Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
-l- 132~774
COSMETIC PREPARATION INCORPORATING STABILIZED
ASCORBIC ACID
Techni~al Field
The present invention relates generally to cosmetic prepara-
tions, and more speci~ically to cosmetic preparations incorpor-
ating stabilized ascorbic acid (Vitamin C) which may topically be
applied to human skin to impart beneficial appearance effects
thereto.
Back~L~nd Art
It is well known that ascorbic acid (or Vitamin C as it is
synonomously referred to herein) is essential to the maintenance
of a healthy and attractive skin appearance in h~mans~ Vitamin C
helps to stimulate and regulate the production of collagen in
human skin tissue thus retarding the formation of wrinkles and
otherwise helping to avoid a prematurely aged look to skin which,
in turn, helps to maintain a healthier and younger looking
appearance longer. Vitamin C also acts to help prevent or mini
mize lipid oxidation and other forms of cellular damage resulting
from prolonged exposure to the sun's ultraviolet rays, further
counteracting premature aging of the skin. It is believed
further still that ascorbic acid helps to inhibit i) ~he forma-
tion of melanin which leads to skin discoloration during the
aging process, and ii) the release of histamine from c~llular
membranes believed to be responsible for many allergenic reac-
tions, particularly among individuals having so-called sensitive
skin.
,
:`
-2- 132~77~
Because o~ these many beneficial effects, it has long been a
desirable objective to percutaneously deliver effective concen-
trations of ascorbic acid directly to the skin's underlying
tissue matrix (i.e. the dermal layer) via a topically applied,
cosmetically elegant carrier or base. Although ascorbic acid isreadily soluble in water, it oxidizes rapidly in aqueous solu-
tions, and thus cannot be stabilized in sufficient concentration
in such media to achieve skin appearance enhancement effects.
Solubility of ascorbic acid in non-aqueous media on the other
hand is quite limited, thereby preventing an anhydrous base from
achieving the desired level of stability and therefore, efficacy.
For these reasons, unstabilized ascorbic acid heretofore has been
used in cosmetic formulas only in trace amounts of less than
about .1~ by weight to serve as an antioxidant, and prior
attempts to develop and market an acceptable cosmetic formulation
containing efficacious concentrations of stabilized ascorbic acid
have failed.
Disclo~ure of th~ Inv~n~ion
Against the foregoing background, it has been discovered
that ascorbic acid, at concentration levels greatly exceeding .1%
by weight, that is, in the range of about 1~ to about 10~ by
weight, may be stabilized indefinitely in a base or carrier
adapted to be topically applied to the skin. The compositions of
the present invention are formed by dissolving the Vitamin C
ingredient in a solution comprising at least two co-solventss one
of which is water, the other of which comprises a non-aqueous,
organic liguid miscible with water, or a combination of one or
- . , . ,. , ~ . -., . ~ . .
- -3~ 2~77~
more such liquids. The organic liquid co-solvent or solvents
preferably are selected to produce a cosmetically elegant formu-
lation which when topically applied to the skin facilitates rapid
percutaneous absorption of the stabilized ascorbic acid contained
S therein whereupon beneficial effects resulting in enhanced skin
appearance may be achieved. The amount of water contained in the
composition may range as high as 12% by weight without adversely
affecting the stability of the ascorbic acid component.
~est ~o~of ~arrying ~ut the Invention
In accordance with the present invention, ascorbic acid
(Vitamin C) is stabilized in a cosmetic composition adapted to be
topically applied to human skin.
Ascorbic acid, L-ascorbic acid, and/or Vitamin C are the
equivalent trivial chemical names given to a white, odorless,
crystalline solid having ~he formula C6H806, a molecular weight:
176.13, and the following structural formula:
' HO~CH20H
1/ \
~ .
HO OH
From Table I below it is seen that ascorbic acid is rela-
tively soluble in aqueous media, but is relatively insoluble in
organic solvents such as alcohol, glycol, fats and oils.
. ` ; , , ,, - ~ ~ , ~ . .
--4--
la~77~
TABLE I
Solubility o~ ~sco~ig_~id (g~m~L
water 0.33
95~ ethanol 0.03
absolute ethanol 0.02
glycerol USP 0.01
propylene glycol 0.05
oils insoluble
fats . insoluble
fat solventsinsoluble
Ascorbic acid is notoriously unstable in the presence of
oxygen and decomposes rapidly to form L-ascorbic acid 2-hydrogen
sulfate and then dehydroascorbic acid. In body cells, the sub-
stance glu~athione helps to reverse the L-ascorbic ~cid - dehy-
droascorbic acid reaction, maintaining a pool of ascorbate dis-
tributed throughout the tissues of the body.
In healthy male adults the ascorbate pool is in excess of
1.5 g which would increase to 2.3 to 2.8 g wi~h daily intakes of
200 mg. This pool is located in the plasma and contains 0.8-1.4
mg/100 ml. If the ascorbate pool were to fall below 600 mg,
physiological changes would start to occur, and levels below 300
mg would initiate clinical signs of the disease known as scurvy.
