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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
Internationa) Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 7 : 
A61K 7/48, 7/42 


Al 


(11) International Publication Number: WO 00/45786 
(43) International Publication Date: 10 August 2000 (10.08.00) 


(21) International Application Number: PCT/USOC/02750 

(22) International Filing Date: 2 February 2000 (02.02.00) 

(30) Priority Data: 

09/246,607 8 February 1999 (08.02.99) US 

(71) Applicant: COLOR ACCESS, INC. [US/US]; 7 Corporate 

Center Drive, Melville, NY 1 1747 (US). 

(72) Inventors: MAES, Daniel, H.; 279A Nassau Road, Huntington, 

NY 11743 (US). MARENUS, Kenneth, D.; 62 McCulloch 
Drive, Dix Hills, NY 11746 (US). FTHENAKIS, Christina, 
G.; 9 Lucille Lane, Dix Hills, NY 1 1746 (US). 

(74) Agent: TSEVDOS, Estelle, J.; Kenyon & Kenyon, One 
Broadway, New York, NY 10004 (US). 


(81) Designated States: AE, AL, AM, AT, AU, AZ, BA, BB. BG, 
BR. BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, 
ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, 
KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, 
MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, 
SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, 
UZ, VN, YU, ZA, ZW, ARIPO patent (GH, GM, KE, LS, 
MW, SD, SL, SZ, TZ, UG, ZW), Eurasian patent (AM, AZ, 
BY, KG, KZ, MD t RU, TJ, TM), European patent (AT, BE, 
CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, 
NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, 
GN, GW, ML, MR, NE, SN, TD, TG). 

Published 

With international search report. 



(54) Title: CHOLESTEROL SULFATE COMPOSITIONS FOR ENHANCEMENT OF STRATUM CORNEUM FUNCTION 



(57) Abstract 



The present invention provides a method of retarding desquamation of the stratum corneum, and maintaining stratum comeum 
thickness, by applying to the skin an effective amount of cholesterol sulfate. Hie retardation of desquamation can be useful in enhancing 
the skin's own UV protection, in prolonging the retention time of a sunless tan, and generally reducing the appearance of lines and wrinkles 
associated with both photo- and chronoaging. 



FOR THE PURPOSES OF INFORMATION ONLY 
Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under die PCX 



