a Nikon 6006
SLR 35 mm model. For each subject at each time interval, a slate
was photographed at 1:6 magnification identifying the subject and
time interval. A frontal photo was taken at 1:6 magnification and
one lateral 45.degree. angle photo of each side of the face was
taken at 1:6 magnification. Subsequently, two frontal UV-light (UV
reflected) photographs were taken at 1:6 magnification employing a
Kodak 18A filter over the lens, a Sunpak MS 4000 Monolight and
T-Max 400 black and white print film. Exposures were taken at f8
and 1/250 sec. shutter speed.
[0117] An expert evaluator graded full facial photos individually
for uniformity of pigmentation at each time-point (baseline and
12-week) grading on a scale of 0 (uniform/even) to 10 (uneven,
blotchy mottled). The expert evaluator also conducted a comparative
assessment of 12-week photos vs. baseline for each individual
subject. Grading scales are from -4 to +4 as follows: [0118] -4
extreme increase in hyper pigmentation [0119] -3 moderate increase
in hyper pigmentation [0120] -2 mild increase in hyper pigmentation
[0121] -1 barely perceptible increase (worsening) in hyper
pigmentation [0122] 0 no difference between baseline and 12-week
[0123] 1 barely perceptible decrease (improvement) in hyper
pigmentation [0124] 2 mild in hyper pigmentation 3 moderate
decrease in hyper pigmentation [0125] 4 extreme decrease in hyper
pigmentation
Example Formulations
[0126] Example sunscreen formulations are prepared according to the
methods described herein with the following ingredients:
TABLE-US-00018 TABLE 17 Ingredient Amount, % w/w Purified Water
45.0-90.0 Homosalate 5.0-15.0 Octocrylene 2.0-10 Oxybenzone 0.5-6.0
Octisalate 5.0 Avobenzone 1.0-3.0 Prolipid 141 2.0-7.0 Butylene
Glycol 2.0-7.0 Microcrystalline Cellulose/ 0.2-5.0
Carboxymethylcellulose Benzyl Alcohol 0.5-2.0 Vitamin E 0.01-3.0
Diethylhexyl syrigylidene malonte 0.01-6.0 Phylanthus Emblica fruit
extract 0.01-1.0 Green Tea Extract 0.01-1.0 Disodium
Lauriminodiproprionate 0.3-3.0 Tocopheryl Phosphates Chlorphenesin
.10-0.20 Butylated PVP .05-.50 Disodium EDTA 0.01-.20 Sodium
Ascorbyl Phosphate .01-1.0 Vitamin A Palmitate .01-1.0
* * * * *