The human body does not manufacture ascorbic acid and must obtain
it from outside sources in order to survive~
Aside from preventing scurvy, ascorbic acid is essential to
many body or biological functions the most notable of which is
the syn~hesis of collagen, the major fibrillar component of
dermal connective tissue, comprising approximately 70-80~ of the
dry weight of the dermis. ~he collagen fibre molecule consists
of amino acid chains wrapped around each other to form a triple
.
,
: - - . ~ : - - .
: ~ . . . - ~.
1325774
helix the formation and stabilization of which depends upon the
presence of hydroxylysine and hydroxyproline.
The essential role played by ascorbic acid in the hydroxyla-
tion of proline and lysine, hence, in the formation and main-
tenance of collagen has been investigated widely and is well
understood. See, for exampl~, Meanning, J.M., and Meister, A.,
"Conversion of Proline to Collagen Hydroxyproline", Biochemis-
try, 5: (4) 1154-1165 (1966); Gottlieb, A.A., Raplan, A.,
Udenfriend, S., "Further Evidence for the Accumulation of a
Hydroxyproline~deficient, Collagenase-degradable Protein during
Collagen Biosynthesis in Vitro", J. Bio. Chem. 2~ (7) 1551-1555
(1966); Barnes, M.J., "Function of Ascorbic Acid in Collagen
Metabolismn~ ~nn. N~. Acad. Sci., ;~: 264-277 (1975); Murad, S.,
Grove, D., Lindberg, K.~., Reynolds, G., Sivarajah, A., Pinnell,
S.R., "Regulation of Collagen Synthesis by Ascorbic ~cid", Proc.
Natl. Acad. Sci. 1~ (5)s 2879-2882 (1981); and Murad, S.,
Sivarajah, A~, Pinnell, S.R., "Prolyl and Lysyl Hydroxylase
Activities of Human Skin Fibroblasts: Effect of Donor Age and
Ascorbaten, J. Invest. Derm. ~: 404-407 ~1980).
More recent studies indicate that ascorbic acid encourages
the synthesis of procollagen by stimulating procollagen mR~A.
See Freiberger, ~., Grove, D., Sivarajah, A.t Pinnell, S.R.,
"Procollagen I Synthesis in Human Skin Fibroblasts: Effect of
Culture Conditions on Biosynthesisn, J. Invest. Derm. ~: 425-430
~1980); Tajima, S., Pinnell, S.R., "Regulation of Collagen Syn-
thesis B., Ascorbic Acid. Ascorbic Acid Increases Type I Procol-
lagen m~ ', Biochem. Biophys. Res. Comm. 106 (2): 632-637
~1982); and Murad, S., Tajima, S., Johnson, G.R., Sivarajah~ A.,
~ .
.. ,. . :: , ~ : . ,
. ..
', ~
--6--
~3~5774
Pinnell, S.R., ~Collagen Synthesis in Cultured Human Skin Fibro-
blasts Effects of Ascorbic Acid and Its Anaolgsn, J. Invest. Derm
81: 158-162 (1983).
Still other studies indicate that ascorbic acid helps in-
crease membrane lipid peroxidation and antioxidation. Girotti,A.W., Thomas, J.P., Jordan, J.E., "Prooxidant and Antioxidant
Effects of Ascorbate on Photosenitized Peroxidation of Lipids in
Erythrocyte Membranes~, photochem. Photobio. 41 (3): 267-276
(1985). Ascorbic acid also may help to inhibit the production of
melanin responsible for skin discolorations. Tomita, Y., Hariu,
A., Mizumo, C., and Seyi, M., "Inactivation of Tyrosinase by
Dopan, J. Invest. Derm. ~ (5): 379-382 (1980). Finally, ascor-
bic acid may prevent the accumulation of histamine in body tis-
sues responsible for many allergenic reactions. Bates, C.J., ~The
Function and Metabolism of Vitamin C in Man" Vit C ~sc~rbiç ~cid,
Counsell, J.N., ~orning, D.H., eds., Applied Sci. Publishers.
Ascorbic levels in humans tend to decrease with age. Colla-
gen levels also decrease accompanied by changes in morphology.
The fibers become more intermittant and thinner resulting inevit-
ably in surface skin effects such as drynessr flaking, wrinkling
and loss of luster and elasticity. See Eyre, D~Ro (1980) ~ "Col-
lagen: Molecular Diversity in the Body's Protein Scaffoldn,
; Science, Vol. ~ 1315-1322; Wright, E.T., Shellow, W.V. ~1973)n,
nThe Histopathology of Wrinklesn, J. Soc. Cosmetic Chem. 24:81-
25 85; and Sinexl F.M. (1968)n, "Role of Collagen in Agingn, In
Treatise on Collagen,Vol 2,Biology of Collagen edited by B.S.
Gould, Part B. Academic Press.
.
