JPH115742A - External preparation containing cholesterol sulfate - Google Patents

Abstract

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.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a dermatological preparation, particularly to an external preparation for skin.

[0002]

BACKGROUND OF THE INVENTION Conventionally, various moisturizing agents and the use of lipids such as cholesterol have been tried in order to treat certain skin diseases and maintain fresh skin (for example, the use of cholesterol). Is G. Lykke
sffldt et al., Lancet, 1983, 1337-1338).

Here, an overview of the findings relating to cholesterol is that it is known that in ichthyosis caused by deficiency of steroid sulfatase, hyperkeratosis is maintained with the accumulation of cholesterol sulfate. ing. It has also been reported that topical application of cholesterol sulfate produces visible drops without erythema (ME Maloney et al., J. Invest. Dermato.
l., 83 (1984), 252-256).

[0004] However, hooves having strong adhesiveness
Is particularly rich in cholesterol sulfate (PM
Elias et al. Clin. Invest.74(1984), 14
14-1421) versus the tightly adherent stratum corneum (hand
There is a significant difference between the stratum corneum (upper arm) and the loosely adherent stratum corneum (upper arm).
(S. Serizawa et al., J. Invest. Dermatol.,99
(1992), 232-236). I
Even if it shifts, the stratum corneum accumulated with cholesterol sulfate
The relationship with is not always clear.

On the other hand, desmosomes in the skin play an important role in the adhesion of keratinocytes, and two serine proteases (trypsin-like enzyme and chymotrypsin-like enzyme)
Degradation of desmosomes has been shown to result in desquamation of the stratum corneum (eg, Y. Suzuki et al., British
J. Dermatol., 134 (1996), 460-46.
4). Based on such knowledge, the person concerned of the present inventor
We have proposed a means to harmonize the formation of the stratum corneum and its physiological exfoliation by promoting the above two types of serine protease activities.

[0006]

By the way, when the activity of the above-mentioned serine protease is abnormally high, on the contrary, it may be necessary to suppress the activity of the serine protease in order to maintain skin homeostasis. .

[0007]

As a result of studies on substances having such an inhibitory action, cholesterol sulfate, which has also been reported to inhibit serine proteases in sperm capacitation experiments, is desmosome Have been found to suppress the enzyme activity.

[0008] Thus, it has been found that cholesterol sulfate can be used significantly to suppress the degradation activity of desmosome, for example, when excessive degradation occurs. Therefore, here, in order to solve the above problems,
Provided is a skin external preparation containing cholesterol sulfate as an active ingredient, and a skin external preparation for suppressing degradation of desmosome containing cholesterol sulfate as an active ingredient. In another aspect, the present invention provides an external preparation for skin for maintaining a normal balance between desmosome degradation and inhibition in skin, comprising cholesterol sulfate as an active ingredient.

[0009] In still another aspect, the present invention provides an external preparation for skin which comprises cholesterol sulfate as an active ingredient, which actively causes skin desquamation and promotes the renewal of the stratum corneum.

[0010] The cholesterol sulfate (that is, cholesterol 3-sulfate) used in the present invention may be of biological origin or may be obtained by semi-synthesis or total synthesis. These are commercially available, and those easily available to those skilled in the art may be used.

Cholesterol sulphate can be used together with diluents or auxiliaries commonly used as cosmetics or external preparations for medical use. These diluents and the like adversely affect the action of cholesterol sulphate. Not be. Representative diluents or auxiliaries include alcohols, water, buffers, chelating agents, urea, surfactants and the like.

[0012] The content of cholesterol sulfate in an external preparation can be varied according to the dosage form and the specific purpose of use.
It may comprise about 0.005 to 20% by weight, preferably 0.5 to 5% by weight, based on the total composition weight. Further, their preparation can be carried out according to a method known as a method for preparing various skin external preparations.

The thus obtained external preparation of the present invention can inhibit the formation and degradation of desmosomes, for example, since cholesterol sulfate as an active ingredient inhibits both activities of trypsin-like and chymotrypsin-like enzymes in desmosomes in a competitive and inhibitory manner. It is possible to balance according to the skin condition.

[0014]

The present invention and its operation and effect will be described more specifically with reference to the following examples. (Effect of Topical Application) Experimental Cholesterol sulfate was obtained from Sigma, and hairless mice (HR-1) were 8-week-old males, three in a group.

On the back of the hairless mouse, 80 μL of a 10 mM cholesterol sulfate (hereinafter, CS) solution in dimethyl sulfoxide (DMSO) was applied once a day. Three days later, the stratum corneum was collected by tape stripping. A biopsy was also performed.

<Histological Observation> A biopsy sample was fixed in 10% formalin and embedded in paraffin. Sections were stained with hematoxylin-eosin. The thickness of the epidermis was measured with an optical microscope (Olympus XL-10) equipped with an image analysis system.

The biopsy sample was fixed with Karnofsky's fixative, treated with reduced 1.0% osmium tetroxide, and embedded in resin. Ultrathin sections were electronically stained with lead citrate and uranium acetate, and then observed with an electron microscope (H7100, Hitachi, Ltd.). The stratum corneum was counted by the bridging method.