AL 


Albania 


ES 


Spain 


LS 


Lesotho 


SI 


Slovenia 


AM 


Armenia 


FI 


Finland 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


FR 


France 


LU 


Luxembourg 


SN 


Senegal 


AU 


Australia 


GA 


Gabon 


LV 


Latvia 


sz 


Swaziland 


AZ 


Azerbaijan 


GB 


United Kingdom 


MC 


Monaco 


TD 


Chad 


BA 


Bosnia and Herzegovina 


GE 


Georgia 


MD 


Republic of Moldova 


TG 


Togo 


BB 


Barbados 


GH 


Ghana 


MG 


Madagascar 


TJ 


Tajikistan 


BB 


Belgium 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


Turkmenistan 


BF 


Burkina Faso 


GR 


Greece 




Republic of Macedonia 


TR 


Turkey 


BG 


Bulgaria 


HU 


Hungary 


ML 


Mali 


TT 


Trinidad and Tobago 


BJ 


Benin 


IE 


Ireland 


MN 


Mongolia 


UA 


Ukraine 


BR 


Brazil 


IL 


Israel 


MR 


Mauritania 


UG 


Uganda 


BY 


Belarus 


IS 


Iceland 


MW 


Malawi 


US 


United States of America 


CA 


Canada 


IT 


Italy 


MX 


Mexico 


uz 


Uzbekistan 


CF 


Central African Republic 


JP 


Japan 


NE 


Niger 


VN 


Viet Nam 


CG 


Congo 


KB 


Kenya 


NL 


Netherlands 


YU 


Yugoslavia 


CH 


Switzerland 


KG 


Kyrgyzslan 


NO 


Norway 


zw 


Zimbabwe 


a 


Cote d'Jvoirc 


KP 


Democratic People's 


NZ 


New Zealand 






CM 


Cameroon 




Republic of Korea 


PL 


Poland 






CN 


China 


KR 


Republic of Korea 


PT 


Portugal 






CU 


Cuba 


KZ 


Kazakstan 


RO 


Romania 






cz 


Czech Republic 


LC 


Saint l^icia 


RU 


Russian Federation 






DE 


Germany 


U 


Liechtenstein 


SD 


Sudan 






DK 


Denmark 


LK 


Sri Lanka 


SE 


Sweden 






EE 


Estonia 


LR 


Liberia 


SG 


Singapore 







WO 00/45786 



PCT/USOO/02750 



CHOLESTEROL SULFATE COMPOSITIONS FOR ENHANCEMENT OF STRATUM 

CORNEUM FUNCTION 

FIELD OF THE INVENTION 

The present invention relates to cosmetic compositions. 
More specifically, the invention relates to topical compositions 
containing cholesterol sulfate and methods of using same in 
treatment of skin. 

BACKGROUND OF THE INVENTION 

The stratum corneum represents the major chemical and 
physical barrier between the body and the environment. It is 
formed by a process in the epidermis which involves the 
transformation of germinative cells into terminally differentiated 
cells; the process of transformation takes approximately one 
month, by which time the terminally differentiated cells are shed 
from the skin surface. The cells at the outermost layer of the 
skin, which are constantly being sloughed off, are replaced by 
cells that are generated by the mitotic activity of the basal 
layer of the epidermis. In the course of their migration from the 
basal layers to the upper levels of the skin, these cells produce 
and accumulate keratin, to the point at which there is virtually 
no cytoplasm remaining; the cell then dies and is shed, to be 
followed by another phalanx of migrating epidermal cells. The 
thickness of the stratum corneum and epidermis in general varies 
in different regions of the body. 

This cornified barrier performs a number of functions. A 
particularly important aspect of its presence is as a physical 
barrier, between the deeper layers of the skin as well as the 
internal organs and the environment. Prevention or attenuation of 
penetration of UV radiation, as well as other harmful stimuli such 
as free radicals, to the deeper skin layers is one very critical 
aspect of this skin layer. Unfortunately, as with many other skin 
functions, the performance capacity of the stratum corneum becomes 
progressively diminished with age. The turnover rate of the 
stratum corneum is considerably decreased in older individuals, 
and the cornified layer gradually becomes much thinner, thereby 



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reducing the efficacy of this physical barrier and permitting 
easier penetration of harmful stimuli such as UV rays. This in 
turn leads to UV-damage to the dermal layers of the skin, 
resulting in degradation of collagen and elastin, finally 
resulting in wrinkling and skin atrophy. Moreover, the thinning 
of the stratum corneum can result in a greater visibility of the 
wrinkling and atrophy, the cause of which is rooted in the dermis. 

Notwithstanding the obvious importance of the stratum 
corneum in maintaining a healthy youthful appearance of the skin, 
rehabilitation and maintenance of the dermis has been a major 
cosmetic focus in preventing the appearance of aging; relatively 
little attention has been paid to developing cosmetic means for 
maintaining a fairly consistent level of stratum corneum function 
into old age. The present invention now provides a means for 
retaining this function, and concurrent uses relating to same. 

Summary of the Invention 

The invention relates to a method of increasing the 
thickness and cohesion of the stratum corneum of the skin, which 
comprises applying to skin an effective amount of cholesterol 
sulfate. The invention also relates to a method of protecting the 
skin against UV radiation comprising applying to the skin an 
effective amount of cholesterol sulfate. In another embodiment, 
application of cholesterol sulfate to the skin reduces 
desquamation, and therefore, skin flakiness. In yet another 
embodiment, the invention provides a method for enhancing a 
sunless tan which comprises applying a self -tanning agent, such as 
DHA in combination with an effective amount of cholesterol 
sulfate. 

Brief Description of the Figures 

Figure 1 illustrates the condition of stratum corneum 
thickness under different cholesterol sulfate treatment regimens, 
as described in Example I: (A) control (no treatment) ; (B) 1% 
ethanol vehicle control; (C) cholesterol sulfate, 0.01fig/ml; 
(D) cholesterol sulfate, .l^g/ml; (E) cholesterol . sulfate, Ijig/ml; 
(F) cholesterol sulfate 10ng/ml. 