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~32S774
Exposure to ultraviolet light also has a tendency to inter-
fere with collagen synthesis causing skin to exhibit theeffects
of premature aging. See Semma, ~., Sasaki, M. (1978), "Aging of
Skin- Changes of the Dermal Connective Tissuen,Cosmeticsand
Toiletries,Vol. 93:29-36; Bisset, D.L. et al (1986), nAn Animal
Model of Solar-Aged Skin",Photochem~nd Photobiol. Abstracts,
Vol. ~, Suppl. pp. 955.
We have found in accordance with the present invention ~hat
replenishment of the dermal tissues wikh ascorbic acid delivered
percutaneously through the stratum corneum can impart beneficial
appearance effects to skin, i.e. improved tone and luster, a
decrease in fine lines and wrinkles, improved elasticity, and so
on. As a suitable base or vehicle for the ascorbic acid, a
stable composition is provided having desirable cosmetic quali-
ties, namely, an agreeable, pleasant feel and appearance when
topically applied to skin.
The stable compositions according to the invention comprise
ascorbic acid dissolved in a solution comprising water and at
least one organic solvent miscible with water.
The organic solvent must be compatible with water, i.e. it
must be polar with one or more hydroxyl groups, and be acceptable
for cosmetic use. There are many mono, di, or polyhydric liquids
suitable for this purpose including, for example, the alcohols,
glycols, and polyols. Without limitation one or more of the
following organic solvents may be employed: ethanol, N-propynol,
isopropyl alcohol, methanol, propylene glycol, butylene glycol~
hexylene glycol, glycerine, sorbitol (polyol), di-propylene gly~
. ~ .
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-8- ~ 132577~
col, and polypropylene glycol. The organic solvent may comprise
up to about 90% by weigh~ of the composition.
Ascorbic acid is ever so slightly soluble in propylene
glycol, i.e. up to about 4% by weight. However, propylene glycol
S by itself is not cosmetically elegant due to its oily feel and
viscous consistency. Accordingly, a blend of propylene glycol
and at least one other organic solvent is preferred with a blend
of propylene glycol and ethanol being mostly preferred. When
such a blend is employed, the composition may comprise ethanol in
the range from about 40% to 90% by weight of the composition and
propylene glycol in the range of about 10% to about 50% by weight
of the composition. A composition comprising ethanol in the
range of about 55~ to about 65~ by weight and propylene glycol in
the range of about 20% to about 25% by weight is especially
preferred because of its excellent cosmetic propertiesr i.e. such
a blend has an elegant feel and appearance.
From Table I it is seen that ascorbic acid is even less
soluble in ethanol than it is in propylene glycol (about 2% by
weight). The effect of blending ethanol with propylene glycol
~0 therefore, is to reduce the solubility level of ascorbic acid in
the organic solvent blend. In accordance with the invention,
water is added to the organic solvent blend to increase the
solubility of ascorbic acid therein. Water may be add~d up to
about 12% by weight without adversely affecting the stability of
the ascorbic acid dissolved in the water/organic solvent solute.
Up to about 10% by weight ascorbic acid may be dissolved
(and stabilized) in the organic solvent/water composition with
the amount of stabilized ascorbic acid varying inversely with
; : :
.
9--
respect to the amount of water, i.e. the greater the
water content, the less stabilized ascorbic acid in
solution.
In a composition comprising by weight about 83%
organic ~olvent blend and about lo~ water, we have found
that about 5% ascorbic acid may be dissolved and remain
stable, and therefore, these proportions are mostly
preferred.
It is quite surprising and unobvious tha~ ascorbic
acid may be stabilized in the compositions of the present
inYention containing such a relatively high water content
or stated otherwise, where water is employed as a co-
solvent. U.S. Patent No. 4,372,874 discloses that
a~corbic acid may be stabilized in a water miscible
organic solvsnt only if wa~er is removed via a desiccant
additive and no mora than 0.5~ by weight residual water
is present.
~he ~oregoing compositions are prepared u~ing
techniques well k~own in the cosmetic arts. The organic
solvent or solvent blend, and water are mixed together at
room temperature in a suitable, standard ~ixing vsssel.
Various othsr cosmetic i.ngre~ients may also be added
including emollients, moisturizers, colorants, fragrance
preservatives and antioxidantsO Finally, ascorbic acid,
pre~erably in powder ~orm, is added to the mixing vessel
under conditions which will avoid contact with air as,
for example, under a hood providing a nitrogen blanket or
atmosphere surrounding the mixing vessel. A suitable
ascorbic ~cid ingredient, in powder ~orm, is commercially
available from Hoffman La Roche, N~ltley, New Jer~ey. The
final composi~ion may then be packaged in containers for
dis~ribution to consumers, preferably
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-lo~ 2 ~ 7 ~ ~
in bottles impervious to U.V. light. If desired, the bottles
may also be provided with a nitrogen filled headspace.
The cosmetic compositions of tha present invention
should provide a stabilizing environment for ascorbic acid,
be percutaneously adsorptive when topically applied to skin,
and impart perceived skin appearance benefits after being
applied.
