JP2003246730A - Tryptase inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a strong and safe tryptase inhibitor. <P>SOLUTION: This tryptase inhibitor contains 6'-amidino-2'-naphthyl4- guanidinobenzoate or its pharmacologically allowable salt as the effective component. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to 6'-amidino-
The present invention relates to a tryptase inhibitor containing 2'-naphthyl 4-guanidinobenzoate or a pharmaceutically acceptable salt thereof as an active ingredient. Also, the present invention provides a proteinase-activated receptor-2 (PA) comprising a compound having a tryptase inhibitory activity or a pharmaceutically acceptable salt thereof as an active ingredient.
R-2: relates to a proteinase-activated receptor-2) inhibitor.

[0002]

BACKGROUND OF THE INVENTION Tryptase is a tetrameric member of the serine proteinase family expressed by mast cells and was isolated and purified from human lungs in 1984. When mast cells are activated, tryptase is released into the extracellular space together with pre-synthesized mediators such as histamine, chymase, and proteoglycan (Schwartz Lewis Seldin, 1981; Cau
ghey Lazarus, 1988). For human tryptase,
It is unique among serine proteinases in that it is sufficiently catalytically active in plasma and extracellular space [Schw
artzBradford, 1986; Goldstein Leong, 1992]. At the cellular level, mast cells (mast
Among the cells, tryptase, chymase, and carboxypeptidase are present in connective-woven mast cells deep in the skin, but it is also reported that tryptase is present in mucosal mast cells in the lung epithelium but not chymase. is there.

Tryptase is a natural antiprotease that regulates the activity of other trypsin-like serine proteinases, such as a mucus protease inhibitor (= antileukoprotease or HUSI-I), antithrombin III, α1.
-Not inhibited by proteinase inhibitors, α2-macroglobulin, or C 1 -esterase inhibitors (A
lterKramps, 1990; Smith Hougland, 1984; Schwartz B
radforld, 1986; Harvima Schechter, 1988; Cromlish
Seidah, 1987).

Regarding the relationship between tryptase and various diseases, it has been reported that the tryptase concentration is higher than usual in patients as described below. Patients with mastocytosis and after systemic anaphylaxis (Schwartz Metcalfe, 1987; Schwartz Yunginge
r, 1989). Patients with aspirin-sensitive asthma during a systemic response after aspirin challenge, especially in their plasma (Bosso
Schwartz, 1991). Patients with asthma (Broide Gleich, 1991; Bousquet Ch
anez, 1991, Wenzel Fowler, 1988). Patients with interstitial lung disease (Walls Bennett, 1991). Patients with interstitial cystitis (Theoharides TC, Kempuraj
D, Sant GR., 2001). Patients with irritable bowel syndrome (Pang X, Boucher W, Tri
adafilopoulos G, Sant GR, Theoharides TC, 1996). Patients with allergic patients following antigen challenge (especially in their bronchoalveolar lavage fluid) (Castells, 1988; Butrus, 199)
0). Patients with atopic and allergic skin (especially
In skin blisters after skin antigen challenge) (Shalit Sch
wartz, 1990; Atkins Schw-artz, 1990). Patients with seasonal allergic rhinitis, especially in nasal washes after topical antigen challenge (Juliusson Holmberg,
1991). Patients with gingivitis and periodontitis, especially in their gingival exudates (Cox Eley, 1989, J Period Res; Eley Cox, 19
92, J Dent). Patients with psoriasis (especially in their lesional skin) (Harvim
a Naukkarinen, 1989).

[0005] Furthermore, it has been confirmed in vitro tests that tryptase is directly involved in the pathogenesis of mast cell-related diseases, which shows that tryptase increases the contractility of airway smooth muscle. (Sekizawa, 1989), it is known that by inactivating a vasoactive intestinal peptide, its bronchodilation effect is destroyed (TamCau
ghey, 1990; TamFranconi, 1990; Franconi, 1989).
This suggests that tryptase is a pathogenic mediator of asthma. In addition, tryptase has been shown to be a potent fibroblast mitogen and has been reported to be associated with asthma and lung fibrosis in interstitial lung disease (Ruoss Hartmann, 1991; Hartmann Ruoss). , 1992). Tryptase activates prostromelysin (= MMP-3), which in turn activates collagenase, which initiates the destruction of cartilage and periodontal connective tissue. It is also said to be related to the etiology of the disease (Gruber Marchese, 198).
9; Gruber Schwartz, 1990; Cox Eley, 1989, J Period
Res; Eley Cox, 1992, J Dent). Tryptase also inactivates the procoagulant (coagulant precursor) function of high molecular weight kininogen (Maier Spragg, 1983) and cleaves fibrinogen (Schwartz Bradfor
d Littman, 1985), which is also known to be capable of promoting blood coagulation disorders.

It has also been reported that tryptase is inhibited by common inhibitors of trypsin-like proteinases such as diisopropylfluorophosphate, phenylmethylsulfonyl fluoride and tosyl-L-lysine chloromethyl ketone (Smith Hougland, 1984; Harvima Sch.
echter, 1988), but these compounds are not suitable for in vivo use because of their high toxicity and poor stability.
It is said to be unsuitable even for testing. Furthermore, peptides-arginine aldehydes known to have tryptase inhibitory action, leupeptin and antipain (Cromlish Seidah, 1987), (Caughey, 19
Also 93), the inhibitory effect of tryptase was weak and its usefulness was limited.

In addition, Japanese Patent Publication No. 9-500532 describes a purified human tryptase inhibitor molecule which is a polypeptide, and a tryptase inhibitor obtained from a leech extract.

Although the relationship between tryptase and various diseases has been described above, the following is also known. For example, it has been reported that mast cells are involved in renal interstitial fibrosis, and tryptase, which is a major proteolytic enzyme thereof, is involved in fibroblast proliferation and promotion of extracellular matrix synthesis. (J Am Soc Nephrol 12: 166
8-1676, 2001).

