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(11 publication number : 11-005742 
(43)Date of publication of application : 12.01.1999 

A61K 31/575 
A61K 7/00 
A61K 7/48 

(21) Application number : 09-218288 

(22) Date of filing : 30.07.1997 

(71) Applicant : SHISEIDO CO LTD 

(72) lnventor : SATO JUNKO 



Priority number : 091 17508 Priority date : 21 .04.1997 Priority country : JP 



PROBLEM TO BE SOLVED: To obtain the subject dermal external preparation for controlling 
decomposition of desmosome in the skin by including a cholesterol sulfate. 
SOLUTION: This external composition is obtained by including a cholesterol sulfate 
(cholesterol 3-sulfate ester) derived from a living body or partially or totally synthesized at 
approximately 0.005 to 20 wt.%, based on the whole composition, preferably 0.5 to 5 wt.%, for 
lotion, cream or the like, and further including a diluent or aid (alcohol, water, chelating agent, 
urea, surfactant or the like) which is commonly used for cosmetics and external medicines. 


[Date of request for examination] 29.05.2002 

[Date of sending the examiner's decision of 28.03.2006 

[Kind of final disposal of application other than 
the examiner's decision of rejection or 
application converted registration] 

[Date of final disposal for application] 1005742Pl.htm 1 1/30/2006 

• Searching P AJ Page 2 of 2 

[Patent number] 

[Date of registration] 

[Number of appeal against examiner's 
decision of rejection]. 

[Date of requesting appeal against examiner's 
decision of rejection] 

[Date of extinction of right] 

http://wwwl9.ipdl.ncipi.go j p/P A 1/result/detail/main/wA A AvIaaZtDA41 1005742Pl.htm 





TITLE: Cholesterol sulphate containing composition - 

used to 

inhibit desmosome decomposition to accelerate 


desquamation and renewal of corneal layer 
PRIORITY-DATA: 1997 JP-0117508 (April 21, 1997) 



JP 11005742 A January 12, 1999 N/A 

004 A61K 031/575 




JP 11005742A N/A 1 997 JP-0218288 

July 30, 1997 

INT-CL (IPC) : A61K007/00, A61K007/48 , A61K031/575 


External dermal composition (especially for inhibition of desmosome 
decomposition, or for maintaining epidermal normal balance between 
decomposition and inhibition of desmosome) contains cholesterol 
sulphate (CS) 
as the active ingredient. 

USE- The CS-containing composition can inhibit desmosome 
decomposition to 

accelerate epidermal desquamation and renewal of corneal layer. 

ADVANTAGE ■•■ CS can antagonistically inhibit activities of both serine 

( trypsin-like enzyme and chymotrypsin-like enzyme) in desmosome to 
balance the 

formation and decomposition of desmosome in accordance with epidermal 

11/30/2006, EAST Version: 

condition . 




CPI-CODES: B01-D02; B14-D07C; B14-N17; D08-B09A; E01; 

Chemical Indexing M5 *01* 
Fragmentation Code 

M781 M903 M904 P943 S005 S032 S131 S133 S134 S142 
S143 S303 S317 S703 S750 S752 S761 S762 U560 U563 
Specfic Compounds 

Chemical Indexing M2 *02* 
Fragmentation Code 

C216 KO K4 K442 M210 M211 M271 M282 M320 M416 

M620 M781 M903 M904 M910 P943 

Specfic Compounds 

00274K 00274U 

Registry Numbers 


Chemical Indexing M3 *02* 
Fragmentation Code 

C216 KO K4 K442 M210 M211 M271 M282 M320 M416 

M620 M781 M903 M904 M910 P943 

Specfic Compounds 

00274K 00274U 

Registry Numbers 




CPI Secondary Accession Numbers: C1999-040366 

1 1/30/2006, EAST Version: 


Page 1 of 4 


JPO and NCIPI are not responsible for any 
damages caused by the use of this translation. 

