TW200306811A - Medicinal compositions for inhibiting tryptase - Google Patents

Abstract

Because of having a potent inhibitory effect on tryptase, 6'-amidino-2' -naphthyl 4-guanidinobenzoate or its pharmaceutically acceptable salt are useful as remedies or preventives for diseases such as systemic anaphylaxis, aspirin-hypersensitive asthma, asthma, interstitial pulmonary diseases, allergic diseases, atopic diseases, skin hydroa, hypersthesia, itch, gingivitis, edema, psoriasis, pulmonary fibrosis, arthritis, periodontal disease, coagulation failure, renal interstitial fibrosis, hyper-vasopermeability or pulmonary edema occurring as side effects of X-ray contrast media, hay fever and so on.

Description

200306811 (1) 发明. Description of the invention [Technical field to which the invention belongs] The present invention relates to a compound containing 6'-fluorenyl-2'-naphthyl-4 4-methyl benzoate or a pharmaceutically acceptable salt thereof. It is a medicinal composition for blocking tryptase which is an active ingredient. In addition, the present invention relates to a medicine for inhibiting proteinase-activated receptor-2 (proteinase-activated receptor-2) using a compound having a tryptase-blocking effect or a pharmaceutically acceptable salt thereof as an active ingredient.组合 物。 Composition. [Prior technology] Tryptase is a tetramer of the serine proteinase family found in giant cells (mast cells). It was isolated and purified from human lungs in 1984. After activation of tryptase by giant cells, it releases extracellular space with pre-synthesized histamine, chymotrypsin (CHYMASE) and glycoprotein (proteoglycan) (Schwartz Lewis Seldin, 1981; Caughey Lazarus , 1 988). Regarding human tryptase, from the viewpoint of sufficient catalytic activity in plasma or extracellular space, it is also specific in serine proteolytic enzymes (Schwartz Bradford, 1 986; Goldstein Leong, 1 992) . It has also been reported that in the cell-level, among giant cells of type 1 allergic reactions, there are woven giant cells deep in the skin, with trypsin, rennet enzyme, and proteolytic enzyme (Carboxypeptidase) There is trypsin in the mucosal giant cells in the lung epithelial tissue, and -6-(2) (2) 200306811 is not present in the curd protein enzyme. Trypsin-like, like other trypsin, is a natural antiproteolytic enzyme that regulates the activity of serine proteolytic enzymes, and will not be caused by mucus proteolytic enzyme blockers (= anti-leucoproteinase or HUSI-I), anticoagulation Antithrombin, αl — proteolytic enzyme blocker, α2-macroglobulin (α2-macroglobulin), or Cl — ester blockers (AlterKramps, 1 990; Smith H ou g 1 and, 1 9 8 4; Schwarts

Brad fo rid, 1986, Harvima Schechter, 1 988; Cromlish

Seidah, 1 987) 〇 On the relationship between tryptase and various diseases, patients with the following trypsin concentration increased than usual: Patients with giant cell disease, and patients with systemic allergic reactions (Schwartz Metcalfe, 1 98 7, Schwartz Yunginger, 1 9 8 9); Patients with aspirin allergic asthma, the patients with the most severe systemic reaction (especially in their plasma) after aspirin attack (Bosso Schwartz, 1991), patients with asthma (Broide Gleich, 1991; Bousquet Chanez, 1991, Wenzel Fowler, 198 8), patients with interstitial lung disease (Walls Bennett, 1991), Patients with essential cystitis (Theoharides TC, Kempuraj D, Sant GR., 200 1) Patients with allergic bowel syndrome (Pang x, Boucher w, (3) (3) 200306811

Triadafilopoulos G, Sant GR, Theoharides TC, 196 9); patients with allergic antigens after challenge with infection (especially in bronchoalveolar wash fluid) (Castells, 1 988; Butriis, 1 990), with Patients with atopic dermatitis and allergic skin (especially in skin blisters after skin antigen attack by antigen challenge) (Shalit Schwarts, 1990, Atkins S chw-art z, 1 9 9 0); with seasonal allergies Patients with rhinitis (especially in nasal washes after topical antigen challenge) (JuliussonHolmberg, 1991); patients with odontitis and periodontal disease (especially in dental exudates) (Cox Eley, 1 989, J Period Res; Eley Cox, 1992, J Dent) Patients with psoriasis (especially in the diseased skin) (Harvima Narkkarinen, 189 9)) In addition, tryptase is directly related to the etiology of giant cell-related diseases, and also It can be confirmed in a test in a test tube (invitr 0) that it can be seen that tryptase increases the contractility of tracheal smooth muscle (Sekizawa, 1 989), deactivates the vasoactive intestinal peptide, and destroys it. The bronchiectasis (Tam Caughey, 1 990; Tam Franconi, 1 990; Franconi, 1 989). It was suggested that tryptase is the pathogenic mediator of asthma. In addition, reports have revealed that tryptase has been shown to be a potent fission promoter for fibroblasts and is associated with pulmonary fibrosis of asthma or interstitial lung disease (Ruoss Hartmann, 1991; Hartmann Ruoss, 1 992). Tryptase activates prostromelysin (= MMP-3, matrix metalloproteinase), and then promatrix lysing enzyme activates collagen-8- (4) (4) 200306811 enzyme and starts to destroy cartilage Or periodontal tissue, so tryptase is considered to be related to the cause of arthritis or periodontal disease, respectively (Gruber Marchese, 1 989, Gruber Schwartz, 1 990, Cox Eley, 1 989, J Period Res, Eley Cox , 1 992, J Dent). In addition, it is also known that tryptase inactivates the function of coagulant precursor (coagulant precursor) of high molecular weight kininogen (HMWK) (Maier Spragg, 1 983) and splits fibrinogen (