The following reports have also been made regarding hay fever. First, when pollen (antigen) in the air clings to the mucous membranes of the nose and eyes and invades the body, the organism regards the antigen as a foreign substance and makes IgE antibodies. Especially in people with hay fever, this IgE antibody is overproduced. IgE antibodies first attach to the surface of cells. When the antigen further enters there, it binds to the antibody to cause an “antigen-antibody reaction” and intracellular release of chemical mediators such as histamine, heparin, and tryptase. These chemical mediators have the action of stimulating the terminals of sensory nerves, causing edema of mucous membranes, and aggravating mucus secretion. This causes excessive runny nose, itchy eyes, and stuffy nose.

The following reports have also been made on the relationship between tryptase and side effects caused by inflammation and X-ray contrast media. Regarding inflammatory cells, multifaceted studies have been conducted on the activation mechanism of mast cells, basophils, eosinophils and respiratory epithelial cells. First, regarding the mechanism of side effects of X-ray contrast agents, hyperosmolar X-ray contrast agents cause histamine release from human basophils, and this release rate is significantly correlated with that due to immunological stimulation. Became clear. It is well known that immunological histamine-releasing ability is enhanced in allergic patients, and the reason why side effects of X-ray contrast agents are high especially in allergic patients is due to such a phenomenon. The possibility was suggested. In addition, as a result of detection of IgE antibody against ioxagluic acid, which is a typical X-ray contrast agent, IgE antibody could be detected in about 30% of patients showing side effects. However, since the amount thereof is very small, the significance of this specific IgE antibody is currently under investigation. Since the concentration of tryptase was increased in the serum of a patient who had a shock symptom with an X-ray contrast medium, it was confirmed that the mediator is released from mast cells in the case of severe side effects. Next, regarding the pathogenic mechanism of aspirin asthma, when an aspirin load test was performed on such patients and urine was collected over time to measure metabolites of various mediators, leukotriene E4, 11b-pGF2a was obtained. And methylhistamine were significantly increased. This result indicates that in aspirin-asthma patients, at least mediator release from mast cells is caused by aspirin, and is considered to contribute to the treatment of aspirin-asthma.

From the above facts, agents inhibiting tryptase are indicated as systemic anaphylactic diseases, aspirin-sensitive asthma, asthma, interstitial lung diseases, allergic diseases, atopic diseases, skin blisters, gingivitis, psoriasis, lung fibers. It is considered to be effective as a therapeutic agent for various diseases caused by tryptase such as infectious diseases, arthritis, periodontal disease, blood coagulation disorders, renal interstitial fibrosis, side effects of X-ray contrast agents, and hay fever.

On the other hand, tryptase has recently been used as a pret.
It has been clarified that it has a specific stimulating action on einase activated receptor-2 (PAR-2). Therefore, hereinafter, proteinase-activated receptor (PAR; prote
inase-activated receptor), especially PAR-2.

Proteinase-activated receptor (PAR)
Is a type of G protein-coupled receptor. The N-terminal extracellular peptide chain of the receptor molecule is cleaved at a specific site by an agonist proteinase, and the exposed receptor activating sequence binds to another site of the receptor molecule. To activate. In 1991, PAR-1 was cloned as a thrombin receptor of human platelets, and its unique activation mechanism was clarified. After that, PAR-2, PAR-3, and PAR-4 have been cloned to date. Of the four PARs, PAR-1 and PAR-
3, PAR-4 is a thrombin receptor, whereas P-4
AR-2 is not activated by thrombin at all, and several proteinases such as trypsin and tryptase have been identified as candidate endogenous agonist enzymes.

PAR-1 plays an important role in the activation of human platelets, is widely distributed in the nervous system, digestive system, respiratory system, vascular system and others and is involved in the control of various functions. Also,
PAR-2, like PAR-1, is widely distributed in the living body, and P
It modifies various functions different from AR-1.

Further, "Japanese Journal of Pharmacology" (Okayama Univ.
According to Masahiro Nishibori, the following report has been made. Along with platelets, important PARs-expressing cells are vascular endothelial cells and vascular smooth muscle. PAR-1 or PA
R-2 stimulation evokes an endothelium (NO) -dependent vasodilatory response. PAR-2 expression in vascular endothelial cells is LPS,
IL-1β and TNF-α markedly increased (J Biol Chem
271,14910-14915,1996), and sep-2 in the septicshock
Have been suggested to be involved (Circulation 99,2590-
2597, 1999). PAR-1 stimulation of vascular endothelium and neutrophils enhances selectin expression in both cells. As described above, the production of thrombin accompanying vascular collapse is mediated by PAR-1 stimulation.
It causes vasodilation and neutrophil accumulation and infiltration, and is closely involved in the progression of inflammatory reactions. The proliferation of cultured vascular endothelial cells and smooth muscle cells is stimulated by PAR-1 and PAR-2 stimulation, but in the coronary balloon restenosis model, PAR-2 receptors are strongly expressed in the intima and media layer, It was suggested that PAR-2 receptor stimulation is involved in proliferative thickening of the intima.
Mast cell tryptase has been identified in addition to trypsin as a PAR-2 receptor stimulating proteinase.

Furthermore, regarding the physiological role of PAR-2, PAR-2 present in respiratory epithelium transmits information through inositol phospholipid metabolism, and especially in respiratory tract epithelial cells, it modifies LPS action and lung epithelium. In cells, various physiologic actions such as promoting adhesion of neutrophils are induced, and PAR-2 present in the sensory nervous system is involved in the expression of inflammatory pain.
It is also reported that gastric mucosa promotes mucus secretion through the release of CGRP and tachykinins, and acts protectively on the gastric mucosa.

