JPH06192085A - Therapeutic agent for mite allergy - Google Patents

Abstract

PURPOSE:To obtain a therapeutic agent, having inhibitory activity against a mite protease and useful for treating and preventing mite allergic diseases (e.g. asthma, allergic rhinitis and atopic dermatitis). CONSTITUTION:This therapeutic agent for mite allergy comprises one or more substances selected from aprotinin, potato protease inhibitors, soybean trypsin inhibitors, antipain, leupeptin, guanidino-fatty acid derivatives, guanidinobenzoic acid derivatives and amidinophenolic derivatives as an active ingredient.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a mite allergy therapeutic agent containing a certain protease inhibitor as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION House dust mites inhaled by humans by breathing are known to cause exogenous asthma, allergic rhinitis and atopic dermatitis [J. Allergy, 3
9 , 325 (1967) and ibid, 42 , 14 (1968)].
Recently, Dell is a kind of house dust mite Dermatophagoides Death Farinae (Dermatophagoides farinae) and Dermatophagoides Death Puteronaishinasu (Dermatophagoides pt
eronyssinus ), major allergen proteins with molecular weights of 25,000 and 15,000 were isolated [J. Immunol., 125 , 5
87 (1980) and Int. Archs Allergy Appl. Immunol.,
81 , 214 (1986)]. In another report, Dermatophagoides pter
onyssinus ), two types of proteases (proteolytic enzymes) have been isolated, and the proteolytic activity of these enzymes is thought to be closely related to mite allergic disease [Lancet, Jul., 15 , 154 (1989)]. In addition, Dermatophagoides fa
It has been experimentally confirmed that Df-protease, which is a protease isolated from Rinae ), has the ability to produce bradykinin that exacerbates the inflammatory response and enhances vascular permeability [Biochem. Biophys. Acta, 1074 , 62
(1991)].

From a comprehensive judgment of these facts, it is speculated that the house dust mite protease promotes the production of bradykinin, resulting in various mite allergic diseases. Therefore, suppressing the protease activity of house dust mite is a treatment for mite allergic diseases (asthma caused by mite, allergic rhinitis, atopic dermatitis, etc.) and / or
Or, it is considered to be useful for prevention.

[0004]

2. Description of the Related Art Up to now, no substance has been reported which inhibits a house dust mite protease. On the other hand, although many inhibitors against proteases other than house dust mite (for example, trypsin, plasmin, thrombin, etc.) are known, it is not known that these inhibitors inhibit the house dust mite protease.

[0005]

OBJECT OF THE INVENTION The present inventor has conducted extensive studies in order to find an inhibitor of the house dust mite protease from the above viewpoints. The present inventor has investigated various mitochondrial proteases (for example, trypsin, plasmin, chymotrypsin, kallikrein, etc.), which are known as inhibitors, and examined the effect on tick mites. It has been found that the agents are not effective and only certain substances have an inhibitory effect. That is, among substances conventionally known as protease inhibitors other than house dust mite, aprotinin, potato protease inhibitor, soybean trypsin inhibitor, antipain, leupeptin, guanidino fatty acid derivative and guanidinobenzoic acid derivative are also potent against mite protease. Anti-trypsin,
alpha ₂ - macroglobulin and ε- amino caproate had little inhibitory effect. More recently, it has been found that amidinophenol derivatives also have a strong mite prosthesis inhibitory effect, whereas those of guanidinophenol derivatives are weak. It was also found that among chemical substances having a guanidino group or an amidino group, an amidinophenol derivative has the most potent mite protease inhibitory effect. Furthermore, substances that have an inhibitory effect on mite protease are
The present invention has been completed by first confirming by actually suppressing the inflammatory reaction (enhancement of vascular permeability) due to a mite protease, which is one of the above systems.

[0006]

The present invention comprises aprotinin, potato protease inhibitor, soybean trypsin inhibitor, antipain, leupeptin, guanidino fatty acid derivative,
The present invention relates to a mite allergy therapeutic agent containing, as an active ingredient, one or more substances selected from guanidinobenzoic acid derivatives and amidinophenol derivatives. Each substance used as an active ingredient in the present invention is well known, but will be described in detail below.

