JPH01305026A - Agent for suppressing formation of enzyme - Google Patents

Abstract

PURPOSE:To obtain an agent capable of suppressing the formation of tyrosinase beforehand in place of inhibiting the action and activity of the enzyme, and useful in the fields of pharmaceuticals, cosmetics, etc., by using propionic acids, butyric acids and valeric acids as active components. CONSTITUTION:The objective agent can be produced by using an active component comprising one or more compounds selected from propionic acids, butyric acids and valeric acids which are tyrosinase-formation inhibiting substances having high safety and stability and forming the active component in the form of a drug by conventional method. The concentration of the tyrosinase-formation inhibiting substance is usually 0.001-1.0M and the pH of the agent is adjusted to 2.5-9.0. The daily dose of the tyrosinase-formation inhibiting substance is 0.001-100g for adult. The tyrosinase-formation inhibiting agent has strong effect to suppress the formation of melanin and gives fair skin. It can be formed in the form of injection, oral drug, external medicine, bathing agent, milky lotion, pack, cream and the like.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is an enzyme production inhibitor, and more specifically, - does not inhibit the action or activity of the enzyme tylosinase once produced, but inhibits the production of the enzyme itself. It involves a completely new type of drug that pre-inhibits the production of the offending enzyme itself.

[Prior Art] Melanin exists in the skin, plays an important role in protecting the body from the harmful effects of ultraviolet rays, and is an important medically and cosmetically important factor. Melanin synthesis occurs through tyrosinase within pigment cells in skin tissue.
tyrosin is converted to dopa and then to dopaquinone by the action of tyrosinase (also called tyrosinase), and 5.6
- It is said to be carried out via sioxindole, etc. Excessive amount of melanin causes dark complexion and uneven distribution.
Both are cosmetic defects.

Traditionally, hydroquinone,
MBE) l (monobenzylether of hydroquinone: monobenzylether of hy
droquinor+e), etc., but although these have a strong skin-whitening effect, their main effect is degeneration and death of pigment cells, impairing the skin's natural physiological functions.
It has the disadvantage of causing side effects such as irreversible vitiligo, pigment abnormalities, and rashes.

Therefore, focusing on tylosinase, an enzyme involved in the production of melanin mentioned above, vitamin C1, glutathione, cysteine, etc. have been used to reduce the amount of melanin in the skin and make the skin fair and beautiful. (Japanese Unexamined Patent Publication No. 142515/1983).

However, all of these drugs are activity inhibitors of the tylosinase enzyme itself, and inhibit the activity and action of tylosinase once produced. As described above, the current state of the art lies in how to inhibit the activity of enzymes that have already been produced.

In contrast, the present invention is intended to prevent the enzyme itself from being produced in advance, and is not intended to treat the enzyme that has already been produced as in the conventional method. In other words,
It targets the most fundamental part of the melanin formation system by focusing on the enzyme (tylosinase) that is the root cause of the problem and suppressing the production of that enzyme itself. Therefore, the present invention is not fundamentally different from the conventional technical idea in terms of the technical idea itself, but rather in the setting of the purpose of the invention itself, and the starting point of the invention itself is new.

In this way, suppressing enzyme production itself means that
This invention has never been conceived before, and the technical problem or purpose of the invention itself is new, and the present invention employs a configuration in which specific organic acids such as propionic acids, butyric acids, and valeric acids are used for this purpose. This is what I did. In addition, the adoption of such a technical idea is completely new, as there is no prior art disclosure or even any suggestion.

In other words, the present invention is novel both in purpose and configuration;
Along with this, the effects are new and remarkable.

[Problems to be Solved by the Invention] As mentioned above, the conventionally used inhibitors of the activity of the tylosinase enzyme itself not only have an insufficient effect of inhibiting the enzyme activity itself, but also all of them Because it is a reducing agent that utilizes the reducing power possessed by , it has poor stability and lacks long-lasting effects, and its skin whitening effect on living cells is insufficient. (Unexamined Japanese Patent Publication No. 53-1425
No. 15, page 1, lower right blade um to page 2, upper left blade ram).