Schwartz Bradforld Littman, 1 98 5) and promote blood coagulation disorders. It has also been reported that although trypsin is a trypsin-like proteolytic enzyme blocker, such as diisopropyl fluor. phosphate (DFP)), phenylmethylsulfonium fluoride and p-toluenesulfonyl-L-lysine chloromethylmethanone (SmithHougland, 1 984, Harvima Schechter, 1 988), these compounds are toxic It is also high and the stability is poor. It is not suitable for use in vivo, even for test in a test tube. In addition, the peptide arginine aldehyde leucine and anti-papaya enzyme (cromlish seidah, 1 987), (Caughey, 1 993), which are known to have trypsin-like blocking effects, also have trypsin-like blocking effects. Weak, limited effectiveness. Japanese Patent Publication No. 9-5 00 532 describes that peptides are purified from human trypsin blocker molecules and trypsin blockers obtained from garlic extracts. The relationship between trypsin and various diseases is as described above. In addition, the following matters are also known. For example, renal interstitial fibrosis is related to giant cells at -9-(5) (5) 200306811, and its main proteolytic enzymes, such as trypsin, are related to fibroblast proliferation or extracellular matrix synthesis (J). Am Soc Nephrol 12: 1668-1676, 2001) ° Pollen disease is reported as follows. First, pollen (antigen) in the air adheres to the mucous membranes of the nose or eyes, and when the body invades the body, the organism treats the antigen as a foreign body and produces an IgE antibody. Especially for people with hay fever, this IgE antibody is overproduced. IgE antibodies first attach to the cell surface. At this time, if there is more antigen invasion, it will bind to the antibody to produce an "antigen-antibody reaction", and release chemically transmitting substances such as histamine, heparin, and tryptase in the cell. These chemical transfer substances have the effect of stimulating sensory nerve ends, causing mucosal edema, and causing severe secretion of mucus. As a result, excessive nasal fluid flows out, causing itchy eyes and causing nasal congestion. The relationship between the side effects caused by inflammation or X-ray contrast agents and tryptase is as follows. Regarding inflammatory cells, a multi-angle review of the activation sequence of giant cells, basophils, eosinophils, and airway epithelial cells was conducted. First, the pathogenesis of side effects of X-ray contrast agents is that high osmotic X-ray contrast agents cause histamine to be liberated from human basophils, and furthermore, this free rate is shown to be meaningfully related to immunological stimuli. The reason is that the histamine free energy of hypersensitive patients is very high, especially the reason that the frequency of side effects of X-ray contrast agents is high in allergic patients, suggesting that this may be caused by this phenomenon. In addition, Igaglic acid, a typical X-ray contrast agent, detected IgE antibodies, and about 30% of patients showing side effects could detect IgE antibodies. However, its amount is very small, so the significance of this specific IgE antibody is now being examined -10- (6) (6) 200306811 is under discussion. In patients with shock symptoms caused by X-ray contrast agents, because the tryptase concentration increases, it is confirmed that the mediator is released from giant cells when the side effects are severe. Secondly, the pathogenesis of aspirin asthma. For such patients, the aspirin saturation test is performed, urine is taken over time, and the metabolites of various media are measured. It is believed that leukotriene E4, lib — PGF2a And methylhistamine increased significantly. The results show that aspirin asthma patients at least cause the medium to be released from giant cells due to aspirin, and also contribute to the treatment of aspirin asthma. Based on the above facts, it is considered that trypsin-blocking agents are considered as a systemic allergic reaction disease, aspirin allergic asthma, asthma, interstitial lung disease, allergic disease, atopic skin disease, skin blisters, and Treatments for various diseases caused by trypsin-like diseases such as sarcoiditis, xerophobia, pulmonary fibrosis, arthritis, periodontal disease, blood clotting disorders, renal interstitial fibrosis, side effects of X-ray contrast agents, and hay fever Effective. On the other hand, recently, it has been shown that tryptase has a specific stimulating effect on proteinase-activated receptor-2 (PAR-2). Therefore, the following is a description of proteinase-activated receptor (PAR; proteinase-activated receptor), especially PAR-20 proteinase-activated receptor (PAR) is G-protein coupled receptor (G-protein coupled receptor (GPCR). The agonist proteinase cuts the n-terminal extracellular peptide chain of the receptor molecule, cuts it at a specific site, and activates the exposed receptor activation sequence by binding to other sites of the receptor molecule. In 1991, -11-(7) (7) 200306811 replicated PAR-1, which is the thrombin receptor of human platelets, and understood its special activation mechanism. Since then, PAR-2, PAR-3, and PAR-4 have been copied. Among the four types of PARs, PAR-1, PAR-3, and PAR-4 are thrombin receptors. In contrast, PAR-2 cannot be activated, and several proteolytic enzymes such as trypsin and tryptase are identified as internal factors. Sexually excited alternate enzymes. Par-1 plays an important role in activating human platelets, and is widely distributed in other nervous systems, digestive systems, respirator systems, and vascular systems, and is related to the control of various functions. In addition, PAR-2, like PAR-1, is widely distributed in living bodies, and it has various functions different from PAR-1. In addition, according to the "Japanese Journal of Pharmacology" (Okayama University • Medicine • Pharmacology Nishiki Masahiro), there are reports as follows. As important as platelets, the PARs-producing cell line vascular endothelial cells and vascular smooth muscle. PAR-1 or PAR-2 stimulation causes an endothelium (NO) -dependent vasodilator response. PAR-2 in vascular endothelial cells significantly increases lipopolysaccharide (LPS), interleukin 1/5 (IL — 1/9), and tumor necrosis factor (TNF — α) (J Biol Chem 271, 1 49 1 0- 1 49 1 5, 1 996), suggesting that PAR-2 is related to septicshock (Circulation 99, 25 90-2597, 1 999). Vascular endothelium and neutrophil PAR-1 stimulated the selectin in both cells to be hyperactive. The thrombin generation accompanying rupture of the blood vessel is thus stimulated by PAR-1, which causes vasodilation and the infiltration of neutrophils, which is closely related to the inflammatory response. Cultured vascular endothelial cells and smooth muscle cells are stimulated by PAR-1 and PAR-2 -12- (8) (8) 200306811 Hypercellular proliferation, in the restenosis mode of coronary balloon catheters, in the intimal and medial layers of blood vessels It is easy to find PAR-2 receptors, suggesting that proliferative hypertrophy of the intima is related to PAR-2 receptor stimulation. As PAR-2 receptor-stimulating proteolytic enzymes, trypsin such as giant cells has been identified in addition to trypsin. In addition, regarding the physiological role of PAR-2, PAR-2 in respiratory epithelial cells conveys information through inositol phospholipid metabolism, especially in the modification of tracheal epithelial cells by LPS, and in pulmonary epithelial cells. Causes various physiological effects such as promoting the attachment of neutrophils. PAR-2 existing in the sensory nervous system is related to the occurrence of inflammatory pain. On the other hand, it has also been reported in the gastric mucosa through CGRP or activin (tachykinins ), Which can promote the secretion of mucus and protect the gastric mucosa. It has been understood in detail that PAR-2 is related to various pathologies such as hyperalgesia, pain, airway contraction, and edema. If it can effectively block trypsin that has a specific stimulating effect on PAR-2, it can be expected to be applicable to PAR-2. -2 receptor diseases, that is, the inhibitory or allergic effect of hypersensitivity, anti-pruritic effect (PAR-2 is located at the end of sensory nerve, stimulated by tryptase, free CGRP or substance P, due to tryptase Or PAR-2 agonist peptides and hyperalgesia), anti-edema effect, anti-wheezing effect (constriction of the trachea by stimulation of the AR-2 in the trachea) and other effects. In particular, great expectations have been placed on it as an antipruritic agent suitable for use in the treatment of atopic dermatitis as an ointment, and as a suppressor for sensory allergies such as neuralgia or postherpetic pain. -13- 200306811 〇) Compounds having trypsin-blocking activity as described above, although diisopropylfluorophosphoric acid, phenylmethylsulfonium fluoride and p-toluenesulfonyl-l-lysine Chloromethyl ketone and the like, but the activity is still insufficient, so compounds having a stronger blocking effect are desired. [Summary of the Invention] The inventors and others, based on related needs, in order to find a more powerful and safe trypsin blocker, and worked hard to find out that 6'-Beryl-2'-naphthyl-4- Pulse benzoate has an amazing blocking effect that no one can predict, and completed the present invention. That is, the present invention relates to a blocker containing a 6'-fluorenyl-2'-naphthyl-4-methylbenzoate or a pharmaceutically acceptable salt thereof as an active ingredient represented by the following formula: Trypsin pharmaceutical composition.

