JPH1180004A - Inhibitor for decomposition of hyaluronic acid, agent for improving and treating disease causing abnormal decomposition of hyaluronic acid and agent for preventing gingivitis and agent for preventing dry skin or rough skin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an inhibitor for decomposition of hyaluronic acid, acting on a cell which exists in human binding tissue and inhibiting decomposition of hyaluronic acid and useful as an agent for improving and treating diseases causing abnormal decomposition of hyaluronic acid such as rheumatism, osteoarthritis and malignant tumor by including heparin. SOLUTION: This inhibitor contains heparin and/or its salt extracted according to ordinary method from intestine or the like of pig or the like, preferably in an amount of 0.001-5 g based on 100 g total amount of medicine. Daily doses of the compound is preferably 1-10 g as total amount of heparin and its salt in the case of oral administration and preferably 0.1-5 g in the case of parenteral administration. The medicine is useful as an agent for improving and treating diseases causing abnormal decomposition of hyaluronic acid, e.g. hepatitis, gingivitis and fibrosis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a hyaluronic acid degradation inhibitor which acts on cells present in human connective tissue and inhibits hyaluronic acid degradation, and more particularly to the improvement and treatment of diseases associated with abnormal degradation of hyaluronic acid. The present invention relates to an excellent agent for ameliorating / treating a disease of abnormal hyaluronic acid degradation, an agent for preventing gingivitis, and an agent for preventing dry or rough skin having an excellent effect on diseases in which hyaluronic acid degradation is physiologically enhanced from normal.

[0002]

2. Description of the Related Art Conventionally, hyaluronic acid retains moisture in intercellular spaces, retains cells based on the formation of a jelly-like matrix in tissues, retains lubricity and flexibility of organ tissues, and impairs mechanical properties. It has many functions such as resistance to external forces such as BIO IND and prevention of bacterial infection.
USRY, 8, 346, 1991).

Further, it has recently been found that hyaluronic acid has various physiological actions depending on its molecular weight.
For example, high-molecular-weight hyaluronic acid (molecular weight of 1,000,000 or more), which is considered to be synthesized in vivo, has an inhibitory action on proteoglycan release, an inhibitory action on bradykinin joint pain, a repair action on damaged cartilage, and an inhibitory action on arthritis. Has an effect (BIO INDUSTRY, Volume 11, 6
32, 1991), and its degradation (low-molecular-weight) product, low-molecular-weight hyaluronic acid, promotes angiogenesis (Scie).
228, pp. 1324, 1985) and leukocyte chemotaxis-promoting action (Japanese Patent Publication No. 6-8323).

[0004] From the above, the low molecular weight of hyaluronic acid can be found in hepatitis and gingivitis (inflammation, Vol. 4, p. 437, 1984),
Rheumatoid arthritis, osteoarthritis (connective tissue, 25, 24
3, 1994), malignant tumor (J. Cellar Physiolog)
y, 160, 275, 1994), and therefore, inhibitors of hyaluronic acid degradation are desired.

In fact, hyaluronidase, which reduces the molecular weight of hyaluronic acid in connective tissue, is supposed to be an enzyme having an inflammation-promoting effect, and its inhibitor was expected to have a pharmacological effect (Inflammation, Vol. 4, p. 437; 1984). But,
There have been no reports of the isolation of hyaluronidase from fibroblasts that form human connective tissue.Therefore, using bovine testis-derived hyaluronidase, bovine testis-derived hyaluronidase has properties similar to those of human fibroblasts. And various inhibitors have been reported (Japanese Patent Publication No.
29271, JP-B-6-4584, JP-A-5-178876, JP-A-6-80553, JP-A-6-80576, JP-A-6-9415
JP, JP-A-6-9416, JP-A-3-685
No. 15) is the current state.