As described above, it is well known that PAR-2 is involved in various pathophysiologies such as hyperalgesia, pain, airway constriction and edema, and PAR-2 specific stimulation. If effective tryptase can be effectively inhibited, it is applied to various diseases involving PAR-2 receptor, that is, suppression of hyperesthesia, analgesic action, and antipruritic action (PAR-2 acts on sensory nerve endings). Yes, tryptase stimulation releases CGRP and substance P, tryptase and PAR-2 agonist peptides cause hyperalgesia, anti-edema action, anti-asthma action (PAR-in the respiratory tract)
Various effects such as tracheal contraction due to 2 stimuli) can be expected. In particular, the application as an ointment as an antipruritic agent in the case of atopic dermatitis and the application as an inhibitor for hyperesthesia such as neuralgia and postherpetic pain can be expected greatly.

As described above, as compounds having tryptase inhibitory activity, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride and tosyl-
Although L-lysine chloromethyl ketone and the like have already been known, a compound having insufficient activity and a stronger inhibitory action has been desired.

[0019]

[Means for Solving the Problems] The inventors of the present invention have conducted diligent research to find a more potent and safe tryptase inhibitor based on such a request, and found that 6'-amidino-
The present invention has been completed by finding that 2′-naphthyl 4-guanidinobenzoate has a surprising inhibitory effect that no one can expect.

That is, the present invention provides the following (1) to (8).
Related to the invention of. (1) A tryptase inhibitor containing 6'-amidino-2'-naphthyl 4-guanidinobenzoate represented by the following formula or a pharmaceutically acceptable salt thereof as an active ingredient. (2) The tryptase inhibitor according to (1) above, wherein the salt is a mesylate salt. (3) The tryptase inhibitor according to (1) or (2) above, wherein the tryptase is human tryptase. (4) Systemic anaphylactic disease, aspirin-sensitive asthma, asthma, interstitial lung disease, containing 6'-amidino-2'-naphthyl 4-guanidinobenzoate or a pharmaceutically acceptable salt thereof as an active ingredient Interstitial cystitis,
Irritable bowel syndrome, allergic disease, atopic disease,
Skin blistering, hypersensitivity, pain, pruritus, gingivitis, edema, psoriasis, pulmonary fibrosis, arthritis, periodontal disease, blood coagulation disorder,
A therapeutic agent for treating or preventing a disease selected from the group consisting of renal interstitial fibrosis, vascular hyperpermeability or pulmonary edema as a side effect of an X-ray contrast agent, and hay fever. (5) The therapeutic agent according to (4) above, wherein the salt is a mesylate salt. (6) The above-mentioned (4) or (5), wherein the disease is pain, pruritus, interstitial cystitis, irritable bowel syndrome, or vascular hyperpermeability or pulmonary edema as a side effect of an X-ray contrast agent. Therapeutic agent. (7) A proteinase-activated receptor-2 inhibitor containing a compound having a tryptase inhibitory action or a pharmaceutically acceptable salt thereof as an active ingredient. (8) The tryptase is human tryptase, and the compound having a tryptase inhibitory action or a pharmaceutically acceptable salt thereof is 6′-amidino-2′-naphthyl-4-guanidinobenzoate or its mesylate salt, (7) ) The described inhibitor.

6'-amidino-2'-naphthyl 4
-Guanidinobenzoate (generic name "nafamostat") is described in Japanese Examined Patent Publication No. 61-1063, which further discloses that the compound has a strong inhibitory activity against trypsin, plasmin, kallikrein and thrombin. It is described that it is useful as an antitrypsin agent, an antiplasmin agent, an antikallikrein agent and an antithrombin agent. Further, the mesylate salt of the above compound is
The generic name is "nafamostat mesilate", which has already been used as a therapeutic agent for "amelioration of acute symptoms of pancreatitis" and "general intravascular coagulation", and for "extracorporeal circulation such as hemodialysis". Is marketed as an anticoagulant.

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION The 6'-amidino-2'-naphthyl 4-guanidinobenzoate used in the present invention can be produced according to the method described in JP-B-61-1063.

PAR-2, a tryptase inhibitor of the present invention
Pharmaceutical preparations such as inhibitors may be parenteral (eg, injection such as intravenous injection, intradermal injection, intramuscular injection or subcutaneous injection, poultice, ointment, inhalant, etc.) However, it is not particularly limited.

The dose depends on the method of administration, the purpose of treatment or prevention, the age and weight of the patient, etc., but is about 0.00
It is preferably 5 to about 100 mg / Kg body weight.

In the case of injections, in addition to the active ingredients, buffers may be added to maintain their pharmaceutical composition usually at pH = about 3.5 to 7, eg phosphate buffers, Furthermore, sodium chloride, mannitol or sorbitol for adjusting the isotonicity may be added. They can be in lyophilized form or in dissolved form, in the latter case containing an antimicrobial preservative, for example 0.2-0.3% 4-hydroxybenzoic acid methyl ester or ethyl ester, in the solution. Is preferred.

Formulations for topical administration may include aqueous solutions, lotions or jellies, oily solutions or suspensions, oily or ointment emulsions. Formulations in the form of aqueous solutions are prepared, for example, by dissolving a compound of the invention or a pharmaceutically useful salt thereof in an aqueous buffer at pH = 4 to 6.5, and if desired, one or more other Prepared by adding substances. The concentration of active ingredient is about 0. 0 in about 10 ml of solution or about 10 g of jelly.
08 to about 1.5 mg, preferably 0.25 to 1.0 mg
Is. As an oily application form for topical administration, for example,
Wetting agents (eg aluminum stearate) and / or surfactants having an HLB value (hydrophilic-lipophilic ratio) of less than 10 (eg glycerol monostearate, sorbitan monolaurate, sorbitan monostearate or sorbitan mono). Fatty acid monoesters of polyhydric alcohols such as oleate) are added to the oil, if desired,
It can be obtained by suspending the compound of the present invention or a pharmaceutically useful salt thereof. The grease-based ointment is, for example, a grease-based ointment base to be spread, to which a surfactant having an HLB value of less than 10 is optionally added,
It is obtained by suspending the compound of the present invention or a salt thereof. Emulsion ointments are obtained by crushing an aqueous solution of the compound of the present invention or a salt thereof in a soft, spreadable grease-based ointment base to which a surfactant having an HLB value of less than 10 is optionally added. To be When used as a preparation for topical administration, it may contain a preservative.

