JPH0669958B2 - Agent for preventing and treating alveolar pyorrhea - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a prophylactic and therapeutic agent for alveolar pyorrhea, and more particularly to a prophylactic and therapeutic agent for alveolar pyorrhea, which contains ovomacroglobulin as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION It is an object of the present invention to provide a novel prophylactic and therapeutic agent for alveolar pyorrhea which has the effects of preventing and healing pyorrhea.

As a result of intensive studies for the above purpose, the present inventors have found that ovomacroglobulin has an excellent preventive effect on alveolar pyorrhea and a healing-promoting effect, and that it has storage stability and safety. Therefore, the present invention has been completed, and the present invention has been completed here.

[0004]

That is, the present invention relates to a prophylactic and therapeutic agent for alveolar pyorrhea, which comprises ovomacroglobulin as an active ingredient.

It is essential that the drug of the present invention contains ovomacroglobulin as its active ingredient. Here, ovomacroglobulin is known as a high molecular weight glycoprotein in egg white, and its preparation method is already known [Feeney, RE, et al., Comparable Biochemistry and・ Physiology (Comp. Biochem. Physiol.), 54A, 281
(1976), Inoki et al., Journal of Biochemistry (J. Biochem.), 92, 1679-168.
2 (1982), 93, 121-127 (198).
3), and Nagase et al., Journal of Biological Chemistry (JBC), vol. 258, No.
12, 7481-7489 (1983)].

The egg white as a raw material for the preparation of the ovomacroglobulin is not particularly limited, and any of various animal species can be used. Generally, easily available egg whites such as chicken, duck, quail, and turkey are preferred.
The method for preparing ovomacroglobulin from the egg white is not particularly limited, and various operations utilizing the physicochemical properties of ovomacroglobulin according to various methods generally used for separating protein components, for example, a protein precipitant Treatment, molecular sieve chromatography (gel filtration), ion exchange chromatography, centrifugation, electrophoresis, dialysis and the like can be performed alone or in combination.

For example, a method may be exemplified in which egg white is mixed with a water-soluble solvent such as Tris-hydrochloric acid buffer or polyethylene glycol or the like is added to remove insoluble proteins such as ovomucin and then subjected to gel filtration. Thus, ovomacroglobulin can be obtained as a glycoprotein having a molecular weight of about 600,000 to 800,000.

The preventive and therapeutic agent for alveolar pyorrhea of the present invention can be put to practical use in the form of a general pharmaceutical preparation containing ovomacroglobulin obtained as described above as an active ingredient. As the pharmaceutical preparation form, various forms can be appropriately selected depending on the intended use of the obtained preparation. Examples thereof include liquid coating agents, lotions, aerosol agents,
In addition to external preparations such as liniments and ointments, injections and the like can be exemplified. For the preparation of various forms, various commonly used diluents, excipients, etc. can be appropriately used. For example, in the preparation of an ointment as an external preparation, a usual hydrophobic or hydrophilic base such as fat, fatty oil, lanolin, petrolatum, paraffin, wax, glycols, higher alcohols, glycerin, water, etc. Can be used. In addition, various additives that are known to be usually added, such as stabilizers, fragrances, and colorants, can be added to the above-mentioned external preparation, if necessary.

The active ingredient to be contained in the drug of the present invention,
That is, the amount of ovomacroglobulin is not particularly limited and may be appropriately selected from a wide range.
It is compounded in the range of ~ 30% by weight.

Further, the dosage and method of the prophylactic and therapeutic agents of the present invention are determined by the form of the preparation, the amount of the active ingredient in the preparation, the age of the patient to whom the preparation is applied, the sex and other conditions, the degree of alveolar pyorrhea and the like. The preventive and therapeutic agent for alveolar pyorrhea of the present invention in the form of an external preparation, for example, is a sufficient amount to spread it over the entire affected area, and is applied to the affected area once or more times a day,
It can be applied by coating or the like. The same applies to other formulations.

As described above, the agent for preventing and treating alveolar pyorrhea of the present invention thus has an excellent effect of preventing and healing pyorrhea and exhibits an excellent effect.

[0012]

EXAMPLES In order to explain the present invention in more detail, preparation examples of ovomacroglobulin as an active ingredient, test examples of their effects and various prescription examples of the formulation of the present invention containing the same will be given below.