In view of the foregoing, and the following description,
it will be understood that in a broad aspect, the present
invention relates to a stable composition for cosmetic use
comprising: ascorbic acid; a first co-solvent; and a second
co-solvent, said first co-solvent being water, said second
co-solvent being an organic solvent, said first and second
co-solvents being miscible with each other, said ascorbic
acid being present in an amount by weight ranging from about
1% to about 10% and said water being present in an amount by
weight ranging up to but no more than about 12%, said second
co-solvent comprisin~ the remainder of the composition and
being selected from the group consisting o~ ethanol, N-
propynol, isopropyl alcohol, methanol, propylene glycol,
butylene glycol, hexylene glycol, glycerine, sorbitol
(polyol), di-propyl~ne glycol, polypropylene glycol, or
mixtures theraof, said second co-solvent comprising up to but
no more than about 90% of the total weight of the composition
in which at least about 40% of the total weight of said
33 '
.
~ i~25~74
-lo(a)-
composition is ethanol, wherein said ascorbic acid is
rendered ~table in said composition thereby permitting said
composition to be topically applied to the skin for
facilitating percutaneous absorption of said ascorbic acid
relative to the skin effective to produce enhanced skin
appearance benefits.
In another broad aspect, the present invention relates
to the method of improving the appearance of skin comprising
the steps of: (a) stabilizing ascorbic acid in a cosmetic
solution which comprises water and up to about 90% by weight
of an organic co-solvent miscible with water wherein at least
about 40% of the total weight of said cosmetic solution is
ethanol; and ~b) topically applying said cosmetic solution to
the area of skin to be affected such that said ascorbic acid
is percutane~usly absorbed by said skin.
The invention will now be further illustrated and
evaluated with respect to meeting the specifications in the
preceding paragraph by the following ~pecific examples which
are not to be construed as limiting.
Example 1
The following preferred composition was formed by mixing
the ingredients together at room temperature under a nitrogen
blanket hood.
B
, . . . ......................... - `
.. . . ...
7 7 ~
-10(b)-
Ingredient %_Wei~ht
ALCOHOL SD 40B 61.05500~
PROPYLENE GLYCOL 21.0000U%
HYDROXYPROPYL CELLULOSE 0.50000%
DEMINERALIZED WATER 10.00000%
ASCORBIC ACID-POWDER 5.00000%
PROPYLENE GLYCOL DIPELARGONATE 1.00000%
OCTYL PELARGONATE 1.00000%
C.S. D&C YELLOW NO.10 (1%)0.02500%
BUTYLATED HYDROXYTOLUENE 0.20000%
BISABOLOL 0.20000%
PANTHENOL-DL-63920 0.20000%
TOTAL: 100.00000%
Example 2
A collagen regulation study was performed in accordance
with the procedures set forth in Pinnell, S.R.: "Regulation
of Collagen Biosynthesis by Ascorbic Acid: A Review", Yale J.
Biol~ Med. 58~ 554-559, (1985); and Tajima, S. and Pinnell,
S.R.: "Collagen ~
B ~
,
13257 1~
Synthesis by Human Skin Fibroblasts in Culture: Studies of
Fibroblasts Explanted From Papillary and Reticular Dermis", J.
Invest. Dermatol. ~:410-412, (1981). Normal human skin fibro-
blasts wer~ seeded into 35 mm tissue culture plates at a density
of 100,000 cells per plate and grown in modified Dulbecco~media
with 20% dialyzed calf serum. Cells were grown to confluent
density for 7 days, the media was removed, the cells were washed
twice with phosphate buffered saline, and experimental conditions
were administered. At this point, cells were density stabilized
by carrying out the remainder of the experiment in 0.5% dialyzed
calf serum. Media was changed daily and all solutions were made
fresh. Conditions were administered for 72 hours and during the
last 6 hours of the experiment 2-3,3H~proline 20 uCi per plate
was added. At the end of the experimentl media and cells were
harvested into a cocktail of protease inhibitors and levels of
collagen and noncollagen protein were determined by digestion -
with highly purified bacterial collagenase. Cell number was
determined by counting in a Coulter~ ounter. Data is expressed
in l'able II below as the average of duplicate determinations
which were conducted on duplicate cultures. The various experi-
mental conditions were:
Condition lo control (untreated cells)
Condition 2: ascorbic acid 100 uM
Condition 3: composition of Example 1 without ascorbic
acid
Condition 4: composition of Example 1 (diluted to finalconcentration of 100 uM ascorbic acid)
-
-12~ 57~
~n~
Condition 1 Condition 2 Condition 3 Condition 4
Collagen synthesis63~8321.2 58.1 230.6
in media
5 cpm/cell
Collagen synthesis~0.429.4 36.9 66.0
in pellet
cpm/cell
~ collagen 39.0 8.3 39.0 22.0
10 in pellet
Total collagen104.2350.6 95.1 2g6.6
synthesis
cpm/cell
Total noncollagen927.81363.0 1357.0 1051.0
15 synthesis
cpm/cell
% Protein synthesis 2.0 4.5 1.3 5.0
devoted to collagen
(corrected for
20 increased proline
content in collagen)
Disc~Lss~-Qn
The results indicate that ascorbic acid enhanced collagen
synthesis 3-fold in human skin fibro~las~s whether or not the
ascorbic acid was dissolved in the vehicle of the composition of
Example 1. In the absence of ascorbic acid, collagen is under-
hydroxylated and incapable of being efficiently secreted from t~e
cell. Vnder these conditions, an appreciable amount remalns in
the cell pellet. In the presence of ascorbic acid it is effic-
iently secreted into the media. This provides a strong indicatorof ascorbate response. Ascorbic acid efficiently led to collagen
secretion from fibroblasts whether or not it was dissolved in
Example 1 vehicle. The study clearly shows that the composition
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of Example 1 is capable of being presented to cells, of entering
cells, and producing the desired response.