1 .This document has been translated by computer. So the translation may not reflect the original 

2 **** s hows the word which can not be translated. 
3. In the drawings, any words are not translated. 


[Detailed Description of the Invention] 

[Field of the Invention] This invention relates to the pharmaceutical preparation for dermatology, 

especially skin external preparations. 


[Background of the Invention] Conventionally, in order to maintain the treatment and the fresh skin of a 
certain fixed skin disease, various kinds of moisturizers and use of lipids including cholesterol have 
been tried (about use of cholesterol, they are G.Lykkesffldt et al., Lancet, 1983, and 1337 -1338 

[0003] If the knowledge relevant to cholesterol is surveyed here, it is known for the ichthyosis caused by 
the deficit of steroid sulfas TAZE that the hyperkeratosis will be held with are recording of a cholesterol 
sulfuric acid. Moreover, the thing of extent which can be viewed without accompanying partial 
spreading of a cholesterol sulfuric acid by erythema for which it can drop and ** is brought about is also 
reported (M. E.Maloney et al, J.Invest.Dermatol., 83 (1984), 252-256). 

[0004] however, especially in the hoof which has a strong adhesive property, a cholesterol sulfuric acid 
exists in abundance (P. - M.Elias et al. — ) J. between the horny layer (palm) pasted up closely and the 
horny layer (overarm) pasted up loosely, to Clin.Invest.74 (1984) and that [ 1414-1421 ] It is reported 
that there is no significant difference (S. Serizawa et al., J.Invest.Dermatol., 99 (1992), 232-236). 
Anyway, the relation between a cholesterol sulfuric acid and the accumulated horny layer is not 
necessarily clear. 

[0005] On the other hand, the desmosome in the skin has the role important for adhesion of a keratin 
cell, and it is checked that decomposition of the desmosome by two sorts of serine proteases (a trypsin 
Mr. enzyme and chymotrypsin Mr. enzyme) brings about the desquamation of a horny layer (for 
example, Y.Suzuki et al., British J.Dermatol., 134 (1996), 460-464). Based on such knowledge, the 
persons involved in this invention person proposed a means to harmonize formation and its 
physiological exfoliation of a horny layer, by promoting the two above-mentioned sorts of serine 
protease activity. 
[0006] • 

[Problem(s) to be Solved by the Invention] By the way, probably, the activity of the above-mentioned 
serine protease may be required when unusually, and control of those activity holds the homeostasis of 
the skin conversely. 

[Means for Solving the Problem] As a result of having examined the matter which has such depressant 
action, in the experiment of the capacitation of a sperm, the cholesterol sulfuric acid with which 
checking those serine proteases is also reported found out controlling the enzyme activity of 

[0008] In this way, the cholesterol sulfuric acid found out that it could be intentionally used for 
controlling the decomposition activity, when superfluous decomposition of desmosome had arisen. 1 1/30/2006 


Page 2 of 4 

Therefore, in order to solve the above-mentioned technical problem here, the skin external preparations 
which come to contain a cholesterol sulfuric acid as an active principle are offered, and the skin external 
preparations for controlling decomposition of the desmosome which comes to contain a cholesterol 
sulfuric acid as an active principle are offered. Moreover, the skin external preparations for maintaining 
the normal balance of the decomposition of desmosome and control in the skin which comes to contain a 
cholesterol sulfuric acid as an active principle as invention of another mode are offered. 
[0009] As invention of further another mode, the positive desquamation in the skin which comes to 
contain a cholesterol sulfuric acid as an active principle is* brought about, and the skin external 
preparations for promoting renewal of a horny layer are offered. 

[0010] As for the cholesterol sulfuric acid (namely, cholesterol 3-sulfate) used by this invention, the 

thing of the living body origin may also be obtained by the semisynthesis and the total synthesis. These 

are marketed and should just use what can come to hand easily to this contractor. 

