JPH09124472A - Inhibitor of collagenase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an inhibitor of collagenases, containing one or more dicarboxylic acids, excellent in stability and safety, capable of manifesting excellent inhibiting actions on the collagenases in a small dose and useful for relieving and treating aging of the skin, metabolic diseases, etc. SOLUTION: This inhibitor of collagenases contains one or more dicarboxylic acids (preferably a 5-12C saturated dibasic acid, e.g. glutaric acid, adipic acid, pimelic acid, etc.) and preferably further one or more metallic chelating agents (e.g. deferoxamine, Na salt of ethylenediaminetetraacetic acid, phosphoric acid or citric acid) used in combination. The dicarboxylic acid is blended in an amount of preferably about 0.01-10.0wt.% based on the total amount of the resultant pharmaceutical preparation.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to collagen, which is a constituent protein of connective tissue, to be specifically cleaved to improve skin aging, metabolic diseases such as osteoporosis, inflammatory diseases, rheumatoid arthritis and osteoarthritis. The present invention relates to a collagenase inhibitor which can be safely used for alleviation and treatment of joint diseases without side effects. More specifically, it relates to a dicarboxylic acid or a collagenase inhibitor containing the same and a metal chelating agent.

[0002]

BACKGROUND OF THE INVENTION Collagen is a major protein that constitutes connective tissue of animals, and in various tissues, type I to XII.
There are 13 known types of I. Collagenase is an enzyme that specifically cleaves such collagen proteins, and causes wrinkles and reduced viscoelasticity of the skin due to aging and ultraviolet rays, metabolic diseases such as osteoporosis, epidermolysis bullosa,
Corneal ulcer, inflammatory diseases such as root disease, rheumatoid arthritis,
It is involved in a wide variety of diseases, including articular diseases such as osteoarthritis.

Therefore, many attempts have been made to alleviate or treat the above-mentioned diseases by inhibiting the collagenase activity, and as a collagenase inhibitor, an amniotic substance-derived substance (JP-A-6-345636) and buckwheat hull protein extract. Substance (International Publication 9420541), oyster extract, peppermint extract (JP-A-6-183990), water-soluble sugar derivative (JP-A-6-1833941), lactoferrin (JP-A-5-186368), caffeic acid or a salt thereof (JP-A-5-183940).
-117145), ascorbic acid, erythorbic acid, and mixtures of these salts (JP-A-4-290819), acetylpolyamide (French Patent 2669629),
Gallotannin (JP-A-4-66524), κ-casein (JP-A-4-41417), cocoa shell extract (JP-A-3-44331), hydroxamic acid tetrapeptide (JP-A-1-160997), 1,3-bis -Substituted-naphthylaminocarbonylphenylurea derivative (JP-B-63-810)
1) etc. are disclosed.

Further, since collagenase is a metalloprotease, diferoxamine and ethylenediamine tetraacetic acid are used.
acid, EDTA) salts, tartaric acid, citric acid and other metal chelating agents are also used as collagenase inhibitors.

However, as a collagenase inhibitor which exhibits a high inhibitory activity even in a small amount and has a small number of side effects and a high stability, at present, a satisfactory one has not yet been obtained.

[0006]

Therefore, the present invention shows high collagenase inhibitory activity even when applied in a small amount, is safe without side effects, has high stability in various drug bases, and has various dosage forms. The purpose is to obtain a collagenase activity inhibitor that can be applied to medicines and cosmetics.

[0007]

[Means for Solving the Problems] In order to solve the above problems, as a result of screening the collagenase inhibitory activity of various substances, the dicarboxylic acids also have a metal chelating action, and the action on the living body is mild and almost no side effects occur. However, they have found that they are stable in various forms of bases, and have completed the present invention.

In the present invention, one kind or two or more kinds are selected from the dicarboxylic acids and mixed in the base. Examples of dicarboxylic acids include glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, 1,9-nonamethylenedicarboxylic acid, and 1,10-decamethylenedicarboxylic acid, saturated dibasic acids having 5 to 12 carbon atoms. Acids are particularly preferred. The suitable amount of the compound is about 0.01% by weight to 10.0% by weight based on the total amount of the preparation.

When one or more dicarboxylic acids are used in combination with one or more metal chelating agents, the collagenase inhibitory activity is synergistically enhanced. As a metal chelating agent, diferoxamine (deferoxami
ne), ethylenediamine tetr
aacetic acid, EDTA) sodium salt, phosphoric acid, citric acid, ascorbic acid, succinic acid, gluconic acid, sodium polyphosphate, sodium metaphosphate, and the like, and one or more of them are selected and blended. .

[0010]

The collagenase inhibitor of the present invention exhibits an excellent collagenase inhibitory action even when added in a small amount, has a mild action on the living body, and is excellent in stability in the preparation. As a result, by applying the collagenase inhibitor of the present invention, skin wrinkling and viscoelasticity deterioration due to aging and ultraviolet rays, metabolic diseases such as osteoporosis, epidermolysis bullosa, corneal ulcer, inflammation of root disease, etc. Can alleviate or treat arterial diseases such as rheumatoid arthritis, rheumatoid arthritis, and osteoarthritis.
It also promotes wound healing.