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Figure 2 illustrates the duration of the self-tanning action 
of DHA with and without cholesterol sulfate. 

Figure 3 illustrates the duration of the self-tanning action 
of DHA with and without cholesterol sulfate and a lipid mix. 

Detailed Description of the Invention 

The present invention, in its various embodiments, is 
predicated on the observation that cholesterol sulfate, when 
applied topically to the skin, enhances the cohesion of the 
stratum corneum resulting in a more prolonged retention of the 
layers of the stratum corneum. Specifically, it has been observed 
that application of cholesterol sulfate to skin cells results in a 
distinct, dose -dependent, increase in the thickness of the layers 
of the stratum corneum, as shown in Figures 1C-F. The observation 
is important for a number of different applications; a 
particularly significant application is in the maintenance of the 
texture of older skin. A current common means of enhancing 
smoothness of the skin is to encourage exfoliation. However, 
exfoliation necessarily involves a high rate of turnover of the 
stratum corneum, and consequent thinning of this layer of the 
skin. While not an issue in youthful skin, desquamation in older 
skin can, in some cases, simply exacerbate a problem already 
established, namely, the natural thinning observed with age. 
Thus, application of cholesterol sulfate to retard desquamation 
and maintain stratum corneum thickness represents an entirely new 
direction in the treatment and maintenance of older, thinning 
skin. A thicker stratum corneum aids in preventing or retarding 
the appearance of fine lines and wrinkles which so frequently 
characterize thinning skin. At the same time, the enhanced 
cohesion of the stratum corneum results in an effective 
strengthening of the protective lipid barrier naturally provided 
therein. . 

To achieve this effect, the cholesterol sulfate or salts 
thereof can be applied in any type of cosmetically or 
pharmaceutically acceptable vehicle for topical application with 
which the active component is compatible, e.g., a gel, a cream, a 
lotion, an ointment, a mousse, a spray, a solid stick, a powder, a 



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suspension, a dispersion, and the like. Preferably, however, the 
cholesterol sulfate is not provided in a liposome formulation, and 
is formulated in a composition containing relatively low levels of 
emulsifiers. Techniques for formulation of various types of 
5 vehicles are well known to those skilled in the art, and can be 
found, for example, in Chemistry and Technology of the Cosmetics 
and Toiletries Industry, Williams and Schmitt, eds., Blackie 
Academic and Professional, Second Edition, 1996, and Remington's 
Pharmaceutical Sciences, 18th Edition, 1990, the contents of which 

10 are incorporated herein by reference. Cholesterol sulfate is 

effective in the claimed function when provided in the composition 
in an amount of from about 0.05 to about 10%, preferably from 
about 0.5 to about 5%, most preferably about 1 to about 3%, all by 
weight of the total composition. 

15 The thickening and cohesion of the stratum corneum also 

provides other benefits, which, in certain specific applications, 
can be appreciated by individuals of all ages. The stratum corneum 
represents an important physical barrier between the environment 
and the deeper skin layers as well as the internal organs. The 

20 presence of this thicker layer thus will provide a greater level 
of protection than is possible with a loose, flaking stratum 
corneum. Although this property can be exploited in a number of 
ways, perhaps the most important is the enhanced self -protection 
from UV rays. The thicker stratum corneum means an increase in 

25 the Minimal Erythemal Dose of UV which will result in sunburn or 
more serious skin damage. In connection with this aspect of the 
invention, cholesterol sulfate may be beneficially combined with 
one or more sunscreens for an enhanced UV protective composition 
which provides both short- and long-term protection. Thus, the 

30 invention provides sunscreen compositions comprising effective 
amounts of cholesterol sulfate and one or more sunscreens. 
Examples of useful sunscreens include, but are not limited to, 
inorganic sunscreens such as titanium dioxide, zinc oxide, and 
iron oxide; and organic sunscreens, such as camphor derivatives, 

35 cinnamates, salicylates, benzophenones , triazines, PABA 

derivatives, diphenylacrylate derivatives, and dibenzoylmethane 
derivatives. In such sunscreen compositions, cholesterol sulfate 



4 



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is present in the amounts described above, and the respective 
sunscreens are present in the amounts normally used for UV 
protection. 