ConclusiQn
The composition of Example 1 stimulates human skin fibroblasts
to preferentially synthesize collagen in a manner similar to
ascorbic acid alone. No evidence was demonstrated tha~ the
Example 1 formulation was toxic to cells or changed the ascor-
bic acid in any way so tha~ it would not be available to exert
its specific effect on collagen synthesis within the cells.
E~mple 3
Two separate cosmetic formulations were prepared by mixing
the below listed ingredients together. Formulation "3~" comprises
an ascorbic acid stabilized composition in accordance with the
invention and was prepared in the same manner as the composition
of Example 1. Formulation "3B" comprises a standard moisturizer
night cream.
% W~ight In~redient
EQ~m~la~ion~ o~m~lation 3B
60.95500 ~ ALCO~OL SD 40B
21.00000 -------- PROPYLENE GLYCOL
.50000 -------- HYDROXYPROPYLCELLULOSE
10.00000 65.40000 DEMINERALIZED WATER
5.00000 ~ ---- ASCORBIC ACID-POWDER
1.00000 -------- PROPYLENE CLYCOL DIPELARGONATE
1.00000 ~ --- OCTYL PELARGONATE
.02500 ~ --- C.S. D&C YELLOW NO,10 ~1%)
.20000 -------- BUTYLATED HYDROXYTOLUENE
.02000 -------- BISABOLOL
.20000 .20000 PANTHENOL-DL-63920
~.10000 .05000 FRAGRANCE
i ~ -------- .50000 CARBOPOL 934
' -------- .20000 XANTHAN GUM
-------- 2.50000 SQUALANE
-------- .50000 SQUALENE - PRESERVED
-------- 8.00000 MINERAL OIL-LIGHT
~ r~ P, P ~ al~
.
.
-14- ~
4.00000 PETROLATUM
-------- .50000 MYRISTYL ~YRISTATE
-- 1.00000 ISOPROPYL MYRISTATE
-------- ~80000 POE FATTY ALCOHOLS
-------- 3.00000 STEARIC ACID
-------- 3.00000 POE (20M) MONOSTEARATE
-------- 3.00000 ETHYLEHEXYL PALMITATE
-------- .40000 METHYLPARABEN
-------- 1.00000 CETYL/STEARYL ALCOHOL (60/40)
-------- .50000 GLYCERYL MONOSTEARATE
-------- .25000 TRIETHANOLAMINE 99%
-------- .20000 PROTEIN COLLAGEN-3% AQ
-------- ~20000 PROTEIN ELASTIN-30% AQ
A -------- .40000 GERMALL ~
.--~ -- .20000 DISODIUM EDTA
-------- 4.00000 LANOLIN ACETATE
~ - .20000 TROPOCOLLAGEN/PROCOLLAGEN-PRES.
================
TOTAL 100.00000% 100.00000~
A clinical study was performed to evaluate skin appearance
benefits imparted as the result of using the composition of the
invention for a period of approximately 3 months. A ~split-face"
procedure was used invclving a panel of fifty-two (52) female
subjects. Each panelist was provided with samples of formula-
tions 3A and 3B and instructed to apply 3~ to one side of the
face and 3B over the entire face, i.e. both sides. The instruc-
tions were designed to provide right/left ba1ance over the entire
test panel. Application of the formulations was required twice
daily, viz.r A.M. and P.M., after cleansing with mild soap and
water and towel drying. Observations were made by an experienced
Clinician at 2 weeks, 4 weeks, 8 weeks and 12 weeks into the
test. A subsequent observation at 14 weeks was performed by an
experienced Dermatologist. The specific parameters observed are
set forth in Table III. Table IV summari2es the results of the
clinical evaluation made at week 12 of the study.
D~
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~able III
~L~ical Te~t Pa~ame~s
Parameter ~Qç~$m~
Visual Texture When observing the person full face,
does one side appear to visually look
better, i.e. smoother/softer, less dry?
Tactile Texture Using the back of the hands and concur-
rently touching each side of the per-
son's face, in an "up/down" motion along
the sides, did one side feel softer/
smoother (less rough) than the other?
Pore Size In persons who had large, visible pores,
did the size of the pore appear smaller
on one side as opposed to the other;
observation areas included the nasal
area, cheekbone, chin.
Elasticity Concurrent, gentle "pinching" of the
suborbital areas; did the skin "spring~
back more quickly on one side as opposed
to the other?
.