However, in recent years, human synovial cells present in joints (connective tissue, Vol. 25, p. 243, 1994) and human uterine cervix cells (FEBS Letters, 347, 95, 1994)
Years), human dermal fibroblasts (BBA, vol. 172, p. 70, 1
990), human lung fibroblasts (J. Clin. Invest. 90,
(Pp. 1492, 1992) report the presence of an endo-type hyaluronan-degrading mechanism that is clearly different from bovine testis-derived hyaluronidase, which degrades hyaluronic acid into tetra- and hexa-saccharides. For these reasons, it is difficult for an inhibitor of testis-derived hyaluronidase to effectively inhibit the degradation of human hyaluronic acid.

[0007]

Accordingly, an object of the present invention is to act on cells present in human connective tissue,
A hyaluronic acid degradation inhibitor that inhibits hyaluronic acid degradation,
Furthermore, it has excellent effects on the treatment and improvement of abnormally degraded disease of hyaluronic acid, which is excellent for the improvement and treatment of diseases associated with abnormally decomposed hyaluronic acid, as well as excellent effects on diseases in which hyaluronic acid degradation is physiologically higher than normal. Another object of the present invention is to provide a gingivitis inhibitor and a dry or rough skin inhibitor having the same.

[0008]

The object of the present invention is to provide an inhibitor of hyaluronic acid degradation characterized by containing heparin and / or a salt thereof, an agent for ameliorating / treating a disease of abnormally decomposing hyaluronic acid, and an agent for preventing gingivitis, and Achieved by dry or rough skin inhibitors. That is, the present invention provides heparin and / or
Or a salt thereof, a hyaluronic acid decomposition inhibitor, an agent for ameliorating or treating a disease of abnormally decomposing hyaluronic acid, a gingivitis inhibitor, and an agent for preventing dry or rough skin.

Hereinafter, the configuration of the present invention will be described in detail.

The heparin used in the present invention may be one extracted from the intestine of a pig or the like by a conventional method.
In addition, it can be used without any particular limitation on the molecular weight.

[0011] The salt of heparin is not particularly limited, but particularly includes pharmaceutically acceptable inorganic acid salts such as sodium salt, ammonium salt and lithium salt.

The hyaluronic acid degradation inhibitor of the present invention is an agent for ameliorating or treating a disease in which hyaluronic acid degradation is abnormally enhanced, or a disease in which hyaluronic acid degradation is physiologically enhanced than normal. It is used as an excellent gingivitis inhibitor and as an agent for preventing dry or rough skin. Furthermore, it can be used as an active ingredient in ordinary pharmaceuticals and cosmetics, and as a research / test reagent by being added to a cultured cell system.

[0013] In the present invention, the disease refers to a condition in which hyaluronic acid degradation is physiologically higher than normal or hyaluronic acid is abnormally degraded.

In the present invention, the disease associated with abnormal degradation of hyaluronic acid includes hepatitis, gingivitis, rheumatism, osteoarthritis and malignant tumors in which the degradation of hyaluronic acid is abnormally enhanced in the affected area, and the amount of hyaluronic acid in serum. It is considered that the degradation of hyaluronic acid is abnormally enhanced in the affected area due to the increase in cirrhosis, transplant refusal, and scleroderma in which organs and skin are hardened by the disease, fibrosis such as cirrhosis, and atherosclerosis. Refers to diseases such as xerosis, dry skin, rough skin, and other diseases such as arteriosclerosis, nephritis, keloids, over-repair, and sepsis, in which the water retention ability in the affected area is reduced as a result of hyaluronic acid degradation.

[0015] The agent for ameliorating / treating a disease caused by abnormal degradation of hyaluronic acid in the present invention refers to a drug applied to a patient with a disease in which the degradation of hyaluronic acid is abnormally enhanced.

Among the above-mentioned diseases accompanied by abnormal degradation of hyaluronic acid, in particular, rheumatism, osteoarthritis, psoriatic arthrosis, gout, polyarthritis, traumatic arthritis, in which the amount of histamine is increased in the serum of patients. For scleroderma, keloids, malignant tumors, and transplant rejection, it is more effective to use the agent for ameliorating and treating abnormal hyaluronic acid degradation disease of the present invention together with an antihistamine.