An additional use of the cholesterol sulfate is in the 
5 enhancement and prolongation of self -tanning products. One of the 
recognized limitations of self -tanners, which are normally based 
on dihydroxyacetone (DHA) as the active component, is that the tan 
on the skin lasts only as long as the skin cells receiving the DHA 
remain in place. In the normal course of events, then, a self- 

10 applied tan usually lasts no more than 5 days, i.e., for as long 
as it takes for the stratum corneum layer to which the DHA was 
applied to fully turn over. When cholesterol sulfate is combined 
with DHA, or any other self -tanning agent, in a typical self- 
tanning formulation, however, the rate of turnover of the stratum 

15 corneum to which the composition is applied is slowed down, 

thereby permitting a longer rate of retention of the "tanned" 
cells, and thus prolonging the length of time the tan remains 
visible on the skin. Thus, the invention provides a self -tanning 
composition comprising an effective amount of cholesterol sulfate 

20 and an effective amount of a self -tanning agent. In a preferred 

embodiment, the self -tanner is DHA, which is usually applied in an 
amount of from about 2.5 to about 10% by weight of the 
formulation. The self -tanner may also be imidazole, preferably in 
combination with DHA, in an amount of about 1-10%, preferably 

25 about 1.5-7.5%. 

In addition to its use in therapeutic products, cholesterol 
sulfate can also be beneficially added to color cosmetic products. 

In this regard, effective amounts of cholesterol sulfate are 
added to makeup formulations such as foundations, blushes, 

30 lipsticks and glosses, eyeliners, eyeshadows, and the like. A 
particular advantage may be obtained with such formulations, in 
that the retardation of desquamation may enhance makeup retention 
on the skin to which it is applied. The sunscreen/cholesterol 
sulfate combination may also be effectively employed in such 

35 products . 

In all formulations in which cholesterol sulfate is 
employed, it is preferred that the cholesterol sulfate be combined 



5 



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with other components of the naturally occurring lipid barrier. 
In a particularly preferred embodiment, the cholesterol sulfate is 
combined with at least one of each of fatty acids, ceramides, and 
a sterol, preferably cholesterol. Fatty acids may be up to 24 
carbon atoms in length. Examples of preferred fatty acids include 
butyric acid, caproic acid, octanoic acid, decanoic acid, 
dodecanoic acid, tetradecanoic acid, palmitic acid, stearic acid, 
linoleic acid and oleic acid. Particularly preferred are fatty 
acids with a C 12 to C ao chain length. 

The ceramides to be employed in the compositions of the 
invention are sphingolipids, having a sphingosine or related 
molecule backbone with fatty acids or co-esterif ied fatty acids 
linked to an amino group on the sphingosine, and in some cases, 
with saccharide moieties linked to the terminal hydroxyl of the 
sphingosine. In particular, the compositions may contain co- 
esterif ied ceramides or acylceramides, cerebrosides, co-esterif ied 
cerebrosides, or acylglycosyl sphingolipids. Particularly 
preferred types of ceramides for the present compositions are 
ceramide III and cerebrosides. 

In those compositions in which cholesterol sulfate is 
combined with these lipids, the lipid components each can be used 
in an amount of from about 0.05 to 10%, preferably 0.5 to about 
5%, most preferably about 1 to about 3%, all by weight of the 
total composition. In a particularly preferred embodiment, the 
cholesterol sulfate and the lipid components are present in 
substantially equal amounts in the composition. It will be 
understood from the foregoing that the lipid component need not be 
pure lipid, but rather may be natural extracts containing one or 
more desirable lipids, and used in amounts consistent with 
attaining the concentrations recommended above. 

The compositions of the invention are applied to the skin in 
a manner appropriate to the intended end result. For example, for 
the general promotion of the appearance of young, healthy skin by 
retardation of desquamation and maintenance of stratum corneum, 
the the best results are achieved after regular application over a 
period of time. A preferred method of obtaining the benefits of 
the composition is via chronic topical application of a safe and 



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effective amount of a composition containing cholesterol sulfate. 
It is suggested as an example that topical application of the 
composition, in an amount of from about 0.1 mg/cm 2 to 2 mg/cm 2 of 
skin, be performed from about once per week to about 4 or 5 times 
daily, preferably from about 3 times a week to about 3 times 
daily, most preferably about once or twice per day. By "chronic" 
application, it is meant herein that the period of topical 
application may be over the lifetime of the user, preferably for a 
period of at least about one month, more preferably from about 
three months to about twenty years, more preferably from about six 
months to about ten years, more preferably still from .about one 
year to about five years, thereby resulting in the treatment or 
prevention of the external signs of photo- or chronoaging. 