[001 1] Although a cholesterol sulfuric acid can be used with the diluent or assistant regularly used as 

cosmetics or medical-application external preparations, these diluents etc. must not have a bad influence 

on an operation of a cholesterol sulfuric acid. As a typical thing of a diluent or an assistant, alcohol, 

water, a buffer, a chelating agent, a urea, a surfactant, etc. can be mentioned. 

[0012] Although a cholesterol sulfuric acid can fluctuate the content to the inside of external 

preparations according to dosage forms and the concrete purpose of use, in the case of a lotion, cream 

pharmaceuticals, etc., it can be included 0.5 to 5% of the weight preferably about 0.005 to 20% of the 

weight per total constituent weight. Moreover, those preparation can be carried out according to the 

well-known approach as the method of preparation of various skin external preparations. 

[0013] In this way, since the cholesterol sulfuric acid as an active principle controls both the activity by 

the trypsin Mr. enzyme and chymotrypsin Mr. enzyme in desmosome in antagonistic inhibition, as for 

the external preparations of this invention obtained, it is possible to make formation and decomposition 

of desmosome balance according to a skin condition. 


[Example] Hereafter, an example explains this invention and its operation effectiveness still more 

(Effect of partial spreading) 

Experiment cholesterol sulfuric acid Sigma What came to hand from the shrine was used, and the 8- 
weeks old male was used for the hair loess mouse (HR-1) three groups. 

[0015] 80micro of 10 cholesterol sulfuric-acid (following, CS) solutions L of mM in dimethyl sulfoxide 
(DMSO) was applied behind the hair loess mouse once per day. The horny layer was collected by tape 
stripping three days after. Moreover, it carried out by uniting a biopsy. 

[0016] The histological observation> biopsy sample was fixed with formalin 10%, and embedding was 
carried out to paraffin. Hematoxylin-eosin staining of the intercept was carried out. The thickness of 
epidermis was measured with the optical microscope (Olympus XL- 10) equipped with the image 
analysis system. 

[0017] It fixed, with the fixing fluid of KARUNOFU skiing, and the biopsy sample was processed with 

1.0% osmium tetroxide of reduction, and carried out embedding to resin. After carrying out electron 

staining of the ultrathin section with citric-acid lead and uranium acetate, it observed with the electron 

microscope (H7100, Hitachi). The horny layer was counted by the bride method. 

[0018] a result -- the epidermis by partial spreading of CS to a mouse, and change (1) of a horny layer 

The scale which will be visible behind [ whole ] a mouse after spreading of CS on the 3rd was 


[0019] (2) A difference was not accepted between the DMSO processing whose thickness of epidermis 
is a basis, and CS processing. 

[0020] (3) The number of layers of the horny layer under an electron microscope was increasing by 
about 1 .5 times by CS processing compared with DMSO processing. 
[0021] (Detection of the desmosome of a horny layer) 

200micro buffer solution containing 0.1M Tris HC1 (pH9), 9M urea, 2%SDS, and 1% mercaptoethanol 1 1/30/2006 


Page 3 of 4 

of buffer solutions per 2mg of horny layers (L) extracted from the horny layer which carried out tape 
stripping and extracted the protein of experiment desmosome at 37 degrees C for 15 hours. Extract 
Laemmli It mixed with the sample buffer solution (Laemmli et al., Nature 227 (1970) 680-685), and 
heated on the water bath for 10 minutes. Supernatant liquid was analyzed by SDS-PAGE (10% gel) after 
centrifugal. Electrophoresis gel was moved to the PVDF film (Applied Biosystems), and desmosome 
protein was detected by the Western blot technique using the antibody to the DESUMO grain L 
The content of the DESUMO grain I in the horny layer of a result CS processing mouse was higher than 
basis DMSO processing. This result shows that decomposition of desmosome is controlled by partial 
spreading of CS. Specifically, please refer to drawing 1 . 
[0022] (Distribution of the cell from a horny layer sheet) 