Results Changes in epidermis and stratum corneum due to topical application of CS to mice (1) On day 3 after application of CS, visible scales developed on the entire back of the mice.

(2) The thickness of the epidermis depends on the base material DMS
No difference was observed between the O treatment and the CS treatment.

(3) The number of stratum corneum under an electron microscope was increased about 1.5 times by the CS treatment as compared with the DMSO treatment.

(Detection of Desmosome in Stratum Corneum) Experimental Desmosome protein was extracted from the stratum corneum collected by tape stripping with 0.1 M Tris HCl (p
H9), a buffer containing 9M urea, 2% SDS and 1% mercaptoethanol (2 mg buffer per 2 mg stratum corneum)
00 μL) and extracted at 37 ° C. for 15 hours. Extract L
aemmli sample buffer (Laemmli et al., Nature 227)
(1970) 680-685) and heated on a water bath for 10 minutes. After centrifugation, the supernatant was subjected to SDS-PAGE (10
% Gel). Run the electrophoresis gel on a PVDF membrane (Appl
ied Biosystems), and desmosome proteins were detected by Western blotting using an antibody against desmoglein I. Results The content of desmoglein I in the stratum corneum of CS-treated mice was higher than that of base DMSO treatment. This result indicates that the degradation of desmosomes is suppressed by topical application of CS. Specifically, please refer to FIG.

[0022] (cell of the variance of the stratum corneum sheets) Experimental angle layer sheet 1 mg, kanamycin 60μg containing detergent mixture [8 mM of dimethyldodecylamine oxide (DMDAO) and 2mM sodium dodecyl sulfate (SDS)] in 1 ml, Incubated at 37 ° C for 24 hours. CS (1 mM, 5 mM), DMS, respectively
After incubation with O and protease inhibitors (0.25 mM chymostatin and 0.25 mM leupeptin), the stratum corneum was vortexed for 2 seconds. The number of cells released into the detergent mixture was counted with a hemocytometer.

Results The dispersion of cells was suppressed to 51.9% when CS was added at 1 mM and to 19.6% when added at 5 mM as compared to when DMSO was added, and the dispersion of cells was inhibited in a concentration-dependent manner. It turned out to be. In addition, the dispersion of cells was suppressed to 1.3% by the addition of the protein inhibitor.

(Effect of CS on trypsin and chymotrypsin) Inhibition behavior of CS was examined using experimental porcine pancreatic trypsin (Wako) and crystalline bovine pancreatic chymotrypsin (Sigma). These proteases were selected because of their extremely high amino acid sequence similarity to desmosome trypsin-like and chymotrypsin-like enzymes, respectively [Suzuki et al., Supra, and Skytt et al., Biochem.
iophys. Res. Comm., 211 (1995), 586-589].

The trypsin activity and chymotrypsin activity were measured using Boc-Phe-Ser-A as a substrate.
rg-MCA (3107-V) and Suc-Leu-
Measured using Len-Val-Tyr-MCA (3120-V) (Peptide Institute). All assays were performed in 0.1 M Tris HCl (pH 8.0) at 37 ° C.

Results CS showed antagonistic inhibition against both trypsin and chymotrypkin, with an inhibition constant of 5.
5 μM, 2.1 μM for chymotrypsin.

To summarize the above, CS is expected to show an inhibitory effect on proteases in the stratum corneum,
ivo confirms that it inhibits desmosome degradation, thickens the stratum corneum and produces scale.

(Prescription example) Composition (% by weight) Stearyl alcohol 6.0 Stearic acid 2.0 Hydrogenated lanolin 4.0 Squalane 9.0 Octyldodecanol 10.0 Cholesterol sulfate 1.0 1,3-butylene glycol 6 2.0 Polyoxyethylene glycol 1500 4.0 Polyoxyethylene (25) cetyl alcohol 3.0 Glycerin monostearate 2.0 Preservatives Appropriate amount Antioxidant Appropriate amount Fragrance Appropriate amount Purified water 54.0 Preparation Add humectant to purified water Heat to 70 ° C. After heating and dissolving the oil, cholesterol sulfate, surfactant, preservative,
Add an antioxidant and fragrance and adjust to 70 ° C. This was added to the above aqueous phase, the emulsified particles were homogenized with a homomixer, deaerated, filtered, and cooled to obtain a cream.

[0029]

As can be understood from the above-mentioned effects of CS, an external preparation for skin containing CS as an active ingredient, when used when excessive desmosome degradation is observed, returns it to a normal state. And could be used to maintain a normal balance between desmosome degradation and suppression.

[Brief description of the drawings]

FIG. 1 is a bar graph showing the effect of cholesterol sulfate on the residual amount of desmosome per stratum corneum weight.

Claims (3)

[Claims] An external preparation for skin comprising cholesterol sulfate as an active ingredient. 2. An external preparation for skin for suppressing the degradation of desmosomes, comprising cholesterol sulfate as an active ingredient. 3. An external preparation for skin, comprising cholesterol sulfate as an active ingredient, for maintaining a normal balance between desmosome degradation and inhibition in skin.