In the present invention, the term "inhibitor" refers to an agent which is applied to a symptom in which the degradation of hyaluronic acid is physiologically higher than normal. Specific symptoms to be prevented are gingivitis, dry skin and rough skin. And the like. In addition, the preventive agent can be used by normal persons for prevention, but it is particularly preferable to apply the preventive agent to a symptom in which the degradation of hyaluronic acid is physiologically higher than normal.

The hyaluronic acid degradation inhibitor of the present invention,
As the form of the therapeutic agent and the preventive agent, tablets, solutions, capsules, granules, powders, ointments, patches, injections, joint injections, suppositories, and the like are prepared using appropriate excipients, carriers, and diluents. Preparations, bath preparations, etc., as well as gels, creams,
Examples include dosage forms such as sprays, patches, lotions, packs, emulsions, powders, and bath salts.

The preparation of such preparations is carried out by a conventional method. For example, for solid preparations, usual pharmaceutical additives such as, for example,
It is formulated using lactose, starch, crystalline cellulose, talc, etc. Capsules are obtained by filling fine granules, powders and the like thus prepared in suitable capsules. Liquid preparations are prepared by dissolving and suspending the agent of the present invention in an aqueous solution containing sucrose, carboxymethyl cellulose and the like. In addition, in the case of preparations such as injections and joint injections, the therapeutic effect is further improved even when formulated together with high molecular weight hyaluronic acid. The high-molecular-weight hyaluronic acid to be blended in the present preparation may be a protein-free high-purity one having a molecular weight of 500,000 or more, preferably 800,000 or more, regardless of the extraction target or production method. 0.1 to 10% by weight, preferably about 1% by weight. Further, the obtained polymer hyaluronic acid may be modified by a crosslinking treatment or the like.

The excipients or auxiliaries used in the hyaluronic acid degradation inhibitor, the improving / therapeutic agent and the inhibitor of the present invention may be those usually used in cosmetics, pharmaceuticals and the like. It is appropriately selected depending on the situation and is not particularly limited. For example, hydrocarbons such as petrolatum, squalane,
Higher alcohols such as stearyl alcohol, higher fatty acid lower alkyl esters such as isopropyl myristate,
Animal fats and oils such as lanolinic acid, polyhydric alcohols such as glycerin and propylene glycol, surfactants such as glycerin fatty acid ester, polyethylene glycol monostearate and polyethylene alkyl ether phosphoric acid, methyl paraoxybenzoate, butyl paraoxybenzoate and the like Examples include preservatives, waxes, resins, various flavors, various colors, various inorganic salts such as sodium citrate, sodium carbonate, various acids such as lactic acid, water, and ethanol.

When the hyaluronic acid degradation inhibitor of the present invention is used as a reagent for research and testing in a cultured cell system, the amount of heparin and its heparin in the culture solution when producing high molecular hyaluronic acid by the cultured cells is determined. The total amount of salt is 1
It is preferably contained in an amount of at least μg / ml, more preferably 10 μg / ml to 10 mg / ml.

The hyaluronic acid degradation inhibitor of the present invention,
The total amount of heparin and its salts in the disease ameliorating / therapeutic agent and in the inhibitor is the type and degree of the target disease,
It varies depending on the age, weight, sex, etc. of the patient and cannot be specified unconditionally.
1 g to 5 g are preferred. If the amount is less than 0.0001 g, the effect of the present invention may not be obtained, and if the amount exceeds 50 g, the effect corresponding to the amount may not be obtained.

The method of administering the hyaluronic acid degradation inhibitor, the agent for improving or treating a disease and the inhibitor of the present invention may be oral or parenteral.

Usually, the daily dose is 0.01 g to 50 g as the total amount of heparin and its salts in oral administration.
g is preferable, and 1 g to 10 g is particularly preferable. For parenteral administration, 0.1 g to 5 g is preferred. If the amount is less than 0.01 g, the effect of the present invention may not be obtained, and if the amount exceeds 50 g, the effect corresponding to the amount may not be obtained.