Aprotinin is a polypeptide existing in animal tissues and blood, and is a substance having a molecular weight of 6511 consisting of 58 amino acids [Biophys. Res. Commun., 18,
255 (1965)], and is known as an inhibitor of proteolytic enzymes such as kallikrein, plasmin, trypsin, and chymotrypsin. In the present invention, any tissue or blood obtained from any animal can be used.

[0008] Potato protease inhibitor is a protease inhibitor present in potato, which strongly inhibits the action of bacterial protease (natto protease),
Molecular weight 38,0 that hardly inhibits the enzymatic action of trypsin
Type I of 00 [Hyogo University of Agriculture, Research report, 6 (1) , 35 (196
3)], type IIa with a molecular weight of 20,000 to 25,000 that strongly inhibits trypsin, chymotrypsin (bovine serum) and bacterial protease (natto bacterial protease) action, and chymotrypsin (bovine serum) and bacterial protease (natto bacterial protease) action. Three types of type IIb [J. Biochem., 70, 817 (1971)] having a molecular weight of 20,000 to 25,000, which strongly inhibit the enzyme action of trypsin, are known, and any inhibitor is used in the present invention. be able to.

The soybean trypsin inhibitor is a polypeptide present in soybean, and is composed of soybean Kunitz trypsin inhibitor (hereinafter abbreviated as Kunitz) having a molecular weight of 22,000 consisting of 181 amino acids and 72 amino acids. A Bowman-Birk inhibitor having a molecular weight of 8,000 (hereinafter abbreviated as BBI) is known [metabolism, 14 (6), 1057 (1977)],
Either can be used.

Antipines are S. michiganensis and S. yokoskanensis ( S. michiganensis ) .
yokosukanensis ), Actinomyces violascens , and other structural formulas: Phe ← Co → Arg → Val-Arginina
It is a substance having 1 and is known as an inhibitor of trypsin, papain, plasmin and the like.

Leupeptin is an oligopeptide produced by various Actinomycetes and has a basic structural formula: R-Leu-Leu-Arg-CHO (where R is acetyl, leupeptin Ac-LL and R are propionyl). Some have leupeptin Pr-LL.). Furthermore, their derivatives, eg 1
It is also known that one or two Leu are replaced with Val or Ile. Leupeptin used in the present invention also includes these derivatives. Leupeptin is known as an inhibitor of proteolytic enzymes such as trypsin and plasmin.

The guanidino fatty acid derivative has a basic structure of a compound having a guanidino group at the ω-terminal of a fatty acid having 4 to 8 carbon atoms, and the guanidino moiety thereof is substituted with various substituents, and / or its acid. A derivative of
For example, various esters and amides are included. Also included are the corresponding thioesters. Further included are their non-toxic acid addition salts. Examples of these guanidino fatty acid derivatives include Japanese Patent Publication Nos. 47-21977, 50-2494 and 45-2.
-33175, 49-2107, JP-A-48-29732, 49-249
The compounds described in Nos. 17, 51-75042 and 54-76532 can be mentioned. Preferably, the compound represented by the general formula (I)

[0013]

[Chemical 1] (Wherein R ¹ represents a hydrogen atom, halogen, nitro, alkyl, alkoxy, carboxy or alkoxycarbonyl group, and n represents an integer of 3 to 6) or a non-toxic acid addition salt thereof. Can be mentioned. More preferable compound (Compound 1)

[0014]

[Chemical 2] 6-guanidinohexanoic acid / p-ethoxycarbonylphenyl ester and its non-toxic acid addition salts are mentioned.

Non-toxic acid addition salts of the compounds included in the present invention include hydrochloride, hydrobromide, hydroiodide,
Inorganic or acetate salts such as sulfates, phosphates, nitrates, lactates, tartrates, citrates, benzoates, methanesulfonates, ethanesulfonates, benzenesulfonates, toluenesulfonates Examples thereof include organic acid salts such as isethionate, glucuronate, and gluconate, and methanesulfonate is preferable. The guanidinobenzoic acid derivative is o-, m- or p- of benzoic acid.
A compound having a guanidino group at the position as a basic structure, and a benzene ring or a guanidino moiety thereof being substituted with various substituents, and / or an acid derivative thereof such as an ester or an amide is included. Also included are the corresponding thioesters. Further included are their non-toxic acid addition salts.

