JPS63267730A - External preparation for treating allergic disease - Google Patents

Abstract

PURPOSE:To obtain the titled external preparation comprising urinostatin as an active ingredient. CONSTITUTION:An external preparation for treating allergic diseases comprising urinastatin (trypsin inhibitor in human urine: acidic glycoprotein having about 67,000mol.wt., pH 2-3 isoelectric point and 5-12% saccharide; inhibiting not only proteolytic enzyme such as trypsin and alpha-chymotrypsin but also enzymes hydrolyzing saccharides and lipids such as hyaluronidase and lipase and showing a wide inhibitory spectrum). The preparation is effective especially against allergic rhinitis and allergic conjunctivitis. A dose is 1,000-100,000 u, preferably 5,000-100,000 u daily. A freeze-dried agent is used as the dosage form, is dissolved when used and sprayed upon nasal cavity by a nebulizer or used as an eye dropper or a nasal drop. Or the preparation is made into capsules and sprayed in powder state.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to an external preparation for treating allergic diseases, particularly allergic rhinitis and allergic conjunctivitis, containing ulinastatin as an active ingredient.

Conventional technology Allergic diseases, typified by allergic rhinitis and allergic conjunctivitis, are caused by so-called type allergy in the nasal and ocular mucosa, and many causative antigens are known, including house dust, pollen, and fungi. ing. To treat these diseases, specific desensitization therapy in which small amounts of antigen are administered, various anti-allergic drugs, or a combination of these are used to suppress allergic reactions caused by antigen-antibody reactions. There is.

Problems to be Solved by the Invention Nebulizer therapy with steroids is useful as an antiallergic agent for allergic rhinitis. However, there are concerns that long-term topical administration of steroids may cause side effects such as bacterial infection. Furthermore, for acute rhinitis caused by a common cold, administration of steroids may actually worsen the symptoms. Therefore, considering that allergic rhinitis and acute rhinitis often occur together, sufficient consideration must be given to the occurrence of side effects when administering steroids.

On the other hand, administration of steroids is also effective for allergic conjunctivitis and is often applied, but the same considerations as above are required.

Means for Solving the Problems The present inventor has conducted repeated research on the development of therapeutic agents that are free from side effects, and has discovered that ulinastatin (human urinary trypsin inhibitor) can be used externally to treat allergic symptoms in the nasal and ocular mucosa. The present invention has been completed based on the discovery that this can be suppressed.

The present invention is an external preparation for treating allergic diseases, particularly allergic rhinitis and allergic conjunctivitis, which contains ulinastatin as an active ingredient.

Urinastatin is a multivalent enzyme inhibitor present in human urine and is a glycoprotein with a molecular weight of approximately 67,000. In order to obtain ulinastatin from urine, for example, the method of Sumi et al. (J, Blochm, 83*141 (1978)) can be used.

In other words, human urine is concentrated using an appropriate method, and arginine and
After passing through a Sepharose column to adsorb ulinastatin, it is eluted with aqueous ammonia containing sodium chloride. Next, gel chromatography is performed using a Sephadex G-200 column in a conventional manner to obtain a ulinastatin fraction.

The molecular weight of ulinastatin thus obtained is approximately 67.0.
00, isoelectric point E) 8 2-3, is an acidic glycoprotein containing 5-12% sugar, and its specific activity is about 2500 u/m
It is g. The activity of ulinastatin was expressed as 1 unit (u), which is the amount of ulinastatin that inhibits the activity of 2 μg of trypsin by 50%.

Urinastatin inhibits not only proteolytic enzymes such as trypsin and α-chymotrypsin, but also sugar and lipid degrading enzymes such as hyaluronidase and lipase, showing a wide spectrum of inhibition.

Next, the action of ulinastatin will be specifically explained using test examples.

Test Example 1 Test method for effect on allergic rhinitis 50,000 u of ulinastatin was dissolved in 10 ml of physiological saline, and 1 ml was sprayed into the nasal cavity at a pressure of IKg/cm2 using a nebulizer manufactured by Flrst. The findings on the nasal mucosa 10 to 30 minutes after the nebulization were evaluated based on the criteria in Tables 1 and 2, and the effectiveness of ulinastatin was determined.

1] Target Patients Twelve patients with allergic rhinitis were targeted. These cases are undergoing specific desensitization therapy using house dust (HD) and/or non-specific desensitization therapy using histaglobin (HG) and neurotropin (NSP).

