JPS6237612B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a therapeutic agent for allergic diseases containing human urinary acid protease as an active ingredient. Many problems remain in the treatment of allergic diseases involving antigen-antibody reactions, such as bronchial asthma, food allergies, and urticaria, and great efforts are being made to treat these diseases. Allergic diseases are classified into four types based on their symptoms or causes. In other words, the type of allergy (anaphylactic type) caused by tissue-fixing antibodies and characterized by increased vascular permeability and smooth muscle contraction (anaphylactic type) is triggered by tissue-fixing antibodies, and the type of allergy (cell type) is induced in the presence of complement and is characterized by cell damage. lytic type), antigen-antibody complexes accumulate on the blood vessel wall, and are triggered by the involvement of complement and polymorphonuclear leukocytes; type allergy characterized by an inflammatory response (Arthus type); triggered by cell-mediated immunity; It is classified as a type of allergy characterized by the appearance of a hypersensitivity reaction, such as a tuberculin reaction. Among human allergic diseases, for example, bronchial asthma is associated with types, types, and types, and each type is thought to induce asthma attacks either independently or in combination. Furthermore, the onset mechanism of these allergic diseases is thought to be as follows. Antigens that have entered the body are processed by macrophages, and information is transmitted to the T cell-B cell lineage.
B cells that receive the information produce immunoglobulin (mainly IgE in type allergy, and mainly IgG in type and type allergy), of which IgE
Antibodies adhere to basophils in blood or mast cells in tissues, creating a sensitized state. After that, the antigen that has invaded again combines with the antibodies that have adhered to the cells, and these cells release histamine, Slow
reacting substance of anaphylaxis (SRS-A)
liberates chemical mediators such as The liberated chemical mediator causes erythema and edema due to smooth muscle spasm and increased capillary permeability, or increased secretion of glandular cells, thereby causing allergic symptoms. On the other hand, IgG antibodies bind to polynuclear leukocytes to achieve sensitization, and secretion of SRS-A is also considered to be a chemical mediator. Antiallergic agents can achieve therapeutic purposes by inhibiting any of these series of processes. For example, xanthine drugs are used to treat asthma.
Beta receptor stimulators or steroids have been used, but these often have undesirable side effects. For example, heart palpitation, tachycardia, etc. have been reported with xanthine drugs and β receptor stimulants. Furthermore, steroids have side effects such as the occurrence of fire tract ulcers and complications of bacterial infection. Furthermore, antihistamines are not effective against asthmatic attacks, and on the contrary, they may make asthma worse because they make it difficult to cough up airway secretions. In view of this background, the present inventors have conducted many years of research in order to develop a drug that can be used in a wide range of treatments for allergic diseases by compensating for the shortcomings of conventional drugs as described above. The present inventors discovered that moderately acidic protease has a strong anti-allergic effect and is suitable for this purpose, leading to the completion of the present invention. Acidic protease, which is the active ingredient of the therapeutic agent for allergic diseases of the present invention, is a known enzyme [Mirsky et al., Journal of Clinical Investigation (J.
Clin. Invest.) Vol. 27, p. 818, 1948], and had never been used as a therapeutic agent for allergic diseases. This acidic protease can be purified using general methods used to purify proteins, such as salting-out method, adsorption chromatography using an inorganic adsorbent, ion exchange chromatography using an ion exchange resin, gel chromatography having a molecular sieving effect, etc. By combining them, they can be collected from human urine. For example, human urine was processed using the method of Seijffers et al. [American Journal of Physiol., Vol. 206, p. 1106, 1964]
After adsorbing acidic protease by passing it through a DEAE-cellulose column equilibrated with 0.1M acetate buffer (PH5.3),
Elute with the same buffer containing 0.3M sodium chloride. After concentrating the eluate, it is further purified by gel chromatography using Sephadex G-100 swollen in 0.9% physiological saline, followed by acid treatment to obtain the acidic protease of the present invention. The acidic protease obtained above had a molecular weight of 32,000-38,000 as a result of analysis by Sephadex G-100 gel chromatography, an isoelectric point of 1 to 3 by ampholine isoelectric focusing, a maximum absorption of 278 nm, and a ninhydrin positive reaction,
Easily soluble in water, insoluble in ether and chloroform. Furthermore, this acidic protease exhibits high hydrolysis activity for hemoglobin in an acidic region of pH 7.0 or lower, but has the property of being significantly inhibited by pepstatin. Furthermore, this acidic protease is stable in an acidic region of pH 7.0 or lower, and unstable in an alkaline region of pH 8.0 or higher. The pharmacological action and toxicity of this acidic protease will be explained below using experimental examples. Experimental Example 1 Anti-ovalbumin IgE antibody production inhibitory effect One group of male Wistar rats weighing 180-200 g
It was used as 10 animals. 0.1 mg of ovalbumin was injected intraperitoneally together with 20 mg of aluminum hydroxide gel, and from the next day, acid protease was injected intravenously once a day for 14 days. Ovalbumin administration 7, 10,
After 14 days, blood was collected, and anti-ovalbumin IgE antibodies in the blood were analyzed by rat homologous PCA reaction (Yutaka Maruyama, Michio Terasawa, Kazuhiro Goto, Takanori Oe, Japanese Pharmacological Journal, Vol. 74, 179)
Page, 1978). The results are shown in Figure 1. Anti-ovalbumin IgE antibody production was significantly suppressed by acid protease administration. Experimental Example 2 Asthma suppressive effect One group of male Wistar rats weighing 180-200g
It was used as 10 animals. 0.1 mg of ovalbumin was injected intraperitoneally together with 20 mg of aluminum hydroxide gel, and from the next day, acid protease was injected intravenously once a day for 14 days. Ovalbumin 25 after 14 days
mg/Kg is administered intravenously to induce an asthma attack, and the resulting airway constriction is controlled by the method of Konzetsu-Resler [Arch. Exptl. Path. Pharmakol. Vol. 195, p. 71,
1940]. Airway constriction in control group
The airway contraction rate of each group was calculated as 100. The results are shown in Table 1.