Here, a mesilate is preferred as the salt, and a human tryptase is preferred as the tryptase. In addition, the present invention relates to a composition containing 6'-fluorenyl-2'-naphthyl-4-pulsylbenzoate or a pharmaceutically acceptable salt thereof as an active ingredient for treating or preventing a systemic allergic reaction. Disease, aspirin allergic asthma, asthma, interstitial lung disease, interstitial cystitis, allergic bowel syndrome, atopic skin disease, skin vesicular disease, hypersensitivity, pain, 搔 -14- (10 ) (10) 200306811 Pruritus, odontitis, edema, dryness, pulmonary fibrosis, arthritis, periodontal disease, hemagglutination disorder, renal interstitial fibrosis, X-ray contrast agent side effects of hypervascular permeability or lung Medical composition for edema and hay fever. Here, the salt is preferably mesilate. In addition, the suitable diseases are pain, pruritus, interstitial cystitis, allergic bowel syndrome, or hypervascular permeability due to side effects of X-ray contrast agents. Pulmonary Edema. In addition, the present invention relates to a medicinal composition for inhibiting a proteolytic enzyme-activated receptor-two using a compound having a tryptase-blocking effect or a pharmaceutically acceptable salt thereof as an active ingredient. Here, tryptase is human tryptase, and the compound having a tryptase-blocking effect or a pharmaceutically acceptable salt thereof is 6′-fluorenyl-2′-naphthyl-4-methyl benzyl Ester or its mesylate is suitable. In addition, 6′-fluorenyl-2′-naphthyl-4′-methyl benzoate (common name is “1 ^ £ & 1! 1〇81 & 1”) is described in Japanese Patent No. 61-1063 The gazette further states that the gist of the gazette states that the compound has a strong blocking activity against trypsin, cytosin, angiotensin, and thrombin, and acts as an antitrypsin agent, an anticytosin agent, an angiotensin agent, and Antithrombin agents are effective. In addition, the general name of the mesylate of the above compounds is "nafamostat mesilate", which has been used as a therapeutic agent for "amelioration of acute symptoms of pancreatitis" and "generalized intravascular hemagglutination", and "hemodialysis" "Extracorporeal circulation" anticoagulant was launched. The best form for implementing the invention -15- (11) (11) 200306811 As the active ingredient of the pharmaceutical composition for blocking tryptase of the present invention, 6'-fluorenyl-2'-naphthyl-4-vein The benzoic acid ester can be produced according to the method described in Japanese Patent Publication No. 61-1063. As the active ingredient of the PAR-2 inhibiting pharmaceutical composition of the present invention, 6'-fluorenyl- 2'-naphthyl- 4 -methyl benzoate is a representative. The active ingredients of the pharmaceutical composition for blocking tryptase of the present invention and the PAR-2 inhibiting pharmaceutical composition can be parenterally or orally administered intravenously, intramuscularly, intradermally, subcutaneously, and locally. Invest in any given route. The dosage is determined according to the method of administration, the purpose of treatment or prevention, the age and weight of the patient, and about 0.005 to about 100 mg / Kg body weight is appropriate. The effective ingredients are intravenously, intramuscularly, intradermally, and subcutaneously. When it is administered via the route, it is administered in the form of an injection. When injecting, add a buffer that usually maintains these pharmaceutical compositions at a pH of about 3.5 to 7 to the active ingredient. For example, a phosphate buffer can be added. In addition, sodium chloride and mannose for adjusting isotonicity can be added. Alcohol or sorbitol. These can be freeze-dried or dissolved. In the latter case, it is preferable to contain an antibacterial preservative in the solution, such as 0.02 to 0.3% of 4-hydroxybenzoic acid methyl ester or ethyl ester. The dosage form for topical administration may be, for example, an aqueous solution, an emulsion or a transparent gel, an oily solution or a suspension, or a fat-like ointment or an emulsion-like ointment. The formulation of an aqueous solution, emulsion, or transparent glue is, for example, the active ingredient of the present invention is dissolved in an aqueous buffer solution with a pH of 4 to 6.5. Since -16- (12) (12) 200306811 is required, one kind can be added to it The concentration of the active ingredient is adjusted in about 10 ml of solution or about 10 g of transparent gum, or about 008 to about 1.5 mg, preferably about 0.25 to 1.0 mg. Oily solutions or suspensions for topical administration, such as humectants (for example, aluminum stearate) and / or HLB 値 (hydrophilic-lipophilic ratio) are less than 10 surfactants (for example, propylene glycol monomer Stearate, sorbitan monolaurate, sorbitan monostearate, sorbitan monooleate, and other mono-fatty acid esters of polyalcohols, etc., should be appropriately added to the oil as needed It can be obtained by dissolving or suspending the effective ingredients of the present invention. The oily ointment can be obtained by suspending the active ingredient of the present invention in a greaseable ointment base which can be applied, by adding a surfactant having an HLB 値 of less than 10, if necessary. The emulsion ointment can be obtained by pulverizing the active ingredient aqueous solution of the present invention, for example, by adding a surfactant having an HLB 値 of less than 10 to make it a soft, greaseable ointment base. When used as a topical preparation, it may contain a preservative. Dosage forms for the effective ingredients of the present invention to be orally administered include, for example, lozenges, granules, fine granules, powders and capsules. Alternatively, it may be in the form of an inhaler or a spray. These preparations can be manufactured by directly applying a general method. In particular, it is suitable as a therapeutic agent for neuralgia or postherpetic pain, and is suitable for oral administration. The evaluation of 6'-fluorenyl-2'-naphthyl-4-imidylbenzoate on tryptase blocking activity evaluation, and 6'-fluorenyl-2'-naphthyl-4-imidylbenzene Effect of formate on PAR-2 and its confirmation-17- (13) (13) 200306811 method. The blocking activity of 6'-fluorenyl-2'-naphthyl-4 4-methyl benzoate to trypsin from humans is based on 151 ^ 71〇 \ 7〇 & 1 * 1) 〇1 ^ 1-1 ^ phenylalanyl-L-seryl-L-arginine-4-methylcoumaryl-7-amide (Boc-Phe-Ser-Arg-MCA) as a matrix, and "Gabexate" (recorded The blocking effect of Compound 1) in WO97 / 037969 was evaluated and compared. Here, in W09 7/03 7969, it is described that one or more kinds are selected from the group consisting of a phosphono fatty acid derivative of the following formula (I), a phosphonobenzoic acid derivative of the formula (II), and a compound of the formula (II) Tryptase blockers such as venylphenol derivatives and fluorenylphenol derivatives of formula (IV) are effective ingredients ...