Examples of the guanidinobenzoic acid derivative included in the present invention include JP-A Nos. 48-29732 and 49-24917 (US Pat. No. 3,824,267), 49-11842 and 50-4038.
No. 50, No. 50-69035, No. 51-16631, No. 52-89640 (US Patent No. 4021472), No. 53-15412, No. 53-147044,
54-70241 and 55-55154 (U.S. Pat.
No. 55-115865 and No. 55-115863 (U.S. Pat. No. 4,283,418) and No. 56-34662 (U.S. Pat. No. 4310533).
No. 62, 111963 and 63-165357 (European Patent Publication No. 222608), 55-100356, 56-110664,
57-53454, 57-142956, 57-142957, 57
-179146, 58-41855, 58-49358, 61-28636
No. 1, 61-286362, 62-103058, 62-155253
Mention may be made of the compounds described in U.S. Pat. Preferably, the general formula (II)

[0017]

[Chemical 3] (In the formula, R ² represents a phenyl group, a natiful group, a substituted phenyl group or a substituted naphthyl group, and R ³ represents various substituents.) A derivative of guanidine benzoic acid or a non-toxic acid addition salt thereof Is mentioned. More preferred compounds include

(Compound 2)

[Chemical 4] p- (p-guanidinobenzoyloxy) phenylacetic acid / N, N-dimethylcarbamoylmethyl ester

(Compound 3)

[Chemical 5] p-guanidinobenzoic acid 1- (N, N-dimethylcarbamoylmethoxycarbonyl) -2-naphthyl ester,

(Compound 4)

[Chemical 6] p-guanidinobenzoic acid / p- (N-phenyl-N-ethoxycarbonylmethylcarbamoylmethyl) phenyl ester,

(Compound 5)

[Chemical 7] p-guanidinobenzoic acid / p-[(2-ethoxycarbonyl-1-indolinyl) carbonylmethyl] phenyl ester,

(Compound 6)

[Chemical 8] p-guanidinobenzoic acid / p-[(3-ethoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-2
-Yl) carbonyl] phenyl ester,

(Compound 7)

[Chemical 9] p-guanidinobenzoic acid / p-[(2-ethoxycarbonyl-1-indolinyl) carbonyl] phenyl ester,

(Compound 8)

[Chemical 10] p-guanidinobenzoic acid / p-[(N-benzyl-N-
Ethoxycarbonylmethyl) sulfamoyl] phenyl ester,

(Compound 9)

[Chemical 11] p-guanidinobenzoic acid-3-chloro-5-hydroxyphenyl ester,

(Compound 10)

[Chemical 12] p-guanidinobenzoic acid-3-chloro-4-pivaloyloxyphenyl ester,

(Compound 11)

[Chemical 13] p-guanidinobenzoic acid / p-sulfamoylphenyl ester,

(Compound 12)

[Chemical 14] p- (p-guanidinobenzoyloxy) phenylacetic acid,

(Compound 13)

[Chemical 15] and p-guanidinobenzoic acid 6-amidinonaphth-2-yl ester and non-toxic acid addition salts thereof.

The amidinophenol derivative has a basic structure of a compound having an amidino group at the 0-, m- or p-position of phenol, and its benzene ring or amidino moiety is substituted with various substituents, and / Also included are esters of the phenol with certain acids or amides of the phenol with certain amines. Further included are their non-toxic acid addition salts. Examples of the amidinophenol derivative included in the present invention include JP-A-58-41855.
Examples thereof include compounds described in Japanese Patent Application Nos. 4-274992 and 5-96758. Preferably, the general formula (II
I)

[0031]

[Chemical 16] (In the formula, R ⁴ represents an organic group.) And a non-toxic acid addition salt thereof. More preferred compounds include

(Compound 14)