In particular, an oral preparation is suitable as a therapeutic drug for neuralgia and postherpetic pain, and an ointment is used as an antipruritic drug.
Further, as an asthma attack therapeutic agent, an inhalant is preferable.

Subsequently, the inhibitory activity of 6'-amidino-2'-naphthyl 4-guanidinobenzoate against tryptase and 6'-amidino-2'-naphthyl 4 were evaluated.
-The action of guanidinobenzoate on PAR-2 and the confirmation method thereof will be described.

The inhibitory activity of 6'-amidino-2'-naphthyl 4-guanidinobenzoate against human tryptase is t-butyloxycarbonyl-L-phenylalanyl-L-sery.
lL-arginine-4-methylcoumaryl-7-amide (Boc-Phe-Se
r-Arg-MCA) as a substrate and a known tryptase inhibitor "gabexate" (WO97 / 037969).
It is evaluated by comparing with the inhibitory effect of compound 1) described in the publication.

Here, in WO97 / 037969, the following guanidino fatty acid derivative of the formula (I) and the formula (I
A tryptase inhibitor containing at least one selected from I) guanidinobenzoic acid derivatives, formula (III) guanidinophenol derivatives, formula (IV) amidinophenol derivatives, etc. is described.

[0031] (In the formula, R1 represents a hydrogen atom, a halogen atom, a nitro group, an alkyl group, an alkoxy group, a carboxy group or an alkoxycarbonyl group, and n represents an integer of 3 to 6.)
A guanidino fatty acid derivative represented by.

[0032] (In the formula, R2 represents a phenyl group, a naphthyl group, a substituted phenyl group or a substituted naphthyl group, and R3 represents various substituents.) A guanidinobenzoic acid derivative.

[0033] (In the formula, R4 and R5 represent various substituents.) A guanidinophenol derivative.

[0034] (In the formula, R6 and R7 represent various substituents.) An amidinophenol derivative.

As mentioned above, the above-mentioned publication describes the following compound 1 as a specific example of the compound represented by the formula (I). Compound 1; 6-guanidinohexanoic acid / p-ethoxycarbonylphenyl ester (generic name "gavexate");

In this publication, as an example of the guanidinobenzoic acid derivative represented by the above formula (II), JP-A-48-2 is disclosed.
9732 and 49-24917 (U.S. Pat. No. 3824267), 4
9-11842, 50-5038, 50-69035, 51-16631
No. 52-89640 (US Patent No. 4021472), No. 53-1541
No. 2, No. 53-147044, No. 54-70241 and No. 55-55154 (U.S. Patent No. 4224342), No. 55-115865 and No. 55-115.
863 (U.S. Patent No. 4283418), No. 56-34662 (U.S. Patent No. 4310533), No. 62-111963 and No. 63-165357 (European Patent Publication No. 222608), No. 55-100356, No. 56-11
0664, 57-53454, 57-142956, 57-142957
No. 57, No. 57-179146, No. 58-41855, No. 58-49358, No. 6
1-286361, 61-286362, 62-103058, 62-155
253 and GB Patent Publication Nos. 2083818 and 2095239 are included.

Further, as more preferable compounds of the above formula (II), the following three compounds 2 to 4 are mentioned. Compound 2: p- (p-guanidinobenzoyloxy) phenylacetic acid N, N-dimethylcarbamoylmethyl ester (generic name "camostat"), Compound 3: p- (p-guanidinobenzoyloxy) phenylacetic acid ("camostat" metabolite), Compound 4: p-guanidinobenzoic acid 6-amidinonaphth-2-yl ester (generic name "nafamostat"),

Furthermore, in the publication, the tryptase inhibitory activity of the methanesulfonates of the above compounds 2 and 3 is
It is stated that the C ₅₀ values were 30.0 and 4.33 nM, respectively. However, for compound 4,
Only the compound names are listed, and the compound 4
Has not been experimentally proved to have a tryptase inhibitory activity similar to that of Compounds 2 and 3,
Nor is there any rationale behind it. Incidentally, Compound 2 is included in Japanese Patent No. 10027969 (Japanese Patent Publication No. 54-41583), while Compound 4 is included in Japanese Patent No. 1332984 (Japanese Patent Publication No. 61-1063).
The two patents are allowed as different patents because they have different invention ideas in terms of chemical structure. Thus, the publication does not describe experimentally or theoretically that compound 4 has tryptase inhibitory activity, and if compound 2 and 3 have tryptase inhibitory activity, compound 4 is inevitably included. There is no theoretical or experimental basis to support the similar effect.

The present inventors have also been studying the mechanism of vascular permeability enhancement and pulmonary edema expression, which are side effects of various iodine contrast agents, and several drugs have been investigated in the process. It was found that it suppresses side effects, but this time, 6'-amidino-2'-naphthyl 4
-We newly found that administration of guanidinobenzoate (3-10 mg / kg, iv) completely suppressed pulmonary vascular hyperpermeability and pulmonary edema.

By the way, as far as PAR-2 is concerned, tryptase is preteinase activated receptor-2 (PA
It has been clarified that it has a specific stimulating action on R-2), but this time, the present inventors have confirmed that tryptase was positive in a permeability measurement experiment using cultured endothelial cells derived from bovine aorta. Has a remarkable effect of increasing protein permeability,
In addition, it was confirmed that the same action was observed in the PAR-2 agonist peptide, and further that the permeability enhancing action by this tryptase was strongly suppressed by 6'-amidino-2'-naphthyl 4-guanidinobenzoate. Found.