[Means for Solving the Problems] The present invention is essential in order to solve the above-mentioned drawbacks all at once, and utilizes a substance that inhibits the activity of the tylosinase enzyme itself, which has been conventionally done. No matter what type of inhibitor is used in this method, no substance has been found that is satisfactory in terms of both activity-inhibiting effect and durability of efficacy.

In other words, although various substances have been extensively tested and various aspects have been thoroughly investigated, once tylosinase has been produced, no matter what drug is used, the intended purpose cannot be completely achieved. I came to the conclusion that it was not possible.

Therefore, we completely changed our way of thinking, and since the cause of these dermatological troubles lies in the production of tylosinase itself, we decided that if we did not allow the production of tylosinase itself, which is the root cause of the troubles, future troubles would not occur. Based on this recognition, we set a completely new technical challenge: to develop a drug that does not produce this tylosinase.

In order to specifically achieve this novel objective, - independently of the direct inhibition of the enzymatic activity of the tylosinase once produced,
The aim is to suppress the production of tylosinase itself by utilizing the autonomous melanin production regulation function of living pigment cells, and to demonstrate the effect of suppressing melanin production, and to produce tylosinase by focusing on highly stable metabolic-related organic acids. We have continued to search for substances that have an inhibitory effect (hereinafter referred to as tylosinase production inhibitors).

As a result, we found for the first time that propionic acids, butyric acids, and valeric acids strongly inhibit the production of tylosinase itself, and confirmed that they are suitable as tylosinase production inhibitors. Furthermore, the inventors have discovered that a tylosinase production inhibitor containing the present substance exhibits the effect of inhibiting melanin production and, in turn, achieves a fair skin effect, thereby completing the present invention.

The propionic acids, butyric acids, and valeric acids referred to in the present invention are:
It refers to propionic acid, orthobutyric acid, isobutyric acid, orthovaleric acid, isovaleric acid, and salts such as alkali metal salts or alkaline earth metal salts of these carboxylic acids, esters, and acid amides.

Although the tylosinase production inhibitor of the present invention has an outstanding tylosinase production inhibiting effect in living cells, it is also unique in that it does not substantially inhibit the activity of the tylosinase enzyme itself.

Various measurement methods referred to in this specification are defined as follows.

(1) The activity inhibitory effect of tylosinase has been reported in the British Journal of Dermatology.
h Journal of Der+satology
) J, Vol. 103, pp. 625-633 (198
Measurement is performed according to the activity measurement method described in 2008). In this method, a test preparation is used and reacted in the same way without changing the pH. If the activity of the tylosinase enzyme itself is decreased compared to the control, it is determined that the activity of tylosinase is inhibited.

(2) The inhibitory effect on melanin production in pigment cells is based on “Cancer Research”.
JN Vol. 42, pp. 1194-2002 (1982
Measure according to the method described in 2010).

That is, 4 x 10' mouse melanoma-derived cells B were placed in Eagle's MEM medium 101 containing 10 v/vX beef tallow serum.
-16 was suspended at 5V/V in a 25cm2 culture Lou bottle.
Culture at 37°C in the presence of XCO2. The main culture solution is replaced with a medium containing a specimen for tylosinase production inhibition test on days 0 and 3, and cultured for 5 days.

Cells were soaked in phosphate buffer (pH
After washing at 7°2), detachment is performed using a solution containing trypsin and EDTA, and the cells are collected by filtration.

After the cells collected on the filter paper are dried, the reflected light at 500 nm is measured using a densitometer, and the total amount of melanin is determined from the reflected absorbance (blackness).

By this method, the treated area is determined to exhibit a melanin production inhibiting effect when the absorbance of the treated area decreases to 80% or less of the absorbance of the control.

(3) The inhibitory effect on tylosinase production is as follows: “Journal of the Japanese Dermatological Association,” Vol. 87, No. 13, pp. 883-901 (
Dopa reaction method described in 1972) and ``
Cancer Research
h), Volume 42, Pages 1194-2002 (198
Judgment was made according to the tylosinase confirmation method described in 2007). That is, mouse melanoma-derived cell B-16
was cultured for 5 days in a culture solution containing the melanin production inhibitor of the present invention according to the above method, and after whitening, the culture solution was discarded, and the cells were fixed with 10v/v% formalin, and then
0.1 w/vl L-dopa containing 0.1 M phosphate buffer (
The cells were immersed in pH 7.3) and maintained at 37°C for 3 hours, and then observed under a microscope.
Acetic acids, lactic acids, and pyruvic acids invented by Shimizu are highly safe and stable tylosinase production inhibitors, so they can be contained as active ingredients in injections, internal medicines, etc. It is used in medicines such as external preparations and bath additives, and cosmetics such as emulsions, bags, and creams, and not only suppresses the production of melanin and has a whitening effect, but also treats pigmentation disorders such as age spots, freckles, and sunburn. Highly effective for prevention.