(In the formula, R1 represents a hydrogen atom, a halogen atom, a nitro group, a thiol group, a oxo group, a oxo group, or a oxo group, and η represents an integer of 3 to 6.) The venyl fatty acid derivative represented by;

(In the formula, 'R2 represents phenyl, naphthyl, substituted phenyl, or substituted Zeyl, and R3 represents various substituents.) Derived from benzoic acid -18- 200306811

(In the formula, R4 and R5 are various substituents.) A quinol phenol derivative represented by;

(In the formula, R6 and R7 are various substituents.) A fluorenylbenzene hydrazone derivative represented by. Therefore, as described above, in the same publication, specific examples of the compound represented by the formula (I) are described as follows: Compound 1: 6-Methylhexanoic acid / p-ethyl phenol ester (common name is "Gabexate ")

H2N HN

(CH2) 5——coo

C00C2H5 In addition, examples of the vasobenzoic acid derivative represented by the above formula (Π) described in the same publication include Japanese Patent Application Laid-Open Nos. 48-29732 and 49-24917 (US Patent No. 3824267) and 49- 118 No. 42, -19 · (15) 200306811 No. 50-4038, No. 50-69035, No. 51-16631, No. 52-89 640 (US Patent No. 4021472), No. 53-15412, No. 53-147044, 54-70241 and 55-55154 (US Patent No. 4234342), 55-115865 and 55-115863 (US Patent No. 4343418), and 56-34662 (US Charter No. 4310533), same as 62-111963 and same 63

—1 6 5 3 5 7 (European Patent Publication No. 222608), the same as 55 — 100356, the same as 56 — 110664, the same as 57 — 53454, the same as 57 — 1 42956, the same as 57 — 1 42957, the same 57 — 1 79 1 46, same as 58 — 4 1 8 5 5, 5 8 — 493 5 8, 6 1 — 2863 6 1, 61 — 286 362, 62 — 103058, 62 — Compounds described in No. 155253 and British Patent Publication Nos. 2083 8 1 8 and 209523 9 Secondly, more suitable compounds than the above formula (Π) include, for example, 3 to 2 of the following compounds : Compound 2: p- (p-P-benzoate) phenylacetic acid N, N-dimethylcarbamate methyl ester (common name is "Camostat"),

CH2C00CH2C0N (CH3) 2 Compound 3: p- (p-P-benzoate) phenylacetic acid ("Camostat" metabolite) -20- (16) 200306811

Compound 4: 6-amidino-2-naphthylethyl p-benzoate (common name "Nafamostat").