Lightening Was pigmentation different, i.e. was one
side or one area lighter than the other;
j areas of observed "lightening~ included
I the whole side of the face or specific
areas such as suborbital area or along-
side cheek.
Lines Areas of observation included crowsfeet
and suborbital only; were lines less
apparent, appear smoother (less depth)
and/or were saborbit areas "less p~ffy~.
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-16- ~ 3 3~!~i7~
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~Y C`l C`l c~ C`l c~ N
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a ~ c~ ~ :~ ~ er
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U~ C~ ~D d~ dQ
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-17~ 2~77~
i~c~io~
At the 2 week exam, a few attributes in favor of the side
treated with Formula 3A emerged, i.e. visual texture, tactile
textuxe, pore siæe, and elasticity. Although the observed bene
~it was slight, the difference appeared to be real. After 4
weeks, treated side benefits were more evident in that the number
of perceived changes in visual/tactile texture, pore siæe and
elasticity increased; however, the degree of change was again
~slight" in the majority of persons. After 8 weeks, visual
texture, pore size and elasticity remained basically the same as
the 4-week observation in terms of number of persons exhibiting
an effect and the degree of change. At this exam, two other
attrihutes emerged, viz. skin lightening and a line effect.
Although these two benefits were noted in a ~air number of per-
sons, the degree of change was slight. At the 12-week and final
exam, most benefits remained basically the same as that observed
at 8 weeks; an increase in the number of effects wer~ noted for
skin lightening, lines and tactile textureO The degree of change
for all parameters remained slight. The evaluation conducted by
the independent Dermatologist at the end of 14 weeks confirmed
the results observed by the Clinician at week 12. A comparison
of the assessments made on the same subjects by both the Clini-
cian and the Dermatologist is set forth in Table V.
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-18 ~ ~32~77~
Table V
Summary o~,Cli~ical Test -
14-Week Derma~ological ~ m v~. 12-Weçk Clini~al ~m
14 WkS. ~
Dermatologist ~N=44) ~linici~n (N-441
~m~n~ ~Q_~ai m~n~ ~reatment No Trea~ment
(Bette~), L~Q~ÇL1 (Better) (Better)
Visual Texture 10 (23%) 6 (14%) 20 (45%) 5 (11%)
Tactile Texture 19 (43%) 11 (25%)** 20 (45%) 2 (5~
Pore Size9 (20%) 10 (23%) 21 (48%) 5 (11%)
. _ .
Ela~ticity, 9 (20%) 2% (5%)* 18 (42%) 3 (7%)
. . _
Lines 18 (41%) 9% (20%)* 26 (59%) 4 ~9%)
Lightening12 (29~) 5 (12~)** 21 (48%) 0
* p = <0.05
** p = 0.06
Conclu~ions
The clinical observations in this test indicate that treat- `
ment by Formulation 3A produces skin benefits in visual/tactile
texture, pore size, elasticity, skin lightening and lines in
approximately 1/2 the panel population; although the degree of
the benefits observed was considered to be slight~ Although the
benefits perceived by the ~erma~ologist were somewhat less than
those observed by the Clinican,.the dermatological assessment did
confirm that the treatment afforded by Formula 3A actually
produced skin benefits with respect to linesr elasticity~ light-
ening and tactile texture.
- , . :.. . -- ; , . , . ~ , . .
. .
~ ~32!~77
Ex ampl e
The effect of Formula 3A (Example 3) on the mechanical
properties of skin was evaluated by a panel of 15 females over a
~ use period of one week employing in vivo extensometry. A Cutech~
S in vivo extensometer was utilized. This device consists of two
arms, one fixed and the other moveable, which are driven apart by
a motor driven lead screw. Flat rectangular tabs at the end of
the two arms are stuck to the skin surface by means of double-
sided adhesive tape and cyanoacrylate. The force exerted on the
skin by the fixed and moveable arms is measured by two strain
guages attached at reduced sections on each of the two arms. The
distance between the tabs is measured by a linear variabla dif-
ferential transformer (LVDT) transducer at the root of the two
arms, respectively.
Pro,cedure
Fifteen (15) female panelists were recruited without regard
to age. These volunteers were instructed to wash both forearms
twice a day with chip bar and allow to air dry over a period of
14 days. Extensibility assessments were taken before, immediate
post-, and 1 hour post-product application. Before and after
measures were also taken after 7 days of 1 X per day product use.
Each panelist was assessed by the in vivo extensometer at two
different sites. Product was applied with the extensometer feet
remaining on the skin surface~ The site was then re-evaluated `~
~or extensibility and this is defined as the "immediate post-
application" point. Ex~ensibility measures were taken again on
an equivalent site one hour post-application.
: . ~ , ':
-20- h ~ 3 2 ~ 7 7 ~
~Eg;L~
As shown in Table VI, the panel demonstrated a significant
increase in the force required to extend the skin 30% after 2
weeks of chip bar washing. These panelists were considered dry
on this basis.