[Table] Airway constriction was significantly suppressed by acid protease administration. Experimental Example 3 Toxicity test 10 ddY male mice per group (body weight 20±1g)
2 g of acidic protease dissolved in physiological saline/
After administering Kg intravenously or intraperitoneally, respectively.
Symptoms were observed for one week, but no abnormality was found. As described above in the experimental examples, the acidic protease of the present invention suppressed IgE antibody production and showed a clear therapeutic effect on experimental asthma. The dose of urinary acid protease that produces these effects is a sufficiently safe dose due to acute toxicity, and since it is a human-derived protein, it does not cause serious side effects such as anaphylaxis due to antigenicity. There is also considered to be little risk of this. Therefore, urinary acid protease is considered to be an extremely useful drug for the treatment of various allergic diseases including asthma. The therapeutic agent of the present invention is usually administered as an injection, intravenously,
It is administered subcutaneously, intramuscularly, intraarticularly, etc., but can also be used as an oral preparation, inhalation preparation, or rectal suppository. The therapeutic dose of acid protease for adults is 1 to 1000 mg per day, preferably 50 to 500 mg, but it can be adjusted as appropriate depending on the symptoms or usage. The acidic protease can be prepared into a pharmaceutical formulation by a conventional method, optionally with a conventional pharmaceutical carrier or excipient. Injections include lyophilized preparations or injection solutions that are dissolved before use; oral preparations include capsules, tablets, granules, powders, or oral liquid preparations; inhalers include lyophilized preparations, and intrarectal preparations. is preferably made into a rectal suppository. Next, examples of the present invention will be shown. Example 1 100 mg of acidic protease is dissolved in 100 ml of physiological saline and filtered aseptically using a membrane filter. Fill 1.0 ml of the liquid into sterilized glass containers, freeze-dry, and seal the containers to obtain a freeze-dried powder preparation. Example 2 Lyophilized acidic protease 100g, lactose 97g
g and 3 g of magnesium stearate are weighed and mixed uniformly. After filling 200 mg of this into No. 2 gelatin capsules, an enteric coating is applied to form enteric-coated capsules.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the results of Experimental Example 1.

Claims (1)

[Claims] 1. A therapeutic agent for allergic diseases containing human urinary acid protease as an active ingredient.