Example 1: Preparation of ovomacroglobulin 20 kg of egg white was mixed with 10 m of 1% NaCl.
It was suspended in M Tris-hydrochloric acid buffer (pH 7.7), polyethylene glycol (molecular weight = 8500, manufactured by Tokyo Kasei Co., Ltd.) was added to the suspension to a concentration of 2.5%, and the mixture was continuously centrifuged (10000 rpm). The supernatant was collected. The obtained supernatant was further supplemented with the same polyethylene glycol as above.
% Until concentration is reached and continuous centrifugation (1000
The precipitated portion was collected at 0 rpm). This was dissolved in the above buffer solution, further centrifuged (10000 rpm, 10 minutes) to collect a supernatant, which was applied to a column (252 × 900 mm) of Sepharose CL-6B (manufactured by Pharmacia). The buffer was eluted at a flow rate of 3.6 L / hour.

For the elution category, the casein-based substrate
The lipsin inhibitory activity can be measured by the method of Kitamoto et al. [T. Kitamot
o, M. Nakashima, and A.K. Ikai, J .; Biochem.,
92, 1679-1682 (1982)].
The trypsin inhibitory activity fractions were collected.

Next, the active fraction thus obtained was buffered with 5 mM Tris-hydrochloric acid buffer (pH 7.7) while being concentrated using a Pellicon cassette (Millipore) equipped with a 100000 molecular sieving membrane. The liquid was replaced. The obtained sample was applied to a DEAE Trisacryl M (Trisacryl M, LKB) column (size 50 × 800 mm) equilibrated with 10 mM Tris-hydrochloric acid buffer solution (pH 7.7) containing 10 mM NaCl. . After thoroughly washing the column with the same buffer, 675 ml of 10 mM Tris-HCl buffer (pH 7.7) containing 150 mM NaCl and 150 m
10 mM Tris-HCl buffer containing M NaCl (pH
7.7) Elution with 675 ml for 2.5 hours each, for a total of 5 hours. Under this condition, the trypsin inhibitory activity fraction was eluted at a salt concentration of 70 mM to 120 mM.

The trypsin-inhibiting activity fractions were collected and collected for 1 m.
It was dialyzed against M phosphate buffer (pH 7.4). After sufficient dialysis, the dialyzed solution was freeze-dried with a freeze drier (manufactured by Rabocon).

From the above, a single ovomacroglobulin purified sample (5.9 to 7.1 g) was obtained.

The purified sample was hydrolyzed with 4N methanesulfonic acid at 110 ° C. for 24 hours (in a sealed tube under reduced pressure), and then analyzed with an amino acid analyzer (Model 835-50, Hitachi high-speed amino acid analyzer, manufactured by Hitachi Ltd.).

The results are shown in Table 1 below.

[0020] Test Example 1 Periodontal Wound Recovery Test (1) Using a 12-week-old rat, a hooked probe was inserted near the central part on the palate side of the left and right upper second maxillary teeth, and when the bone was reached, it was moved along the crown several times. And reciprocate and peel to form a wound surface.
Then, K file No. 40 for root canal treatment is inserted just below the contact point of the left second molar, and the buccal side and the palate side are communicated, and silk thread (blade silk) 4-0 is once around the second molar. Wrap and knot on the buccal side. After confirming hemostasis, apply the reagent. All the above operations were performed under an intraperitoneal anesthesia of ketalal.

(2) A dental # 12 bird's beak replacement blade knife was used to make an incision from the distal third molar to the mesial center of the first molar, and a hooked probe was inserted to reciprocate to remove the gingiva. The bone surface was exposed about 0.5 mm.

(3) The following agents were used as the reagents.

Experiment Group 1: Hydrophilic Vaseline Ointment (Hydrophilic Vaseline Base Only) Sarah Beeswax 8 g Stearyl Alcohol 3 g Cholesterol 3 g White Vaseline 86 g Propylparaben 0.0625 g Experimental Group 2: Hydrophilic Vaseline Ointment Containing 0.1% Ovomacroglobulin 0. 5% aqueous ovomacroglobulin solution 20g (containing 40mg methylparaben) Hydrophilic Vaseline base 80g Experimental section 3: 0.005% Hydrophilic Vaseline ointment containing 0.005% ovomacroglobulin 0.025% Ovomacroglobulin aqueous solution 20g (containing 40mg Methylparaben) Hydrophilic petrolatum base 80 g Further, each of the above-mentioned test reagents is placed on a rat tray and applied once a day for 15 minutes to be used. The coating amount is about 0.32 g on average. Do not give drinking water for 2 hours after application,
The first application was performed after hemostasis was confirmed. All the above operations were performed under anesthesia with Nembutal (0.1 ml) in the abdominal cavity.