As is clear from the test results shown in Table 3, ulinastatin was effective in reducing nasal mucosal swelling and nasal discharge.

The nasal symptoms of case 12, which showed a significant response, are shown in Figure 1 (before using ulinastatin) and Figure 2 (after using ulinastatin).

(Hereinafter, blank space) Test Example 2 Effect on allergic conjunctivitis 50,000 u of ulinastatin was dissolved in 1 ml of physiological saline to prepare an eye drop, and the solution was filled into an eye drop container. One drop was instilled from this container into the negative eye, and one drop of physiological saline was instilled into the opposite eye as a control.

Case IH1K (female) 43 had odontobial dermatitis, and occasionally experienced itching in the eyes, conjunctival hyperemia, and swelling in the lower eyelids. I occasionally take Zagiten and Celestamine.

Urinastatin was administered to the right eye, and physiological saline was administered to the left eye, and the conjunctiva was observed immediately, 10 minutes, 30 minutes, and 8 hours after administration.

Immediately after administration, a slight increase in hyperemia was observed in both eyes, but
After 0 minutes, hyperemia on the ulinastatin-administered side disappeared. However, on the side to which the physiological saline had been administered, hyperemia remained even after 10 minutes. Thirty minutes later, both eyes were no longer hyperemic. The itching sensation disappeared 30 minutes after administration. Even 8 hours after administration, hyperemia in both eyes remained resolved. Also,
No lacrimation or purulent secretion was observed during the entire observation period.In this case, the itching sensation disappeared 30 minutes after ulinastatin was administered, suggesting that ulinastatin was effective.

Case 2 T, K (male), 22 years old, had allergic rhinitis (house dust allergy).
Itching in the eyes and congestion in the conjunctiva were observed. Azeptln 2mg was administered for allergic rhinitis. Urinastatin was administered to the right eye and physiological saline was administered to the left eye. Observations were made before administration, immediately after administration, 10 minutes, 30 minutes, 3 hours, and 13 hours and 30 minutes after administration.

Before administration, mild hyperemia of the conjunctiva and dilation and dilatation of blood vessels were observed in both eyes (see Figures 3 and 4), and mild itching was observed in the eyes. Immediately after administration, no irritating pain was reported in both eyes. A transient increase in conjunctival hyperemia was observed on the side treated with ulinastatin. 10 minutes after administration, the hyperemia on the ulinastatin administration side had decreased (Figure 5 (10 minutes after ulinastatin administration) and Figure 6 (physiological saline administration 1).
After 30 minutes, hyperemia was observed on the side to which physiological saline had been administered, but only slight hyperemia was observed on the side to which ulinastatin had been administered. Three hours later, slight hyperemia was observed in the internal beak on the side to which ulinastatin had been administered. 13 hours 3
After 0 minutes, hyperemia on the ulinastatin administration side was observed as before administration. Throughout the entire observation period, the itching sensation disappeared until 3 hours after ulinastatin was administered, but after 13 hours and 30 hours.
Minutes later, the itching sensation appeared again.

No lacrimation or purulent secretion was observed due to administration of ulinastatin. In this case, the itching sensation and hyperemia disappeared after administration of ulinastatin, so ulinastatin seems to have been effective.

Test Example 3 Acute Toxicity 1 group of 10 ddY male mice (body weight 20-22 g)
4 g/kg of ulinastatin was administered intravenously or intraperitoneally, and symptoms and weight changes were observed for one week. Body weight changes during the observation period were similar to those in the control group, and no deaths were observed.

Test Example 4 Administration test to domestic rabbits One drop each of ulinastatin 50,000 u/m I eye drops was instilled into the right eye of a male rabbit weighing 3.5 kg, and one drop of physiological saline was injected into the left eye, immediately after the instillation. , 10 minutes later,
The conjunctiva and general condition were observed 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, and 24 hours later.

Conjunctival hyperemia, edema, lacrimation, and pus secretion were not observed during the entire course of treatment after ulinastatin instillation. Similarly, no significant changes were observed in the left eye treated with physiological saline.

Therapeutic dose of ulinastatin is 1,000 to 100 doses per day.
．． 000u, preferably 5,000~100゜000u
However, the dose can be increased or decreased as appropriate depending on the symptoms or usage.