[Chemical 17] 5- [p- (p-amidinophenoxycarbonyl) benzylidene] -3-ethoxycarbonylmethylrodanine,

(Compound 15)

[Chemical 18] 1- [p- (p-amidinophenoxycarbonyl) benzyl] -2-isopropylimidazole,

(Compound 16)

[Chemical 19] p- (p-amidinophenoxycarbonyl) benzoic acid
N- [1-ethoxycarbonyl-2- (3-indolyl) ethyl] amide,

(Compound 17)

[Chemical 20] p- (p-amidinophenoxycarbonyl) benzoic acid
2S-ethoxycarbonyl-1-pyrrolidinylamide,

(Compound 18)

[Chemical 21] p- (p-amidinophenoxycarbonyl) benzoic acid
2R-benzyloxycarbonyl-1-pyrrolidinylamide,

(Compound 19)

[Chemical formula 22] 4-[(4-amidino-2-methoxycarbonyl) phenoxycarbonyl] benzoic acid N-phenyl-N-ethoxycarbonylmethylamide,

(Compound 20)

[Chemical formula 23] p- (p-amidinophenoxycarbonyl) -α-methylcinnamic acid / N- [1R-ethoxycarbonyl-2- (3
-Indolyl) ethyl] amides and their non-toxic acid addition salts. In the present invention, each active ingredient may be used alone, but two or more types of active ingredients may be blended to form a single preparation.

[0039]

INDUSTRIAL APPLICABILITY Each substance used in the present invention has a strong inhibitory activity against mite protease and an inhibitory action on inflammation caused by mite protease, and has very little toxicity. It is useful for the treatment and / or prevention of mite allergic diseases for mammals, especially humans, such as asthma, allergic rhinitis and atopic dermatitis caused by mite.

It has been confirmed that the toxicity of each substance used in the present invention is extremely low. For example, the LD ₅₀ value of aprotinin is 2.5 × 10 ⁶ kallikrein inhibitor unit / kg (mouse, iv administration), and the LD ₅₀ value of gabexate mesylate (mesylate salt of Compound 1) is 24.
8 mg / kg (mouse, iv administration), the LD ₅₀ value of camostat mesylate (mesylate salt of said compound B) 210 mg
/ Kg (mouse, iv administration). Therefore, it can be judged that all the substances used in the present invention are sufficiently safe and suitable for use as a medicine.

The substance used in the present invention is usually administered systemically or locally, orally or parenterally. The dose varies depending on the age, body weight, symptom, therapeutic effect, administration method, treatment time, etc., but is usually in the range of 1 mg to 1000 mg, preferably 50 mg to 500 mg per adult per dose.
Orally administered once to several times a day, or in an amount of 0.1 mg to 100 mg per adult once a day, or once to several times a day, or 5 to 20% ointment It is applied to the affected area once to several times a day. Of course, as described above, since the dose varies depending on various conditions, a dose smaller than the above dose range may be sufficient in some cases, or a dose exceeding the range may be necessary in some cases.

Solid compositions for oral administration according to the present invention include tablets, powders, granules and the like. In such solid compositions, the one or more active substances are at least one inert diluent such as lactose, mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, aluminometasilicate. Mixed with magnesium acid. According to a conventional method, the composition comprises additives other than an inert diluent, for example, a lubricant such as magnesium stearate, a disintegrant such as calcium fibrin glycolate, a stabilizer such as lactose, glutamic acid or asparagine. It may contain a solubilizing agent such as an acid. If necessary, the tablets or pills may be coated with a film of a gastric or enteric substance such as sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, or may be coated with two or more layers. Further, it may be a capsule of an absorbable substance such as gelatin.

Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs and the like, and generally used inert diluents. For example, it contains purified water and ethanol. This composition may contain, in addition to an inert diluent, auxiliary agents such as a wetting agent and a suspending agent, a sweetening agent, a flavoring agent, an aromatic agent and a preservative.
Other compositions for oral administration include spray formulations which contain one or more active substances and are formulated in a manner known per se.