In addition, in accordance with the gist described in the gazette, the IC50 of the melansulfonate blocking activity of the mesylates of the above compounds 2 and 3 was 30.0 and 4 3 3 nM, respectively. However, compound 4 only includes its compound name. Compound 4 does not have the same experimental evidence to block the activity of pancreatic eggs as compounds 2 and 3, and there is no theoretical basis to prove it. Incidentally, Compound 2 is included in Patent No. 1 002769 (Japanese Patent Publication No. 54-4 1583), while Compound 4 is included in Japanese Patent No. 13329S4 (Japanese Patent Publication No. 61-1063), see both Their chemical structures and invention concepts are different, and their licenses are different. Thus, in the same bulletin, there is no record of its experimental or theoretical proof that compound 4 has the ability to block the activity of pancreatic eggs. There is almost no theoretical or experimental evidence showing that compounds 2 and 3 have a blockade. Every time the pancreas-like egg is active, it is proved that compound 4 must have the same effect. The present inventors have also studied the mechanism of the occurrence of hypervascular permeability and pulmonary edema related to the side effects caused by various iodine contrast agents in the past -21-(17) (17) 200306811. Several agents can suppress the side effects, but this time, 6 'monomethyl-2'-naphthyl-4 monomethyl benzoate (3-10mg / kg, iv) was newly found to completely inhibit Pulmonary vascular hyperpermeability and pulmonary edema. Having said that, regarding PAR-2, although tryptase has been shown to have a specific stimulating effect on proteinase-activated receptor-2 (proteinase-activated receptor-2), this time, the present inventors and others, using Permeability measurement of endothelial cells cultured from bovine aorta showed that trypsin-like protein had significant hyperpermeability. In addition, it was confirmed that PAR-2 agonist peptide also had the same effect. In addition, trypsin-like permeability was found. The hyperactivity is strongly inhibited by 6'-fluorenyl-2'-naphthyl-4 -methyl benzoate. Similarly, the present inventors have found that tryptase directly stimulates PAR-2 of vascular endothelial cells, causes protein permeability, and side effects such as edema of contrast agents are related to tryptase / PAR-2. As described above, the inhibitory effect of '6--Pyridyl-2', Pyridyl-4, and Pyridyl basic formate on par-2 is very profound. The inventors et al. The effect of 6'-amyl-2'-naphthyl-4-methyl benzoate caused by reduced pulmonary function and pulmonary edema was investigated by an in vitro test (invivo). In addition, when the test was performed, the index of decreased pulmonary function was a decrease in the partial pressure of oxygen (Pa02) in the arterial blood, and the index of pulmonary edema was an increase in the amount of water in the lung tissue, an increase in the content of Na +, and a decrease in the content of K +. As a result, when ioxaglic acid, an iodine contrast agent, was administered, it was found that Pa02 decreased, the water content in the lung tissue increased, and the Na + content increased. Ren-22- (18) (18) 200306811 Any of these reduced lung functions and pulmonary edema were found to be completely acceptable. It is inhibited by 6'-fluorenyl-2'-naphthyl-4 4-methyl benzoate. The above description, together with the examples shown below, makes it very clear that 6'-fluorenyl-2'-naphthyl-4-pulsylbenzoate can block tryptase very strongly, so It is thought that for diseases caused by too much tryptase, such as systemic allergic diseases, aspirin allergic asthma, asthma, interstitial lung disease, interstitial cystitis, allergic bowel syndrome, allergic diseases, allergic diseases Orthotopic dermatosis, cutaneous vesicular disease, odontitis, xerobia, pulmonary fibrosis, arthritis, periodontal disease, blood clotting disorders, renal interstitial fibrosis, side effects of X-ray contrast agents, and hay fever Trypsin-like various therapeutic agents are extremely effective. In addition, as shown in this experimental system, it is thought that tryptase stimulates proteolytic enzyme-activated receptor-2 (PAR-2) and increases permeability, on the other hand, PAR-2 also exists in sensory nerves or bronchi Due to the tryptase-induced stimulation of pain, hyperalgesia, pruritus, and tracheal contraction, the 6'-amyl group-2'-naphthyl-4-methyl benzoate mesylate is considered to be related to pancreas Protease / PAR-2 related disorders are particularly effective as a therapeutic agent for hypersensitivity, pain, pruritus, edema and wheezing. [Embodiment] The following describes the present invention in more detail based on examples. First, the medicines used were prepared from human lung trypsin-like (EC 3 4.21.59, cat No 202-14471, biochemical, Wako Pure-23- (19) (19) 200306811 medicine), 6 'a Fluorenyl-2'-naphthyl-4 monomethyl benzoate methanesulfonate (nafamo stat mesilat e) and gabexat e mesilat e and t-butyloxycarbonyl-L-phenylalanyl-L-seryl-L-arginine- 4-methyl coumaryl-7-amide (Boc-Phe-Ser-Arg-MCA) (Sigma). : Boc-Phe-Ser-Arg-MCA is dissolved in DMSO (75 // Μ), and finally diluted 20 times for use. Examples Example 1 Measurement of blocking human tryptase activity The blocking effect of nafamostat mesilate on the activity of purified tryptase from human lungs was determined. A known gabexate mesilate was used as a control. Dissolve nafamostat mesilate in Tris-HCl buffer solution, add tryptase (final concentration is InM), and leave at 37 ° C for 5 minutes. Use this mixture as a matrix solution, add Boc-Phe-Ser-Arg-MCA (final concentration is 3.75 // M), and react at 371 for 5 minutes, then in a hot water bath at 100 ° C , Soak for 30 to 60 seconds to stop the reaction. After centrifuging the mixed solution (1 300 Or pm, 5 minutes), the fluorescence intensity of the decomposition product was measured at an excitation wavelength of 370 nm and a fluorescence wavelength of 460 nm as an indicator of enzyme activity. About gabexate mesilate for control, the same measurement was performed. Figure 1 shows the block diagram of nafamostat mesilate and mesilate for purified tryptase activity from human lungs, as shown in Figure 1. In the figure, each line is represented by the average line of soil -24- (20) (20) 200306811 SE (N = 4-5, but when 〇1 and lnM, N = 2) As shown in Figure 1, the nafamostat mesilate system is extremely Strong and concentration-dependent inhibition of purified tryptase activity from human lungs with IC5 () 値 of 0 016nM. On the other hand, the IC50 of gabexate mesilate used as a control was 194 nM. It is understood that nafamostat mesilate has been shown to have an amazing blocking activity of about 1 2000 times that of the known gabexate mesilate. Example 2 Trypsin-like hyperpermeability in cultured vascular endothelial cells Cultured vascular endothelial cells (bovine aortic endothelial cells) were used to investigate tryptase and Serine-Leucine-Isoleucine-Glycine-Lysine known as PAR-2 agonist -The hyperpermeability of protein (bovine blood gluten albumin) by the bow of Valine (SLIGKV). In addition, experiments on the inhibitory effects of nafamostat mesilate were also performed. For the measurement of protein permeability, the apparatus shown in Fig. 2 was used. The device is formed by a plastic outer container made of plastic and a plug-in board which can be installed therein. The bottom of the plug-in board is provided with a flat polycarbonate film (pore diameter is 3.0-// m and area is 1.0 cm2). Cultured cells of bovine aortic endothelial cells (40 X 104 cells / cm2) were inoculated on a polycarbonate membrane with 10% FBS, 100U / ml penicillin, and # g / ml Streptomycin Dulbecco's modified Eagle. 's Medium (DMEM) is a culture medium, which is cultured for 4 to 5 days under conditions of 95% 02 and 5% C02, and then used for permeability experiments. In addition, the culture medium was changed every 2 days. Krebs es- (21) (21) 200306811 is used for the osmotic protein to bind bovine blood albumin (Ab-EB) identified by Evans Blue.