Table VI
Comparison of E~tensibility Valu~s Betwçen Baseline and
Chip Bar Washed Fo~earm Sit~s
Mean Force ~ecorded
Baseline 1~ day Significance
~h~L
Control 4.02 6.84 p < 0.001
Group
Formula 3A3.81 7.24 p < 0.001
Table VII illustrates the results of a single product appli-
cation. Prior to product application both sets of sites were
statistically identical after 1 application of Formula 3Af how-
ever, the treated sites require signi~icantly less force for a
30% extension.
~hl~ YlI
Comparison of ~xtensibility Values Be~wee~ Fo~mula 3
Tr~ated and Control ~ites ~fter 1 Applicati
Mean Force RecoLded
Pre- ImmQ~ 1 Hour
treatmen~ PQa~ tre~tPost-Treat
Control 6.84 6.30 6.77
Group
Formula 3A7.25 4.85 5.19
Significance p > 0.05 p < 0.05 p < 0.02
., .
: . .. ~ .
-21-~jt 132~
The results of 7 days of treatment are given in Table VIII.
As shown by the data in this table, there is a significant
decrease in the force required for the extension after the eighth
product application. There was no difference between the pre-
treatment value on Day 7 and the pretreatment value Day l, ~hichwould have demonstrated a persistent improvement.
Table VII~
Comparison of ~xtensibility Yalues Between Fo~mula 3A
Treated and Co~t~ol Si~e~ ~f~er 7 and 8 Application$
Mean Force Recorded
Pre-trea~men~ Immediate
(7 applicantions)Post ~eatment
Control 5.75 5.67
Group
Formula 3A 5.30 3.l9
Significance p > 0.05 p < O.OOl
Tables VII and VIII illustrate that at least a 25% improve-
ment in the force required to deform the skin 30% was achieved
for each measured application timepoint.
Conclusions
The results of this study demonstrate that application of
Formula 3A results in a change in the skin's biomechanical
properties. Comparison of the force required to extend the skin
30% shows a statistically significant decrease after single and
multiple applications. The extensometer used in this study is an
established method for assessing the biomechanical properties of
the skin. See Gunner, C.W., ~utton, W.C., and T.E. Burlin,
(1979), "The Mechanical Properties of Skin In ViYo - A Portable
, . : : ,. .,: .
. . .: : ,................... ..
:: -
-22~ 32~77~
~and-Held Ex~nsometern, Bri~. J. Derm. lOQ:161-163; and Marks,
R. (1983), ~Techniques for Measuring the Mechanical Properties of
Skinn, J. Soc. Cosmet. Chem. 34:429-437. The decrease in the
force required to extend the skin 30~ found with Formula 3A
treatment indicates that the skin has become more flexible i.e.
is less strained by a deformation.
~m~
A study was conducted to determine whether the composition
of Example 1 is percutaneously absorptive when topically
applied to human skin.
PrQC~edUr~ !
Normal human breast skin was obtained from three unselected
patients undergoing mammary reduction as a cosmetic procedure.
Breast skin was obtained within one hour of excision and immedi-
lS ately prepared as follows. All subcutaneous fat was removed byscissor excision and the skin was cut with a scalpel into approx-
imately 1.5 x 1~5 cm squares. Each piece of skin was mounted
into a glass diffusion chamber having a diameter of 0.9 cm and an
available diffusion area of approximately 0.63 cm2. The skin,
epidermal side up, was clamped between the upper and lower half
of the chamber with a spring loaded column clamp. The dermal
side of the chamber was bathed in a solution of 0.9% NaCl~ 0.01%
sodium azide~ 0.4 mM KH2PO4, 2.0 mM K2HPO4, pH 7.4. The volume
of the lower chamber is standardized for each chamber and varies
from 2.6 to 3.4 ml. An attempt was made to minimize all vari-
ables by examining all skin specimens carefully for defects
during their harvesting. The subcutaneous fak was carefully
.. : . ., ~ - : :. .
, . . ~ . . .
.. . . . : : .................................. ::.
:. : .. . . ... .
-23-
257~1~
removed and the skin re-examined at the end of the experiment.
Attention was paid to the layering of the solution onto the
epidermis, maintenance of hydration of the skin, and prevention
of evaporation from the lower chamber.
~ 1 mL sample of the composition of Example 1 (5% ascorbic
acid solution) was mixed with 5 uCi of L-(carboxyl-C14) ascorbic
acid, Amersham code CFA.620 batch 12 with a purity ranging from
96 to 98%. Twenty microliters of ~he ascorbic acid mixture then
was placed on the epidermal surface of the skin mounted in the
percutaneous absorption chamber. The upper chamber was covered
by parafilm and placed in a~shaking water bath at 37C. In some
cases the chambers were placed in a 37C incubator and stirred
with a teflon coated bar by a magnetic stirrer. Results of these
two methods were found to be similar. At 72 hours the chambers
were removed and duplicate 0.5 ml aliquots were removed from the
bottom chamber through the side port. 2.5 ml of scintillation
fluid cocktail Aquasol-2~as added and the samples counted in a
liquid scintillation counter. An identical 20 lambda of the
original solution was counted in 0.5 ml of the bathing solution
and 2.5 ml Aquasol-2 by similar means. Cutaneous absorption was
calculated as the percentage of radioactivity placed on the
epidermal side of the skin which eventually found its way into
the bottom chamber bathing fluid.