The injections for parenteral administration according to the present invention include aseptic aqueous or non-aqueous solutions, suspensions and emulsions. Examples of the aqueous solution and suspension include distilled water for injection and physiological saline. Examples of the non-aqueous solution and suspension include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, alcohols such as ethanol, polysorbate 80 and the like. Such compositions may further contain adjuvants such as preservatives, wetting agents, emulsifiers, dispersants, stabilizers (eg lactose), solubilizers (eg glutamic acid, aspartic acid). These are sterilized by, for example, filtration through a bacteria-retaining filter, blending of a bactericide, or irradiation. These can also be used by preparing a sterile solid composition and dissolving it in sterile water or a sterile solvent for injection before use. The ointment for parenteral administration according to the present invention can be manufactured by a conventional method.

[0045]

The present invention will be described in more detail with reference to experimental examples and examples, but the present invention is not limited to these experimental examples and examples.

Experimental Example 1: Inhibitory effect on house dust mite protease Test method: Int. Arch. Allergy Appl. Immunol., 91, 8 from a culture extract of house dust mite ( Dermatophagoides farinae ) containing house dust mite protease (Df-protease).
0 (1990). The purified enzyme [150 units / mg (37 ° C, pH 8)] was stored at -80 ° C in a phosphate buffer solution (PBS) (1 mg / ml). The stored enzyme solution was diluted with cold 1 mM hydrochloric acid before use. The activity of the enzyme is determined by the synthetic substrate (Boc-Gln-Gl
y-Arg-MCA) at 37 ° C. according to the method of Kawabata et al. [Eur. J. Biochem., 172, 17 (1988)]. 50 mM Tris-HCl buffer (pH 8.0, 1880 μ) containing various compounds (0 to 0.2 μM)
l), a substrate (0 to 0) dissolved in dimethyl sulfoxide.
50 μM, 100 μl) was added. After adding the enzyme (1.1 nM, 20 μl) dissolved in 1 mM hydrochloric acid, the initial value of hydrolysis was excited at 380 nm, emitted at 440 nm, and measured by a fluorescence spectrophotometer.

The inhibitory effect of each compound on Df-protease was evaluated by changing the molar ratio of each compound to Df-protease. That is, a mixture of Df-protease and each compound was incubated at 37 ° C. for 1 to 3 minutes (the incubation time was changed depending on each compound), and the residual enzyme activity was measured under the same conditions as above. Ki value is Lineweave
Calculated from r-Burk plot.

Results: The results are shown in Table 1.

[0049]

[Table 1]

[0050]

[Table 2]

It is understood from Table 1 that not all proteolytic enzyme inhibitors have an inhibitory effect on Df-protease, and only specific substances exhibit an inhibitory effect.

Experimental Example 2: Inhibitory effect on enhancement of vascular permeability by Df-protease Experimental method: The experiment was carried out using guinea pigs (350 to 400 g). That is, a 1% solution of Evans blue dissolved in physiological saline was intravenously administered at a rate of 20 mg / kg of animal body weight, and immediately thereafter, Df-protease (0.01-0.8
nmol) in PBS solution (100 μl) or Df-protease (0.4 nmol) and aprotinin (0.1-0.6 nm)
ol) was administered intradermally. 30 minutes after the administration, the vascular permeability was evaluated by the size of the spot of Evans blue that penetrated the skin surface at the administration site.

Results: The results are shown in FIG. Lane A in the figure
From the left, Df-protease 0.01, 0.03, 0.09, 0.27
And vascular permeability when a PBS solution containing 0.80 nmol is administered. It can be seen that vascular permeability is enhanced depending on the concentration of Df-protease. Lane B shows Df-protease (0.4 nmol) and aprotinin (0.1 from the left,
It shows vascular permeability when a mixed solution of 0.2, 0.3, 0.4 and 0.6 nmol is administered. It can be seen that the action of enhancing vascular permeability by Df-protease is suppressed depending on the concentration of aprotinin. The enhancement of vascular permeability is a phenomenon of inflammation, which indicates that one of the pathological conditions of mite allergy was revealed by Df-protease. Therefore, in this experiment, it was confirmed in vivo that administration of aprotinin, which is one of the substances used in the present invention, suppresses the mite allergic symptom.