Similarly, the present inventors have found that tryptase
Directly stimulating PAR-2 in vascular endothelial cells causes protein permeation enhancement, and side effects such as edema due to contrast medium tryptase / PAR-2
It was found that the system was involved.

As described above, the inhibitory effect of 6'-amidino-2'-naphthyl 4-guanidinobenzoate on PAR-2 is very interesting. The effects of 6'-amidino-2'-naphthyl 4-guanidinobenzoate on hypopulmonary function and pulmonary edema were examined by in vivo data. In conducting the test, the index of decreased lung function is a decrease in arterial blood oxygen partial pressure (PaO ₂ ), and the index of pulmonary edema is an increase in water content in lung tissue, Na ^+.
The content was increased and the K ⁺ content was decreased. As a result, administration of the iodine contrast agent ioxaglic acid showed a decrease in PaO _2, an increase in lung tissue water content and an increase in Na ⁺ content, and these decreased lung function and pulmonary edema were both 6′-amidino-2′-.
It was found to be completely suppressed by naphthyl 4-guanidinobenzoate.

In addition to the above description, as is apparent from the examples shown below, 6'-amidino-2'-naphthyl 4-guanidinobenzoate very strongly inhibits tryptase, so that it cannot be attributed to excessive tryptase. Arou diseases such as systemic anaphylaxis disease, aspirin hypersensitivity asthma, asthma, interstitial lung disease, interstitial cystitis, irritable bowel syndrome, allergic disease, atopic disease, skin blisters, gingivitis, psoriasis , Pulmonary fibrosis, arthritis, periodontal disease, blood coagulation disorder, renal interstitial fibrosis, side effect of X-ray contrast agent, pollinosis, etc., and is considered to be extremely effective as a therapeutic agent for various diseases caused by tryptase.

As is clear from this experimental system, tryptase is a proteinase-activating receptor (PAR-2).
It is considered that the permeability is enhanced by stimulating the action of erythrocyte. On the other hand, PAR-2 is also present in sensory nerves and bronchi, and the stimulation of tryptase causes pain, hyperalgesia, pruritus, and airway constriction. The mesylate salt of'-amidino-2'-naphthyl 4-guanidinobenzoate is considered to be involved in the tryptase / PAR-2 system,
In particular, it is useful as a therapeutic drug for hyperesthesia, pain, pruritus, edema, asthma and the like.

[0045]

Example First, as a drug to be used, human lung-derived purified tryptase (EC 3.4.21.59, cat No. 202-14471, for biochemistry, Wako Pure Chemical Industries, Ltd.), 6'-amidino-2'-naphthyl 4-guanidino was used. Benzoate mesylate (nafamostat mesylate) and gabexate mesylate, and t-bu
tyloxycarbonyl-L-phenylalanyl-L-seryl-L-arginine-
4-methylcoumaryl-7-amide (Boc-Phe-Ser-Arg-MCA) (S
igma company) was prepared. Boc-Phe-Ser-Arg-MCA is DMS
It was dissolved in O (75 μM) and finally diluted 20-fold for use.

Example 1 Measurement of Human Tryptase Inhibitory Activity The inhibitory action of nafamostat mesylate on the purified tryptase activity derived from human lung was measured. A known gabexate mesylate was used as a control.

Nafamostat mesylate was dissolved in Tris-HCl buffer, tryptase (final concentration: 1 nM) was added, and the mixture was allowed to stand at 37 ° C. for 5 minutes. Boc-Phe-Ser-Arg-MCA (final concentration 3.75 μM) was added to this mixture as a substrate solution, reacted at 37 ° C for 5 minutes, and then immersed in a 100 ° C water bath for 30-60 seconds. The reaction was stopped by.
After centrifugation of this mixed solution (13,000 rotations, 5 minutes), the fluorescence intensity of the degradation product was measured at an excitation wavelength of 370 nm and a fluorescence wavelength of 460 nm, and used as an index of enzyme activity. The same measurement was performed for the control gabexate mesylate. A graph showing the inhibitory effect of nafamostat mesylate and gabexate mesylate on the purified tryptase activity derived from human lung is shown in FIG. In the graph, each value is the average value ± S. E. (N = 4-5, where N = 0.1 and 1 nM
2).

As shown in FIG. 1, nafamostat mesylate extremely potently suppressed the purified activity of human lung-derived purified tryptase in a concentration-dependent manner, and its IC ₅₀ value was 0.01.
It was 6 nM. On the other hand, the IC ₅₀ value of gabexate mesylate used as a control was 194 nM. Nafamostat mesylate is about 1% of the known gabexate mesylate.
It can be seen that it shows a surprising inhibitory activity of 2000 times.

(Example 2: Permeability enhancing action of tryptase in cultured vascular endothelial cells) Using cultured vascular endothelial cells (bovine aortic endothelial cells), tryptase and PAR-
Serine-Leucine-Isoleucin known as 2 agonists
The protein (bovine serum albumin) permeability enhancing effect of e-Glycine-Lysine-Valine (SLIGKV) was examined. In addition, an experiment was also conducted on the inhibitory effect of nafamostat mesylate.

The apparatus shown in FIG. 2 was used for the measurement of protein permeability. This device consists of a plastic outer plate container and an insert that can be placed inside, and the bottom of the insert is fitted with a planar polycarbonate membrane (pore diameter 3.0-μm, area 1.0 cm ² ). .
Cultured bovine aortic endothelial cells (4.0 × 10 ⁴ cells / cm ² ) were seeded on a polycarbonate membrane, and 10% FBS,
Dulbecco's Modified Eagle's Me containing 100 U / ml penicillin and 100 μg / ml streptomycin
After culturing with dium (DMEM) as a medium under conditions of 95% humidity and 5% CO ₂ for 4 to 5 days, the medium was subjected to a permeability experiment. The medium was replaced every two days. The protein for permeation was bovine serum albumin (Ab-E
The Krebs-Ringer solution of B) was used.