Ringer solution. In the permeability test, bovine vascular endothelial cells cultured on the insert plate were fully washed with Krebs-Ringei * solution (PH7.4) and immersed in the tray. The tray is filled with 1.5ml of Krebs-Ringer solution in advance, and 0.5ml of Krebs-Ringer solution is added to the insert plate, so that the liquid levels of the two are consistent. Trypsin in a certain concentration, SLIGKV as PAR-2 agonist, and bovine blood albumin (Ab-EB; Elastin blue in final concentration, 4% in Evans blue). 67mg / ml) —Add to the insert plate, and measure the Ab-EB concentration (absorbance at 620nm) leaking from the external liquid as an indicator of protein permeability. Figure 3 shows the effect of tryptase and proteolytic enzyme activated receptor (PAR-2) agonist peptide (SLIGKV) on the hyperpermeability of protein in cultured vascular endothelial cells. In the figure, each actinide represents an average of ± S. E. 〇

As shown in Figure 3, both tryptase and Serine-Leucine-Isoleucine-Glycine-Lysine-Valine (SLIGKV) of PAR-2 agonist peptides can cause the protein permeability of vascular endothelial cells to increase. The effect of tryptase is about 10,000 times the PAR-2 agonistic peptide (SLIGKV) Example 3 nafamostat mesilate inhibits tryptase-induced hyperprotein permeability. As shown in Example 2 above, the presence of tryptase allows culture- 26- (22) (22) 200306811 Hyperpermeability of protein in vascular endothelial cells. In this embodiment, the same method as in Example 2 is followed, and the experiment of nafaniostat mesilate for its suppression is used. Nafamostat mesilate was added to the insert plate together with trypsin. In the same manner as in Example 2, the amount of tryptase was added so that the final concentration was 1 nM. The results are shown in Figure 4. Figure 4 is a graph showing the inhibitory effect of nafamostat mesilate on the hyperpermeability of proteins caused by trypsin-like cells in cultured vascular endothelial cells. In the figure, each line is represented by the average 値 ± S.E. (N = 4 to 7). According to this test, tryptase-like hyperpermeability was inhibited by the concentration of nafamostat mesilate. Its 1C 5.値 is 0 02 9nM. Example 4 Inhibition of nafamostat mesilate and gabexate mesilate on hypervascular permeability in the lung caused by iodine contrast agent ioxaglic acid. Rats were anesthetized by intraperitoneal administration of sodium pentobarbital (50 mg / kg), and ioxaglic acid was anesthetized. (Hexabrix320, Tanabe Pharmaceutical) (4g iodine / kg) and Evans blue physiological saline solution (20mg / kg) were injected into the thigh vein. The injection capacity is 16 m 1 / k g and the injection speed is 1.5 m 1 / min. Ten minutes after the injection, the blood was slaughtered, the pulmonary artery was perfused with physiological saline, and the lung tissue was removed and weighed. Evans blue leaked from lung tissue was extracted with formamidine (4ml / g tissue wet weight) and quantified based on 620nm absorbance. On the other hand, the lung weave was dried at 60 ° C for 24 hours, and then its dry weight was measured -27- (23) (23) 200306811. Vascular permeability is expressed as the dry weight per unit of Evans Blue leaked from lung tissue. In addition, for the purpose of investigating pulmonary function, the partial pressure of oxygen (Pa0 2), partial pressure of carbon dioxide (PaC02), and pH in arterial blood were measured at 5, 10, 20, 40, and 60 minutes after the administration of the contrast agent. STAT (i-STAT Cooperation, New Jersey, USA) 'is monitored by the femoral artery. In addition, for the purpose of investigating pulmonary edema, one hour after the administration of the contrast agent, the lungs of the rats were removed, and the water content and the Na + and K + contents in the lung tissue were measured. Lung water content (%) is expressed as 100X (wet weight-dry weight) / wet weight. On the other hand, the Na + and K + contents were measured by an atomic absorption spectrophotometer (flame method). The results are shown in Figures 5, 6 and 7. Figure 5 is a graph showing the inhibitory effects of nafamostat mesilate and gabexate me si late on the vascular permeability gradual effect in rat lungs caused by the iodine contrast agent ioxaglic acid. In the figure, each line is represented by mean 値 ± SE (() Figures within; number of cases, * P < 0.05, ** P <0.01; comparison with control group (Dunnett's test)). As shown in Fig. 5, the administration of iodine contrast agent ioxaglic acid significantly replenishes vascular permeability in the lungs of rats. nafamostat mesilate (3-10mg / kg) inhibits the vascular hyperpermeability of ioxaglic acid meaningfully and strongly, especially in the 10 mg / kg administration group, which is considered to be almost free of i ο X ag 1 ic acid Caused by vascular hyperpermeability. On the other hand, although gabexate mesilate (50mg / kg) significantly inhibits the vascular hyperpermeability induced by ioxaglic aci d, it is not considered to be significantly inhibited as nafamostat mesilate. In addition, FIG. 6 is a graph showing the inhibitory effect of nafamostat mesilate on -28- (24) (24) 200306811 arterial blood oxygen partial pressure, carbon dioxide partial pressure and pH changes caused by iodine contrast agent iOXagiic acid. (Each is the mean soil SE, each group uses 4 to 6 rats, * P < 0 05; compared with the non-administration group (Dunnett's test)), the results shown in Figure 6, because When ioxaglic acid was administered, it was found that the partial pressure of arterial blood oxygen caused by a decrease in pulmonary function was significantly reduced, and it was understood that nafamostat mesilate (10 mg / kg) could completely suppress the decrease of the partial pressure of oxygen. In addition, the inhibitory effect of nafamostat mesilate on rat pulmonary edema (increased water content, increased Na + content, and decreased K + content) and nafamostat mesilate induced by the iodine contrast agent ioxaglic acid shown in Figure 7 SE, 4 to 6 rats in each group, * P <0.05; comparison with non-administered group (Dunnett's test), t <0.05; ioxaglic acid alone-administered group comparison (Dunnett's test)), showing increased lung water content and increased Na + content in lung tissue caused by ioxaglic acid, that is, the so-called pulmonary edema was found to be inhibited by administration of nafamostat mesilate (10 mg / kg). φ Formulation Example 1 An injection was prepared by the usual method using the following ingredients. nafamostat mesilate 5 0ml. Physiological saline for injection 2ml Formulation example 2 The following ingredients are dissolved in distilled water for injection and freeze-dried '-29- (25) 200306811 Injectables that are dissolved during use. nafamostat mesilate 10.0ml boric acid 1.0 mg dextrose mannitol 20.Omg Formulation Example 3 The following ingredients were dissolved in distilled water for injection and then freeze-dried @ $ into the injection used when dissolved. nafamostat mesilate 10.0ml succinic acid 1.0mg dextrose mannitol 20.Omg Formulation Example 4 The following ingredients were dissolved in distilled water for injection and then freeze-dried to produce an injection for dissolution when used.