The specific organic acids used in the present invention may be used alone or in combination of two or more, and when used in combination, a synergistic effect is expected.

It should be noted that these organic acids are also contained in food, albeit in small amounts, and are simply ingested with food on a daily basis, as they are known to be produced by microorganisms in the intestines. As is clear, there is virtually no toxicity, and there are no safety issues.

Hereinafter, the present invention will be explained in detail through experiments.

Fruit 91. Search for tylosinase production inhibitors 1st
Using the metabolism-related organic acids shown in the table, the inhibitory effect on solute production was investigated. Each organic acid has a predetermined p)
I was previously neutralized with sodium hydroxide and used at a concentration of 10 mM.

The results are shown in Table 1.

No. 1 Table (Note 1) For those with optical isomers, L type was used.

(Note 2) + means showing an inhibitory or suppressing effect, - means not showing an inhibitory or suppressing effect.

As is clear from the results in Table 1, unlike other organic acids, propionic acid, orthobutyric acid, and orthovaleric acid do not inhibit the activity of tylosinase, but they do exhibit an inhibitory effect on tylosinase production. It has been found. It has also been found that propionic acid, orthobutyric acid, and orthovaleric acid have an inhibitory effect on melanin production due to their inhibitory effect on tylosinase production.

On the other hand, although kojic acid exhibits an inhibitory effect on melanin production, it has been found that this is due to an inhibitory effect on the activity of tylosinase and is not due to an inhibitory effect on tylosinase production.

Experiment 2. Effect of salt type and concentration on tylosinase production inhibitory effect According to the method of Experiment 1, propionic acid, orthobutyric acid, isobutyric acid, orthovaleric acid, and isovaleric acid were used to determine the effect of salt type and concentration on tylosinase production inhibitory effect. The effect of concentration was investigated.

The results are shown in Table 2.

Table 2 (Note) + means showing an inhibitory effect, - means not showing an inhibitory effect.

As is clear from the results in Table 2, propionic acid, orthobutyric acid, isobutyric acid, orthovaleric acid, and isovaleric acid are alkali metal salts such as lithium, sodium, and potassium, and alkaline earth metal salts such as calcium and magnesium. It shows the same inhibitory effect on tylosinase production, and its concentration is about 2.
It was found that the inhibitory effect was exhibited at 5 mM or more.

Experiment 3. Enhancement of tylosinase production inhibitory effect According to the method of Experiment 1, the combined effect of propionic acid, orthobutyric acid, and orthovaleric acid on tylosinase production inhibitory effect was investigated. Each organic acid was neutralized in advance with sodium hydroxide to a predetermined p)l, and used in a predetermined combination (molar ratio) and predetermined concentration.

The results are shown in Table 3.

Table 3 (Note) + means showing an inhibitory effect, - means not showing an inhibitory effect.

As is clear from the results in Table 3, propionic acid, orthobutyric acid, and orthovaleric acid are more effective in suppressing tyrosinase production when used in combination with two or more selected from them than when each is used alone. It turned out that it could be strengthened.

Experiment 4. Each of the tylosinase production inhibitors prepared by the methods in Clinical Test Examples 4.6.8 and 10 was tested on 60 randomly selected men and women (30 men, 30 women) aged 20 to 55 who were suffering from age spots and freckles. , average age for men: 32.6 years, for women: 36.5 years
We asked children aged 3 to 3 years old to use the product and conducted a questionnaire survey regarding its safety and effectiveness.

The results are shown in Table 4.

Table 4 As is clear from the results in Table 4, the tylosinase production inhibitor of the present invention can be used safely without any side effects, and is highly effective in treating and preventing age spots and freckles. .