In the permeability experiment, bovine vascular endothelial cells cultured on the insert were thoroughly washed with Krebs-Ringer solution (pH 7.4) and immersed in the plate. Krebs on the plate
-1.5 ml of Ringer's solution was filled in advance, and 0.5 ml of Krebs-Ringer's solution was added to the insert so that the liquid surfaces of both were in agreement. SLIGKV as tryptase or PAR-2 agonist at a given concentration
To Evans blue-conjugated bovine serum albumin (A
b-EB; final albumin concentration was 4%, Evans blue concentration was used as 0.67 mg / ml) and was added into the insert, and Ab-EB concentration leaked into the external solution was measured (absorbance at 620 nm), It was used as an index of protein permeability. Tryptase and proteolytic enzyme activated receptor (PAR-2) agonist peptide (SLIGKV)
FIG. 3 shows a graph showing the protein permeability-enhancing effect of cultured vascular endothelial cells. In the graph, each value is the average value ±
S. E. Indicates.

As shown in FIG. 3, tryptase and P
AR-2 agonist peptide Serine-Leucine-Isoleu
Both cine-Glycine-Lysine-Valine (SLIGKV) cause protein permeability enhancement in vascular endothelial cells,
The potency of tryptase was about 10,000 times that of the PAR-2 agonist peptide (SLIGKV).

Example 3 Inhibitory Action of Tryptase-Promoted Protein Permeability by Nafamostat Mesylate The presence of tryptase enhances protein permeability in cultured vascular endothelial cells. Is clear from. In this example, an experiment was conducted on the inhibitory effect of nafamostat mesylate according to the same method as in Example 2. The same method as in Example 2 was performed except that nafamostat mesylate was added together with tryptase into the insert, and the amount of tryptase added was adjusted to a final concentration of 1 nM.

The results are shown in FIG. FIG. 4 is a graph showing the inhibitory effect of nafamostat mesylate on protein permeation enhancement by tryptase in cultured vascular endothelial cells, and each value in the graph is an average value ± S. E. Is shown (N = 4-7). According to this test, the protein permeability-enhancing effect of tryptase was suppressed by nafamostat mesylate in a concentration-dependent manner, and its IC ₅₀ value was 0.02.
It was 9 nM.

(Example 4: Inhibitory effect of nafamost mesylate and gabexate mesylate on vascular permeability enhancement in rat lung by iodine contrast medium) Rats were anesthetized by intraperitoneal administration of pentobarbital Na (50 mg / kg) to obtain ioxaglic acid. (Hexa Brix 320,
Tanabe Seiyaku Co., Ltd. (4 g iodine / kg) was infused through the femoral vein with Evans blue physiological saline solution (20 mg / kg). The injection volume was 16 ml / kg, and the injection rate was 1.
It was 5 ml / min. The blood was killed 10 minutes after the injection, and the pulmonary artery was perfused with physiological saline, and the lung tissue was extracted and weighed.
Evans blue leaked to lung tissue was extracted with formamide (4 ml / g tissue wet weight) and quantified by absorbance at 620 nm. On the other hand, lung tissue is 24 at 60 ° C.
After drying for an hour, the dry weight was measured. The vascular permeability was expressed as the amount of Evans blue leaked into lung tissue per unit dry weight. Also, for the purpose of investigating lung function, oxygen partial pressure (PaO ₂ ) and carbon dioxide partial pressure (PaCO ₂ ) in arterial blood.
And pH, contrast agent administration 5, 10, 20, 40 and 60
Minutes later, the femoral artery was monitored using i-STAT (i-STAT Cooperation, New Jersey, USA). Furthermore, for the purpose of investigating pulmonary edema, the rat lung was removed 1 hour after the administration of the contrast medium, and the lung tissue water content and Na ⁺ and K ⁺
The content was measured. The lung water content (%) was expressed as 100 × (wet weight−dry weight) / wet weight. On the other hand, the Na ⁺ and K ⁺ contents were measured by an atomic absorption photometer (flame method). The results are shown in Figures 5, 6 and 7.

FIG. 5 is a graph showing the inhibitory effect of nafamostat mesilate and gabexate mesylate on the vascular permeability-enhancing effect of the iodine contrast agent ioxaglate in rat lungs, and each value in the graph is the mean value ± S. ． E.
(Number in (); number of cases, * P <0.05, **
P <0.01; comparison with control group (Dunnett's test)).
As shown in FIG. 5, administration of the iodine contrast agent ioxaglic acid markedly enhanced vascular permeability in rat lungs.
Nafamostat mesilate (3-10 mg / kg) significantly increased the vascular permeability-enhancing effect of ioxaglic acid, and
It was strongly suppressed, and in particular, no increase in vascular permeability due to ioxaglic acid was observed in the 10 mg / kg administration group.
On the other hand, gavexate mesylate (50 mg / kg) significantly suppressed the vascular permeability-enhancing effect of ioxaglic acid, but not as markedly as nafamostat mesylate was observed.

Further, FIG. 6 shows rat arterial blood oxygen partial pressure, carbon dioxide partial pressure, and p by the iodine contrast agent ioxaglic acid.
It is a graph showing the inhibitory effect of nafamostat mesylate on changes in H (each value is mean ± SE, 4-6 rats in each group, * P <0.05; non-administration group) (Dunnett's test)), the results shown in FIG. 6 showed that administration of ioxaglic acid caused a significant decrease in arterial oxygen partial pressure due to lung function decline, and that nafamostat mesilate (10 mg / kg) It can be seen that the decrease in oxygen partial pressure is completely suppressed.