When the composition is used in conjunction with a 
sunscreen, it is applied in the same amounts as specified above, 
on an as -needed basis, to mitigate the effects of exposure to the 
sun. When used in combination with a self -tanner, the composition 
is also applied in similar amounts, on the portion of the skin to 
be tanned, with repetition, again, on an as-needed basis. 

The invention is further illustrated by the following non- 
limiting examples: 

Example I 

This example illustrates the ability of cholesterol sulfate 
to retard desquamation and maintain stratum corneum thickness. 

Matek skin equivalents are obtained and prepared for use in 
accordance with the supplier's protocol. Equilibrated skins are 
treated topically with dilutions of cholesterol sulfate. 
Cholesterol sulfate is solubilized 1 mg/ml in ethanol, and 
serially diluted 10-fold to yield doses of 0.01, .1, 1 and 
lOpg/ml. Each dose is added topically to an equivalent, using a 
100*il volume. Treatment is repeated daily along with media 
replacement over a three day period. One sample is treated with 1% 
ethanol, representing a vehicle control. Following treatment, 
equivalents are fixed according to standard protocol, and sent for 
histological preparations. Figures 1A-F show stained sections of 
the two controls plus* the treatment samples . The figures show a 



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very loose organization of the stratum corneum in the media 
control, with a gradual increase in organization and cohesion of 
the stratum corneum seen in the treatment samples, which increases 
with the amount of cholesterol sulfate in the treatment. Some 
5 compaction is seen in the ethanol treated sample, which is 

believed due to dehydration of the sample combined with lipid 
removal . 

Example II 

10 The following is a composition according to the present 

invention: 



Material ; ; Weight % 

Phase I 

15 isocetyl alcohol 2.5 

octyl hydroxystearate 2 . 0 

alpha hydroxylauric acid 0.5 
Phase II 

purified water QS 

20 cyclodextrin 1 . o 

Phase III 

ethoxydiglycol 5 . 0 

laureth-23 1.5 

25 dipropylene glycol 1.0 

sodium hyaluronate (1%) 1.2 

pantethine 0 . 1 

PhaselV 

30 sucrose 2.0 

dihydroxyacetone 5 . o 

Phase V 

cyclomethicone 12 . 0 

35 dimethicone 3.0 

cyclomethicone/dimethicone 2 . 0 

tricaprylyl citrate 1.5 



8 



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dimethicone 3.0 
Phase VI 

malvaceae extract 0.2 

fragrance 0.4 

tocopheryl acetate 0.1 

wheat bran extract 0.2 

linoleic acid 0.2 

sodium cholesterol sulfate 0.2 

Phase VII 

nylon- 12 2.0 
Phase VIII 

polyquaternium-37/propylene glycol 1.2 



Example III 

A study is conducted to determine the effect of cholesterol 
sulfate on skin flakiness, as an indicator of its effect in 
reducing desquamation. Fifteen subjects between the ages of 21 
and 65 years are selected for the study. The subjects report for 
the study without moisturizers or any other products on their 
hands and their baseline measurements are taken. The subjects are 
given a product containing 0.5% cholesterol sulfate in a water and 
oil emulsion base to take home and self -administer on their right 
hands only, twice a day in the morning after washing and in the 
evening at least 15 minutes before bedtime for four weeks. The 
left hand serves as the untreated control site. The subjects are 
only allowed to use the test product and specifically log its use 
in a daily diary. At the end of two and four weeks the subjects 
return for testing without applying the product for at least 12 
hours and they are re -evaluated under the same conditions. 