As is clear from the above experiments, these tylosinase production-inhibiting substances exhibit a tylosinase production-inhibiting effect, exhibit a strong melanin production-inhibiting effect, and can achieve a high skin-fairing effect. Furthermore, the safety of these tylosinase production-inhibiting substances is extremely high considering their effective amounts.

In the tylosinase production inhibitor of the present invention containing one or more than 281 selected from propionic acids, butyric acids, and valeric acids as an active ingredient, these tylosinase production inhibitors can be used as such, but Usually, the concentration is between 0.001 and 1.0, preferably 0.0
05 to 0.5M, and its pH is 2.5 to 9.0, preferably 3.0 to 8.0. The daily dose for adults is 0.001 to 100 grams as a tylosinase production inhibitor, preferably 0.01 to 10 grams for systemic use such as intramuscular injection, and preferably 0.01 to 10 grams for oral use such as oral administration. 0.5 to 10
0g, for external use, bath preparations, and for transdermal or transmucosal use of cosmetics such as emulsions and creams.
The amount is 0.01 to 100 grams, but this amount can be increased or decreased as appropriate depending on usage, symptoms, etc.

In order to prevent or treat local pigmentation disorders such as age spots, freckles, and sunburn, as well as systemic pigmentation disorders such as addisonism, a tylosinase production inhibitor may be used alone or, if necessary, in combination with a tylosinase production inhibitor. Tylosinase production inhibitors combined with materials such as physiologically active substances, nutrients, base materials, excipients, etc. can be used as pharmaceuticals, cosmetics, etc., depending on the purpose, or
The shape can be freely selected depending on the usage pattern.

For example, injectables are liquid or solid injections that are dissolved in distilled water for injection, and oral drugs are liquid,
Transdermal preparations are available in the form of powders, granules, capsules, tablets, etc., and external preparations such as liquid preparations, emulsions, creams, ointments, poultices with cream applied on one side, bath preparations, and even lotions. It can be used in cosmetics such as emulsions, bags, and creams. In addition, when using a transdermal method, in order to increase the tylosinase production inhibitory effect and strengthen the melanin production inhibitory effect, it is also necessary to promote the penetration of the tylosinase production inhibitor into the skin tissue by iontophoresis. For example, it is advantageous to use a supporting electrolyte in combination to enhance the effect of ion introduction.

In addition to the tylosinase production inhibitor of the present invention, other organic acids such as acetic acids, lactic acids, and pyruvic acids, as well as vitamin C1, vitamin E1, glutathione, cysteine, kojic acid, placenta extract, and glucosamine derivatives may be added as necessary. , Photosensor No. 201 (Okayama Cloth, manufactured by Nippon Kanko Shiki Kenkyusho Co., Ltd.), colloidal sulfur, hydroquinone derivatives, etc. can be used in combination with one or more of them to suppress melanin production and further enhance the whitening effect. can also be selected as appropriate. Furthermore, it is optional to use a sunscreen agent in combination to enhance the main whitening effect.

Next, Examples of the tylosinase production inhibitor of the present invention will be described.

Example 1 Injection Sodium propionate 8.0 parts by weight, L-lactic acid 2.0
parts by weight, 10 parts by weight of sodium acetate, 5 parts by weight of sodium chloride
．． Dissolve 3 parts by weight of potassium chloride, 0.3 parts by weight of potassium chloride, and 0.2 parts by weight of calcium chloride in 1,000 parts by weight of water, purify and filter according to a conventional method, and add 21 parts of this a solution to a sterilized glass container. The solution was filled and sealed to produce an injection.

This product is suitable as a tylosinase production inhibitor,
It can be advantageously used for the treatment and prevention of local pigmentation disorders such as spots, freckles, and sunburn, as well as systemic pigmentation disorders such as adisonism.

Example 2 Oral preparation (tablet) 40 parts of calcium orthobutyrate pentahydrate and 45 parts of maltose!
! 61 parts by weight of cornstarch, 4 parts by weight of sucrose fatty acid ester, and tableted according to a conventional method.
Tablets each weighing 150 μg were manufactured.

This product is suitable as a tylosinase production inhibitor,
As in the case of Example 1, it can be advantageously used for the treatment and prevention of pigmentation.