Furthermore, a graph showing the inhibitory effect of nafamostat mesylate on rat lung edema (increased water content, increased Na ⁺ content and decreased K ⁺ content) by the iodine contrast agent ioxaglic acid in FIG. 7 (each value is an average value ± S.
E. , 4-6 rats in each group, * P <0.05;
Comparison with non-administered group (Dunnett's test), † P <0.0
5; Comparison with ioxaglic acid alone administration group (Dunnett's te
From st)), it was revealed that the administration of nafamostat mesylate (10 mg / kg) suppressed the development of so-called pulmonary edema such as increase in lung water content and increase in Na ⁺ content in lung tissue due to ioxaglic acid. It was

(Formulation Example 1) Using the following components, an injection is prepared by a conventional method. Nafamostat mesylate 5.0 mg Distilled water for injection 2 ml

(Formulation Example 2) The following components are dissolved in distilled water for injection and lyophilized to prepare an injection to be dissolved before use. Nafamostat mesylate 10.0 mg Boric acid 1.0 mg D-mannitol 20.0 mg

(Formulation Example 3) The following components are dissolved in distilled water for injection and freeze-dried to prepare an injectable solution to be dissolved before use. Nafamostat mesylate 10.0 mg Succinic acid 1.0 mg D-mannitol 20.0 mg

(Formulation Example 4) The following components are dissolved in distilled water for injection and freeze-dried to prepare an injection to be dissolved before use. Nafamostat mesylate 10.0 mg Benzoic acid 1.0 mg D-mannitol 20.0 mg

(Formulation Example 5) The following ingredients were prepared so that 100.0 mg of nafamostat mesylate was contained in one capsule.
The amounts are mixed and a capsule is manufactured by a conventional method. Nafamostat mesylate 100.0 mg Lactose 59.0 mg Crystalline cellulose 33.4 mg Calcium carboxymethyl cellulose 3.6 mg Magnesium stearate 4.0 mg Total 200.0 mg

(Formulation Example 6) The following components and amounts were mixed so that 50.0 mg of nafamostat mesylate was contained in 500 mg of the preparation, and a fine granule was prepared by a conventional method. Nafamostat mesylate 50.0 mg Lactose 249.0 mg D-mannitol 75.0 mg Corn starch 110.0 mg Hydroxypropylcellulose 16.0 mg Total 500.0 mg

(Formulation Example 7) The following ingredients and amounts were weighed so that 1 mg of ointment contained 10 mg of nafamostat mesylate, and an ointment was prepared by a conventional method. Nafamostat mesylate 1.0 g Ethylene glycol 20.0 g Liquid paraffin 5.0 g White petrolatum 74.0 g Total 100.0 g

(Formulation Example 8) The following ingredients and amounts were weighed out so that 1 mg of cream contained 10 mg of nafamostat mesylate, and a cream was prepared by a conventional method. Nafamostat mesylate 1.0 g Ethylene glycol 20.0 g Sorbitan sesquioleate 4.0 g Vaseline 30.0 g Paraffin 7.0 g Lanolin 4.0 g Methyl paraoxybenzoate 0.6 g Purified water 33.4 g Total 100.0 g

(Formulation Example 9) The following ingredients and amounts were weighed so that 1 mg of cream contained 10 mg of nafamostat mesylate, and a cream was prepared by a conventional method. Nafamostat mesylate 1.0 g Ethylene glycol 30.0 g Whale wax 5.0 g Lanolin 5.0 g Cetanol 25.0 g Glycerin monostearate 3.5 g Sorbitan monostearate 1.5 g Glycerin 3.0 g Carboxyvinyl polymer (1% aqueous solution) 5.0 g Hydroxide Potassium 0.2 g Methyl paraoxybenzoate 0.6 g Purified water 20.2 g Total 100.0 g

Formulation Example 10 The following ingredients and amounts were weighed out so that 1 g of lotion contained 10 mg of nafamostat mesylate, and a lotion was prepared by a conventional method. Nafamostat mesylate 1.0 g Ethylene glycol 30.0 g Propylene glycol 64.0 g Isopropyl alcohol 5.0 g Total 100.0 g

[0069]

Curiously, no tryptase inhibitory action of tryptase of 6'-amidino-2'-naphthyl 4-guanidinobenzoate or a pharmaceutically acceptable salt thereof has been reported to date. Surprisingly, as is apparent from the above experimental results, 6'-amidino-2'-naphthyl 4-guanidinobenzoate or a pharmaceutically acceptable salt thereof, particularly 6'-amidino-2'-
Mesylate salt of naphthyl 4-guanidino benzoate (nafamostat mesylate), it has a much stronger tryptase inhibitory effect than known gabexate mesylate (IC ₅₀ value is 194 nm) (IC ₅₀ values 0.016 nm) But it became clear here for the first time. And, surprisingly, the efficacy of nafamostat mesylate was more than 12000 times that of gabexate mesylate.

Therefore, the present tryptase inhibitor is applicable to systemic anaphylaxis disease, aspirin hypersensitivity asthma, asthma, interstitial lung disease, interstitial cystitis, irritable bowel syndrome, allergic disease, atopic disease, cutaneous bullosa. , Hypersensitivity,
Pain, pruritus, gingivitis, edema, psoriasis, pulmonary fibrosis, arthritis, periodontal disease, blood coagulation disorder, renal interstitial fibrosis, vascular hyperpermeability or pulmonary edema as a side effect of X-ray contrast agent, and pollen There are great expectations for future research and development as therapeutic agents for the treatment or prevention of diseases selected from the group consisting of diseases.