nafamostat mesilate 10 0ml benzoic acid l.Omg dextrose mannitol 20.Omg Formulation Example 5 Mix the following ingredients and amounts to make capsules according to the usual method, so that each capsule contains lOO.Omg of nafamostat mesilate. -30- (26) 200306811 nafamo st at mesilat e 1 0 0 0ml lactose 59.0mg crystalline cellulose 334mg carboxymethyl cellulose calcium salt 3.6mg magnesium stearate_4.0mg count 2 0 0 0 m g

Formulation Example 6 The following ingredients and portions were mixed, and a fine granule was prepared according to a conventional method so that 50 mg of nafamostat mesilate was contained per 500 mg of the formulation. nafamostat mesilate 50.0r ng lactose 249.0π d-mannitol 7 5 · Omg corn starch 1 10.On hydroxypropyl cellulose 16 Or ng meter 5 00 On

Formulation Example 7 Weigh out the following ingredients and amounts, and make ointments according to the usual method, so that each mg of ointment contains 10 mg of nafamostat mesilate. -31-(27) 200306811 nafamo st at mesilat e 1. 0g ethylene glycol 20 Og flowing paraffin 5 Og white petrolatum_74.Og 5 ten 1 0 0.0 g

Formulation Example 8 Weigh the following ingredients and amounts, and make a cream according to the usual method, so that each 1 g of cream contains 10 mg of nafamostat mesilate. nafamostat mesilate 1.0 mg ethylene glycol 20.00 g sorbitan sesquioleate 4.0 g petroleum jelly 30.00 g paraffin wax 7.0 g aqueous lanolin 4.0 g methyl parahydroxybenzoate 0 6 g purified water 33.4 g 100.Og

Formulation Example 9 The following ingredients and amounts were weighed out, and a cream was manufactured according to a conventional method so that each 1 g of the cream contained 10 mg of nafamostat mesilate. -32- (28) 200306811 nafamo st at mesilate 1.0 g ethylene glycol 3 0 0 g whale 5.0 g hydrous lanolin 50 g g cetyl alcohol 2 5.0 g self-emulsifying glyceryl monostearate 3.5 g mono 1.5g sorbitan stearate

Glycerin 3.0 g Carboxyvinyl polymer (1% aqueous solution) 5.0 g Potassium hydroxide 0 2 g Methyl parahydroxybenzoate 0 6 g Refined water_20.2 g Total 1 0 0.0 g Formulation example 1 0