The present invention has revealed that fibroblasts present in connective tissue have a mechanism of degrading hyaluronic acid, and that this mechanism is inhibited by heparin.

As a result, the present drug can be expected to sufficiently treat allergic diseases as well as diseases involving abnormal degradation of hyaluronic acid in connective tissues and joints.

[0027]

The present invention will be described in more detail with reference to the following examples and comparative examples. Prior to Examples, an evaluation system for examining the effect of a hyaluronic acid degradation inhibitor will be described. The amounts shown in Tables 3 to 12 all represent% by weight.

(1) Method of preparing MEM medium Minimum Essential Medium
(Dai Nippon Pharmaceutical Co., Ltd., 10-101) 10.6 (V / V) Non Essenti as final concentration in 10.6 g.
al Amino Acid (Dainippon Pharmaceutical Co., Ltd., 16-
810), 1 mM sodium pyruvate (16-820, manufactured by Dainippon Pharmaceutical Co., Ltd.), 1.2% (W / V) sodium bicarbonate, and distilled water were added to make 1 liter. 7 (hereinafter abbreviated as MEM medium).

(2) Inactivation of Fetal Bovine Serum (FBS) FBS (Irvine Scientific) was heated at 56 ° C. for 30 minutes.

(3) Preparation of high molecular tritium hyaluronic acid for cell addition Normal human fibroblast cell line [Detroit 551 (ATCC
CCL 110)] was adjusted to 2 × 10 ⁵ cells / ml in a MEM medium containing 10% (V / V) inactivated FBS, and 50 ml was placed in a 225 cm ² flask, and cultured for 3 days to obtain a confluent state. I made it. Then tritium glucosamine, a precursor of hyaluronic acid (American R
adiolabeled Chemicals Inc.) was added to the culture system (10 μCi / ml), and after further culturing for 3 days, tritium-labeled hyaluronic acid was removed from the culture solution to Underh.
ill et al. (J. Cell Biology, 82, 475, 1).
979), and a high molecular weight tritium hyaluronic acid (specific radioactivity, 0.1 μCi / μg) having a molecular weight of 1,000,000 or more was prepared using a gel filtration column. This was used as a polymer tritium hyaluronic acid for addition to a cell culture system.

(4) Culture of Tritiated Hyaluronic Acid Added to Normal Human Fibroblasts Normal human fibroblast cell line [Detroit 551 (ATCC
CCL 110)] in a MEM medium containing 10% (V / V) inactivated FBS at 1.5 × 10 ⁵ cells / m 2.
to a 12-well plate (Falcon).
8 ml each, and 95% (V / V) air-5% (V / V)
V) Static culture at 37 ° C. for 3 days in an atmosphere of carbon dioxide,
Further, the medium was replaced with only a MEM medium, and the cells were cultured for one day. Then, the polymer contains tritium hyaluronic acid (1
(4000 DPM / ml) MEM medium was prepared, the medium was replaced, and culturing was performed for 3 days. At the time of medium exchange, various drugs were added.

(5) Addition of high-molecular tritium hyaluronic acid to rheumatoid synovial cells Culture of human rheumatoid synovial cells was increased by 1.5 × in MEM medium containing 10% (V / V) inactivated FBS. 10 ⁵ pieces / m
to a 12-well plate (Falcon).
8 ml each, and 95% (V / V) air-5% (V / V)
V) Static culture at 37 ° C. for 3 days in an atmosphere of carbon dioxide,
Further, the medium was replaced only with the MEM medium containing 1% FBS, and the cells were cultured for one day. Thereafter, a MEM medium containing high molecular tritium hyaluronic acid (14000 DPM / ml) was prepared, the medium was replaced, and the cells were cultured for 3 days. At the time of medium exchange, various drugs were added.