Formulation Example 1 Aprotinin (10 mg) was sufficiently dissolved in physiological saline (500 ml), and the obtained solution was sterilized and sterilized by a conventional method, 5 ml each was filled in ampoules, and then freeze-dried to obtain 100 μg in one ampoule. 1000 injections containing the active ingredient were obtained.

Formulation Example 2 100 g of camostat mesylate, 2 g of magnesium stearate, and 18 g of lactose were mixed well until homogeneous, and then tableted by a conventional method to give tablets 1000 each containing 100 mg of the active substance. I got a lock. The obtained tablets are coated with hydroxypropyl cellulose by a conventional method,
The target tablet for oral administration was prepared.

[Brief description of drawings]

FIG. 1 is a photograph showing the morphology of an organism when observing the effect of Df-protease on vascular permeability enhancement in guinea pigs.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Hiroshi Kodera 4-49-5 Izumi, Suginami-ku, Tokyo Casa Izumi 204

Claims (4)

[Claims] 1. A mite containing, as an active ingredient, one or more substances selected from aprotinin, potato protease inhibitor, soybean trypsin inhibitor, antipain, leupeptin, guanidino fatty acid derivative, guanidinobenzoic acid derivative, and amidinophenol derivative. Allergy remedy. 2. The active ingredient is 6-guanidinohexanoic acid.
The therapeutic agent according to claim 1, which is p-ethoxycarbonylphenyl ester or an acid addition salt thereof. 3. The active ingredient is p- (p-guanidinobenzoyloxy) phenylacetic acid.N, N-dimethylcarbamoylmethyl ester, p-guanidinobenzoic acid.1-
(N, N-dimethylcarbamoylmethoxycarbonyl)
-2-naphthyl ester, p-guanidinobenzoic acid, p
-(N-phenyl-N-ethoxycarbonylmethylcarbamoylmethyl) phenyl ester, p-guanidinobenzoic acid / p-[(2-ethoxycarbonyl-1-indolinyl) carbonylmethyl] phenyl ester, p-guanidinobenzoic acid / p- [(3-Ethoxycarbonyl-
1,2,3,4-Tetrahydroisoquinolin-2-yl) carbonyl] phenyl ester, p-guanidinobenzoic acid / p-[(2-ethoxycarbonyl-1-indolinyl) carbonyl] phenyl ester, p-guanidinobenzoic acid / p-[(N-benzyl-N-ethoxycarbonylmethyl) sulfamoyl] phenyl ester, p
-Guanidinobenzoic acid-3-chloro-5-hydroxyphenyl ester, p-guanidinobenzoic acid-3-chloro-4-pivaloyloxyphenyl ester, p-guanidinobenzoic acid-p-sulfamoylphenyl ester,
p- (p-guanidinobenzoyloxy) phenylacetic acid, p-guanidinobenzoic acid-6-amidinonaphtho-2
-The therapeutic agent according to claim 1, which is one or more substances selected from yl esters and acid addition salts thereof. 4. The active ingredient is 5- [p- (p-amidinophenoxycarbonyl) benzylidene] -3-ethoxycarbonylmethylrhodanine, 1- [p- (p-amidinophenoxycarbonyl) benzyl] -2-isopropyl. Imidazole, p- (p-amidinophenoxycarbonyl) benzoic acid N- [1-ethoxycarbonyl-2-
(3-Indolyl) ethyl] amide, p- (p-amidinophenoxycarbonyl) benzoic acid.2S-ethoxycarbonyl-1-pyrrolidinylamide, p- (p-amidinophenoxycarbonyl) benzoic acid.2R-benzyloxycarbonyl -1-pyrrolidinylamide, 4-[(4
-Amidino-2-methoxycarbonyl) phenoxycarbonyl] benzoic acid / N-phenyl-N-ethoxycarbonylmethylamide, p- (p-amidinophenoxycarbonyl) -α-methylcinnamic acid / N- [1R-ethoxycarbonyl-2] The therapeutic agent according to claim 1, which is one or more substances selected from-(3-indolyl) ethyl] amide and acid addition salts thereof.