(4) The evaluation method is as follows. That is,
On the 1st, 2nd, and 7th days after the start of the experiment, the treated rats were sacrificed, the lesions were taken, fixed with Bouin, dechlorinated with trichloroacetic acid and embedded in paraffin, and then hematoxylin-eosin staining (HE staining) or Azan staining was performed, and changes in the number of inflammatory cells and collagen fibers were observed.

(5) The results obtained (histological findings) are shown below.

In the control group (experimental group 1), inflammatory cell infiltration and bleeding in the tissue showed strong dilation of capillaries and bleeding images in the gingival lamina propria from the gingival crevicular epithelium to the alveolar crest and around it Infiltration of fibrin layer and scattered neutrophils was observed.

On the other hand, in Experiment Zone 2 and Experiment Zone 3,
Although there was dilation of capillaries in the lamina propria, the bleeding tendency was not so strong and the lamina propria tended to be already covered by the fibrin layer. In addition, the infiltration of neutrophils in the lamina propria observed in Experimental Group 1 was small. But the gingival cleft epithelium,
A localized strong neutrophil infiltration image was observed on the surface of the fibrin layer of the adherent epithelium. In comparison between the experimental section 2 and the experimental section 3, the experimental section 2 was found to have less expansion of capillaries in the lamina propria and less bleeding image, and less neutrophil infiltration in the epithelium.

Regarding the change in collagen fibers, as a result of HE staining, in Experimental Group 1, rupture, degeneration, etc. were observed in the collagen fibers near and around the wound site, whereas in Experimental Groups 2 and 3, Although the collagen fibers near the wound were torn, they did not spread to the surrounding area, and this tendency was stronger in experimental section 2 than in experimental section 3.

The preparation and formulation examples of the preparation of the present invention are shown below. In each example, “part” means “part by weight”.

Formulation Example 1: Formulation of a preventive and therapeutic agent for alveolar pyorrhea of the present invention Ovomacroglobulin 0.01 g Preservative Appropriate amount Fragrance Appropriate amount Distilled water is added to bring the total amount to 100 ml.

Distilled water was added to the above ovomacroglobulin, preservative and flavor to bring the total volume to 100 ml, followed by sterilization.
A prophylactic and therapeutic agent for alveolar pyorrhea of the present invention in the form of a spray solution was prepared.

Prescription Example 2: Formulation of a preventive and therapeutic agent for alveolar pyorrhea of the present invention Ovomacroglobulin 0.05 g Preservative proper amount flavoring proper amount Distilled water is added to bring the total volume to 100 ml.

In the same manner as in Formulation Example 1, a preventive and therapeutic agent for alveolar pyorrhea of the present invention in the form of a solution for spraying having the above composition was prepared.

Prescription Example 3: Formulation of a preventive and therapeutic agent for alveolar pyorrhea of the present invention Preparation of hydrophilic ointment Ovomacroglobulin 0.5 g White petrolatum 250 g Stearyl alcohol 220 g Propylene glycol 120 g Sodium lauryl sulphate 15 g Ethyl paraoxybenzoate or paraoxybenzoic acid Methyl 0.25 g Propyl paraoxybenzoate 0.15 g Purified water Appropriate amount Total amount 1000 g The above components are blended to prepare a prophylactic and therapeutic agent for alveolar pyorrhea in the form of a hydrophilic ointment containing ovomacroglobulin of the present invention. did.

Formulation Examples 4 to 7: Formulation of a preventive and therapeutic agent for alveolar pyorrhea of the present invention Preparation of hydrophilic ointment In Formulation Example 3, 0.01 g, 0.05 g, 0.1 g and 1 g of ovomacroglobulin were added. Instead, a prophylactic and therapeutic agent for alveolar pyorrhea in the form of a hydrophilic ointment of the present invention was prepared in the same manner.

Claims (1)

[Claims] 1. A preventive and therapeutic agent for alveolar pyorrhea, which contains ovomacroglobulin as an active ingredient.