(6) Evaluation of Decomposition of Polymer Tritium Hyaluronic Acid by Cells After completion of the culture, the culture solution was collected and subjected to a heat treatment at 100 ° C. for 5 minutes, and then 1 ml of the medium was added to a Sepharose CL-2B column (1 cm inner diameter, (Length 60 cm) and gel filtration was performed under the following conditions. Flow rate: 0.6 ml / min Fractionation: 4 ml / 1 fraction Total number of fractions: 25 Further, 16 fractions of 10 to 25 fractions in which hyaluronic acid having a molecular weight of 100,000 or less eluted were collected, and [ ³ H] radioactivity was measured and decomposed. The amount of hyaluronic acid was determined.

Example 1, Comparative Examples 1-4 Chondroitin sulfate A (Comparative Example 2), chondroitin sulfate B (Comparative Example 3), heparin (Example 1), and heparan sulfate (Comparative Example 4) at a final concentration of 1 mg / It was dissolved in MEM medium to make the volume of the solution. Only MEM medium was used as Comparative Example 1.

Test Example 1 (Decomposition of hyaluronic acid by human normal skin fibroblasts) Using Example 1 and Comparative Examples 1-4, the amount of decomposed high molecular tritium hyaluronic acid was calculated by the method (6) described above. did. Table 1 shows the results.

[0036]

[Table 1]

As a result, the decomposition of hyaluronic acid without addition of heparin was completely suppressed by the addition of heparin (Example 1).
The degradation was not suppressed by any of the other glycosaminoglycans, chondroitin sulfate A, chondroitin sulfate B, and heparan sulfate (Comparative Examples 1 to 4).
4).

From these results, it was found that heparin was effective as a human hyaluronic acid degradation inhibitor. Further, the hyaluronic acid degradation inhibitor of the present invention can be used as an active ingredient of a therapeutic agent for a disease in which hyaluronic acid degradation is abnormally enhanced.

Examples 2 to 4 Chondroitin sulfate C at a concentration of 10 mg / m in a medium
l (Example 2), 100 mg / ml (Example 3), 50
The MEM was dissolved to a concentration of 0 mg / ml (Example 4).

Test Example 2 (Decomposition of hyaluronic acid by human normal skin fibroblasts) Using Examples 2 to 4 and Comparative Example 1 (MEM medium only),
Decomposition of the polymer tritium hyaluronic acid was examined by the method (6) described above, and the amount of decomposed hyaluronic acid was calculated.
Table 2 shows the results.

[0041]

[Table 2]

As a result, heparin was found to inhibit the degradation of hyaluronic acid in a dose-dependent manner (Examples 2 to 4).
4).

From these results, heparin is effective as a human hyaluronic acid degradation inhibitor. Moreover, the therapeutic agent for abnormally promoting hyaluronic acid degradation containing the hyaluronic acid degradation inhibitor of the present invention is considered to be effective for treating rough skin in which hyaluronic acid degradation is abnormally enhanced.

Test Example 3 (Decomposition of hyaluronic acid by rheumatoid synovial cells) Using Examples 2-3 and Comparative Example 1 (MEM medium only),
Decomposition of the polymer tritium hyaluronic acid was examined by the method (6) described above, and the amount of decomposed hyaluronic acid was calculated.
Table 3 shows the results.

[0045]

[Table 3]

As a result, it was found that heparin also inhibited the degradation of hyaluronic acid in rheumatoid synovial cells in a dose-dependent manner (Examples 2-3).

From these results, heparin is effective as a human hyaluronic acid degradation inhibitor. In addition, the therapeutic agent for a disease with abnormally enhanced hyaluronic acid degradation containing the hyaluronic acid degradation inhibitor of the present invention is considered to be effective for the treatment of rheumatism or the like in which hyaluronic acid degradation is abnormally enhanced.

Examples 5 to 7 (tablets)

[0049]

[Table 4]

Each of the components shown in Table 4 was uniformly mixed, and the mixture was tableted in a usual manner to give a tablet of 300 mg.

Examples 8 to 10 (capsules)

[0052]

[Table 5]

The components shown in Table 5 were uniformly mixed, and 200 mg of the mixture was filled into No. 3 hard capsules according to a conventional method.