Evaluation of flakiness is determined via the D-Squame Discs 
Method and Image Analysis. Briefly, four D-Squame discs are 
firmly pressed on the back of each hand with hand held uniform 



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pressure device and removed by gently pulling away from the skin. 
The D-Squame discs are mounted on clear microscope slides and 
labeled according to subject's name and visit. Desquamation is 
evaluated from the D-Squame discs via the image analyzer. Skin 
evaluation is carried out before treatment, and after two and four 
weeks of treatment . 

An OPTIMA image analyzer is used to evaluate skin flakiness. 
The D-Squame samples containing the stratum corneocytes are placed 
under a camera on top of a light table and each image is imported 
into the image analyzer. The average Gray Value corresponding to 
the sample density is measured. The denser the sample, the higher 
the Gray Value difference. The treated skin shows a 22.5% 
decrease. in flakiness relative to baseline after two weeks, and a 
24.1% decrease after 4 weeks. The decrease in flakiness is 
apparently due to the observed effect on cohesion of the stratum 
corneum . 

Example IV 

This example illustrates the efficacy of the addition of 
cholesterol sulfate to DHA in enhancing duration of self -tanning. 
Two products are prepared for testing, one a control formulation 
containing 5% DHA, and the second the test formulation containing 
5% DHA and 0.2% sodium cholesterol sulfate. A total of 10 
panelists participate in the study. The control formulation is 
applied to the right arm and the test formulation on the other. 
Equal amounts of the products (800^1) are dispensed and blended in 
until absorbed. 

Color measurements are obtained with a Chromameter before 
treatment, 24 hours after treatment, and 4 days and 5 days. 
Decrease in reflectance and increase in red coloration and yellow 
coloration (AL\ Aa*, Ab*) obtained from the Chromameter are 
calculated as compare to baseline skin color. Total color change 
AE* is calculated for each time point as follows: 

AE*= square root of (AL*) 2 + (Aa*) 2 + (Ab*) 2 



10 



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The results, shown in Figure 2, demonstrate that there is a 
decrease in skin reflectance and an increase in skin redness and 
yellow coloration due to the self- tanning effect of the products. 
Total change in * color values (AE*) observed from the graph show 
that there is 10% darker color on the arms treated with the 
formulation containing the cholesterol sulfate as compared with 
the one treated with DHA alone. After 4 and 5 days, there is 
still 20% and 25% darker tan on the site treated with DHA and 
cholesterol sulfate, as compared with the site treated with DHA 
alone. These data show that the addition of cholesterol sulfate 
to DHA results in a longer lasting tan. 

Example V 

This example illustrates the efficacy of a composition 
containing DHA combined with cholesterol sulfate and a lipid mix 
in enhancing the intensity and duration of self -tanning. 
Two products are tested: a test product containing 5% DHA, 0.2% 
sodium cholesterol sulfate, 0.2% linoleic acid and 0.2% SC 
complex, containing wheat bran extract and olive oil extract, and 
a standard self -tanning product containing only 5% DHA as control. 

A total of 22 panelists participate in the study, divided 
into two groups of eleven each. The control is applied on the 
right arm, and the test product is applied on the left arm. Equal 
amounts of the product (800ul) are dispensed and blended in until 
absorbed . 

Color measurements are taken as described in Example IV. The 
results, shown in Figure 3, demonstrate that there is a decrease 
in skin reflectance and an increase in skin redness and yellow 
coloration due to the self -tanning effect of the products. The 5% 
DHA product with the lipid mix appears to retain color more 
efficiently during the entire seven-day study period when compared 
with the control product. 



11 



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What is claimed is: 

1. A method of retarding desquamation in the stratum corneum of 
the skin comprising applying to the skin a composition comprising 
an effective amount of cholesterol sulfate. 

2. The method of claim 1 in which the composition comprises from 
about 0.05% to about 10% cholesterol sulfate. 

3. The method of claim 1 in which the composition comprises about 
1% to about 3% cholesterol sulfate. 

4 . A method of treating or preventing the thinning of the stratum 
corneum of the skin comprising applying to the skin a composition 
comprising an effective amount of cholesterol sulfate. 

5. The method of claim 4 in which the composition comprises from 
about 0.05% to about 10% cholesterol sulfate. 

6. The method of claim 4 in which the composition comprises about 
1% to about 3% cholesterol sulfate. 

7. A method of protecting the skin from the effects of UV 
radiation which comprises applying to the skin a composition 
comprising effective amount of cholesterol sulfate. 