The dosage form is, for example, a freeze-dried preparation, which is dissolved before use and sprayed into the nasal cavity with a nebulizer, or instilled into the nose or eyes. Alternatively, it may be sprayed in powder form as a capsule.

Next, the method for obtaining ulinastatin will be explained using a production example, but the method for obtaining ulinastatin is not limited to this example, and it goes without saying that general biochemical methods for purifying proteins can be used. .

Production Example 1 Prokshe method (J, Lab, C
I In, Med, 19, 491 (1971)). Healthy adult urine 6501 was concentrated and dialyzed against deionized water, then adjusted to pH 7.8 with IN sodium hydroxide solution, and then diluted with 0.05 M Tris-HCl buffer (pH
A DEAE cellulose column (20
x80cm) to adsorb ulinastatin. After washing this column with the same buffer solution 401,
The adsorbed ulinastatin was eluted using the same buffer containing M sodium chloride, and the eluate was incubated at 60°C.
Heat treatment was performed for 20 minutes to inactivate the contaminating protease to obtain 16 g of crude ulinastatin. This crude ulinastatin was adsorbed onto a DEAE cellulose column (8 x 60 cm) equilibrated with 0.02 M glycine-hydrochloric acid buffer (pH 3, 4). After sequentially washing this column with the same buffer solution 101 and then with the same buffer solution 101 containing 0.2M sodium chloride, ulinastatin was eluted with the same buffer solution 81 containing 0.4M sodium chloride.

After concentrating the eluate by ultraconcentration, Sephadex G-1
Purified ulinastatin was obtained by gel chromatography using a column (10×95 cm) packed with 0.00 and physiological saline as a developing solvent.

The specific activity of ulinastatin obtained here is 2500 u
/ mg.

Example 1 40 g of lyophilized ulinastatin is dissolved in 2000 ml of physiological saline and aseptically filtered using a membrane filter. Fill 1 ml of the filtrate into sterilized glass containers,
Freeze-dry the product using a conventional method to obtain a freeze-dried preparation.

Example 2 Capsules After thoroughly mixing 20 g of ulinastatin with 60 g of lactose, each capsule was filled in a quantity of 40 mg using a conventional method to form capsules.

Example 3 Eye drops ulinastatin 10g Benzalkonium chloride 1g Sodium chloride 4.8g Sodium hydrogen phosphate 4.73 g Sodium dihydrogen phosphate
4g Each of the above components was dissolved in 10100O of sterile distilled water, filtered aseptically, and filled in 10ml portions into sterilized ophthalmological containers.
Use as eye drops.

Example 4 Nasal drops ulinastatin 10g Benzethonium chloride 1g Sodium chloride 9g Each of the above components was dissolved in 10100O of sterile distilled water, filtered aseptically, and filled in 10ml portions into sterilized nasal spray containers,
Use as nasal spray.

[Brief explanation of the drawing]

Figures 1 and 2 are photographs showing the effects of ulinastatin in Test Example 1 and Case 12; Figures 3 and 4 are photographs showing the state before administration of ulinastatin in Test Example 2 and Case 2; The figure and FIG. 6 are photographs showing the effect of ulinastatin. ``T continuation - 7 #13 Original text (method) % formula % 1. Indication of the case 1988 Patent order No. 101281 2. Name of the invention External preparation for the treatment of allergic diseases 3. Person making the amendment 1 Related: Patent applicant 4, Agent Address: 1-6 Kanda Surugadai, Chiyoda-ku, Tokyo (and 2 others) 5. Date of amendment order: July 1, 1982 (Shipping date: July 28, 1982) 6. Subject of amendment IJI A detailed description of the invention and brief drawings 7,
Contents of the amendment (1) From the last line of page 5 of the specification to the second line of page 6 of the same specification, “In addition,
······It was shown to. ” to be deleted. (2) “(See Figures 3 and 4) on page 9, line 13.
” to be deleted. (3) Lines 3 to 1 from the bottom of page m9, “(Figure 5...
・Reference)" is deleted. (0, page 14, line 4 from the bottom to page 15, line fJSZ)
4. A simple explanation of the drawing, old photo. ” to be deleted. (5) Delete the drawing.

Claims (1)

[Claims] External preparation for the treatment of allergic diseases containing ulinastatin as an active ingredient