Examples 11 to 12 (Liquid)

[0055]

[Table 6]

Each component shown in Table 6 was dissolved in purified water, and the mixture was stirred and homogenized to obtain a liquid preparation.

Examples 13 and 14 (lotion)

[0058]

[Table 7]

Each component shown in Table 7 was mixed and dissolved to prepare a lotion.

Examples 15 to 16 (bath additives)

[0061]

[Table 8]

The components shown in Table 8 were mixed to prepare a bath agent. In addition, this bath agent is diluted about 3000 times at the time of use.

Examples 17 to 20 (Ointment)

[0064]

[Table 9]

In Table 9 above, each component of (B) was mixed in a hot water bath while heating to 80 ° C., and the mixture was stirred in a mixture of each component of (A) heated to 80 ° C. While slowly adding. Next, a homogenizer (Tokusyuk)
Each component was sufficiently emulsified and dispersed by stirring vigorously (2500 rpm) for 2.5 minutes with Ika Kogyo Co., Ltd., and then gradually cooled while stirring to obtain an ointment containing heparin.

Examples 21 to 23 (injections)

[0067]

[Table 10]

The components shown in Table 10 above were placed in a measuring cylinder, dissolved in distilled water for injection, filtered through a membrane filter (0.22 μm), and dispensed into glass ampules.
An injection was prepared.

Examples 24 to 26 (Toothpaste)

[0070]

[Table 11]

Purified water, glycerin, carrageenan, saccharin, butyl parahydroxybenzoate, chlorhexidine diglyconate, fragrance, and heparin were weighed and mixed according to the formulation in Table 11 to swell the binder, and then the second binder was used.
After adding calcium phosphate and sodium lauryl sulfate, further mixing and defoaming, the mixture was filled into a tube by a conventional method to obtain toothpaste.

Examples 27 to 29 (joint injections)

[0073]

[Table 12]

Aqueous solutions of the components listed in Table 12 above were prepared in a conventional manner, heated or sterilized by filtration, and dispensed into injection syringes in an amount of 2.5 ml each.

[0075]

As described above, according to the present invention, a hyaluronic acid degradation inhibitor which acts on cells present in human connective tissue and inhibits hyaluronic acid degradation, and further improves or improves diseases associated with abnormal hyaluronic acid degradation. Provide a therapeutic and / or therapeutic agent for abnormally degraded hyaluronic acid degradation, and a gingivitis inhibitor, an agent for preventing dry or rough skin which has an excellent effect on diseases in which hyaluronic acid degradation is physiologically enhanced from normal. Clearly what you can do.

────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code FI A61K 31/725 ACK A61K 31/725 ACK ACS ACS ADA ADA ADU ADU // A61K 7/00 7/00 JW 7/50 7 / 50 9/08 9/08 E 9/20 9/20 B 9/48 9/48 A C08B 37/10 C08B 37/10

Claims (8)

[Claims] 1. A hyaluronic acid degradation inhibitor comprising heparin and / or a salt thereof. 2. An agent for ameliorating / treating a disease of abnormally decomposing hyaluronic acid, comprising the hyaluronic acid decomposition inhibitor according to claim 1. 3. The method according to claim 2, wherein the abnormal hyaluronic acid degradation disease is hepatitis, gingivitis, rheumatism, osteoarthritis, or malignant tumor. 4. The method for improving or treating arthropathy according to claim 2, wherein the abnormal hyaluronic acid degradation disease is rheumatism or osteoarthritis. 5. The method according to claim 2, wherein the abnormal hyaluronic acid degradation disease is fibrosis. 6. The method according to claim 6, wherein the abnormal hyaluronic acid degradation disease is psoriasis,
The agent for ameliorating / treating an abnormally degraded hyaluronic acid disease according to claim 2, wherein the agent is xeroderma or rough skin. 7. A gingivitis inhibitor comprising the hyaluronic acid degradation inhibitor according to claim 1. 8. A dry or rough skin inhibitor comprising the hyaluronic acid degradation inhibitor according to claim 1.