Example 3 Oral preparation (capsule) 10 parts by weight of propionic acid monohydrate, magnesium L-lactate.
50 parts by weight of trihydrate, 87 parts by weight of maltose, and 4 parts by weight of sucrose fatty acid ester are uniformly mixed, processed into granules using a granule molding machine, and then encapsulated in gelatin capsules according to a conventional method to produce 150 mg per capsule. Manufactured human capsules.

This product is suitable as a tylosinase production inhibitor,
As in the case of Example 1, it can be advantageously used for the treatment and prevention of hyperpigmentation.

Example 4 External preparation (ointment) 1.0 parts by weight of sodium orthovalerate trihydrate and 4.0 parts by weight of calcium isobutyrate were uniformly mixed with 10.0 parts by weight of glycerin, and this mixture was mixed with 50.0 parts by weight of petrolatum. 0 parts by weight,
The mixture was added to a mixture of 10.0 parts by weight of wood wax, 10.0 parts by weight of lanolin, 14.5 parts by weight of sesame oil and 0.5 parts by weight of peppermint oil, and further mixed uniformly to prepare an ointment.

As in Example 2, this product is suitable as a tylosinase production inhibitor, and can be advantageously used for the treatment of hyperpigmentation, as well as for the prevention of sunburn.

Example 5 External preparation (emulsion) 2.0 parts by weight of sodium propionate, 3.0 parts by weight of isovaleric acid.
0 parts by weight, 12.0 parts by weight of liquid paraffin, 4.0 parts by weight of lanolin, 3.5 parts by weight of oleic acid, 1.0 parts by weight of triethanolamine, and 3.0 parts by weight of octyldodecyl myristate. Mix evenly with
To this was added a preservative, an appropriate amount of perfume, and 71.5 parts by weight of purified water, and the mixture was run through a homogenizer to produce an emulsion.

This product is suitable as a tylosinase production inhibitor,
As in the case of Example 4, it can be advantageously used for the treatment and prevention of pigmentation disorders.

Example 6 External preparation (lotion) 2.0 parts by weight of sodium propionate, 1.0 parts by weight of polyoxyethylene hydrogenated castor oil, 15.0 parts by weight of ethanol, 0.1 part by weight of DL-lactic acid, sodium orthobutyrate 0°3 parts by weight, 1,
4.0 parts by weight of 3-butylene glycol, 0.5 parts by weight of sodium pyrrolidone carboxylate, a preservative, an appropriate amount of fragrance, and purified water 77, hat! A lotion was prepared by uniformly mixing the two parts according to a conventional method.

This product is suitable as a tylosinase production inhibitor,
As in the case of Example 5, it can be advantageously used for the treatment and prevention of pigmentation disorders such as age spots, freckles, and sunburn.

Example 7 Bath Agent DL - 50 parts by weight of sodium lactate and 15 parts by weight of sodium propionate were mixed with 35 parts by weight of purified water and appropriate amounts of coloring and fragrance to produce a bath agent.

``Ice crystals are suitable as a tylosinase production inhibitor, and are diluted 100 to 10,000 times in bathing water.
It is suitable for the treatment and prevention of local or systemic hyperpigmentation. In addition, ice crystals can exhibit the effect of suppressing melanin production even when used diluted in facial cleansing water, lotion, etc., as in the case of bathing water.

Example 8 Cosmetics (Milk'e) 0.5 parts by weight of polyoxyethylene behenyl ether, 1.0 parts by weight of polyoxyethylene sorbitol tetraoleate
Parts by weight, 1.0 parts by weight of lipophilic glyceryl monostearate, 0.5 parts by weight of propionic acid, 0 parts by weight of behenyl alcohol
．． 5 parts by weight, 1.0 parts by weight of avocado oil, and appropriate amounts of vitamin E1 preservative and fragrance are heated and dissolved according to a conventional method,
To this, 1.0 parts by weight of sodium isobutyrate, 5.0 parts by weight of 1,3-butylene glycol, 0.1 parts by weight of carboxyvinyl polymer and 85.3 parts by weight of purified water were added,
A milky lotion was prepared using a homozonizer.

Ice crystals are suitable as tylosinase production inhibitors,
It can be advantageously used for the treatment and prevention of pigmentation disorders such as age spots, freckles, and sunburn.