On the other hand, tryptase selectively activates proteolytic enzyme-activated receptors (PAR-2) widely distributed in the living body such as vascular endothelial cells, digestive tract, sensory nerves, bronchi, etc. Contracts airway smooth muscle,
Pain via tachykinin release from sensory nerves, the known fact that it causes hyperalgesia, pruritus, inflammation, edema, and, just as tryptase enhanced protein permeability in cultured vascular endothelial cells, Based on the results of this test in which PAR-2 agonist peptides also showed similar effects, nafamostat mesylate strongly and concentration-dependently suppresses the protein permeability-enhancing effect of tryptase in cultured vascular endothelial cells. Became clear.

On the other hand, the lung failure and anaphylactoid action as a side effect of the iodine contrast agent are considered to be mainly based on the actions of histamine and tryptase derived from mast cells. In this experiment, the iodine contrast agent caused a marked increase in pulmonary vascular permeability, and this effect was almost completely suppressed by nafamostat mesylate. On the other hand, gabexate mesylate significantly suppressed pulmonary vascular hyperpermeability, but its effect was incomplete.

From the above, nafamostat mesylate strongly inhibits the tryptase activity, so that the tryptase / PAR-2 system is considered to be involved in the above-mentioned diseases, in particular, the perception of neuralgia and postherpetic pain. A therapeutic and preventive effect can be expected for diseases such as hypersensitivity, pruritus, edema, and asthma.

Therefore, the inhibitors and suppressors of the present invention can be used for systemic anaphylaxis disease, aspirin hypersensitivity asthma, asthma, interstitial lung disease, allergic disease, atopic disease, cutaneous bullosa, hypersensitivity, and pruritus. , Gingivitis, edema,
Treatment of a disease selected from the group consisting of psoriasis, pulmonary fibrosis, arthritis, periodontal disease, blood coagulation disorder, renal interstitial fibrosis, vascular hyperpermeability or pulmonary edema as a side effect of X-ray contrast agent, and hay fever Alternatively, it is useful as a therapeutic agent for prevention.

[Brief description of drawings]

FIG. 1 is a graph showing the inhibitory effect of nafamostat mesylate and gabexate mesylate on purified tryptase activity derived from human lung.

FIG. 2 is a device for measuring protein permeability in cultured vascular endothelial cells.

FIG. 3 is a graph showing an action of enhancing protein permeability in cultured vascular endothelial cells by tryptase and proteolytic enzyme activated receptor (PAR-2) agonist peptide (SLIGKV).

FIG. 4 is a graph showing the inhibitory effect of nafamostat mesylate on the increase in protein permeability by tryptase in cultured vascular endothelial cells.

FIG. 5 is a graph showing the inhibitory effect of nafamostat mesylate and gabexate mesylate on the vascular permeability-enhancing effect in the rat lung by the iodine contrast agent ioxaglic acid.

FIG. 6 is a graph showing the inhibitory effect of nafamostat mesylate on changes in rat arterial blood oxygen partial pressure, carbon dioxide partial pressure, and pH by the iodine contrast agent ioxaglic acid.

FIG. 7 is a graph showing the inhibitory effect of nafamostat mesylate on rat lung edema (increased water content, increased Na ⁺ content and decreased K ⁺ content) by the iodine contrast agent ioxaglic acid.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 7/10 A61P 7/10 9/14 9/14 11/00 11/00 11/06 11/06 13 / 10 13/10 13/12 13/12 17/00 17/00 17/04 17/04 17/06 17/06 19/02 19/02 25/00 101 25/00 101 25/02 101 25/02 101 25/04 25/04 27/16 27/16 37/08 37/08 39/00 39/00 43/00 111 43/00 111 (72) Inventor Toshiaki Sendou 2-Minamiohashi, Minami-ku, Fukuoka-shi, Fukuoka 20-17 F-term (reference) 4C084 AA02 AA17 NA05 NA06 NA14 ZA081 ZA211 ZA361 ZA531 ZA591 ZA661 ZA671 ZA811 ZA891 ZA961 ZB131 ZC202 ZC211 ZC422 4C206 ZA21 ZA ZA ZA ZA ZA ZA ZA ZA ZA ZA ZA ZA ZC42

Claims (8)

[Claims] 1. A 6'-amidino-2 'represented by the following formula:
-A tryptase inhibitor containing naphthyl 4-guanidinobenzoate or a pharmaceutically acceptable salt thereof as an active ingredient. 2. The tryptase inhibitor according to claim 1, wherein the salt is a mesylate salt. 3. The tryptase inhibitor according to claim 1 or 2, wherein the tryptase is human tryptase. 4. A systemic anaphylactic disease, which comprises 6'-amidino-2'-naphthyl 4-guanidinobenzoate or a pharmaceutically acceptable salt thereof as an active ingredient.
Aspirin hypersensitivity asthma, asthma, interstitial lung disease, interstitial cystitis, irritable bowel syndrome, allergic disease, atopic disease, bullous skin, hyperesthesia, pain, pruritus, gingivitis, edema, Treatment of a disease selected from the group consisting of psoriasis, pulmonary fibrosis, arthritis, periodontal disease, blood coagulation disorder, renal interstitial fibrosis, vascular hyperpermeability or pulmonary edema as a side effect of X-ray contrast agent, and hay fever Or a therapeutic agent for prevention. 5. The therapeutic agent according to claim 4, wherein the salt is a mesylate salt. 6. The disease is pain, pruritus, interstitial cystitis,
The therapeutic agent according to claim 4 or 5, which is irritable bowel syndrome or vascular hyperpermeability or pulmonary edema as a side effect of an X-ray contrast agent. 7. A proteinase-activated receptor-2 inhibitor containing a compound having a tryptase inhibitory action or a pharmaceutically acceptable salt thereof as an active ingredient. 8. The tryptase is human tryptase, and the compound having a tryptase inhibitory activity or a pharmaceutically acceptable salt thereof is 6′-amidino-2′-naphthyl 4
A guanidinobenzoate or its mesylate salt,
The inhibitor according to claim 7.