~esulta and niscuss~n
Based on a weight of 17.2 mg per ~wenty microliters of the
` composition of Example 1 and a content of 5% w/w ascorbic acid,
~ approximately 860 ugm ascorbic acid was applied per 0.63 cm2
:'
A~P~ R~
, - ~
:;.`. , ~ ': ~ . ' ; . . ' ' !
.'.` ' '' ' '`'; '` ' " . ' ' '
'~' . , . `. ~`: ` ' " ' ' ~ ' ' ' '
~32577~
-24-
area. A mean value of 12.8~ absorbed or an average of 175 ugm
ascorbic acid per cm2 of skin was absorbed as measured over
twelve samples for each of the three patients. This study not
only demonstrates that the ascorbic acid of the Example 1 compos-
ition penetrates human skin and is absorbed into the dermallayer, but furthermore indicates that the amount of ascorbic acid
absorbed could be expected to have an impact on dermal collagen
synthesis.
~xamplQ-~
To evaluate the stability of the composition of Example 1,
duplicate samples thereof were placed in separate 2 oz. sealed
jars with the headspace volumes filled with 3 different
atmospheres ~at three different volumes, viz. oxygen, air, and
nitrogen, at 5~, 25% and 50~, respectively (for a total of 18
lS samples). Ascorbate content in each jar was assayed in
accordance with the methodology set forth in the United States
Pharmacopia, Vol. XXI, p. 75, "Ascorbic Acidn. The jars were
sealed after headspace filling under a hood, and stored for one
month at room temperature and 110Fo The jars were then opened
and assayed again using the same method. The results are set
forth in Table IX below.
~. . . . . .
It' ~2~77~
-25-
Ascorbic Acid ~kility ~
Ascorhate Stability
S~o~Laqe _~ ~eiqh~ __
5 ~mQ~ Q~ ~n~ l month
2 50 RT 5.07 3.93
110F -- 3.81
RT 5.07 4.55
110F -- 4-3~
RT 5.07 4.86
110F -- 4.79
Air 50 RT 5.07 --
110F -- 4.80
~ RT 5.07 -~
110F -- 4.93
RT 5.08 --
110F -- 4-95
N2 50 RT 5.07 5.08
~ 110F -- 5 05
RT 5.07 --
110F
RT 5.07 --
- 110F
Table IX clearly shows that, as expected, under an oxygen envir-
onment, ascorbic acid is unstable and decomposes rapidly.
However, the Table further shows ~hat when exposed to air or
ni~rogen, even at elevated temperatures, the a~corbic acid con~
tent of the composition studied remained essentially unchanged
after one month ~hereby indicating ~hat the compositions are
stable under such conditions.
In practice, the compositions of the present invention may
be applied topicallyr preferably after cleansing the skin area to
be affected with mild soap and warm water~ It has been found
that after application, a standard moisturizing lotion or cream
, ~ , . ,, - ~,.......................... . .
, . . ,~ - - . .
:; ... . , . ~ , , , . . ~ .
-2~-
~L32~77~
may be applied to the same skin area without affecting the effi-
cacy of the ascorbic acid composition which tends to be rapidly
percutaneously absorbed by the skin. In fact, it has been found
that the use of a moisturizer immediately following topical
S application of the ascorbic acid composition of the present
invention, as described above, actually increases the rate of
percutaneous absorption of the ascorbic acid ingredient. A pre-
ferred moisturizer suitable for this purpose is "Accolade Night
Treatment", marketed by the independent ~epresentatives of Avon
Products, Inc., New ~ork, New York~
From the foregoing, it will be appreciated that the present
invention discloses stable compositions comprising ascorbic acid
which are cosmetically elegant, and which when topically applied
to human skin produces actual perceived skin appearance benefits
including, but not limited to improvements in luster, tone, and
elasticity, and r~ductions in fine lines, wrinkles, and pore
size. In accordance with the invention, ascorbic acid is stabil-
ized by dissolving the latter in at least two co-solvents, one of
which is water, and the other of which is an organic solvent
miscible with water or a blend of such organic solvents. The
resulting compositions as disclosed herein are unobvious, ad~an-
tageous, and efficacious.
As used herein the terms "solution", "preparation"~
"composition~, "formulation~, and "formula" are to be construed
in~erchangeably.
The terms ~cosmetic" or "cosmetic preparation" or "cosm~tic
composition" as used herein, means (i) articles intended to be
rubbed, poured, sprinkled, or sprayed on, introduced into, or
. ~-. ' ', '
~-.
: .
- 1325774
-27-
otherwise applied to the human or animal body or any part
thereof for cleaning, beautifying, promoting
attractiveness, or altering the appearance, and (ii)
articles intended for use as a component of any such
articles, e.g. sun screening compositions, medicinal or
first aid creams, and so on.
It is desired that the present invention be limited
only by the true spirit and scope o~ the appended claims.
'' ~
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