Example 9 Cosmetics (pack) 2.0 parts by weight of sodium propionate were mixed with 1.5 parts by weight of squalane, 0.5 parts by weight of polyoxyethylene hydrogenated castor oil, and 4.0 parts by weight of glycerin according to a conventional method. 15.0 parts by weight, 10.0 parts by weight of ethanol, and 67.0 parts by weight of purified water were uniformly mixed to produce a pack.

Ice crystals are suitable as tylosinase production inhibitors,
As in the case of Example 8, it can be advantageously used for the treatment and prevention of pigmentation disorders.

Example 10 Cosmetics (Cream) 2 parts by weight of polyoxyethylene glycol monostearate, 5 parts by weight of self-emulsifying glycerin monostearate, 5 parts by weight of propionic acid, 1 part by weight of behenyl alcohol, 1 part by weight of liquid paraffin, trioctanoic acid 10 parts by weight of glyceryl, 3 parts by weight of sodium hydrogen carbonate, and appropriate amounts of preservatives and fragrances are heated and dissolved according to a conventional method, and 2 parts by weight of orthobutyric acid and 1 part by weight are added to the solution.
, 5 parts by weight of 3-butylene glycol and 75 parts by weight of purified water were added, and the mixture was passed through a homogenizer to produce a cream.

For ice crystals, it is suitable as a tylosinase production inhibitor, and as in Example 8, it can be advantageously used for the treatment and prevention of hyperpigmentation.

[Effects of the Invention] As stated in the main text, the present invention provides that one or more substances selected from propionic acids, butyric acids, and valeric acids do not substantially inhibit the activity of the tylosinase enzyme itself. Although it does not, we have newly discovered that it exhibits a strong inhibitory effect on tylosinase production itself in pigment cells,
The present invention aims to establish a tylosinase production inhibitor containing the tylosinase production inhibitor as an active ingredient.

In addition, this tylosinase production inhibitor exhibits a strong melanin production suppressing effect and may even achieve a fair skin effect, so it is used in pharmaceuticals such as injections, oral preparations, external preparations, and bath preparations, as well as cosmetics such as emulsions, packs, and creams. It can be advantageously used for the treatment and prevention of local pigmentation disorders such as age spots, freckles, and sunburn, as well as systemic pigmentation disorders such as adisonism.

This tylosinase production inhibitor is only contained in small amounts in food, and it is also known to be a substance produced by microorganisms in the intestines. Considering its effective amount, it is extremely safe and can be used with peace of mind. Available. Furthermore, it is highly stable against heat and pH, and is easy to heat sterilize and store for long periods of time.
It has extremely advantageous conditions for industrial production and use, and is of great industrial significance in fields such as pharmaceuticals and cosmetics.

Procedural amendment July 6, 1999 Yoshi, Commissioner of the Patent Office 1) Takeshi Moon 1. Number of people involved in the incident 1986 Patent Application No. 134482 2 Name of the invention Enzyme production inhibitor 3 Person making the amendment Relationship to the case Patent applicant 1-2-3 Shimoishi, Okayama City, Okayama Prefecture Hayashibara Biochemical Research Institute Co., Ltd. Representative Ken Hayashi Hara (2 others) 4 agents 6 Contents of amendment (1) "Insufficient" stated on page 4, line 15 of the specification.

” will be corrected to “insufficient.”

(2) "Pages 1194 to 2002 (1982)" stated in page 7, line 14 of the specification is replaced with "Pages 1994 to 200
2 pages (1982)”.

(3) “Pages 1194 to 2002 (1982)” stated on page 8, line 15 of the specification is replaced with “Pages 1994 to 2002”
2 pages (1982)”.

(4) "Melanin production inhibiting substance of the present invention" described on pages 18 to 19 of page 8 of the specification is amended to "tylosinase production inhibiting substance of the present invention."

(5) "For example, for injections" written on page 18, line 15 of the specification will be amended to "For example, for injections."

(6) Insert "for example," after "[Also, as necessary,"] on page 19, line 8 of the specification.

(7) "4.0 parts by weight of glycerin" described on page 8, lines 18-19 of the specification is corrected to "4.0 parts by weight of glycerin."

Claims (1)

[Claims] (1) A drug that suppresses the production of tylosinase itself, which contains as an active ingredient one or more selected from propionic acids, butyric acids, and valeric acids.