8. The method of claim 7 in which the composition comprises from 
about 0.05% to about 10% cholesterol sulfate. 

9. The method of claim 7 in which the composition comprises about 
1% to about 3% cholesterol sulfate. 

10. The method of claim 7 in which cholesterol sulfate is applied 
in combination with at least one sunscreen. 



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11. A composition for protection of the skin from the effects of 
UV radiation comprising an effective amount of cholesterol sulfate 
and at least one sunscreen. 

12. The composition of claim 11 in which the sunscreen is 
selected from the group consisting of titanium dioxide, zinc 
oxide, iron oxide, camphor derivatives, cinnamates, salicylates, 
benzophenones, triazines, PABA derivatives, diphenylacrylate 
derivatives, dibenzoylmethane derivatives, and combinations 
thereof . 

13. A method for artificially tanning the skin comprising 
applying to the skin a composition comprising applying to the skin 
an effective amount of cholesterol sulfate and an effective amount 
of at least one self -tanning agent. 

14. A composition for artificially tanning the skin comprising an 
effective amount of cholesterol sulfate and an effective amount of 
at least one self -tanning agent. 

15. The composition of claim 14 which comprises DHA as the self- 
tanning agent. 

16. The composition of claim 15, which also contains an 
imidazole. 

17 . A composition for retarding desquamation and enhancing the 
thickness of the stratum corneum comprising an effective amount of 
cholesterol sulfate and effective amounts of at least one of each 
of fatty acids, ceramides and a sterol. 

18. The composition of. claim 17 in which the fatty acid is a C l2 - 
C 20 fatty acid. 

19. The composition of claim 17 in which the ceramide is ceramide 
III or a cerebroside. 

20. The composition of claim 17 which comprises cholesterol 
sulfate, a C l2 -C 20 fatty acid, ceramide III or a cerebroside, and 
cholesterol . 



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21. A method of preventing or retarding the appearance on the 
skin of fine lines and wrinkles associated with aging which 
comprises applying to the skin an effective amount of cholesterol 
sulfate. 



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FIG. 1C 

SUBSTITUTE SHEET (RULE 26) 



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FIG. 1D 




FIG. 1E 




FIG. IF 

SUBSTITUTE SHEET (RULE 26) 



WO 00/45786 PCT/USOO/02750 

3/4 



FIG. 2 



in 
a> 

"5 5.0- 

> 

* 4.5- 

LU 

< 4.0- 
_2 3.5- 
U 3.0- 
•E 2.5- 
&2.0- 
J 1.5- 
U 1.0- 
J0.5- 

H o.o- 1 - 



10% 



1 day 



20% 



4 day 



HDHA alone 
□ DHA + CS 



25% 



m 



5 day 



SUBSTITUTE SHEET (RULE 26) 



WO 00/45786 PCT/USOO/02750 

4/4 



FIG. 3 



O 

u 



<D 
00 

c 

CO 

U 

■ 4— » 



4.00-1 
3.50- 
3.00- 
2.50- 
2.00- 
1.50- 
1.00- 
0.50- 
0.00- 





M 24 Hour 
EODay 3 

□ Day 4 

□ Day 7 



DHA ALONE 



DHA, CS AND LIPID 



SUBSTITUTE SHEET (RULE 26) 



INTERNATIONAL SEARCH REPORT 



Intt JJonal Application No 

PCT/US 00/02750 



A. CLASSIFICATION OF SUBJECT MATTER 

IPC 7 A61K7/48 A61K7/42 



According to Internationa} Patent Classif fcatton (IPC) or to both national classification and IPC 



B. FIELDS SEARCHED 



Minimum documentation searched (classification system followed by classification symbols) 

IPC 7 A61K 



Documentatton searched other than rrtirUrmim o^cumantatton to the extent that such documents are Included In the fields searched 



Electronic data base consulted during the international search (name of data base and, where practical, search terms used) 



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Category • Citation of document, with IrtcScation, where appropriate, of the relevant passages 



Relevant to claim No. 