Weigh out the following ingredients and amounts to make an emulsion according to the usual method, so that each lg of the emulsion contains 10 mg of nafamostat mesilate. nafamo st at mesilate 1 0 g ethylene glycol 3 0.0 g propylene glycol 6 4.0 g isopropanol_5.0 g total 1 0 0.0 g -33- (29) (29) 200306811 industrial availability '—Naphthyl-4-methyl benzoate or its pharmaceutically acceptable salt for blocking tryptase action is unbelievable.' There have been no reports so far. Surprisingly, from the above experimental results, it is shown that 6'-fluorenyl-2'-naphthyl-4 -carboxylbenzoate or its pharmaceutically acceptable salts, especially 6'-fluorenyl-2 'Nafamostat mesilate, a naphthyl-4-methyl benzoate, has a more potent trypsin-blocking effect (IC50) than the known gabexate mesilate (IC5 () (is 194nM). (0.016nM), this is the first time it is unveiled. Secondly, what's surprising is that nafamostat mesilate is more than 12,000 times more potent than gabexate mesilate. Therefore, the pharmaceutical composition for blocking tryptase of the present invention is selected from the group consisting of systemic allergic reaction disease, aspirin allergic asthma, asthma, interstitial lung disease, interstitial cystitis, allergic bowel syndrome, Atopic skin disease, skin vesicular disease, hypersensitivity, pain, pruritus, odontitis, edema, psoriasis, pulmonary fibrosis, arthritis, periodontal disease, blood clotting disorders, renal interstitial fibrosis, X-rays Contrast agent side effects such as hypervascular permeability or pulmonary edema and hay fever are a therapeutic agent for the treatment or prevention of diseases, which are highly anticipated for future research and development. On the other hand, tryptase selectively activates proteolytic enzyme-activated bodies (PAR-2) that are widely distributed in vascular endothelial cells, digestive tracts, sensory nerves, and bronchi, and so on. Known facts of pain, hyperalgesia, pruritus, inflammation, and edema caused by activin released by sensory nerves. In addition, tryptase increases -34- (30) (30) 200306811 protein permeability of cultured vascular endothelial cells Similarly, the PAR-2 agonist peptide also showed the same effect in this test result, showing that nafamostat mesilate can potently and strongly inhibit trypsin-like effects on the protein permeability of cultured vascular endothelial cells. On the other hand, the side effects of iodine contrast agent-like pulmonary insufficiency or allergic disease are thought to be mainly based on the action of histamine and tryptase from giant cells. In this experiment, iodine contrast agents obviously caused pulmonary vascular permeability to penetrate, and this effect was almost completely inhibited by nafamostat mesilate. On the other hand, although gab ex ate mesilate significantly inhibits pulmonary vascular hyperpermeability, its effect is not complete. The above-mentioned nafamostat mesilate strongly blocks tryptase, and can be expected to be allergic to the above-mentioned diseases that are considered to be related to the tryptase / PAR-2 system, especially neuralgia or postherpetic pain, Treatment and prevention of pruritus, edema and asthma. Therefore, the pharmaceutical composition for blocking and the pharmaceutical composition for inhibition of the present invention are selected from the group consisting of systemic allergic reaction disease, aspirin allergic asthma, asthma, interstitial lung disease, interstitial cystitis, and allergic bowel Symptoms, atopic dermatosis, vesicular dermatitis, hypersensitivity, pain, pruritus, odontitis, edema, psoriasis, pulmonary fibrosis, arthritis, periodontal disease, blood clotting disorders, renal interstitial fibrosis, X-ray contrast agents are effective in treating or preventing diseases such as hypervascular permeability or pulmonary edema and hay fever. [Schematic description] -35- (31) (31) 200306811 Figure 1 shows the block effect of nafamostat mesilate and gabexate mesilate on trypsin-like activity purified from human lungs. Fig. 2 shows a device for measuring protein permeability in cultured vascular endothelial cells. Fig. 3 is a graph showing the effect of tryptase and proteolytic enzyme activated receptor (PAR-2) agonist peptide (SLIGKV) on protein permeability in cultured vascular endothelial cells. Figure 4 is a graph showing the inhibitory effect of nafamostat mesilate on protein permeability invasion by trypsin-like culture in vascular endothelial cells. Fig. 5 is a graph showing the inhibitory effect of nafamostat mesilate on pulmonary permeability in the lungs caused by the iodine contrast agent ioxaglic acid. Figure 6 is a graph showing the inhibitory effects of nafamostat mesilate on the changes in partial pressure of oxygen in the blood of the arteries, the partial pressure of carbon dioxide and the pH caused by the iodine contrast agent ioxaglic acid. Figure 7 is a graph showing the inhibitory effect of nafamostat mesilate on pulmonary edema (increased water content, increased Na + content, and decreased K + content) in rats caused by iodine contrast agent ioxaglic acid. -36-

Claims (1)

(1) (1) 200306811 Patent application scope 1 A pharmaceutical composition for blocking tryptase, characterized in that it contains 6′-pulse-2′-Caiji-4- as represented by the structural formula Methylbenzoate or a pharmaceutically acceptable salt thereof is an active ingredient. 2 The pharmaceutical composition according to item 1 of the patent application, wherein the salt is mesylate. 3 The pharmaceutical composition according to item 1 or item 2 of the patent application scope, wherein the tryptase is human tryptase. 4 A medicinal composition, characterized in that it contains 6'-fluorenyl-2'-naphthyl-4-pulse benzoate or a pharmaceutically acceptable salt thereof as an active ingredient, and is selected for treatment or prevention. From systemic allergic reaction disease, aspirin allergic asthma, asthma, interstitial lung disease, interstitial cystitis, allergic bowel syndrome, atopic skin disease, skin blistering, hypersensitivity, pain, itching Disease, odontitis, edema, psoriasis, pulmonary fibrosis, arthritis, periodontal disease, hemagglutination disorder, renal interstitial fibrosis, x-ray contrast agent side effects of hypervascular permeability or pulmonary edema and hay fever Disease. 5 The pharmaceutical composition according to item 4 of the patent application, wherein the salt is mesylate. 6 If the pharmaceutical composition of item 4 or item 5 of the scope of patent application, -37- (2) (2) 200306811 where the disease is pain, pruritus, interstitial cystitis or allergic bowel syndrome, X-ray contrast agent Side effects include hypervascular permeability or pulmonary edema. 7. —Dilute inhibitory protein-degrading enzyme-activated receptor-2 medicinal composition, which is characterized in that it contains a compound that has a trypsin-blocking effect and forms a pharmaceutically acceptable salt as an effective ingredient. 8. The pharmaceutical composition according to item 7 of the patent application, characterized in that the tryptase is human tryptase, the compound having a blocking effect of tryptase or a pharmaceutically acceptable salt thereof is 6 '-脒-2'-naphthyl-4 monomethyl benzoate or its methanesulfonate. -38-