As an example, the collagenase inhibitory action of azelaic acid will be shown below. Azelaic acid was added to an aqueous solution containing collagenase in a final concentration of 0 to 15 μg / ml so that the final concentrations were 8 mM and 16 mM, and then collagen labeled with fluorescein isothiocyanate (FITC) was added to a final concentration of 40 μg / ml. In addition to 3
Incubated for 1 hour at 7 ° C. After stopping the enzymatic reaction by adding ethanol, centrifugation was performed to remove unreacted FITC-labeled collagen, and the fluorescence intensity in the supernatant was measured at an excitation wavelength of 495 nm and a fluorescence wavelength of 520 nm. The fluorescence intensity was similarly measured for the system to which only the solvent not containing azelaic acid was added, and used as a control. The results are shown in Table 1.

[0012]

[Table 1] Table 1 also shows the collagenase inhibition rate determined by the formula (1). From this, azelaic acid was about 48 to 58% at 8 mM with respect to the release of FITC by collagenase,
It was shown that it was suppressed to about 28 to 31% at 16 mM. That is, the collagenase inhibition rate increased depending on the concentration of azelaic acid, and was 41.5 to 52.3, 16 at 8 mM.
It was 68.9 to 71.8% in mM.

(Equation 1)

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The collagenase inhibitor according to the present invention can be provided in the form of powder, aqueous suspension, solubilized in an aqueous base containing a polar organic solvent such as alcohol, or in the form of an emulsion. It can be used by adding to or blending with beverages, pharmaceuticals, cosmetics and the like. In foods, it can be added to oils and fats products, emulsified products, soft drinks and the like. In the case of pharmaceuticals, addition to oral preparations, injections, external skin preparations, etc. is considered. As the cosmetics, in addition to lotion, milky lotion, cream, etc., addition to oral compositions such as mouth rinses is possible.

[0014]

EXAMPLES Further, the features of the present invention will be described in detail with reference to examples. First, as Examples 1 to 3 of the present invention, Table 2 shows an ethanol-containing aqueous preparation type collagenase inhibitor. In Table 2, dicarboxylic acid is dissolved in ethanol and mixed with purified water in which a chelating agent is dissolved to solubilize it.

[Table 2]

Table 3 shows the formulations of Examples 4 to 6 which are emulsion type collagenase inhibitors. In Table 3,
Add dicarboxylic acid to the oil phase and heat to 75 ° C to dissolve,
Homogenize. On the other hand, adding a chelating agent to the water phase and heating,
It melts and maintains at 75 degreeC, the said oil phase component is gradually added to this, and after emulsifying, it cools and prepares.

[Table 3]

Table 4 shows the formulations of Examples 7 to 9 which are cream type collagenase inhibitors. Table 4
Medium, dicarboxylic acid is added to the oil phase and heated to 75 ° C. to dissolve and homogenize. On the other hand, a chelating agent is added to the aqueous phase, heated and dissolved to maintain the temperature at 75 ° C., and the oil phase component is gradually added to this to emulsify and then cooled to prepare.

[Table 4]

The above-mentioned examples of the present invention were evaluated for their healing effect on inflammatory ulcers. Five mice were made into one group, and an inflammatory ulcer was caused on the back by treatment with a surfactant. The sample was applied to this site twice a day by 0.5 g each for 7 days, and the healing status of the ulcer was observed 7 days later. The healing status was evaluated in three grades of "completely healed", "almost healed" and "incompletely healed", and the number of mice for each evaluation was shown. In addition,
In Tables 2 to 4, dicarboxylic acid is converted to gallotannin,
Those using ascorbic acid as the chelating agent were designated as Comparative Examples 1 to 3, respectively.

Further, the stability and skin irritation when the preparation was stored at 25 ° C. for 3 months were also evaluated. The storage stability was evaluated as "○; no change in state is observed", "△: Discoloration or discoloration or separation of components is slightly recognized", and "x: Discoloration or discoloration or separation of components is significantly recognized", The skin irritation was evaluated by obtaining a skin irritation index according to the criteria shown in Table 5 by a 48-hour occlusion patch test by 20 male panelists and calculating an average value of 20 individuals. The above results are summarized in Table 6.

[Table 5]

[0019]

[Table 6] From Table 6, in the group to which the present invention was applied, the tendency of skin ulcer healing was observed in all of the groups, and incompletely cured mice were not seen. Particularly good healing was observed in the application groups of Example 8 and Example 9. On the other hand, in the comparative application group, none of the mice showed complete healing, and almost half of the mice had incomplete healing.

Further, in the examples of the present invention, no change was observed in the state of the preparation even after 3 months. On the other hand, discoloration was observed in all of the comparative examples. For skin irritation, ethanol is 50
The skin irritation index was slightly higher in Examples 1 to 3 in which the composition was blended in a weight percentage, but it was still practically no problem. On the other hand, in each of the comparative examples, the skin irritation index was significantly higher than that of each example.

[0021]

INDUSTRIAL APPLICABILITY As described above in detail, according to the present invention, a collagenase inhibitor having excellent stability and safety and having a good inhibitory activity can be provided.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12N 9/99 C12N 9/99

Claims (3)

[Claims] 1. A collagenase inhibitor comprising one or more dicarboxylic acids. 2. A collagenase inhibitor comprising one or more dicarboxylic acids and one or more metal chelating agents. 3. The dicarboxylic acid has 5 to 12 carbon atoms.
3. The collagenase inhibitor according to claim 1 or 2, which is a saturated dibasic acid.