DE 196 42 872 C (HENKEL KGAA) 
12 February 1998 ( 1998-02-12) 
page 3, line 13 
page 5, line 12 - line 23 
page 5 
claims 

WO 90 01323 A (BERNSTEIN JOEL E) 
22 February 1990 (1990-02-22) 
page 2, line 12 - line 23 



1-12 



1,2,5, 
17,18,21 



~X] Further documents are listed in the continuation of box C. 



Patent family members are listed In annex. 



Special categories of cted documents : 

'A" document defining the general state of the art which is not 

considered to be of particular relevance 
"E" earlier document but published on or after the international 
filing date 

V document which may throw doubts on priority ctaim(s)or 
which is cited to establish the publication date of another 
citation or other special reason (as specified) 
"O" document referring to an oral disclosure, use, exhfoitionor 
other means 

"P- document pUfened prior to the irrtematJonaJ fDing date but 
later than the priority date claimed 



T later document published after the International filing date 
or priority date and not in conffict with the applcationbut 
cited to understand the principle or theory underlying the 
Invention 

■X" document of particular relevance; the claimed invention 
cannot be considered novel or cannot be considered to 
involve an Inventive step when the document is taken alone 

"Y- document of particular relevance; the claimed Invention 
cannot be considered to Involve an Inventive step when the 
document Is combined with one or more other such docu- 
ments, such combination being obvious to a person skilled 
n the art 



Dateoftheex^rorripletto 

19 April 2000 


Date of mailing of the fntematlona) search report 

09/05/2000 


Name and malting address of the ISA 

European Patent Office, PB. 5818 Patenttaan2 
NL-22B0HVRQswfjk 
Tel (+31-70) 340-2040, Tx. 31 651 epo* 
Fax (+31-70) 340-3016 


Authorized officer 

Pel 11 Wablat, B 



page 1 of 2 



INTERNATIONAL SEARCH REPORT 



Mi lional Application No 



PCT/US 00/02750 



C.(Contln 


uatton) DOCUMENTS CONSIDERED TO BE RELEVANT ~ ' 


Category ' 


Citation of document, with indication, where appropriate, of the relevant passages 


Relevant to claim No. 


E 

X 
A 


DE 198 34 812 A (BEIERSDORF AG) 

3 February 2000 (2000-02-03) 

page 3, line 27 
page 3, line 40 - line 44 
page 3, line 61 
page 6, line 30 - line 37 
page 7, line 4,5 
page 7; example 2 
page 9, line 9 
claims 1,2,4 

CHEMICAL ABSTRACTS, vol. 132, 

Columbus, Ohio, US; 

abstract no. 170820, 

WILDEN, MIM VAN DER ET AL: "A 

skin-Identical lipid concentrate for an 

Improved sk1n-barr1er function" 

XP002135991 

abstract 

4 FRAGRANCE J. (1999), 27(10), 71-74 , 
1999, 

CHEMICAL ABSTRACTS, vol. 104, 
Columbus, Ohio, US; 
abstract no. 10400, 

ABE, TAKASHI ET AL: "Cosmetics containing 

cholesteryl sulfates" 

XP002135992 

abstract 

& JP 60 161911 A (KANEBO, LTD., JAPAN) 
23 August 1985 (1985-08-23) 


1,2.5,7, 
8,10-12, 
17-21 

1,2,4,5 

1-6. 
17-21 



page 2 of 2 



INTERNATIONAL SEARCH REPORT 





tflfOfl 


nation on patent family members 


Ink jlonaJ Application No 






PCT/US 00/02750 


1 Patent document 
cited in search report 


Publication 
date 


Patent family 


PiibBcation 
date 


DE 19642872 


c 


12-02-1998 


AU 4780897 A 
W0 9817244 A 
EP 0941054 A 


15-05-1998 
30-04-1998 
15-09-1999 


WO 9001323 


A 


22-02-1990 


AU 4216089 A 
CA 1336762 A 
US 5508034 A 


05-03-1990 
22-08^1995 
16-04-1996 


DE 19834812 


A 


03-02-2000 


NONE 






OP 60161911 


A 


23-08-1985 


JP 1433601 C 
JP 62041684 B 


07-04-1988 
04-09-1987 





Rum PCT/tSAOlO (patanl fantfy trmx) (Jtfy 1882)