experiment horny layer sheet lmg kanamycin 60micro - 37 degrees C incubated for 24 hours in g 
content cleaning agent mixed liquor [dimethyl dodecyl amine oxide [ of 8mM ] (DMDAO) and 2mM 
sodium dodecyl sulfate (SDS)] 1ml. Respectively, the horny layer sheet was agitated for 2 seconds with 
the vortex mixer after the incubation performed including CS (ImM, 5mM), DMSO, and a protease 
inhibitor (0.25mM chymostatin and 0.25mM leupeptin). The number of the cells which have separated 
in cleaning agent mixed liquor was measured by the haemacytometer. 

[0023] Compared with the time of distribution of a result cell adding DMSO, it became clear that it 
would be controlled to 19.6% if it adds by 5mM to 51.9%, and distribution of a cell would be controlled 
by the concentration dependence target in CS if it adds by ImM. In addition, in addition of a protein 
inhibitor, distribution of a cell was suppressed to 1.3%. 
[0024] (Operation over the trypsin and chymotrypsin of CS) 

The experiment crystal Buta pancreas trypsin (Wako) and the crystal cow pancreas chymotrypsin 
(Sigma) were used, and the inhibition behavior of CS was investigated. In addition, the homologous of 
the trypsin Mr. enzyme of desmosome and a chymotrypsin Mr. enzyme, and an amino acid sequence 
chose these proteases from the very high thing, respectively [above-mentioned Suzuki's and others 
reference and Skytt et al., Biochem.Biophys.Res.Comm., 211 (1995), and 586 -589 reference]. 
[0025] Trypsin activity and chymotrypsin activity were measured as a substrate using Boc-Phe-Ser-Arg- 
MCA (3107-V) and Suc-Leu-Len-Val-Tyr-MCA (3120-V) (peptide lab), respectively. All assays were 
performed at 37 degrees C among 0.1M Tris HC1 (pH8.0). 

[0026] Result CS showed antagonistic inhibition also to which of a trypsin and KIMOTORIPUKIN, and 

the inhibition constant was 2.1microM to 5.5microM and a chymotrypsin to the trypsin. 

[0027] If the above is summarized, it is expected that inhibitory action is shown to the protease in a 

horny layer, and CS is in vivo. Producing a scale is checked, while controlling decomposition of 

desmosome then and thickening a horny layer. 


(Example of a formula) 
Presentation (% of the weight) 

Stearyl alcohol 6.0 Stearin acid 2.0 Hydrogenation lanolin 4.0 Squalane 9.0 An octyl dodecanol 10.0 
Cholesterol sulfuric acid 1.0 1, 3-butylene glycol 6.0 polyoxy ethylene glycol 1500 4.0 Polyoxyethylene 
(25) cetyl alcohol 3.0 Glyceryl monostearate 2.0 antiseptics Optimum dose Antioxidant Optimum dose 
Perfume Optimum dose Purified water A moisturizer is added to 54.0 preparation purified water, and 
heating adjustment is carried out at 70 degrees C. After the heating dissolution, a cholesterol sulfuric 
acid, a surfactant, antiseptics, an antioxidant, and perfume are added, and oil is adjusted to 70 degrees C. 
This was added to the previous aqueous phase, it makes an emulsification particle into homogeneity and 
cooled [ deaerated, filtered and ] in the homomixer, and the cream was obtained. 

[Effect of the Invention] If it is used when decomposition of superfluous desmosome is obtained, 
returning it to a normal condition is expected and the skin external preparations which include CS as an 
active principle will be able to be used for maintaining the normal balance of decomposition of 
desmosome, and control, so that I may be understood from an operation of the above CS. 1 1/30/2006 

JP,1 1-005742, A [DETAILED DESCRIPTION] Page 4 of 4 

[Translation done.] 1 1/30/2006